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Doctorial thesis from the Department of Immunology, the Wenner-Gren Institute, Stockholm University, Stockholm, Sweden Early-life gut microbiota and breast milk oligosaccharides in relation to childhood immune maturation and allergy Ylva Margareta Sjögren Stockholm 2009 1 Cover illustration: © Taina Litwak All previously published papers were reproduced with permission from the publishers. Distributed by Stockholm University Library. Printed in Sweden by Universitetsservice AB, Stockholm 2009 © Ylva Margareta Sjögren ISBN 978-91-7155-854-1 2 To all of us who dreamt about owning a pet but never could… 3 4 SUMMARY Today, atopic allergy is the most common chronic disease among children in the developed world. This high prevalence could be associated with low microbial exposure due to changed lifestyle factors. The neonatal gut is immediately colonised by various bacterial species during and after birth. This gut microbiota is the most numerous bacterial stimuli in infancy and appears to be important for immune maturation. Previous studies also indicate that an altered gut microbiota might be associated with infant allergy development. Immunomodulatory components in human milk might differ between mothers and could therefore explain the contradictory results seen regarding breastfeeding and allergy development. Oligosaccharides, the third most abundant solid component in human milk, survive the passage through the stomach and can be utilised by the gut microbiota. We analysed nine abundant neutral oligosaccharides in colostrum samples from allergic and nonallergic women and related the consumption of these oligosaccharides to subsequent allergy development in the children. We found a considerable variation in the concentration of neutral oligosaccharides in colostrum, which was not to be explained by the allergic status of the women. Neither was the consumption of neutral colostrum oligosaccharides related to allergy development in the children. We further studied the early infant gut microbiota in relation to allergy development during the first five years of life, thus with a longer follow up than in previous studies. Also, we investigated gut microbiota at species level rather than genera level. Faecal samples were collected at one week, one month and/or two months after birth and presence and amounts of Clostridium difficile, Bifidobacterium (B.) adolescentis, B. longum, B. bifidum, B. breve, Lactobacilli (L.) group I (comprising L. rhamnosus, L. casei and L. paracasei), Lactobacilli group II (comprising L. gasseri and L. johnsonii) and Bacteroides fragilis were investigated. Infants who harboured Lactobacilli group I and B. adolescentis at one week of age developed allergic disease less frequently, during their first five years, than infants who did not harbour these bacteria at the same time. Further, we investigated whether early colonization with these bacteria was associated with lifestyle factors inversely related to allergy development. Actually, levels of house dust endotoxin and family size were associated with number of Bifidobacterium species in the early faecal samples. Levels of salivary secretory IgA (SIgA) might be associated with allergy development and SIgA levels as well as the expression of Toll like receptors (TLR) on peripheral blood mononuclear cells (PBMC), could be influenced by microbial exposure. Thus, we investigated if early colonization with the above mentioned bacterial species influences childhood levels of salivary SIgA and spontaneous expression of TLR2 and TLR4 mRNA in PBMC collected twelve months after birth. In addition, we studied whether the early colonization associates with LPS-induced cytokine and chemokine production from the PBMCs. The early Bifidobacterium flora influenced levels of salivary SIgA at six and twelve months. In addition, the intensity of early Bacteroides fragilis colonization was inversely associated with spontaneous TLR4 mRNA expression in the PBMCs and to LPS-induced IL-6 and CCL4 production. In conclusion, the work presented in this thesis indicates that the early gut microbiota might be altered in infants developing allergic disease. In addition, Bifidobacterium species influence early mucosal immune responses and colonization with these species is associated with house dust endotoxin levels and family size. Thus, the early gut microbiota could, to some extent, explain the inverse relationship between family size/endotoxin exposure and allergy development. However, consumption of colostrum neutral oligosaccharides does not protect against early allergy development. 5 SAMMANFATTNING Idag är den vanligaste kroniska sjukdomen bland barn i västvärlden atopisk allergi. En förklaringsmodell är att vi idag utsätts för mikrobiella stimuli i mindre utsträckning än tidigare, vilket troligtvis beror på förändrade livsstilsfaktorer. Spädbarn koloniseras med bakterier under och efter födseln. Det höga antal bakterier som koloniserar tarmen verkar spela roll för immunförsvarets mognad. Tidigare studier har påvisat vissa skillnader i den tidiga tarmfloran mellan barn som utvecklar allergi och barn som inte utvecklar allergi under sina första två år. I bröstmjölk finns ett flertal komponenter som är viktiga för spädbarns immunförsvar. Dessa ämnen varierar mellan kvinnor och är beroende av såväl miljö- som genetiska faktorer. Några av dessa substanser skiljer sig dessutom åt mellan allergiska och icke-allergiska kvinnor. Denna variation i bröstmjölkens sammansättning skulle kunna förklara varför det är svårt att dra någon slutsats när det gäller amningens effekt på allergiutveckling. Efter fett och laktos förekommer oligosackarider i högst koncentration i bröstmjölk. Dessa bryts inte ned i magsäcken utan når tarmen intakta och kan där användas som näring till tarmbakterier. Vi analyserade nio av de vanligaste oligosackariderna i kolostrum från allergiska och icke-allergiska mammor, samt relaterade denna tidiga oligosackarid-konsumtion till allergiutveckling hos barn som följdes till 18 månader. Det var stor skillnad i oligosackaridkoncentration mellan olika kvinnors kolostrum. Däremot kunde inte kvinnornas allergistatus förklara detta och det var inte heller några skillnader i oligosackaridkoncentration mellan vad de allergiska och icke-allergiska barnen konsumerat under sina första dagar. Vidare studerade vi tarmfloran under de första två månaderna hos spädbarn som under sina första fem år antingen utvecklade eller inte utvecklade allergi. Uppföljningen var därför längre än i tidigare studier. Förekomst och mängder av Clostridium difficile, Bifidobacterium (B.) adolescentis, B. longum, B. bifidum, B. breve, Lactobacilli (L.) grupp I (bestående av L. rhamnosus, L. casei och L. paracasei), Lactobacilli grupp II (bestående av L. gasseri och L. johnsonii) samt Bacteroides fragilis analyserades i de tidiga fecesproverna. De barn som vid en veckas ålder var koloniserade med Lactobacilli grupp I eller B. adolescentis utvecklade inte allergi i samma utsträckning som de barn som saknade dessa bakterier vid samma ålder. Vi undersökte även om livsstilsfaktorer påverkar koloniseringen av dessa bakterier. Både familjestorlek och endotoxin i damm från barnens hem korrelerade med antalet bifidobakterier i de tidiga fecesproverna. Koncentrationen av sekretoriskt IgA (SIgA) i saliv kan vara associerat med allergiutveckling. Mikrobiella stimuli antas påverka SIgA nivåer och uttrycket av några av de receptorer som känner igen mikrober (TLR2 och TLR4). Därför undersökte vi hur de ovan nämnda tarmbakterierna påverkar nivåerna av SIgA i saliven under de första fem åren samt uttrycket av TLR2 och TLR4 på perifiera blodceller vid tolv månaders ålder. Den tidiga bifidobakteriefloran hade en tydlig förhöjande effekt på nivåerna av SIgA under det första året. Dessutom korrelerade mängden Bacteroides fragilis negativt med det spontanta uttrycket av TLR4 vid tolv månader. Vidare producerade dessa perifera blodceller lägre IL-6 och CCL4 efter stimuli med TLR4 liganden LPS. De resultat som sammanfattas i denna avhandling tyder på att den tidiga tarmfloran är annorlunda hos barn som utvecklar allergi. Dessutom påverkar den tidiga bifidobakteriefloran SIgA i saliven under det första året. Bifidobakteriefloran verkar i sin tur vara påverkad av familjestorlek och endotoxin-exponering. Därför skulle tarmfloran till viss del kunna förklara det negativa sambandet mellan familjestorlek/endotoxin-exponering och allergiutveckling. Däremot verkar inte konsumtion av höga nivåer av oligosackarider i kolostrum skydda mot allergiutveckling under de första 18 månaderna. 6 LIST OF PAPERS This thesis is based on the following original articles, referred to in the text by their Roman numerals: I Sjögren Y. M, Duchén K, Lindh F, Björkstén B, Sverremark-Ekström E. Neutral oligosaccharides in colostrum in relation to maternal allergy and allergy development in children up to 18 months of age. Pediatr Allergy Immunol 2007;18:20-26. II Sjögren Y. M, Jenmalm M. C, Böttcher MF, Björkstén B, Sverremark-Ekström E. Altered early infant gut microbiota in children developing allergy up to 5 years of age. Clin Exp Allergy 2009;39:518-526. III Sjögren Y. M, Tomicic S, Lundberg A, Böttcher M.F, Björkstén B, SverremarkEkström E, Jenmalm M.C. Influence of early gut microbiota on the maturation of childhood mucosal and systemic immune responses. Under revision 7 Om snöret inte håller, utan går av, är det bara att försöka med ett annat snöre Nalle Puh (A.A Milne) 8 TABLE OF CONTENTS TABLE OF CONTENTS........................................................................................................................................9 ABBREVIATIONS .............................................................................................................................................. 10 INTRODUCTION ................................................................................................................................................ 11 INNATE IMMUNITY ...................................................................................................................................... 12 Mononuclear phagocytes and antigen presenting cells................................................................................. 12 Pathogen recognition receptors..................................................................................................................... 13 Chemokines and cytokines in innate responses............................................................................................ 16 Granulocytes and Mast cells ......................................................................................................................... 18 Neutrophils ................................................................................................................................................... 18 Natural Killer cells........................................................................................................................................ 18 ADAPTIVE IMMUNITY ................................................................................................................................. 20 T cells............................................................................................................................................................ 20 CD8+ T cells ........................................................................................................................................... 20 CD4+ T cells ........................................................................................................................................... 20 γδ T cells .................................................................................................................................................. 23 B cells ........................................................................................................................................................... 23 Antibodies ................................................................................................................................................. 24 THE GUT ASSOCIATED IMMUNE SYSTEM ............................................................................................. 26 Antimicrobial peptides.................................................................................................................................. 27 Immunoglobulin A........................................................................................................................................ 28 Oral tolerance................................................................................................................................................ 29 Intestinal T cells and oral tolerance........................................................................................................ 30 Intestinal dendritic cells and oral tolerance............................................................................................ 31 ALLERGY ......................................................................................................................................................... 33 Factors influencing allergy development...................................................................................................... 34 Epidemiology of the hygiene hypothesis.................................................................................................. 36 Neonatal and infant immune responses ........................................................................................................ 37 Neonatal and infant immune responses in relation to subsequent allergy development .............................. 38 THE COMMENSAL GUT MICROBIOTA..................................................................................................... 41 Factors influencing the composition of the gut microbiota .......................................................................... 41 Infant gut microbiota .................................................................................................................................... 43 Fermentation, prebiotics and probiotics ....................................................................................................... 44 Methods for studying the gut microbiota...................................................................................................... 45 Gut microbiota and immune responses......................................................................................................... 46 Gut microbiota and allergy ........................................................................................................................... 48 IMMUNOLOGY OF HUMAN MILK ............................................................................................................. 50 Breast feeding in relation to allergy development ........................................................................................ 51 Human milk oligosaccharides....................................................................................................................... 52 PRESENT STUDY ............................................................................................................................................... 54 AIMS ................................................................................................................................................................. 54 MATERIAL AND METHODS ........................................................................................................................ 55 Study population study I ............................................................................................................................... 56 Study population study II and III.................................................................................................................. 57 RESULTS AND DISCUSSION ....................................................................................................................... 59 Human milk oligosaccharides in colostrum of allergic and non-allergic women (I) ................................... 59 Colostrum oligosaccharide concentrations in relation to subsequent allergy development (I) .................... 60 Early infant gut microbiota (II and III)......................................................................................................... 60 Early infant gut microbiota in relation to allergy development (II) ............................................................. 62 Environmental factors influence the early infant gut microbiota (II)........................................................... 65 Early infant gut microbiota in relation to maturation of immune responses (III)......................................... 67 Clinical relevance ......................................................................................................................................... 72 CONCLUDING REMARKS ............................................................................................................................ 74 FUTURE PERSPECTIVES ................................................................................................................................ 75 ACKNOWLEDGEMENTS................................................................................................................................. 77 REFERENCES ..................................................................................................................................................... 79 9 ABBREVIATIONS 2´-FL 3-FL AB AD AMP APC APRIL ARC B. BAFF C. CBMC CD CFU CoNS CTL DC DC-SIGN DGGE DNA E. ELISA Fel d 1 FISH GG+ GALT GF HMGB1 HMO HPLC IEC IEL IFNγ Ig IL ILF L. LAL LBP LDFT LNDFH I 2´-Fucosyllactose 3-Fucosyllactose Asthma bronchialis Atopic dermatitis Antimicrobial peptide Antigen presenting cell A proliferation-inducing factor Allergic rhinitis/conjunctivitis Bifidobacterium B cell activating factor Clostridium Cord blood mononuclear cell Cluster of differentiation Colony forming unit Coagulase-negative Staphyloccocus Cytotoxic T lymphocyte Dendritic cell Dendritic cell-specific intracellular adhesion molecule 3-grabbing integrin Denaturing gradient gel electrophoreses Deoxyribonucleic acid Escherichia Enzyme-linked immunosorbent assay major allergen of cat (Felis domesticus) Fluorescent in situ hybridization Gram negative bacteria Gram positive bacteria Gut associated lymphoid tissue Germ-free High mobility group box protein 1 Human milk oligosaccharides High performance liquid chromat Intestinal epithelial cell Intraepithelial lymphocyte Interferon γ Immunoglobulin Interleukin Isolated lymphoid follicle Lactobacilli Limulus Amebocyte Lysate LPS binding protein Lactodifucotetraose Lacto-N-difucohexaose I LNFP I LNFP II LNFP III LNnT LNT LP LPS MACs MALT MAMP M cell mDC MHC MLN NK cell NKT cell NOD OVA PBMC PCR pDC PGN pIgR PP PPR PSA RA ROS S. SCFA SIgA SPT STAT TCR TFAN TGFβ TLR Th TNF T reg TSLP 10 Lacto-N-fucotetraose I Lacto-N-fucotetraose II Lacto-N-fucotetraose III Lacto-N-neotetraose Lacto-N-tetraose Lamina propia Lipopolysaccharide Microflora associated characteristics Mucosa associated lymphoid tissue Microbial associated molecular pattern Microfold cell myeloid dendritic cell Major histocompatebility complex Mesenteric lymph node Natural killer cell Natural killer T cell Nucleotide-binding oligomerization domain Ovalbumin Peripheral blood mononuclear cell Polymerase chain reaction Plasmacytoid dendritic cell Peptidoglycan Polymeric immunoglobulin receptor Peyer´s patches Pathogen recognition receptor Polysaccharide A Retinoic acid Reactive oxygen species Staphylococcus Short chain fatty acids Secretory IgA Skin prick test Signal transducer and activator of transcription T cell receptor 4-Triflouroacetamidoaniline Transforming growth factor β Toll-like receptor T helper cell Tumor necrosis factor T regulatory cell thymic stromal-derived lymphoprotein INTRODUCTION The immune system in mammals consists of innate and adaptive branches. An innate immune response is the first defence against pathogens and involves anatomical and physiological barriers, i.e. skin and temperature, as well as innate immune cells, like granulocytes, monocytes and macrophages. These cells respond rapidly by phagocytosing pathogens and by secreting substances like chemokines and cytokines which attract and activate other cells. The adaptive branch is slower, but reacts to specific antigens and generates immunological memory. Adaptive immunity is characterised by lymphocytes, i.e. T and B cells, their cytokines as well as antibodies (immunoglobulins) produced by the B cells1. T and B cell clones have receptors which are specific for diverse antigens on e.g. pathogens. On B cells, these receptors are called antibodies. Secreted antibodies have several different functions i.e. preventing entry of pathogens, facilitating opsonisation and activating complement. In addition, antibodies can bind to Fc receptors on other cells than B cells. In allergic individuals, IgE antibodies, towards innocuous antigens, have bound to e.g. mast cells and the cross-linking of these antibodies leads to release of mediators causing allergic symptoms. There is a constant collaboration between the innate and the adaptive immune system. Antigen presenting cells (APCs), like macrophages and dendritic cells (DCs) recognize microbial associated molecular patterns (MAMPs) via pathogen recognition receptors (PRRs)2. The APCs instruct naïve CD4+ T cells to differentiate into either T helper (Th) 1, Th2, Th17 or T regulatory cells (T regs) which are characterized primarily by their cytokine profile. The cytokines secreted by Th2 cells facilitate the production of IgE antibodies by B cells3. However, cytokines secreted by Th1 cells dampen the Th2 responses. The binding of diverse MAMPs to PRRs on DCs generally favours Th1 differentiation1. We have studied the most numerous microbial stimuli in infancy, the early infant gut microbiota, in relation to allergy development. In addition, we investigated how it relates to the maturation of the immune system and whether it is influenced by environmental factors inversely related to allergy development. We also studied whether consumption of human milk oligosaccharides, postulated to support the growth of certain gut microbiota species, is associated with allergy development. 11 INNATE IMMUNITY Mononuclear phagocytes and antigen presenting cells Monocytes develop from the myeloid lineage in the bone marrow and they constitute 5-10 percent of peripheral leukocytes in human adults4. Blood monocytes are precursors for macrophages and DCs and they also contribute directly to immune responses against pathogens5 by secreting cytokines and reactive nitrogen and oxygen intermediates. Human circulating monocytes are divided into two subsets, CD14hiCD16- and CD14loCD16+, depending on their expression of CD14 and CD165. CD14 is involved in recognition of lipopolysaccharide (LPS) from Gram negative bacteria (G-) whereas CD16 is an immunoglobulin G receptor - FcγRIII. The CD14hiCD16-subset represents the majority of circulating monocytes (80-90%). They are large and highly phagocytic with several granula and often called classical or inflammatory monocytes, as they migrate to inflamed tissue6. The CD14loCD16+ population is smaller and often called resident monocytes since they migrate to non-inflamed tissue under homeostatic conditions. Yet, they are most often called proinflammatory due to their production of high levels of tumour necrosis factor (TNF) after stimulation with PRR ligands5. The difference in migratory properties depends on the expression of different chemokine receptors on the two subsets. In example, the CD14hiCD16subset expresses high levels of CCR2 and thus responds to monocyte chemoattractant protein (MCP-1 i.e. CCL2). The expression of the CCR2 receptor is low in CD14loCD16+ monocytes and these cells rather migrate in the presence of CX3CL1. Furthermore, the subsets express different immunoglobulin, adhesion and scavenger receptors: CD14loCD16+ expresses higher levels of major histocompatibility complex (MHC) II and FcγRII and III7. Blood monocytes can be matured into macrophages and dendritic cells, in vitro, when cultured in the presence of M-CSF and GM-CSF + interleukin (IL) 4, respectively8. Less is known about this differentiation in vivo6. Monocytes circulate in blood for approximately 3 days5 and thereafter migrate to peripheral tissue where they mature into macrophages or DCs6. Macrophages are a highly heterogeneous population mainly found in peripheral tissue9. They are phagocytic cells that also release several cytokines and growth factors once they are activated. Therefore, macrophages are important for clearance of altered cells and tissue repair in addition to their bactericidal activity6. Macrophage differentiation is influenced by 12 the microenvironment in the actual tissue6. In example, macrophages in the gut meet high amounts of cytokines with regulatory activity like transforming growth factor (TGF) β and IL-10. Thus when maturing into intestinal macrophages they lose several receptors and adhesion molecules. As they express co-stimulatory molecules, e.g. CD40, CD80 and CD86 at low levels, intestinal macrophages are poor APCs6. Also, they lack CD14 expression10 and are therefore non-responding to inflammatory stimuli like LPS. Still, the intestinal macrophages are efficient phagocytic cells with bactericidal activity6. There are also resident macrophages in other tissues, exemplified by Langerhans cells in the skin, Kuppfer cells in the liver, osteoclasts in bone and microglia cells in the brain7. Dendritic cells are of major importance for immune responses11. They stem from the myeloid or plasmacytoid lineage in the bone marrow and are initially divided into myeloid (conventional) DCs or plasmacytoid DCs, respectively8. They are situated at sites where pathogens enter, e.g the mucosal tissue. Like monocytes and macrophages, DCs recognize pathogens via PRRs (see below)11. Furthermore, DCs initiate adaptive immune responses by activating T cells. The DCs are the most potent APCs, generally assumed as the only cells able to differentiate naive T cells into Th1, Th2, Th17 or Tregs11. Activated dendritic cells frequently migrate from peripheral tissue to lymphoid organs in order to interact with T cells4. Dendritic cells can further be divided into two subtypes depending on their location, blood DCs and tissue DCs11. In addition, several different subtypes of DCs have been identified in different tissues and also within different tissues, e.g. within the intestines (see below). Pathogen recognition receptors PRRs are essential for innate immunity. They recognize MAMPs that are shared by many different microorganisms and accordingly PRRs have broad specificity against pathogens1. Toll-like receptors (TLRs) is the best characterized group of PRRs. They are trans-membrane receptors that recognize e.g. bacterial cell wall components and viral nucleic acids12. The human TLRs identified so far (TLR 1-11) and their ligands are summarized in Table 1. For example, the flagellin of flagellated bacteria is recognized by TLR5 and unmethylated CpG dinucleotides in bacterial and viral DNA are recognized by TLR9. TLR2 recognizes several different lipid proteins, e.g. peptidoglycan (PGN) and lipotechoic acid13. These molecules are abundant in the cell layer of gram positive (G+) bacteria. The cell wall of G- bacteria contains LPS (also referred to as endotoxin) which is recognized by TLR4. TLR4 also recognizes 13 envelope proteins from viruses and the endogenous heat-shock protein 60 and high mobility group box 1 protein12, 14. For effective down-stream signaling, LPS binding protein (LBP) binds to LPS. Thereafter, the LPS-LBP complex is transferred to CD14 which further transfers LPS to MD-215. Eventually this complex directs TLR4 rearrangement and recruitment of other adaptor proteins. Finally, intracellular signaling, either MyD88 dependent or independent, occurs. The ligation of most TLRs leads to downstream signaling via MyD88 and eventually to the activation of transcription factors like NFκB12. This results in transcription of genes (e.g. cytokines and chemokines) involved in the inflammatory response. Not only immune cells express TLRs. They can also be found on/in epithelial cells and fibroblasts. Importantly, different TLRs are found on different cells. The intracellular TLR7 and TLR9 are expressed by pDCs, while mDC express TLR2, TLR3, TLR4, TLR5, and TLR816. Monocytes express TLR1, TLR2, TLR4, TLR5, TLR817. Epithelial cells in the gut appear to have a low expression of TLRs (e.g. TLR 2-5) or only express them on the basolateral surface (i.e. the surface not facing the lumen containing the microbiota)13. PRRs on innate cells are not only important for innate immune responses but also for adaptive immune responses1. DCs recognize microbes and further internalize and present the antigens of the pathogens to Th cells. Simultaneously the DCs receive signals via its PRRs. The DCs share these signals with the Th cells via cytokines. Most often MAMPs binding to TLRs lead to IL-12 production by the DC which directs the naïve T cell to differentiation into a Th1 cell1. Another group of PRRs is the nucleotide-binding oligomerization domain (NOD) family1. It consists of two intracellular receptors, NOD1 and NOD2. They recognize different bacterial PGNs in the cytosol12. As with TLR engagement, NOD engagement leads to the production of pro-inflammatory cytokines and chemokines resulting in e.g. neutrophil recruitment1. 14 Table. 1. The different human toll like receptors and their common ligands. TLR receptor Ligand Pathogen containing the ligand TLR1 Tri-acyl lipopeptides Bacteria and mycobacteria TLR2 Lipoteichoic acid G+ bacteria Peptidoglycan G+ bacteria (mainly) Zymosan Fungi Heat shock proteins Host TLR3 Double stranded RNA Viruses TLR4 Lipopolysaccharide G- bacteria Envelope proteins Virus Heat shock proteins Host HMGB1 Host TLR5 Flagellin Flagellated bacteria TLR6 Di-acyl lipoproteins Mycoplasma TLR7,8 Single stranded RNA Virus TLR9 CpG dinucleotides Bacteria Hemozoin Plasmodium TLR10 Unknown Unknown TLR11 Unknown Uropathogenic bacteria Constructed from references 12-14. HMGB1= High mobility group box 1 protein, G+= Gram positive bacteria, G-= Gram negative bacteria. TLR regulation The expression of TLRs can change following stimulation and is dose and time dependent12, 18 . Our group showed that the TLR4 monocyte expression was down regulated shortly after LPS stimulation while TLR2 expression was up regulated after PGN stimuli in human peripheral blood cells (PBMCs)19. Pro-inflammatory cytokines e.g. IFNγ can also influence TLR expression14. It has been shown that high LPS exposure induces endotoxin (LPS) tolerance18. Thus, endotoxin tolerant mice have decreased splenic TLR2 and TLR4 expression20. Also, these mice do not produce IL-1β when retreated with LPS. In addition, Nomura et al showed that mouse macrophages produce less IL-6 and IL-12 when preincubated with high doses of LPS18. Intravenous endotoxin administration also influences 15 human CD14+ cells to produce less IL-1β, TNF, IL-6 and CXCL8 (IL-8) after LPS stimuli in vitro21. Chemokines and cytokines in innate responses Chemokines are small proteins that are important for directing immune cells to sites of inflammation22. In addition, they participate in organ development and angiogenesis. Chemokines are classified according to the arrangement of cysteine residues in the N-terminal region of the protein and thus belong to the C, CC, CXC or CX3C subclasses. Chemokines are produced by different cell types and can either be produced constitutively (homeostatic chemokines) or in response to inflammatory stimuli (inflammatory chemokines). The complexity of chemokines is due to their binding to receptors i.e. one chemokine can bind several different receptors and the receptors can also have several ligands. CXCL8 (IL-8) is a neutrophil attractant which is released by e.g. monocytes and epithelial cells in response to bacterial stimuli22, 23 . In addition, CXCL8 (IL-8) and CCL2 (MCP-1) are important for angiogenesis22. Furthermore, CCL2 (MCP-1) is a monocyte, T cell, natural killer (NK) cell and basophil attractant which also causes smooth muscle cells to proliferate. CCL4 (MIP-1β) is another inflammatory cytokine released by monocytes in response to microbial stimuli. It attracts several different leukocytes e.g. monocytes, T cells and DCs. Cytokines are secreted proteins24 which mainly act locally. That is, they bind to receptors on nearby cells (paracrine effect) or to receptors on the cell that produced them (autocrine effect). They are involved in both innate and adaptive immunity and cause for example cells to grow, differentiate and/or secrete substances like other cytokines or antibodies. Monocytes can secrete several cytokines, like TNF, IL-1β, IL-6, IL-12, IL-15, IL-18, IL-23 and IL-2724. IL-15 activates NK cells1 and IL-27 is a pro-inflammatory cytokine which up regulates the production of interferon (IFN) γ from NK cells and Th1 cells24. The type 1 interferons, IFNα and IFNβ, are important for protection against viruses1. Some of the mentioned cytokines are of particular importance for this thesis and thus described below and/or in specific sections. Tumor necrosis factor TNF is a pro-inflammatory cytokine which is rapidly produced by macrophages and monocytes in response to e.g. TLR ligands. TNF exerts its functions by binding to TNF receptor-1 or TNF receptor-2 and further activates cells like neutrophils, platelets and macrophages25. It is also important for sleep regulation and embryonic development. 16 Furthermore, TNF could induce apoptotic or necrotic cell death and might thus prevent tumor growth. However, excess production of TNF is linked to autoimmune diseases and the wasting syndrome, cachexia. Interleukin 1β IL-1β is produced by several cell types e.g. monocytes, macrophages and neutrophils26. The related IL-1α is also produced by these cells but IL-1α has an autocrine effect and is not secreted like IL-1β. Interleukin 1β is also a pro-inflammatory cytokine. It is responsible for fever induction and, together with IL-6, responsible for the acute phase response23. Interleukin 6 IL-6 is another pro-inflammatory cytokine. It is produced by APCs but can also be produced by fibroblasts and epithelial cells27. IL-6 binds to the receptor complex of the IL-6 receptor and gp130. The IL-6 receptor is present on several different leukocytes but also on hepatocytes. As previously mentioned it induces acute phase responses but IL-6 has several other functions and is considered as a pleiotropic cytokine28. It is a growth factor for B cells and important for differentiation and IgA secretion (see below). IL-6 is also important for macrophage differentiation, especially osteoclast differentiation28. It protects CD4 T cells from cell death27 but also inhibits the suppressive effect of T regs29. Furthermore, it can promote Th2 responses in addition to promotion of Th17 differentiation27, 30. Thus, IL-6 has been linked to autoimmune diseases28. Interleukin 10 IL-10 is an anti-inflammatory cytokine produced by a variety of cells including macrophages, DCs, B cells and T regs31. It exerts its anti-inflammatory functions by decreasing cytokine production and expression of co-stimulatory molecules on macrophages and DCs. Furthermore, it is important for IgA production by B cells. However, IL-10 can also activate mast cells, NK cells and CD8+ T cells. IL-10 deficient mice develop colitis and systemically administrated IL-10 might be beneficial as therapy for psoriasis and Crohn’s disease31. Interleukin 12 IL-12 is produced by monocytes, macrophages, DCs and neutrophils32. It consists of the two subunits p35 and p40. The p35 together with p19 also forms the related cytokine IL-23 (see the section about T cells). IL-12 binds to the receptor complex of IL12-R β1 and IL12Rβ2. 17 This leads to IFNγ production by T and NK cells. Thus IL-12 is important for Th1 differentiation. Granulocytes and Mast cells Neutrophils, eosinophils and basophils are all polymorphonuclear leukocytes. Neutrophils are by far the most abundant of the granulocytes and are described in more detail below. Eosinophils, basophils and mast cells are especially important for the protection against parasites1. Mast cells are situated in mucosal and connective tissue whereas eosinophils and basophils move from blood to the site of infection. A cell wall component of parasites, chitin, appears to be able to recruit eosinophils. These cells are also involved in the allergic response and further described in the allergy section. Neutrophils Neutrophils are phagocytic cells with antimicrobial activity1. Neutrophils express opsonin receptors which facilitates the phagocytosis. They constitute approximately 60% of leukocytes in blood33. Upon infection they are one of the first cells to be recruited. Once a pathogen is phagocytosed the phagosome fuses with the granules in the neutrophil. These granules contain different antimicrobial peptides (AMPs), reactive oxygen species (ROS) and enzymes which kill the pathogen. Thereafter the neutrophil often undergoes apoptosis and is cleared by macrophages. Neutrophils can also release their antimicrobial substances when sensing infection34. This destroys surrounding tissue and pus is formed. Apart from being effective killers, neutrophils are also important for wound healing. This is due to their ability to sterilize the wound, to abolish the recruitment of additional neutrohils and to attract macrophages. Furthermore, these macrophages release growth factors for epithelial cells. Natural Killer cells NK cells are important for the protection against intracellular pathogens1 and they also kill transformed malignant cells35. Human blood NK cells can be divided according to their CD56 expression36. CD56dim express lower levels of CD56 and have high cytotoxic activity. In contrast, CD56bright express higher levels of CD56 and produce more cytokines than CD56dim. NK cells have activating and inhibitory receptors which recognize ligands on target cells. The missing self hypothesis suggests that infected cells no longer express MHC class I and thus 18 not the ligand for the inhibitory receptors (e.g. KIR2DL1). If activating receptors (e.g. NKG2D) simultaneously bind its ligands (e.g. MICA) the target cell will be killed by the NK cell (for description of the killing see the part about CD8+ T cells). Lately, the importance of signals from accessory cells for effective NK cell function has been proposed35. These accessory cells are often antigen presenting cells which produces cytokines or signals via cellcell contact. 19 ADAPTIVE IMMUNITY T cells T cells originate in the bone marrow but develop in the thymus, hence their name. They recognize antigens via their CD3-associated receptor37 and they are initially divided according to the structure of the receptor. The T cell receptor (TCR) is either a complex of αβ subunits or γδ subunits. The thymus exports six different populations of T cells; γδ T cells, naïve CD4+ αβ T cells, naïve CD8+ αβ T cells, NKT cells, T regulatory cells and intraepithelial lymphocytes (IELs)38. During the maturation in the thymus, the loci of the αβ receptor undergo V(D)J recombination, leading to the specificity of the TCR38. The expression of the β subunit allows the CD4-CD8- (double negative) T cells to express both the CD4 and the CD8 co-receptors (double positive)39. These co-receptors recognize MHC class II and I, respectively. After positive selection via MHC interactions the αβ CD4+ CD8+ T cells are negatively selected to avoid strong self-reactive T cells. The αβ T cells then become single positive CD4+ or CD8+ T cells. The single positive αβ T cell leaves the thymus when certain surface molecules, e.g. S1p1, are up regulated allowing the cell to proliferate when the receptor interacts with its antigen38. CD8+ T cells The CD8+ T cells are also called cytotoxic T lymphocytes (CTL). They are responsible for killing of virus-infected cells and tumor cells40. When cells are infected, peptide antigens will be expressed on MHC class I, which are expressed on virtually all nucleated cells, and thus presented to CTLs. Just like NK cells, the CTLs contain granules. These granules contain lytic enzymes e.g perforin. Perforin formes pores in the cell membrane of the infected cell. The granula also contains granzymes which trigger apoptosis of the infected cell. CD4+ T cells The CD4+ T cells are also called Th cells. APCs, which express MHC II, present antigens to Th cells. The central role of Th cells is then to coordinate adaptive immune responses by secreting cytokines and chemokines which attract and activate target cells41. Accordingly, 20 when B cells present antigens to Th cells, the Th cells produce cytokines activating the B cells which thereby differentiate into antibody secreting plasma cells. DCs are, in general, the only APC that can direct naïve CD4+ T cells into Th1, Th2, Th17 or T regs cells11. For a functional differentiation, the TCR needs to encounter its antigen, signals from co-stimulatory molecules have to occur (see the part about B cells) and cytokines have to be present. The development of Th1 and Th2 cells appears to be induced mainly by IL-12 and IL-4, respectively41 (see Fig. 1). The transcription factors T-bet and Gata-3 induce cytokine production in Th1 and Th2 cells, respectively. They also appear to be important for the differentiation of the two subsets. Another factor that is important for differentiation of Th1 cells is the IL-12 transducer Stat4. The IL-4 transducer Stat6 and the IL-2 transducer Stat5 are involved in Th2 differentiation. In mice, is Th17 cell differentiation induced by IL-6 and TGFβ. The cytokines important for human Th17 cell differentiation are more controversial. A study in 2007 showed that IL-23 and IL-1β but not TGFβ were important for the differentiation42. However, others postulate that the serum used in this study contained TGFβ and thus some TGFβ is important for differentiation of human Th17 cells but excess amounts of this cytokine inhibit Th17 differentiation43. The transcription factor involved in Th17 differentiation is RORγt and the IL-6, IL-21 and IL-23 signal transducer Stat3 is also of importance. The T helper cells are classified primarily according to their cytokine production (Fig. 1). T helper 1 cells produce high levels of IFNγ and IL-2 after encountering an antigen. IL-2 is important for formation of CD4+ and CD8+ memory cells41. IFNγ has antiviral activity, up regulates the production of IL-12 and also activates macrophages41, 44. Th2 cells produce IL-4, IL-5, IL-9, IL-10, IL-13 and IL-2541. As mentioned above, IL-4 is important for Th2 differentiation. It is also important for IgE class switching in B cells. IL-5 recruits eosinophils and IL-9 activates mast cells and induces mucin production in epithelial cells. Further, IL-13 is important for IgE class switching and immune responses against helminths. IL-25 (IL-17 E) induces several genes in Th2 cells leading to increased levels of Th2 cytokines24, 41. Human Th17 cells secrete IL-17A and F, IL-22, IL-26, IFNγ and CCL2042. IL-17 consists of six subsets, IL-17 A-F. The A and F subset causes epithelial, endothelial and fibroblasts to release IL-6 and CXCL8 (IL-8) and thus induces neutrophil migration. 21 The Th1 cells are particularly important for immune responses towards intracellular pathogens41. For example, their production of IFNγ increases the microbicidal activity by macrophages. The immune responses mediated by Th2 cells combats parasite infections45 whereas Th17 cells appear to be important for immune responses towards extracellular pathogens41. However, Th cells also play major roles in autoimmune diseases (Th1 and Th17 cells) and allergic diseases (Th2 cells). Figure 1. The different subsets of CD4+ T cells. The cytokines and transcription factors important for differentiation are shown as well as the cytokines produced by the diverse subsets. This picture was originally published in Blood. CD4 T cells: fates, functions and faults. Zhu J and Paul WE. Blood 2008;112:1557-69. © the American Society of Hematology. Regulatory T cells Several different subsets of T regs exist. The CD4+ T regs can be divided into natural T regs and induced T regs41, 46. The natural T regs differentiate in the thymus and these cells express the transcription factor Foxp3 and high levels of the IL-2 receptor, CD2547. As with Th2 differentiation, activation of Stat5 is involved in differentiation of T regs41. The natural T regs exert their regulatory effect through cell-cell contact but also produce the regulatory cytokines TGFβ and IL-1041, 46. Induced T regs are Foxp3 positive as well as Foxp3 negative and they differentiate in the periphery. Human and mice CD25-Foxp3- T cells can become Foxp3+ after TGFβ stimuli (when pro-inflammatory cytokines are absent)46. In humans, there is no clear marker that can distinguish T regs from effector T cells let alone natural T regs from induced T regs46, 48 . This is because Foxp3 expression can be up regulated after TGFβ stimuli. 22 However, the Foxp3 loci in natural T regs could be more de-acetylated than the Foxp3 loci in TGFβ-stimulated T cells, postulating a new approach to identify natural T regs48. Th3 cells are one example of induced T regs. They exert their regulatory effect by secreting mainly TGFβ but also IL-10. Th3 cells are important for oral tolerance. T regulatory 1 cells (Tr1) produce IL-10 and it is debated whether they are a lineage of T regs or merely a state that each of the other existing lineages can reside in41. The main function of T regs is to suppress inflammatory responses. Inadequate T regulatory responses are thus involved in the pathogenesis of autoimmune disorders and allergy development41. However, T regs might suppress immune responses which are important for combating human papillomavirus-induced cancer49. Also T cells express TLRs. TLR5 is strongly expressed on isolated human T cells50. Activation via the TCR appears to be important for surface expression of some TLRs e.g. TLR2 and TLR4. However, TCR activation down-regulates TLR5 expression. Interestingly, the ligation of TLR2 on T regs enhances their suppressive activity50. γδ T cells The majority of the γδ T cells that migrate from the thymus reside in epithelial tissue, including the skin and gut51. The antigens that stimulate epithelial γδ T cells appear to be lipid antigens and phosphorylated microbial metabolites rather than the protein antigens that αβ T cells recognize37. Presentation of antigens via MHC I or II is not needed for γδ T cell activation. γδ T cells can be activated after stress or transformation of surrounding cells51. Activated γδ T cells play an important role in tissue repair and can release the growth factors, insulin growth factor-1 and keratinocyte growth factor. They can also lyse infected cells like macrophages and epithelial cells. In addition, γδ T cells can secrete various cytokines and chemokines including IFNγ, TNF and IL-437. B cells B cells are necessary for the humoral response since they produce antibodies. They develop in the bone marrow in mammals but actually got their name from the Bursa of Fabricus, where they develop in birds52. The B cells fully mature in secondary lymphoid organs e.g. lymph 23 nodes or the spleen. In these organs, the germinal center is the follicular environment where humoral immune responses are initiated53. A B cell that recognizes an antigen internalizes and presents it, via MCH II, to a CD4+ T cell52. Further CD40 on the B cell interacts with CD40 ligand (CD154) on the T cell. Then other co stimulatory molecules are expressed i.e. B7-1 or B7-2 (CD80 or CD86) on the B cell which binds CD28 or CTLA-4 on the T cell. If CD28 is bound there is a positive signal activating the T cell that then produces cytokines52. These cytokines further support B cell proliferation and its differentiation into plasma or memory cells, in addition to class switching (see below). Plasma cells secrete antibodies and reside in organs which are exposed to antigens, e.g. the gut. Memory B cells either remain in their follicle or circulate. They respond rapidly when encountering its antigen once more. B cells also express TLRs, however the expression is somewhat altered during differentiation54. Human memory B cells express TLR1, 5, 6, 7, 9, 10 with a low expression of TLR2. The TLR ligation can induce polyclonal memory B cell proliferation and thus could TLR ligation be important for preserving the memory B cell pool. The ligation of TLR 7 and 9 in human B cells contribute to induction of antibody production. One fundamental difference between murine and human B cells is the lack of TLR4 on human B cells. Antibodies Antibodies recognize antigens, e.g. on pathogens, and can thus facilitate the clearance of pathogens52. They function as receptors on B cells (when bound to Fc-receptors) and secreted antibodies can also bind to Fc-receptors on other cells e.g. monocytes, macrophages, neutrophils and mast cells. When antigen-antibody complexes bind to receptors on macrophages and neutrophils the complexes are phagocytosed resulting in clearance of the pathogen. IgG and IgM also activate complement which primarily results in lyses of cells and pathogens as well as phagocytosis of immune complexes. Secreted antibodies also prevent pathogens from entering the body (see the part about IgA). An antibody consists of two heavy chains and two light chains (κ or λ). These chains have constant and variable parts where the variable part determines the specificity of the binding to a certain antigen. This specificity is achieved through VDJ recombination which occurs during maturation in the bone marrow52. The heavy chain determines the class of the antibody i.e. IgM, IgD, IgA, IgG or IgE. Mature B cells express membrane bound IgM and IgD. IgM is the first antibody produced when the mature B cell encounters an antigen. Class switching is needed for production of IgG, IgA or IgE55. It occurs at gene level where the gene for the constant part of the heavy chain, usually IgM, is replaced by the gene encoding the constant part of IgG isotypes, IgA isotypes or IgE. 24 The DNA in between is deleted. Immunoglobulin G consists of four isotypes, IgG1, IgG2, IgG3 and IgG4, in humans52. IgA and IgE are abundant in mucosal tissue55 and are further discussed in the gut immunology and allergy section, respectively. 25 THE GUT ASSOCIATED IMMUNE SYSTEM The gastrointestinal tract consists of the oesophagus, the stomach, the small intestine and the large intestine (colon)56. The small intestine can further be divided into duodenum, jejunum and ileum. The different compartments have different physiological functions important for food digestion (the stomach and duodenum), absorption of nutrients (small intestine) and water and electrolytes (small intestine and colon). Mucosal epithelia are primary sites for antigen entry57. Therefore mucosal associated lymphoid tissue (MALT) is of vast importance for mounting immune responses towards foreign antigens. There are more lymphocytes in the gut associated lymphoid tissue (GALT) than in all other secondary lymphoid tissues combined58. The GALT is an immune privileged organ with the difficult task to mount immune responses towards pathogens while at the same time being non-responsive to commensal bacteria and food antigens. The primary barrier of the gut mucosa is the single layer of epithelium. It consists of 5 types of cells, enterocytes, goblet cells, enteroendocrine cells, M cells (see below) and Paneth cells17. The enterocytes absorb nutrients in the small intestine. Paneth cells are only present in the small intestine and produce antimicrobial peptides (AMPs). The goblet cells produce mucin, containing highly glycosylated proteins, which protects the epithelium59. Beneath the epithelial cells lies the connective tissue, lamina propia (LP)60. The GALT has its main site in the LP. The GALT consists of Peyer´s patches (PP) in the small intestine and of isolated lymphoid follicles (ILF) in the colon (Fig. 2)58. The most concentrated number of PP is in the terminal ileum11. Both PP and ILF are aggregates of B-cell follicles with intervening T cells. PPs and ILF are the inductive sites where activation occurs whereas the diffuse tissues of the lamina propia are effector sites where high amounts of IgA are produced61. There are mainly two types of cells in the gut mucosa which are able to transfer luminal antigens to cells in PP and ILF; DCs and Microfold (M) cells. The M cells are integrated in the epithelial layer and transport live microbes and microbial material from the lumen. DCs, present in the LP, are able to open tight junctions in the epithelium making it possible for the DC to send their dendrites into the gut lumen and sample antigens (Fig. 2)60. The DCs can then present antigens to lymphocytes in PP and MLNs, but appear to never stray beyond this lymphoid tissue and thus systemic infection is prevented60. 26 Figure 2. The structure of the gut associated lymphoid tissue (GALT). Adapted from60. © Taina Litwak 2004. Reprinted by permission from Taina Litwak. Antimicrobial peptides AMPs are a part of the innate branch and produced by virtually all multi-cellular organisms62. In mammals, they are produced by leukocytes (especially neutrophils) and epithelium63. One type of AMPs is enzymes that kill bacteria by disrupting their membrane, e.g. lysozyme59. Another group is C-type lectins, e.g. RegIIIγ. Both these groups are especially efficient in killing G+ bacteria. Yet, defensins is the most common group of AMPs in mammals63. Alfa and β-defensins are the most abundant defensins in humans. The defensins are cationic peptides which disrupt the membranes of pathogens. They are active against both G+ and Gbacteria but also against fungi, protozoa and viruses. Alfa-defensins dominate in the small intestine. They are produced by paneth cells in the crypts of small intestinal villi. Enterocytes 27 in the colon produce mainly β-defensins. Upon infection or inflammation, β-defensin production is induced63. The fourth type of AMPs, cathelicidins (e.g. LL-37), is produced in the colon and also kills bacteria by membrane disruption59. Due to their structure, different AMPs have activity against different pathogens. It has been postulated that the diverse AMPs produced by an individual can shape the colonizing intestinal microbiota63. Apart from killing pathogens, regulating the number of bacteria in the gut and preventing bacteria from entering, AMPs might also protect the stem cells in the gut63. In addition, LL-37 is chemotactic and might also induce Th1 cytokine secretion by dendritic cells59. Defensin β1 and Clara cell 16-kD protein also act on DCs and inhibits Th2 differentiation64. Several AMPs are produced constitutively also in germ-free animals59. However, the ligation of PRRs is important for the production of other AMPs. In example, production of RegIIIγ is initiated by MyD88 signalling and NOD2 signalling is important for α-defensin production59. NOD2 mutations have been observed in patients with Crohn’s disease. Thus, it is postulated that these mutations lead to lower α-defensin production facilitating the pathogeneses of Crohn’s disease59. Immunoglobulin A Secretory IgA (SIgA) is of immense importance for mucosal immunity as it prevents the invasion of pathogens e.g. Salmonella typimurium65, influenza virus66 and respiratory syncytial virus67. It binds to adhesins and other receptors of pathogens thus preventing them from penetrating the mucosal barrier68. In addition, SIgA can also bind to flagella of bacteria and neutralize toxins. Furthermore, SIgA facilitates antigen sampling by binding to M cells69. This leads to controlled antigen sampling which is suggested to induce further IgA production without causing inflammation69. Since IgA does not activate the classical complement pathway it is considered as an anti-inflammatory antibody70. Antigens that reach the lamina propia can be bound by IgA and either excreted to the lumen or cleared by binding to FcαRI on e.g. DCs or neutrophils71. Humans produce two IgA subclasses; IgA1 and IgA270. Immunoglobulin A1 predominates in the circulation while both IgA1 and IgA2 are abundant at mucosal surfaces. In the gastrointestinal tract, IgA1 is more frequently produced in the upper digestive tract while IgA2 is produced at higher levels in the colon11. The hinge region of IgA is longer in IgA1, 28 than IgA2, which makes IgA1 more susceptible to bacterial proteases70. Immunoglobulin A mostly occurs as oligomers at mucosal surfaces in humans. The J chain joins the monomers together and is also important for the transport of IgA across the surface and into the lumen. The polymeric immunoglobulin receptor (pIgR) at epithelial surfaces interacts with the J chain and shuttles IgA through the epithelial cell. Eventually pIgR is cleaved leaving IgA with a secretory component. The seceretory component protects SIgA from proteolytic degradation72. Large amounts of SIgA, 40-60 mg/kg, are produced in the gut lumen every day61. Class switching to IgA is either T cell dependent or T cell independent70. The secretion of IL4 and TGFβ1 by antigen-primed CD4+Th2 cells make the B cells undergo µ α switching in PP73. IgA+ B cells differentiate further in the mesenteric lymph nodes before they enter the blood stream61. As GALT DCs produce retinoic acid (RA), gut homing receptors (α4β7 and CCR9) on lymphocytes are up regulated, which facilitates B cell-trafficking to the lamina propia73. There, the IgA primed B cells finally become IgA secreting plasma cells by the help of IL-5 and IL-6 produced by CD4+Th2 cells73. In addition the secretion of B cell activating factor (BAFF) and a proliferation-inducing factor (APRIL) by DCs is important for fully functional IgA secreting B cells70. Lately a T cell independent pathway for the activation of IgA producing B cells has been postulated. Work by Mora and colleagues show that the production of IL-5, IL-6 and RA by DCs in the PP provide a milieu for the B cells to start producing IgA as well as up regulating gut homing receptors74. Also, others postulate that the release of APRIL from epithelial cells can cause IgD+ B cells to directly class switch to IgA75 The release of TGFβ1, APRIL and BAFF by DC is probably also needed for this to function properly70. Oral tolerance The intestinal immune system encounters more antigens than any other part of the body76. Antigens from pathogens should mount a strong immune response but on the other hand food and commensal antigens should be tolerated. When not tolerated, disorders like inflammatory bowel disease, coeliac disease and food allergy could occur. Mice undergoing mesenteric lymphadenectomy cannot induce oral tolerance which postulates a central role for MLNs in oral tolerance induction58. The predominant site for naive T cell activation takes place in the MLNs77. It is not known whether tolerance to commensals and food antigens is acquired in 29 the same way78. The commensal microbiota can, in contrast to food antigens, signal through TLRs which might be important for tolerance induction. Food antigens can also be found in systemic peripheral lymph organs; however when the MLNs are intact commensals never stray beyond the MLNs. Several factors influence the outcome of an immune response towards orally administered antigens77. The maturation of DCs is influenced by the surrounding cytokine mileu. In addition, the differentiation of T cells, after antigen presentation, is influenced by the maturity status of the DC. This and how T cell subsets react to antigens are described below. The amount of antigen available (see also factors influencing allergy development) and how it is processed also plays a substantial role for tolerance induction77. The acidic environment and the proteolytic enzymes in the gut facilitates antigen breakdown. An intact barrier decreases excess immune responses to the antigen. Inflammation and low secretion of SIgA are two factors that reduce the integrity of the barrier. Intestinal T cells and oral tolerance As mentioned above, lymphocytes that are primed in the PP migrate to the MLN for further differentiation, then enter the bloodstream and accumulate in the mucosa. As with B lymphocytes, T lymphocytes up regulate the adhesion molecules CCR9 and α4β7 which facilitate the migration to the gut mucosa. Lymphocytes located between intestinal epithelial cells are called intraepithelial lymphocytes (IELs)79. Their primary role is to eliminate infected and damaged epithelial cells, thus sustaining the integrity of the epithelial barrier. The IEL population holds NK cells, γδ T cells and CD4+ and CD8+ αβ T cells. The different compartments of the gut harbour different IELs. In humans, the small intestine IELs are primarily CD8+ αβ T cells while the colon mainly harbour CD4+ αβ IELs79. The LP is inhabited by both CD4+ and CD8+ T cells76. Of importance for oral tolerance are clonal deletion (apoptosis) and clonal anergy of antigenspecific CD4+ T cells58. This appears to happen after high-dose antigen feeding. An anergic cell is not activated when its receptor binds the antigen due to lack of costimulation. Immature DCs express low levels of CD80 and CD8677 and are thus unable to induce signals via CD28 on T cells. CD80 and CD86 can also bind to CTLA-4 on T cells leading to an inhibitory signal. The molecule CTLA-4 appears to be important for oral tolerance77. Administration of multiple doses of low concentrations of antigen probably induces active suppression by T 30 regs. Several different subsets of T regs exist in the gut76. When IL-10 and TGFβ were depleted from LP T cells, they lost their unresponsiveness towards commensal bacteria indicating that T regs are involved in the tolerance towards commensal antigens. The Th3 cells that produce TGFβ have been isolated from mice MLNs after feeding small doses of antigen for tolerance induction76. Tr1 cells, which produce IL-10, are induced in PPs and MLN after feeding77. In addition, mice deficient in IL-10 develop inflammatory bowel disease, a finding which postulate a role for these cells in controlling immune responses. Furthermore, CD4+CD25+ T cells could also be important for tolerance induction as children who outgrow their cow´s milk allergy have allergen-specific CD4+CD25+ T cells in their blood80. In mice, it has been shown that different compartments of the body harbour different T regs81. The T regs in spleen and lymph nodes are mainly Foxp3+IL-10- and arise from thymic precursor cells whereas the most abundant T regs in the small intestine and PPs are Foxp3-IL-10+ and resemble Tr1 cells81. Also, the large intestine T regs differ from the small intestinal T regs, as they are merely Foxp3+IL-10+. Intestinal dendritic cells and oral tolerance The functional properties of DCs differ due to their different locations10. In presence of TGFβ, DCs from LP and MLN are better at inducing Foxp3 in naive T cells, than are splenic DCs. Activated DC subsets from the PP produce higher levels of IL-10 than activated DCs from the spleen. These PP DCs induce naive T cells to produce higher IL-10 and Th2 cytokines than T cells activated by splenic DCs. This appears to be especially attributed to the CD11chiCD11b+CD8α-10. Other subsets in PPs are CD11chiCD11b-CD8α+ and CD11chiCD11b-CD8α-. These three subtypes of DCs also appear to be present in LP of the small intestine. Few DCs are present in the LP in the colon. Instead they are mostly located in the ILFs10. The MLNs contain DCs that have migrated from the LP but also DCs that are derived from blood-borne precursors. Also, plasmacytoid DC have been identified in the PPs and MLNs10. Plasmacytoid DCs have been described to be tolerogenic in mice76. Why do mucosal DCs differ from systemic DCs and preferably secrete anti-inflammatory cytokines and induce the differentiation of Tregs, Th2 and Th3 cells during homeostasis? Lately, the intestinal epithelial cells have been shown to play a fundamental role in conditioning DCs. The thymic stromal-derived lymphoprotein (TLSP) released by epithelial cells is able to condition DCs into a non-inflammatory state (Fig. 3). Further these DCs induce naive T cells to differentiate into Th2 cells. In addition, TSLP can convert Foxp331 thymocytes into Foxp3+ thymocytes82. Other factors that are important for conditioning of DCs, is the presence of IL-10 and TGFβ, either produced by other DCs10 or T cells11. These GALT DCs also are important for IgA production as previously mentioned. Figure 3. Proposed mechanism for conditioning of dendritic cells (DCs). Epithelial cells produce retinoic acid (RA) and in response to commensal bacteria; thymic stromal-derived lymphoprotein (TSLP). These substances together with interleukin (IL) 10 and transforming growth factor (TGF) β, influence DCs to mature into conditioning DCs which produce little IL-12. Instead they produce RA, TGFβ and a proliferation inducing factor (APRIL) which are important for IgA production and regulatory responses in the gut. Reprinted by permission from Macmillan Publishers LtD: [Nature Reviews Immunology] 2008;8:435-446. When pathogens invade the mucosal tissue an inflammatory response needs to be initiated. This is postulated to occur when pathogens invade mucosal epithelial cells11. Compared to commensal bacteria, the pathogens produce virulence factors, like toxins and adherence 32 factors, which make it possible to invade the epithelial cell. Once invaded the epithelial cells produce inflammatory cytokines and chemokines, e.g. CXCL8, which attracts and activates other cells. Neutrophils, macrophages and monocytes are examples of cells that are activated by the inflammatory cytokines/chemokines and the pathogens. Possibly DCs from the MLNs induce Th1 responses when meeting the pathogens10. ALLERGY Today, atopic allergy is the most common chronic disease among children in the developed world. The current prevalence of the allergic symptoms asthma, eczema and allergic rhinoconjunctivitis has been investigated in the ISAAC studies. Thus, among 6-7 year-old Swedish children, 10% have asthma, 13% have eczema and 10% have symptoms of allergic rhinoconjunctivitis83. Exposure to common environmental antigens such as plant pollen, house dust and animal and food proteins can cause an allergic response. However, it is not known why some individuals develop allergic disorders. Atopic allergies are IgE-mediated and also called hypersensitivity type 1 or immediate hypersensitivity reactions. The above prevalence data only demonstrates symptoms, however it is estimated that approximately 25% of the population in the developed world have IgE-mediated allergic disease3. The term atopy means that a person has a hereditary or personal predisposition to produce IgE antibodies towards allergens and to develop allergic symptoms84. The risk of developing allergic disease is much higher if both parents are allergic compared to if they are non-allergic85. Since the prevalence of atopic allergy has increased during the last decades, genetics alone cannot explain the development of the disorder86. The current view about atopic allergy is that both genetic and environmental factors seem to interact with each other, leading to the production of IL-477. IL-4 plays an important role in developing naive T cells into CD4+ Th2 cells (Fig. 4a). The cytokines produced by Th2 cells induce IgE class switching3. Further allergen encounter makes the B cells secrete IgE. Mast cells, basophils and eosinophils have receptors for IgE55. The immediate allergic response occurs when the allergen crosslinks IgE bound to Fcε receptor 1 on the mast cell3 (Fig. 4b). This leads to mast cell degranulation followed by the release of histamine, heparin and proteases within minutes, all leading to the clinical symptoms. The clinical symptoms arise from the contraction of intestinal and bronchial smooth muscles, increased mucus production, vasodilatation and increased vascular permeability. The eosinophils and neutrophils are recruited to the inflammatory site by 33 cytokines and chemokines released by e.g. the mast cells55. The allergen-specific T cells as well as the eosinophils and neutrophils are responsible for the late allergic response (Fig. 4c). This response occurs hours to days after contact with the allergen and the mediator´s, leukotrienes and prostaglandines, effects are more pronounced and long-lasting than those of histamine. Figure 4. The allergic reaction. The naïve Th cell is presented to an allergen (a). In an IL-4 rich milieu the Th cell develops into a Th2 cell. The B cell encounters the allergen and presents it to the Th2 cell which then produces IL-4, IL-5 and IL-13. IgE class switching is then induced. The IgE antibodies attach to Fcε receptors on mast cells and eosinophils and the individual has become sensitized. Once the individual encounters an allergen again this leads to crosslinking of the Fcε receptors and release of inflammatory mediators causing the allergic response (b and c). The substances released from mast cells cause the immediate reaction (b) while the substances released from eosinophils cause the late reaction (c). Reprinted by permission from Macmillan Publishers LtD: [Nature Reviews Immunology] 2002;2:446-453 Factors influencing allergy development Parental allergy is a strong risk factor for allergy development87. Both complete chromosome regions and gene polymorphisms have been linked to allergic disease64. The linkage region in 34 chromosome 5q31-33 includes 14 genes associated with asthma and atopy e.g the genes for IL-4, IL-13 and CD14. Polymorphisms in genes involved in microbial recognition and innate immune responses as well as Th2 differentiation are also linked to allergic disease, as are polymorphisms in genes for epithelial-produced chemokines, i.e. the eosinophilic attractants CCL11, CCL24 and CCL26. Interestingly, phenotypes might respond differently depending on their environmental exposure postulating that gene-environment interactions are of importance. It has been shown that the CC allele, of CD14 C-260, protects against high specific IgE when children are exposed to high levels of endotoxin or stable animals88. Children bearing the TT allele rather have high total IgE levels when exposed to high endotoxin levels and stable animals but low IgE levels when living with a dog or a cat. The dose, timing and route of allergen exposure seem to influence allergy development. The foetus can be exposed to allergens already in utero89. Allergens cross the placenta90 and consumption of food allergens correlate with the levels in cord blood91. However, the IgE levels in cord blood do not correlate with the IgE levels at six month92 indicating that allergen exposure in utero does not lead to any persistent IgE-sensitization. The exposure to high birch pollen in utero is not significantly associated with sensitisation later in life93. However, the exposure during the first months of life significantly increases the risk for allergic asthma and positive skin prick test (SPT)94, postulating that the exposure in infancy might be of importance. Yet, exposure to pets during the first year of life has, in contrast, been shown to be associated with lower frequency of allergic rhinitis and asthma later in life95. Previously, the American Academy of Pediatrics recommended pregnant and lactating women to avoid peanuts. In addition, they also recommended a delayed introduction of dairy products, egg, fish and nuts to the infants. However, these recommendations were not enough evidence based and today no organisation recommends food avoidance due to its allergenicity96. Others have instead suggested the opposite i.e. that earlier introduction of complementary foods (with continued breastfeeding) increases tolerance induction97. This is due to the postulated time window of opportunity for tolerance induction97. Theoretically, the fetus can be exposed to allergen-specific IgE antibodies from its mother, but it is controversial whether IgE crosses the placenta98. IgE antibodies are readily detected on fetal cells in the placenta, regardless of maternal allergy99 and it is likely that this IgE actually originates from the mother100. 35 Figure 5. Allergic heredity is associated with allergic disease and also the timing and dose of allergen exposure appear to influence allergy development. Environmental factors increase or decrease the risk of developing allergic disease. Importantly, the interaction between heredity, allergen and environmental exposure contribute to the development of allergic disease. Epidemiology of the hygiene hypothesis In 1989 David Strachan described that children with older siblings developed allergy to a lower extent101. It was postulated that the lower incidence of atopic disease among these children was due to higher microbial exposure of these children, the so called hygiene hypothesis. Since then, several epidemiologic studies have confirmed the inverse relationship between number of siblings and allergy development102. Also, certain infections are associated with less allergic disease. Matricardi et al showed that seropositivity to several food and orofecal pathogens was associated with 60% lower incidence of allergic disease than seronegativity to these pathogens103. Another recent study showed that seropositivity, against different infectious pathogens, decreased the incidence of different atopic disorders104. Supporting this, seropositivity towards the herpesvirus Epstain Barr Virus (EBV) was negatively associated with IgE sensitization and this was further strengthened if the subjects also were seropositive for cytomegalovirus85. The same study could not show any association between seropositivity against respiratory viruses and IgE sensitization, however85. Actually, severe infections with respiratory syncytial virus might instead be associated with increased risk of asthma development105. So far it cannot be concluded that childhood infections protect from atopic disease106. 36 There is a lower prevalence of atopic allergy among children growing up on farm107. Children growing up in this environment are exposed to high levels of microbial products, i.e. endotoxin and bacterial DNA108. Furthermore, children exposed to higher levels of endotoxin are less frequently allergic109. In addition, prenatal exposure to farming environment might be particularly protective110. Consequently, it might be the microbial products and not the infections per se that prevent allergic disease. Neonatal and infant immune responses The immune system of the newborn is immature111. The structure of the lymph nodes and spleen is underdeveloped. In addition, the number of B and T cells is low. Infant and neonatal B cells rather differentiate into memory B cells than plasma B cells, due to low expression of CD21 on naïve B cells112. Class switching is also incomplete113 and neonates respond poorly to thymus independent antigens like polysaccharides on encapsulated bacteria112. Actually, IgG antibody responses to polysaccharides are limited during the first 18-24 months whereas antibody responses to protein antigens are limited during the first twelve months112. Fortunately, maternal IgG is actively transferred across the placenta during pregnancy to compensate for the impaired production of Ig isotypes at birth114. However, these antibodies are catabolised during the first two to six months after birth112, 113. As shown in formula-fed infants, the infants own production of intestinal SIgA is rare during the first ten days after birth115. Actually, the isolated lymphoid follicles in the colon do not develop during fetal life116. Instead they develop when the gut is colonized. It takes up to five years before the levels of salivary SIgA reach adult levels (Tomicic et al unpublished). Also, the transport of IgA appears to be immature in infants. That is, the levels of salivary SIgA are low in infancy whereas the levels of total IgA are higher117. This immaturity seems to be more pronounced in Swedish compared to Estonian infants (Tomicic et al unpublished). With a rather immature adaptive immune system the innate branch is of increased importance. However, also the innate part is somewhat immature in neonates. There are fewer DCs in newborns and they express lower levels of co-stimulatory molecules111. Possibly, the production of Th1 associated cytokines is suppressed23. Actually, cord blood mononuclear cells (CBMCs) produce less IFNγ, IL-12 and probably also less IL-1β after TLR ligation than adult PBMC23, 118. The neonatal production of IL-6, IL-10 and IL-23 is possibly higher, after 37 TLR ligation in monocytes and APCs, compared to adult levels23, 118. This does not appear to be attributed to the level of TLR expression, however, as neonatal and adult PBMC have similar basal expression of TLRs19, 118 . Neither does the type of delivery influence the cytokine production by CBMC after TLR ligation118. The IL-6/TNF ratio produced by CMBC is higher, after TLR ligation, than from PBMC119. This appears to be especially attributed to factors in the serum of cord blood i.e. adenosine. It is not known for how long the circulating levels of adenosine persist, but blood cells from 2 month- and 12 month-old infants produce much lower levels of IL-6, IL-10 and TNF, after TLR ligation, compared to the cord blood levels118. The high production of IL-6 at birth could be important for increasing acute phase proteins23. This, together with the role of IL-6 in preventing neutrophil recruitment could prevent too much inflammation when the neonate is exposed to colonizing microbes. At birth, infants are coated with a waxy coating, the vernix caseosa23. As this waxy layer contains several AMPs it is thought to be the first protection against colonizing microbes. A majority of infants are colonized with coagulase-negative Staphyloccocus (CoNS) one day after birth120. Not surprisingly, approximately half of all infants respond immunologically to the colonization of their skin23. The benign rashes they develop are probably caused by the CoNS, which penetrate the hair follicles, and the macrophages, eosinphils, neutrophils and mast cells that then are recruited120. Macrophages produce IL-6 which further leads to the production of acute phase proteins23. In all, this response is believed to be beneficial for the maturation of the neonatal immune system. Neonatal and infant immune responses in relation to subsequent allergy development It is believed that a somewhat Th2 skewed immune system predisposes allergy development121. Th1 cells secrete IFNγ which is able to counter-regulate Th2 responses. A lower amount of Th1 cytokines therefore creates a milieu for allergic sensitisation. As the neonatal immune system appears to be somewhat Th2 skewed (see above) the exposure to microbial products early in life is central since it leads to activation of TLRs on e.g. innate cells and subsequently to the development of Th1 cells1. The receptor Notch is expressed on naïve T cells. When DCs encounter TLR ligands, they express Delta which binds to Notch leading to Th1 differentiation122. It is assumed that more immature dendritic cells instead express Jagged, leading to Th2 differentiation. It has been shown that substances mimicking microbial products switch the allergen-specific response from Th2 to Th1 by increasing the 38 production of Th1 cytokines123. Also, mice with a deficient TLR4 receptor responded with high IgE responses and anaphylactic shock when a food allergen coupled to an adjuvant was administered 124. However, if the mice simultaneously were given CpG oligodeoxynucleotides (mimicking bacterial DNA) no anaphylactic reactions were seen. T regulatory cells dampen both Th1 and Th2 responses121. CD4+CD25+ T cells suppress proliferation of CD4+ and CD8+ T cells probably via cell-cell contact77. Tr1 cells also suppress T cell proliferation and appear to switch antibody production away from IgE towards IgG4121. In addition, TGFβ inhibits proliferation and differentiation of naive T cells77. Insufficient microbial stimulation can also lead to poorer development of T regs. Ligation of TLR2 on T regs enhances their suppressive activity50. There might be a time window of opportunity when the exposure to microbes is essential for a proper down regulation of Th2 responses125. Results from animal experiments support this theory. In a murine model, Bifidobacterium (B.) infantis could restore oral tolerance in neonatal ex-germ-free mice but not in adult mice126. Also, sensitisation at 2 years has been shown to correlate with high amounts of Th2 cytokines produced by PBMC from 6 month-old infants after allergen stimulation127. Although the above mentioned study could not show it, there might also be an imbalance in cytokine and chemokine production in children developing allergic disease already prenatally. One study showed a significantly lower proportion of IL-12 producing CBMCs following allergen stimulation in children who became IgE-sensitized at two years of age when compared to CMBC from non-sensitized children128. Also, the ratio of CCL22 (a Th2 associated chemokine) to the Th1 associated cytokines CXCL10 and CXCL11, is higher in cord blood of children who develop allergy at two years of age129. Furthermore, the phytohemaglutamin (PHA)-induced expression of the transcription factor GATA3 is higher in CBMC from infants who develop IgE-associated allergic disease compared to CBMC from infants who remain non-allergic130. Studies from our group also indicate an early immaturity of anti-microbial immune responses by monocytes in children with allergic mothers, an impairment which seems to persist during the first 2 years of life19, 131 . Yet, it is rather the allergic status of the child, that influences antimicrobial immune responses in monocytes at five years of age132. Others have shown that exposure to house dust endotoxin and pets are associated with higher IFNγ production by CMBC and infant PBMC after phorbol 12-myristate 13-acetate and Concavilin A stimuli133. Probably are both the in utero environment and early repeated microbial stimuli important for priming of immune responses. 39 Sensitized infants who develop allergic symptoms have lower levels of salivary SIgA compared to sensitized infants who do not develop allergic symptoms (117, Tomicic et al unpublished). Also, infants with IgA deficiency might be at increased risk for developing allergic disease134. Furthermore, the allergen-specific IgA levels in feces are lower in a mouse model with food allergy compared to tolerant mice135. 40 THE COMMENSAL GUT MICROBIOTA It is estimated that the gut harbours 500-1000 different species of bacteria136. These species belong to nine different phyla of bacteria with Firmicutes and Bacteroides being the most predominant137. The total amount of gut bacteria is estimated to weigh approximately one kg and outnumbers the number of cells in the human body by a factor of ten138, 139. The bacteria are differently distributed along the gastrointestinal tract (Fig. 6), with few species in the acidic stomach. The bacterial density increases in the small intestine and the colon harbours the highest number of bacteria, 1012 colony forming units (CFU)/ml137. The gut harbours both anaerobic and facultative anaerobic genera of bacteria but the anaerobes constitute the largest proportion, approximately 99% in adults140. Bifidobacterium, Clostridium (C.), Bacteroides, Lactobacilli (L.) and Eubacterium are common anaerobic genera of gut bacteria141. Streptococcus, Enterococcus, Staphylococcus (S.) and Escherichia (E.), together with other Enterobacteriaceae, are facultative anaerobic genera of gut bacteria. A polysaccharide rich mucus gel layer is overlying the gut epithelium. This mucus layer provides carbohydrate motifs that enable microbes to attach to the mucus and form biofilms136. The biofilm promotes several of the functions exerted by the microbiota136. It also makes the microbes resist the forces of gut peristalsis58. The gut microbiota has several functions. It ferments non-digestible carbohydrates (see below) and synthesizes vitamins, e.g. vitamin K and biotin. Apart from being important for immune system development (see below), the commensal microbiota also protects the host from pathogens in additional ways. Lactic acid producing bacteria, e.g. lactobacilli and bifidobacteria, lower the pH in the gut and create a milieu less friendly for pathogens. The commensal microbiota also competes with pathogens for nutrients and receptors. Factors influencing the composition of the gut microbiota The composition of the intestinal microbiota is rather stable in healthy individuals144. Thus, adults are colonization resistant meaning that it is difficult for new strains to establish. This is probably due to the vast amount of obligate anaerobes in the adult intestine140. However, the gut microbiota can be altered by several factors like drugs, diet, stress, disease and aging. Compared to the adult flora, the infant gut microbiota is less diverse. Mitsouka has studied the 41 gut microbiota in relation to age and shown that bifidobacteria, E. coli and Streptococcus are most numerous in infancy (see below for further discussion on gut microbiota in infancy)144. Figure 6. Distribution of bacterial genera in different locations of the gastrointestinal tract. CFU = colony forming units. Reprinted by permission from Macmillan Publishers LtD: [EMBO reports] 2006;7:688-693. Modulated by references 142, 143. In elderly people, Bifidobacterium numbers are decreased, however other bacteria like lactobacilli, clostridia, E. coli and Streptococcus increase in numbers. The consumption of various antibiotics alters the gut microbiota145, 146. C. difficile is regarded as a pathogen but can persist in the lumen without causing disease. However, when the existing community is changed e.g. by antibiotics it can give rise to pseudomembranousus colitis137. The host genotype also appears to influence the diversity of the gut microbiota137. Differing genotypes could give rise to different attachment sites for microbes. Also, the MHC genotype has been reported to influence the murine fecal microbiota. The possibility that there is a host selection for specific bacteria indicates cooperation between the host and the bacteria. Indeed, as mentioned above, the gut microbiota exerts several functions of benefit for its host. Thus, the term commensal, which means that one part benefits while the other part is unaffected, is therefore misleading and one would rather refer to the relationship as mutualistic147. 42 Infant gut microbiota The foetus is sterile in utero but the colonisation starts immediately after birth148. The first colonizers are facultative anaerobes, e.g. E. coli and staphylococci, which consume oxygen140. Anaerobes colonize the gut a few days after birth. These are e.g. Bifidobacterium, Bacteroides, Clostridium and Lactobacillus species. It has been shown that type of delivery influences the gut microbiota. Vaginally delivered infants are exposed to the faecal, peritoneal and vaginal flora of the mother. Neonates born with caesarean section are initially mainly exposed to bacteria in the hospital environment and of nursing staff. Indeed, neonates born with caesarean section often harbour less bifidobacteria and bacteroides145 and are colonised later with E. coli, lactobacilli, Bacteroides and Bifidobacterium species149, 150. It can take up to over one month before infants born with caesarean section have similar levels of these bacteria compared to those of vaginally delivered infants150. A long-lasting vaginal delivery appears to increase the chance of finding live bacteria in the mouth and stomach of newborn infants148. Infants raised in developed compared to developing countries appear to have a different microbiota. A higher prevalence of Ethiopian and Estonian infants, compared to Swedish infants, harbour lactobacilli151, 152. Adlerbeth et al showed that Pakistani infants were colonised earlier than Swedish infants and also harboured a more diverse enterobacterial microbiota153. The hygienic procedures in developed countries might be responsible for these differences which also might have unfavourable consequences (see below). Bifidobacteria constitute a large proportion of the gut microbiota in infants154. Once fully weaned, infants harbour a lower proportion of bifidobacteria. Studies investigating differences in gut microbiota between breastfed and formula fed infants indicate that breastfed infants harbour higher numbers of bifidobacteria155. However, there are less clear differences between studies today compared to studies performed decades ago, suggesting that the composition of formula has improved140. Clostridia have been found in higher numbers in formula-fed infants. Streptococcus, Bacteroides and Enterobacteriaceae might also be higher in formula-fed infants140, 148. In addition, microbiota associated characteristics appear to differ between breast-fed and formula-fed infants156. 43 Fermentation, prebiotics and probiotics Several attempts have been made to change the composition of the gut microbiota. As mentioned above the diet could influence the gut microbiota. Non-digested carbohydrates like dietary fibres, resistant starch and oligosaccharides reach the colon and are fermented by the gut microbiota157. The end products produced following this fermentation are mainly short chained fatty acids (SCFA) and gases (e.g. methane and CO2)158. The different degradation enzymes and adhesion factors in various bacterial species determine their ability to ferment different substrates. Lactobacilli, which occur in higher numbers in the small intestine, preferentially ferment mono-, di- and tri-saccharides159. Bacteroides and bifidobacteria, which are more abundant in the colon, are efficient at degrading complex carbohydrates. Genes involved in poly/oligosaccharide metabolism have been shown to account for 8% of the genome of B. longum while an even larger part of the genome of Bacteroides thetaiotaomicron contains genes responsible for polysaccharide breakdown160. Bifidobacteria and lactobacilli are producers of lactate and therefore often called lactic acid producing bacteria144. Bifidobacteria also produce acetate. About 90% of the SCFAs in faeces of infants below three months are acetic acid156. Prebiotics are defined as “non-digestible food ingredients that beneficially affect the host by selectively stimulating one or a limited number of bacterial species already resident in the human colon”161. Prebiotics such as inulin and fructo-oligosaccharides have been shown to function as substrates for bifidobacteria and lactobacilli and thereby increasing their growth, but investigations on how the whole gut microbiota is affected by prebiotics are lacking. In addition, much is still unknown about the substrate preferences by many bacterial species162. Thus, prebiotics are probably not only degraded by the target bacteria but also by other species. Furthermore, some species are unable to degrade complex carbohydrates and can instead ferment the products produced by other species. This is termed cross-feeding. Also, factors in the gut environment, e.g. pH, influence the metabolism of different bacteria. Nevertheless, prebiotics have been shown to improve gut health by increasing faecal weight and thereby increase gut peristalsis counteracting constipation163. Probiotic bacteria are live orally administered bacteria with potential health benefits164. Mainly species of bifidobacteria and lactobacilli have been used as probiotics. Probiotics have been postulated to have many effects but are so far only concluded to have effects on lactose 44 intolerance (since they increase the concentration of lactase) and some types of diarrhoea. However, there is no conclusive evidence that probiotics actually colonise the gut154. Methods for studying the gut microbiota Culture-dependent methods have been extensively used for analyses of the gut microbiota. However, these methods are time-consuming and, importantly, not all species in the gut can be cultivated159. In addition, it might be difficult to analyse different species, within a genus, by cultivation. Sequencing of bacterial genomes made the use of molecular methods feasible. It was shown that the genes for 16SrRNA contained conserved and variable regions. The conserved regions have made it possible to design probes or primers to study domains of bacteria while the variable regions made it possible to investigate different genus or species of bacteria159. Several different techniques use the 16SrRNA genes. These include terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoreses (DGGE). These techniques are useful for analysis of known species of bacteria but also for analysis of diversity as they provide molecular fingerprints of the gut microbiota. However, they have a rather low sensitivity and do not quantify bacterial numbers165. Fluorescent in situ hybridisation (FISH) and Quantitative (Real-time) PCR uses probes and primers to detect and quantify known species, groups or genus of bacteria. Quantitative PCR can detect and quantify bacteria present in low numbers165. Yet, one disadvantage with molecular methods is that they also detect dead bacteria. Faecal samples are most widely used when studying the gut microbiota. However, different bacteria adhere at various sites within the diverse compartments of the gut. Faecal samples might thus not reflect the ecology in different parts of the gut165. Also, aerobe bacteria might appear to exist in higher numbers when faeces are studied instead of biopsies from the more anaerobe gut166. Another useful tool, for studying differences in the gut microbiota between two populations, is to use microbiota-associated characteristics (MACs). This method measures the function of the microbes since metabolic products, like short chained fatty acids, are studied. Still, this method does not provide information of the presence and numbers of certain bacteria. 45 Gut microbiota and immune responses Germ-free (GF) animals have been used to investigate the role of the commensal microbiota. These animals have alterations in their intestinal morphology and thicker muscle cell wall compared to animals reared conventionally139,167. The large intestine of GF animals accumulates mucus due to the lack of mucus-degrading enzymes and they therefore have a larger caecum. Importantly, the immune system in these animals is altered compared to conventionally colonized animals. Especially the GALT is less developed in GF animals with few IgA producing B cells, less T cells and smaller, underdeveloped PPs and MLN´s61, 168. In addition, CD4+CD25+ T cells from the GALT of GF mice elicits lower suppressor activity than CD4+CD25+ T cells from conventional mice169. Other secondary lymphoid organs are also affected. For example, the spleen of GF mice has been shown to contain fewer numbers of CD4+ T cells170. In fact, the systemic immunity is affected i.e. with lower immunoglobulin levels and GF animals are thus more susceptible to infections168. Gnotobiotic animals are GF animals selectively colonized with one or more known bacterial species168. The use of these animals makes it possible to study the effect of the commensal microbiota or certain gut microbes on the immune system. After colonisation with commensal bacteria, increased populations of IgA+ B cells can be detected and thus also increased production of IgA167, 168.Germ-free mice appear to be somewhat Th2 skewed. They produce low amounts of IFNγ and IgG2a but high amounts of IgE, IgG1 and IL-4 after tolerogenic doses of ovalbumin (OVA) followed by a systemic challenge with OVA126. After introduction with B. infantis, the IFNγ and IgG2a response is restored in neonatal mice but not in adult mice126. This postulates a role for the commensal microbiota in early tolerance induction. Interestingly, a polysaccharide from Bacteroides fragilis restores the proportion of CD4+ T cells in the spleen of GF mice and drives the T cell development towards a Th1 phenotype170. The commensal microbiota also appears to be important for intestinal homeostatsis171. Following gut injury, mice with a disturbed gut microbiota produce less cytokines compared to mice with a normal gut microbiota171. Also, in the same study it was shown that mice deficient in MyD88 produce significantly less cytokines than wild type mice after gut injury. Thus the activation of TLRs is central in the recognition of the commensal microbiota and important for gut homestasis. The immune modulatory capacity of probiotics has been studied in both mice and humans. Yet, it is important to remember that humans and conventional mice are not gnotobiotic and it 46 can not be concluded whether the probiotic bacterium exerts the immune effects or whether it alters the existing microbiota which in turn is responsible for the effects. As this thesis focuses on how the very early gut microbiota influences immunity and allergy development, I only discuss studies that have given the probiotic bacterium early. Mice given L. rhamnosus GG or B. lactis directly from birth had higher numbers of TGFβ-secreting CD4+ cells in their MLN´s and more Foxp3-expressing cells in their peribronchial lymph nodes compared to untreated mice172. These mice also produced less total IgE and less OVA-specific IgE och IgG2a. They also responded with lower production of IL-4, IL-5, IFNα and IL-10 to OVA. Infants who received pro- and prebiotics showed higher regulatory responses, measured as plasma IL-10, compared to placebo-treated infants. In addition, the pro- and prebiotic-treated infants had higher plasma concentrations of C-reactive protein, total IgA and total IgE at six months compared to the placebo group173. However, PBMC from six month old infants receiving L. acidophilus since birth did not respond differently to allergens than PBMC from placebo-treated infants174. Conflicting results could be due to the studies being performed in mice versus humans. In addition, different stimuli and bacterial strains have been used. Also, most studies have not investigated if the probiotic bacterium actually colonizes the infant gut. It might thus be more relevant to investigate some of the gut bacteria species actually colonizing the gut in relation to immune responses. Actually, early S. aureus colonization has been shown to increase the plasma levels of sCD14175. Low levels of sCD14 have been shown to be associated with allergic disease175. Both in vivo and in vitro studies are necessary to study effects on the immune system mediated by probiotics. However, when performing in vitro studies one might avoid the confounding factors associated with in vivo studies. Commensal G+ and G- bacteria induce different amounts of pro-inflammatory cytokines from human monocytes176. G+ bacteria induce higher amounts of TNF whereas G- bacteria are better at inducing IL-6 and IL-8. In comparison to monocytes, dendritic cells respond less to G+ bacteria177. Importantly, the commensal bacteria are in contact with dendritic cells rather than monocytes. A study on CBMCs reveals that both G+ and G- commensal bacteria are efficient in inducing sCD14 and sCD83 production from dendritic cells175. Several studies show that certain strains of lactobacilli (L. casei and L. rhamnosus) are less efficient in inducing activation markers on dendritic cells compared to E. coli178 and Klebsiella pneumoniae179. Yet, within the genera of lactobacilli strain-specific responses might occur as L. casei and L. reuteri, but not L. plantarum, bind to the PRR DC-SIGN (dendritic cell-specific intercellular adhesion molecule 47 3-grabbing nonintegrin) and induce the maturation of T regulatory cells180. Also, several strains of lactic acid bacteria, but not all, induce IL-10 production from PBMC and downregulate Th2 cytokine production after PHA stimulation181. Diverse Bifidobacterium species have also been shown to induce activation markers (CD83, CD86) on DCs generated from PBMCs182. Also, B. bifidum, B. longum and B. pseudocatenulatum induce IL-10 production182. Gut microbiota and allergy The gut microbiota could have changed according to increased hygienic conditions. There appears to be an early colonisation with “skin bacteria” like Staphyloccous today compared to decades ago183. Also, differences in gut microbiota between children from affluent countries compared to children from developing countries postulates a role for increased hygiene as a modulator of the gut microbiota151-153. Differences in gut microbiota have also been shown between children raised with an antroposofic lifestyle with more fermented food and less use of antibiotics146. Antroposophic children less often develop allergies compared to children raised during conventional conditions184. In the late 1990´s studies postulated a role for the gut microbiota in relation to allergic disease. Children who were allergic at two years of age were less often colonised with bifidobacteria and lactobacilli whereas they had higher counts of coliforms and S. aureus185. Another study found that colonisation with B. adolescentis, was more common in children with atopic allergies as compared to non-allergic children who were colonised with B. infantis, B. bifidum and B. breve to a higher extent186. However, Murray et al did not see any differences, regarding species of bifidobacteria, when they compared the faecal microbiota between sensitized wheezy and non-sensitised non-wheezy children187. Yet, they found that children with eczema had lower counts of bifidobacteria. Thus, there might be a difference in the gut microbiota between allergic and non-allergic children. Nevertheless, these studies do not address whether it is potential differences in the infant gut microbiota that drives development of allergic disease or if allergic disease might influence the gut microbiota. Therefore it is relevant to study the infant gut microbiota in relation to subsequent allergy development. Such prospective studies have mainly focused on genera of bacteria and show interesting but not quite concurrent results. One study showed that children 48 who had become allergic at the age of two years were less often colonised with enterococci and bifidobacteria but more frequently colonised with S. aureus and had higher counts of clostridia already as infants138. Kalliomäki et al also showed that children who had become allergic at 12 months of age had higher counts of clostridia at three weeks of age188. No other genera of bacteria appeared to differ but the fatty acid profile of bacteria were different between the two groups suggesting that children who develop allergic disease have a different gut microbiota composition. A large study by Adlerbeth et al showed that increased total IgE in serum at 18 months correlated with later colonisation by lactobacilli species149. However, no other bacteria were associated with allergy development or total IgE. Lately, the KOALA study showed alterations in C. difficle and E. coli colonisation at 1 month in children who developed allergy during their first 2 years189. However, no alterations in total bifidobacteria189 or at Bifidobacterium species level190 were seen between infants who developed allergy compared to infants who did not. Evaluation of allergic outcome was performed early in all of these studies (at 12, 18 or 24 months). During this time allergic disease is mainly manisfested in the skin and gut and later allergic disease, i.e. respiratory allergy, was not considered in these studies. 49 IMMUNOLOGY OF HUMAN MILK Human milk is the ideal nutrition for infants. It contains optimal proportions of the macronutrients; carbohydrates, lipids and proteins, as well as micronutrients such as vitamins and minerals. Apart from containing nutrients important for the growth of the infant, human milk also contains several immunomodulatory components191. As the immune system of the infant is far from developed at birth (see above), human milk IgA, lactoferrin, oligosaccharides, leukocytes, cytokines and lysozyme play a substantial role in protecting the infant from pathogens192. Since the lactating mammary glands belong to the mucosal immune system, the human milk antibodies reflect maternal MALT-encountered antigens193. Therefore, the SIgA in human milk protects the infant from the antigens the mother has been exposed to. Several different leukocytes are present in human milk192. They are most predominant in colostrum and decrease during the course of lactation. Macrophages and neutrophils are most common but also lymphocytes, mainly T cells, are present. The human milk macrophages express activation markers and could thus theoretically activate the infant’s lymphocytes, while the role of the neutrophils in human milk is not known. Both anti-inflammatory (e.g. IL-10, TGFβ) and pro-inflammatory (e.g. IL-1β, IL-6, IL-8, TNF) cytokines are present in human milk191, 192. They mainly arise from production in the mammary gland, although the leukocytes in human milk contribute to the production of cytokines192. The physiological role of these cytokines is not completely understood since it is not known to which extent the cytokines survive the passage through the stomach. Still the cytokines in human milk could be of importance for the development of the immune system in infants (see below). Lactoferrin is a major protein in human milk194. It chelates free iron leading to increased absorption of iron for the infant as well as making this nutrient unavailable for bacteria. Another AMP in human milk is lysozyme which is able to break bonds in peptidoglycan. 50 Exosomes have also been found in human milk195. These human milk exosomes have been found to increase the development of Foxp3+CD4+CD25+ T regulatory cells from PBMCs and also to regulate the production of several pro-inflammatory cytokines. Breast feeding in relation to allergy development It has been estimated that 13% of the world deaths of children below five could be prevented if the World Health Organisation’s breast-feeding guidelines (exclusive breastfeeding during the first 6 months) were followed191. However, the role of breastfeeding in allergy prevention is controversial. One meta-analysis, regarding exclusive breastfeeding for the first 3 months, pointed at a decreased risk of breastfeeding on development of allergic rhinitis196. The effect was however not that pronounced for children with atopic heredity. Kull et al showed that exclusive breastfeeding for 4 months or more decreased the risk of developing eczema and asthma197, 198. However, another large Swedish study demonstrated that breastfeeding did not protect children from atopic dermatitis during their first year199. Therefore, it is debated whether the different immunomodulatory components in human milk differ between mothers. Indeed, some studies show that low levels of SIgA are associated with increased risk of cow’s milk allergy in infants, however not all studies confirm this200. The composition of polyunsaturated fatty acids in milk might differ between allergic and non-allergic mothers and could also be related to allergy outcome in children201. The levels of cytokines might vary between allergic and non-allergic mothers with significantly higher IL-4 concentrations in the milk from allergic mothers202. A recent study from our group indicates that mothers with higher exposure to infections, before the age of ten, have increased levels of TGFβ1, IL-6 and IL-8 in their breast milk203. Ovalbumin allergen has been found in the milk of lactating mice exposed to OVA204. Interestingly, the mice fed by OVA exposed mothers were protected against asthma as shown by e.g. decreased airway hyperreactivity, lower number of eosinophils in the broncoalveolar lavage and less IL-4 producing CD4+ T cells. Both the presence of TGFβ in the milk and the TGFβ signalling by T cells appeared to be responsible for the tolerance induction. Levels of allergens like casein, egg proteins and peanut proteins have also been detected in human milk. However whether they cause tolerance induction in humans is not known200. 51 Human milk oligosaccharides Human milk oligosaccharides (HMOs) are the third most abundant solid component in human milk after lactose and lipids205. Colostrum contains roughly 20g HMOs/L while the concentration in mature milk is lower, 12-14g/L206. HMOs are complex carbohydrate structures (3-10 monosaccharide units) that are synthesised in the mammary gland by glycosyl- and fucosyltransferases207. HMOs contain lactose in the reducing end206, 208. Lactose consists of the two monomers D-glucose and D-galactose. Other monomers of HMOs are Nacetylglucosamine, L-fucose and sialic acid. Different enzymes combine these monomers into manifold structures. Approximately 130 different HMOs have been characterised so far209. Of these are roughly 90 % neutral and the rest acidic. It is the secretor status and the Lewis blood group of the mother that determines the pattern of oligosaccharides in the breast milk and at least four different HMO patterns exist209. Approximately 77% of Caucasians are secretors meaning they have the enzyme α1-2 fucosyltransferase208. This enzyme is needed for the formation of 2-fucosyllatose (2-FL) and lacto-N-fucopentaose I (LNFP I). Non-secretors therefore lack these HMOs. Other common HMOs are Lacto-N-tetraose (LNT) and Lacto-Nneo-tetraose (LNnT) which form core structures important for longer HMOs like LNFP I, II, III and Lacto-N-difuco-hexaose I (LNDFH I). 2-FL together with 3-Fucosyllactose (3-FL) and Lactodifucotetraose (LDFT) are shorter oligosaccharides. The HMOs have been shown to be resistant towards digestive enzymes and therefore reaches the intestines intact210. The high concentrations of HMOs combined with the fact that they are resistant to digestion have raised the question of the function of these glycans. Several studies postulate two important functions of these oligosaccharides194, 209. The HMOs are assumed to be the major components responsible for the differences seen between breastfed and formula fed infants (see above)209. HMOs function as the first prebiotics since they are fermented by bacteria like B. infantis211. The N-acetyl-glucosamine containing oligosaccharides have been shown to be essential for the growth of a subspecies of B. bifidum212. Also Coppa and collegues have shown that high concentrations of oligosaccharides in milk correlate with a higher diversity of Bifidobacterium species207. Interestingly, HMOs also appear to act as anti-adhesion agents inhibiting pathogens from binding to gut epithelial surfaces123, 205. The glycosyl- and fucosyltranferases that synthesise HMOs are also responsible for the formation of the glycans present on different cell types. Therefore HMOs resemble glycans on human cells. Pathogens use these glycans to adhere to 52 epithelial cells. When free HMOs are present in the gut lumen the pathogens bind to these and therefore HMOs decrease the infection load of orofeacal pathogens. Indeed, HMOs have been shown to protect infants from infectious diarrhoea213. High concentrations of α1,2-linked HMOs have been shown to be most protective. 53 PRESENT STUDY AIMS Previous studies suggest that the infant gut microbiota is important for maturation of the immune system and subsequent allergy development. The overall aim of this thesis was therefore to further investigate how the infant gut microbiota influences childhood immune responses and allergy development. In addition, the role of breast feeding in allergy prevention is debated. As human milk oligosaccharides might stimulate the growth of certain species of the gut microbiota, the consumption of these was investigated for their role in allergy development. Furthermore, other factors, negatively associated with allergy development, were investigated for their influence on certain species of the early infant gut microbiota. The specific aims of each study were to: Study I: • Investigate whether the colostrum from allergic and non-allergic mothers differ in composition regarding neutral oligosaccharides. • Study if there is a difference in the consumption of colostrum oligosaccharides between neonates who at the age of 18 months had developed allergic disease compared to neonates who had not developed allergy at 18 months. Study II: • Examine the presence and amounts of four Bifidobacterium species, 2 groups of Lactobacilli, C. difficile and Bacteroides fragilis in infant faecal samples collected at one week, one month and two months of age and relate to the development of allergic disease in these children up to five years of age. • Investigate whether these early infant gut microbiota species are influenced by the exposure to environmental factors i.e. endotoxin, Fel d 1 and family size. Study III: • Examine whether early colonization with the above mentioned species of bacteria influences childhood mucosal immune responses i.e. salivary SIgA. • Investigate whether early colonization with these bacteria influence systemic immune responses in terms of spontaneous TLR2 and TLR4 mRNA expression and LPSinduced cytokine production by PBMC collected 12 months after birth. 54 MATERIAL AND METHODS The methods used are listed below and further discussed in each paper: Study I: • Gel permeation chromatography for separation of colostrum carbohydrates according to size. • High performance liquid chromatography (HPLC) for analysis of neutral HMOs (trito hexasaccharides). Study II and III: • Extraction of nucleic acids from faecal samples collected one week, one month and two months after birth. • Real-time PCR for analysis of C. difficile, B. adolescentis, B. longum, B. bifidum, B. breve, lactobacilli group I (comprising L. rhamnosus, L. casei and L. paracasei), lactobacilli group II (comprising L. gasseri and L. johnsonii) and Bacteroides fragilis. Study II: • Limulus Amebocyte Lysate (LAL) assay for quantification of house dust endotoxin. The house dust was collected from carpets once when the children were between 3-12 months old. • Enzyme-linked immune sorbent assay (ELISA) for quantification of house dust Fel d 1. Study III: • ELISA for quantification of salivary SIgA. The saliva was collected at age six and twelve months and two and five years. • Real-time PCR for quantification of TLR2 and TLR4 mRNA expression in PBMC collected 12 months after birth. • Luminex for analysis of LPS-induced cytokine and chemokine production (IL-6, CXCL8 (IL-8), IL-10, IL12p70, IL-17, IL-1β, CCL2 (MCP-1), CCL4 (MIP-1β), TNF) from the above mentioned PBMC. The statistical analyses are described in each paper. 55 The different study populations are described below. The studies were approved by the Regional Ethics Committee for Human Research at Linköping University. Study population study I Mothers attending the Antenatal Health Care Centres in Linköping August 1993 to March 1996 and in the beginning of 2004 were invited to participate in a prospective study of the development of atopic symptoms in relation to environmental factors and maternal immunity. The children were born between January 1994 to July 1997 and June to October 2004. All the children were delivered at term and they had an uncomplicated perinatal period. Mothers who breastfed their babies for less than 3 months were excluded. Clinical examinations and SPT against fresh hen’s egg, milk and extracts from cat and peanut (ALK, Denmark) were carried out at 6, 12 and 18 months of age, and whenever allergic symptoms were suspected. At these appointments the parents also completed a questionnaire regarding e.g. clinical symptoms. In all studies, SPT was considered to be positive if the mean diameter of the wheal reaction was ≥3 mm. Atopic eczema was defined as pruritic chronic or chronically relapsing dermatitis with typical morphology and distribution214. Food allergy was defined as a positive skin prick test, combined with a positive clinical history of immediate skin and/or gastrointestinal reactions or atopic symptoms upon exposure to a certain food and clinical remission of atopic symptoms on an exclusion diet. Breast-feeding was defined as exclusive when all cow’s milk formula, except for extensively hydrolysed formula (i.e. Nutramigen™, Bristol Meyers), were avoided. The diagnosis of atopy in the parents was based on a convincing clinical history of bronchial asthma, allergic rhinoconjunctivitis, atopic eczema and/or food allergy. The mothers were classified as allergic if they showed clinical symptoms of allergic disease and had circulating IgE antibodies against a panel of common allergens, measured with allergy screen test (Magic Lite™, ALK, Hørsholm, Denmark) or Phadiatop (Pharmacia CAP System RAST FEIA, Pharmacia Diagnostics AB, Uppsala, Sweden). Mothers not reporting clinical symptoms of allergy and having a negative allergy screen test or Phadiatop were classified as non-allergic. Children were classified as non-allergic when they did not show any symptoms of allergy and had a negative SPT. Children were classified as allergic if they showed clinical symptoms of allergic disease. 56 From the original study population, we randomly selected 20 women (11 allergic and 9 nonallergic) and 20 children. Eleven of these children were non-allergic and nine showed symptoms of atopic eczema, food allergy or both (see paper I). Study population study II and III The study population, including 123 Swedish children, has been described in detail by Voor et al215. The children were born between March 1996 and October 1999 in Linköping, Sweden. All were born at term and they had an uncomplicated perinatal period. A clinical examination of the babies was made at three, six and twelve months and at two and five years. At these occasions, skin prick tests were performed, and questionnaires were completed regarding e.g. symptoms of allergy, use of antibiotics and family size. Atopic dermatitis was defined as in study I. Asthma was defined as three or more episodes of bronchial obstruction during the last 12-month period, of which at least one should be verified by a physician. Allergic rhinitis/conjunctivitis was defined as rhinitis and/or conjunctivitis following at least twice in one hour after allergen exposure and not in relation to an obvious infection e.g. fever or a cold. SPTs, with fresh skimmed cow’s milk and thawed egg white, were performed at all follow ups. From 12 months, the children were tested with cat allergen extract and at two and five years also with birch- and timothy extracts (ALK, Hørsholm, Denmark). Inclusion in study II was based on availability of faecal samples at one week, one month and/or at two months of age and known allergy status up to the age of five. The children were classified as allergic and non-allergic based on symptoms and SPT. Thus, the allergic children had shown symptoms of allergic disease and at least one positive SPT during their first five years of life, while the non-allergic children had not shown symptoms of allergic disease, nor had a positive SPT. Therefore, 47 infants were included. Sixteen children were classified as allergic and 31 as non-allergic. For demographic data of these children see table 1 in paper II. The demographic data did not differ between the allergic and non-allergic group. Inclusion in study III was based on availability of faecal samples at one week, one month and/or at two months of age as well as blood samples collected at twelve months, and/or 57 salivary samples, collected at six months, twelve months, two years and/or five years. As known allergic status during the first five years was not an inclusion criterion, additional children, compared to study II, could be included. In all, 64 infants were included; this group included all 47 subjects in study II. The demographic data of all 64 children is shown in table 2. Table 2. Demographic data of the subjects. Subjects n=64 32 3 50 53 2 11 3 29 21 10 7 17 Female subjects Born with caesarean section Any atopic heredity Exclusively breastfed ≥ 3 months Oral antibiotics ≤ 3 months Pets Number of family members Household area/individual (m2) AD cumulative five years AB cumulative five years ARC cumulative five years SPT cumulative five years % (50%) (5%) (78%) (83%)a (3%) (17%) (3-8)b (18-75)b (33%)c (16%)c (13%)c (27%)c AD= Atopic dermatitis, AB= Asthma bronchialis, ARC= Allergic rhinitis/conjunctivitis, SPT= Skin prick test a = missing data for one subject regarding exclusive breastfeeding. b = missing data for three subjects regarding number of family members and thus household area/individual. c = six children had left the study at five years of age. A majority of the children had a history of atopic disease in the immediate family (78%, Table 2). In total, three children were delivered with caesarean section. Most infants were exclusively breastfed during their first three months (83%) and only two infants received oral antibiotics during this time. Seventeen children (27%) were sensitized during their first five years of life. A majority of these children showed SPT reactivity to food allergens before five years (12 children). Also, at five years of age, ten of the sensitized infants were SPT positive to inhalant allergens. Twenty-one children had atopic dermatitis during their first five years of life. At age five, seven children had allergic rhinitis/conjunctivitis either in combination with asthma (four) or without (three). Two children had asthma in combination with atopic dermatitis and four children had asthma alone. 58 RESULTS AND DISCUSSION Human milk oligosaccharides in colostrum of allergic and non-allergic women (I) Some components in human milk might differ between allergic and non-allergic mothers201, 202 . Oligosaccharides are the third most abundant solid component in human milk205 and appear to be resistant towards digestive enzymes210. Thus, they reach the intestines intact which postulate a significant role for the HMOs in the intestine. In order to investigate whether maternal allergy influences the content of these, the concentration of the neutral oligosaccharides LNDFH I, LNFP I, LNFP II, LNFP III, LNT, LNnT, 3-FL, 2´-FL and LDFT were analysed in colostrum samples from allergic (n=11) and non-allergic (n=9) women. The colostrum samples were collected at the same time of the day and at the same time in the lactation period; 2-4 days post partum. In accordance with previous studies213, 216, 217, 2´-FL and LNFP I were the most abundant neutral oligosaccharides. There was no difference in the amount of neutral oligosaccharides in the colostrum from allergic compared to non-allergic mothers (see table 2 in study I). This suggests that the amount of neutral oligosaccharides in human milk is not influenced by the allergy status of the mother. Although there was no difference in HMO concentration between colostrum of allergic and non-allergic women, the variation in oligosaccharide concentration in milk from different women was immense. This variation is in accordance with previous studies and partly depends on the secretor status, the ABO- and Lewis blood group types218. Actually, the production of HMOs is catalyzed by the same enzymes, glycosyltransferases including fucosyltransferases, which are involved in the production of cell surface glycoconjugates. On erythrocytes these conjugates determine blood group type218 and on other cell types they function as receptors for particles and pathogens. Due to their structural resemblance with these cell surface glycoconjugates, the HMOs might prevent the binding of certain pathogens to the mucosa, thus lowering the infection rate by these pathogens205, 210 . Indeed, high amounts of α1,2-linked fucosylated oligosaccharides appears to protect the infant from several types of diarrhoea213. Therefore, variation in HMO concentrations might be of importance for other diseases. 59 Colostrum oligosaccharide concentrations in relation to subsequent allergy development (I) The HMOs might function as the first prebiotics by increasing the growth of certain commensal gut bacteria209, 211. Furthermore, the gut microbiota might be altered in children developing allergic disease138, 188, 189 . Thus, we studied the concentrations of the above mentioned oligosaccharides in colostrum samples and related these data to allergy development at 18 months. Our results indicate that children who had developed allergic symptoms at the age of 18 months tended to have consumed colostrum with higher concentrations of neutral oligosaccharides in total, although the difference did not reach statistical significance (p=0.12). This tendency could however not be attributed to any specific neutral oligosaccharide. The fact that oligosaccharides in human milk act as receptor homologues213, 218 and thus prevent pathogens from binding to the cells in the mucosa implies that a high amount of neutral oligosaccharides protects against orofecal pathogens. Interestingly, it has been shown that infections with food and orofecal pathogens were able to reduce the risk of atopy with 60%103. Accordingly, the ability of human milk oligosaccharides to bind pathogens and serve as anti-infective agents might influence allergy development more than their ability to stimulate gut microbiota-immune interactions. Still, others have shown that infants who received formula supplemented with prebiotic galacto- and fructooligosaccharides had less atopic dermatitis during their first six months compared to infants who received standard formula219. In addition, the prebiotic group had more bifidobacteria in their faeces. Probably, additional studies are of importance for investiging the role of HMOs and prebiotics on allergy development. Early infant gut microbiota (II and III) The commensal gut microbiota appears to be important for oral tolerance induction and gut homeostasis126, 171 . Previous studies have shown differences in the infant gut microbiota between children who develop allergic disease compared to children who do not138, 188, 189 . However, this has not been confirmed by others149. In order to investigate whether early infant gut microbiota is related to allergy development and immune maturation, faecal samples were collected from infants (n=64) at one week, one month and two months after birth. Real-time PCR was employed for analysis of the following bacterial species; C. difficile, B. bifidum, B. longum, B. adolescentis, B. breve, Bacteroides fragilis, Lactobacilli group I (L. rhamnosus, L. paracasei, L. casei) and Lactobacilli group II (L. gasseri, L. johnsonii). The Lactobacilli and 60 Bifidobacterium species were chosen due to their abundance in early faecal samples from infants220, 221 and their postulated role in allergy development222. Also, C. difficile has been associated with allergy development189. Furthermore, a component from Bacteroides fragilis has recently been shown to be a potent immunomodulator170 and thus this bacteria was also included. Table 3. Proportion of infants (%) colonized with Bifidobacterium (B.), Lactobacillus, Clostridium (C.) difficile and Bacteroides fragilis during the first two months of life. The numbers in the headers show the number of infants with available faecal samples at the different time points. The figures, after the different bacteria, show the percent of infants who were colonized with the bacteria. 1 week 1 month 2 months Bacteria n=52 n=56 n=56 B. longum 88 91 91 B. adolescentis 71 66 75 B. bifidum 56 63 63 B. breve 40 43 50 Lactobacilli group I 31 57 57 Lactobacilli group II 33 43 54 C. difficile 6 11 21 Bacteroides fragilis 60 55 55 Bifidobacteria were detected in all infants in at least one of the three samples collected during the first two months of life (Table 3). Compared to studies using cultivation to detect bacteria this prevalence of bifidobacteria is high138. However, other studies using molecular methods find bifidobacteria in most faecal samples from one- month old infants189, 223. Bifidobacterium longum, which includes B. infantis, was the most prevalent Bifidobacterium species in the infant faecal samples, which is in accordance with previous findings221, adolescentis was common, which is in agreement with some 223, 225 224 . Also B. but not other previous studies221, 224. Bifidobacterium breve was the least common of the Bifidobacterium species. Both Lactobacilli group I and Lactobacilli group II were present in the early faecal samples. Others have shown that Swedish infants harbour lactobacilli species, belonging to these groups, during their first two months220. Few infants were colonised with C. difficile. Yet, C. difficile appears to be more common in later infancy as 59% of 12 month- old infants harbour this bacterium (Johansson et al unpublished). The two infants who received antibiotics (Table 2) harboured few Bifidobacterium species. However, DNA from Bacteroides fragilis constituted a major fraction of the nucleic acids purified from the faecal samples of these children (close to one percent). Actually, strains of Bacteroides fragilis are resistant to several 61 different antibiotics226. Two of the three children who were born with caesarean section harboured C. difficile. Others have reported that infants born with caesarean section harbour C. difficile more often than vaginally delivered infants227. The samples from the children who were not exclusively breastfed did not appear to differ from the samples from exclusively breastfed children. Early infant gut microbiota in relation to allergy development (II) Infants who developed allergic disease were, as one-week old infants, less often colonized with Lactobacilli gr. I and B. adolescentis compared to infants who remained non-allergic (p=0.02 and p=0.03, Fig. 7). Figure 7. Proportion of infants (%) colonized with Lactobacilli gr. I and Bifidobacterium (B.) adolescentis during the first two months of life. Allergic infants developed allergy during their first five years while nonallergic infants did not. Also, B. adolescentis, Lactobacilli group I, and C. difficile were less often detected in the three samples, obtained during the first two months of life, from allergic infants than in the samples from non-allergic infants (p=0.045, p=0.02 and p=0.03 respectively, Fig. 8). This suggests that persistent colonisation with these species are of importance. The colonization pattern of the other bacteria was similar in infants who did and did not develop allergy (see Table 3 in study II). 62 Figure 8. Number of occasions with detectable Bifidobacterium (B.) adolescentis, Lactobacilli group I and Clostridium (C.) difficile during the first two months of life in relation to allergy development up to age five. The y-axis shows the percentage of infants who did (A, n=11) or did not develop allergy (NA, n=21) during their first five years of life. The bacteria were studied on three occasions during the first two months and the x-axis illustrates the number of occasions with detectable bacteria. As calculated with Mann-Whitney U test, the nonallergic children harboured B. adolescentis, Lactobacilli gr. I and C. difficile on more occasions than the allergic group (p=0.045, p=0.02 and p=0.03 respectively). None of the other investigated bacteria occurred at different occasions between the allergic and the non-allergic group. Children are only included if faecal samples from all occasions were available. The early infant gut microbiota has been related to allergy development during the first two years of life, in most previous studies138, 149, 189. At this age, allergic manifestations are mostly limited to the skin and gut. The children in our study were followed to the age of five, i.e. to an age when respiratory allergy and sensitization to inhaled allergens have become the major clinical manifestation of allergy. We further subdivided the allergic children (who were all SPT+ as previously mentioned) according to their clinical symptoms. Nine children were included in the atopic dermatitis only group (AD only) and seven children in the allergic rhinitis/conjunctivitis and/or asthma ever group (ARC/AB ever). As shown for the total allergic group, the AD only group were less frequently colonized with Lactobacilli group I one week after birth compared with the non-allergic group (p=0.03). This should be considered in the context of clinical studies, in which different strains of probiotic lactobacilli reduced the incidence of infant eczema228 and IgE-associated eczema229, 230 . Importantly, preliminary data from a cohort from Sachs’ Children’s hospital also indicate that children 63 who are sensitized and develop allergic symptoms before the age of five are less frequently colonized with Lactobacilli gr. I during their first two months, compared to non-allergic children (p=0.06-0.13) (Johansson et al unpublished). In addition, infants to allergic mothers have a delayed colonization with Lactobacilli gr. I (Johansson et al unpublished). The primer Lactobacilli gr. I detects L. rhamnosus, L. casei and L. paracasei. Interestingly, Lactobacillus rhamnosus stimulated DCs have been shown to induce hyporesponsive T cells231. Also, L. reuteri and L. casei bind to the PRR DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) on DCs leading to priming of IL-10 producing T regs180. These studies thus provide mechanisms supporting our findings. Yet, bacterial colonization in the seven children with respiratory allergy did not differ from bacterial colonization in the non-allergic children. It is difficult to interpret the role of Lactobacilli group I on respiratory allergies as only one child developed respiratory allergy without previous atopic dermatitis. Furthermore, only seven children had developed allergic asthma and rhinitis/conjunctivitis at age five. Children belonging to the AD only group, at age five, might develop respiratory allergy later as a substantial proportion of sensitized children with atopic eczema develop respiratory allergy before age seven232. Bifidobacterium adolescentis was more commonly detected in infants not developing allergic disease. In addition, the AD only group tended to be less frequently colonized with B. adolescentis one week after birth compared with the non-allergic group (p=0.06). This is in contrast to findings by others where no difference was observed regarding early B. adolescentis colonization between the infants who did or did not develop allergic disease190, 225 . Colonization with B. catenulatum (at one month) and B. bifidum (at three and six months) was however more common among the infants who developed allergic disease225. However, in these reports, allergy development was investigated early, at six225 and twelve months190, and a follow up of the children in these studies might show different results. Others have shown differences in Bifidobacterium composition between allergic and non-allergic infants186, 233 and children234, where B. adolescentis186, 234 and B. pseudocatenulatum233 were more frequently detected in the allergic individuals. However, these are all investigations of the gut microbiota in children with an already manifested allergic disease in contrast to our prospective study. Different Bifidobacterium species elicit distinct immune responses from diverse immune cells182, 235 . Hence, the early Bifidobacterium colonization could thus have the potential to influence immune responses during the postulated short period early in life when the immune system might be matured in order to prevent allergy development125. 64 Studies investigating gut microbiota composition when the disease is already established might primarily indicate that individuals with allergic disease provide a different gastrointestinal milieu, favouring growth and attachment of certain species of bacteria. Certain bacterial species appear to be associated with allergic disease when the gut microbiota is less diverse, exemplified by C. difficile. Previous studies suggest that colonization early in life with C. difficile is more common among infants who develop allergic disease than among infants who do not189. We could not confirm this, as infants who developed allergy were less likely to be colonized with C. difficile during their first two months. The low rate of C. difficile colonization combined with the relatively few number of allergic children may explain the lack of a reverse association in our study. Nevertheless, several studies, in which clostridia is more abundant in allergic infants, also indicate low prevalence of some other commensal bacteria in children developing or suffering from allergy138, 188, 236. Prevalence of non-toxigenic and toxigenic C. difficile colonization at one month is associated with eczema and wheeze, respectively227. Interestingly, both non-toxigenic and toxigenic C. difficile colonization is associated with formula feeding and caesarean section, early factors which are associated with a less diverse microbiota. Therefore, it might rather be a gut microbiota with lower diversity that is associated with allergy development. Interestingly, a large recent study did not find any relationship between the presence of different bacteria and allergy development up to 18 months of age149, but indeed showed that infants who developed allergy had a lower diversity in their gut microbiota237. Additional prospective studies investigating the microbiota before the onset of allergic disease in combination with mechanistic studies are important for understanding the role of the gut microbiota, or certain gut microbiota species, on allergy development. Environmental factors influence the early infant gut microbiota (II) Type of delivery, diet and country of birth influence the infant gut microbiota145, 149, 151-153. Yet, other factors might also contribute to the colonization of the infant gut. We investigated whether lifestyle factors, inversely related to allergy development, influence the early colonization of the above mentioned species. Information about number of family members was collected through questioners. Dust samples were collected from the infant’s homes and analysed for endotoxin and Fel d 1 with LAL and ELISA, respectively. Number of Bifidobacterium species at one week and two months correlated significantly with number of 65 family members (Fig. 9). Considering that higher counts of bifidobacteria145 and later acquisition of Clostridium species in infants with siblings149 have been reported, family members and siblings may rapidly contaminate the infant with a commensal microbiota. Figure 9. Larger household size was associated with additional number of Bifidobacterium species in the faecal samples collected one week and two months after birth (a and b). Although displaying the same trend, no significance was seen in the one month samples (see study II). The x-axis shows number of family members and the y-axis the number of Bifidobacterium species in the infant faecal samples. The p-values indicate the trend after Cuzick´s non-parametric test for trend. Colonization with several (3-4) Bifidobacterium species, at one week and two months of age tended to be associated with exposure to higher levels of endotoxin than colonization with low numbers (0-2) of Bifidobacterium species (Fig. 10). This was particularly obvious during the first week of life (Fig. 10b). There was no similar correlation with the Fel d 1 levels in house dust. Endotoxin is a component in G- bacteria and should probably be viewed as a marker of total bacterial exposure in the homes and not as a factor directly related to the G+ bifidobacterial microbiota. However, postnatal colonization with the G- and facultative aerobic enterobacterial microbiota, is important to establish an appropriate environment for anaerobic bacteria, such as bifidobacteria and bacteroides140. Today, staphylococci are acquired the first days after birth among Swedish infants, while it takes up to two months before most infants have achieved an enterobacterial microbiota183. Speculatively, high bacterial exposure may lead to early colonization with appropriate facultative aerobic bacteria, which in turn would facilitate colonization of commensal anaerobic bacteria. Thus, lifestyle factors might influence the colonization in infants. In addition, our group has shown that the maternal faecal flora influences the colonization of some species of bacteria, i.e. B. bifidum (Johansson et al unpublished). 66 Figure 10. Number of faecal Bifidobacterium species (B. sp.) in relation to endotoxin levels in house dust (EU/mg dust). The white boxes symbolize samples with two or less B. sp. and the grey boxes those with three or more. In figure a and b, the number of B. sp. is measured in faecal samples collected at one week after birth. In figure c and d, the faecal samples were collected at one month and two months, respectively. Early infant gut microbiota in relation to maturation of immune responses (III) In study II, we showed that children who developed allergic disease were less often colonized with Lactobacilli gr. I, B. adolescentis and C. difficile. To explore potential mechanisms behind this association we investigated whether early colonization with the gut microbiota species, analyzed in study II, associates with childhood immune maturation, specifically salivary SIgA and the spontaneous TLR2 and TLR4 mRNA expression in PBMC collected 12 months after birth. The possible allergy-protective effects from the increased pre- and postnatal microbial exposure in farming environments might increase the expression of several PRRs e.g. TLR2 and TLR4110, 238 . Thus, we hypothesized that early exposure to certain intestinal microbes modulates TLR expression and LPS responsiveness at twelve months. The spontaneous expression of TLR4 mRNA, which is important for responses to G- bacteria, correlated inversely to the intensity (i.e. the relative amounts) of one week/one month colonization with the G- bacteria Bacteroides fragilis (r=-0.47, p=0.02 and Figure 11a). The expression of TLR2, which is important for responses to G+ bacteria was not affected, 67 however, nor were the G+ bifidobacteria, lactobacilli and C. difficile associated with TLR2 or TLR4 mRNA expression. Fig 11. The spontaneous expression of TLR4 mRNA (a) at twelve months and the LPS-induced production of CCL4 (MIP-1β) (b) and IL-6 (c) are inversely correlated to the intensity of Bacteroides fragilis colonization at one month after birth. The y-axis shows in figure 11a, the relative TLR4 mRNA expression/rRNA in PBMC collected twelve months after birth. In figure 11b and c, the y-axis shows the concentration of CCL4 (b) and IL-6 (c) in supernatants from the PBMCs stimulated with LPS for 24 hours. The x-axis shows the relative amount of Bacteroides fragilis at one month expressed as percent Bacteroides fragilis DNA of total amount nucleic acids.. The dotted line shows the detection limit at 5*10-6 % Bacteroides fragilis DNA of total amount nucleic acids. n.d. = not detectable. Following this observation we wanted to investigate whether the intensity of Bacteroides fragilis colonization influences LPS responsiveness. In concordance with the TLR4 mRNA findings, the intensity of Bacteroides fragilis colonization at one week, one month and two month were all inversely related to the LPS-induced production of IL-6 and CCL4 (MIP-1β) (r=-0.62 to -0.79, p=0.002 to 0.04 and r=-0.75 to -0.86, p=0.001 to 0.005; Fig. 11b and c exemplifies one month colonization). Also, LPS-induced CXCL8 (IL-8) and IL-17 were inversely related to the intensity of Bacteroides fragilis colonization at one week after birth (r=-0.75, p=0.008 and r=-0.68, p=0.02). In contrast, the intensity of B. adolescentis colonization at one/two month correlated or tended to correlate to TNF, IL-10 and IL-12p70 (see study III). Partially supporting our findings, it has been observed that increased exposure to muramic acid from G+ bacteria in house dust, correlated with IL-6 and TNF production in mitogen-stimulated PBMC239. Conversely, the same study also showed that house dust levels 68 of 3-hydroxy fatty acids, from G- bacteria, were inversely associated with the production of IL-6 and TNF after mitogen stimulation239. High LPS exposure induces endotoxin (LPS) tolerance18 and endotoxin tolerant mice have decreased splenic TLR2 and TLR4 expression20. Speculatively, the observed decreased LPS responsiveness in infants colonized with high levels of Bacteroides fragilis could thus be due to endotoxin tolerance caused by the high exposure to the G- Bacteroides fragilis. Also, PSA, which is another component from Bacteroides fragilis has immunomodulatory properties240. Apart from restoring the proportion of CD4+ T cells in the spleen of ex-GF mice, this component is also able to balance the production of inflammatory cytokines, e.g. TNF and IL-17, in mice with induced colitis241. Hypothetically, exposure to increased levels of gut Bacteroides fragilis PSA might regulate the production of inflammatory cytokines systemically. Interestingly, Round and collegues have shown that administration of PSA to GF mice prevents induced systemic septic shock (Personal communication with June L Round at Keystone symposia A6 2009). In addition, IL-6 and TNF production is lower in PSA treated mice. This fits well with our results that early colonization with high numbers of Bacteroides fragilis reduces spontaneous TLR4 expression. The levels of salivary SIgA is higher in sensitized infants who show no symptoms of allergic disease compared to the SIgA levels in infants with allergic disease117 (Tomicic et al unpublished). Further, the salivary SIgA levels reflect the antigen-specific SIgA levels in the gut (after low antigen administration)242 and thus we hypothesized that early colonization with certain gut microbes influences SIgA levels in the gut and in additional compartments of the MALT (salivary glands). The number of Bifidobacterium species in faeces collected one week, one month and two months after birth correlated with salivary SIgA at six months (r=0.51 to 0.58, p=0.02 to 0.045; Fig. 12a exemplifies the one month colonization). Furthermore, the intensity of one/two month B. adolescentis colonization were associated with higher SIgA at six months (Fig. 12b and r=0.55, p=0.03, respectively). The intensity of B. bifidum colonization at one week, one month and two months after birth were correlated with the SIgA production at twelve months (r=0.41 to 0.47, p=0.01 to 0.04, figure 12c exemplifies one month colonization). In addition, early colonization with B. adolescentis, B. breve and B. bifidum tended to be associated with higher levels of SIgA at six and twelve months (study III). Thus, Bifidobacterium colonization appears to influence salivary SIgA levels up to twelve months, but not later in infancy nor at five years of age. The later SIgA formation might be influenced by other factors i.e. infections (Sandin et al unpublished). In 69 addition, the levels of SIgA at six months and five years were significantly associated with the colonisation with lactobacilli group I at one month after birth (see Figure 2 a and d in study III). Gut microbes appear to induce APRIL and TSLP production from IEC75. These factors are involved in IgA2 class switching by B cells. Possibly could our results indicate that early Bifidobacterium colonization influences APRIL and TSLP production leading to increased SIgA production. Yet, others did not show that microbial colonization during the first two months was associated with plasma levels of APRIL at four months243. The gut microbiota might be more efficient in inducing APRIL production locally, however. Since the GALT and the salivary glands are part of the common mucosal immune system244 the IgA production by gut B cells might be reflected in saliva. Figure 12. Total salivary secretory IgA (SIgA) at six months is associated with a more diverse Bifidobacterium (B.) flora (a) and the intensity of B. adolescentis colonization (b) at one month. Total SIgA at twelve months is associated with the intensity of B. bifidum colonization at one month (c). The y-axis shows total salivary secretory IgA (SIgA) (µg/ml) at six and twelve months and the x-axis shows the number of Bifidobacterium species (a) and the intensity of B. adolescentis (b) and B. bifidum (c) colonization. The intensity is expressed as percent B. adolescentis or B. bifidum DNA of total amount nucleic acids. The dotted line shows the detection limit at 5*10-6 % B. adolescentis or B. bifidum DNA of total amount nucleic acids. n.d. = not detectable. As higher levels of SIgA might protect sensitized infants from allergy development117, it is tempting to speculate that a more diverse Bifidobacterium flora could prevent allergy development partially by stimulating salivary SIgA. I have summarized findings from us and others regarding environmental factors associated with allergy development in figure 13. 70 There is a strong negative association between family size/endotoxin exposure and allergy development102, 109. The other associations in this figure are weaker. However, what we and others have shown is that these factors associate with each other. Family size and endotoxin exposure appear to influence the diversity of the early Bifidobacterium flora (II). Others have shown that a less diverse early gut microbiota is associated with allergy development237. In addition, we showed that infants who developed allergic disease were less often colonized with Lactobacilli gr. I, B. adolescentis and C. difficile during their first two months. Thus, a more diverse gut microbiota might explain the inverse relationship between family size/endotoxin exposure and allergy development. Children with older siblings have higher SIgA (Sandin et al unpublished). Although endotoxin exposure was not associated with SIgA levels (Tomicic et al unpublished), Estonian infants, who are exposed to higher levels of endotoxin, have higher levels of SIgA than Swedish infants117, (Tomicic et al unpublished). These results fit with the speculation above that a more diverse Bifidobacterium flora could prevent allergy development partially by stimulating salivary SIgA. Conversely could the observed association between the early Bifidobacterium flora and SIgA levels during the first year indicate that an overall more diverse gut microbiota, in general, increases salivary SIgA. Actually, also colonization with lactobacilli gr. I, at one month, was associated with higher levels of salivary SIgA at six months and five years (see study III). Interestingly, Estonian infants, who are more commonly colonized with lactobacilli152, have higher levels of SIgA, compared to Swedish infants (Tomicic et al unpublished). Further studies are needed to demonstrate if my speculations are true. Figure 13. Associations between environmental factors, SIgA and allergy development. The green arrows indicate positive associations whereas the black arrows indicate negative associations. References to different studies are shown. 71 Clinical relevance By studying the infant gut microbiota in relation to allergy development we might understand which bacteria that could be of importance in allergy prevention and thus appropriate probiotic candidates. Studies have investigated how very early administration of probiotics (from birth or prenataly to pregnant mothers) associates with subsequent allergy development. Interestingly a strain of L. rhamnosus, given to pregnant and breastfeeding mothers and to their infants reduced the frequency of eczema in the children up to age seven indicating that this species might be important in eczema prevention228, 245. However, SPT positivity did not differ between the two groups. Another double-blind randomised placebo-controlled trial investigated the role of L. reuteri administration to mothers prenatally and to their infants for their first 12 months229. The prevalence of eczema outcome and asthma did not differ between the two groups, however the probiotic group had significantly lower proportion of IgEassociated eczema at two years. Recently, L. rhamnosus administration but not B. lactis administration was shown to decrease the incidence of IgE-associated eczema230. Trials showing no association between probiotic administration and allergy development have also been completed246. Importantly, administration of L. acidophilus during the first six months and an increased risk for sensitization has also been reported247. The different strains, when they are administered (prenatally and/or from birth), number of participants and the follow up period are all factors which could influence the outcome. The largest study performed, which used several probiotic strains (2 strains of L. rhamnosus, B. breve and Propionibacterium freudenreichii) and prebiotic galacto-oligosaccharides, showed promising results regarding eczema and atopic eczema outcome at two years of age 248. Yet, the five-year follow up could not find any difference in allergic disease between the probiotic- or placebo-treated group249. This somewhat fit with our results where early Lactobacilli gr. I colonization and B. adolescentis colonization was associated with decreased development of atopic eczema but not later allergy development. Importantly, much is still unknown about the gut microbiota. The solution might not be to administer some strains of probiotic bacteria but rather to increase the diversity of the gut microbiota as a whole. In paper II we showed that lifestyle factors appear to influence the Bifidobacterium flora. Promoting vaginal delivery could be another approach. Caesareandelivered infants appear to have a delayed colonization with several different species of bacteria150. In addition these infants are at higher risk for developing allergic disease250. 72 Probiotic therapy might be beneficial when vaginal delivery is not an option. Actually, the five-year follow up mentioned above showed that infants delivered with ceaserean section were at decreased risk of developing atopic disease if they were given probiotics from birth compared to if they received placebo249. Our study regarding consumption of colostrum oligosaccharides did not show any significant association with allergy development at 18 months of age. Yet, additional substances in human milk might also stimulate species of the gut microbiota206. Also, human milk contains Bifidobacterium species223 and has been shown to modulate TLR responses251. Thus, it might be of relevance to promote breastfeeding also for its ability to contribute to infant colonization and stimulation of gut microbiota-immune interactions. 73 CONCLUDING REMARKS • The content of neutral oligosaccharides in colostrum did not depend on maternal allergy, nor was consumption of colostrum with high amounts of oligosaccharides associated with less allergy development up to 18 months. • Infants who harboured Lactobacilli group I (L. rhamnosus, L. paracasei, L. casei) and B. adolescentis at one week of age less frequently developed allergic disease compared to infants who did not harbour these bacteria at the same time. This might imply the importance of a diverse gut microbiota early in life for the development of a fully functional immune system. • Family size and endotoxin exposure influenced the early Bifidobacterium flora. Thus, a more diverse gut microbiota might possibly explain the inverse relationship between family size/endotoxin exposure and allergy development. • The early Bifidobacterium flora influenced levels of salivary SIgA at six and twelve months. Speculatively, this provides a potential mechanism to why early B. adolescentis colonization could be associated with less allergy development. • The intensity of early Bacteroides fragilis colonization influenced LPS responsiveness of PBMCs collected at 12 month. Thus, this bacterium might influence systemic immune responses. 74 FUTURE PERSPECTIVES In our second study we investigated the early infant gut microbiota in relation to allergy development up to five years of age and found differences at species level. Since human milk is the only food during the first months (in our study population) it might influence the early gut microbiota. Although we could not show that consumption of colostrum oligosaccharides influences allergy development (study I) the later milk oligosaccharides might be of importance. As breast milk consumed by the children in study II has been collected we can relate the neutral oligosaccharide composition in these samples to the presence of amounts of the bacterial species investigated in paper II and probably understand more about the prebiotic function of HMOs. In our third study we showed that infants who harbour a more diverse Bifidobacterium flora during their first two months have increased levels of salivary SIgA at six months. After encountering bacterial components, the intestinal epithelial cells (IECs) appear to be able to produce cytokines important for IgA class switching e.g. APRIL75. How certain gut microbiota species influences the production of these cytokines and thus IgA class switching in the gut needs to be further investigated. In addition, IECs produce other cytokines which are important for conditioning of DCs and further for differentiation of regulatory T cells as described previously10. Thus, it would be interesting to study whether lactic acid bacteria induce production of regulatory and conditioning cytokines e.g. TGFβ and TSLP from IECs. These cytokines also influence IgA production. To further understand the role of IgA in development of allergic disease it would be interesting to measure fecal levels of SIgA and relate to later allergy development. Actually, high levels of SIgA in faeces are associated with tolerance in mice135. However, this would have to be performed in age-matched infants not receiving breast milk. Although IECs appear to be somewhat non-responding towards lactic acid bacteria, regarding IL-8 production252, pre-incubation with lactic acid bacteria reduces the pro-inflammatory responses mediated by Salmonella typhimurium or flagellin and abolishes DC maturation induced by G- bacteria252, 253. In study II we showed that infants who developed allergy were less often colonized with C. difficile during their first two months. In contrast others show that early C. difficile colonization is more common in infants who develop allergic disease compared to infants who remain non-allergic138, 75 189 We speculate that it is not C. difficile colonization per se that is associated with allergy development. However, C. difficile induces unfavorable immune responses when the gut microbiota is less diverse. A diverse lactic acid flora might decrease these responses and it would be of interest to study if pre-incubation with lactic acid bacteria influences IL-8 production of IEC after C. difficile stimulation. We have also shown that IECs are rather non-responsive to stimulation with breast milk or LPS alone (Holmlund et al unpublished). However, simultaneous stimulation with LPS and breast milk induces high IL-8 production. Speculatively, these responses could cause too much inflammation and it would thus be interesting to study whether pre-incubation with lactic acid bacteria decreases these responses. We have investigated the gut microbiota in relation to allergy development. In addition, we show that certain gut microbiota species appear to influence systemic and mucosal immunity (III). Consequently, the observed associations could also be attributable to other disorders. Increased infant SIgA in saliva and feces would prevent both allergens and pathogens from entering the mucosa thus increasing the integrity of the barrier. The gut microbiota has also been linked to inflammatory bowel disease, obesity and type 1 diabetes254-256 The gut microbiota could probably influence these diseases by modulating immunological factors. However, other physiological factors could also be influenced by the gut microbiota257. Future studies will have to reveal how the vast amounts of genes present among the bacterial strains in the gut, the gut microbiome147, relates to the physiology and health of the host. 76 ACKNOWLEDGEMENTS I am most grateful to my supervisor Eva Sverremark Ekström for giving me the opportunity to do a PhD. You have been an excellent supervisor and given me a lot of support during these past four years. You have always listened to my ideas and then pushed me in the right direction for them to succeed. Your belief in me has made me believe more in myself. I am very thankful to my co-author Maria Jenmalm at Linköping University for all your great ideas and help with the manuscripts. You always take your time and it is wonderful to work with people who are so dedicated to their work. I would also especially like to thank professor Bengt “Muck” Björkstén for all your help and your enthusiasm. Your previous work has been such an inspiration and I have learnt a lot working with you. I am also grateful to you for introducing us to the Linköping staff. Another person without whom this work would not have been possible is Frank “Zeke” Lindh. Thank you for bringing me to Uppsala and Isosep and for teaching me the procedure of HMO analysis! Thanks also for the fun we had there and for keeping the “godisskål” filled! I am also very grateful to my other co-authors from Linköping: Karel Duchén, Malin Böttcher, Sara Tomicic and Anna Lundberg for all your help. Your previous work has been an inspiration. I would also like to thank you all (and Maria Je) for being excellent soccer supporters. I appreciate all the effort Lena Lindell and Ann-Marie Fornander at Linköping Universty hospital have put into collecting and keeping track of samples. Other persons who have helped me in my work by simply being there and eager to discuss are my previous and present roommates and group members: Yvonne Sundström, Nora Bachmayer, Petra Amoudruz, Ulrika Holmlund, Maria Johansson, Ebba Sohlberg, Shanie Hedengren and Elin Hultgren. I have so enjoyed sharing room and laughs with you. Thank you for letting me ask “stupid” questions and for being great friends! I am also grateful for all the fun we have had during conferences. We have seen too much (Yvonne and me on the built island in Vienna). We have danced in Bangkok (Petra and Shanie) and Taos (Maria) and enjoyed sunbathing and skiing. Many thanks to Nora for your cute notes, for teaching me the importance of a plan and for enjoying a glass of wine! I am grateful for the help I have got from my “slaves” that is my 10- and 20-points project workers: Caroline “Carro” Nissan, Karin Sundström and Maria Johansson. Thanks for sharing the interest of my work with me but also for sharing other important things in life like chocolate (Karin), gossip (Caroline) and exercise (Maria)! Thank you, Gelana Yadeta, Margareta “Maggan” Hagstedt, Ann Sjölund and AnnaLena Jarva for your help with everyday issues like keeping track of my LADOK points and answering my stupid questions about where the different chemicals are! Thanks to all the seniors at the department, professors Carmen Fernandez, Klavs Berzins, Marita Troye-Blomberg and Eva Severinsson, for interesting discussions! 77 I would like to thank all present and former students at the Immunology department for making the department a nice place to come in to. John Arko-Mensah, Nancy Away, Halima Balogun, Jubayer Rahman, Amre Nasr, Salah Farouk, Nnaemeka Iriemenam, Anna Tjärnlund, Camilla Rydström, Jacob Minang, Khosro Masjedi, Manijeh Vafa Homann, Fernando Sosa, Olivia Simone, Stefania Verani, Jacqueline Calla, Geshanti Arambepola, Nicky Thrupp, Susanna Kearsly, Charles Arama, Lucia Liszka, Russel AlBalaghi, Stephanie Boström and Olga Daniela Chuquima. I would especially like to thank Lisa Israelsson and Pablo Giusti for keeping the spirit up with your laughs and crazy ideas and Anna-Karin Sigfrinius for teaching me how the QPCR works. Thank you, Anette, Thomas, Ulrika, Björn, Pär and Håkan at Isosep for nice lunches, fun parties and all the gossip! Mina underbara vänner, tack! För att ni aldrig tröttnar på att prata. För allt galet och alla skratt. Särskilt tack till: Carro! Vi har kämpat oss igenom gymnasiet, ett flertal universitetskurser och kan (förhoppningsvis) båda två kalla oss doktorer när 2010 nalkas. Du är en underbar vän som har gett mig en massa stöd! Vi har delat skrattanfall, en minisäng och mer eller mindre lyckade nyårsaftnar och jag hoppas vi får dela så mycket mer i framtiden. Kia! För att du alltid finns där när jag behöver och för att man aldrig vet var man kan hamna när man är med dig. Kanske i ett hustält i Venedig eller på kändisspan i Halmstad. Tack också för att du lät mig hänga i korridoren! Jossan, Saad och Isak! För att ni är så gulliga, för mysiga middagar och alla gånger jag fått skjuts. Och Jossan, visst kan man ha väldigt kul i krogkön? Marie! För att du gjorde studierna så mycket roligare och var en superb labbpartner. Min andra familj som jag har fått under dessa fyra år; Ingvar, Monica, Robin och Karin. Tack för alla härliga skiddagar och middagar vi delat! Jag är så väldigt tacksam för de möjligheter min underbara familj har gett mig. Tack mamma och pappa för att ni alltid funnits där och tragglat glosor, fysik, matte och kemi med mig! Se nu var det har lett… Tack moster Maggan för att du som familjens första doktor har gett mig fotspår att vandra i! Tack Sven och Hanna för allt kul vi hade under barndomen och nu fortsätter ha när vi blivit vuxna! Även om vi alltid kommer vara barnen... Jacob, du har skänkt mig så mycket glädje, stöd och kärlek under dessa år. Jag är väldigt lycklig över att du finns i mitt liv och alltid får mig att le. Det värmer så att du försöker förstå ”de där bakterierna” och tack vare din smittsamma entusiasm har du fått mig att kämpa på när det har varit som tyngst. Jag älskar dig! This work was supported by: The Ekhaga foundation, the Cancer and Allergy foundation, Mjölkdroppen, the Magnus Bergvall foundation, the Konsul Th C Berghs foundation, the Swedish Research Council (57X-1516005-2 and 74X-20146-01-2), the National Swedish Association against Allergic Diseases, the National Heart and Lung Association and the Swedish Foundation for Health Care Sciences and Allergy Research. 78 REFERENCES 1. Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature. 2007; 449(7164):819-826. 2. Kaisho T, Akira S. Toll-like receptor function and signaling. J Allergy Clin Immunol. 2006; 117(5):979-87. 3. Valenta R. The future of antigen-specific immunotherapy of allergy. Nat Rev Immunol. 2002; 2(6):446453. 4. Tacke F, Randolph GJ. Migratory fate and differentiation of blood monocyte subsets. Immunobiology. 2006; 211(6-8):609-618. 5. Serbina NV, Jia T, Hohl TM, Pamer EG. Monocyte-mediated defense against microbial pathogens. 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