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Licentiate thesis from the Department of Immunology, Wenner-Gren Institute, Stockholm University, Sweden Innate immune responses in cord blood, and the influence of pathological pregnancy Ebba Sohlberg Stockholm 2011 SUMMARY Innate immune cells, such as monocytes, are important for early-life immune responses, as adaptive immunity in the newborn is suboptimal. Immaturity of infection control in newborns has been related to defective antimicrobial responses and impaired toll-like receptor (TLR) signaling, findings illustrating the importance of understanding immune maturation in the neonatal period. Our overall aim was to study innate immunity in the newborn, with focus on monocytes and monocyte-NK cell collaboration, and how it is affected by exposure to the pregnancy-related disorder preeclampsia. In the first study we investigated the two major monocyte subsets, the CD14++CD16- and CD14+CD16+ cells, in cord blood (CB). Surface expression of activation-related receptors was assessed. Further, CB and adult peripheral blood (PB) mononuclear cells (MC) were stimulated in vitro with peptidoglycan (PGN), and phosphorylation of MAPK and production of intracellular and secreted cytokines was measured. CB monocyte subsets produced high levels of the early-response cytokines with TNF and IL-12p70 exceeding adult levels, and also showed high phosphorylation of p38-MAPK. Thus, the monocyte response to a common bacterial ligand at birth did not appear immature. However, the CD14+CD16+ cells were present in a higher frequency in PGN-stimulated CBMC cultures and this could reflect a failure to further differentiate into effector cells following bacterial stimulation. We concluded that the monocyte subset cytokine responses to PGN in CB are potent, but that the behavior of CD14+CD16+ cells following PGN stimulation might contribute to a qualitative difference of the neonatal antimicrobial response as compared to the adult. In the second study we examined whether the immune-mediated pregnancy disorder preeclampsia would affect CB monocyte and NK-cell activation. Levels of cytokines were quantified in maternal and CB serum, CBMC were analyzed for expression of activationrelated surface receptors, and intracellular and secreted cytokines after stimulation with PGN or PGN+IL-15. Preeclampsia induced an inflammatory serum cytokine profile with differences for mild and severe preeclamptic subgroups in both mothers and newborns. The CB monocyte compartment was not affected by preeclampsia, however CB NK cells showed an altered expression of the activating receptors NKG2D and NKp30. Also, unstimulated CB NK cells from the preeclamptic group expressed more intracellular TNF and IFN-γ. Together this suggests that in preeclampsia, an in utero inflammatory priming of neonatal innate immunity takes place, and possibly indicates an effect on NK-cell function in newborns of preeclamptic mothers. 2 LIST OF PAPERS This thesis is based on the original papers listed below, which will be referred to by their roman numerals in the text: I. Sohlberg E, Saghafian-Hedengren S, Bremme K, Sverremark-Ekström E. Cord blood monocyte subsets are similar to adult and show potent peptidoglycan-stimulated cytokine responses. Revised and resubmitted. II. Sohlberg E, Saghafian-Hedengren S, Bachmayer N, Rafik Hamad R, Bremme K, Sverremark-Ekström E. Preeclampsia affects cord blood NK-cell activation patterns. Manuscript. 3 TABLE OF CONTENTS SUMMARY............................................................................................................................................................. 2 LIST OF PAPERS.................................................................................................................................................. 3 TABLE OF CONTENTS....................................................................................................................................... 4 ABBREVIATIONS ................................................................................................................................................ 5 THE IMMUNE SYSTEM...................................................................................................................................... 6 THE INFLAMMATORY RESPONSE ................................................................................................................ 7 INNATE SENSORS ............................................................................................................................................... 8 INTRACELLULAR SIGNALING FOLLOWING TLR ACTIVATION ......................................................... 10 CYTOKINES ........................................................................................................................................................ 11 EARLY RESPONSE CYTOKINES.................................................................................................................. 12 MONOCYTES / MACROPHAGES................................................................................................................... 15 MONOCYTE HETEROGENEITY................................................................................................................... 15 NK CELLS............................................................................................................................................................ 17 MONOCYTE AND NK-CELL COLLABORATION ...................................................................................... 19 IMMUNE FUNCTION EARLY IN LIFE ......................................................................................................... 19 PREGNANCY AND IMMUNE ACTIVATION ............................................................................................... 21 IN UTERO INFLUENCES ON MATURATION OF NEONATAL IMMUNE RESPONSES ......................... 22 PREECLAMPSIA ................................................................................................................................................ 23 CONTRIBUTING FACTORS TO DEVELOPMENT OF PREECLAMPSIA ................................................. 23 EFFECTS OF PREECLAMPSIA ON OFFSPRING......................................................................................... 25 PRESENT STUDY ............................................................................................................................................... 26 OBJECTIVES.................................................................................................................................................... 26 SUBJECTS AND METHODS .......................................................................................................................... 26 RESULTS AND DISCUSSION ........................................................................................................................ 27 Paper I .......................................................................................................................................................... 27 Paper II ......................................................................................................................................................... 31 GENERAL CONCLUSIONS.............................................................................................................................. 34 FUTURE PERSPECTIVES ................................................................................................................................ 35 PREECLAMPSIA ........................................................................... ERROR! BOOKMARK NOT DEFINED. NEONATAL IMMUNITY AND VIRAL INFECTIONS ...................................ERROR! BOOKMARK NOT DEFINED. ACKNOWLEDGEMENTS................................................................................................................................. 37 REFERENCES ..................................................................................................................................................... 38 4 ABBREVIATIONS APC antigen-presenting cell CB cord blood CBA cytometric bead array CBMC cord blood mononuclear cells CLR C-type lectin receptor DAMP danger-associated molecular pattern DC dendritic cell ERK extracellular signal-regulated protein kinase FCS fetal calf serum IFN interferon IL interleukin IDO indoleamine 2,3-dioxygenase LPS lipopolysaccharide MAPK mitogen-activated protein kinase MHC major histocompatibility complex MICA/B MHC class I chain-related proteins A/B NK cell natural killer cell NLR nucleotide oligomerization domain-like receptor NOD nucleotide oligomerization domain PAMP pathogen-associated molecular pattern PBMC peripheral blood mononuclear cells PGN peptidoglycan PRR pattern recognition receptor RLR retinoic-acid inducible gene like receptor ROS reactive oxygen species Tc T cytotoxic cell Th1 T helper type 1 Th2 T helper type 2 TLR toll-like receptor TNF tumor necrosis factor Treg regulatory T cell ULBP UL-16 binding proteins 5 THE IMMUNE SYSTEM The mammalian immune system is very complex and processes vast amounts of information. It is capable of distinguishing self from non-self and thus, protects against a multitude of environmental microbes that we face from the moment of birth. The immune system also has to be able to tell pathogenic from non-pathogenic organisms, as organs like the gut are colonized rapidly at birth with a commensal microbial flora. Further, the immune system can mediate homeostasis and eliminate damaged and/or altered cells. Physical barriers such as skin and mucosa act in concert with antibacterial molecules and specialized cells to preserve immunity of the host. Immune cells are present throughout the body in the circulating blood and lymphatic tissues and in specialized immune compartments such as the thymus, spleen and lymph nodes. Two intertwined branches of immunity cooperate to eliminate threats to the host, the innate and adaptive immune system. Constitutive innate defenses ensure rapid limitation of microbial invasion through preexisting molecules, such as complement factors and acute phase proteins, and phagocytic cells. Innate recognition of microbial presence can be mediated through the expression of germ-line encoded pattern recognition receptors (PRRs) or Natural Killer (NK)-cell associated receptors. The principal innate effector cells are neutrophils, NK cells, monocytes, macrophages and dendritic cells (DCs). Neutrophils are professional phagocytes that engulf and eliminate microbes by production of reactive oxygen species (ROS). NK cells display cytotoxic activity against virus-infected or tumor cells, and are potent producers of cytokines that regulate other immune cells. Monocytes are precursors of and replenish macrophages and DCs in tissues, as well as being potent cytokine producers upon microbial encounter. Macrophages are specialized in the uptake, digestion and presentation of antigens to adaptive cells. DCs are the most effective antigen-presenting cells (APCs), they are present in peripheral tissues where they sample antigen to later migrate to lymph nodes to prime naïve T cells and activate B cells and NK cells. Innate defenses may be sufficient to clear invading pathogens, but if not they can limit infection until cells of the adaptive immune branch have expanded enough to ensure elimination of the threat. The initial quality of the innate response, and which cytokines are produced therein, will direct subsequent adaptive functions.1 The adaptive effector cells are T and B lymphocytes. They display a high degree of diversity and specificity to pathogens, due to somatic rearrangements of genes encoding the B cell- and T cell-receptors. Adaptive immunity is divided into humoral responses, which lead to the release of antigen-specific 6 antibodies from plasma B cells, or cell-mediated responses where the effectors are antigenspecific T cells. The major T cell populations are the CD4+ T helper (Th) cells and the CD8+ T cytotoxic (Tc) cells. For T and B cells to become activated they need to recognize antigens presented on major histocompatibility complex (MHC) class I or II, and get additional signals from co-stimulatory molecules and cytokines produced by innate cells. Upon activation, clonal proliferation of T- and B cells generates highly specific effector cells but also longlasting memory cells. THE INFLAMMATORY RESPONSE Inflammation is critical for both innate and adaptive immunity. It is the process that first takes place upon infection or tissue injury and is characterized by alterations in vascularity, secretion of soluble mediators and the migration of leukocytes. Infiltration of leukocytes to an inflamed site is promoted both by dilation and increased permeability of blood vessels, and up-regulation of adhesion molecules like selectins and inter-cellular adhesion molecules (ICAMs) on endothelial cells. Tissue resident cells such as macrophages, DCs and mast cells secrete inflammatory factors that promote immune cell function in inflamed tissues. Further, local production of chemoattractants such as the chemokine interleukin (IL)-8 is important in mediating adherence and leukocyte infiltration. The acute inflammatory response is characterized by arrival of neutrophils and subsequent recruitment of monocytes that differentiate into macrophages and DCs on site. Both neutrophils and macrophages phagocytose microbes, but macrophages have a longer life span. They also have an important role in orchestrating the immune response through the production of IL-1, IL-6 and tumor necrosis factor (TNF), cytokines that mediate many of the systemic acute-phase effects. Once pathogen clearance is achieved, feedback inhibitory loops are initiated to restrain immune activation and prevent tissue damage associated with excessive inflammation. One major inhibitory pathway induced by inflammation and microbial recognition, is expression of suppressor of cytokine signaling (SOCS) proteins that inhibit signal transduction.2 For resolution of inflammation the immune effector cells should themselves be removed from the site. This can happen either by recirculation or by uptake by macrophages that in turn die locally by programmed cell-death. A failure of these or other inhibitory mechanisms to develop may lead to chronic inflammation and disease.3 7 INNATE SENSORS Innate immune cells possess a variety of sensors that enable them to distinguish between different classes of pathogens. They are found both on the cell surface and either free or membrane-bound in the cytosol, ensuring detection of microbes that employ different modes of infection. The innate sensors are commonly referred to as pattern-recognition receptors (PRRs). Manipulation of the PRR signaling pathways seems to be a common theme for microbial interference with immune responses underlining their importance for host defense.4 PRRs recognize structures that microbes cannot easily change without affecting their fitness such as components of the bacterial cell wall, or viral nucleic acids. Collectively these molecules are referred to as pathogen-associated molecular patterns (PAMPs). Also nonimmune cells like epithelial cells express PRRs and can produce factors upon PRR ligation that stimulate the immune response.5 Therefore it may be possible that an initial sensing of microbes occurs in tissues that thereafter direct the activation of immune cells.6 Innate sensors can also respond to endogenous danger-associated molecular patterns (DAMPs) like heat shock proteins, self nucleic acids or uric acid. The release of DAMPs is a common event when cells have been exposed to damage, pathogens or toxins or are undergoing necrosis. The discovery of immune responses initiated by danger signals sparked the debate on whether the immune system responds to substances that cause damage, rather than to those that are simply foreign.7 There are several varieties of innate sensors and deficient expression or function of many of them has been associated with increased susceptibility to infectious and chronic inflammatory diseases.8-10 The major families of PRRs are C-type lectin receptors (CLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), retinoic acid inducible gene-like receptors (RLRs) and Toll-like receptors (TLRs). CLRs, like Dectins or DC-SIGN, are expressed on the cell surface and recognize mannose, fucose and glucan carbohydrate structures from fungi, but also from mycobacteria and viruses. Their ligation primarily modulates NF-κB activity and thus production of inflammatory cytokines.11 NLRs are present in the cytosol and act as sensors of bacterial components. For instance NOD1/NOD2 recognize peptides derived from the degradation of PGN, a bacterial cell wall polymer,12 although it is somewhat unclear how extracellular PGN reaches cytoplasmic NODs.13 Some of the NLRs are associated with large cytoplasmic complexes called inflammasomes, like the prototype NALP3, that sense PAMPs and/or DAMPs and metabolic stress. NLR ligation results in activation of inflammatory caspases, that leads to proteolytic activation of the 8 inflammatory cytokines IL-1β and IL-18.14 RLRs, like RIG-I or MDA5, are present in the cytosol and recognize viral RNA which leads to production of type I interferons and inflammatory cytokines.15 TLRs are transmembrane receptors found on the cell surface or on intracellular vesicles. There are 10 TLRs so far in the mammalian genome that together display a wide specificity.16 For instance TLR4 recognizes lipopolysaccharide (LPS), TLR5 bacterial flagellin, TLR7 and TLR8 single stranded RNA and TLR9 unmethylated CpG motifs in DNA. TLR2 recognizes bacterial lipoproteins but also lipoteichoic acid (LTA) and PGN together with NOD1/NOD2.12 Figure 1. Expression of PRRs at different cellular locations enables detection of pathogens employing different infection modes. Modified from Werts, Girardin & Philpott.17 9 INTRACELLULAR SIGNALING FOLLOWING TLR ACTIVATION The importance of the TLR pathway is evident considering the clinical consequences of defects in TLR-associated signaling molecules, reviewed in Ku et al.18 TLR activation initiates distinct intracellular signaling cascades, which result in transcription of genes encoding inflammatory cytokines and chemokines, co-stimulatory molecules and antimicrobial compounds. TLRs are composed of various domains including extracellular ligand-binding leucine-rich-repeats and an intracellular Toll/IL-1 receptor (TIR) signaling domain. TLRs are continuously recycled from the cell surface in absence of ligand. Upon ligation the TLRs are stabilized, dimerize and association of adaptor proteins to the TIRdomains is triggered. The adaptors include MyD88, IRAK, TRIF, TRAF and TAK1 and they serve to transduce and amplify the signal. All TLRs except TLR3 activate the MyD88dependent pathway, making MyD88 one of the key adaptors. Downstream of TAK1, mitogen-activated protein kinases (MAPKs) are activated through phosphorylation. MAPKs in turn phosphorylate the transcription factor AP-1 that switches on genes encoding the inflammatory cytokines.19 There are three major families of MAPKs; the extracellular signalregulated protein kinases (ERK) and the c-Jun NH2-terminal kinases (JNK) and p38-MAPK that show some overlap in function.20 Stimulation or inhibition of MAPK members can affect cytokine release and/or expression of co-stimulatory molecules by APCs. For example, inhibition of p38-MAPK and ERK has been shown to suppress PGN-induced production of IL-6, IL-10, TNF and IL-12p70 by DCs.21 MAPKs have been under investigation in connection to inflammatory diseases and implicated as potential targets for therapeutics.22,23 Activation of TLRs is essential for the innate immune response against pathogens, however signaling needs to be strictly regulated in order to avoid inappropriate inflammatory responses. Many negative regulators exist including splice variants for adaptor proteins,24 MAPK-phosphatases25 and microRNAs,26 that all target different steps in the signaling cascade to suppress inflammation. 10 Figure 2. TLR signaling elicits production of inflammatory cytokines and type I interferons. Adapted from Kumar, Kawai & Akira.19 Reprinted with the permission of Elsevier. CYTOKINES In order to coordinate immune responses, cells need to communicate. For this purpose, the production and secretion of soluble proteins called cytokines is of major importance. Cytokines bind to a variety of receptors and induce cell-specific responses to regulate the intensity and duration of an immune response. A given cytokine can have different actions depending on antagonistic or synergistic effects with other cytokines and whether the target cell has prior antigen priming. There are different families of cytokines based on function; the prototype cytokines are called interleukins (ILs), since they are produced by, and deliver signals to leukocytes. Among other functions, ILs are involved in perpetuation of inflammation together with the tumor necrosis factor (TNF) family. Interferons (IFNs) are key molecules in the early defense against viruses and induce an antiviral state in cells. Chemokines are cytokines that influence the migratory behavior of leukocytes. A brief description of the cytokines that are produced early on during an immune response, and by innate cells such as monocytes and NK cells will follow. 11 EARLY RESPONSE CYTOKINES IL-1β and TNF Some of the earliest cytokines initiated during inflammation are IL-1β and TNF that are produced primarily by activated monocytes, macrophages and DCs. In addition NK cells, B and T cells also produce TNF. Both IL-1β and TNF are very potent pro-inflammatory cytokines and are therefore not normally found in high levels in circulation in healthy individuals. Synthesis of IL-1β is tightly regulated by the inflammasome and occurs upon recognition of PAMPs and DAMPs. This leads to activation of caspase-1 and proteolytic cleavage of pro-IL-1β into IL-1β.14 TNF is also produced in response to PRR activation by PAMPs. Further, both IL-1β and TNF induce their own synthesis and that of each other. The two cytokines synergize to mediate the systemic acute phase response that follows local inflammation including induction of fever, production of acute phase proteins such as Creactive protein by the liver, and activation of the vascular endothelium. IL-1 signals through the type 1 IL-1 receptor (IL-1R1) and TNF through TFNR1/TNFR2. Both cytokines act immunostimulatory and ligation to their cognate receptors launches downstream pathways that are similar to those elicited by TLR ligands. Overproduction of IL-1β and TNF is involved in several pathologies such as septic shock, and various autoinflammatory and autoimmune disorders, reviewed in Kastner et al. and Bradley et al.27,28 Blockade of IL-1 and TNF signaling through antibodies, soluble receptors or receptor antagonists are currently being used as therapeutic agents for inflammatory disorders including rheumatoid arthritis (RA) and Crohns disease.27,29 IL-6 Many of the systemic acute phase effects are mediated by IL-1β and TNF in combination with IL-6. Further, IL-6 has an important role in the shift from the early innate response to subsequent adaptive immunity.30 Mainly monocytes, macrophages, DCs, endothelial cells and B cells produce IL-6 in response to PRR and/or IL-1β/TNF stimulation. IL-6 acts pro- or antiinflammatory depending on the stage of the immune response. In the initial phase of infection or tissue injury, IL-6 induces acute-phase proteins, activation of macrophages and mediates lymphocyte arrival to the site of inflammation. In the resolution phase of inflammation, IL-6 favors the onset of acquired responses by e.g. inducing neutrophil apoptosis, whilst acting as a lymphocyte stimulatory factor. Also, IL-6 can block further pro-inflammatory cytokine and chemokine production thus affecting leukocyte recruitment.30,31 12 IL-6 signaling is propagated by the glycoprotein (gp) 130 homodimer. It forms a complex with the membrane-bound IL-6 receptor (IL-6R) or with the soluble (s) IL-6R, when either is bound to IL-6. The cellular distribution of the IL-6R is rather restricted but the sIL-6R renders cells that lack expression responsive to IL-6.32 IL-6 driven inflammation is associated with autoimmune diseases such as RA, multiple sclerosis (MS), and inflammatory bowel disease, reviewed in Kishimoto et al.33 Recent studies have demonstrated that IL-6 has an important role in regulating the balance between IL-17-producing Th17 cells and regulatory T cells (Treg),33,34 thereby possibly providing an important link to the involvement of IL-6 in pathophysiology. IL-8 IL-8 (CXCL8) is a chemokine produced during inflammation by a range of cells, e.g. monocytes, granulocytes, lymphocytes, fibroblasts and endothelial cells. Neutrophils are the primary targets for IL-8 and respond by chemotaxis and subsequent upregulation of surface adhesion molecules. IL-8 also enhances generation of ROS and delays neutrophil apoptosis.35 IL-8 signals through two G-protein coupled receptors, CXCR1 and CXCR2, which also mediate the effect of many other chemokines. CXCR1 and CXCR2 are expressed mostly on neutrophils and myeloid cells such as monocytes but also on Tc, endothelial and epithelial cells.36 CXCR1 and CXCR2 have been implicated in various inflammatory disorders such as RA, MS and asthma.37 IL-12p70 IL-12p70, from here on referred to as IL-12, is a pro-inflammatory cytokine that is closely involved in both innate and adaptive immune responses. It is composed of two subunits, p40 and p35,38 that together form the biologically active cytokine. Expression of the two subunits is independently regulated and the rate-limiting step is production of the p35 subunit.39,40 IL12 is predominantly produced by monocytes, macrophages, DCs and neutrophils in response to activation of PRRs, and/or co-stimulatory signals and cytokines from activated T cells and NK cells. IL-12 signals through the IL-12 receptor that is expressed mainly by NK cells and T cells. IL-12 has a crucial role for establishing antigen-specific Th1 responses and enhancing activation of B cells and the production of Th1-related antibody isotypes, which are essential to control infections with many microbial pathogens. Although IL-12 might not be required for Th1 differentiation,41 it induces clonal proliferation of, and maintains already committed IFN-γ secreting Th1 cells. 13 IFN-γ IFN-γ belongs to the interferon family whose members are key molecules in the early antiviral defense. Further, IFN-γ is essential for control of intracellular bacteria and tumor malignancies. The main IFN-γ producers are NK cells, NKT cells and T cells. IFN-γ directly promotes antiviral mechanisms by induction of antiviral enzymes but its main function is in immunomodulation. Over 300 genes are estimated to be upregulated by interferon signaling including genes coding for molecules involved in adhesion, apoptosis, cell-signaling and transcription.42 IFN-γ distinguishes Th1 type of immune responses and IFN-γ signaling, through the interferon gamma receptors IFNGR1/2, is fundamental in the maintenance of these responses.43 Its production is induced by mitogenic/antigenic stimuli and cytokines that are secreted by PRR-activated myeloid cells. IFN-γ is also a major activator of APCs and enhances responses to microbial inducers such as TLR ligands and antigen processing and presentation capabilities.44 IL-10 IL-10 is a unique cytokine with regulatory properties that can act as a negative feedback switch on inflammatory responses. This is accomplished by the fact that the same cells that initiate inflammation also start to produce IL-10. These cells include monocytes and B cells, but also the majority of T cell subpopulations, DCs and NK cells, which underscores its role as a critical negative regulator. IL-10 has diverse effects on most hematopoietic cell types for instance in growth and/or differentiation of T cells, B cells and NK cells. IL-10 dampens the inflammatory response through down-regulation of genes encoding inflammatory mediators, and by enhancing expression of molecules that antagonize the inflammatory response.45 IL-10 signals through the IL-10 receptor composed of at least two subunits members of the IFNR family. IL-10 suppresses a broad spectrum of activated macrophage/monocyte functions, including cytokine synthesis (for example IL-1β, IL-6, IL-12, TNF) by inhibition of NF-κB, expression of MHC II molecules and co-stimulatory molecules CD80 and CD86.45 Another way in which IL-10 puts the brakes on inflammatory responses is by enhancing the differentiation of IL-10 secreting Tregs, which contribute to control of immune responses and tolerance in vivo.46 Administration of recombinant IL-10 has only had modest success in protecting against inflammatory diseases, most probably due to the fine balance between immuno-suppression and immunopathology. The dose and cellular location is likely to be important for future attempts to modulate IL-10 activity.47 In the context of vaccination and 14 adjuvancy it is interesting to note that certain TLR stimuli do not elicit IL-10 production whilst the production of inflammatory cytokines is preserved.48 MONOCYTES / MACROPHAGES Monocytes are mononuclear phagocytes that belong to innate immunity. They play a crucial role not only in innate responses but also in priming and maintenance of adaptive responses. Besides acting as an important source of early cytokines following infection, circulating monocytes are precursors of macrophages and DCs that act as highly efficient APCs.49 Monocytes originate from hematopoietic progenitor cells in the bone marrow. Their growth and differentiation depend on lineage determining cytokines such as M-CSF and GM-CSF, and upon interactions with cells in the local niche. After differentiation, monocytes enter the blood stream where they circulate for a few days and constitute about 5-10% of the leukocyte pool. Under steady-state conditions, monocytes can replenish tissue-resident macrophages by homing to different tissues, although to what extent this occurs has been debated.49,50 Differentiation of tissue-specific macrophages or DCs is to a large extent dictated by the local microenvironment, signals from neighboring cells and the extracellular matrix. Stromal cells within tissues can exert immune regulatory functions by directing development towards regulatory phenotypes.51 When pathogen invasion occurs, there is a shift in the homeostatic pathway and monocytes act as efficient effector cells with antimicrobial activities. They are drafted from the blood into inflamed tissues promoted by signals such as monocyte chemotactic protein-1 (MCP-1/CCL2) and adhesion molecules, such as integrins and selectins that control migration. At the inflamed site monocytes will differentiate into recruited macrophages and/or DCs. MONOCYTE HETEROGENEITY The blood monocyte population is heterogeneous and the subpopulations most likely play different roles in antimicrobial responses. The two major subsets are distinguished by the presence or absence of CD16 (FCγRIII) and the degree of expression of CD14 (LPS coreceptor). The CD14++CD16– subpopulation constitutes 80-90% of total monocytes in comparison to the minor subset of CD14+CD16+ monocytes.52 15 CD14++CD16– monocytes and CD14+CD16+ monocytes have distinct trafficking potential due to differential expression of chemokine receptors, and are recruited into tissues via distinct molecular mechanisms. CD14+CD16+ cells express high levels of CX3CR1 and migrate towards the chemokine CX3CL1. CD14++CD16− cells express CCR2 and CD62L and can migrate in response to CCL2.49,53-55 With regard to PRRs, monocytes in general express high levels of NOD1/NOD2 and TLR2 and TLR4.56,57 CD14+CD16+ cells in turn display the highest levels of TLR2 and TLR4.58,59 In response to the TLR4 ligand LPS, CD14++CD16– cells produce high levels of the cytokines IL-6, IL-8, CCL2 and CCL3, whereas CD14+CD16+ cells produce IL-1β, TNF, IL-6 and CCL3. Both CD16+ and CD16- cells produce IL-10.58,60,61 Further, the CD14+CD16+ cells have increased capacity for antigen presentation and phagocytosis.62,63 The two subsets may also differentiate into macrophage and DC subpopulations with distinct phenotypes. One study found that TLR2/1-induced differentiation of monocytes into CD1b+ DCs was restricted to CD14+CD16+ cells, while differentiation into DC-SIGN+ cells was enhanced in the CD14++CD16– subset.64 The frequency of CD14+CD16+ cells is increased in adult and neonatal sepsis65 and other inflammatory and infectious conditions.66-68 Table 1. Some receptors differentially expressed by the two major monocyte subsets. The developmental relationship between CD14++CD16− and CD14+CD16+ cells is currently unknown but the two subsets have been suggested to represent sequential stages of monocyte differentiation.69,70 Support for this theory has come from transcriptome analysis69 and studies where CD16−CX3CR1− monocytes became CD16+CX3CR1+ under the influence of TGF-β, IL-10, M-CSF and CCL2.71,72 Thus the CD14++CD16− cells could give rise to the more mature macrophage and DC-like CD14+CD16+ cells,72,73 although this remains to be proven. 16 To make matters more complicated, CD14+CD16+ cells can be further subdivided into CD14highCD16+ and CD14dimCD16+ cells.60 A recent proposal suggests a common nomenclature for monocytes naming these 3 types classical (CD14++CD16-), intermediate (CD14hiCD16+), and nonclassical monocytes (CD14dimCD16++).74 In contrast to classical and intermediate monocytes, CD14dimCD16++ cells produce low amounts of inflammatory cytokines in response to bacterial ligands, but high amounts to measles virus and TLR7/8 ligands and could therefore be involved in the inflammatory response to viruses.60 NK CELLS NK cells are innate cells that are widely distributed in lymphoid and non-lymphoid tissues and constitute approximately 5-20% of the blood lymphocyte population.75 NK cells are CD3CD56+ cells and express a range of other receptors, of which NKp46 has also been suggested to define NK cells in order to facilitate cross-species comparisons.76 The NK-cell developmental pathway is not fully clear but commitment to the NK cell lineage, education of NK cells towards self-markers and establishment of functional competence occurs in the bone marrow.77 NK cells may also develop in peripheral sites such as secondary lymphoid organs.78 NK cells contribute to host defense through cytolysis-mediated killing of virusinfected cells and tumor cells. This is accomplished by release of pre-formed cytoplasmic granules that contain lytic proteins like granzyme and perforin, and/or by FAS ligand-FAS interactions. NK cells are also an important source of cytokines and chemokines, like IFN-γ, GM-CSF, IL-10, IL-13 and TNF, which contribute to inflammation and polarization of T-cell responses. The NK cell population is heterogeneous and there are two major NK-cell subsets with functional differences and distribution between anatomical sites. In blood, the majority of NK cells belong to the CD56dimCD16+ cytolytic NK subset and express homing receptors for inflamed sites. The minor circulating CD56brightCD16– NK cell subset displays low cytolytic activity but high production of IFN-γ and express homing receptors for secondary lymphoid tissues.79 NK cells are not simply ‘on’ or ‘off’; rather they vary in their responsiveness. Activation status is regulated in a rather complex fashion, and is the net result of simultaneous signals from a set of inhibitory and activating receptors. Central to NK-cell recognition of aberrant cells is the presence or absence of MHC class I molecules on the cell surface (“missing-self” 17 recognition).80 The two major families of MHC-specific inhibitory receptors are the Ig superfamily (KIR and LIR) and the C-type lectin (CD94/NKG2A) receptor superfamily. These prevent NK-cell activation upon encounter of cells displaying normal MHC class I expression. Stressed, transformed or virally infected cells can lack normal MHC expression and thus fail to provide the inhibitory signal. These cells will be lysed provided that they also express activating ligands. NK-cell activation receptors include CD16, NKG2D, 2B4, and NKp80 and the natural cytotoxic receptors (NCRs) NKp46, NKp44, and NKp30. The main activating receptors constitutively found on all NK cells in peripheral blood are NKG2D and the natural cytotoxicity receptors (NCRs) NKp30 and NKp46.81 NKG2D and NKp30 are highly expressed on cord blood NK cells,82 suggesting that they play important roles for NK-cell activation very early in life. NKG2D recognizes molecules that are upregulated on stressed and/or transformed cells such as MHC class I chain-related proteins A and B (MICA/MICB) and the UL-16 binding proteins (ULBP) that are induced by DNA damage, oxidative stress and inflammation.83 NKG2D-ligand interaction triggers cytotoxicity, cytokine production and cell proliferation.84,85 The ligand specificity of the activating receptor NKp30 is currently unknown, but has been speculated to include viral and apoptosis-related proteins.86,87 Ligation of NKp30 is involved in NK-cell induced DC maturation through NKcell killing of immature (i)DCs, and induction of DC maturity by IFN-γ and TNF secretion, suggesting an immunoregulatory role.88,89 18 MONOCYTE AND NK-CELL COLLABORATION Accessory cells are required to provide both soluble and contact-dependent activation signals for proper pathogen mediated NK-cell activation. NK cells become fully active only when additional stimuli such as monocyte secreted IL-12, IL-15 and IL-18 are present. IL-12 induces proliferation in pre-activated NK cells and enhances NK-cell cytotoxicity and IFN-γ secretion. Further, myeloid cell activation by IFN-γ amplifies production of IL-12/IL-18, which in turn potentiates IFN-γ secretion thus creating a feed forward loop.43 IL-15 is important to maintain NK cell development and proliferation.90,91 IL-18 seems to direct differentiation into a distinct NK-cell “helper” pathway characterized by expression of several mature DC-associated surface markers.92 Moreover, it seems there is close relation between the three cytokines in mediating activation of NK-cell effector functions. For example, in combination with IL-12, both IL-18 and IL-15 induce potent IFN-γ production.93-95 Interestingly, the monocyte subsets seem to have the capacity to differentially affect NK-cell activation. Gene set enrichment analysis has shown that CD14+CD16+ cells are enriched in genes related to NK-cell mediated cytotoxicity,69 indicating a close relationship between these cells in battling infection. Further, our group have found that PGN-stimulated CD14+CD16+ cells induce higher NK-cell IFN-γ production than CD14++CD16- cells,96 most probably connected to a higher capacity for IL-12 production (Paper I). IMMUNE FUNCTION EARLY IN LIFE The development of the immune system starts in utero in an environment normally free from microbes. The immune system of the newborn is therefore largely antigen inexperienced and lacks memory cells, something that may contribute to the high frequency and severity of microbial infections that afflict both newborns and infants. Although it is becoming increasingly clear that neonatal cells are capable of mounting fully mature immune responses to certain antigens, the neonatal immune system does exhibit a number of functional immaturities as compared to the adult. Naïve B cells express lower levels of activation-related markers such as CD40 and CD80/CD86, and there is limited production of IgG to a range of T-cell dependent and – independent antigens. Further, there seems to be a bias for formation of memory instead of plasma B cells all together hampering efforts for early-life immunization, reviewed in 19 Siegrist.97 With regard to the neonatal T cell compartment, Tc cells can mount mature cytotoxic responses under certain circumstances,98,99 but both Th1 and Th2 cell responses are often reduced in magnitude. The most pronounced and recognized defect is in T-cell production of IFN-γ.100,101 Nonetheless, the tuberculosis vaccine BCG can be administered at birth and induces potent Th1 responses.102,103 This seems to depend on BCGs’ capacity to engage multiple TLRs on DCs.104 Immaturities in DCs and their ability to further support e.g. Th1 differentiation may be an important factor underlying neonatal susceptibility.105 This is probably related to a reduced DC production of IL-12 in response to LPS and a number of other microbial stimuli.106-108 Neonatal monocytes have been extensively studied, as they are an integral part of the early innate defense, and have been described to give both immature and mature responses. Still, very little is known regarding the developmental expression of RLRs, NLRs and CLRs or their signaling intermediates, however TLR2 and TLR4 are expressed at an adult level on neonatal monocytes.109,110 Regarding various downstream TLR adaptors, findings have included lower111 or adult-level112 basal MyD88 expression and adult-level110-112 or deficient110,112 ERK1/2 and p38-MAPK activation in response to LPS, LTA and/or R-848 (a TLR7/8 ligand). At birth there are adult-level NK-cell percentages, including the CD56dim and CD56bright subpopulations.113 Cytotoxicity and capacity for production of IFN-γ after IL-12+IL-18 stimulation are at adult levels.114 However, neonatal NK cells have high expression of inhibitory NKG2A/CD94 receptors, and low expression of granzyme B that might contribute to low cytolytic activity of cord blood NK cells without prior cytokine stimulation.115 Overall the neonatal immune response exhibits a qualitatively different cytokine production profile than that of adults, which in turn could affect infection susceptibility of the newborn. The dogma has long been that newborns have a Th2-biased immunity and this has recently developed to incorporate a Th2/Th17-type of immunity over Th1-type of immunity.116-118 Although Th1 type of immunity appears to be down-regulated it is also clear that the neonatal response strongly depends on the stimulus used and the setting in which the cells are cultured,116,118-121 and a global Th2 bias at birth is not evident.122 During ontogeny maturation of various immune response takes place and a recent longitudinal study showed slow agerelated development, from cord blood to the age of 5, in cytokine production capacity to multiple TLR stimuli.123 Further, upregulation of CD80 and HLA-DR on monocytes and DCs following TLR stimulation is also increased during the first year of life.119 20 PREGNANCY AND IMMUNE ACTIVATION Much has happened since Medawar, Billingham and Brent suggested mechanisms behind the tolerance to the semi-allogeneic fetus.124 The thought of the fetus as a graft probably hampered progress in the field of reproductive immunology as from an evolutionary perspective, the co-existence of mother and fetus should in fact be desirable. Immunological detection of pregnancy is now recognized to be important for maintaining gestation successfully. The maternal immune system changes both locally in the intrauterine environment and in the peripheral circulation. The implantation site is filled with immune cells such as NK cells, macrophages and T cells. A phenotypical and functionally unique NKcell subset named uterine (u)NKs are the pre-dominant cell population in the uterus and make up some 70% of all decidual leukocytes. The uNKs contribute to vascularisation early in pregnancy by production of angiogenic cytokines.125 Some factors that are proposed contribute to tolerance to the allogeneic fetus include trophoblast expression of Fas ligand and subsequent clonal deletion of activated T cells, and suppression of T-cell activation in placenta by local production of indoleamine 2,3dioxygenase (IDO) by APCs.126 In the presence of IDO, Treg are promoted and in its absence there is a re-programming towards more inflammatory Th17 cells.127,128 Interestingly recent findings reveal that CD14+ myelomonocytic cells communicate with NK cells in the decidua and that the crosstalk results in the induction of IDO and subsequent induction of Tregs.129 Moreover, macrophages in decidua secrete high spontaneous levels of IL-10 and thus contribute to regulatory environment.130 In addition, placental exosomes found in maternal circulation can act immunosuppressive by inhibition of T- and NK cell signaling.131,132 In circulation the maternal adaptive immune system is suppressed with a down-regulation of T-cell functions whereas components of innate immunity are systemically activated, and this dysregulation is thought to be crucial for gestation.133 What type of immune response is beneficial overall seems to depend on the location, e.g. blood or fetal-maternal interface and on the stage of pregnancy. As the T cell repertoire is expanding, so is the debate surrounding the role of different T cells for reproductive success, discussed in Saito et al.134 In contrast to prior belief, a strict Th2 dominance throughout gestation does not appear to be essential. IFNγ was traditionally considered harmful to pregnancy, but has been shown in murine models to be needed for proper vascularization in the early stages of placental development.135 In addition, there are increased numbers of Tregs in circulation in pregnancy.136 With regard to the innate system, monocytes are activated and display increased surface expression of 21 molecules mediating adhesion (CD11a and CD11b, CD54) and phagocytosis (CD64), and have an increased ability to produce pro-inflammatory cytokines (IL-1β, IL-6, IL-12) and also IL-10.137,138 NK cells numbers are reduced, and so is cytotoxicity and IFN-γ production.133 IN UTERO INFLUENCES ON MATURATION OF NEONATAL IMMUNE RESPONSES That the conditions we face in the womb may influence future health and disease is at the heart of an emerging scientific frontier focused on the intra-uterine environment. In pregnancy there is a continuous exchange of maternal and fetal factors. Maternal antibodies, mainly IgG, readily cross the placenta to enhance neonatal immunity.139,140 Whether there is trans-placental cytokine transfer is less clear but at least IL-6 has been reported to cross the placenta.141,142 It was shown early on that fetal cells like trophoblasts and leukocytes can be found in the maternal circulation.143,144 Also maternal cells can be found in the fetal circulation, including most subsets of immune cells.145 Tregs, that can influence other effector cell responses, may play an important role in maintaining the balance of maternal and fetal immune responses. One study found that maternal cells that had crossed the placenta induced generation of Tregs that suppressed fetal antimaternal responses. This finding reveals a form of antigen-specific tolerance induced in utero.145 In addition, it appears that in utero exposure to allergens or infectious pathogens can lead to priming of antigen-specific fetal T lymphocytes.146,147 Moreover, maternal immune activation and development of neonatal immunity could be intertwined. Maternal allergy affects functional responses of cord blood mononuclear cells (CBMC) in terms of a lower monocyte derived IL-6 production,109 and this defective response persists into childhood.148 This connection was not seen for paternal allergy indicative of a pre-natal influence, thereby raising the question if other maternal immunological conditions may also affect neonatal immunity. 22 PREECLAMPSIA Preeclampsia is the most common complication associated with pregnancy. This multisystem disorder is potentially life threatening and occurs in around 3-5% of all pregnancies. The pathophysiology is not completely understood, but is characterized by insufficient placental development during early pregnancy, endothelial-cell dysfunction and an excessive maternal systemic inflammatory response. There are many risk factors for developing preeclampsia; first and foremost having had preeclampsia in a previous pregnancy but also pre-existing hypertension, diabetes, autoimmune disease, family history of preeclampsia, obesity and multiple gestations, reviewed in Redman et al.149 Preeclampsia is a heterogeneous disorder and the varied manifestations may arise from maternal, placental, or shared etiologies.150,151 The clinical stage of preeclampsia manifests after 20 weeks of gestation with hypertension (above 140/90 mmHg) and proteinuria (above 0.3 g/l).152,153 Based on these two clinical parameters preeclampsia can be divided into mild and severe subgroups. Severe preeclampsia is often, but not always, related to an early onset of the disorder (delivery necessary before 34 weeks). Severe and early onset preeclampsia is associated with more adverse maternal and perinatal outcomes.154-156 CONTRIBUTING FACTORS TO DEVELOPMENT OF PREECLAMPSIA Preeclampsia is believed to progress in three stages with the last stage representing the fullblown clinical disorder. The first stage involves abnormal placentation due to maternal immune maladaptation at the implantation site.157 Normally fetal trophoblasts invade deep into the maternal spiral arteries to create a high flow, low resistance vascular bed that can supply the fetus with nutrients, oxygen and carry out waste exchange. In preeclampsia this process is impaired leading to narrow spiral arteries of the placenta and ensuing oxidative stress.151 The trophoblast expresses non-classical HLA-E, -G and –C. HLA-C is the dominant ligand for inhibitory KIR receptors expressed by uNK cells, and some KIR/HLA-C combinations have been found to be unfavorable to trophoblast invasion due to the overall signals that the NK cell receives.150 The second stage of preeclampsia may result from the endoplasmic reticulum and oxidative stress of the preeclamptic placenta occurring in addition to the already inflammatory environment of pregnancy.157 Placental immune cells are activated; for instance there is increased IL-6 production by decidual cells and increased number and altered receptor 23 expression of placental NK cells.158-160 The preeclamptic placenta further releases proinflammatory debris such as microparticles and cell-free fetal DNA into the maternal circulation, which provokes elevation of the systemic inflammatory response.161,162 In general, a Th1 type of immune profile is predominant in preeclampsia and is believed to be part of the immunopathology.151,163,164 In circulation there is a higher percentage of IFN-γ+ NK cells161 and monocytes spontaneously produce IL-1β, IL-6 and IL-8.165 Further, secreted IL-18 and IL-12 that drive IFN-γ production and Th1 responses are also enhanced.161,166 Enhanced Th1 type of immunity may affect the tolerance system. Circulating Treg cells are reported to be significantly lower than those in normal pregnancy, however their suppressive function is unaltered.136 Recently, molecules related to maternal endothelial cell dysfunction have been suggested as biomarkers for predicting and diagnosing preeclampsia. The best studied is FMS-related tyrosine kinase 1 (sFlt-1), a soluble form of the vascular endothelial growth factor (VEGF) receptor. Increased levels of sFlt-1 appear in circulation before clinical manifestation of preeclampsia.167 Figure 3. The different stages and contributing factors for the development of preeclampsia. Adapted from Redman & Sargent.151 Reprinted with the permission of AAAS. 24 EFFECTS OF PREECLAMPSIA ON OFFSPRING Preeclampsia is the most common reason for pre-mature births as the only treatment is delivery and removal of the placenta. Apart from being born too early, children of preeclamptic mothers run an increased risk of intra-uterine growth restriction due the underdeveloped placenta. Placental morphology and growth has been identified as a predictor of cardio-vascular health in the offspring,168 and later in life the children of preeclamptic mothers more often suffer from childhood hypertension.169 They also have a higher incidence of conditions such as arthritis, asthma and diabetes.170-172 From previous studies it has been hard to separate the effect of being born too early from that of having been exposed to the inflammatory environment in a preeclamptic pregnancy. However, a recent study by Wu et al. showed that these adverse effects were indeed related to preeclampsia itself and not prematurity by using a cohort of children born at term to preeclamptic mothers.170 Still very little is so far known regarding the possible underlying mechanisms and the state of the immune system in these children. 25 PRESENT STUDY OBJECTIVES The overall aim of this work was to examine the response capacity of the neonatal innate immune system, with particular focus on monocytes and monocyte-NK cell collaboration. We also wanted to investigate how the neonatal monocyte and NK-cell compartments were influenced by a maternal immune-mediated disorder like preeclampsia. More specifically we aimed to: • Carry out a comprehensive analysis of monocyte subsets in cord blood and investigate cytokine responses to a common bacterial ligand at birth (Paper I). • Examine production of inflammatory mediators by, and activation-related receptors on, monocytes and NK cells in cord blood obtained after healthy or preeclamptic pregnancies (Paper II). SUBJECTS AND METHODS Subject demographics and methods are found in detail in the corresponding papers. In this section there will however be a brief description of the experimental procedure. Peripheral venous blood was collected from healthy adult volunteers, mothers or from the umbilical cord right after delivery. Serum was separated and mononuclear cells were isolated from blood by Ficoll-Paque gradient centrifugation. Cells and serum were frozen until simultaneous analysis of adult/neonatal or healthy/preeclamptic groups. Upon analysis, cytokine levels in serum were assessed by cytometric bead array (CBA). Cells were cultured in medium or were stimulated with the bacterial ligand PGN or PGN+IL-15. Expression of surface markers, MAPK-phosphorylation, intracellular and secreted cytokines were assessed by flow cytometry, ELISA and/or CBA. Also, experiments were carried out substituting fetal calf serum (FSC) in culture medium for neonatal/adult or healthy/preeclamptic serum prior to phenotypic or functional assessment. 26 RESULTS AND DISCUSSION Paper I Immunological responses at birth are under intense investigation to shed light on neonatal infection susceptibility. Besides suboptimal adaptive immunity,173 the reduced ability to cope with infectious challenge appears to be associated with qualitative differences in neonatal innate immune responses that seem to preferentially favor Th2- and Th17- over Th1-type of immunity.116-118 Monocytes can be divided into two major subpopulations, CD14++CD16- and CD14+CD16+ cells, which are suggested to play different roles in antimicrobial responses. Among other characteristics, CD14+CD16+ cells have a higher expression of TLR2 and TLR4 and secrete more inflammatory cytokines upon stimulation with certain TLR ligands,58,59 than CD14++CD16- cells. The monocyte subsets have not previously been investigated in newborns, and we hypothesized that the characteristics of the neonatal monocyte compartment might differ from the adult. Altered ratios of the monocyte subsets, and/or behavioral diversity of these cells, could contribute to the qualitative difference between the neonatal and adult cytokine profile. Firstly, we found that the monocyte subsets in CB and adults were present in similar frequencies, with CD14++CD16- representing approximately 93% and CD14+CD16+ cells 7% of the total monocyte population. Secondly, in both groups high expression of CD11c, CD80/CD86 and CD163 characterized the CD14+CD16+ cells, and in adults also high expression of HLA-DR. There were no differences in the levels of expression of any of the markers between CB and adult monocytes indicating an overall similar basal phenotype. Innate immune responses in the newborn are not generally impaired, as CBMC are capable of producing sizeable amounts of selected cytokines when stimulated with a variety of TLR agonists.116,118-120 In this study a limited amount of CBMC necessitated the choice of one stimulus for functional studies. We chose the bacterial ligand PGN that is a common target for immune recognition, since it is an essential part of the cell wall of both Gram positive and Gram negative bacteria.12 PGN is recognized by the PRRs TLR2 and NOD1/NOD2 that are highly expressed by monocytes.56,57 When CBMC were stimulated with PGN we noted secretion of equal (IL-1β, IL-6, IL-8, IL-10) or even greater (TNF) cytokine amounts compared with adult levels. Also intracellular CB monocyte TNF levels exceeded adult levels along with higher intracellular IL-12p70. With regard to the monocyte subsets, the CD14+CD16+ cells expressed more IL-12p70, but not more TNF, than CD14++CD16- cells. In a previous study we provided indirect evidence for increased IL-12 from CD14+CD16+ cells 27 as they induced higher NK-cell IFN-γ production than CD14++CD16- cells,96 and here we show it directly in both CB and adults. The CD14+CD16+ have previously been described as the major TNF producers, however this was not the case in our study. The TNF response may be stimulus-specific or data could be confounded by the fact that the CD14+CD16+ cells can be further subdivided into CD14highCD16+ and CD14dimCD16+ cells, where the latter produce lower amounts of TLR2-induced TNF than CD14highCD16+ cells.60 After initial recognition of PGN, a cascade of intracellular events culminate in the production of cytokines and differences in regulation and/or activation level of mediators in this pathway could have an impact on the level of cytokine production. PGN-induced signal transduction is well known to activate the three major families of MAPKs.174 As TLR expression on CB monocytes is at an adult level,109,110 we searched for an intracellular explanation for elevated TNF and IL-12p70 production from CB monocytes by investigating activation of two MAPK, ERK1/2 and p38-MAPK. ERK1/2 was activated by PGN but we saw no difference in phosphorylation between CB and adults. However, CB monocytes had a higher phosphorylation of p38-MAPK that correlated with levels of intracellular IL-12p70 production. Even though both MAPK are activated following PGN recognition, they appear to differentially regulate APC maturation pathways. Inhibition of p38-MAPK seems to confer the most pronounced effects on cytokine release by APCs and thus in development of Th1 responses,21,175 whereas inhibition of ERK1/2 seems to have only partial or negative effects on DC maturation.176,177 Cytokine responses of neonatal cells to different innate and adaptive ligands have been extensively studied. Although some common patterns have emerged, results are often inconsistent between studies using whole blood or MCs as the source of cells. A contributing factor could be that levels of adenosine are reported to be high in neonatal serum. Adenosine is believed to preferentially down-regulate certain cytokines and neonatal cells are described as more sensitive to adenosine-mediated cytokine suppression than adult cells.121 Therefore one explanation for the divergent findings on neonatal cytokine production could lie in the distinction between the inherent capacities of neonatal cells as opposed to the influence of external factors. In our study we tried to separate the two and firstly showed that the inherent capacity of CBMC for production of many inflammatory cytokines was not impaired. Secondly, we added CB and adult sera to both CBMC and PBMC to investigate whether this would influence cytokine production. In comparison to FCS, both types of human sera conferred changes in cytokine production, with an increase in production of IL-6 and IL-8 but a decrease in TNF and IL-10. Overall it did not matter if the cells came from CB or adults for 28 the observed effects of serum substitution. Only TNF production from PBMC was differentially regulated by factors in CB and adult serum, as CB serum was far more suppressive. This was not observed with CBMC indicating that adult cells were more sensitive to these effects in contrast to previous observations.121 That TNF production in particular is down-regulated in monocytes, might be due to adenosine-induced accumulation of cAMP that preferentially targets TNF through posttranscriptional mechanisms.178 The benefit of in utero suppression of neonatal and/or maternal TNF responses is perhaps easy to conceive given the connection of TNF to spontaneous abortion and miscarriage.179 In connection to this, TNF blockade has been suggested as a promising treatment for patients with immune-mediated fertility problems.180 As newborns have a heightened susceptibility to infections and a poor ability to mount protective vaccine responses, immune maturation is of great importance to study. Newborn immunity seems unable to support Th1-type of responses which has been related to impaired synthesis of e.g. IL-12.106-108 However results regarding IL-12, and other pro-inflammatory cytokines, from our study and others suggests no impairment in neonatal production.116,118-120 If CB monocyte activation of p38-MAPK and production of IL-12p70 is not deficient but in fact higher than in adult cells, then why should neonatal innate immunity be unable to generate successful Th1 responses? The answer might be found within the neonatal DC compartment, as DCs are the primary cells that activate T lymphocytes. Early experiments showed that CB DCs were poor stimulators of the mixed lymphocyte reaction whereas CB T cells responded normally to adult allogeneic DCs.181 It was subsequently shown that CB DCs have lower levels of co-stimulatory CD80/CD86 and MHC II181 and incomplete maturation in response to stimulation with single TLR ligands as compared to adult cells.182 Although DC capacity for IL-12 synthesis is reduced,106-108 it was shown that this impairment was not an inherent property of CB monocyte-derived DCs, as they synthesized adult-level IL-12 when matured with GM-CSF and IL-4.106 This might implicate that precursors of neonatal DCs may lack the appropriate maturation signals to allow for proper differentiation. Collectively, one might speculate that the neonatal deficiency in infection control stems mainly from suboptimal interactions between DCs and (also immature) T cells, which leaves the newborn vulnerable to sustained infections. The monocyte response per se could on the other hand be effective to certain strong immune stimuli such as PGN, and contribute to initial protection. Also, PGN-stimulated CB monocytes are able to induce CB NK-cell IFN-γ (Paper II), indicating that this important monocyte-NK cell collaboration is also functional at birth. 29 Some limitations in the study design should also be discussed here. Although we conclude that inherent CB monocyte subset responses to the bacterial ligand PGN are potent and not suppressed, other innate ligands such as viruses, may elicit a different response from neonatal monocyte subsets, which remains to be defined. Further, we assessed cytokine production after 24 hrs to ensure maximal production, particularly with IL-12p70 in mind. However, TNF is one of the first cytokines to appear, peaking around 4-6 hrs, and its production then declines.110 Any differences in kinetics of CB and adult TNF production are not visible when only assessing one time point, and thus adult levels could be higher earlier on as suggested by previous studies.110 Nonetheless, the intracellular cytokine assay shows accumulated levels over 24 hrs and therefore we clearly show that when stimulated with PGN, the CB monocyte TNF response is not deficient. In this study we provide a comprehensive analysis of monocyte subsets in the human newborn – a field where very little is known. These findings suggest that at birth, monocytes have a high inherent cytokine production capacity when challenged with a common bacterial ligand. However their TNF and IL-10 responses appear to be suppressed by factors in serum. Interestingly, we found that the percentage of the CD14+CD16+ monocyte subset after PGN stimulation was higher in CB than in adult cultures, suggesting that they may fail to undergo further differentiation into macrophage or DC effector cells. Previous studies of monocyte subpopulations are mainly from mice, where the subsets do not directly correspond to human, or from adults, and therefore these data may give insights for future understanding of neonatal monocyte responses at birth. 30 Paper II During pregnancy the maternal immune system goes through complex adaptations with the establishment of the hemochorial placenta and tolerance generation to a semi-allogeneic fetus. In preeclampsia, a major pregnancy-related disorder, immune maladaptation at the implantation site leads to a poorly developed placenta unable to sustain the needs of the growing fetus.157 Pregnancy in itself is characterized by a mild inflammatory state with a systemic activation of innate immune components, and in preeclampsia this response is excessive.137,183 A relation between maternal immune status and neonatal immunity has been suggested, but the impact of a preeclamptic pregnancy on the developing neonatal immune system has not been well studied. Our hypothesis was that the increased inflammation and exaggerated activation of immune cells at the fetal-maternal interfaces in preeclampsia might affect activation status of neonatal innate cells. We started by confirming that preeclampsia induces a maternal inflammatory serum cytokine profile, by finding elevated systemic IL-1β, IL-6, IL-8 and IFN-γ levels. Depending on the two major features used for clinical diagnosis, blood pressure and the amount of protein in the urine,152,153 preeclampsia can further be divided into mild and severe subgroups. The two subgroups of mothers showed different cytokine profiles, where IL-8 was higher in the severe group and IL-1β in the mild. For CB there were higher soluble (s)CD163 levels in the group with mild preeclamptic mothers whereas IL-8 and IL-10 levels were elevated in the severe group. Inflammation and hypoxia both drive IL-8 production, and the increased levels in CB serum could originate from epithelial and/or endothelial (placental) cells or CB leukocytes. Increased IL-10 levels might indicate a regulatory response to inflammation in these neonates. This theory was supported by the fact that the highest individual CB serum levels of IL-8 and IL-10 coincided. Furthermore, there was a correlation between levels of IL-6 and IL-10 between healthy and mild preeclamptic mothers and respective CB group, something that could suggest similar responses to an unknown pregnancy-related factor or possibly selective transfer over placental surfaces akin to transport of IgG antibodies.139,140 If so, either of these mechanisms could be disrupted by the exaggerated abnormalities in placental morphology observed in severe preeclamptic pregnancies, however this remains a speculation. As monocytes and NK cells are activated in the preeclamptic mother,161,165,166 we more closely examined these cells in the newborn. The CB monocyte compartment seemed generally unaffected by preeclampsia, and there were similar expression levels of CD11c, CD80/CD86, HLA-DR and CD163 on monocytes in the two CB groups. The expansion of 31 CD14+CD16+ cells is associated with ongoing inflammation65 and in the preeclamptic CB group CD14+CD16+ cells were slightly increased although the difference was not significant. We also assessed production of IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF, but spontaneous and PGN-stimulated secretion from MCs (all cytokines) and intracellular monocyte production (IL-12p70 and TNF) was similar for the two CB groups. The major finding of this study was the observed alterations in the CB NK-cell compartment. Firstly we examined surface levels of the constitutively expressed NK-cell activation receptors NKG2D and NKp30. Their levels are elevated on CB NK cells,82 indicating that they could be important for NK-cell activation early in life. In the mild preeclamptic group we observed lower NKG2D and higher NKp30 expression. In terms of factors that could affect these receptors, TGF-β and IFN-γ can down-regulate NKG2D,184,185 and NKp30 can be upregulated by IL-15.186 Levels of IL-15 and TGF-β were not measured directly in serum, but there were no differences in IFN-γ between the mild preeclamptic CB group and the severe or healthy group. Interestingly, both NKG2D ligands (MICA/MICB and ULBP) are expressed in placenta and released on placental exosomes.131 These exosomes are mainly secreted towards the maternal side of the placenta but are also observed on the fetal side (Lucia MinchevaNilsson, personal communication). In vitro experiments showed that they exerted immunosuppressive functions by down-regulating NK-cell NKG2D expression and subsequent NK-cell cytotoxicity.131 Furthermore, an increase in shedding of microparticles from the preeclamptic placenta has been reported,161,187 together suggesting that an eventual increase in microparticle burden in CB serum (and/or alterations in cytokine levels), may provide a link to altered preeclamptic CB NK-cell receptor expression. To test this hypothesis we incubated healthy CBMC with medium containing CB sera from the healthy, mild or severe preeclamptic group, and subsequently measured expression of NKG2D and NKp30. However, levels of the two receptors were similar in all three settings suggesting that there were no differences in serum factors that could influence NK-cell receptors between the three CB groups. One might therefore speculate that the observed changes in NK-cell receptor expression may persist after birth and the immediate effect of the in utero environment, thus potentially affecting the delicate regulation of NK-cell activation in response to NKG2D and NKp30 ligands in these neonates. Secondly, we examined cytokine production and found that unstimulated CB NK cells from the mild preeclamptic group expressed more intracellular TNF and IFN-γ than CB NK cells from the healthy group. That both receptor expression and pre-activation of CB NK cells was 32 seen primarily in the mild preeclamptic group might be a result from common factors acting on these cells. Monocyte-induced NK-cell production of TNF and IFN-γ was not influenced by preeclampsia, however this does not rule out that cytokine production and/or cytotoxicity induced via NKG2D or NKp30 by their cognate ligands might be altered. This and other aspects of the functional relevance of altered CB NK-cell characteristics remain to be investigated. In preeclamptic mothers both the monocyte and the NK-cell compartment are activated, however in CB only NK-cell activation was observed. The factors that are proposed to drive inflammatory changes in the mother, like systemic oxidative stress, growth factors, leptin and syncytiotrophoblast debris,188 are not likely to be as prominently present in the circulation of her developing fetus. Since monocytes and NK cells are activated in very different ways, one could envision that their triggering factors might not be uniformly present in preeclamptic mothers and CB alike. One could also speculate that NK cells might be more sensitive to inflammation-induced changes or reacting to an up-regulation of stress ligands e.g. MICA/MICB and ULBP in the preeclamptic placenta, although this has not been substantiated. One interesting finding from this study was the pronounced difference between the mild and severe subgroups of preeclampsia, both for mothers and newborns, something that could relate to potential differences in management of these patients. To our knowledge however there were no major differences in administration of medications for mild or severe preeclamptic patients. Cortisol is given to women who develop early onset preeclampsia in order to promote fetal lung development. As cortisol is an immunosuppressive drug it could have effects on the immune activation status of mothers and possibly also of their neonates. In our study population only 3 women were characterized as early preeclampsia, and the mothers did not significantly differ from the other preeclamptic cases in our data set. CB was obtained from only one of these deliveries but showed no difference to other preeclamptic CB in our assays. The pronounced difference between the subgroups that we observed here should warrant future studies to observe this sub-grouping when attempting to elucidate how preeclampsia affects immune parameters both in infants and mothers. In summary the findings from this study show that an in utero inflammatory priming of neonatal innate immunity takes place in preeclamptic pregnancies, as shown by altered systemic and NK-cell specific cytokine levels and altered NK-cell activation receptor expression. The fetal origin of adult diseases hypothesis states that the health and disease of an individual is influenced by intrauterine life, and indeed children born after preeclamptic 33 pregnancies have an increased incidence of various diseases in childhood. To elucidate potential functional consequences of changes in the NK-cell compartment, or whether preeclampsia-associated inflammatory activation is observed into childhood, would require more studies and a follow-up of the same children. GENERAL CONCLUSIONS Although the newborn immune response is deficient in many aspects and shows an altered cytokine profile compared to adults after stimulation with various TLR ligands, the CB monocyte response to PGN, a cell-wall component present in virtually all bacteria, is potent, in particular with regards to TNF and IL-12p70 production. Moreover, cytokine production in CB and adult cells is under the regulation of serum factors that favor certain cytokine responses over others. Monocyte capacity for production of early-response cytokines does not seem to be altered in newborns of preeclamptic mothers despite the increased inflammatory propensity of their mothers. However, CB NK cells show altered phenotype and increased spontaneous cytokine production after preeclamptic pregnancies. Further, preeclampsia was associated with an inflammatory serum cytokine profile in both mothers and newborns. Therefore a pathological pregnancy may skew maturation of neonatal immunity and should be considered as a factor influencing innate responses and/or immune cell activation during the earliest years of life. 34 FUTURE PERSPECTIVES PREECLAMPSIA With respect to the findings from Paper II, we will continue to investigate CB NK-cell functionality in terms of cytotoxicity and cytokine production induced through NKG2D and/or NKp30 ligation in vitro. Investigations of the same parameters in children later in the neonatal/infant period should also be carried out to further clarify the relevance of the alterations in NK-cell characteristics at birth. A follow-up of a cohort of children born to preeclamptic mothers would also enable examination of whether the inflammatory serum milieu induced by preeclampsia persists. However, at present we are unable to collect more samples or enroll patients for long-time studies. Therefore future work will focus on monocytes and monocyte-NK cell functions and collaboration, particularly in relation to viral exposures. NEONATAL IMMUNITY AND VIRAL INFECTIONS In paper I and II we investigated CB antibacterial responses with focus on monocytes and NK cells. However newborns are also subjected to viral infections early in life, towards which NK cells are important effector cells. Members of the herpesvirus family are widely spread viruses in human populations. Primary infection with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) often occur during early childhood and the viruses then persist in a latent form. In a previous study of a cohort of 2-year old children we examined the relation between herpesvirus seropositivity and the quality of innate responses.96 At this age only half of the children had acquired EBV and/or CMV infection although all should most likely have been exposed. Further, the innate response was markedly different, with seropositive children showing lower NK-cell IFN-γ production as well as lower serum IFN-γ levels. Also adaptive responses were modulated by herpesvirus infection, as PBMC from 2-year-old EBV seropositive children tended to have an overall increased cytokine production after PHA stimulation.189 In connection to this, we have found that early acquisition of EBV infection protects against persistent IgE sensitization, suggesting that early-life microbial exposure could contribute to an allergy protective immune profile.190 Possibly an initial difference exists between individuals in immune response factors which could contribute to viral susceptibility, thus explaining why not all exposed children were 35 seropositive. Also, herpesvirus infection might modulate the immune system during early life, thus explaining the differences in immune responses we observed in 2-year old children. To elucidate this we plan to infect CBMC, and possibly cells from children later in childhood, with EBV and determine differences in antiviral responses, both innate and adaptive. Further, we will relate serostatus to immune function at different ages in children. 36 ACKNOWLEDGEMENTS To Eva Sverremark-Ekström for excellent supervision and for allowing me to work freely yet never stray. To my co-authors and collaborators Shanie Saghafian-Hedengren, Nora Bachmayer, Rangeen Rafik Hamad and Katarina Bremme. To all midwifes and nurses, especially to Mari Jonsson, at the delivery ward and Agneta Nordwall at the Department of Cardiology, both at the Karolinska University Hospital, Solna for their kind assistance with sample collection. To the seniors scientists, my co-supervisor Marita Troye-Blomberg, Carmen Fernandez, Eva Severinson and Klavs Berzins. Maggan, Gelana and Students at the department for being friendly and helpful. 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