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The influence of chronic stress on T cell immunity Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) des Fachbereiches für Biologie an der Universität Konstanz vorgelegt von Annette Sommershof April 2010 Table of contents SUMMARY ............................................................................................................................ 1 CHAPTER 1: INTRODUCTION ............................................................................................... 4 THE IMMUNE SYSTEM IN RESPONSE TO STRESS ...................................................................... 5 TCD8+ CELLS IN VIRAL INFECTIONS ...........................................................................................5 DENDRITIC CELLS ....................................................................................................................5 SKIN DENDRITIC CELL TYPES ...................................................................................................6 LANGERHANS CELL MIGRATION...............................................................................................6 LANGERHANS CELL MIGRATION: ROLE OF CYTOKINES .........................................................7 LANGERHANS CELL MIGRATION: ROLE OF CHEMOKINES AND CHEMOKINE RECEPTORS.........8 ANTIGEN PROCESSING AND PRESENTATION BY DENDRITIC CELLS ............................................9 DC SIGNALS FOR CLONAL EXPANSION AND DIFFERENTIATION OF NAÏVE T CELLS ...................11 ROLE OF TCR MEDIATED SIGNALING – SIGNAL 1.................................................................11 COSTIMULATION BY THE B7 FAMILY – SIGNAL 2 ................................................................12 ROLE OF OTHER CORRECEPTORS ........................................................................................13 ROLE OF CYTOKINES - SIGNAL 3.........................................................................................13 EFFECTOR FUNCTION OF TCD8+ CELLS.....................................................................................14 MIGRATION OF T LYMPHOCYTES ...........................................................................................14 MOVING INTO SECONDARY LYMPHOID ORGANS .................................................................15 MOVING OUT – ROLE OF CHEMOKINE RECEPTOR CCR7 AND ADHESION MOLECULES...........15 DIVERSITY OF THE MEMORY T CELL POOL ..............................................................................16 LINEAGE RELATIONSHIP BETWEEN NAIVE, EFFECTOR, AND MEMORY T CELLS ........................17 LYMPHOCYTIC CHORIOMENINGITIS VIRUS (LCMV) .................................................................17 STRESS AND IMMUNITY ....................................................................................................... 19 INTERACTION OF THE IMMUNE SYSTEM AND THE BRAIN - THE HPA AND SNS AXIS ..................19 THE SYMPATHETIC NERVOUS SYSTEM (SNS) .......................................................................19 THE HYPOTHALAMUS-PITUITARY-ADRENAL (HPA) AXIS .....................................................20 BIDIRECTIONAL COMMUNICATION BETWEEN THE CNS AND THE IMMUNE SYSTEM ..............21 GLUCOCORTICOID HORMONES ...............................................................................................22 MOLECULAR MECHANISM OF GC-INDUCED IMMUNOSUPPRESSION .....................................22 CELLULAR MECHANISM OF GC-INDUCED IMMUNOSUPPRESSION .........................................23 REGULATION OF THE HPA AXIS AND GC ACTION UNDER CHRONIC STRESS EXPOSURE..............25 ANIMAL MODELS OF CHRONIC STRESS AND IMMUNITY...........................................................26 SOCIAL STRESS ..................................................................................................................26 THE SOCIAL DISRUPTION STRESS (SDR) MODEL .......................................................................26 I Table of contents CHAPTER 2: ATTENUATION OF THE CYTOTOXIC T LYMPHOCYTE RESPONSE TO LYMPHOCYTIC CHORIOMENINGITIS VIRUS IN MICE SUBJECTED TO CHRONIC SOCIAL STRESS ................................................................................................................................ 28 ABSTRACT .......................................................................................................................... 29 INTRODUCTION ................................................................................................................... 29 RESULTS ............................................................................................................................. 30 DISCUSSION ........................................................................................................................ 40 MATERIALS AND METHODS ................................................................................................ 43 CHAPTER 3: IMPAIRED MIGRATION OF SKIN DENDRITIC CELLS IN RESPONSE TO CONTACT SENSITISATION IN MICE SUBJECTED TO CHRONIC SOCIAL STRESS .................. 48 ABSTRACT .......................................................................................................................... 49 INTRODUCTION ................................................................................................................... 49 RESULTS ............................................................................................................................. 50 DISCUSSION ........................................................................................................................ 54 MATERIAL AND METHODS:................................................................................................. 57 CHAPTER 4: SUBSTANTIAL REDUCTION OF NAÏVE AND REGULATORY T CELLS FOLLOWING TRAUMATIC STRESS ...................................................................................... 59 ABSTRACT .......................................................................................................................... 60 INTRODUCTION ................................................................................................................... 60 RESULTS ............................................................................................................................. 62 DISCUSSION ........................................................................................................................ 67 MATERIAL AND METHODS .................................................................................................. 69 CHAPTER 5: DISCUSSION .................................................................................................. 72 REFERENCES ...................................................................................................................... 78 APPENDIX........................................................................................................................... 97 ABBREVIATIONS ................................................................................................................. 98 RECORD OF ACHIEVEMENT / EIGENABGRENZUNG ............................................................ 100 ACKNOWLEDGMENT ........................................................................................................ 101 II Summary Summary Chronic environmental and psychological stress has long been suspected to increase the susceptibility and outcome of numerous infectious and inflammatory diseases. The release of neurotransmitters (catecholamines) and adrenal hormones (glucocorticoids) has been well documented as the basis for a connection between the central nervous system and peripheral components of the immune system. Glucocorticoids, the end products of stress-induced neuroendocrine pathways and the hypothalamic-pituitary-adrenal (HPA) axis, belong to the most potent anti-inflammatory hormones in the body and serve to control immune responses for example during an infection. However, prolonged or excessive elevation of glucocorticoids as occurring during recurrent or chronic stress can negatively impact various aspects of immune cell functions and may therefore contribute to disease development and progression. The precise mechanisms and course of events leading to the suppression of immune functions during chronic stress and how these effects result in certain diseases remains poorly understood. The aim of the present thesis was to further analyze underlying mechanisms of chronic stressrelated immunosuppression focusing on T cell-mediated immunity. In order to mimic recurrent stress experiences, a well-established mouse model of chronic social stress termed “social disruption stress” (SDR) was chosen for the experiments conducted in chapters 2 and 3. Pharmacological intervention (i.e. the blockage of the respective receptors) in these experiments allowed us to differentiate between the impact of glucocorticoids and catecholamines, the main mediators of stress responses during chronic exposure. Chapter 2 focuses on the impact of chronic social stress on the outcome of virus-specific cytotoxic TCD8+ cell (CTL) responses in mice after infection with lymphocytic choriomeningitis virus (LCMV). Taking into account that the duration of stress exposure and also the timing of stress relative to an immune challenge can greatly impact the outcome of immune alterations, we directly compare different stress procedures. We show that social stress impacts the generation of IFN-γ-producing, virus-specific TCD8+ splenocytes only when applied prior to virus infection. We further demonstrate that during a prolonged stress exposure glucocorticoid hormones strongly impact the proliferation capacity of TCD8+ cells in the spleen of infected mice. This impairment results most probably from reduced TCD8+ activation as well as an impaired cytokine secretion profile. The reduced expansion of TCD8+ cells appears to be organ specific, as we found no such alterations in the inguinal lymph node or in the blood or peripheral tissues such as the liver and lung. A possible explanation for the organ-specific decline in TCD8+ cell expansion could be an altered migration capacity of splenic TCD8+ cells as demonstrated by adoptive T cell transfer experiments. Chapter 3 describes the impact of chronic social stress on the migratory capability of skin dendritic cells (DCs). Skin DC and in particular Langerhans cells are known as critical inducers of cutaneous immune responses. Glucocorticoid-mediated impairment of DC function has been in the focus of recent investigations. To directly investigate the consequences of chronic stress on skin DC function, we performed contact allergen-induced skin sensitization assays using the fluorescent dye fluorescein isothiocyanate (FITC), which 1 Summary allowed us to trace the migration of skin CD11c+ DCs in vivo. Our data reveal that chronic social stress applied prior to skin sensitization suppresses the migratory capability of epidermal CD11c+ DCs to regional lymph nodes. Using an ex vivo ear skin explant model of skin DC migration we further show that the altered migration is presumably a result of an impaired mobilisation of CD11c+ DCs from the skin. In chapter 4 we characterize phenotypic changes in T lymphocyte subsets in the peripheral blood of severely traumatized human patients. Posttraumatic stress disorder (PTSD) is associated with an enhanced susceptibility to various somatic diseases although the exact mechanisms linking traumatic stress to subsequent physical health problems have remained elusive. Our results demonstrate that PTSD patients exhibit a profoundly altered composition of the peripheral T cell compartment characterized by a reduction in naive T lymphocytes, and increased proportion of central (TCM) and effector memory (TEM) cells. Furthermore, we show that subjects with PTSD display a substantial reduction of peripheral regulatory T cells (Treg). To a smaller extent, these findings are also observed in trauma-exposed non-PTSD individuals, indicating a cumulative effect of traumatic stress on T cell distribution. Zusammenfassung Chronischer Stress kann durch Umwelteinflüsse oder psychologische Faktoren hervorgerufen werden und steht seit langem im Verdacht, die Anfälligkeit und den Verlauf von infektiösen und entzündlichen Erkrankungen zu verstärken. Die Vermittlung des Stress-Reizes zwischen dem zentralen Nervensystem (ZNS) und den periphären Komponenten des Immunsystems wird durch die Freisetzung von Neurotransmittern wie beispielsweise den Catecholaminen und adrenergen Hormonen wie den Glucocorticoiden erreicht. Glucocorticoide als Endprodukte der stress-induzierten neuroendocrinen Antwort und der HypothalamusHypophysen-Nebennierenrinden-Achse (HPA) gehören zu den potentesten antiinflammatorischen Hormonen des Körpers und dienen zur Kontrolle von Immunantworten beisspielsweise während einer Infektion. Durch chronischen oder wiederholten Stress kann eine systemische Erhöhung der Glucocorticoid-Konzentrationen eintreten und zu einer Unterdrückung verschiedener Immunfunktionen führen und damit die Entwicklung und den Verlauf von Krankheiten negativ beeinflussen. Allerdings sind die genauen Mechanismen, die zu einer Unterdrückung der Immunfunktionen führen bisher nur wenig verstanden. Das Ziel der vorliegenden Arbeit bestand in der Untersuchung der Mechanismen, die zu einer Unterdrückung des Immunsystems und insbesondere der T-Zell-vermittelten Immunantworten durch chronischen Stress führen. In Kapitel 2 und 3 wurde der Einfluss von wiederholtem, chronischen Stress in einem etablierten Mausmodell (dem so genannten „social disruption stress“ Modell, SDR) untersucht. Die spezifische Hemmung der jeweiligen Rezeptoren erlaubte es, den Einfluß von Glucocorticoiden und Catecholaminen, den bedeutensten Vermittlern der Stressantwort, zu unterscheiden. Kapitel 2 beschreibt den Einfluss von chronischem, sozialen Stress auf die Virus-spezifische, TCD8+-Zell-vermittelte Antwort nach Infektion mit dem lymphozytären ChoriomeningitisVirus (LCMV). Da bereits bekannt ist, dass sowohl die Dauer wie auch der Zeitpunkt der 2 Summary Stress-Prozedur eine erhebliche Rolle bei der Auswirkung auf die Immunfunktionen spielt, wurde in unserem Modell der Einfluss verschiedener Stress-Protokolle direkt miteinander verglichen. Wir konnten zeigen, dass andauernder sozialer Stress die Bildung von Virusspezifischen, IFN-γ-sekretierendenen TCD8+-Zellen beeinträchtigt, allerding nur wenn die Stress-Applikation vor der Virusinfektion erfolgt. Ausserdem konnte gezeigt werden, dass im Falle einer verlängerten Stress-Prozedur Glucocorticoide die Virus-induzierte Proliferation von TCD8+ Zellen in der Milz hemmen. Dabei ist die verringerte Proliferation wahrscheinlich auf eine reduzierte Aktivierung der TCD8+-Zellen sowie eine Hemmung ihrer ZytokinSekretion zurückzuführen. Die gehemmte Proliferation der TCD8+-Zellen scheint dabei Organspezifisch zu sein, da keine Veränderungen in den inguinalen Lymphknoten, im Blut oder periphären Geweben wie der Leber oder Lunge beobachten werden konnte. Ein möglicher Grund hierfür kann in einer veränderten Fähigkeit zur Migration der TCD8+ Zellen liegen, wie in adaptiven TCD8+-Zell-Transfer-Experimenten demonstriert werden konnte. Kapitel 3 beschreibt den Einfluss von chronischem, sozialem Stress auf das Migrationsverhalten von dendritischen Zellen in der Haut. Epidermale und Dermale dendritische Zellen, insbesondere epidermale Langerhans-Zellen, sind bekannt für ihre Beteiligung an der Initiierung von T-Zell-vermittelten Immunantworten in der Haut. In neueren Studien wurde vermehrt eine Glucocorticoid-vermittelte Beeinflussung der Funktion von dentritischen Zellen untersucht. Um den direkten Einfluss von chronischem Stress auf die Funktionen der dendritischen Zellen der Haut zu untersuchen, haben wir eine durch ein Kontakt-Allergen (den Fluoreszenz-Farbstoff FITC) induzierte Haut-Sensibilisierung durchgeführt, was eine Untersuchung der Migration von dendritischen CD11c+-Zellen in vivo erlaubte. Unsere Ergebnisse zeigen, dass sozialer Stress, wenn vor der Haut-Sensibilisierung appliziert, die Migrationsfähigkeit von epidermalen, dendritischen CD11c+-Zellen zu den regionalen Lymphknoten hemmt. Durch die Verwendung eines ex vivo-Ohrhaut Explantations-Modells zur Untersuchung der Migration dendritischer Zellen der Haut konnten wir weiterhin zeigen, dass die veränderte Migration vorwiegend das Ergebniss einer geminderten Mobilisierung von dendritischen CD11c+-Zellen in der Haut ist. In Kapitel 4 beschreiben wir Veränderungen in T-Lymphozyten-Populationen im Blut von schwer traumatisierten, humanen Patienten. Die posttraumatische Belastungsstörung (PTBS bzw. PTSD, aus engl. „posttraumatic stress disorder“) ist mit einer erhöhten Anfälligkeit für diverse somatische Erkrankungen assoziert. Allerdings sind Mechanismen, welche die traumatischen Stressbelastungen mit den physischen Gesundheitsproblemen verbinden bisher nur schwer fassbar. Unsere Ergebnisse zeigen, dass PTSD-Patienten eine erhebliche Veränderung der Zusammensetzung der peripheren T-Zell-Population aufweisen, charakterisiert durch eine Reduzierung von naiven T-Lymphozyten und einen erhöhten Anteil von zentralen und Effektor-Gedächtniszellen (TCM und TEM). Desweiteren konnten wir zeigen, dass PTSD-Patienten eine erhebliche Reduzierung von regulatorischen T-Zellen (Treg) aufweisen. Etwas weniger ausgeprägt konnten diese Befunde auch in traumatisierten, nichtPTSD-Patienten nachgewiesen werden, was auf einen kumulativen Effekt von traumatischen Erlebnissen auf die Veränderungen der T-Zell-Populationen schließen lässt. 3 Chapter 1 Introduction Introduction The immune system in response to stress Innate and adaptive components of our immune system are evolved to protect the body from invading pathogens like disease-causing bacteria, fungi, viruses and parasites. The immune system is not operating autonomously but influences and is influenced by the central nervous system (CNS) through a complex, interacting network of nerves, hormones, and neuropeptides. This interaction is thought to serve as a mechanism to fine-tune many immune responses, for instance to prevent the body from harmful excessive immune responses. CNSimmune interactions also play an important role during stress responses. The stress response is essential for the organism since it facilitates all necessary changes (physiological and behavioral) that allow the body to cope with threatful situations (“fight or flight-response”). However, the same mediators that promote these adaptive responses can negatively influence various functions of the innate and adaptive immune system when this responsiveness becomes excessive or inadequate. Such a dysregulation of the immune system that often occurs under chronic or recurrent stress can have significant implications for infectious and inflammatory disease susceptibility and progression. TCD8+ cells in viral infections Although in vertebrates the innate immune system controls viruses upon initial encounter, it is the specific adaptive immune response that effectively mediates the clearance of viral pathogens. This is mainly achieved by the activation and expansion of antigen-specific effector TCD8+ lymphocytes that are exquisitely refined in their ability to recognise and lyse infected cells presenting viral antigenic peptides on surface major histocompatibility (MHC) class I molecules (1-2). The generation of cytotoxic TCD8+ cells (CTLs) from naïve TCD8+ precursors in turn depends on the interaction with professional antigen-presenting cells (APCs) that in addition to the MHC I/antigen complex, express high levels of costimulatory molecules required for effective activation of naïve T cells (3). In general, antiviral responses to fast-replicating viruses involve the initial activation of TCD8+ cells in secondary lymphoid organs, followed by exponential proliferation and differentiation into effector TCD8+ cells with high cytotoxic capacity (day 3-6 post infection). During the subsequent phase of antiviral immune responses (day 6-8) activated cytotoxic TCD8+ cells leave the secondary lymphatic organs and migrate to the site of infection where they specifically kill infected target cells that express their cognate antigen (1). In the late course of a viral infection (day 8-10) when the majority of infected cells are eliminated and viral clearance is achieved, the effector TCD8+ population diminishes rapidly through extensive cell death, while maintaining a pool of longlived antigen specific memory cells (4). Dendritic cells Dendritic cells (DCs) are a family of highly specialized antigen-presenting cells (APCs), most capable of efficiently activating naïve T cells to initiate antigen-specific TCD8+ cell responses (3, 5). Their functional capacity to capture, process and present antigenic peptides on MHC I molecules is a critical property that is modulated during DC maturation process (3, 5). In most 5 Introduction tissues, DCs are present in a so-called ‘immature’ state, display high endocytic activity but express low levels of MHC and costimulatory molecules and are therefore weak stimulators of naïve T cells (3). Once they have acquired and processed foreign antigens, DCs undergo a number of phenotypic and functional changes resulting in a down-regulation of their phagocytic ability and up-regulation of their antigen-presenting as well as T cell-stimulatory competence (e.g. redistribution of MHC molecules from intracellular endocytic compartments to the DC surface, increase in the surface expression of costimulatory molecules such as CD80 and CD86) (3). Skin dendritic cell types Langerhans cells (LC) are a subtype of dendritic cells (DC), residing in an immature state within the epidermis where they comprise between 1-3% of total epidermal cells to form a dense intraepithelial dendritic network (6). Phenotypically, LCs are distinct from conventional DCs in that they express high levels of langerin, a C-type lectin receptor (CD207) that is a potent inducer of the LC-characteristic Birbeck granules (7-8). LCs also constitutively express the epithelial-cell adhesion molecule EpCAM as well as E-cadherin (CDH1), a homotypic adhesion molecule that anchors LCs to neighboring keratinocytes (9). CD205 (also known as DEC205) is another molecule that is constitutively expressed by LCs and is implicated in antigen capture and processing (10). While LCs represent the most prominent DC population in the epidermis, a number of other DC subsets, including small numbers of plasmacytoid DCs, dermal myeloid DCs (dDC), and the recently described dermal langerin+ DCs (langerin+ dDC) populate the dermal layer of the skin (11-13). Phenotypic characterization of these latter subsets has shown that they are distinct from epidermal LCs, in that they express low levels of the epithelial-cell adhesion molecule EpCAM (14). The two dermal DC populations can be further distinguished phenotypically by their differential expression of CD11b and CD103 (αE-integrin), both expressed at high levels on LC. Taken together, the existence of at least three major subpopulations of DCs in the skin has been established: epidermal langerin+ LCs (EpCAMhigh CD11b+ CD103-), dermal langerin- DCs (EpCAM- CD11bhigh CD103-), and dermal langerin+ DCs (EpCAM- CD11blow CD103+) (11-13). Langerhans cell migration Topical exposure to contact allergens and skin irritants initiates the activation and subsequent maturation of LCs in the epidermis. The maturation of LCs is accompanied by phenotypic and functional alterations, including increased expression of costimulatory molecules such as CD83 and CD86, and MHC II antigens enhancing their respective antigen presenting capability (15). On the other hand, altered expression of various adhesion molecules and chemokine receptors enable their mobilisation from the epidermis and homing to skindraining lymph nodes (LNs) (16). The ability of LCs to migrate from the site of Ag encounter to the area of T cell priming is fundamental for the initiation of cutaneous immune responses 6 Introduction (17-19). Consequently, epidermal mobilisation of LCs and their subsequent LN migration are integral processes, orchestrated by a variety of cutaneous cytokines and chemokines. Langerhans cell migration: role of cytokines IL-1β and TNF-α are thought to be the most important cytokines for the mobilisation and migration of LC towards the skin-draining LNs (16). Intradermal injection of mice with each cytokine results in a rapid egress of LCs from the epidermis and their subsequent accumulation in the draining LNs (20-22). Moreover, systemic administration of either TNFα- or IL-1β-neutralizing antibodies prior to skin sensitization causes an almost complete inhibition of allergen-induced LC accumulation in the LN (23-24). Contact sensitization and skin irritation is known to induce an up-regulation of IL-1β and the de novo secretion of TNFα (25). The current opinion is that topical exposure to allergens induces the secretion of IL-1β by LCs, which in turn stimulates de novo production of TNF-α by keratinocytes (26-27). This view is supported by the fact that the speed of migration induced by IL-1β is slower than that observed with TNF-α (20, 28), as is the observation that IL-1β-induced LC migration can be inhibited by neutralizing anti-TNF-α antibodies (24). Furthermore, IL-1β is believed to provide a second mandatory signal for migration, independent of its role to induce TNF-α secretion. This idea is supported by the observation that TNF-α-induced LC migration is repressed by treatment with neutralizing anti-IL-β antibodies (24). The mechanism by which IL-1β and TNF-α prompt LC migration include altered expression of adhesion molecules and chemokine receptors on LCs along with a differential responsiveness to the relevant chemotactic ligands in the local microenvironment, which pave the way for LCs to traffic to downstream LNs (16). TNF-α-driven processes include the down-regulation of CCR6 (26-27) and E-cadherin (29) on LCs, allowing their detachment from keratinocytes. Once these mobilisation signals have triggered LC detachment, the cells must migrate through the extracellular matrix and traverse a basement membrane before gaining access to the dermal afferent lymphatics. TNF-α induced up-regulation and activation of adhesion molecules, including α6β1 (30) and CD44 (31) which are important for LC interaction with extracellular matrix proteins such as epidermal laminin are suggested to be involved in this process. Other cytokines induced by contact allergens in the epidermis, such as IL-10 have been reported to repress the maturation and mobilisation of LCs by inhibiting the production of IL1β and TNF-α by cells (32-33) and may therefore facilitate LC retention in the epidermis, providing a counterbalance for controlling cutaneous immune responses and inflammation (32-34). 7 Introduction Langerhans cell migration: role of chemokines and chemokine receptors In addition to acquiring the capacity to detach from their epidermal environment, maturing LCs must also alter their chemokine receptor repertoire to one endowing their movement towards skin draining LNs. Immature LCs express a number of chemokine receptors (CCR1, CCR2, CCR5, CCR6 and CXCR1) by which they are attracted to sites of inflammation where they capture and process antigens (26). Upon maturation, LCs lose their responsiveness to most of the inflammatory chemokines through receptor down-regulation but up-regulate other G protein-coupled chemokine receptors, such as CCR4 and CCR7 (35). By up-regulation of CCR7, LCs acquire responsiveness to the constitutively expressed and selective ligands SLC (secondary lymphoid organ chemokine or CCL21/6C-kine) and ELC (Epstein-Barr virus-induced molecule 1 (EBI-1) ligand chemokine or CCL19/MIP-3β) that allow their homing to skin-draining LNs. The important role of CCR7 in the migration of LCs to the draining LNs is well established. Evidence has been drawn from the observation that LCs fail to migrate to LNs in CCR7-deficient (36) and plt (paucity of lymph node T cells) mutant mice, which lack both CCR7 chemotactic ligands SLC/CCL21 and ELC/CCL19 (37). Although plt mice exhibit a substantial reduction of LC migration to the skin-draining LNs after allergen sensitization, their emigration from the epidermis is not compromised (36-38). Furthermore, antagonizing the CCR7 ligands SLC/CCL21 and ELC/CCL19 in WT mice does not abrogate migration from the epidermis to the dermis (35). Interestingly, CCR7 ligands are differentially expressed in mice, with both CCL21 isoforms, CCL21-Ser and CCL21-Leu expressed predominantly in the afferent lymphatics, whereas CCL19 and CCL21-Ser localized to the LN paracortex (39). Therefore, it seems reasonable to assume that CCR7 is not only involved in guiding LCs into the dermal lymphatic vessels but might also control LC access deeper into the LN cortex (16). Taken together, these findings have lead to the following conclusions: First, CCR7 regulates the entry of LCs into the dermal lymphatic vessels and their migration to the LN paracortex rather than their mobilisation from the epidermis. Second, CCR7 mobilisation of LCs from the epidermis and the subsequent migration to the dermis occurs in a CCR7-independent manner. Recent evidence suggests that CXCR4 and its ligand CXCL12 are crucial for LC migration from the epidermis to the dermis (35, 40). Consistent with this idea, migration of LCs from the epidermis to the dermis is abrogated by CXCR4 and CXCL12-blocking antibodies (35). Moreover, epicutaneous sensitization increases CXCR4 expression on LCs, whereas the CXCR4 ligand, CXCL12, is concomitantly up-regulated in dermal lymphatics (40). Based on these findings, a two-step model for LC migration has been proposed: in a first step LCs exit the epidermis and migrate to the dermis, a process that is CXCR4-CXCL12 dependent. In a second CCR7-regulated step, LCs gain access to the dermal lymphatic vessels and migrate towards the LN (41) (Figure 1). 8 Introduction Figure 1: Migration of epidermal Langerhans cells and dermal DCs via afferent lymphatics to draining LNs The scheme illustrates a proposed model for the migration of epidermal LC via afferent lymphatics en route to the LN. The migratory cascade can be divided into discrete steps, initiated by an inflammatory mobilizing signal (e.g. by epicutaneous contact allergen application) that induces the secretion of IL-1β by LCs, which in turn stimulates TNF-α production by keratinocytes resulting (1) in the detachment of LC from epidermal keratinocytes, (2) interstitial trafficking from the epidermis via the basement membrane to the dermis, (3) transit via intercellular spaces between afferent lymphatic endothelium cells, and (4) migration through the afferent lymph vessels towards draining LNs. CXCR4-CXCL12 interaction controls LC migration from the epidermis to the dermis, while CCR7-CCL21 triggers the recruitment of LCs and both dermal DC populations towards lymphatic vessels. Furthermore, the transit of each DC population via the afferent lymphatics is mediated by a CCL19/21 gradient. Antigen processing and presentation by dendritic cells Virus-derived peptides presented by DCs to TCD8+ cells via MHC I molecules result from either cytoplasmatically synthesized proteins (i.e. by viral protein synthesis in the cytoplasm) or endocytosed exogenous proteins that are released from the endosomal compartment into the cytoplasm (3, 42). These peptides are marked in the cytosol for degradation via ubiquitin conjugation and are subsequently digested by the proteasome. The proteasome is a multisubunit ATP-dependent protease that plays a major role in the degradation of foreign antigens (e.g. viral, bacterial, or fungal), but is also important for normal turnover of cell proteins in the cytosol (43). Only a small percentage of the peptides released after proteasomal digestion in the cytosol has an optimal binding length for MHC I molecules and therefore further processing by cytosolic aminopeptidases such as the leucine aminopeptidases (LAPs) (44), puromycin-sensitive aminopeptidases (PSAs) and the bleomycin hydrolase (BH) takes place (45). The generated peptides are then taken up in the cytosol by an endoplasmic reticulum-resident heterodimeric ATP-dependent peptide transporter associated with antigen processing (TAP) and are 9 Introduction translocated into the lumen of the endoplasmic reticulum (ER). The TAP transporter constitutes only one subunit of the macromolecular peptide-loading complex composed of TAP1/2, tapasin, and the assisting proteins calreticulin and ERp57. Tapasin acts as a bridging molecule linking TAP and MHC I complexes, but also functions by stabilizing the TAP complex, retaining unloaded MHC I molecules in the ER, and controlling the quality of MHC I-bound peptides (46). Some precursor peptides are further trimmed in the ER by the IFN-γinducible aminopeptidase ERAP or ERAAP (47-48). The peptides are then loaded onto newly synthesized MHC I/β2-microglobulin dimers and are rapidly transported via the Golgi complex to the cell surface, where they are presented to the T cell receptor (TCR) on TCD8+ cells (Figure 2). Compared to the numerous peptides encoded by viral pathogens only a small subset is able to generate a specific TCD8+ cell response, with various factors determining whether a specific antigenic epitope is immunodominant or subdominant, including the efficiency of antigen processing, the binding affinity of the peptide for the MHC I molecule, and the magnitude of naïve TCD8+ cells responding to its cognate MHC/peptide complex (49). In the presence of the inflammatory cytokines interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) the proteasomal activity and antigenic peptide production is altered, resulting in a gradual replacement of three constitutive catalytic β-subunits β1 (δ), β2 (MC14), and β5 (MB1) of the 20S proteasome by their homologous subunits β1i (LMP2), β2i (MECL1) and β5i (LMP7) to form so-called ‘immunoproteasomes’ (50). As a consequence, cleavage preferences of the proteasome are markedly changed (51-52), resulting in an altered spectrum of epitopes presented to TCD8+ cells. With regard to one of the most studied viral model systems, the murine lymphocytic choriomeningitis virus (LCMV) infection, formation of the immunoproteasome is well established to induce an altered TCD8+ cell response, favoring the generation of TCD8+ cells specific for the LCMV glycoprotein-derived epitope GP33-41, whereas for instance GP276-286 specific TCD8+ cells are less efficiently generated (53-54). 10 Introduction Figure 2: Scheme of the classical MHC class I-restricted antigen processing and presentation by DCs Cytosolic viral proteins are marked for degradation by ubiquitination and are subsequently degraded into peptide fragments by the proteasome. The peptides, partially further processed by cytosolic aminopeptidases, are then transported via the TAP into the lumen of the endoplasmatic reticulum (ER) where they can bind to nascent MHC class I molecules associated with β2-microglobulin (β2m). Assisting proteins of the peptide-loading complex like tapasin, calreticulin and ERp57 increase the efficiency of peptide loading. The complex of MHC I heavy chain, β2m and peptide fragment then leaves the ER and is transported via the Golgi to the cell surface, where it can be recognized by the TCR of TCD8+ cells. The combinatory signal of cognate MHC/peptide complex and costimulatory molecules provided primarily by B7 molecules induces the stimulation of naive TCD8+ which become activated and initiate a program of clonal expansion and effector differentiation. Fully activated cytotoxic effector TCD8+ (CTL) cells mediate their function by secretion of effector molecules like perforin and granzymes to induce apoptosis in their target cells. DC signals for clonal expansion and differentiation of naïve T cells Role of TCR mediated signaling – signal 1 The activation of naive T cells is triggered through engagement of the TCR/CD3 complex and its cognate MHC/peptide complexes on DCs. One of the most immediate consequences of T cell receptor (TCR) stimulation is the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAM) within the associated cytosolic TCR-ζ and CD3-γ,-δ, -ε chains by the src family protein kinases p56lck and p59fyn (55). While p59fyn is weakly associated with the cyoplasmatic domain of the ζ and CD3 chains, p56lck is associated with the cytoplasmatic domain of the co-receptor molecules CD4 or CD8. Binding of the MHC/peptide complex to its cognate TCR complex and co-receptors results in the clustering of the latter and helps to stimulate signal transduction by bringing p56lck together with ITAMS in the cytoplasmatic domain of the TCR complex. The lymphocyte common antigen CD45, a receptor-linked protein tyrosine phosphatase plays a crucial role for supporting signal transduction from 11 Introduction TCRs by catalysing the tyrosine dephosphorylation of the positive regulatory autophosphorylation sites of p56lck and p59fyn, thereby activating the protein kinases (56-57). TCR-ζ chain phosphorylation by p56lck and p59fyn then recruits the protein kinase ZAP-70 to the receptor complex. ZAP-70 becomes activated and in turn phosphorylates the scaffold proteins LAT (linker of activated T cells) and SLP-76. Phospholipase C-γ (PCL-γ) is one of the key signalling molecules recruited by the phosphorylation of LAT and SLP. Activated PCL-γ initiates different key downstream signalling pathways including 1) protein kinase C (PKC) which subsequently activates the transcription factor NFκB; 2) a Ras/MAP kinase cascade and downstream induction of the transcription factor AP-1, and 3) the phosphatase calcineurin pathway that activates the nuclear factor of activated T cells (NFAT) (58). All three transcription factors (NFκB, AP-1, and NFAT) can bind to distinct cis-regulatory elements in the promotor region of the interleukin-2 (IL-2) gene and are essential to activate its transcription (59-60). IL-2 is one of two major growth hormones for T cells and it sustains proliferation of effector TCD8+ cells in an autocrine manner when produced by TCD8+ cells (61). IL-2 is also provided by activated TCD4+ cells to support clonal expansion and acquisition of effector function by the TCD8+ cells (27-29). Activation of PKC through stimulation of the TCR complex induces expression of CD69 (Leu-23) via NFκB activation (62). CD69 is one of the earliest inducible cell surface molecules newly synthesized and expressed during T cell activation. The induction of CD69 results in increased transcription of IL-2, TNF-α, INF-γ, and up-regulation of CD25 expression (62-65), the IL-2Rα chain that enables high-affinity binding of IL-2. Costimulation by the B7 family – signal 2 Stimulation of the TCR complex alone is not sufficient for induction of T cell activation, proliferation, and survival but is controlled by several factors that are provided by the presence of costimulatory signals (66). In the earliest stages of TCD8+ activation this costimulatory signalling is provided through B7.1 (CD80) and B7.2 (CD86) molecules expressed on mature DCs that bind the transmembrane glycoprotein CD28 which is constitutively expressed on T cells (67). The importance for costimulation is emphasized by findings that stimulation of TCD4+ Th1 or TCD8+ cells in the absence of CD28 induces a state of anergy. Anergic T cells are characterized by reduced cytokine synthesis (including IL-2), a lack of proliferation, and failure to differentiate into effector cells when reencountered with their cognate antigen (68-69). The biological consequences of B7/CD28 costimulation are numerous and include control of the T cell cycle, survival and differentiation, as well as amplification of the membraneproximal signalling generated by TCR ligation (70). B7/CD28 costimulation in the presence of a TCR signal results in increased transcription of the IL-2 gene and up-regulation of CD25 expression (71-73). TCD8+ cells from CD28-deficient mice exhibit an impaired proliferative response and IL-2 secretion in response to mitogen stimulation, which is only partially restored by the addition of exogenous IL-2 (74). B7/CD28 signalling further promotes T cell 12 Introduction division by mediating the subsequent progression of T cells into the S phase via both IL-2dependent and IL-2-independent regulatory mechanisms (75). In addition, CD28 can promote T cell survival by enhancing the nuclear translocation of NF-κB, which positively effects the expression of anti-apoptotic genes including Bcl-xl (76). Role of other correceptors In addition, inducible costimulatory molecules including CD40, 4-1BB, OX40, inducible costimulator (ICOS), and LFA-1 prolong or sustain activation of T cells (57). CD40/CD40Ldependent signaling for instance enhances B7 expression on DCs (11–13), thereby increasing the level of costimulation available to the T cells. OX40 (CD134) and 4-1BB (CD137) members of the TNFR family provide costimulation upon engagement with their ligands OX40L and 4-1BBL, and the latter preferentially induces TCD8+ proliferation and production of IFN-γ (77). Other counter-receptors such as cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) provide inhibitory signals, thereby limiting the expansion and activation of T cells. CTLA-4 is engaged by both B7-1 and B7-2 ligands and its function involves negative signalling (78-79) and competitive antagonism of B7/CD28-mediated costimulation (80), resulting in a decrease in proliferation and IL-2 production (81). Role of cytokines - signal 3 Several studies support that the strength and duration of TCR and costimulatory signalling determines the extent of subsequent TCD8+ clonal expansion (82-85). This concept is supported by the finding that T cell proliferation can be induced within 6 hours of stimulation if naïve T cells are stimulated with high dose of antigen in the presence of co-stimulation, but requires as long as 40 hours if stimulation occurs in the presence of low doses of antigen and costimulation (86). However, after an initial single period of TCR/CD28 stimulation, it is assumed that TCD8+ cells initiate a program for their autonomous clonal expansion and development into functional effector TCD8+ cells without a requirement for continued signaling through the Ag receptor (87). This view is emphasized by the observation that in vitroactivated TCD8+ cells undergo further short-term expansion and acquire full effector function when these cells are transferred in vivo into an antigen-free environment (88). Inflammatory cytokines such as IL-12 or type I IFN (IFN-α or IFN-β) produced by macrophages and/or activated DCs respectively synergize with signals from the TCR and costimulatory receptors, thereby critically influencing the continued TCD8+ cell division and even more important their development into fully differentiated effector cells (83, 88-92). In the absence of these additional signals (“signal 3 cytokine”), TCD8+ clonal expansion can be compromised due to poor survival of the expanding cells that also do not acquire cytolytic activity or the ability to produce IFN-γ. For example, type I IFNs act directly on TCD8+ cells to allow clonal expansion in response to LCMV infection. This was demonstrated by examining the responses of adoptively transferred TCR transgenic TCD8+ cells specific to the GP33-41 13 Introduction epitope of LCMV. In comparison to wildtype TCD8+ cells, clonal expansion of cells that were deficient for the type I IFN receptor was reduced by greater than 99%, and this outcome was shown to be due to poor survival instead of a proliferation defect (93-94). Other investigators have highlighted the role of IL-2 for effector differentiation in the later phase of clonal expansion. For instance, expansion of IL-2Rα-/- TCD8+ cells appears to be normal in secondary lymphoid organs after viral stimulation while being impaired in peripheral, non-lymphoid tissues (61). Effector function of TCD8+ cells Fully differentiated effector TCD8+ cells control virus infections by secreting effector cytokines such as TNF-α (95) and in particular IFN-γ (96) and by using either or both of two cytolytic pathways (97-98). The first, a granule exocytosis pathway, is mediated by perforin and two serine proteases (granzyme A and granzyme B) that act by initiating a caspase cascade in the target cell, leading to apoptotic death (99-100). Perforin mainly exerts its function of causing cell lysis by penetrating the target cell membrane (101). Recent work however has suggested that perforin rather functions by enabling the granzymes to escape from endosomes into the cytosol of the target cell (102-103). The second is a Fas-mediated apoptotic pathway that functions by binding of the Fas ligand (FasL) on TCD8+ cells to the Fas-receptor on target cells. Perforin-dependent cytotoxicity mediated by effector TCD8+ cells has been shown to be crucial for clearance of non-cytopathic viruses such as LCMV (104-105), while the Fas-dependent pathway seems not essential for clearance of this virus (106). The precise mechanisms how IFN-γ exerts its antiviral effects are not fully understood. It is thought that IFN-γ acts mainly by restricting intracellular viral replication (107), thereby reducing the rate with which a virus establishes productive infection in new host cells. A role for TCD8+ cell-derived IFN-γ as an essential effector mechanism in the control of acute LCMV infection has been confirmed by the observation that viral clearance is delayed in IFN-γ receptor-deficient mice (62). Similarly, treatment with neutralizing anti-IFN-γ mAb has been demonstrated to result in reduced virus elimination from the spleen and liver, which is accompanied by a decreased generation of effector TCD8+ cells (108-109). Migration of T lymphocytes Under steady state non-inflammatory conditions naïve T cells constantly circulate through the bloodstream, the secondary lymphoid organs including spleen, mesenteric and peripheral lymph nodes (LNs), and then subsequently return to the bloodstream. This process is directed by various adhesion molecules and chemokine receptors (110). The circulatory path continues until activation and differentiation into effector TCD8+ cells in secondary lymphoid tissues. Effector TCD8+ cells then acquire the capacity to traffic to inflammatory peripheral tissues. 14 Introduction Moving into secondary lymphoid organs Role of adhesion molecules Naïve T cells leave the blood and enter peripheral lymph nodes via specialized postcapillary vessels called high endothelial venules (HEV) in a complex process that involves rolling, adhesion and extravasation through the endothelium. The interactions of the lymphocyte adhesion receptor L-selectin (CD62L) and the β2 integrin LFA-1 (CD11a/CD18) with their respective ligands, the vascular adressins (e.g. GlyCAM-1 and CD34) and intercellular adhesion molecule ICAM-1/-2 play a key role in this process (110). In contrast to LNs, HEVlike vessels are not present in the spleen. T cells access the splenic red pulp directly from the blood while subsequently traversing the boundary marginal sinus and entering the T cell zones (the periarteriolar lymphatic sheath - PALS) of the white pulp in a CD62L-independent manner (111-113). Role of the chemokine receptor CCR7 The chemokine receptor CCR7, expressed at high levels on naïve T cells and activated mature DCs plays a key role in the recruitment of T cells into lymph nodes and the splenic white pulp (114-115). The secondary lymphoid organ chemokine SLC/CCL21 is constitutively expressed by cells of the HEVs and at lower levels by stromal cells within T cell zones of the spleen, lymph nodes, and Peyer's patches (114, 116-117). Expression of ELC/CCL19, the second ligand for CCR7 is present in cells throughout the T cell zones of secondary lymphoid tissues but does not appear to be produced by HEVs (118). The importance of CCR7 and its ligands in vivo has been demonstrated in mice lacking expression of CCR7 and “paucity of lymph node T cell” (plt/plt) mice that lack the SLC/CCL21 and ELC/CCL19 genes expressed in lymphoid organs. Both animal models exhibit greatly reduced migration of T cells into LNs and have further shown to display an abnormal distribution in secondary lymphoid organs (36, 119). For instance, T cells do not accumulate in the white pulp of the spleen where T cells are normally located, instead they accumulate around the sinuses in the red pulp where red blood cells are usually found. Once present in T cell zones of secondary lymphoid organs, naïve T cells continue to migrate through this area, which enables them to screen DCs for their cognate antigen. The expression of ELC/CCL19 by activated DCs within the T cell zone is thought to enhance T-cell attraction, thereby providing an optimal co-localization of antigen-presenting DCs and naïve T cells resulting in a more efficient T cell priming and effector generation (120). Moving out – Role of chemokine receptor CCR7 and adhesion molecules Once fully activated TCD8+ cells lose their attraction for the lymph node and splenic T cell compartments by simultaneous down-regulation of secondary lymphoid organ homing receptors such as CD62L and CCR7 (121). The up-regulation of a new range of surface molecules such as CD44, LFA-1 and/or α4β1 integrin enables them to migrate to sites of inflammation in non-lymphoid tissues like the liver and lung. These traffic signals that direct 15 Introduction effector TCD8+ to peripheral tissues are highly complex and are implicated to be organ-specific (121). The distinct migration pattern of naïve and effector TCD8+ cells in correlation to their CCR7 expression profile has been confirmed in adaptive transfer experiments using TCR-transgenic TCD8+ cells from mice specific for the GP33 epitope of LCMV. Only naïve (CCR7+), but neither memory nor effector TCD8+ (CCR7- CD62low CD44high) cells homed to LNs of noninfected recipient mice, while similar numbers of naïve, memory, and effector cells entered the spleen, although with distinct splenic locations: naïve cells predominantly localised in the PALS, while effector cells were completely excluded from these areas but instead accumulated in the red pulp (122). Diversity of the memory T cell pool When TCD8+ responses have occurred and the antigen is cleared from the system, most of the Ag-specific cytotoxic TCD8+ cells (90-95%) undergo programmed cell death and only a small fraction (5-10%) survives and differentiates into long-lived, antigen-specific memory TCD8+ cells. These memory cells rapidly re-acquire effector functions without needing a third signal (90, 123). Memory T cells are heterogeneous and can be divided in terms of their surface phenotype, development, effector function, and trafficking properties (124). The existence of at least three different subpopulations of memory cells has been established: central memory T cells (TCM), and two distinct types of effector memory cells that are distinct from central memory T cells by expressing low levels of the lymph node homing receptor CCR7. These two populations of effector memory cells can further be distinguished by their differential surface expression of CD45RA, the high molecular weight isoform of the receptor-type protein tyrosine phosphatase CD45 (124). Central memory cells (TCM) are CD45RO+ memory cells that constitutively express CCR7 and CD62L; both receptors are required for their extravasation through HEVs and the migration to T cell areas of secondary lymphoid organs. They recirculate between the blood and the lymphoid compartment and are thought to provide a pool of antigen-experienced cells with a high proliferative capacity but a lower activation threshold than naïve cells, thus allowing for a more rapid generation of effector cells during recall responses (125). In contrast, effector memory T (TEM) cells have lost the constitutive expression of CCR7 but have acquired chemokine receptors and adhesion molecules, allowing their homing to peripheral tissues and to sites of inflammation. TEM cells are thought to enable a recall response in the tissue, as these cells survey non-lymphoid organs in search of cognate antigens and, while able to produce IFN-γ upon antigen recognition, are only moderately cytotoxic (125). The peripheral TEMRA population (124) is most prominent within TCD8+ cells, while within TCD4+ cells only few TEMRA cells (1-2% of TCD4+ cells) can be detect. Like the TEM population, TEMRA cells have lost constitutive expression of CCR7, but re-express CD45RA. Functionally they 16 Introduction share properties with the TEM population in that they display an immediate effector function and low proliferative capacity. Compared to TEM cells they exhibit an even lower expansion potential and higher expression of perforin and are therefore considered to be “terminally differentiated” effector memory cells (124). Lineage relationship between naive, effector, and memory T cells Longitudinal studies performed in mice have suggested a linear differentiation pathway for T cell development, whereby memory T cells are direct offspring of effector cells (126). In light of this opinion, a linear differentiation pathway with TNaïve Æ TEffector Æ TEM Æ TCM differentiation has been demonstrated following acute LCMV infection in mice (127-128). This model proposes that memory T cell development does not occur until the antigen is removed or greatly decreased in concentration. These results are in contrast to the finding of a persistence and stability of both TEM and TCM subsets in humans (129). Figure 3: Schematic model of T cell differentiation according to the “progressive differentiation model” According to the model of progressive T cell differentiation, the strength and duration of antigenic stimulation as well as the type and amount of cytokines offered to naïve T cells during their priming phase will either favour their differentiation into short-lived effector T cells (TEff) or into cells with a memory phenotype that are devoid of immediate effector function - with strong signals (as occurs during the early stage of immune responses) turning differentiation towards (TEff) and weak signals (late stages of immune responses) imprinting central memory T cells (TCM) (dashed arrows indicate potential contributions of cytokines). After elimination of their cognate antigen some of the effector cells (TEff) will persist as effector memory cells (TEM). Thus, both memory cell types (TCM and TEM) are maintained in the memory pool, with TEM mediating immediate protection in non-lymphoid tissues, and TCM cells mediating reactive memory and home to T cell areas of secondary lymphoid organs. In humans and in some settings of viral infection in mouse models, a “progressive differentiation model” has been proposed in which naïve T cells can directly develop a memory stage without traversing an effector state (85, 124, 130). According to this model the 17 Introduction differentiation depends on the applied signal strength and duration of the stimulation during the priming period (Figure 3). Short duration of antigenic stimulation is thought to favour the development of an intermediate level of differentiation that will persist as TCM cells, whereas a longer duration of stimulation favours the differentiation into TEffector cells (124). Next, at the end of the immune response, intermediates give rise to TCM cells, whereas TEM arise from fully differentiated TEffector cells. This model postulates that memory progenitor cells are already detectable early during immune responses concomitant with the development of an effector T cell pool and that TCM cells can further differentiate into TEM cells following Ag stimulation and into TEMRA in response to homeostatic cytokines (131). Lymphocytic Choriomeningitis Virus (LCMV) The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a prominent model to study immunological mechanisms of both acute and persistent viral infection. The natural host of the noncytopathic virus is the mouse. LCMV is an enveloped virus with a bisegmented negative-strand (NS) RNA genome. The viral genome encodes four proteins on two ambisense RNA sequences, the L (long) and S (short) RNA segments which have an approximate size of 7.2 and 3.4 kb, respectively (132-133). The S RNA encodes the 63 kDa nucleoprotein (NP) and the 75 kDa glycoprotein precursor (134). The glycoprotein precursor (GPC) is further processed to a 6 kDa signal peptide and the two glycoproteins GP-1 (40 to 46 kDa) and GP-2 (35 kDa), which build the spikes on the virion envelope as trimer or tetramerstructures (135). Host cell infection involves the interaction with the α-dystroglycan receptor (136) and internalisation of the virions within vesicles, followed by the GP-mediated fusion with cellular membranes and delivery of the nucleocapsids into the cytoplasm. The L RNA segment codes for the 200 kDa virus-specific RNA polymerase (L) and an 11 kDa RING finger protein (Z) (133). Infection of C57BL/6 (H-2b) mice with LCMV induces a strong and protective cytotoxic T cell (CTL) response. In C57BL/6 mice, this response is strongly dominated by CTLs specific for the GP-derived epitopes GP33-41/Db and GP34-41/Kb, the NP-derived NP396-404/Db epitope (137), and the RNA polymerase (L)-derived epitope L455-463 (138). In addition the generation of CTLs against the subdominant epitopes GP276-286/Db, GP92-101/Db, GP118125/Kb (137) and the recently identified GP70-77, NP166-175, NP235-243 (139), GP166173, GP221-228, GP365-372 and GP44-52 (138) have been characterized. Intraperitoneal (i.p.) or intravenous (i.v.) infection of adult immunocompetent mice with low doses of LCMV-WE (200pfu i.v.) results in a well-characterized acute infection during which the virus replicates systemically in tissues including the spleen, leading to a peak virus titer on day 4 after infection. Both type I (α and β) IFNs as well as IFN-γ are induced early after infection and play a role in controlling viral replication. IFN-α and IFN-β secretion occurs in close correlation with the activation of NK-cells. However, several lines of evidence indicate that NK cells are not capable of controlling the virus infection in vivo (140-141). Experiments with CD4-deficient or CD4-depleted mice further demonstrated that T help is not important 18 Introduction for the LCMV-specific CTL response in the acute phase (142-143). Virus-specific antibody production occurs early after infection, whereas neutralising antibodies can be detected 20 to 60 days after infection and are important for a long-term elimination of the virus (144). LCMV-specific CTL response peaks between day 7 to day 9 after infection, leading to effective elimination of virus infected cells (145). The indispensable role of CD11c+ DCs during TCD8+ cell priming is well established and very limited DC numbers (as few as 1000) are sufficient to generate potent CTL activation (146). Moreover, conventional CD11chigh CD8+ splenic DCs (sDC) are thought to be the most important DC subpopulation for TCD8+ cell priming and have been shown to efficiently present Ag to and stimulate the proliferation of naive LCMV-specific TCD8+ cells (147). Stress and immunity The pioneering experimental report on an interaction between psychological stress and the immune system was published by Selye in 1936, showing that chronic stress results in an atrophy of the thymus (148). Since then, numerous epidemiological studies have revealed that severe psychological stress has strong suppressive effects on the immune system (149). Such suppression of the immune system has significant implications on disease susceptibility and progression. Investigations in humans have revealed that chronic stress contributes to many illnesses including cardiovascular diseases and cancer (150-151). Moreover, substantial evidence has linked chronic or recurrent exposure to stress with exacerbation of inflammatory and autoimmunity diseases such asthma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and psoriasis (152-153) and an increased susceptibility to infectious diseases. The relationship between psychological stress and a higher vulnerability to infectious diseases has also been confirmed experimentally by laboratory stressors demonstrating an impaired responsiveness to Hepatitis B (154-157) and influenza virus vaccination (158-159). In addition, it was shown that human volunteers who were inoculated with five different strains of respiratory viruses showed a dose-dependent relationship between stress and clinical symptoms after infection (160). Interaction of the immune system and the brain - the HPA and SNS axis The hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS) are the two major pathways by which the immune system is modulated during psychological stress (Figure 4). The sympathetic nervous system (SNS) The sympathetic nervous system, a major component of the autonomic nervous system, is a fast-acting response to stress that can be detected within seconds after a stress stimulus. The SNS exerts its function by the release of the sympathetic neurotransmitters, the catecholamines. The SNS originates in the central nervous system (CNS) (in nuclei within the brain stem and spinal cord) and gives rise to preganglionic efferent fibres that leave the CNS through the thoracolumbar region of the spinal cord (161). Most of the sympathetic 19 Introduction preganglionic fibres travel to ganglia located in the paravertebral chains, where they synapse with postganglionic neurons. From there, the long postganglionic neurons extend across most of the body and their nerve terminals innervate nearly every organ in the body including primary and secondary lymphoid organs (162-163). In general, T cell zones, macrophages and plasma cells are richly innervated, while follicular zones of B cells are poorly innervated (162). These postganglionic sympathetic neurons are noradrenergic fibres, meaning that they act by locally releasing norepinephrine (NE). Additionally, preganglionic sympathetic fibres that end in the adrenal medulla secrete acetylcholine, which activates the secretion of epinephrine (adrenaline) and norepinephrine (NE, noradrenaline) directly into the blood stream, where they exert their action as circulating hormones (161). The adrenal medulla, unlike the postganglionic sympathetic nerve terminals, releases mainly epinephrine, and to a much lesser extend NE. This response is also known as the sympathetic adreno-medullary response of the SNS. Thus, the principal end products of the SNS are catecholamines (NE and epinephrine) with norepinephrine acting both as a neurotransmitter when released via nerve fibres and as a hormone when it is released by the adrenal medulla into the blood along with adrenalin (161). The hypothalamus-pituitary-adrenal (HPA) axis The activation of the hypothalamus-pituitary-adrenal (HPA) axis takes place somewhat slower (usually within 3-5 min of stress onset). The main components of the HPA axis are the paraventricular nucleus (PVN) in the hypothalamus, the anterior pituitary gland located at the base of the brain, and the adrenal glands (164). Upon activation of the HPA axis corticotrophin-releasing hormone (CRH) and arginine-vasopressin (AVP) are secreted from the PVN of the hypothalamus into the hypophyseal portal blood, which in turn stimulate the expression of adrenocorticotropin hormone (ACTH) in the anterior pituitary gland. CRH seems to play a permissive role in ACTH secretion, whereas AVP has synergistic or additive effects, but very little ACTH secretory activity on its own. ACTH then circulates in the bloodstream where it acts on the adrenal cortex to induce the expression and release of adrenal steroid hormones, in particular glucocorticoids (GCs) into the blood (for an overview see (165)). Glucocorticoids (cortisol in humans and most mammals, corticosterone in rats and mice) represent the final effector molecules of the HPA axis that mediate their function primarily by acting directly on immune cells, which they access via the blood. In addition, GCs play an important role in regulating the activity of the HPA axis under basal and stress conditions, by exerting a negative feedback control directly on the pituitary and also on the synthesis and secretion of CRH and AVP. For example GCs potently inhibit pituitary ACTH secretion and down-regulate the action of CRH through binding to their receptors (166). Moreover the secretion and effects of CRH and AVP are influenced by neurotransmitters in the hypothalamus. 20 Introduction Figure 4: Signalling between the central nervous system (CNS) and the immune system through the HPA axis and the SNS The hypothalamus-pituitary-adrenal (HPA) axis exerts most of its influence systemically through a release of glucocorticoid hormones (GC). The first part of this axis, the hypothalamus, is located in the forebrain and neuroendocrine cells in the hypothalamus release corticotropinreleasing hormone (CRH) that stimulates the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary. The adrenal cortex responds to ACTH by releasing glucocorticoid hormones into the circulation. The sympathetic nervous system (SNS) transmits sympathetic information to peripheral targets by releasing norepinephrine (NE) from noradrenergic nerve terminals that end in all primary and secondary lymphoid organs, and/or systemically by releasing epinephrine (along with some NE) from the adrenal medulla into the circulation. Dotted lines represent negative regulatory GC feedback pathways, blue lines represent bi-directional communication of peripheral immune events to the brain that involve the secretion of inflammatory cytokines like TNF-α, IL-1 and IL-6. (Figure modified from Esther M. Sternberg, Nature Reviews Immunology, 2006.) Summing up, the CNS regulates the immune system through two major classes of effector molecules: glucocorticoids, which are regulated in the hormonal stress response by the HPA axis, and the catecholamines norepinephrine and epinephrine, which are released either by the sympathetic adreno-medullary system or via postganglionic nerve fibres. Both branches closely interact with each other and have positive reverberating feedback loops at different levels. For instance, reciprocal neural connections exist between the CRH and noradrenergic neurons, with CRH and NE stimulating each other. Bidirectional communication between the CNS and the immune system Bidirectional communication between the CNS and the immune system allows the immune system to signal to the brain through neural and humoral routes. In fact, certain cytokines and in particular the pro-inflammatory cytokines TNF-α, IL-1 and IL-6 are known to activate both the SNS and the HPA-axis (167-168). For example TNF-α, IL-1 and IL-6 stimulate hypothalamic CRH and/or AVP secretion, resulting in the secretion of GCs. How these inflammatory cytokines pass the blood-brain barrier to reach the hypothalamic CRH and AVP neurons is unclear. However, this feedback loop constitutes an important mechanism by 21 Introduction which GCs serve as a regulatory mechanism to prevent excessive activation of the immune response during infection (169-170). Studies in mice using viruses that elicit both strong early pro-inflammatory and later T cell responses (LCMV clones 13 and WE, influenza, HSV-1) have confirmed the release of endogenous GCs, whereas viruses that induce little or no inflammation do not stimulate significant GC induction (171). In this context it has been demonstrated that endogenous secretion of GCs protect the host against cytokine-mediated pathologies during murine CMV infection (172). More specifically it has been shown that if GCs are removed by adrenalectomy, IL-12, IFN-γ, TNF-α, and IL-6 production increases and the mice die due to septic shock. Moreover, experimental evidence in mice have confirmed that an increase of endogenous GC levels plays a protective role for the host during experimentally induced autoimmune encephalomyelitis (173) and arthritis (174). Glucocorticoid hormones GC hormones are long known for their immunosuppressive effects and clinically, GCs and their synthetic analogues are used as potent immunosuppressive agents. Many of the immunomodulatory effects of stress have been attributed to the action of GC stress hormones, therefore the cellular and molecular mechanisms of GC-mediated immunosuppression will be introduced in the following paragraphs. Molecular mechanism of GC-induced immunosuppression GCs belong to the family of steroid hormones and their action is mainly mediated through binding to the respective cytoplasmic receptors. There are two main receptors in the cytoplasm for GCs, the glucocorticoid receptors (GR) and the mineralocorticoid receptor (MR). GCs have a higher affinity for MR than for GR (175), thus at low levels, GCs bind preferentially to the MR, only at high levels, e.g. during stress exposure, the GRs are occupied (176). The most accepted mechanism by which GCs enter the cell is through passive diffusion facilitated by their relative small size and lipophilic nature. However, it has also been proposed that GCs can mediate their action by binding to membrane-associated glucocorticoid response receptors (mGCR) and that this interaction might participate in a GCmediated apoptosis (177-179), but the precise mechanism has not yet been identified. The glucocorticoid receptors (GRs) belong to the nuclear hormone receptor superfamily that are present in the cytoplasm in an inactive state and form multi-protein complexes with hsp90 and other chaperons. Upon GC binding, GRs dissociate from this complex and translocate as a homodimer to the nucleus where they bind via a zinc finger motif in their DNA-binding domain to the glucocorticoid response element (GRE) (180). The bound GR homodimer then modulates gene expression directly by either up-regulation or down-regulation of target genes, depending on the GRE sequence and promoter context. A direct down-regulation of gene expression occurs mainly via binding to so-called negative glucocorticoid response elements (nGRE) (Figure 5). 22 Introduction Figure 5: Molecular mechanisms of GC action Glucocorticoid hormones (GCs) passively diffuse into the cell and exert their effects by binding to membrane-bound glucocorticoid receptors (GR). Alternatively GCs bind to membrane-bound GR receptors. In the cytosol GCs bind to the GRs resulting in the dissociation of the heat shock protein complex and a translocation of the ligand-bound GR into the nucleus. In the nucleus GRs modulate e.g. cytokine transcription either by directly binding to glucocorticoid response elements (GRE, nGRE) or via interaction with other transcription factors like NFκB. GRs can also modulate gene expression through protein-protein interaction with other trancription factors such as NFκB (181-182), activator protein 1 (AP-1) (183-185), STAT and nuclear factor of activated T cells (NFAT) (186-188). Different mechanisms have been demonstrated by which GRs act to down-regulate gene expression via repression of NFκB activity. For example, GRs can induce the expression of the inhibitory protein IκB that in turn sequesters NFκB in the cytoplasm, thereby preventing its translocation into the nucleus (189190). In addition, direct interaction between NFκB and GRs has also been shown to repress NFκB-dependent gene expression (189, 191-193) as well as a competition between GRs and NFκB for limited cofactors such as CREB-binding protein (CBP) (194). Cellular mechanism of GC-induced immunosuppression The most general effect of GCs is to inhibit synthesis and/or release of cytokines that promote inflammatory reactions. For example, GCs have been demonstrated to suppress proinflammatory cytokines like IL-1β, IL-6, IL-8, IL-12 and TNF-α (170, 195) and up-regulate anti-inflammatory cytokines like IL-4 and IL-10 (195-196). GCs can induce a shift from TCD4+ Th1 cytokine responses (with predominant secretion of IL-2, IFN-γ and TNF-β) to a Th2 23 Introduction pattern (with predominant secretion of IL-10 and other anti-inflammatory cytokines) (164, 197-199). Beside their capacity to modulate cytokine secretion, GCs have been shown to affect nearly all other aspects of immune cell functions such as cell trafficking, maturation and differentiation, antigen presentation, proliferation, and effector function (170). For instance, GCs can substantially impair the function of T cells. They have been shown to reduce TCD8+ cell activation and proliferation (200). In particular, it has been suggested that GCs inhibit early events in T cell activation. For instance, short pre-treatment of Jurkat T cells with the synthetic GC dexamethasone (DEX) has been shown to inhibit the tyrosine-phosphorylation of ZAP-70, stimulated by CD3 cross-linking, whereas high doses of DEX showed opposite effects (201). Inhibition of T cell proliferation by GCs is accompanied by reduced IL-2 production and is thought to be mediated by GR interference with the transcription factor AP1 (183, 202-203). Modulation of IL-2 receptor α and β chain expression by GCs is also discussed as a potential mechanism how GCs inhibit the proliferation of T cells (204). While most reports indicate that GCs suppress T cell function, other studies demonstrate an enhancement of T cell function after GC exposure. For instance, in vitro treatment of splenic lymphocytes with corticosterone resulted in increased expression of IL-2 receptors after TCR stimulation when corticosterone was added within the first hour of the stimulus (139). GCs are also potent inducers of apoptosis, and physiological GC concentrations as well as concentrations achieved during pharmacological GC treatment or chronic stress responses can cause the death of immature thymocytes being most prominent within the CD4+ CD8+ TCRlow thymocyte population (205). Massive GC-induced thymocyte apoptosis is a well-known phenomenon in response to chronic stress that contributes to thymic involution under stress (206-207). In contrast, resting peripheral T lymphocytes are comparatively resistant to GCinduced apoptosis (208). Furthermore, GCs are also discussed to inhibit antigen-presenting cells by suppressing the generation, maturation, and immunostimulatory properties of DCs. For example GCs have been shown to selectively inhibit the expression of the costimulatory molecules CD80 and CD86 and to down-regulate MHC II expression. (209-210). Physiologically relevant GC levels have also been demonstrated to suppress the efficiency of presentation of MHC I/peptide complexes by virus-infected DCs by decreasing the production of antigenic peptides (209, 211). Many of the above mentioned findings have been elicited experimentally in in vitro systems or by exogenous administration of GCs and their synthetic analogs. It is important to note that pharmacological doses or forms of GCs exert different effects on immune functions than they do under physiological stress conditions. For example, synthetic versions of GCs (e.g dexamethasone) have a significantly longer half-life (212-213) as well as higher affinity for GC receptors (214), therefore exerting much stronger effects than endogenous GCs. 24 Introduction Moreover, synthetic GCs do not bind to corticosteroid-binding globulin (CBG), a plasma protein that binds a large proportion of circulating GCs, thereby preventing its translocation to the nucleus (170). Therefore these approaches may not mirror the complex situations during a physiological stress response, making it difficult to extrapolate the outcome of chronic stressinduced alterations on immune reactions from these data. Regulation of the HPA axis and GC action under chronic stress exposure Recurrent or prolonged activation of the HPA axis as it occurs under chronic stress exposure can create an environment in which systemically elevated levels of GCs suppress various immune cell functions (169-170). Enhanced circulating GC levels may directly occur through chronic activation of the HPA axis or secondary through a reduced sensitivity of the HPA axis to negative feedback. However, the action of endogenous GCs is not solely determined by their systemic concentration in the circulation but various factors selectively modulate the sensitivity of immune cells or tissues to GCs under basal conditions and during physiological stress responses (170). Under basal conditions there is a considerable heterogeneity among immune cells and tissues with respect to the expression of GC receptors (GRs), suggesting that different immune compartments and cells exhibit different responsiveness and sensitivity to GCs. Whereas the thymus and lymph nodes display high levels of GRs (with the thymus expressing the highest amounts), GR expression in the spleen is much lower and thymic T cells were found to express higher levels of GRs than T cells isolated from the spleen (215216). GR activation can also be modulated selectively during stress exposure. For instance, in a study investigating the effect of restraint stress on GR activation it was found that acute, stress-induced elevation of GCs results in GR activation in the thymus and LNs, whereas receptors in the spleen were not activated (217). Changes in plasma corticosteroid-binding globulin (CBG) in response to high levels of GCs or catecholamines play an important role by modulating GC action under chronic stress exposure. For example chronic stress exposure can be associated with an impaired production of corticosteroid-binding globulin (CBG) and can also reduce the GC binding capacity of CBG, resulting in increased levels of free and biologically active GC hormones (218-219). Rats exposed to chronic social stress were shown to exhibit a significant decrease in plasma CBG levels (218). This decrease in CBG levels led to greater access of free GC hormones to GRs in the spleen than is typically found under basal or acute stress conditions, providing one mechanism by which chronic stress has a greater impact than acute stress on splenic immune functions (218). The accessibility of GCs to its cytoplasmatic receptors can be further modulated after GCs have entered the cell. This occurs through the conversion of GCs from their active 11β-hydroxy- into its inactive 11-keto-form by the 11β-hydroxysteroid dehydrogenase (11β-HSD). The expression of 11β-HSD is known to be tissue-specific and its activity may also be altered distinctively during chronic GC exposure, providing a further mechanism by which tissue-specific GC sensitivity occurs 25 Introduction (220). Consideration of these factors that regulate GR activity is important in order to understand the differential impact of GCs on immune responses during stress and how GCs can achieve selectivity in influencing specific immune functions in some tissues or cells but not in others. Animal models of chronic stress and immunity A key question is to whether and how the immunosuppressive effects of prolonged GC elevation promote and exacerbate the pathophysiology of infectious and other diseases under physiological stress responses. In this regard, numerous studies in animals have investigated the effect of a variety of psychological and physical chronic stress stimuli on the outcome of infectious diseases, with some studies reporting immunosuppressive effects and others showing enhancing effects (170, 221). These studies also provide evidence that a variety of conditions determine the magnitude of stress-related changes on immunity: (a) the duration of the stress-exposure (acute vs. chronic), (b) the timing of the stress exposure relative to immune activation (c) the nature of the stressor, and (d) the level of GCs secreted during a stress challenge (which to some extent in turn dependents on a and c) (221-222). Social stress In general, based on the nature of the stress stimulus, stressors can be grouped into different broad categories: (a) physical (e.g. electric, chemical), (b) psychological (e.g. restraint, exposure to novel environment, forced swimming) or (c) social (222). Animal models based on social stress are considered to be a behaviorally valid system to study the effects of relatively natural stressful situations since social stress is a chronic or recurring factor in the life of virtually all humans. Another reason why social stressors are implicated to be highly relevant in comparison to the other categories (223-224) is the observation that a social stressor, because of an often unpredictable nature, will not induce habituation of the stress response and will therefore not induce adaptation (225-228). Chronic social stress is known to stimulate both activation of the pituitary-adrenocortical system (HPA) and the sympathetic adreno-medullary system, resulting in elevated concentrations of free GCs and the catecholamines norepinephrine and epinephrine, respectively (219, 229). The social disruption stress (SDR) model ‘Social disruption’ stress is a model of chronic social stress and mirrors a daily recurrent experience of stress. This stress model is based on the disruption of an established social hierarchy of group-housed male mice (residents) in their home cage, which is experimentally induced by daily confrontation with an unfamiliar aggressive intruder mouse (230-231). Social disruption (SDR) is typically conducted for the duration of 2 hours daily and is repeated for 6 consecutive days to mimic a recurring or chronic stressor. During the daily stress procedure, the aggressive intruder repeatedly attacks and defeats the residents, which show typical behavioral signs of fear and submissiveness (231). 26 Introduction Effects of social disruption stress (SDR) on immune functions Similar to other chronic stress models, social disruption stress activates the HPA-axis, resulting increased levels of circulating GCs (225, 231-232). Repeated social defeat in SDR mice has been demonstrated to cause thymus atrophy (233), decreased T cell numbers in the bone marrow and spleen (234), and impaired T cell functions (235). However, in contrast to other chronic stressors, mice challenged with the social disruption paradigm develop a functional GC insensitivity (231, 233, 236). This functional GC resistance develops under chronically increased levels of GCs and is regarded as a form of protection against the immunosuppressive effects of GCs. For instance splenic CD11b+ monocytes exhibit an increased secretion of the pro-inflammatory cytokines IL-1α, IL-6, and TNF-α upon mitogen stimulation (236-237). GC resistance in splenic monocytes has been shown to be a function of decreased GR translocation from cytoplasm to nucleus with associated decreased inhibition of NF-kB (238). GC insensitivity has also been supposed for splenic CD11c+ cells, as has been demonstrated by SDR-induced CD11c+ activation and an increased production of TNF-α, IL6, and IL-10 in response to TLR stimulation (239). Immune alterations induced by social defeat can exacerbate viral and bacterial pathogenesis. For example, mice exposed to SDR have been demonstrated to be more susceptible to reactivation of a latent HSV-1 infection, influenza A virus infection, and to an LPS-induced endotoxic shock (240-242). Moreover, it has been shown that SDR increased the susceptibility to a Theiler virus-induced CNS inflammation (a model of multiple sclerosis) (243), an effect that was dependent on the precise timing between virus inoculation and stress exposure. It is important to note that higher disease susceptibility in mice exposed to SDR is not a constant finding. For instance, SDR showed no impact on an ongoing BCG-infection (244-245) and was found to improve the resistance to a bacterial infection with Escherichia coli (246). Taken together, the heterogeneity of effects on immune functions observed upon chronic stress exposure and the complexity of the underlying mechanisms necessitate further studies along these lines. Aim of the thesis The aim of the present thesis was to characterise underlying mechanisms of chronic stressrelated immunosuppression focusing on T cell-mediated immunity. Chapter 2 elucidates the mechanisms by which chronic social stress affects the outcome of virus-specific cytotoxic TCD8+ cell (CTL) responses in mice after infection with lymphocytic choriomeningitis virus (LCMV). Chapter 3 focusses on the impact of chronic social stress on the migratory capacity of skin dendritic cells. For this purpose, we performed contact allergen-induced skin sensitisation assays using the fluorescent dye fluorescein isothiocyanate (FITC), which allowed us to trace the migration of skin CD11c+ DCs in vivo. In chapter 4 we analysed stressassociated alterations in peripheral T cell subsets in a group of severely traumatised human patients. 27 Chapter 2 Attenuation of the cytotoxic T lymphocyte response to lymphocytic choriomeningitis virus in mice subjected to chronic social stress Annette Sommershof, Michael Basler, Carsten Riether, Harald Engler and Marcus Groettrup Submitted to Journal of Immunology Chapter 2 Abstract Chronic stress is suspected to increase the susceptibility to infections but experimental evidence is scarce. We examined the effects of chronic social stress on virus-specific cytotoxic T lymphocyte (CTL) responses in mice after infection with lymphocytic choriomeningitis virus (LCMV). Mice subjected to social stress on six consecutive days prior to infection showed a significant reduction of IFN-γ producing TCD8+ splenocytes and markedly lowered plasma concentrations of IFN-γ during the late stage of the infection. In contrast, the generation of LCMV-specific CTL responses was not altered in mice undergoing the same stress procedure concurrently with infection. Furthermore, stress exposure six days before and additional three days after LCMV infection profoundly reduced the expansion of TCD8+ cells in the spleen, due to a diminished in vivo proliferation capacity as shown by BrdU incorporation. Pharmacological blockade of glucocorticoid receptors with RU-486 completely abrogated the stress-associated decline of TCD8+ expansion. Stressed mice showed a significantly reduced expression of the early T cell activation marker CD69 as well as impaired in vitro cytokine secretion of IFN-γ and IL-2. Additionally, social stress led to an altered migration capacity of TCD8+ cells as demonstrated by adoptive T cell transfer experiments. Taken together, this study shows that chronic social stress fundamentally suppresses the functional capacities of T cells during LCMV infection providing a mechanism by which stress can increase the susceptibility to viral infections. Introduction Chronic stress is suspected to increase the susceptibility to infectious diseases by affecting the function of cells of the innate and adaptive immune system. For example, studies in humans have shown that stressful life conditions were associated with an increased risk for influenza virus infection (247) and rhinovirus infection (248) as well as herpes simplex virus recurrences (249). However, experimental evidence is scarce that chronic stress indeed enhances the susceptibility to viral infections, e.g., by suppressing anti-viral immune responses. Only few studies have investigated the effects of chronic stress on T cell-mediated immunity during viral infection. For example, mice exposed to chronic restraint displayed a decreased generation of virus-specific T-lymphocytes (CTL) in response to primary HSV-1 infection (250), and altered memory cytotoxic T lymphocyte activation (251-253). However, most of the studies investigating the impact of chronic stress on antiviral responses were performed using stressors without relevant behavioral context. Although these studies provided important insights into the immunmodulatory capacities of chronic stress, animal models of social stress are considered to be biologically more relevant, also with respect to stressful situations in humans. In this context, some recently performed studies examined the impact of social stress on the pathophysiological outcome of viral infections such as Theiler’s virus (243, 246) and influenza virus (240) or mycobacterial BCG (bacillus Calmette-Guérin)- 29 Chapter 2 infection (244) but very little is known about the impact of chronic social stress on T cell responses to viral infections. A series of studies has investigated the consequences of reapeated social stress exposure on T cell number and function. For instance, it has been demonstrated that social stress is accompanied by reduced numbers of T cells in the blood, spleen, and bone marrow of mice (234). In this context, it has been shown that social defeat in rats lead to decreased homing of adoptively transferred peripheral blood T cells into lymphoid organs, suggesting altered migration properties (254). Finally, it has been reported that social stress influences proliferation, cytokine production and T cell mediated cytotoxicity. For example, socially stressed mice exhibit suppressed T cell proliferation responses to the mitogen ConA (235) and increased production of pro-inflammatory cytokines IL-6 and IFN-γ as well as decreased production of anti-inflammatory cytokine IL-10 (235). The aforementioned results indicated that social stress substantially alters the trafficking and functional capacities of T lymphocytes. However, it is important to note that these results were observed in the absence of viral infection, thus information on whether and how social stress affects the outcome of TCD8+ cell-mediated responses during a virus challenge are largely unknown. To analyse the effects of chronic social stress on an anti-viral T cell response, we performed a systemic infection using lymphocytic choriomeningitis virus (LCMV). LCMV is a natural mouse pathogen inducing a strong cytotoxic T cell (CTL) response that is responsible for virus elimination (255-256). The CTL response of C57BL/6 mice is focused on the three dominant GP33-41/Db, GP34-41/Kb and NP396-404/Db as well as several sub-dominant (GP276-286/Db, GP92-101/Db, GP118-125/Kb and NP205-212/Kb) T cell epitopes (2, 257). In addition, antigen processing and T cell epitope production are well characterized in the LCMV infection model and therefore it represents an optimal model to study potential T cell alterations in response to social stress. In the present study we provide evidence that chronic social stress compromises the activation and expansion of LCMV-specifc TCD8+ cells in the spleen. We further show that glucocorticoids play a fundamental role in these alterations by intrinsically inhibiting TCD8+ cell cytokine production and proliferation. Additionally, we extend previous findings that social stress leads to a profoundly impaired migration pattern of TCD8+ cells. Results Experimental design To analyze the impact of social disruption stress on the LCMV-specific cytotoxic T cell response, we compared different stress protocols (Figure 1). In a first trial, the stress procedure was either applied concurrently with LCMV infection (experiment 1a) or the experimental mice were first subjected to the stressor and received the infection afterwards (experiment 1b). In a second set of experiments, we examined the effect of a prolonged stress 30 Chapter 2 procedure on the outcome of LCMV infection, including T cell expansion and migration as well as antigen presentation. For this purpose, mice were exposed to six consecutive days of SDR followed by LCMV infection and three additional days of SDR (experiment 2). Figure 1. Scheme of the experimental design Thymus atrophy and adrenal hypertrophy Organ masses of thymus and adrenal glands were determined after six days of SDR (Figure 2 A, B). Repeated exposure to the social stressor resulted in atrophy of the thymus and hypertrophy of the adrenals as shown by a significant reduction of thymus mass (SDR: 42.5±1.4 mg vs. control: 69.6±2.2 mg; p≤0.001) and a marked enlargement of the adrenal glands (SDR: 4.9±0.1 mg vs. control: 4.0±0.1 mg; p≤0.001). Both thymic atrophy and adrenal hypertrophy are consequences of frequent or persistent adrenocortical activation and are classical indicators of chronic stress (225). Figure 2. Social disruption stress (SDR) caused thymic atrophy and adrenal hypertrophy Organ masses of thymus (A) and adrenal glands (B) after six consecutive days of SDR. Data represent mean ± SEM of one out of two experiments with at least five mice per experiment. Effects of social stress on the LCMV-specific T cell response In a first set of experiments (Figure 3 A), the effect of SDR on the LCMV-specific CTL response was examined in two different timing schedules, comparing the impact of a six-day stress procedure applied prior to virus infection (experiment 1b) with an experimental set-up in which SDR was administered concurrently with the infection (experiment 1a). At the peak of the primary response (day 8), intracellular cytokine staining (ICS) was performed to 31 Chapter 2 determine the frequency of IFN-γ producing TCD8+ cells specific for six defined LCMV epitopes: two dominant (GP33-41/Db/Kb and NP396-404/Db) and four subdominant epitopes (GP276-286/Db, GP92-101/Db, GP118-125/Kb and NP205-212/Kb). Mice that were subjected to social stress prior to virus infection (experiment 1b) exhibited a significant reduction of IFN-γ producing TCD8+ cells specific for the dominant LCMV epitopes GP33-41/Db/Kb (SDR: 2.5±0.1% vs. control: 4.6±0.4% of total TCD8+ cells; p≤0.001) and NP396-404/Db (SDR: 1.9±0.1% vs. control: 3.1±0.2%; p≤0.001) compared to control mice. The CTL-responses to the subdominant epitopes GP276-286/Db, NP205-212/Kb, GP92101/Db and GP118-125/Kb were not significantly altered. We further compared these results with the quantification of GP33-specific TCD8+ cells by MHC-tetramer staining. As shown in Figure 3 D, SDR mice exhibited similar percentages of TCD8+/GP33-tetramer+ cells compared to control mice (SDR: 16.8 ± 0.5 % vs. control: 15.7 ± 0.8% of total TCD8+ cells), while the frequency of IFN-γ producing TCD8+ cells was reduced (SDR: 6.3 ± 0.3% vs. control: 10.0 ± 0.8%; p≤0.001). These results demonstrate that SDR profoundly impacts the function of splenic TCD8+ cells by inhibiting antigen-specific IFN-γ secretion. In contrast, the LCMV-specific CTL response was not significantly altered in mice receiving the same stress procedure concurrently with the infection (experiment 1a). The frequency of IFN-γ producing TCD8+ cells specific for GP33-41/Db/Kb (SDR: 8.5±0.2% vs. control: 7.0±0.8%), NP396-404/Db (SDR: 6.2±0.5% vs. control: 4.8±0.7%) and GP276-286/Db (SDR: 3.8±0.2% vs. control: 3.2±0.2%) were slightly but not significantly increased. Effects of social stress on plasma IFN-γ levels in LCMV-infected mice We further determined plasma levels of IFN-γ in SDR and control mice on days 2, 4, and 6 post LCMV infection. As shown in Figure 3 E, plasma concentrations of IFN-γ increased gradually in both groups during the course of LCMV infection. However, while the concentration of IFN-γ was not altered in SDR mice οn day 4 post infection, we observed an almost 50% reduction in circulating IFN-γ levels on day 6 of infection (SDR: 306±64 pg/ml vs. control: 514±65 pg/ml; p=0.06) compared to controls. 32 Chapter 2 Figure 3: Epitope-specific CTL responses in LCMV-infected control and SDR mice Mice were subjected to six days of SDR and were subsequently infected with 200 pfu LCMV i.v. Epitopespecific CD8+ T cell responses were quantified on day 8 after infection by intracellular cytokine assay and tetramer staining. (A) Scheme of the experimental design. (B, C) Percentage of IFN-γ secreting CD8+ cells from LCMV-infected SDR and control mice after 5h of in vitro re-stimulation in the presence of indicated peptides. ∅ represents background values of splenocytes without peptide stimulation. (C) All values represent the mean percentage of IFN-γ positive cells of CD8+ cells ± SEM of 5 mice. (D) Production of IFN-γ by GP33-specific CD8+ cells after peptide-stimulation is compared with the percentage of CD8+/GP33-tetramer+ cells. (E) Plasma levels of IFN-γ in LCMV-infected SDR and control mice. On day 2, 4, and 6 post-infection peripheral blood was taken from SDR and control groups and cytokine levels for IFN-γ were determined using a multiplexed bead-based assay. All data are representative of one out of two experiments with at least five mice per experiment. Prolonged stressor exposure leads to reduced expansion of TCD8+ splenocytes During the course of an LCMV infection, TCD8+ lymphocytes undergo multiple cycles of cell division after exposure to the antigen, as indicated by a progressive increase of the TCD8+ population within 8 days. Prolongation of the stress procedure for three additional days after LCMV infection caused a pronounced reduction of TCD8+ cells in the spleen of SDR mice on day 6 (SDR: 15.8±0.2% vs. control: 20.2±0.7% of splenocytes; p≤0.001) and day 8 (SDR: 31.3±2.5% vs. control: 52.4±0.6%; p≤0.001) of infection (Figure 4 B). In contrast, there were no significant differences in the TCD8+ population on day 2 (SDR: 9.3±0.2% vs. control: 9.6±0.2%) and day 4 (SDR: 7.3±0.2% vs. control: 7.8±0.1%) after infection, indicating that 33 Chapter 2 the alterations occur during the antigen-driven T cell expansion phase and are not due to initially reduced TCD8+ cell numbers. In contrast to the profound reduction of splenic TCD8+ cells, alterations in the LN were much less pronounced and not significant (SDR: 30.1±0.9% vs. control: 35.4±1% of splenocytes; p≤0.001), whereas no differences were observed in the blood (SDR: 55.8±1.9% vs. control: 56.7±2%), liver (SDR: 47.2±0.5% vs. control: 48.9±2.2%) or lung (SDR: 54.8±2.6% vs. control: 55.2±3.8%). Social stress affects the proliferation capacity of TCD8+ lymphocytes In order to determine whether the decreased TCD8+ cell expansion found in SDR mice is due to a higher propensity of TCD8+ cells to undergo apoptosis, we performed stainings with Annexin V and propidium iodide (PI). The frequency of early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptotic cells was determined by flow cytometry on days 2 and 8 post infection. Analyses revealed no differences in the frequency of TCD8+ cells and total splenocytes undergoing apoptosis between SDR mice and non-stressed control mice (see Table I). Table I: Percentages of apoptotic cells in SDR and control mice Percentages of total and TCD8+ apoptotic splenocytes on day 2 and 8 post infection in SDR and control mice. All values represent the mean±SEM of five individual mice and are representative of one out of two experiments. To further examine the deficit in TCD8+ cell expansion, we analysed in vivo T cell proliferation by performing a BrdU pulse-labeling assay. Mice were treated with 1 mg BrdU i.p. on day 5 of LCMV infection and BrdU incorporation in splenocytes was assessed by intracellular staining 24 hours later. Analysis of BrdU staining in control mice demonstrated that approximately 50% of TCD8+ cells divide between day 5 and day 6 of LCMV infection (Figure 4 C). In contrast, the frequency of BrdU positive TCD8+ splenocytes in SDR mice was significantly reduced by 28% (SDR: 34.2±1.0% vs. control: 47.2±1.1% of TCD8+ cells; p≤0.001). Our findings strongly suggest that the massive decrease in TCD8+ splenocyte expansion observed in SDR mice is predominantly a result of diminished proliferation capacity rather than an altered death rate of TCD8+ cells. Stress reduces expression of the T cell activation marker CD69 on TCD8+ cells Next, we investigated whether the reduced proliferation of TCD8+ cells is accompanied by inefficient T cell activation. Therefore, we quantified the expression of different T cell activation markers throughout the early stage of LCMV infection by flow cytometry. Our data revealed that SDR and control mice exhibited comparable surface expression of CD44 and CD62L (day 2 and day 4 post infection, data not shown). However, we found significantly 34 Chapter 2 lowered expression of the early T cell activation marker CD69 on day 2 post LCMV-infection in SDR compared to control mice (Figure 4 D; SDR: 35.7±1.1% vs. control: 51.3±2.1% of TCD8+ cells; p≤0.001). Figure 4: Effects of social stress on TCD8+ cell expansion Mice remained undisturbed or were subjected to six days of SDR, then infected with 200 pfu of LCMV i.v., and SDR mice were stressed for additional three days post infection. (A) Scheme of the experimental design (B) The percentage of TCD8+ lymphocytes was determined on days 2, 4, 6, and 8 post LCMV infection by flow cytometry. (C) Reduced TCD8+ proliferation in LCMV-infected SDR mice compared to control mice. On day 5 after LCMV infection, both groups received an in vivo pulse labelling with BrdU. Twenty-four hours later, spleens were removed and BrdU incorporation was assessed by flow cytometry after CD8 surface staining and intracellular staining for BrdU. Representative BrdU pulse profiles are illustrated for SDR and control mice obtained at day 6 of LCMV-infection. ∅ represents background values of splenocytes from mice that did not receive BrdU injection. (D) CD69 surface expression on TCD8+ splenocytes in SDR compared to control mice. On day 2 post infection, TCD8+ splenocytes were analysed for CD69 surface expression by flow cytometry. The histogram represents CD69 expression of TCD8+ splenocytes from SDR (black line) and control (grey line) mice compared to non-infected naïve mice (dotted line). The negative control represents background values of the TCD8+ splenocyte population that was not stained for CD69 (dashed line). Data are presented as the mean percentage of CD69-positive cells ± SEM of TCD8+ cell population. (E, F) TCD8+ cell expansion on day 8 post-infection in SDR and control mice treated with slow-release pellets of RU486 (E), nadolol (F) or placebo. Data are presented as the mean percentage of CD8-positive cells ± SEM of the lymphocyte population. Two-factor ANOVA revealed a significant stress × drug interaction for RU486 (p < 0.001). All data are representative of one out of two experiments with five mice per experiment. 35 Chapter 2 Glucocorticoids mediate the stress-induced decrease in TCD8+ cell expansion To identify the endocrine factors mediating the stress-induced decrease in TCD8+ cell expansion in the spleen, SDR and control mice were implanted with continuous release pellets containing either the glucocorticoid (GC) receptor antagonist RU486 or the β-adrenergic receptor antagonist nadolol. Unstressed control mice treated with RU486 or nadolol exhibited similar TCD8+ cell expansion in the spleen on day 8 of LCMV-infection compared to mice receiving placebo pellets (Figure 4 E, F), demonstrating that neither of the two drugs influenced the LCMV-infection by itself. More importantly, splenic TCD8+ cell numbers in RU486-treated SDR mice did not differ from placebo- or RU486-treated control mice (Figure 4 E, SDR RU486: 51.6±1.4%, control RU486: 53.5±1.3%, control placebo: 53.2±1.8%), indicating that treatment with the GC receptor antagonist completely abolished the stressinduced effects on TCD8+ cell expansion. In contrast, the frequency of TCD8+ splenocytes in nadolol-treated SDR mice was not significantly altered compared to placebo-treated SDR mice (Figure 4 F, SDR nadolol: 32.7±5.6% vs. SDR placebo: 29.6±3.4%), demonstrating that β-adrenergic receptor blockade was not effective in preventing the stress-associated changes of TCD8+ cell expansion in the spleen. Viral clearance In order to clarify whether the impaired generation of TCD8+ cells in the spleen resulted in an altered viral clearance, mice were infected with 200 pfu LCMV and virus titers were determined in the spleen of stressed and non-stressed mice on day 2, day 4, day 6 and day 8 post-infection. Figure 5: Effects of SDR on viral titers in the spleen of LCMV-infected mice After 6 days of SDR exposure mice were infected with 200 pfu LCMV and subsequently stressed for three further days. On days 2, 4, 6 and 8 post infection, virus titers were determined in the spleen. Viral titers are shown as log10 plaque forming units (pfu) per spleen. Symbols represent individual mice. Our results demonstrated that splenic viral titers were not altered at early time points of infection (day 2 and day 4 p.i.) in SDR mice (Figure 5). We noted for both groups an extensive decrease of the viral load during the later time course of the infection period (day 6 and day 8 p.i.). However, viral titers in the spleen of SDR mice were not consistently altered 36 Chapter 2 compared to control mice on day 8 p.i., although we noticed a tendency for enhanced virus titers among stressed mice (SDR: 18.5±7.4*103 pfu vs. control: 5.6±1.2*103 pfu). Social stress did not alter phenotype or antigen presentation of DCs in the spleen To explore whether the reduced activation of TCD8+ cells in the spleen is the result of an altered antigen presentation, we compared the frequencies of splenic CD11c+ DCs and F4/80+ macrophages on day 2 and day 4 post-infection. As shown in Figure 6 A, SDR mice exhibited similar percentages of CD11c (SDR: 5.4±0.1% vs. control: 5.7±0.2%) and F4/80 (SDR: 8.9±0.4% vs. control: 9.7±0.6%) positive cells compared to unstressed control mice. Further analysis of the activation state of CD11c+ cells, analysed by MHC class I (H-2Db and H-2Kb) expression and CD80, CD86 surface expression revealed no differences between SDR and control mice (Figure 6 B), indicating that SDR neither affected the percentage of antigen presenting cells in the spleen nor the activation of dendritic cells. Figure 6: Effects of social stress on DCs and antigen presentation (A) Percentages of splenic CD11c+ DCs and F4/80+ macrophages in control and SDR mice on day 4 post infection as determined by flow cytometry. (B) Activation state of CD11c+ cells in SDR compared to control mice. On day 4 post infection, CD11c+ splenocytes were assessed for surface expression of H-2Db, H-2Kb, CD80 and CD86 by flow cytometry. Representative histogram of each staining is shown for SDR (black line) and control (grey filled) mice compared to non-infected naïve mice (dotted line). Dashed lines represent staining with isotype control. Data in the bar plot are presented as the mean percentage of positive cells ± SEM of CD11c+ cells. (C, D) Antigen presentation in the spleen of SDR mice compared to control mice. On day 4 of LCMV-infection, splenocytes from stressed (squares) and unstressed (diamonds) mice were used either (C) directly as antigen presenting cells for a GP33-specific CTL line or (D) were pulsed with GP33 peptide and then used as stimulator cells. Activation of the Thy1.1+ CTL line was analyzed at different E:S ratios (CTLs:splenocytes) by staining for Thy1.1+ effector cells and intracellular IFN-γ. Data are presented as the mean percentage of IFN-γ positive cells ± SEM of Thy1.1+ cells. All data are representative of one out of two experiments with five mice per experiment. 37 Chapter 2 Recent findings have suggested that glucocorticoids impair the competence of DCs to process and present virally expressed antigens, thereby altering their ability to activate T cells (211). To further address the possibility that splenocytes from SDR-mice present LCMV-derived epitopes less efficiently, we performed an ex vivo antigen presentation assay. GP33-41/Db/Kb presentation of splenocytes (day 4 post-infection) was assessed by their capacity to activate a GP33-specific CTL line. Splenocytes from non-infected naïve mice were used as a negative control. As shown in Fig. 6 C, splenocytes from SDR and control mice presented the LCMVderived GP33-41/Db/Kb epitope to a similar extent, indicating that antigen presentation was not affected in SDR mice. To exclude that the diminished TCD8+ activation observed in SDR mice was due to some other functional defects of splenic antigen presenting cells to prime naïve T cells, we further analysed the ability of pulsed GP33 splenocytes to stimulate naive GP33-specific CTLs. For this purpose splenocytes (day 4 post-infection) were pulsed with indicated concentrations of GP33-peptide in vitro and then used as stimulator cells for GP33specific CTLs. As shown in Fig. 6 D, GP33-pulsed splenocytes from control or SDR-mice could stimulate the GP33-specific CTLs to the same extend at all concentrations used. Taken together, our results provide evidence that the diminished TCD8+ splenocyte activation observed in SDR mice cannot be explained by an indirect mechanism via Ag-presenting DCs. Composition of 20S proteasome in LCMV-infected SDR and control mice Since the proteasome is the key protease generating peptides for the MHC class I antigen presentation pathway, we hypothesized that SDR may impair the induction of the IFN-γinducible proteasome subunits LMP2 and LMP7 thereby altering the repertoire of LCMVpeptides presented to CTLs (53, 258-259). To address this question, we examined the subunit composition of the 20S proteasome in livers of stressed and unstressed mice on day 8 of LCMV infection (Figure 7). Comparison of the 20S proteasome composition in liver of stressed and non-stressed control mice revealed equal amounts of the subunits LMP2 and LMP7, thus demonstrating that the cellular content of the immunoproteasome was not altered. Figure 7: Composition of 20S proteasome subunits purified from the pooled livers (n=5) of control mice (upper panel) and SDR mice (lower panel) after infection with LCMV After 6 days of SDR exposure, mice were i.v. infected with 200 pfu LCMV-WE and subsequently stressed for three further days. On day 8 post infection, livers were removed and pooled, 20S proteasomes were isolated and the subunits of isolated 20S proteasomes were analysed on Coomassie-stained two-dimensional NEPHGE/SDS PAGE. The positions of the proteasome subunits LMP2 and LMP7 are indicated. 38 Chapter 2 Impaired secretion of IFN-γ and IL-2 by TCD8+ splenocytes of stressed mice In order to elucidate whether the decreased TCD8+ cell proliferation in SDR mice was attributable to an altered cytokine production, we further analyzed IFN-γ and IL-2 secretion of splenocytes, and purified splenic TCD8+ cells in response to anti-CD3/CD28 mAb stimulation. As shown in Figure 8 A stimulated splenocytes from SDR mice consistently demonstrated severely reduced IFN-γ (SDR: 579±51 vs. control: 896±157 ng/ml; p=0.16) and IL-2 (SDR: 289±19 vs. control: 524±73 pg/ml; p≤0.05) secretion compared to splenocytes from control mice. Purified TCD8+ cells from SDR-mice also demonstrated a significant reduction of IFNγ (SDR: 1948±234 vs. control: 2831±224 ng/ml; p≤0.05) and IL-2 (SDR: 428±15 vs. control: 596±53 pg/ml; p≤0.05) secretion when compared with those derived from control mice. Therfore we suggest that the limited in vivo TCD8+ proliferation is due to a lack of T-cell stimulating cytokines, particularly of IL-2 secretion. Effects of social stress on the homing capacity of splenic TCD8+cells Adoptive transfer experiments with magnetically purified T cells were performed to examine whether social stress altered the homing pattern of these cells. Equal numbers of infected control (Thy1.2-, Ly5.2+) and SDR (Thy1.2+, Ly5.2+) TCD8+ cells were mixed and i.p. transferred into infected Ly5.1 (Thy1.2+, Ly5.2-) recipient mice (Figure 8 B: experimental design). Twenty-four hours after the adoptive transfer, the ratio of recovered donor-derived TCD8+ cells was analysed in the spleen, LN and blood (Figure 8 C, D). Reduced frequencies of TCD8+ cells from SDR mice were recovered from the spleen (SDR: 38.8±2.0% vs. control: 61.2±2.0%; p≤0.001), whereas the frequency of SDR TCD8+ cells in the blood was increased compared to control TCD8+ cells (SDR: 53.1±1.7% vs. control: 46.9± 1.7%; p≤0.05). The homing of adoptively transferred SDR TCD8+ cells towards the LN was slightly but not significantly decreased (SDR: 46.3±5.1% vs. control: 53.8±5.1%; p=0.34). We further compared these results with intravenously transferred TCD8+ cells and found a lower ratio of recovered SDR to control TCD8+ cells in the spleen (SDR: 40.3±1.6% vs. control: 59.7±1.6%; p≤0.001) and blood (SDR: 43.1±1.3% vs. control: 56.9±1.3%; p≤0.001), whereas the frequency of recovered cells in the LN was not altered (SDR: 51.1±1.3% vs. control: 48.9±1.3%). These results demonstrate that TCD8+ cells from SDR mice exhibit a decreased homing to the spleen compared to TCD8+ cells from control mice. 39 Chapter 2 Figure 8: Effects of social stress on cytokine secretion and migration capacity of TCD8+ splenocytes (A) Splenocytes (SPL) or purified TCD8+ cells recovered on day 4 post infection were stimulated for 24 h with plate-bound CD3/CD28 mAb. Supernatants were analyzed for IFN-γ and IL-2 secretion by ELISA. Data are presented as the mean percentage of cytokine secretion of triplicate cultures ± SEM of one out of two experiments with at least four mice per experiment. (B, C, D) Homing pattern of TCD8+ cells from SDR and control mice after adoptive transfer. Equal numbers of sorted TCD8+ cells (5*106 cells) isolated either from LCMV-infected control (Thy-1.1) or SDR (Thy-1.2) mice were adoptively co-transferred into infected recipient mice (Ly5.1). Twenty-four hours after transfer, the ratio of donor-derived TCD8+ cells was analysed in the spleen, LN and blood by flow cytometry. (B) Scheme of the experimental design. (C) Representative flow cytometric plots of recovered control (Thy1.2-, Ly5.2+) and SDR (Thy1.2+, Ly5.2+) TCD8+ cells from indicated host (Thy1.2+, Ly5.2-) organs are shown. (D) Data are presented as the ratio ± SEM of recovered SDR to control TCD8+ cells in the spleen, LN, and blood. The data are representative of one out of three experiments with five mice per experiment. Discussion The present study demonstrates that chronic social stress compromises the TCD8+ cellmediated response to LCMV-infection. LCMV infection leads to a rapid expansion of IFN-γproducing CTLs visible around day 5 and peaks on day 8 post infection. When mice were exposed to a six-day social disruption procedure, we noted a significant reduction of IFN-γ producing TCD8+ cells in the spleen on day 8 post infection, indicating that social stress profoundly impairs the function of TCD8+ cells by inhibiting antigen-specific IFN-γ secretion. 40 Chapter 2 Therefore, we further determined plasma levels of IFN-γ in mice throughout the course of LCMV infection. We found that on day 6 post-infection, when levels of IFN-γ were maximal in the control group, SDR mice showed an almost 50% reduction in circulating IFN-γ. These results support previous reports, demonstrating that social stress alters the production of pro- and anti-inflammtory cytokines including IFN-γ, TNF-α and IL-10 (235, 260). Interestingly, these alterations were only found when the stress procedure was applied prior to virus infection, whereas LCMV infection at the onset of the stress procedure had only marginal effects on the CTL response. The divergent impact on the CTL response in the two timing schedules clearly demonstrates that the timing of the stressor relative to virus inoculation is critical for the consequences of stress on the antiviral immune response. These observations are compatible with recent reports demonstrating that SDR exacerbates the pathophysiology of Theiler’s virus infection when the stress was applied prior but not concurrently with infection (243, 246). Similar results have been obtained in a BCG-infection model, showing that social stress did not alter the course of an ongoing infection when applied several days after the infection (244). These data are in line with the insight that priming of T cells occurs during the first two days after virus encounter, i.e. at a time point when stress-related immune suppression needs to be established in order to show strong effects. Prolongation of the SDR procedure for three additional days after LCMV infection led to a profound reduction of TCD8+ cells in the spleen of SDR-treated mice on day 6 and day 8 post infection. In contrast, there were no differences in the TCD8+ population on day 2 and day 4 after infection, indicating that a modulation of TCD8+ population occurs during the antigendriven T cell expansion phase and is not due to initially reduced TCD8+ cell numbers. The stress-induced decrease in the TCD8+ cell population could have been the result of enhanced apoptosis and/or reduced proliferation of TCD8+ cells in the spleen. We tested both possibilities by measuring apoptosis by Annexin V/PI staining and cell proliferation with a BrdU incorporation assay on relevant time points after LCMV infection. Apoptosis analyses were performed on day 2 post infection, to test whether SDR initially leads to an altered cell death at early stages of the immune response, as well as on day 8 post infection, to take into account possible alterations in the apoptosis rate occurring during the antigen driven T cell expansion phase. Comparison of apoptotic cells revealed no differences in the frequency of apoptosis within the TCD8+ population and total splenocytes, respectively, between SDR mice and nonstressed control mice. On day 5 of infection, when the expansion of LCMV specific TCD8+ cells is most vigorous, we performed an in vivo BrdU proliferation assay and found that the proliferation rate was significantly reduced by 28%. Our results clearly show that the diminished TCD8+ population in SDR mice results from a decreased proliferation capacity of TCD8+ cells rather than an increased apoptosis rate. These results strongly support previous reports that noted suppressed in vitro proliferation of T cells upon ConA stimulation (235). Similar results have been obtained in a tetanus toxin vaccine model demonstrating that restraint stress diminished the proliferating rate of lymphocytes following stimulation with 41 Chapter 2 antigen (261). However, all reported data were based on in vitro stimulation of splenocytes either with relevant antigen or mitogen. Hence, apart from the present study, altered in vivo T cell proliferation in response to stress has not been demonstrated yet. We assumed that the reduced TCD8+ proliferation is probably accompanied by inefficient T cell activation. Indeed, we found significantly lowered expression of the early T-cell activation marker CD69 on day 2 of LCMV-infection in SDR mice compared to the control group, while other activation markers, like CD44 and CD62L, were not affected. Although the function of CD69 has not been ultimately defined, cross-linking studies have suggested a role for CD69 in regulating IL-2 and IFN-γ production, enhancement of IL-2 receptor (CD25) expression, and IL-2-dependent T cell proliferation (262).Therefore, the stress-induced perturbation of T cell proliferation is likely initiated by an early disturbance of TCD8+ activation. Previous studies have shown that glucocorticoids modulate the Ag-presenting capability of dendritic cells (DCs) by altering their maturation (210, 263-264), MHC surface expression (210), and the formation of MHC–antigen complexes (209, 211). Therefore, we examined the possibility that the reduced TCD8+ activation in SDR mice is the consequence of altered antigen-presentation in the spleen. However, SDR and control mice did not differ in the overall percentage of splenic CD11c+ DCs or F4/80+ macrophages throughout the early stage of LCMV infection. Furthermore, DCs from SDR mice expressed similar levels of costimulatory and MHC class I molecules compared to control mice, suggesting that stress did not affect the maturation process of DCs in vivo. Applying an ex vivo antigen presentation assay, we could also exclude the possibility that APCs derived from SDR mice exhibit an impaired efficiency of MHC class I antigen presentation. The discrepancies to previous results are likely due to different experimental approaches, namely the application of exogenous GCs in contrast to the more complex secretion of endogenous GCs during the physiological stress response. However, these latter findings indicate that the diminished TCD8+ splenocyte activation observed in SDR mice could not be explained by an indirect mechanism via Ag-presenting DCs. Based on the above findings it was tempting to speculate that the suppressed T cell proliferation might be mediated by T-cell extrinsic factors. Indeed, the limited capability of TCD8+ splenocytes to proliferate in vivo was accompanied by a profoundly attenuated IFN-γ and IL-2 secretion upon ex vivo restimulation with anti-CD3/CD28. Given the fundamental role of IL-2 in promoting the expansion, survival, and maturation of T cell, we further propose that the decreased in vivo proliferation capacity of TCD8+ cells and the subsequently diminished TCD8+ expansion of SDR mice was primary caused by an intrinsic defect of SDR TCD8+ cells in IL-2 production. To identify the endocrine factors mediating the stress-induced decrease of TCD8+ cell expansion in the spleen, mice were either treated with the GC receptor blocker RU486 or the 42 Chapter 2 β-adrenergic receptor antagonist nadolol. We found that in vivo blockade of GC receptors completely abrogated the stress-associated TCD8+ cell decline in the spleen of SDR mice. In contrast, treatment of SDR mice with nadolol resulted in a TCD8+ cell reduction similar to that obtained from placebo-treated SDR mice, suggesting that the reduction in TCD8+ cell numbers was not related to systemic increases of the catecholamines adrenaline and noradrenaline. In conclusion, these data strongly indicate that suppression of TCD8+ cell expansion in response to stress is mediated by glucocorticoid hormones. Using in vivo T cell transfer experiments, we could finally demonstrate that adoptively transferred TCD8+ splenocytes derived from SDR mice exhibit a reduced in vivo homing potential to the spleen. Because regulation of CCR7 expression and its ligands (ELC/CCL19 and SLC/CCL21) constitutes a crucial factor for T cell migration and retention in the T cell zones of secondary lymphoid organs, we further tested whether an altered chemokine receptor profile was responsible for the different migration pattern. We observed neither a difference in CCR7-expression between TCD8+ cells from SDR and control mice nor in the chemotactic migration towards CCL21/CCL19 in vitro, arguing against a potential impairment of CCR7mediated signaling. However, our observations may reflect an elevated splenic egress of SDR TCD8+ cells. This mechanism would contribute to our observation that SDR mice exhibit a sizeable TCD8+ cell population in peripheral compartments like the liver, lung and blood on day 8 post infection which was comparable to that of control mice. Our results support the concept that glucocorticoids play a major role in modifying T lymphocyte trafficking into secondary lymphoid organs as demonstrated by previous cell migration studies (234, 254). Taken together, this study provides novel evidence that social stress negatively influences the primary immune response to LCMV-infection characterized by a reduced number of IFN-γ producing TCD8+ cells as well as profoundly decreased expansion of activated TCD8+ splenocytes in case of a prolonged stress exposure. We propose a mechanism whereby glucocorticoids inhibit cytokine production and proliferation of TCD8+ cells thereby altering their functional capacity. In conclusion, our data imply that social stress is associated with a profound suppression of T cell function, which may represent a key factor in the increased susceptibility of socially stressed individuals to viral infections. Materials and Methods Animals Male C57BL/6 mice (H-2b) as well as B6.SJL-PtprcaPep3b/BoyJ (also referred to as “Ly5.1 congenic mice”) used in this study were originally obtained from Charles River Laboratories. B6.PL (Thy1.1) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Experimental mice were used at 7-8 weeks of age and were kept in a specified pathogen-free facility on a 12/12 h light/dark cycle with ad libitum access to food and water. Both stressed and control mice were housed in groups of 5 mice per cage. All animal experiments were approved by the reviewing board of the Regierungspräsidium Freiburg. 43 Chapter 2 Stress procedure The social disruption (SDR) procedure was previously described elsewhere and has been shown to induce chronic social stress in mice (225, 230-231). The stress paradigm is based on the disruption of an established social hierarchy of group-housed male mice (residents) which is experimentally induced by daily confrontations with an unfamiliar aggressive intruder mouse. For the stress procedure, an intruder was introduced into the residents cage for 2 h daily over a period of six (experiment 1) or nine (experiment 2) consecutive days. To prevent habituation, a different aggressor was used for each stress cycle. The stress procedure always started at the beginning of the dark period when animals display increased activity and naturally rising glucocorticoid levels. Control mice were left undisturbed in their home cages throughout the entire experiment. Administration of hormone receptor antagonists Mice were subcutaneously implanted with time release pellets (Innovative Research of America, Sarasota, FL) containing either 0.5 mg of the non-selective β-adrenergic receptor antagonist nadolol (21-day release) or 30 mg of the glucocorticoid type II receptor antagonist RU486 (12-day release). The pellets were implanted in the neck region of the animals two days prior to the stress procedure under ketamine/xylazine anaesthesia. Placebo pellets comprising only the inert carrier substance were used as a control. The optimal drug dosage for RU486 was determined in a preliminary experiment based on the ability to effectively prevent stress-induced thymic atrophy. Viruses and media LCMV-WE was obtained originally from F. Lehmann-Grube (Heinrich Pette Institute, University of Hamburg, Germany) and propagated on the fibroblast line L929. Mice were infected i.v. with 200 pfu of LCMV-WE. All media were purchased from Invitrogen Life Technologies (Karlsruhe, Germany) and were supplemented with 5% or 10% FCS, and 100 U/ml penicillin/streptomycin. Flow cytometry Single cell suspensions derived from organs of control and SDR mice (2 × 105 cells) were incubated with fluorochrome-conjugated mAbs for 20 min at 4°C. Following antibody staining, samples were washed twice and acquired on a FACScan flow cytometer (BD Immunocytometry Systems, San Jose, CA) and analyzed by the FlowJo software (Tree Star, San Carlos, CA). Tetramer staining was carried out by staining 2 × 105 splenocytes in 100 µl medium with 2 µl GP33-specific tetramers for 30 min at 37°C. Subsequently, samples were stained with anti-CD8α for 20 min at 4°C, washed twice and acquired on a FACScan flow cytometer (BD Immunocytometry Systems). The following anti-mouse mAbs were used: CD8α (clone 53-6.7), CD69 (clone H1.2F3), Thy1.2 (clone 53-2.1), CD11c (clone HL3), 44 Chapter 2 CD86 (clone B7-2), CD80 (clone B7-1), H-2Db (clone KH95), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), Ly5.2 (clone 104), F4/80 (clone BM8). Intracellular staining (ICS) for IFN-γ Splenocytes (2×106 cells) were incubated in 96-well round-bottom plates with 10-7 M of the specific peptide in 100 µl IMDM /10% FCS in the presence of brefeldin A (10 µg/ml) for 5 h at 37°C. Cells were stained with Cy5-conjugated anti-mouse CD8α (clone 53-6.7, BD PharMingen, San Diego, CA) for 20 min at 4°C. Following fixation with 4% paraformaldehyde at 4°C for 5 min, cells were incubated overnight with FITC-conjugated anti-mouse IFN-γ (clone XMG1.2 BD PharMingen) in PBS containing 2% FCS and 0.1% (w/v) saponin (Sigma, Germany). The following day, samples were washed twice and acquired on of FACScan flow cytometer and analyzed using FlowJo software. BrdU in vivo proliferation assay For the determination of TCD8+ splenocyte proliferation, SDR and control mice were injected intraperitoneally with 1 mg of the thymidine analogue BrdU (Sigma) on day 5 post LCMV infection. Twenty-four hours later, spleens were removed and stained for 20 min at 4°C with anti-CD8α. Incorporation of BrdU was assessed by intracellular staining with FITCconjugated mouse anti-BrdU by using the FITC BrdU Flow Kit (BD PharMingen) according to the manufacturer’s instructions. Antigen-presentation assay Spleen cells were collected from control and SDR mice on day 4 post-infection and were used as stimulator cells in an antigen-presentation assay. LCMV-infected (LCMV GP33-specific CTL line was generated from Thy1.1 mice as previously described (54). CTLs were used in the antigen-presentation assay at an effector-to-stimulator (E:S) ratio of 1:6 in the first dilution, and serial threefold dilutions of stimulators were performed. In a second approach, GP33-pulsed splenocytes from control or SDR-mice were used as stimulator cells for GP33specific CTLs. Briefly, splenocytes were pre-incubated with different concentrations of GP33-peptide [10-7-10-11] for 1h at 37 ºC and washed three times with PBS-. 3x106 stimulator cells were incubated in round-bottom 96-well plates with 5x105 CTLs in 200 µL IMDM 10% + brefeldin A (BFA) (10 µg/mL) for 4 h at 37°C. The staining, fixation and permeabilization of the cells were performed as detailed for the ICS IFN-γ. Adoptive T cell transfer Spleen cells were collected from control and SDR mice on day 6 post-infection and T cells were purified by negative selection with the Pan T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). LCMV-infected (day 6 post-infection) Ly5.1 mice were used as recipients. A total of 1x107 mixed T cells from control (5x106) and SDR (5x106) mice were intraperitoneally (i.p.) or intravenously (i.v.) transferred in a ratio of 1:1 into Ly5.1 recipient 45 Chapter 2 mice. To ensure co-injection of equal numbers of TCD8+ cells from each donor, the ratio of TCD8+ cells was analyzed by flow cytometry before transfer. The ratio of recovered donor derived cells (controlLy5.2+, Thy1.1+; SDRLy5.2+, Thy1.2+) in the spleen, LN and blood was analysed 24 h after the transfer by flow cytometry. Plasma IFN-γ concentration Blood for the quantification of IFN-γ in the plasma was collected from the retro-orbital capillary plexus on days 2, 4, and 6 after LCMV infection. IFN-γ was quantified using a commercially available bead-based assay (Bio-Plex Mouse IFN-γ Assays, Bio-Rad Laboratories, Reinach, Switzerland). Samples were prepared according to the manufacturer’s instructions and were analyzed on a flow cytometer (LSR II, BD Immunocytometry Systems, San Jose, CA) using FACSDiva Software. Absolute cytokine concentration was calculated based on the median fluorescence intensity of the cytokine standard dilution. Stimulation and cytokine secretion of splenocytes Spleens were collected from control and SDR mice on day 4 post-infection, and 4x105 splenocytes, or magnetically enriched TCD8+ cells (negative selection with TCD8+ cells MACS microbeads) were stimulated in 200 µl IMDM 10% for 24 h with 5 µg/ml coated anti-mouse CD3/CD28 mAb (clone 145-2C11, clone 37.51; BD) in flat-bottom 96-well plates. The supernatants were analyzed for IFN-γ and IL-2 secretion in triplicates using commercial ELISA kits for murine IFN-γ and IL-2 according to the manufacturer's instructions (BD). Virus titer To determine viral clearance, spleens were removed on day 2, day 4, day 6 and day 8 post infection, homogenized in MEM containing 5% FCS and stored at –80°C. Supernatants were titrated by 10-fold serial dilutions onto monolayers of the MC57 fibroblast line. LCMV infected MC57 cells were detected after 48 h of incubation at 37°C by immunofocus assay using the LCMV NP-specific mAB (VL-4) as previously described (265). Purification of 20S proteasome from mouse organs and fluorogenic assay For proteasome preparation, pooled livers of SDR and control mice, respectively, were removed on day 8 of LCMV infection and stored at -70°C until proteasome preparation. The proteasome purification procedure including DEAE sepharose chromatography, ammonium chloride precipitation, sucrose gradient centrifugation and MonoQ FPLC was performed as described previously (52). Subunits of isolated 20S proteasomes were analysed on Coomassie-stained two-dimensional NEPHGE/SDS-Page. Statistical analysis Data are expressed as mean ± S.E.M. Means of two independent groups were analyzed using Student’s t-test for independent pairs. Two-factor ANOVA was used to test for the effects of 46 Chapter 2 stress and drug treatment as well as the interaction of both factors. The level of significance was set at p<0.05. All statistics were calculated using GraphPad InStat 3 for Windows (GraphPad Software, La Jolla, CA). 47 Chapter 3 Impaired migration of skin dendritic cells in response to contact sensitisation in mice subjected to chronic social stress Annette Sommershof & Marcus Groettrup Chapter 3 Abstract Chronic stress is not only implicated to increase the susceptibility to infectious diseases but substantial evidence has linked chronic or recurrent exposure to stress with exacerbation of several inflammatory and autoimmune skin disorders. Skin DCs and in particular Langerhans cells (LCs) are known as critical inducers of cutaneous immune responses, however their fate under chronic social stress situations is less documented. We examined the effects of chronic social stress on the migratory capability of skin DCs after epicutaneous skin sensitisation with the contact allergen FITC. Mice subjected to social stress on six consecutive days prior to contact sensitisation showed a significantly reduced migration of FITC-bearing CD11c+ DCs to regional lymph nodes. Pharmacological blockade of β-adrenergic receptors with nadolol did not reverse the stress-associated decline of CD11c+ DCs migration. Furthermore, in vitro migration assays using ear skin explants showed that CD11c+ DCs from SDR mice emigrated less efficiently out of the skin. These results point at a potential mechanism of how stress could negatively influence cutaneous immune responses. Introduction Psychological stress has long been suspected to alter skin immunity and to play an important role in the pathophysiology of numerous inflammatory skin disorders. For example, human epidemiological studies have shown that stressful life conditions are associated with exacerbated symptoms of psoriasis and atopic or allergic contact dermatitis (266-268). Experimental evidence that chronic psychological stress can indeed affect cutaneous immune responses, for example to contact allergens, arouse from mouse models investigating the impact of various stress conditions on the outcome of experimentally induced allergic contact dermatitis (contact hypersensitivity - CHS or delayed-type hypersensitivity - DTH reactions). Moreover, recent studies analysed the contribution of stress hormones on the outcome of DTH reactions, demonstrating that exposure to high levels of corticosterone or prolonged exposure to moderate levels suppresses skin DTH reactions in response to the contact allergens dinitrofluorobenzene (DNFB) or oxazolone (OXA), suggesting that GC hormones are major mediators of the stress-induced suppression of DTH reactions (221). Other studies suggest that GC-independent mechanisms also participate in the stress-related suppression of contact sensitivity. For instance, it has been demonstrated that local intradermal injection of epinephrine at the time of allergen-induced sensitisation inhibited the induction of a DTH response to epicutenously administered DNFB (269). Evidence that LCs are major contributors in CHS reactions aroused from the observation that, during sensitisation of the epidermis they are able to recognise, internalise, and process reactive haptens encountered at the skin surfaces, and transport them from the skin via afferent lymphatics to T cell areas of regional lymph nodes (32). Moreover, recent studies demonstrated that the inducible ablation of LCs in adult Langerin-DTR mice results in a diminished CHS response to different haptens (18) and suboptimal priming of CHS-effector T cells due to inefficient transport of the Ag from the epidermis (19). In this context it has been 49 Chapter 3 shown that in vitro pre-treatment of LCs with epinephrine or norepinephrine resulted in an impaired antigen presentation of these cells, leading to a weakened DTH reaction when reinjected into earlier immunised mice. These results may provide a mechanism for the suppressive effects of stress on DTH responses via inhibition of Ag presentation (269). Studies in mice investigating the effects of GCs on LC function showed that topical corticoisteroid treatment induces apoptosis in LCs and inhibit the expression of costimulatory cytokines (270). Although there is now increasing evidence that both neuroendocrine systems, the HPA axis and the SNS, are able to modulate contact allergen-induced cutaneous immune responses, little is known about their respective contributions under physiological chronic stress conditions (271). Moreover, the underlying mechanisms by which GCs and/or catecholamines may exert their function to influence certain immune cells engaged in contact hypersensitivity reactions are almost lacking. In this regard, skin DCs, which are implicated to play a pivotal role in the initiation of cutaneous immune responses to contact allergens and in skin allergic diseases, have become the focus of recent research. There is novel evidence that a perturbation of the migratory capability of skin DCs and in particular epidermal LCs is involved in pathologies like psoriasis (272) and autoimmune dermatitis (273). Given the undisputable role of stress in the pathogenesis of skin disorders, it is important to understand whether and how chronic stress alters skin DC function, particularly in context of their migration capacity, and how these alterations contribute to skin disorders. Our primary aim was to investigate a potential stress-induced modulation of skin DC migration and to identify neuroendocrine modulators mediating these alterations. Results Diminished in vivo skin DC migration in SDR mice In order to elucidate the impact of chronic social stress on the migratory capability of skin DCs, we performed a FITC-induced in vivo migration assay. Fluorescein isothiocyanate (FITC) is a fluorescent marker for migratory DCs and has been used in in vivo epidermal LC migration assays since the 1980ies (274-276). Topical FITC application has been shown to preferentially induce LCs in the epidermis, and FITC-bearing cells in the draining LNs were found to be primarily derived from LCs (277-278). Mice were stressed for six days and the FITC-solution was applied 20 hours after the last stress exposure to the abdomen of SDR and control mice, and the presence of FITC-bearing CD11c+ cells in the inguinal LNs was analysed 24 hours after sensitisation. 50 Chapter 3 Figure 1: Effects of social stress on the migratory capability of skin DCs to draining LNs SDR and control mice were painted with 30 µl of 1% FITC in acetone/dibutylphalate (1/1) on the abdomen. 24 h later, inguinal LNs were collected and single-cell suspensions were stained with APC-labelled antiCD11c antibody and analysed by flow cytometry for the percentage of FITC+ DCs. (A) Dot plot of CD11c staining and histograms for control and SDR mice are shown, representing the percentage of recovered FITC+ positive cells pre-gated on CD11c+ cells. Negative control represents the FITC background fluorescence obtained from naïve mice. (B) Data are presented as the mean percentage of FITC-positive cells ± SEM of the CD11c+ population. As shown in Figure 1, 24 hours after FITC-treatment, the proportion of FITC+ CD11c+ cells in the inguinal LN of control mice was markedly higher compared to SDR mice (control: 13.3±2.1% vs. SDR: 4.1±0.5%), indicating that exposure to chronic stress prior to hapten stimulation results in a substantial impairment of CD11c+ cell migration from the epidermis into skin draining LNs. Although we did not provide direct evidence that the immigrated FITC+ cells in the LN originate from the epidermis, hence reflecting LCs, it has been demonstrated previously that after epicutaneous FITC sensitisation the majority of FITCbearing cells are derived from epidermal LCs (277-278). Phenotype of migrated CD11c+ skin DCs In order to correlate the migration of CD11c+ cells with their maturation induced by FITC sensitisation, we analysed the expression of CD86 and CCR7 on recovered FITC+ CD11c+ cells in the inguinal LNs. As shown in Figure 2 A, immigrated FITC-bearing DCs exhibit a strong expression of the costimulatory molecule CD86 and the chemokine receptor CCR7, compared to resident FITCDCs exhibiting a semi-mature phenotype with intermediate levels of both molecules. Moreover, migrated FITC+ CD11c+ from stressed mice did not significantly differ in their expression of CD86 (Figure 2 B) and CCR7 (Figure 2 C), compared with those of unstressed control mice. These data demonstrate that by the time they reach the draining LNs, skin derived DCs apparently display a mature phenotype with respect to CD86 and CCR7 expression. These observations are in line with previous reports, demonstrating that full maturation of skin DCs is a fundamental requirement for their migratory competence under inflammatory conditions (15). 51 Chapter 3 Figure 2: Phenotype of migrated FITC+ CD11c+ cells in the LN The activation state of migrated FITC+ CD11c+ cells in the inguinal LNs was assessed 24h after abdominal FITC-sensitisation by their expression level of CD86 and CCR7. (A) Representative dot plots of CD11c staining and contour plots, representing FITC versus CD86 and CCR7 profiles of the pre-gated CD11c+ population are shown. (B, C) Data in the bar plot are presented as mean fluorescence of CD86 (B) and CCR7 (C) expression ± SEM of migrated FITC+ CD11c+ cells in control and SDR mice. Role of catecholamines in the modulation of skin DC migration To identify the endocrine factors mediating the stress-induced decrease of skin DC migration, SDR and control mice were implanted two days prior to the stress procedure with continuous release-pellets containing the β-adrenergic receptor antagonist nadolol. Figure 3: Skin DC migration in SDR and control mice treated with slow-release pellets of nadolol or placebo The frequency of FITC+ CD11c+ cells in the inguinal LNs of placebo-treated SDR mice was not different from that in nadolol-treated mice. Data are presented as the mean percentage of FITC-positive cells ± SEM of the CD11c+ population. Unstressed control mice treated with nadolol exhibited similar percentage of FITC-positive cells in the inguinal LNs compared to mice receiving placebo pellets, demonstrating that nadolol did not influence the skin DC migration by itself. Notably, the frequency of FITC+ CD11c+ cells in nadolol-treated SDR mice was not significantly altered compared to placebo52 Chapter 3 treated SDR mice (SDR nadolol: 10.3±1.4% vs. SDR placebo: 10.7±1.6%), indicating that βadrenergic receptor blockade was not effective in preventing the stress-associated changes of CD11c+ migration into the LN (Figure 3). Impaired ex vivo emigration of skin DCs in SDR mice Two possibilities could explain the reduced migration and accumulation of CD11c+ in the skin draining LNs of SDR mice. First, inefficient DC migration is caused by an inability of adequate DC numbers to emigrate out of the skin. The other possibility is that skin DCs are mobilised to the same degree, but fail to enter the afferent lymphatics and/or transit to a lesser extent via lymph to the LN. The latter scenario might involve a defective synthesis of chemotactic signals important for LN homing such as the CCR7 ligands ELC/CCL19 and SLC/CCL21. To test whether the impaired migration of skin derived CD11c+ observed in SDR mice reflect a diminished egress of activated DCs from the skin, we evaluated the migratory potential from SDR and control mice ex vivo in a skin ear explant model. Mouse skin cultures are commonly used to study skin DC migration in ear skin cultures as an "ex vivo chemotaxis assay" (279) and previous studies have demonstrated that the migration of DCs out of ear skin explants into the culture medium reflect, in part, their in vivo migratory capacity (280). For example in vitro DCs migrate into the dermis to form so called “dermal cords” in the region of dermal lymphatics as they do under in vivo conditions, before continuing to migrate spontaneously into the medium (280). Moreover, it has been demonstrated that the vast majority of skin-emigrated DCs in the medium represent epidermal LCs (32, 280) and that SLC/CCL21 and ELC/CCL19 induce egress of LCs from explanted skin (279). Stressed and control mice were sensitised with FITC-solution on the dorsal and ventral side of each ear. Twenty-four hours later, ears were excised and ventral ear sheets were cultured in presence or absence of relevant chemokines to stimulate the release of DCs from the skintissues. As illustrated in Figure 5 A, an average of about 2.300 DCs, identified as CD11c+ cells spontaneously emigrated out of one ventral ear half into the culture medium in the absence of exogenous cytokines. In contrast, the number of ex vivo emigrated CD11c+ cells was markedly reduced in skin explants from SDR mice (control: 2236±446 vs. SDR: 1082±95), indicating that DCs from SDR mice exhibit a reduced capability to egress outof the skin. 53 Chapter 3 Figure 4: Effects of social stress on the mobilisation of skin DCs in ear explants Mice were subjected to six days of SDR and 20h after the last stress exposure mice were contact-sensitised with 1% FITC solution on both ears. 24 hours after contact sensitisation ears were removed and split into dorsal and ventral halves. Ventral ear halves were incubated dermis-side down in the (A) absence (w/o) or presence of (B) SLC and (C) ELC. After 24 hours, cells that migrated ex vivo from the tissue into the culture medium were collected and stained for CD11c expression. The number of migrated CD11c+ cells was enumerated using an Accuri flow cytometer. Because DCs are attracted to lymphatic vessels by CCR7-SLC and ELC interaction (281282), we further determined the magnitude of CD11c+ egress in response to both chemoattractants. As shown in Figure 4 B, C, the migratory potential of skin DCs from SDR mice was also significantly lower in the presence of SLC/CCL21 (Figure 4 B; control: 3193±546 vs. SDR: 1558±44) and ELC/CCL19 (Figure 4 C; control: 3020±238 vs. SDR: 1171±228) with respect to the control counterparts, suggesting that the impaired skin DC mobilisation in SDR mice is not due to a lack of these chemokines. Since both SLC/CCL21 and ELC/CCL19 are known to be potent chemoattractants for LCs in skin explant cultures and the presence of exogenously added chemokines is established to significantly increase emigration (283-284), absolute numbers of emigrated CD11c+ cells in our experiments cannot be directly compared since the experiments were performed independently and hence vary in absolute cell numbers. Discussion In this study we demonstrate that chronic social stress suppresses the migratory capability of skin DCs to regional LNs after contact sensitisation with FITC. These findings were associated with an impaired mobilisation of CD11c+ cells from the skin, as revealed by the frequency of CD11c+ cells migrated ex vivo out of ear skin explants. Moreover, we could show that an action of catecholamines at peripheral β-adrenergic receptors is not involved in the suppressive effect of stress, since in vivo blockage of these receptors by the specific antagonist nadolol did not reverse the stress-associated decline of skin DC migration. We are currently investigating whether GCs are the major mediators of the stress-induced altered skin CD11c+ migration. Although we did not distinguish between the dermal DC populations and epidermal LCs in our experiments, it is likely that the reduced migration of skin CD11c+ cells reflects an 54 Chapter 3 inhibition of epidermal LCs. Previous studies demonstrated that topical FITC application preferentially induces LC migration in the epidermis, and FITC-bearing cells in the draining LNs were found to be primarily derived from LCs (277-278). Thus skin DC migration in this model is mainly independent of other dermal DCs and allows conclusion of the involvement of epidermal LCs. Nevertheless, we cannot rule out the possibility that FITC passes through the epidermis and penetrates the dermis to bind to dermal DCs, therefore at least some of the FITC-labeled cells recovered in the draining LNs may represent dermal DCs. The precise mechanism by which neuroendocrine factors act to inhibit the mobilisation of CD11c+ DCs from the skin is not yet clear. Upon skin sensitisation a rapid release of inflammatory cytokines such as TNF-α and IL-1β induces a variety of mechanisms ensuring the mobilisation of LCs from their environment as well as their homing towards regional LNs (16). Consistent with this idea, it was demonstrated that in response to epicutaneous FITCapplication, mRNA levels of TNF-α and IL-1β in epidermal cells are rapidly up-regulated (32). Moreover, inhibition of contact allergen-induced LC migration by prior treatment of mice with neutralizing TNF-α mAbs or an IL-1β antagonist is associated with both impaired epidermal egress and LN accumulation (23, 32). On the one hand, both TNF-α and IL-1β are needed for breaking up E-cadherin bonds endowing the detachment of LCs from neighbouring keratinocytes and their subsequent mobilisation within the epidermis (26-27). On the other hand, the migratory capability of LCs is tightly linked with the concomitant upregulation of the chemokine receptors CXCR4 and CCR7 (15) that attract and guide migrating LCs from the epidermis into the lymphatic vessels and further on into the lymph nodes (41). In this context, TNF-α has been shown to induce the up-regulation of CCR7 in LCs (283). Thus a reduced TNF-α and/or IL-1β de novo synthesis or secretion will result in decreased LC detachment and accumulation of LCs in the epidermis, and such mechanism could contribute to our observation. Indeed, there is compelling evidence that GCs are generally able to negatively regulate TNF-α and IL-1β expression both at transcriptional and post-transcriptional levels (285-287). Moreover a previous study demonstrated that the synthetic glucocorticoid DEX affects allergen- and IL-1β −mediated but not TNF-α-induced LC migration and accumulation in the LNs, suggesting that the impaired LC migration in this model is predominantly caused by inhibition of TNF-α secretion by keratinocytes (288). Since the stress procedure occurs prior to the contact sensitisation, an alternate explanation for the observed impairment of skin DC migration could be a stress-induced decrease of absolute CD11c+ DC numbers in the skin. This reduction could in turn be the result of a GC-mediated apoptosis or a redistribution of DCs in response to the stress procedure, which has occurred prior to the contact sensitisation. In this context it is of importance to note that physiological stress responses are generally associated with significant changes in the distribution of leukocytes within the body (234, 289). For instance, it has been shown that chronic social stress in rats results in decreased numbers of TCD8+ and TCD4+ cells in the blood of subordinate animals (289). Moreover, exposure of mice to social disruption stress is associated with a recruitment of CD11b+ leukocytes from the bone marrow to the blood and the spleen as well 55 Chapter 3 as a reduction of total T cell numbers in the spleen (234). Social stress-induced reduction of T cells in the blood and spleen could also be due to a redistribution of T cells to other immune compartments. This view is supported by a study demonstrating that radiolabeled, adoptively transferred blood T cells accumulate in the bone marrow of defeated recipient rats, wherease their recirculation in the spleen and lymph nodes is decreased (254). Such changes in leukocyte distribution may have significant implications for the outcome of chronic stress exposure on cutaneous immune responses after contact sensitisation (290). Thus the experiments conducted in this study are yet preliminary and factors triggering the impaired skin CD11c+ DC accumulation in the LNs of SDR mice need to be further elucidated. Moreover our experiments clearly contain several potential limitations, as phenotypic characterization of migrated CD11c+ cells needs to be conducted, allowing an ultimate differentiation between epidermal EpCAM+ LC and the different subsets of EpCAMdermal DCs. It has been demonstrated previously that the majority of FITC-bearing cells derive from epidermal LCs (277-278). Moreover, additional analysis time points after FITCsensitisation are required to elucidate the kinetics and magnitude of CD11c+ arrival in the LN, and to determine whether the reduced accumulation observed in SDR mice reflect a delayed arrival or a general migration deficiency. Various studies have examined the effects of psychological stress on Ag-specific skin immunity by analyzing the outcome of delayed type hypersensitivity reactions (DTH) in response to contact allergens. DTH and CHS (contact hypersensitivity) reactions are T cellmediated immune reactions that in humans manifest as an inflammatory skin disease referred to as contact dermatitis. DTH reactions have been shown to be either enhanced (291-292) or reduced (292-294), depending on the time point and duration of the stress exposure. For example, a short duration stressor administered immediately before sensitisation significantly enhanced DTH reaction in response to DNFB or OXA (291), whereas chronic stress significantly suppressed the DTH reaction (290). Moreover it has been suggested that GC hormones are major mediators of the stress-induced suppression of DTH reactions (295). Given the role of LCs in CHS and DTH responses, we propose that impaired skin DCs may represent a mechanism contributing to altered skin immunity after contact sensitisation in stressed mice. In conclusion, the data presented here show that chronic social stress is able to diminish skin CD11c+ cell migration toward the LN and this inhibition is not attributable to an action of catecholamines at peripheral β-adrenergic receptors. Skin DCs are gaining increasing interest because of their involvement in several skin diseases such as psoriasis (272) or autoimmune dermatitis (273). Moreover, a deficient migratory property is suggested to play an important component in these diseases. Therefore, further investigations are needed in order to understand the mechanisms underlying stress-induced enhancement of skin diseases at the level of DCs. 56 Chapter 3 Material and Methods: Animals Male C57BL/6 mice (H-2b) used in this study were originally obtained from Charles River Laboratories. Experimental mice were used at 7-8 weeks of age and were kept in a specified pathogen-free facility on a 12/12 h light/dark cycle with ad libitum access to food and water. Both stressed and control mice were housed in groups of 5 mice per cage. Stress procedure For the stress procedure, an intruder was introduced into the residents cage for 2 h daily over a period of six consecutive days. To prevent habituation, a different aggressor was used for each stress cycle. The stress procedure always started at the beginning of the dark period when animals display increased activity and naturally rising glucocorticoid levels. Control mice were left undisturbed in their home cages throughout the entire experiment. Experimental design To analyze the impact of social disruption stress on the migratory capacity of DCs, the experimental mice were subjected to the stress procedure prior to contact sensitisation. For this purpose, mice were exposed to six consecutive days of SDR and both groups (stressed and control mice) were challenged by epicutaneous contact sensitisation, 20 h after the last stress procedure. The corresponding assays were carried out as described in the text. Figure 5: Scheme of the experimental design Administration of hormone receptor antagonists Mice were subcutaneously implanted with time-release pellets (Innovative Research of America, Sarasota, FL) containing 0.5 mg of the non-selective β-adrenergic receptor antagonist nadolol (21-day release). The pellets were implanted in the neck region of the animals two days prior to the stress procedure under ketamine/xylazine anaesthesia. Placebo pellets comprising only the inert carrier substance were used as a control. Flow cytometry Single cell suspensions derived from LNs of control and SDR mice (2 × 105 cells) were incubated with fluorochrome-conjugated mAbs for 20 min at 4°C. Following antibody 57 Chapter 3 staining, samples were washed twice and acquired on a FACS Calibur or Aria flow cytometer (BD Immunocytometry Systems, San Jose, CA) and analyzed by the FlowJo software (Tree Star, San Carlos, CA). The following anti-mouse mAbs were used: CD11c (clone HL3), CD86 (clone B7-2), CCR7 (clone 4B12). Assay for hapten-induced DC migration (FITC painting) The FITC painting assay is a model of hapten-induced DC activation and is widely used to trace the in vivo migration of LCs. Mice were painted on the shaved abdomens with 30 µl of 1 mg/ml FITC dissolved in 1:1 acetone: dibutylphalate. 24 hours later, draining inguinal LNs were collected and gently disrupted. As a control, inguinal LNs were taken from naïve mice. Single cell suspensions were stained for CD11c. Following antibody staining, samples were washed twice, acquired on a FACS Calibur or Aria flow cytometer (BD Immunocytometry Systems, San Jose, CA) and analyzed by the FlowJo software. Results are expressed as the percentage of FITC positive cells of CD11c+ cells in the lymph nodes. Ear skin explant cultures and DC migration 20h after the last stress exposure, SDR and control mice were contact sensitised with 1% FITC solution on both ears. 24 hours after contact sensitisation ears were excised, washed with 70% ethanol and dried for 10 min. Ears were split into dorsal and ventral halves and ventral halves were used for the emigration assay. For elimination of non-DCs that are initially released into the culture, ear halves were floated dermal side down in a six-well plate containing 2 ml RPMI and incubated for 30 min at 37º C. After incubation ventral halves were transferred on new six well plates containing 1,5 ml RPMI and incubated for 24 h at 37º C in the presence or absence of SLC or ELC [200 ng/ml]. Cells that migrated ex vivo from the ear tissue into the culture medium were collected, stained for CD11c expression and resuspended in 200 µl FACS buffer. The total number of migrated CD11c+ cells from each ventral ear halve was assessed by acquiring 100 µl of the single-cell suspensions on a FACS Accuri (Accuri Cytometers, Inc. Ann Arbor, MI). The data are expressed as total number of CD11c+ cells emigrated from each ventral ear halve. Statistical analysis Data are expressed as mean ± S.E.M. Means of two independent groups were analyzed using Student’s t-test for independent pairs. The level of significance was set at p<0.05. All statistics were calculated using GraphPad InStat 3 for Windows (GraphPad Software, La Jolla, CA). 58 Chapter 4 Substantial reduction of naïve and regulatory T cells following traumatic stress Annette Sommershof, Hannah Aichinger, Harald Engler, Hannah Adenauer, Claudia Catani, Eva-Maria Boneberg, Thomas Elbert, Marcus Groettrup, Iris-Tatjana Kolassa Published in Brain, Behaviour and Immunity 2009, 23 (8), 1117-24 Chapter 4 Abstract Posttraumatic stress disorder (PTSD) is associated with an enhanced susceptibility to various somatic diseases. However, the exact mechanisms linking traumatic stress to subsequent physical health problems have remained unclear. This study investigated peripheral T lymphocyte differentiation subsets in 19 individuals with war and torture related PTSD compared to 27 non-PTSD controls (n = 14 trauma-exposed controls; n = 13 non-exposed controls). Peripheral T cell subpopulations were classified by their characteristic expression of the lineage markers CD45RA and CCR7 into: naïve (CD45RA+ CCR7+), central memory (TCM: CD45RA- CCR7+) and effector memory (TEM: CD45RA- CCR7- and TEMRA: CD45RA+ CCR7-) cells. Furthermore, we analyzed regulatory T cells (CD4+CD25+FoxP3+) and ex vivo proliferation responses of peripheral blood mononuclear cells after stimulation with anti-CD3 monoclonal antibody. Results show, that naïve CD8+ T lymphocytes were reduced by 32% (p = 0.01), whereas CD3+ central (p = 0.02) and effector (p = 0.01) memory T lymphocytes were significantly enhanced (+22% and +34%, respectively) in PTSD patients compared to nonPTSD individuals. To a smaller extent, this effect was also observed in trauma-exposed nonPTSD individuals, indicating a cumulative effect of traumatic stress on T cell distribution. Moreover, PTSD patients displayed a 48% reduction in regulatory T cells (p < 0.001). Functionally, these alterations were accompanied by a significantly enhanced (+34%) ex vivo proliferation of anti-CD3 stimulated T cells (p = 0.05). The profoundly altered composition of the peripheral T cell compartment might cause a state of compromised immune responsiveness, which may explain why PTSD patients show an increased susceptibility to infections, and inflammatory and autoimmune diseases. Introduction Exposure to traumatic stressors such as life-threatening accidents, physical assaults, sexual abuse, or combat experience poses a risk for severe mental disorders, and in particular for the development of posttraumatic stress disorder (PTSD). PTSD is characterized by reexperiencing the traumatic event (in form of intrusive recollections, nightmares or flashbacks), by persistent avoidance of stimuli associated with the trauma and emotional numbing, as well as a constant state of heightened alertness and increased arousal (American Psychiatric296). Since the risk for developing PTSD increases with the number of traumatic stressors experienced (297-298), PTSD is a serious mental health problem in war and conflict regions, where exposure rates are high (299). In addition to psychiatric morbidity, numerous studies have shown that traumatic stress and especially PTSD are associated with poor selfreported physical health (e.g., heightened rate of infectious diseases), increased health care use and costs, and an elevated risk for multiple comorbid medical disorders such as cardiovascular, respiratory, gastrointestinal, musculoskeletal or inflammatory and autoimmune diseases (300-302). Peripheral T lymphocytes consist of a range of functionally different subpopulations, i.e., naïve, effector and memory T cells, which provide effective protection against a wide range 60 Chapter 4 of viruses and other pathogens. Fine regulation of generation, maintenance and function of the peripheral T cell compartment is crucial for an optimal balance between immunity and peripheral tolerance (303). Dysregulation within the peripheral T cell compartment, e.g., as a consequence of thymic involution and altered T cell activation or homeostasis, is involved in a variety of immunopathologies such as rheumatoid arthritis (304) and multiple sclerosis (305-306). Regarding the fundamental role of T cells in infectious diseases and inflammatory or autoimmune disorders, we hypothesized that the enhanced susceptibility to such diseases in PTSD patients could be linked to changes in the composition of the peripheral T cell pool. Indeed, major T cell populations in PTSD patients have been evaluated in several studies, but results obtained so far are contradictory. For instance, it has been reported that PTSD patients exhibit higher numbers of circulating T lymphocytes (302, 307) whereas other studies reported no differences (308-309) or even lower T cell numbers (310). A similar picture emerges with respect to the T helper cell population, with one study reporting an increase (307) of circulating T helper lymphocytes and others showing a decreased proportion (310311) or no differences (308-309, 312-313). Regarding cytotoxic T cells, the majority of studies found no differences between PTSD patients and controls (308-309, 312-313) while two studies reported lower levels (310-311). In addition, a higher ratio of CD4/CD8 lymphocytes has been suggested in PTSD patients versus controls (314). Considering the structural diversity among the peripheral T cell pool, we assume that it is inappropriate to compare bulk T cell populations since aberrations may occur in the activation and differentiation states of T cells. Therefore, we decided to provide a detailed characterisation of T cell maturation subsets in a sample of PTSD patients, applying a differentiation model of T cells defined by changes in the expression of the lineage markers CD45RA and CCR7. CD45RA is a high molecular weight isoform of the receptor-type protein tyrosine phosphatase CD45, also known as the common leukocyte antigen, which is required for the regulation of signal transduction pathways involved in T cell activation. According to this differentiation model, naïve T cells (CD45RA+ CCR7+) become activated after antigen stimulation, then differentiate into memory cells, and partly develop into effector cells with a strong cytolytic capability (125, 315). Memory T cells are a heterogeneous population and can be divided into distinct subsets of central memory (TCM) and effector memory cells, respectively, characterized by the presence or absence of the chemokine receptor CCR7 (125). TCM cells predominantly home to secondary lymphoid organs and lack immediate effector function but rapidly proliferate and gain cytolytic activity upon antigen stimulation. Conversely, the effector memory subset displays immediate effector function, has a low proliferative capacity and migrates to peripheral tissues (124). The effector memory T cells can be further subdivided into CD45RA- (TEM) and CD45RA+ (TEMRA) cells, which have been shown to differ in their expansion potential and the expression of perforin (124). 61 Chapter 4 Peripheral CD4+CD25+ regulatory T (Treg) cells are crucial for controlling immune responses and maintaining self-tolerance by inhibiting autoreactive T cells (316). The transcription factor FoxP3 (forkhead box P3) has been shown to be essential for the development and suppressive function of peripheral Tregs and is used as an intracellular marker for the identification of Tregs (317-319). Genetic defects in FoxP3 have been shown to cause the severe, systemic autoimmune syndrome IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) in humans (320). Additionally, there is growing evidence that a decrease in number or function of peripheral Tregs might contribute to the development of inflammatory and autoimmune diseases, such us multiple sclerosis, asthma, type 1 diabetes, psoriasis, and rheumatoid arthritis (321). Considering the fundamental role of Tregs in the regulation of immune responses and the increased prevalence of PTSD to inflammatory or autoimmune disorders (302), we further analyzed the frequencies of peripheral Tregs in PTSD patients and non-PTSD subjects. In order to clarify whether changes in the peripheral T cell pool are accompanied by functional alterations such as an altered T cell proliferation capacity, we further investigated the responsiveness of T lymphocytes after T cell receptor (TCR) stimulation with anti-CD3 monoclonal antibody. In the present study, we present a differentiated characterisation of the differentation state of T lymphocytes in a group of severely traumatized PTSD patients. We demonstrate that PTSD patients exhibit a profoundly altered composition of the peripheral T cell compartment, as indicated by a marked reduction in naïve and an increase in CD45RA- memory T cells, compared to control individuals. Furthermore, this is the first study showing that subjects with PTSD display a substantial reduction of peripheral regulatory T cells, which could be a cause of the increased susceptibility to inflammatory and autoimmune diseases in those with PTSD Results Quantification of naïve and memory T lymphocytes As shown in Table 1, the PTSD group had experienced a significantly greater number of different traumatic event types than the non-PTSD participants and reported significantly higher CAPS and HAM-D scores. Table 1: Clinical characteristics of PTSD patients and non-PTSD subjects CAPS, Clinician Administered PTSD Scale; HAM-D, Hamilton Depression Rating Scale. Significant pvalues and their correspondent group means are displayed in bold. 62 Chapter 4 PTSD patients and control individuals did not differ with respect to absolute numbers of lymphocytes (PTSD: 2028.9 ± 405,7, n = 18; non-PTSD: 1936,7 ± 455,9, n = 20; F = 0.43, p = 0.52), or their overall percentage of B lymphocytes (PTSD: 3.0 ± 1.3% of leukocytes, n = 18; non-PTSD: 3.0, ± 1.3% of leukocytes n = 24; F = 0.0, p = 0.98) and CD3+ T lymphocytes (see table 2). However, as presented in Table 2 and Figure 1A-D, the percentage of CD3+ T cells of the naïve (CD45RA+ CCR7+) phenotype was reduced in individuals with PTSD compared to non-PTSD subjects, whereas the percentage of CD45RA- memory phenotype was increased. This was due to an increased frequency of both the TCM (CD45RA- CCR7+) and TEM (CD45RA- CCR7-) populations in PTSD individuals. No significant group differences were observed for the CD3+ TEMRA population (CD45RA+ CCR7- ). Figure 1: PTSD patients display an altered peripheral T lymphocyte subset distribution (A) Representative flow cytometric analysis of the whole T cell (CD3+) population and subset distribution. (B-D) Data are presented as the mean percentages + SEM of (B) total, (C) naïve and CD45RA- memory T cells, or (D) central memory (TCM) and effector memory (TEM) cells from PTSD patients and non-PTSD individuals. We further examined whether these alterations occurred in both the cytotoxic (CD8+) and T helper (CD4+) lymphocyte populations. As shown in Table 2, PTSD patients had a significantly lower percentage of CD8+ T lymphocytes compared to control individuals. Further subdivision revealed a massive reduction of naïve CD8+ T cells. The CD8+ TEM population was significantly increased in the PTSD group compared to the control group, whereas no differences were observed for the TCM and the TEMRA subsets. 63 Chapter 4 No significant group differences were detected for the percentage of CD4+ T cells and the naïve or memory CD4+ T cell subpopulations (see Table 2). Table 2: T cell maturation subsets in PTSD patients vs. non-PTSD individuals Significant p-values and the correspondent group means are displayed in bold. To clarify whether the above-mentioned alterations are a specific feature of PTSD, or rather constitute a general consequence of trauma exposure, we repeated these analyses after subdividing the non-PTSD group into a group with substantial exposure to traumatic stressors and a control group with no or few traumatic experiences. With respect to the reduction in naïve T cells and enhancement of memory T cells, the trauma-exposed non-PTSD group displayed an intermediate phenotype positioned between the PTSD group and the nonexposed controls, indicating a cumulative effect of exposure to traumatic stressors on T cell distribution (see Figure 2). 64 Chapter 4 Figure 2: Cumulative effect of traumatic stress on peripheral T lymphocyte subset distribution Data are presented as mean percentages + SEM of naïve and CD45RA- memory subsets within the total (A) CD3+, (B) CD8+ and (C) CD4+ population in PTSD patients, as well as trauma-exposed and control individuals. (D) PTSD symptom severity, (E) number of experienced war and torture event types. Quantification of FoxP3 expressing T cells and proliferation capacity of T cells Regarding the immunoregulatory function of CD4+CD25+FoxP3+ regulatory T cells (Tregs) and their role in maintaining self tolerance (316), we further compared the frequencies of peripheral Tregs in PTSD patients and non-PTSD subjects. Strikingly, we found a 48% reduction in the percentage of peripheral Tregs in PTSD individuals compared to non-PTSD individuals (PTSD: 1.2 ± 0.6%, n = 15; non-PTSD: 2.3 ± 0.9%, n = 20; F = 17.5, p < 0.001, see Figure 3A-B). To further investigate the proliferative capacity of T cells we performed a CFSE-based ex vivo proliferation assay. As presented in Figure 3C-D, peripheral blood T lymphocytes of PTSD patients displayed higher ex vivo proliferation responses when stimulated with anti-CD3 mAb (PTSD: 46.5 ± 14.8%, n = 12; non-PTSD: 34.7 ± 15.3%, n = 15; F = 4.1, p = 0.05). 65 Chapter 4 Figure 3: PTSD patients exhibit a lack of peripheral Tregs and increased ex vivo T cell proliferation (A) Representative flow cytometric analysis of CD4+CD25+FoxP3+ Tregs. (B) Mean percentages + SEM of peripheral Tregs in PTSD vs. without intracellular FoxP3 staining. (C) Representative proliferation profile of PBMCs after ex vivo stimulation with anti-CD3 or without stimulation (Neg). (D) Mean percentages + SEM of proliferation response in PTSD vs. non-PTSD individuals. The negative control (Neg.) represents gated CD4+ cells Moderating variables Since our sample consisted of male and female participants as well as of smokers (PTSD: N = 7 vs. non-PTSD: N = 5) and non-smokers, we repeated all analyses with gender or smoking as additional between-factors, to control for the possible influence of these variables on the immune alterations reported here. For the different immunological variables, no significant main effects of gender and no significant group × gender interactions could be identified. After introducing gender as additional factor, all group differences reported above remained statistically significant, except the ex-vivo proliferation response (p =.13). Similarly, we could not identify significant main effects of smoking and no significant group × smoking interactions. After introducing smoking as additional factor, all group differences reported above remained statistically significant, except the overall percentage of CD8+ T lymphocytes (p =.21), the percentage of CD3+ TEM cells (p =.06) and the ex-vivo proliferation response (p =.16). 66 Chapter 4 Discussion In the present study we characterize phenotypic changes in T lymphocyte subsets in the peripheral blood of severely traumatized PTSD patients compared to non-PTSD individuals. Our results demonstrate that PTSD patients exhibit a profound reduction in CD3+ naïve T lymphocytes, accompanied by an increased proportion of central (TCM) and effector memory (TEM) cells. Interestingly, to a smaller, albeit not statistically significant extent, this effect could also be observed in trauma-exposed non-PTSD individuals, indicating a cumulative effect of exposure to traumatic stressors on T cell distribution. The reduction of naïve and the increase of TEM cells were most pronounced within the CD8+ T cell population, whereas CD4+ T cells were not significantly altered. Furthermore, regulatory T cells were reduced by 48% in PTSD patients compared to non-PTSD individuals. Functionally, these alterations were accompanied by a significantly enhanced proliferation of anti-CD3 stimulated T cells ex vivo. These stress-related alterations of the peripheral T cell compartment might constitute a key factor in the enhanced susceptibility of persons with PTSD to a range of physical diseases. More specifically, it has been observed that a shrinking repertoire of naïve T cells may correlate with an enhanced susceptibility to infectious diseases. Therefore, we propose that the reduction of the naïve CD8+ T cell pool in PTSD patients could compromise their ability to mount an effective T cell response to various pathogens and thus might be a key factor in the enhanced susceptibility to infectious diseases. This impairment has been confirmed in immunocompromised individuals such as elderly persons where the progressive loss of naïve T lymphocytes is known to be a major reason for the increased risk for age-related diseases (322-323). Moreover an accumulation of CD45RA- effector-memory cells is characteristic of an aging immune system (324) and thus is consistent with other reports showing that psychological stress is associated with immunological aging (325-327). Interestingly, the enhanced proportion of memory cells in PTSD patients only occurred within the CD45RAmemory pool, i.e. in the TEM and TCM subpopulations, being most prominent in the TEM population. In contrast, the TEMRA population, which re-expresses the CD45RA isoform, did not differ between PTSD and control individuals. CD45RA+ memory cells functionally differ from the CD45RA- memory pool by their predominantly high lytic potential, their very low expansion potential and their increased sensitivity to apoptosis (124). In accordance with our finding of increased CD45RA- memory T cells in the PTSD group, enhanced T cell mediated memory responses to various pathogens, as measured by delayed-type hypersensitivity (DTH) reaction, have been reported in PTSD patients in earlier studies (302, 312). The most striking alterations appear in the percentage of peripheral regulatory T cells (Tregs), with almost a 50% reduction in PTSD patients compared to non-PTSD individuals. Tregs play a pivotal role in maintaining self-tolerance and are essential for the suppression of autoimmune diseases. Deficiency or dysfunction of Tregs in humans has been linked to several inflammatory and autoimmune diseases including multiple sclerosis, asthma, type 1 diabetes, psoriasis, and rheumatoid arthritis (321). We therefore propose that the percental reduction of 67 Chapter 4 Tregs in the blood of individuals with PTSD reported here could be related to the increased risk of PTSD patients for autoimmune diseases in general, and for rheumatoid arthritis, psoriasis, hypothyroidism, and diabetes in particular (302, 328-330). In addition, Tregs are crucial players in controlling both inflammation and virus-specific T lymphocyte responses. During acute and chronic infections, Tregs suppress inflammation to limit immunopathological side effects of inflammation (331). The substantial reduction of peripheral Tregs in individuals with PTSD could bear the risk of excessive inflammation due to suboptimum control of the immune response. This view is supported by studies reporting enhanced levels of proinflammatory cytokines in PTSD patients (332). Assuming that the increased memory population might be accompanied by an altered T cell proliferation capacity, we analyzed the proliferation response of T lymphocytes ex-vivo after stimulation with anti-CD3 mAb. We found significantly increased proliferation of PBMCs isolated from blood of PTSD patients compared to non-PTSD individuals. It has been shown that memory T cells exhibit a lower activation threshold and a higher proliferative capacity after in vitro stimulation (125), thus it is possible that the enhanced ratio of memory T cells found in PTSD patients is responsible for the augmentation in T cell proliferation. Recently, it has been proposed that Tregs are involved in the suppression of naïve and memory T cell proliferation, thereby altering the quantity of the memory T cell pool (333). Therefore the increased proliferation capability of T lymphocytes in response to T-cell receptor (TCR)triggering could be associated with the reduced proportion of Treg cells since the latter have been shown to inhibit naïve and memory T cell proliferation (333-335). Whether the observed changes in the distribution of T cell maturation subsets are due to alterations in the thymic output of naïve T cells or peripheral T cell turnover needs to be established in future studies. Many researchers have reported that individuals with PTSD show anomalies in their neuroendorine profile, which is characterized by elevated norepinephrine levels (336). With respect to cortisol, result indicate frequently lower (337) but in some studies also elevated (338) hormone levels in PTSD patients. However, cortisol levels of most of the patients seem to vary during their circadian rhythm within the normal range (339). Since lymphocytes express both glucocorticoid receptors and functional adrenergic receptors (170, 340), it might be speculated that an altered neuroendocrine profile could have mediated between traumatic stress or PTSD on the one hand and the immune outcomes reported here on the other hand. Future studies need to elucidate, if T cells subsets differ in their sensitivity to stress hormones maybe by differentially expressing neuroendocrine receptors. Given the considerable prevalence of traumatic stress, and in particular the high prevalence of PTSD in populations affected by conflict, terror and combat (297, 299), the results of this study are of high societal and economic relevance for health care. A considerable body of clinical investigations has revealed that a variety of therapeutic interventions may effectively reduce trauma-related mental suffering (National Collaborating Centre for Mental 341). In a recent study, we demonstrated that successful treatment – in this case by means of Narrative 68 Chapter 4 Exposure Therapy – also significantly reduced cough, diarrhoea, and fever (342). This leads us to suggest that successful psychotherapeutic intervention may improve immune function, possibly through alterations of the T cell compartment. Given the importance of these associations for a broad range of trauma-affected individuals from victims of violence and abuse to peacekeeping forces and rescue workers, more attention should be given to the potential for improving physical, in addition to mental health, through trauma treatment. Material and Methods Participants We examined the distribution of blood T lymphocyte subsets in 19 individuals with current PTSD (12 male, 7 female; mean age = 33.6 years, SD = 7.1, range 21-48) according to the DSM-IV (American Psychiatric 296) and 27 non-PTSD control subjects (9 male, 18 female; mean age = 29.1 years, SD = 8.3, range 19-50). PTSD patients were refugees (4 Africa, 1 Balkan, 14 Middle East and Afghanistan), with chronic (mean symptom duration = 7.2 years, SD = 4.4) and severe (mean sum score in the Clinician Administered PTSD Scale [CAPS] (343) = 79.6, SD = 18.6) forms of PTSD due to multiple highly stressful war and torture experiences. On average, patients have lived in Germany for 4.9 years (SD = 3.6). All patients were recruited from the Psychotrauma Research and Outpatient Clinic for Refugees, University of Konstanz, located at the Centre for Psychiatry Reichenau, Germany. The non-PTSD group was recruited through advertisement and was matched to the patient group with regard to age and region of origin (3 Africa, 11 Balkan, 13 Middle East and Afghanistan). Since this control group varied with respect to the number of traumatic event types experienced (range: 0 - 9) some of the analyses were repeated with a three group (PTSD, trauma-exposed and non-exposed controls) design. For this purpose we divided the non-PTSD group by median split into a group with substantial exposure to traumatic stressors (4 - 9 different traumatic event types; n = 14) and a control group with no or few traumatic experiences (0 - 3 traumatic event types; n = 13) based on the number of past traumatic event types assessed with the event checklist of the CAPS (343). Subjects were excluded if they reported intake of glucocorticoids, had acute or chronic somatic illnesses, or met criteria for additional mental disorders other than stress-related affective or anxiety disorders. Fourteen PTSD patients and 2 trauma-exposed controls met the DSM-IV criteria for a current major depressive episode. Eight PTSD patients and 2 traumaexposed controls reported current intake of psychotropic medication (PTSD: 2 hypnotics, 3 anxiolytics, 5 antidepressants and 2 neuroleptics; non-PTSD: 1 hypnotic, 1 antidepressant). Since the pattern of results did not change if we excluded all medicated participants from the statistical analysis, we only report the original analysis here. 69 Chapter 4 Clinical interviews All participants underwent an extensive standardized clinical interview administered by experienced psychologists and trained translators. PTSD symptoms and the number of traumatic event types experienced were assessed with the CAPS (343). The vivo checklist of war, detention and torture events (344), which assesses common traumatic experiences in conflict regions and during torture, allowed for a detailed evaluation of the number of traumatic event types experienced. The Mini International Neuropsychiatric Interview (M.I.N.I.) (345), was used to screen for potential comorbid mental disorders. In addition, the severity of depressive symptoms was assessed with the Hamilton Depression Rating Scale (HAM-D) (346). After complete description of the study to the subjects, written informed consent was obtained. All procedures were approved by the Ethics Committee of the University of Konstanz. Blood sampling Blood was collected between 10 and 11 a.m. in EDTA-treated tubes for T cell phenotyping and in sodium citrate-treated cell preparation tubes for proliferation assays (BD Vacutainer, Franklin Lakes, NY). In order to control for possible HIV and hepatitis A, B and C infections, an additional blood sample was sent to a diagnostic laboratory for standard hepatitis and HIV tests. All samples were negative for HIV or hepatitis C. Subjects classified with acute or chronic hepatitis A or B (n = 3) were excluded from the study. Two patients and one traumatized control showed an infection history for hepatitis B (as indicated by a positive result for hepatitis B core IgG antibody). Since the pattern of results did not change if we excluded them from the statistical analysis, they remained in the sample. Lymphocyte phenotyping and T cell proliferation Whole blood was analysed for the percentage of total T cells (CD3+), cytotoxic T cells (CD3+ CD8+) and T helper cells (CD3+ CD4+) as well as B cells (CD45+CD19+), by flow cytometry. T cell maturation subsets were determined according to their expression profile of the surface molecules CD45RA and CCR7. For quantification of T cell phenotypes, 100 µl whole blood was incubated for 20 min at room temperature with either APC-conjugated anti-CD3 (clone SK7) or a combination of PerCP-conjugated anti-CD3 and APC-conjugated anti-CD8 (clone SK1) or APC-conjugated anti-CD4 (clone RPA-T4), and PE-conjugated anti-CD45RA (clone HI100) and FITC-conjugated anti-CCR7 (clone 150503) monoclonal antibodies (mAbs). For quantification of B-lymphocytes 100 µl blood was stained with PerCP-conjugated anti-CD45 (clone 2D1) and APC-conjugated anti-CD19 (clone HIB19). For quantification of Treg cells, blood samples were stained with PerCP-conjugated anti-CD3, APC-conjugated anti-CD4, FITC-conjugated anti-CD25 (clone M-A251), and intracellular FoxP3 expression was detected using the PE anti-human FoxP3 staining kit (eBioscience, San Diego, CA). Following antibody staining, standard lyse-wash was performed using BD FACS lysing solution; samples were washed twice, and 1×105 cells were acquired on a FACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose, CA), and analyzed with FlowJo 70 Chapter 4 software (Tree Star, San Carlos, CA). All monoclonal antibodies were purchased from BD PharMingen (San Diego, CA), except CCR7 mAb from R&D Systems (Minneapolis, MN). Absolute lymphocyte numbers (cells/µl) were measured, using an automated hematology analyzer (XT-2000i, Sysmex, Horgen, Switzerland). For the proliferation assay, 1×105 CFSE-labeled peripheral blood mononuclear cells (PBMCs) were suspended in RPMI medium containing 10% FCS and stimulated for 72h in 96-well flatbottom microtiter plates coated with anti-human CD3 mAb (2 µg/ml, clone OKT3, eBioscience), and cell proliferation was measured by flow cytometry in triplicates. The investigator who performed the immunological analyses was blind for the group assignment of the probes. Statistical analyses Group differences in the immunological parameters were analyzed using ANOVAs. The independent variables were either two (PTSD, non-PTSD) or three groups (PTSD, traumaexposed and non-exposed controls). Statistical significance for the immune measures was assessed by nonparametric permutation tests, using 1000 random permutations of group labels (347). Throughout the text and the tables all data are presented as mean ± standard deviation. In the figures data are displayed as mean + standard errors. 71 Chapter 5 Discussion Discussion Associations between stress and immune functions have been carefully documented in the past, but the specific mechanisms by which chronic stress influences disease susceptibility and outcome remain not fully understood. The aim of the present thesis was to further analyse underlying mechanisms of stress-induced immunosuppression. In chapter 2 we characterised the mechanisms by which chronic social stress affects the outcome of TCD8+ cell-mediated responses during a virus infection in a mouse model. Chapter 3 describes the impact of chronic social stress on the migratory capacity of skin dendritic cells after contact allergen sensitisation in mice. In chapter 4 we describe traumatic stress-associated alterations in peripheral T cell subsets in humans. Chapter 2 focuses on the impact of chronic social stress on TCD8+ cell-mediated immune responses in a mouse model of LCMV-infection. Unlike most previous studies we directly compared the outcome of different stress protocols in our model. Mice that were subjected to social stress prior to virus infection exhibited a significant reduction of IFN-γ-producing TCD8+ cells specific for the dominant LCMV-derived epitopes GP33 and NP396. Comparison of different readout systems allowed the conclusion that the stress exposure mainly impacts the function of splenic TCD8+ cells by inhibiting antigen-specific IFN-γ secretion. In contrast, the generation of IFN-γ producing TCD8+ cells was not significantly altered in mice receiving the same stress procedure concurrently with the infection. Previous studies in mice have clearly demonstrated that chronic social stress exposure can suppress the generation of TCD8+ cells during the course of a viral infection under some conditions, but it does not do so under all conditions. Our results extend previous work by demonstrating that the timing of the stress exposure is an important factor in determining the direction of the stress-induced immune alterations (246) and add new insight under which circumstances chronic stress results in an immunosuppressive reaction. We also found that prolonging the stress procedure for three additional days after LCMVinfection decreased the expansion of activated TCD8+ splenocytes. Pharmacological blockage of GC receptors in stressed and control mice revealed that these alterations were mediated by GCs. Although GCs are known to induce apoptosis in T cells, we found no differences in the propensity of TCD8+ cells to undergo apoptosis whereas the in vivo proliferation of TCD8+ cells was markedly reduced. Analysis whether the reduced expansion of TCD8+ cells correlates with an inefficient initial activation showed that TCD8+ cells of stressed mice display a significantly lowered expression of the early T cell activation marker CD69. These results suggest that the impairment of TCD8+ cells occurs at the earliest stages of a viral infection. Induction of CD69 on TCD8+ cells is known to induce IL-2, IFN-γ and CD25 expression (62-65), thereby promoting the proliferation of effector TCD8+ cells in an autocrine manner. Analysis of the cytokine secretion capacity of isolated TCD8+ cells proved that TCD8+ cells from stressed mice exhibit an impaired IL-2 and IFN-γ secretion when restimulated in vitro. Previous studies have shown that GCs suppress the activation, proliferation and cytokine production of TCD8+ cells in vitro. These effects have also been confirmed in splenic TCD8+ cells isolated from stressed mice that underwent the social disruption stress procedure. Our findings place these 73 Discussion previously reported cellular mechanisms of GC-mediated impairment of TCD8+ cell function into the context of a relevant in vivo viral infection model. It was previously reported that GCs could impair DC maturation and Ag presentation in vitro (209-210). A very recent report has also demonstrated that DCs are the main targets of stress/glucocorticoids in vivo in a model of HSV-1 infection (348). We excluded the possibility of an altered maturation state by analyzing the expression of the costimulatory molecules CD80 and CD86 on splenic DCs. Moreover, we found no differences in their competence to present LCMV-derived epitopes in vitro. We also excluded the possibility that an altered composition of the 20S proteasome contributes to the reduced generation of TCD8+ cells in stressed mice. The impaired TCD8+ responses in stressed mice thus cannot be assigned to an alteration of dendritic cells in our model. Therefore we propose that GCs act directly on TCD8+ cells early during their activation phase. The controversial outcome compared to the previous report (348) could be due to the different stress models used to induce chronic stress in mice (e.g. restraint stress versus social stress; daily recurrent short exposure versus recurrent long exposure). In this context it is important to note that only social disruption stress has been shown to induce an insensitivity of splenic CD11b+ monocytes and CD11c+ DCs to the inhibitory effects of GCs (236). Although the GC insensitivity of CD11c+ DCs is less well documented, it is feasible that this mechanism may contribute to our observation of an unimpaired DC function. Other observations in our model are less well understood. We cannot yet provide a reasonable explanation why the reduced expansion of TCD8+ cells appears only in the spleen but not in the inguinal lymph node or peripheral tissues. In general, the expression of GC receptors (GRs) is distinct in different tissues and immune cell types and reflects their unique sensitivity to GCs (215). Adoptive transfer experiments showed that splenic TCD8+ cells derived from SDR mice exhibit an impaired in vivo migration to the spleen. An elevated egress of TCD8+ cells might compensate for the reduced generation in the spleen and could provide the peripheral compartments with sufficient effector cells. Chronic stress is not only implicated to play an important role in the susceptibility to infectious diseases but numerous studies in humans and mice provide accumulative evidence that chronic stress can impact the pathophysiology of several skin inflammatory and autoimmune disorders. In the past, the impact of chronic stress on skin immunity in mice has been focused on antigen-specific T cell-mediated immune reactions in response to contact allergens (DTH reactions). The precise mechanisms how stress hormones (GCs and catecholamines) down-regulate cutaneous immune functions are yet poorly understood. Although skin DCs and in particular LCs are known as critical inducers of cutaneous immune responses, their fate under chronic stress situations is less documented. In chapter 3 we focused on the effects of social disruption stress on the migratory capability of skin DCs. We show that chronic social stress exposure prior to skin sensitisation with the contact allergen FITC strongly impacts the migration of epidermal FITC-bearing CD11c+ DCs to regional 74 Discussion lymph nodes. The impaired migration capacity of skin CD11c+ DCs could not be assigned to an action of catecholamines at peripheral β-adrenergic receptors since in vivo blockage of these receptors did not reverse the effect. This finding is of particular interest since previous reports have suggested a role for the sympathetic neurotransmitter norepinephrine (NE) on the migratory function of LCs (271). However, data on whether NE exerts its effects on LC migration by binding to α- or β-adrenergic receptors are less clear with one report pointing towards an involvement of α-adrenergic receptors (349) while another study suggested signaling through β-adrenergic receptors (350). With regard to stress-induced modulation of leukocyte trafficking it is well established that chronic social stress-induced increase in circulating neutrophils, monocytes and NK cells as well as the decrease in blood T and B cell numbers is at least partly mediated by stimulation of α- and β-adrenergic receptors (229). Therefore further analyses are needed to elucidate whether catecholamine-mediated αadrenergic signaling contributes to the altered CD11c+ DCs migration observed in our model. In vitro assays using ear skin explants demonstrated that CD11c+ DCs from SDR mice emigrated less efficiently out of the skin, even in the presence of the CCR7-relevant chemokines ELC/CCL19 and SLC/CCL21. We conclude that the severe decrease in the accumulation of skin-derived CD11c+ DCs in the lymph nodes of SDR mice may occur through their retention in the skin. These results also suggest that the impaired epidermal DC mobilisation in SDR mice is not due to a lack of ELC/CCL19 and SLC/CCL21 production. It has been shown that exposure to high as well as prolonged moderate levels of exogenous corticosterone suppressed skin DTH reactions in response to the contact allergens DNFB or OXA (295). Related to chronic stress previous reports demonstrated a diminished DTH reaction in response to DNFB in mice that were exposed to restraint stress (290). The interaction of allergen-bearing skin CD11c+ DCs with naïve T cells in the lymph nodes is pivotal for T cell priming and initiation of cutaneous immune responses induced by contact allergens. Vice versa, a deficiency or alteration of skin DC migration is associated with a less effective transport of the Ag to draining lymph nodes and presumably a less effective induction of the DTH response (278, 351-352). The defective migration of allergen-bearing CD11c+ DCs as observed in our model may therefore represent an initial event resulting in the previously described lack of DTH elicitation in stressed mice. Although these initial findings do not yet deliver sufficient mechanistic insights, further studies along these lines are of high interest, especially with regard to the high prevalence of inflammatory and autoimmune skin disorders associated with stress. Recent reports have highlighted the important role of skin DCs and specifically LCs in the pathogenesis of skin diseases such as psoriasis and autoimmune dermatitis. Different mechanisms have been proposed how defective migration of LCs may contribute to the onset and progression of inflammatory and autoimmune skin disorders. For instance, LCs that retain within the epidermis could present antigens locally to sustain or exacerbate cutaneous inflammatory reactions (272). Another proposed mechanism aroused from the concept that LCs are not only important for protective T cell mediated immunity, but play also an important role in the 75 Discussion maintenance of peripheral tolerance to self-antigens (353). Thus a reduced migration of LCs to skin draining lymph nodes could also result in a loss of self-tolerance. For instance in a mouse model of lupus dermatitis (autoimmune dermatitis-prone mice) FITC-activated LCs appear to accumulate in the skin, which was shown to precede the onset of dermatitis and correlated with the development and severity of skin inflammation (273). An improved understanding of the mechanism and consequences of impaired skin DC migration in response to chronic stress exposure may help to understand how stress increases the onset and progression of inflammatory and autoimmune skin disorders. In chapter 4 we describe the phenotypic changes in T lymphocyte subsets in the peripheral blood of severely traumatised human patients. Posttraumatic stress disorder (PTSD) is associated with an enhanced susceptibility to various somatic diseases (300-302). In the past contradictory results were obtained when addressing the question whether an altered composition of the peripheral T cell compartment could be linked to the enhanced susceptibility of PTSD patients to infectious diseases and inflammatory or autoimmune disorders (302). In this thesis, we provide a more differentiated characterization of peripheral T cell subsets with regard to the high diversity of memory cells present among the peripheral T cell pool. Our results demonstrate that PTSD patients exhibit a reduction of naïve TCD3+ lymphocytes that was accompanied by an increased proportion of central (TCM) and effector memory (TEM) cells. Interestingly, the reduction of naïve and the increase of TEM cells were most prominent within the TCD8+ cell population, whereas no significant changes were observed for TCD4+ cells. The same tendency was also observed in trauma-exposed non-PTSD individuals, indicating a cumulative effect of exposure to traumatic stressors on T cell distribution. We could also show that PTSD patients exhibit nearly 50% reduction of regulatory T cells (Tregs) compared to non-PTSD individuals. This new finding is highly important given the fundamental role of regulatory T cells (Tregs) in suppressing immune responses to self-antigens and prevention of autoimmune diseases. These stress-related alterations of the peripheral T cell compartment described in our study might constitute a key factor in the enhanced susceptibility of individuals suffering from PTSD to a range of physical diseases. Further studies are needed to clarify whether the observed changes in the distribution of T cell maturation subsets are due to alterations in the thymic output of naïve T cells or whether an altered peripheral T cell turnover contributes to the observed changes. A reduced emigration of naive T cells from the thymus may contribute to the reduction of naïve TCD3+ lymphocytes observed in PTSD patients. Such diminished thymic output could be analysed by quantification of peripheral recent thymic emigrants (RTE) and we are currently testing this possiblilty. On the other hand, the maintenance of naïve and memory T cells is distinctively regulated in the periphery. The peripheral naïve TCD8+ and TCD4+ cell pool is maintained through combinatory signals provided by TCR interactions with self-MHCclass II and class I ligands and by cytokines (354). Both IL-4 and IL-7 have been implicated to be important for the survival of naïve T cells in the periphery, while the role of IL-7 in supporting both 76 Discussion survival and homeostatic expansion of naïve T cells in vivo is more evident (354). For TCD8+ memory cells, it has been suggested that IL-4 plays a role in the early phases of memory generation, IL-7 in the intermediate stages and IL-15 in long-term maintenance (130). The nature of survival signals for TCD4+ memory cells is less well understood. In conclusion it would be important to determine whether an altered cytokine secretion profile may contribute to the observed reduction of naïve and the increase of TCM and TEM cells in PTSD patients. The reduction of naïve TCD8+ cells in PTSD patients could compromise their ability to mount an effective T cell response to various pathogens and may also impair their responsiveness to virus vaccination. Therefore we are planning to evaluate hepatitis B vaccine responses in PTSD patients and control individuals by quantifying HBsAg-specific T cell responses after vaccination. Figure 1: Of mice and men. 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ICS IFN IL ITAM LC LCMV LN LMP LPS adrenocorticotropin hormone antigen activator protein-1 antigen presenting cell arginine-vasopressin beta 2-microglobuline brefeldin A bromodeoxyuridine corticosteroid binding globulin chemokine receptor clusters of differentiation 5-(6)- Carboxyfluorescein diacetate N-succinimidyl ester contact hypersensitivity cytomegalovirus central nervous system concanavalin A corticotrophin-releasing hormone cytotoxic T lymphocytes dendritic cell dexamethasone delayed-type hypersensitivity EBI1-ligand chemokine endoplasmic reticulum ER-associated aminopeptidases fluorescence activated cell sorting fetal calf serum fluorescin-isothiocyanat fluorescence forward scatter fast protein liquid chromatography glucocorticoid glycoprotein (LCMV) glucocorticoid receptor glucocorticoid response element high endothelial venules hypothalamus-pituitary-adrenal intra-peritoneal intra-venous intracellular cytokine staining interferon interleukine immunoreceptor tyrosine-based activation motifs Langerhans cell lymphocytic choriomeningitis virus lymph node low molecular weight protein lipopolysaccharide 98 Appendix mAb MCMV MECL MHC MR NE NEPHGE NFAT NFκB NK NP PALS PBS PE PFU PTSD PVN SDS SDR SNS SLC TAP TCR Th TCM TEff TEM TNF Treg Ub WE µM ZAP monoclonal antibody mouse cytomegalovirus multicatalytic endopeptidase complex subunit major histocompatibility mineralocorticoid receptor norepinephrine non-equilibrium pH gradient gel electrophoresis nuclear factor of activated T cells nuclear factor 'kappa-light-chain-enhancer' of activated B-cells natural killer cells nucleoprotein (LCMV) periarteriolar lymphatic sheath phosphate-buffer saline phycoerythrin plaque forming unit post traumatic stress disorder paraventricular nucleus sodium dodecyl sulphate social disruption stress sympathetic nervous system secondary lymphoid tissue chemokine transporter associated with antigen processing T cell receptor T helper cells central memory T cells effector T cells effector memory T cells tumor necrosis factor regulatory T cells ubiquitin LCMV-WE strain micro molar zeta-chain-associated protein kinase 70 99 Appendix Record of achievement / Eigenabgrenzung Chapter 2: Results described in these chapters were all performed by myself apart from quantification of IFN-γ in the plasma, which was carried out by Carsten Riether. Chapter 3: Results described in these chapters were exclusively performed by myself or under my direct supervision. Chapter 4: I performed all immunophenotyping and functional assays described in the chapter and wrote the manuscript the manuscript of this publication together with Hannah Aichinger. 100 Appendix Acknowledgment First of all I would like to thank Prof. Dr. Marcus Groettrup for giving me the opportunity to perform my PhD in his group. Thank you for all the excellent support and scientific guidance over the past years, for fruitful discussions when new results made scientific work exciting but also when experimental difficulties appeared. I thank Prof. Dr. Alexander Bürkle and Prof. Dr. Volker Stefanski for their kind agreement to serve as reviewer of this thesis. I would like to express my special gratitude to my supervisor Dr. Michael Basler for sharing his broad knowledge, supporting me with constructive criticism and always helpful advice during my whole thesis. My sincere thanks go to Harald Engler for his essential support with all problems coming along with the stress procedure. This PhD project profitted a lot from our interesting discussions and the many new impulses I received from him. I wish to thank Hannah Aichinger for the enlightening discussions about PTSD. It was a pleasure to work so closely together when analysing data and I appreciated very much writing the manuscript together with her. I would also like to thank the staff of the animal housing facility (TFA), especially Birgitt and Andrea for their cooperation and kindness to meet my special wishes concerning mouse orders. Special thanks go to Ulli Beck and Gerardo Alvarez for helpfulness whenever needed and for sharing reagents and other material with me. Special thanks also to Tina Wünsch for her highly valuable assistance with the blood stainings. I also want to thank Bernd Kress for his invaluable support during the last year of my thesis. Thanks to all other present and past members of the Groettrup and the Öhlschläger lab for their constant helpfulness, their entertaining company and for the great atmosphere in our group. The friendly and supportive atmosphere inherent to the whole group contributed essentially to the final outcome of my studies. In particular I would like to thank Marc Müller, Karin Schäuble from the BITG and Kathrin Kluge who became good friends. Special thanks to Marc for his support with pellet implantation and taking blood samples, his admirable calmness even in stressful phases and, most important, for his entertainment and humour. It was a pleasure for me to share the lab and also countless evenings with you. I also thank my friends in Bonn (Betti, Tine, Eva, Minka) for providing support and friendship. Although we see each other very rarely these days, you are important to me. Ich möchte mich von ganzem Herzen bei meiner Familie bedanken. Insbesondere bei meinen Eltern, die mich jederzeit mit Liebe bei allen meinen Entscheidungen unterstützt haben. Zum Schluss möchte ich mich ganz besonders bei Jörg bedanken für all die Unterstützung und Liebe. Danke. 101