Microbial–immune cross-talk and regulation of the immune system

Abstract

We are all born germ-free. Following birth we enter into a lifelong relationship with microbes residing on our body's surfaces. The lower intestine is home to the highest microbial density in our body, which is also the highest microbial density known on Earth (up to 10¹²/g of luminal contents). With our indigenous microbial cells outnumbering our human cells by an order of magnitude our body is more microbial than human. Numerous immune adaptations confine these microbes within the mucosa, enabling most of us to live in peaceful homeostasis with our intestinal symbionts. Intestinal epithelial cells not only form a physical barrier between the bacteria-laden lumen and the rest of the body but also function as multi-tasking immune cells that sense the prevailing microbial (apical) and immune (basolateral) milieus, instruct the underlying immune cells, and adapt functionally. In the constant effort to ensure intestinal homeostasis, the immune system becomes educated to respond appropriately and in turn immune status can shape the microbial consortia. Here we review how the dynamic immune–microbial dialogue underlies maturation and regulation of the immune system and discuss recent findings on the impact of diet on both microbial ecology and immune function.

Introduction

A 30-μm-thick single cell intestinal epithelial layer physically segregates not only 100 trillion bacteria but also foreign antigens and sporadic pathogens from our body.^1–3 We are therefore constantly living dangerously, exposed to the threat of being invaded by our indigenous bacteria. Long co-evolution between host and bacteria has led to sophisticated immune protection mechanisms at the intestinal epithelial interface so our blissful relationship with our microbial partners does not stem from ignorance.^4,5 Rather, our immune system engages in a constant yet controlled sampling of luminal antigens through specialized cells including M cells or CX₃CR1⁺ dendritic cells (DC).^6,7 Although such mechanisms contribute to the symbiotic relationship with intestinal commensals, they explain neither how pathogens are sensed nor how patients suffering from inflammatory bowel disease over-react against their commensals. Investigating the establishment of intestinal homeostasis ensuing after bacterial encounter is key towards the understanding of this complex immune–microbial cross-talk.

The majority of the microbiota is currently unculturable but advances in non-culture-based techniques offer exciting avenues to glimpse the enormous complexity of the intestinal ecosystem.⁸ Such metagenomic techniques rely on the genetic sequences of the 16S ribosomal DNA, which are highly conserved yet sufficiently varied among species.

With the birth of each newborn, a new microbial ecosystem is also born. Microbial communities progressively colonize mucosal and epithelial surfaces and eventually converge towards a stable generic adult-like profile at around 3 years of age.⁹ This transitional intestinal colonization follows a highly dynamic pattern characterized by a unique combination of bacterial species, which appear or disappear over time. It is generally accepted that infants acquire a microbiota through opportunistic colonization by available surrounding environmental bacteria, which would explain this ‘trial and error’ colonization.^9–13 Supporting this, the mode of delivery dictates the founding bacterial ecosystem with a predominance of either maternal faecal–vaginal or environmental bacteria following delivery by the birth canal or Caesarean section, respectively.¹⁴ The adult microbiota is represented by an estimated 1000 bacterial species classified in only 8–10 of the 55 known bacterial divisions.^15–18 Each individual possesses an idiosyncratic microbial fingerprint that remains stable over months.^15,19,20 Sequencing of faecal metagenomes from 22 individuals from four countries of three different continents revealed that the human gut microbes may group into three distinct combinations at the community level, termed ‘enterotypes’, with high levels of bacteria from the genus Bacteroides (enterotype 1), Prevotella (enterotype 2) or Ruminococcus (enterotype 3).²¹ Whether these enterotypes are stable over time or confounded by common transient states of health, environmental conditions, or nutrition is still unclear. More recent work suggests nutrition as a major enterotype-determining factor.^22,23 Another study explored microbiota dynamics in greater detail using deep-sequencing analysis of human microbiota composition at multiple time-points, showing pronounced variability in an individual's microbiota across months, weeks and even days.²⁴

In this review we will discuss the dynamic microbial–immune cross-talk and emphasize the impact of microbial composition on the educational cues conveyed to the immune system. Finally, we will summarize recent findings on diet on the microbial–immune axis.

Intestinal microbial education of the immune system

Because bacterial colonization and immune system maturation occur simultaneously, studies in germ-free animals represent invaluable tools to uncouple these two postnatal events.²⁵ Studying the relationship between the host and its microbiota began with Louis Pasteur, who postulated that life was dependent on microbial colonization.²⁶ Although this was proven wrong 10 years later by Nuttal and Thierfelder²⁷ who undertook the challenging task to raise guinea pigs under germ-free conditions, this opened new scientific questions that have fascinated scientists to-date. Germ-free animals are born and raised under absolute sterile conditions within flexible-film isolators maintained under positive pressure with HEPA-filtered sterile air.²⁸ Deliberate association of germ-free animals with a defined microbiota can experimentally address the formation of a new ecosystem and its direct impact on the host. Technically, colonization experiments are performed by simple co-housing of a germ-free mouse with a mouse harbouring a bacterial flora or by oral gavage with single bacterial species or defined consortia of bacterial species. The power of such colonization experiments resides in the capability to experimentally control both host genotype and bacterial species, the latter ranging from undefined floras to a single defined bacterial species. Such landmark experiments could discern developmental changes from microbe-driven immune changes and formally prove that indigenous bacteria are true educators of the immune system both at mucosal and systemic sites.

Whereas few IgA-expressing B cells are detected in the lamina propria of germ-free mice, B-cell proliferation and induction of IgA follows bacterial encounter.^29,30 Moreover, proliferating B cells promote an increase in the size and number of the germinal centres in the Peyer's patches³¹ and maturation of cryptopatches into isolated lymphoid follicles.³² The numbers of intestinal lamina propria CD4⁺ T cells³³ and intra-epithelial lymphocytes expressing the αβ T-cell receptor are increased.³⁴ We have also shown that colonization with a benign microbiota specifically induces an increase in colonic regulatory T cells.³⁵ Furthermore, genes involved in host–microbe interactions such as nutrient absorption, epithelial barrier strengthening, angiogenesis and xenobiotic metabolism are actively transcribed.³⁶ Despite compartmentalization of intestinal microbes to the mucosa, their impact on the immune system reaches beyond this physical boundary through less well-understood mechanisms.³³ While the germinal centres in the spleen and peripheral lymph nodes gain cellularity,³³ total serum IgA, and also IgM and IgG, levels increase. Accumulating evidence suggests that intestinal microbes influence the readiness of the extra-intestinal immune system to mount protective responses towards pathogens. For example, the microbiota has been shown to be important for antiviral immune responses in the respiratory mucosa of mice. Antibiotic treatments resulted in defective CD4⁺ T-cell, CD8⁺ T-cell and B-cell immunity following intranasal infection with influenza virus.³⁷ Conversely, influenza virus infection increased the risk for Listeria monocytogenes infection through a general glucocorticoid-induced immune suppression.³⁸

Lessons from anatomy

In the gut-associated lymphoid tissue, distinct lymphoid inductive and effector sites integrate stimuli directly from the gut lumen. Peyer's patches, isolated lymphoid follicles and mesenteric lymph nodes (MLN) are the main inductive sites whereas the lamina propria is the main effector site. The afferent lymphatics converging to the MLN drain the Peyer's patches, isolated lymphoid follicles and the lamina propria whereas the efferent lymphatics enter into the bloodstream via the thoracic duct. Following MLN adenectomy indigenous intestinal bacteria infiltrate into the bloodstream, demonstrating that the MLN are true microbial firewalls at the mucosal–systemic junction.³⁹ Sharing anatomical and functional similarities, Peyer's patches and isolated lymphoid follicles are lymphoid follicles dispersed along the small intestine that sample bacteria through overlaying microfold (M) cells intercalated within the epithelial cell layer. Underneath the M cells DC endocytose and present intestinal antigens whereby bacteria-specific B-cell and T-cell responses are induced. Alternatively, luminal antigens can also be sampled by transepithelial dendrites from subepithelial CX₃CR1⁺ resident DC.^6,7 A fraction of these intestinal resident DC migrate to the MLN where specific B-cell and T-cell responses are induced. In contrast to systemic DC, intestinal DC have a poor bactericidal activity and a rapid turnover, which allow engulfed live bacteria to reach but not travel past the MLN.⁴⁰ Activated T and B cells (but not DC) exit the MLN via efferent lymphatics and eventually home back to their effector site, the lamina propria. Activated B and T lymphocytes are imprinted for mucosal homing to the small intestine by the expression of integrin α₄β₇, which mediates vascular adhesion and binding to the mucosal addressin molecule MAdCAM-1.⁴¹ In addition, B and T cells often use different receptor combinations, especially chemokine receptors, to home to the small or large intestines. Surface expression of CCR9 endows homing to the small intestine where its ligand, CCL25, is produced.^42,43 Induction of CCR9 on T cells is enhanced by a CD103⁺ DC subset that is abundant in the lamina propria of the small intestine and capable of rapidly inducing retinoic acid signalling events upon T-cell priming.⁴⁴ A large proportion of IgA⁺ B cells in the small intestine and all in the colon express CCR10, which allows them to respond to the widely expressed mucosal epithelial chemokine CCL28.^43,45

The immuno-epithelome at the interface between innate and adaptive immunity

Beyond their role as physical barriers, intestinal epithelial cells have been re-evaluated as frontline immune cells. The orchestrated triad between epithelial, innate and adaptive cells (the ‘immuno-epithelome’)⁴⁶ may underlie the readiness of the immune system to constantly sense and adapt to luminal antigens (Fig. 1a).^46–48

Figure 1.

The cross-talk between microbes, dietary compounds and the immune system at epithelial surfaces. (a) Luminal microbial cues are sensed and relayed by intestinal epithelial cells expressing Toll like receptors (TLR). Gram-positive bacteria induce secretion of anti-bacterial RegIIIγ in an epithelial intrinsic manner, a process that is crucial for the limitation of bacterial adherence to the epithelium. Epithelium secreted cytokines including interleukin-17C (IL-17C), IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) modulate the mucosal immune milieu. Whereas IL-17C acts in an autocrine manner to induce anti-microbial peptides and pro-inflammatory cytokines, IL-25, IL-33 and TSLP have been associated with protective mucosal T helper type 2 responses such as against helminth infections. On the other hand epithelial cells respond to IL-22 produced by the underlying innate lymphocytes (as well as T helper type 17 cells and γδ T cells) by increasing secretion of anti-microbial peptides, a process that is further enhanced by the presence of IL-17C. (b) Dietary compounds bind to immune cells and provide immune protective mechanisms. The microbiota metabolizes dietary fibres into short-chain fatty acids such as butyrate or acetate, which strengthen epithelial barrier integrity and promote resolution of intestinal inflammation. Dietary compounds contained within cruciferous vegetables bind to aryl hydrocarbon receptor (AHR), a process that is necessary for the formation of isolated lymphoid follicles (ILF) and intra-epithelial lymphocytes (IEL).

Innate sensing and dynamic adaptations to luminal contents

Following birth, intestinal epithelial cells (IEC) become exposed to microbes. The IEC express Toll-like receptors (TLR) that relay innate immune signals following encounter with conserved microbial structures called pathogen-associated molecular patterns. One mechanism by which IEC induce and maintain tolerance to the colonizing bacteria is by repressing the TLR signalling molecule IRAK1 (interleukin-1 receptor-associated kinase 1) via microRNA-146a.⁴⁹ Intrinsic TLR signalling directly modulates the expression of Reg3g in a microbe-dependent manner,^50,51 a process supported by epithelium-derived cytokines including interleukin-22 (IL-22) and IL-17C.^52,53 RegIIIγ is a C-type lectin that is mostly expressed in the small intestine by enterocytes and Paneth cells and specifically targets Gram-positive bacteria. Mice deficient in RegIIIγ or myeloid differentiation primary response gene (88) (MyD88; the adaptor protein shared by all TLRs except TLR3) in IEC fail to maintain the ∼50 μm spatial segregation between IEC and luminal bacteria.⁵¹ As a result, RegIIIγ is necessary and sufficient to limit interaction of Gram-positive bacteria with the epithelial cell layer and may provide an explanation as to why patients suffering from inflammatory bowel diseases have increased expression of Reg proteins at mucosal sites.⁵⁴ As a compensatory mechanism to limit bacterial–epithelial interaction, RegIIIγ^−/− mice show increased IgA-producing plasma cells and increased T helper type 1 (Th1) cells in the lamina propria. Supporting a microbially malleable Reg3g expression, anti-microbial expression patterns are highly dynamic in neonates.^55,56

IL-22, a messenger to epithelial cells

Innate immune cells include myeloid and innate lymphocytes and, in terms of evolution, appeared before adaptive immune cells. We have begun to appreciate that innate lymphocytes (ILC) are not confined to natural killer (NK) cells but encompass a much broader cellular diversity. Innate lymphocytes have been recognized to be important mucosal effector cells and include lymphoid tissue inducer cells (LTi or NKR⁻ RORγt⁺ ILC), a subset of NK-like cells (NKR⁺ RORγt⁺ ILC) and the newly discovered Lin⁻ c-kit⁺ cells termed natural helper cells, which include multi-potent progenitor type 2 (MPP^type2) cells, nuocytes, innate type 2 helper cells and natural helper cells.^57–61 These cells all belong to the lymphoid lineage as shown by their absence in Rag^−/−Il2rγ^−/− mice, which do not support the development of lymphoid cells.^62,63

Until recently LTi cells have been exclusively studied in the context of embryonic lymph node development. Lymph node anlagen are initiated via the interaction of lymphotoxin-α₁β₂ expressed on LTi and lymphotoxin-β-R expressed on stromal cells. Beyond their pivotal role in the genesis of lymph nodes, LTi cells have been recognized as influential intestinal immune effector cells in postnatal life. The LTi cells depend on the orphan transcription factor retinoic acid-related orphan receptor γt (RORγt) and express copious amounts of IL-17 and IL-22. They are phenotypically heterogeneous with subpopulations of CD4⁺ CD127^high CD117^high, CD4⁻ CD127^high CD117^high and CD4⁻ CD127^low CD117^low.

Another subset of cells expressing RORγt and NK receptors (NKR) has been identified in the small intestine, colon, MLN and liver.^52,64 As in LTi cells, these NKR⁺ RORγt⁺ ILC express IL-22 (but not IL-17). The microbiota provides important cues for the production of IL-22 and germ-free mice fail to develop IL-22-producing NKR⁺ RORγt⁺ ILC but not NK and LTi cells.^52,64 In addition to NKR⁺ RORγt⁺ ILC and NKR⁻ RORγt⁺ ILC, IL-22 is also produced by Th17 cells⁶⁵ and γδ T cells, all represented at mucosal surfaces. Intriguingly, the receptor for IL-22 is exclusively expressed on IEC, indicating that IL-22-producing cells in the mucosa directly instruct epithelial cells.^66–68

Intestinal epithelial cells respond to IL-22 by secreting anti-microbial molecules including RegIIIβ/γ and gauge the expression and secretion of these molecules to the amount of IL-22.⁶⁸ Therefore, IL-22 represents a messenger of the immuno-epithelome cross-talk. Whereas IL-22 is increased following intestinal inflammation, it remains constitutively expressed during homeostasis. Following infection with the attaching/effacing pathogen Citrobacter rodentium, IL-22 production increased leading to enhanced epithelial anti-microbial peptide secretion.^68,69 Whereas Rag^−/−Il2rγ^−/− mice succumbed after C. rodentium infection, Rag^−/− mice could control the infection during the first weeks in an IL-22-dependent manner.⁶⁴ Although NKR⁻ RORγt⁺ ILC seem to be the crucial IL-22 source in this model as suggested by disease exacerbation following in vivo depletion, both NKR⁺ RORγt⁺ ILC and NKR⁻ RORγt⁺ ILC up-regulate IL-22 production early after C. rodentium infection.^64,69 A protective role for innate IL-22 was also shown in models of colitis induced by transfer of naive CD4⁺ CD45RB^high T cells into lymphopenic Rag^−/− hosts or administration of oral dextran sulphate sodium (DSS).^70,71 Conversely, in Helicobacter hepaticus-induced colitis innate cells promoted disease, but in this case this could be traced back to their production of IL-17.⁷²

Autocrine epithelial IL-17C

Three reports have characterized the biological functions and the receptor for IL-17C, which consists of the heterodimer formed by IL-17RA and IL-17RE.^53,73,74 Both IL-17C and its receptor IL-17RE are highly expressed in colonic IEC and together orchestrate innate anti-bacterial responses. Mice with impaired IL-17C–IL-17RE signalling showed pronounced inflammation following infection with C. rodentium⁵³ or DSS-induced colitis⁷³ resulting from impaired secretion of IEC anti-bacterial peptide, pro-inflammatory cytokines and chemokines. Moreover, IL-17C and IL-22 were demonstrated to act in synergy and enhanced the expression of anti-bacterial peptides including S100A8/9 and RegIIIβ/γ. Although the mechanisms whereby IL-17C is regulated at homeostasis and its impact on indigenous bacteria remain to be tested, this newly uncovered IL-17–IL-17RE axis joins the ‘immuno-epithelome’ dialogue.

Thymic stromal lymphopoietin

Thymic stromal lymphopoietin (TSLP) belongs to the IL-7 family and was discovered more than a decade ago. It owes its name to its identification in conditioned supernatant of a mouse thymic stromal cell line as a B-cell growth factor.⁷⁵ Epithelial cells constitutively express Tslp and its expression can be increased following breach of the epithelial barrier. The receptor for TSLP is expressed on B cells, monocytes, DC, mast cells, activated CD4⁺ T cells and IEC from jejunum, ileum and colon.^76,77 In addition to its key role in Th2 responses, such as instances of allergic diseases and helminth infections, TSLP appears to be critical in the resolution of intestinal inflammation and homeostasis.⁷⁶ At steady-state, epithelial-derived TSLP has been shown to condition DC towards a non-inflammatory Th2 type.^78,79 In helminth infections, TSLP enhanced protective Th2 responses following infection with Trichuris muris⁸⁰ but was dispensable for control of Heligmosomoides polygyrus or Nippostrongylus brasiliensis infection.⁸¹ In DSS-induced colitis TSLP has an immunoregulatory role. Taylor et al. ⁸² reported that absence of TSLP signalling led to higher inflammation marked by IL-12 and interferon-γ. Consistent with those results, genetic ablation of intrinsic intestinal epithelial cell TLR signalling via nuclear factor-κB lowered TSLP production and increased IL-12-producing DC.⁸³ On the other hand, Reardon et al.⁷⁶ did not observe elevated inflammation but rather an impaired resolution. In this study epithelial breach led to increased TSLP, which up-regulated secretory leucocyte proteinase inhibitor (SLPI), a serine protease inhibitor, antimicrobial peptide and inhibitor of nuclear factor-κB. Interestingly TSLP could act in an epithelial autocrine manner to induce SLPI. Hence, TSLP is a critical regulator of intestinal immune homeostasis and modulation of TSLP concentration may serve as an indicator of intestinal epithelial cell integrity.

IL-25 and IL-33

IEC are also a source of IL-25 (also known as IL17-E) and IL-33, and, in a similar maner to TSLP, have been implicated in intestinal homeostasis and Th2 responses. Increased expression of these cytokines has been associated with exposure to allergens or helminths.^84–87 Recently, four independent laboratories newly identified IL-25 and IL-33 responsive Lin⁻ c-kit⁺ natural helper cells.^57–60 Whether these cells (MPP^type2, nuocytes, innate type 2 helper cells, and natural helper cells) represent distinct populations or whether they are related remains to be carefully determined. However, they share similar biological functions in that they elicit Th2 responses and have been implicated in anti-helminth responses. Interestingly the IL-5-producing natural helper cells found in the mesentery and in fatty deposits in the peritoneal cavity and surrounding the kidney promote B-1 cell renewal and production of IgA.⁶⁰ The biological relevance of IL-25 and IL-33 at homeostasis and whether bacterial stimuli from commensals set the baseline expression of these epithelial-derived cytokines remains to be elucidated.

Adaptive immune adaptations

Regulatory T cells–Th17

In the thymus, self-reactive T cells are eliminated or differentiated into regulatory T (Treg) cells. In addition to the thymus-derived natural Treg (nTreg) cells, naive T cells can differentiate into Treg cells in the periphery and provide tolerance to foreign antigens as induced T reg (iTreg) cells.⁸⁸ Expression of Helios, an Ikaros transcription factor family member, is a potential surrogate marker that seems to be able to distinguish nTreg (Helios⁺) from iTreg (Helios⁻) cells in vivo.⁸⁹

Mucosal surfaces are exposed to large amounts of foreign innocuous antigens from ingested foods and commensals. Induction of oral tolerance prevents pro-inflammatory responses to such antigens by mechanisms involving T-cell anergy or clonal deletion. Additionally, the co-ordinated induction, migration and maintenance of Treg cells in the lamina propria plays a pivotal role in the establishment of oral tolerance. Ten years ago it was established that neonatal colonization with a diversified intestinal microbiota, but not a single bacterium, is required for successful induction of oral tolerance to ovalbumin.^90,91 Later it was shown that oral tolerance to ovalbumin involved the induction of Treg cells in the MLN in a process dependent on antigenic presentation by CD103⁺ DC and favoured by retinoic acid and transforming growth factor-β. Homeostasis of Treg cells required IL-10 produced by intestinal CX₃CR1⁺ intestinal resident macrophage-like cells.^92–94

Although commensal bacteria are innocuous to an immunocompetent host, real-life microbiotas harbour species ranging from true commensals to pathobionts. Accordingly, immune responses are carefully shaped and adapted to the prevailing commensal–pathobiont scale.^95,96 Colonization with defined commensals (non-pathobionts) contained within the altered Schaedler flora leads to induction and accumulation of Treg cells in the colonic lamina propria, critical for the establishment of homeostasis at the mucosa.³⁵ Induction of Treg cells has also been associated with colonization with Clostridium species or polysaccharide A from Bacteroides fragilis.^97–99 Conversely, if the microbiota contains pathobionts, such as the prototypic epithelial-associated segmented filamentous bacteria,¹⁰⁰ Th17 cells probably bolster Treg cells to prevent bacterial invasion.^101,102 As expected, fewer Treg cells can be found in the lamina propria of germ-free hosts and Th17 cells are virtually absent.^{35,97,103,104} Hence, while Treg cells induced by commensals are critical for intestinal homeostasis, Th17 cells induced by pathobionts are likely to be critical for combating invasive pathobionts and their presence is critical for intestinal homeostasis. Based on the finding that the T-cell receptor repertoire from Treg cells at mucosal sites was distinct from other compartments, a recent study suggested that intestinal bacteria directly shape the Treg-cell repertoire in the intestine.¹⁰³

IgA-secreting plasma cells

Undoubtedly one of the most striking differences between germ-free and colonized animals is the prominent induction of IgA at mucosal surfaces.^105,106 At least 80% of all plasma cells are located in the lamina propria, and together they produce more IgA (in humans 40–60 mg/kg/day) than all other immunoglobulin isotypes combined, indicating strong evolutionary pressure for the maintenance of this energy demanding output. Serum IgA is monomeric, whereas mucosal secretory IgA is dimeric through covalent interaction with a joining (J) chain attached to the two constant regions. Dimeric IgA is produced at mucosal sites and secreted into the lumen via transcytosis mediated by the polymeric immunoglobulin receptor pIgR expressed on the basolateral site of intestinal epithelial cells.¹⁰⁷ This is a sacrificial transport because a portion of the receptor (the secretory component) undergoes proteolytic cleavage to release dimeric IgA into the intestine.^108,109 In humans (but not in mice), there are two subclasses of IgA – IgA1 and IgA2 – encoded by two separate constant chains with IgA2 most commonly found at mucosal surfaces.

Paradoxically, the biological functions of IgA still remain partially enigmatic. Astonishingly, selective IgA deficiency is the most common humoral immunodeficiency in humans, occurring at a frequency of about 1 in 500 to 1 in 2000.¹¹⁰ IgA-deficient individuals are not overtly symptomatic and the same is recapitulated in IgA-deficient mice.¹¹¹ The mild phenotype is traced back to compensation of secretory IgM, illustrating the sophisticated immune flexibility at mucosal surfaces to ensure homeostasis. Notwithstanding this, experimental evidence supports a key role for IgA at mucosal surfaces. Specific secretory IgA has been shown to inactivate rotavirus intracellularly during epithelial transcytosis,¹¹² protect against influenza virus,¹¹³ and neutralize the potent mucosal immunogen cholera toxin.¹¹⁴ The observation that secretory IgA is induced by and binds to commensals strongly suggests that IgA limits epithelial adherence, a process referred to as immune exclusion.¹¹⁵ IgA has been described as ‘polyspecific’ with low affinity to bacteria but this could reflect technical limitations stemming from the inherent vast diversity of intestinal microbes. Indeed we have shown that colonization with a single bacterium induces IgA that specifically binds to the colonizing bacterium and that IgA specificity dynamically adapts to changes in intestinal microbial composition.³⁰ Moreover, administration of exogenous Bacteroides thetaiotaomicron-specific IgA antibodies in lymphopenic Rag1^−/− hosts limited pro-inflammatory responses following B. thetaiotaomicron monocolonization supporting an additional anti-inflammatory role for specific IgA.¹¹⁶ Moreover, specific IgA has been suggested to play a role in regulating luminal bacterial composition. Aberrant anaerobic expansion is observed in lymphopenic Rag2^−/− and SCID mice. In this case the whole adaptive arm of immunity is absent, which makes it difficult to conclude on the direct implications of IgA.^100,117 Mice deficient in activation-induced cytidine deaminase (AID), an enzyme involved in class switch recombination and somatic hypermutation, showed increased expansion of anaerobic bacteria in the proximal intestine.¹¹⁸ Recently, the same authors characterized mice carrying a knock-in mutation in AID (AID^G23S) that, while maintaining class switch recombination, had impaired somatic hypermutation and therefore absence of high-affinity IgA.¹¹⁹ Similarly to AID^−/−, a dysbiosis was observed in AID^G23S mice demonstrating that IgA specificity directly shapes intestinal microbial composition.¹¹⁹ Furthermore, antibody-deficient (JH^−/−) and AID^G23S mice have increased bacterial translocation into the MLN, indicating that mucosal antibodies prevent bacterial breach of the epithelial barrier.^39,119 Taken together these studies indicate that mucosal IgA specificity to indigenous microbes confers immune exclusion and regulates bacterial composition.

Diet effects on microbial ecology and immune function

The inter-relationship between gut microbes and metabolic processing of nutrients is well appreciated. While intestinal microbes thrive in a safe and nutrient-rich niche, the host profits from their metabolic activity, which renders otherwise indigestible foods available. Exemplifying this, animals harbouring a microbiota require 30% less caloric intake to maintain their body weight than their germ-free counterparts.¹²⁰ Emerging studies indicate that beyond its caloric contribution, diet plays a dynamic and previously unrecognized role in shaping both bacterial ecology and the immune system.¹²¹ Pattern recognition receptors including TLRs, inflammasomes, C-type lectins such as dectin-1 and RNA-sensing retinoic acid-inducible gene (RIG) -like helicases such as RIG-I and melanoma differentiation-associated gene-5 (MDA5), relay microbial cues to the immune system. Interestingly free fatty acids and ATP induce downstream signalling of TLR2/4 and the inflammasome, respectively.^122,123 In addition to pattern recognition receptors other receptors expressed on immune cells can sense the metabolic environment including the serine/threonine kinase mammalian target of rapamycin (mTOR),¹²⁴ double-stranded RNA-activated protein kinase (PKR),¹²⁵ the aryl hydrocarbon receptor (AHR),¹²⁶ and diverse nuclear hormone receptors.¹²⁷ The metabolism from otherwise indigestible plant polysaccharides into short-chain fatty acids (SCFA) by the microbiota and its impact on immune system function has been extensively investigated (Fig. 1b). Intake of fibres dictates the amount of metabolized SCFA available in the lumen and this in turn shapes intestinal microbial ecology. Conversely, SCFA have an impact on the immune system. For example, low abundance of the SCFA butyrate diminishes T-cell derived cytokines¹²⁸ and reinforces intestinal barrier integrity.¹²⁹ Another microbial-processed SCFA, acetate binds to the G-protein coupled receptor 43 (GPR43), which is essential for resolution of intestinal inflammation.¹³⁰ In another study, acetate has been linked to promote and maintain intestinal epithelial integrity and so protect against the enteropathogen Escherichia coli (O157 : H7).¹³¹ Experiments conducted in gnotobiotic mice associated with prototypic human microbes have demonstrated that dietary habits have a rapid, profound and predictable impact on the relative abundance of species and genes making up the microbiota and the microbial metagenome, respectively.^23,132,133 Supporting this, microbial communities were shared among mammalian species and humans with similar dietary habits.²² However, a direct relationship between dietary compounds and maturation of the immune system has only recently been shown. Physiological signalling through AHR, which was originally discovered for its role in detoxification processes in the liver, has been shown to be crucial for the formation of intra-epithelial lymphocytes¹³⁴ and isolated lymphoid follicles¹³⁵ and AHR signalling promotes the accumulation of innate lymphoid cells.¹³⁶ Cruciferous vegetables, such as broccoli, contain a physiological ligand for the AHR and its deprivation recapitulated the phenotype observed in Ahr^−/− mice. Defective AHR signalling resulted in impaired mucosal protective mechanisms as shown by exacerbated DSS-induced colitis or increased susceptibility to C. rodentium infection. Future studies should offer exciting new avenues into the cross-talk between the host, microbes, metabolism and immunity.

Immune disorders

Autoimmune and allergic immune disorders such as inflammatory bowel disease, multiple sclerosis or asthma are rapidly increasing in westernized countries.^137–139 Genetic background certainly plays a role in disease predisposition in some individuals but genetics alone does not offer a satisfactory explanation for the observed increase in incidence. In 1989 Strachan¹⁴⁰ formulated the hygiene hypothesis based on the observation that hay fever was less prevalent in children with older siblings. The hygiene hypothesis argues that increased sanitation in industrialized countries led to decreased infections with common pathogens and a concomitant rise in allergic disorders. In the last decades westernized countries undertook drastic measures to increase hygiene including water decontamination, food pasteurization and sterilization, uninterrupted cold chain, vaccination and wide-use of antibiotics. Accumulating evidence indicates a shift in the composition of indigenous intestinal microbes in westernized countries.¹⁴¹ Changes in lifestyle in industrialized countries such as antibiotic usage and dietary habits undoubtedly impact the microbiota composition. In line with this, the microbial communities of European children are dramatically dissimilar from those of rural Africans as demonstrated in a metagenomic study.¹⁴² In addition to a greater microbial diversity, the cellulose-hydrolysing and xylan-hydrolysing bacteria Prevotella and Xylanibacter were only detected in children from Africa and this was consistent with increased SCFA. However experimental evidence that changes in type and level of microbial stimulation can impact disease outcome, are mainly supported by animal models. The non-obese diabetic (NOD) mouse and the biobreeding diabetes-prone (BB-DP) rat develop a disease that shares many similarities with Type 1 diabetes and are therefore a good animal model for the study of Type 1 diabetes. Interestingly, the incidence of Type 1 diabetes in these animals was correlated to the hygiene conditions prevailing in the animal facility. The incidence of diabetes was lower in animals raised under conventional status compared with animals raised under specific pathogen-free (SPF) status.^143,144 A direct link between microbiota and Type 1 diabetes was shown in NOD mice deficient for the TLR adaptor molecule MyD88. Whereas germ-free MyD88^−/− NOD mice had a high diabetes incidence, their SPF counterparts were protected from disease.¹⁴⁵ The authors concluded that the normal microbial stimuli protect from diabetes in a MyD88-independent manner. In a model of ovalbumin-induced asthma, an increased number of infiltrating lymphocytes and eosinophils with more pronounced secretion of Th2 cytokines was observed in airways of germ-free mice compared with SPF mice.¹⁴⁶ Similarly, in a model of peanut allergy, mice treated with antibiotics or deficient in TLR4 underwent anaphylaxis, suggesting that bacterial-induced TLR4 signalling is critical in limiting inflammation.¹⁴⁷ Clinical studies comparing the microbiota from healthy controls and patients suffering from allergic or autoimmune disease are in agreement with the animal models.^138,148 For instance, patients suffering from Crohn's disease had a reduced diversity in the microbiota especially for the Firmicutes phylum.^149,150 Administration of Faecalibacterium prausnitzii to mice, a species of Firmicutes that was markedly depleted in Crohn's disease patients, could ameliorate disease outcome in experimental 2,4,6-trinitrobenzenesulphonic acid-induced colitis.¹⁵¹ Although some studies comparing the microbial composition of atopic allergic individuals with that of healthy individuals observed differences,^152,153 others could not reproduce this finding.¹⁵⁴ Such comparative studies have to be interpreted with caution because dysbiosis could be a consequence rather than the cause of inflammatory disease. Nonetheless evidence provided from animal models and clinical studies is accumulating to support the concept that immune regulation can be influenced by the composition of intestinal microbes.

Concluding remarks

It is undisputable that immune education and indigenous microbes influence each other and form an entity. In the last decade our understanding of mucosal immunology has flourished with the appearance of high throughput sequencing of the microbiota. The immune system is moulded through its indigenous microbial communities and no longer can mucosal immunologists study a microbiota without specifying its composition. Conversely the immune pressure exerted by the host influences microbial communities. Dietary compounds have been shown to act on both the immune system and microbial composition. Hence, the neonatal immune system may sample environmental cues provided by both the founding colonizing bacteria and the ingested dietary compounds and mature accordingly. Changes in lifestyle in westernized countries may have altered or temporally delayed the type and level of such stimuli and provide some mechanistic insight into the increasing levels of immune-mediated disorders.

Disclosure

The authors have nothing to disclose.