An Expanding Stage for Commensal Microbes in Host Immune Regulation

REVIEW

An expanding stage for commensal microbes in host immune regulation

Yan Shi1,2 and Libing Mu1

Gastrointestinal commensal microbiota is a concentrated mix of microbial life forms, including bacteria, fungi, archaea and viruses. These life forms are targets of host antimicrobial defense in order to establish a homeostatic symbiosis inside the host. However, they are also instrumental in shaping the functions of our immune system via a diverse set of communication mechanisms. In the gut, T helper 17, regulatory T and B cells are continuously tuned by specific microbial strains and metabolic processes. These cells in return help to establish a mutually beneficial exchange with the gut microbial contents. Imbalances in this symbiosis lead to dysregulations in the host’s ability to control infections and the development of autoimmune diseases. In addition, the commensal microbiota has a significant and obligatory role in shaping both gut intrinsic and distal lymphoid organs, casting a large impact on the overall immune landscape in the host. This review discusses the major components of the microbial community in the gut and how its members collectively and individually exert regulatory roles in the host immune system and lymphoid structure development, as well as the functions of several major immune cell types. Cellular & Molecular Immunology (2017) 14, 339–348; doi:10.1038/cmi.2016.64; published online 9 January 2017

Keywords: commensal fungi; gut microbiota; peripheral lymphoid organs; T cell subtypes

INTRODUCTION quantitative measurements, the list of immune regulatory factors Symbiosis is common in biology.1 Although in the cases of large and cells that are most abundantly found in the gut includes T species, this co-existence is often a visual wonder, host microbe helper 17 (TH17) cells, γδ T cells and immunoglobulin A (IgA)- symbiosisisdeeplyrootedinbiologicalsystemsformore producing B cells. The containment of microbiota is accom- fundamental reasons. Making up 60% of fecal mass,2 gastro- plished at several levels. The epithelia of the intestinal lumen is intestinal colonization of microbial species in higher animals is covered by a mucus layer. Some proteins in this layer, such as likely to first serve as a metabolic complement in food Mucin2, play important roles in downregulating intestinal processing, content fermentation and detoxification.3 The diges- dendritic cells’ (DC) inflammatory responses.8 In the lower tion of complex sugars by gut microbiota provides not only an intestine where the microbial burden is relatively heavy, this additional source of energy to maintain the microbial commu- protective layer is particularly thick. The occasional microbial nity but also essential nutrients for gut epithelial cells.4 Germ- breach of defenses results in microbes that are either phagocy- free (GF) animals are resistant to high calorie food intake- tosed by macrophages or taken into the lamina propria (LP) by induced weight gain5 and, accordingly, require 30% more energy DCs for prolonged storage and antigen sampling.9 In addition to to maintain a similar body mass.6 In addition, biochemical the barrier function, gut-associated lymphoid organs (GALTs) evidence indicates that specific bacterial strains located in the produce large quantities of IgA, often targeting antigens that are intestine promote lipid storage.7 Therefore, energy harvesting is unique in the microbiota.10 In addition, TLR ligands in the gut thedrivingforceformammalianhostsandsetsthestagefor stimulate Reg3β and Reg3γ antimicrobial molecule production their microbial companions in the gastrointestinal tract. by epithelial cells,11 although in the case of intestinal Paneth cells Whilethissalubriousecologyisbeneficial, the host exerts an (specialized in secretion of antimicrobial substances), these enormous effort to contain this cohort of 'guest workers.' By molecules are constitutively produced.12

1Institute of Immunology, Department of Basic Medical Sciences, Center for Life Sciences, Tsinghua University, Beijing 100084, China and 2Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary T2N 4N1, Canada Correspondence: Dr Y Shi, Institute of Immunology, Department of Basic Medical Sciences, Center for Life Sciences, Tsinghua University, D301 Medical Sciences Bldg., Beijing 100084, China. E-mail: [email protected] Received: 7 September 2016; Revised: 12 October 2016; Accepted: 12 October 2016 Commensal microbes in host immune regulation YShiandLMu

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For the host, this defense mechanism is not a zero sum dysbiosis in diseases. In this writing, we intend to emphasize game. Our immune system develops out of the backdrop of a two particular points: how the microbiota impacts several large number of microbial species. This deep interconnection important immune cell types (Figure 1) and how it helps to was discovered soon after germ-free animals were developed. establish secondary lymphoid structures (Figure 2). While GF animals have life spans that are similar or slightly longer than normally reared counterparts,13 their immune The microbes in the gut systems are underdeveloped.14 They were found to have very Amniotic fluid is sterile. While the fetus is exposed to minute few 'reaction centers' and lymphocytes. Bauer et al.14 reported amounts of released microbial antigens in gestation, the first that in GF Swiss Webster mice, there was a universal reduction inoculation does not take place until environmental exposure in the size of lymph nodes, number of macrophages and after birth. Newborns by vaginal delivery receive the first seeds 'immune competent cells.' It is now well established that a of fecal microbiota from their mother. In comparison, Cesarean healthy immune system relies on a well-maintained microbiota. delivery is associated with a slow acquisition of two dominant In fact, evolution has very likely applied selection pressure to bacterial divisions, Firmicutes and Bacteroidetes,withthegut enrich the diversity of microbiota in higher animals.15 One inoculation mainly consisting of maternal skin-resident species. hypothesis even speculates that the extreme diversity in the gut This difference leads to significantly higher rates of asthma, of more complex animals is linked to the development of connective tissue disorders, arthritis, leukemia and so on, per adaptive immunity. In contrast, small and short-lived life forms population statistics over lifetime in the latter.17,18 After birth, have no such need for immune memory functions.16 Addi- the microbial composition undergoes a period of variation until tional investigation is required to determine the veracity of this it reaches a relatively stable mixture. It should be noted that the hypothesis. However, the work in the last decade has revealed a presence of stable microbiota by itself conveys the generic complex pattern of host microbiota exchange, including local 'colonization resistance.' In other words, the preexistence of immune regulations, overall immune development and these microbes has the natural ability to limit the expansion of

Figure 1 Gut microbial environment impacts resident lymphocytes. In the steady state, gut microbial contents polarize and ﬁne-tune several types of lymphocytes. The presence of bacterial strains such as segmented ﬁlamentous bacteria (SFB) drives B cells to produce IgA, likely assisted by RALDH+ dendritic cells. The resulting immunoglobulin A (IgA) restricts the otherwise unchecked expansion of several bacterial species. Treg cells are induced by Clostridia members and are also strongly linked to the availability of fermentation products such as short-chain fatty acids (SCFAs), while T helper 17 (TH17) cells are mostly driven by SFB and the high concentration of ATP in the intestinal lumen. In parallel to these events, gut microbial antigens are sampled by dendritic cells (DCs) and presented to various types of lymphocytes, leading to a complex balance of activation/inhibition and gut immune homeostasis.

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Figure 2 Peripheral lymphoid volume and gut microbiota. Dendritic cells (DCs) entering secondary lymphoid organs (SLOs) is a critical event to maintaining the proper structure of lymph nodes (LNs). We have found that fungal species drive a group of CD103+CD11b +RALDH+ DCs to leave the gut and enter distal LNs. These fungi mediate the MAdCAM-1 to PNAd addressin profile switch to initiate the adult pattern of lymphocyte homing, resulting in SLO volume expansion. This switch gradually reduces the migration of these DCs to peripheral LNs. In the early weeks, lymphocytes circulating through these LNs are further routed back to the gut, a phenomenon that gradually disappears in the later weeks. The small presence of these DCs is critical to preventing structure attrition in adult LNs. later species, potentially covering pathogenic strains.19 In antibody) responses against influenza infection. However, their contrast, antigens present in the microbiota at times act as functions could be replaced with a provision of TLR ligands.25 facilitating triggers to induce autoimmune attacks, such as the This result suggested that canonical PAMP sensing is a case of experimental autoimmune encephalomyelitis (EAE).20 contributing factor to the regulation by gut microbiota. Beyond In neonates, members of the Actinobacteria, Proteobacteria infection models, a great deal of research has focused on the and Bacteroidetes dominate first.21 Slowly, the community connection between non-infectious/autoimmune diseases and profile changes and individual differences become evident. In individual species/strains of commensal bacteria. Locally, the adult human gut, Firmicutes and Bacteroidetes phyla are microbiota dysbiosis has been linked to two forms of inflam- highly represented.22 In addition, there are several 'core' phyla matory bowel diseases (IBD): Crohn’sdisease(CD)and that persist in individuals, although the exact composition of ulcerative colitis (UC). IBDs do not develop in GF mice until the community also evolves over time. As our gut commensals the first inoculation, and in some cases, antibiotic treatment are made of large numbers of anaerobic bacteria and existing helped to reduce the pathological severity.26,27 Acommon culture media are not tailored for individual needs, it is difficult feature of both types of diseases is the limited diversity in gut to culture most of them ex vivo, making studies on their commensal bacteria. At the phylum level, there was also a metabolism incomplete.23 However, PCR technologies have characteristic decrease in the normally dominant Bacteroidetes greatly facilitated our understanding of these distributions. and Firmicutes with a rise in other relatively underrepresented Earlier work was mostly based on 16S RNA sequences, as groups, such as Proteobacteria and Actinobacteria.28–30 Systemic bacterial rRNAs were sufficiently conserved for common impact by gut microbiota on autoimmunity is also common. probes, and in addition, they exhibited sufficient taxonomic One proposal suggests that the hyperallergic state in modern differences for identification. Then, next generation sequencing populations is a result of an overemphasis on 'hygiene,' which (NGS) based on 16S RNA pre-amplification followed by limits the diversity of commensals in the gut.31 For more severe shotgun analysis became available, as did direct sequencing of aliments in distal organs, a clear link has been established genomes. The sequences can be clustered and compared to between intestinal bacteria and neurological autoimmunity. several collected sources, allowing an unprecedented power of EAE models developed limited pathology in GF mice, and resolution. These metagenomic profiles provide rich genetic antibiotic treatment was beneficial.32 The presence of micro- insights into the diversity of these guest workers. However, biota and autoantigens in EAE were two critical factors driving mechanism-wise, metabolic function rather than genetic infor- disease development.20 In particular, the presence of segmented mation is more tightly associated with immune regulations. filamentous bacteria (SFB) was sufficient to trigger EAE via a This area of research is more difficult and has only recently process involving TH17 cells.33 However, protection against moved toward the center of focus. EAE has been associated with Clostridium spp. and Bacteroides Overall, depletion of gut bacteria has a significant impact on fragilis.34–36 SFB-driven TH17 cells were also involved in the the immune response to pathogens. Antibiotic treatment development of autoimmune arthritis.37 Unexpectedly, the reduced both innate and adaptive immune responses against same strain was found to suppress type I diabetes.38 These experimental LCMV infection.24 Neomycin-sensitive bacteria examples clearly indicate the vital yet complex role of gut were associated with strong antigen-specific(Tcelland bacteria in the development of autoimmunity.

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Although bacteria are the prototypical residents in the As research moves deeper into gut microbiota-mediated host intestinal tract and we are marveled by their sheer magnitude immune regulation, the field has moved from simple descriptive of clonal diversity, a stable microbiota also contains archaea analyses to metabolic/molecular investigations of inner mechan- and fungi, as well as viruses. As bacteria are the dominant isms. The inclusion of regulators from other biological kingdoms species in terms of numbers, they attract a lion's share of vastly increases the complexity and depth of these investigations. attention and have received more advanced/dedicated research. For instance, human gut fungi were first identified almost a B cells/IgA century ago,39 but analyses of these residents did not take place GF animals have a reduced number of GALTs.54,55 Peyer’s until the end of the last century.40 In contrast, commercial patches (PP) and isolated lymphoid follicles (ILF) are also preparations for bacterial culture were manufactured in the diminished.56 An important function of these local structures is 1800s. A large and comprehensive survey of existing literature to set the stage for IgA production. The significance can be on human gut sequencing analyses suggested that approxi- appreciated by two known facts. First, the majority of total mately 1000 species of bacteria inhabit the gut, with a log antibodies produced in the host is IgA, which is secreted into higher number of strains,15,41 while the eukaryotic species are the intestinal lumen to target gut microbes. Second, the close to 100. Archaea, mostly methanogenic, are still 10-fold microbiota significantly expands in the absence of IgA, lower.42 Some argue that the metabolic contributions from compensated by increased antimicrobial epithelial functions,57 non-bacterial organisms might have been underestimated. For indicating an essential role performed by secreted IgA in gut instance, as fungi are, in general, larger in size, the metabolic/ immune homeostasis. Microbes in the gut were often found to regulatory impact may be larger than what would be expected be heavily coated by IgA,10 and the coated microbes exhibited from simply considering the species number.43 In fact, clinical enhanced translocation into lymphoid tissues, facilitating anti- reports often cite fungal imbalance as one of the most common gen presentation.58 These findings may have implications findings in association with gastrointestinal pathologies. In the beyond local immunity. Peterson et al.59 showed that inocula- human colon, the most common fungi are Candida, Aspergil- tion of GF mice with the single strain Bacteroides thetaiotao- lus, Saccharomyces and Cladosporium.44 The abundance of micron induced a strong innate response, and simultaneous Candida albicans has been positively associated with CD, an introduction of a hybridoma secreting a specific IgA signifi- observation that was made after stool samples were analyzed cantly altered the landscape of innate immunity, ranging from from those affected in comparison with their disease-free complement to FcγRtoinflammatory cytokines. The central relatives.45 Candida dominance was also found in UC patients, message is that the IgA response to the gut microbiota sets the whose symptoms appeared to be controlled by anti-fungal threshold for the entire immune system.59 SFB is a well- treatment.46 Fungal β-glucan recognition by C-type lectin characterized IgA target. AID (activation-induced cytidine receptor Dectin-1 resulted in activation of CARD9 (caspase deaminase)-deficient mice produce reduced quantities of anti- recruitment domain–containing protein 9), which in turn body due to limited somatic hypermutation and class switch. − − drove TH17 polarization in the gut.47,48 Iliev et al.49 reported Parabiosis of regular mice with AID / mice significantly that in mice, deficiencies in this pathway led to an increased suppressed gut SFB outgrowth in the latter.60 IgA production susceptibility to DSS (dextran sodium sulfate)-induced colitis, in humans appears to be linked to genetics and food intake. accompanied by the expansion of Candida and Trichosporon. In Recently, a comprehensive survey in human twins up to 2 years humans, a single-nucleotide polymorphism in the CLEC7A of age found that the transition from receiving IgA in the breast gene (Dectin-1), rs2078178, is strongly associated with the milk to their own production was affected by several factors. severity of UC (medically refractory UC), suggesting a role of Genetics had the largest impact at the beginning, although this anti-fungal immunity in intestinal inflammation.49 correlation became weaker with age. To complement the The gut virome is also an interesting area of development. human study, some particular strains were isolated and The human gut contains ~ 109/g virions.50 Currently, the direct introduced into GF mice; IgA responses that developed there- link between gut viral imbalance and immunological disorders after were impacted by food selection.61 is not well established. One interesting hypothesis is that the For IgA production, intestinal DCs sample the microbial phages present in the gut act as a feedback mechanism to content and translocate to draining MLNs (mesenteric LNs) for control other microbiota. For example, they may kill off the the induction of B cell activation. These B cells then move to excess expansion of dominant bacterial strains to maintain the PP for antibody secretion.9 The secretion is mediated by population balance.51 Several studies have been published to polymeric Ig receptor (pIgR), which binds and transports IgA highlight the importance of gut viruses in shaping the land- from the basolateral to the apical interface.62 DCs from the gut scape of the microbiome.52 More recently, it was reported that were different from the conventional DCs in their unique in IBD patients, the gut virome was altered. In contrast to the ability to induce IgA from activated B cells, an ability at that decreased genetic diversity of bacteria in these patients, time attributed to their interleukin-6 (IL-6) production.63 Caudovirales bacteriophages showed significant expansion, In recent years, particularly with the proper classification of along with a more diversified virome. At this stage, whether innate lymphoid cells (ILC),64 increased consideration has been these changes contribute to the disease state or are only given to a group of retinoic acid (RA)-producing RALDH+ secondary consequences of the disease is not clear.53 (retinaldehyde dehydrogenase) DCs, as they may serve as the

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343 conduit to introduce RA to lymphoid tissues. Intake restriction from conventional peripheral T cells. TCRs specific for gut of vitamin A, the precursor of RA, reduced the IgA levels in the bacterial antigens such as those from Parabacteroides distasonis small intestine.65,66 RA itself promoted splenocyte IgA produc- mediated Treg induction only in the gut but not in the tion in vitro;67,68 however, it appeared that in vivo,other thymus,85 cementing the essential role of the gut environment cytokines including IL-6 and transforming growth factor-β in Treg induction. Using a chloroform extraction protocol, there (TGFβ) were required for the production as well.63,66,69 There has been a series of studies that link Clostridia members to Treg is also evidence that RA may serve as a specific inducer of IgA inductioninthecolonbutnotinthesmallintestineorother class switch.70 Interestingly, in a flagellin-specific T cell receptor lymphoid tissues in the gut. Clostridium clusters IV and XIVa can (TCR)-transgenic system, depletion of CD4+CD25+ Tcells increase the number of Tregs. This increase was associated with blocked IgA production and introduction of Foxp3+ CD4+ TGFβ and indoleamine 2,3-dioxygenase, both known to help T cells restored the response, suggesting that Tregs can work as pTreg induction.34 Based on this information, the same group conventional CD4+ helper T cells in IgA production.71 isolated 17 strains of clusters IV, XIVa and XVIII of Clostridia Kawamoto et al.andConget al. similarly reported that Tregs from human fecal specimens and inoculated GF mice. In entered germinal centers to not only increase the quantity of comparison with non-discriminatory human fecal transplant, this IgA but also to expand the diversity in their BCR genes.71,72 selection increased the promotion of Tregs in colonic LP. This Unexpectedly, the microbiota may have built-in mechanisms to inoculation was horizontally transmissible to other GF mice.82 fine-tune IgA production in MLNs. Diehl et al.73 reported that Remarkably, the reduction of those strains was found in multiple signaling via Myd88 on a group of CX3CR1hi DCs in the LP sclerosis (MS) patients,35 suggesting the biological relevance of inhibited their ability to transport gut bacteria into MLNs, thereby these bacteria in forming a balanced gut immune response. reducing T cell activation and IgA production. This finding likely In a non-mutually exclusive form, Treg induction in the gut adds another layer of regulatory complexity to these LNs. can be accomplished by particular bacterial products. Short- chain fatty acids (SCFAs) are fermentation products of com- Regulatory T cells plex sugars that are digestible by bacteria present in the fecal Tregs are from two developmental origins with characteristic content.86 Butyrate, acetate and propionate are the main expression of Foxp3 as the master switch. Thymic migrants species with the ability to shape gut immune homeostasis. (tTregs) represent a population of self-reactive T cells that This production stream leads to a local SCFA concentration – escape negative selection74 77 and provide a steady pool of approaching 100 mM, a level that is much higher than that Tregs available in the periphery. In situ induction of peripheral required to modulate T cell polarization. In 2013, Smith et al.87 Tregs (pTregs) is more tuned to local availability of antigens reported that SCFAs increased the number of Foxp3+/IL-10+ and the state of inflammation and is dynamically regulated.78 colonic Treg cells. The authors suggested that this effect was These two populations together form the immune suppressive mediated by SCFAs, such as propionate signaling via GPR43 network mediated by a plethora of mechanisms, such as (G-protein-coupled receptor), which in turn inhibited HDAC6 constitutive expression of CTLA-4 and secretion of IL-10 and (histone deacetylase) and HDAC9 activation.87 Arpaia et al.88 TGFβ. In a seminal study, Shimon Sakaguchi reported that extracted SCFAs from gut contents and found mainly that transfer of CD25+ cell-depleted lymphocytes into Balb/c nude butyrate potentiated the extrathymic de novo transformation mice led to broad-spectrum autoimmunity, where gastritis was from CD4+ T cells to Tregs driven by TGFβ/IL-2 and anti- among the most overt.79 Early work by Mottet et al.80 showed CD3. Apparently, butyrate also stabilized Foxp3 expression as a that in a CD4+CD45RB+ T cell transfer colitis model, the consequence of HDAC inhibition.88 Similar findings have been presence of CD25+ Tregs was essential to suppressing bowel reported by Furusawa et al.,89 which further indicated that inflammation. These reports all suggested the inhibitory role of these induced Tregs were capable of suppressing the CD4+ Treg-mediated suppression in gut autoimmunity. CD45RB+ T cell transfer colitis model. In addition, propionate Helios and neuropilin 1-negative Tregs, the quintessential was found to increase splenic Treg conversion as well.88 pTregs, are hard to detect in other organs but are abundant in Interestingly, in another report, propionate and acetate, but the LP of the small intestine.81 Conversely, the number of these not butyrate, were found to increase gut tTreg accumulation peripherally induced Tregs was reduced in the absence of rather than the conversion from CD4+ Tcells.87 These microbiota or dietary antigens.82,83 In the presence of micro- differences may require further analysis to identify subtle biota, the gut environment can generally be described as anti- discrepancies in experimental protocols. In addition to the inflammatory. Under this general setting, there are a few direct induction of Tregs, butyrate can signal via GPR109a distinct pathways via which Tregs can be induced. In one (also known as the niacin receptor) on macrophages and DCs. modality, DCs are the main driving force behind Treg These activated cells then mediate the induction of Tregs. This induction. Coombes et al.84 reported that there was a steady regulation apparently relies on the induction of RALDH1 stream of gut CD103+ DCs that moved to the MLNs, where expression in DCs.90 Independent of GPRs, PSA (polysacchar- they induced Tregs in an RA-dependent fashion.84 How this ide A) produced by B. fragiles ameliorated TNBS and Helico- induction was linked to gut microbiota was not studied in that bacter hepaticus-induced colitis. This effect was mediated by report. From the angle of microbial antigen-specific induction IL-10-producing pTreg cells and required signaling via of pTregs, it was found that gut Treg TCR usage was different TLR2.91–93 Therefore, microbial products and metabolites

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modulate a list of regulatory functions to suppress gut appeared to be induced via CD11clow CD70high DCs that inflammation, mainly via the induction of Treg cells. produced IL-6 and TGFβ. These DCs promoted TH17 differentiation by activating STAT3 in the targeted cells.109 Thelper17cells It was discovered later that the ATP metabolic enzymes ecto- TH17 cells are a CD4+ T cell polarization in addition to TH1/ nucleoside triphosphate diphosphohydrolases (ENTPD1, TH2, characterized by the lineage marker RORγt, in contrast to CD39) were essential for controlling the expansion of TH17 T-bet and GATA3, which are required for the other two, cells. Entpd7-deficient mice were resistant to oral infection with respectively. This particular lineage strongly produces IL-17A Citrobacter rodentium but had a higher tendency to develop and IL-17F and possesses a greater plasticity in conversion to EAE.111 The roles of ATP and the P2X7 receptor have been the TH1 phenotype in vivo.94,95 IL-17 family cytokines and confirmed in the lesions of human psoriatic skin where ATP- their signaling via a diverse set of receptors are a prominent stimulated DCs induced a biased TH17 immune response.110 feature of gut mucosal immunity. They suppress the outgrowth In addition, acetate and propionate can induce TH17 cell of several strains of bacteria and fungi and are involved in differentiation independent of GPRs. In vitro, acetate and autoimmune regulation. Additional information was recently propionate triggered the transcription of IL-17A, IL-17F, RORα, published in a recent expert review on the matter.96 TH1 RORγt, T-bet and interferon-γ (IFN-γ), which are genes that polarization is in general considered proinflammatory. One of are important for TH1 and TH17 T cell differentiation.112 the early surprises was that some autoimmunities such as MS Although TH17 and Tregs are considered functionally antag- and RA (rheumatoid arthritis) were not consistently associated onistic, Tregs in the gut can express Rorγt. In two Science articles, with TH1 polarization.23,97 It was found that the p40 subunit of it was reported that the presence of microbiota expanded a IL-12 could be paired with p19 instead of p35 to form the population of Helios-neuropilin- Rorγt+ Tregs, mainly in the cytokine IL-23, which was sufficient to drive EAE 98 as well as colon.113,114 The deficiency of this gene was associated with IBDs.99,100 Most of these effects were found to be regulated by reduced pTregs. In one study, the rise of these cells required RA TH17 T cells. Although IL-23 is critical to sustaining the and dendritic cells. The surface expression of CTLA4 on these phenotype, it is actually not the cytokine that drives the Tregs suppressed DCs via downregulation of CD80 and CD86, differentiation. The commitment to this polarization appears reducing TH2 polarization.114 This suppression is similar to one to be highly environmentally dependent and can be induced by of the proposed common inhibitory mechanisms by Tregs.115,116 several combinations of stimulatory factors. TGF-β+IL-6, IL-21 The other study also found that Rorc was highly upregulated in alone and IL-1β+IL-6+IL-23 have all been reported to induce colonic CD4+Foxp3+ Tregs. However, in contrast to the last TH17 polarization, and resulting populations displayed inter- study, the authors described a critical regulation on TH1/TH17 esting functional variations.101 With the molecular regulation cells with no apparent impact on TH2 differentiation.113 Distinct of TH17 cells becoming clear, its role in gut homeostasis has from this population and driven mainly by GATA3, a population come into focus. of Helios+ Tregs was induced by IL-33 signaling via ST2 (Il1rl1) TH17 T cells are a significant presence in the LP of the on their surface.117 This induction was inhibited by IL-23. They colon. In GF mice, the overall T cell number does not differ significantly expanded when Rorvt+ Tregs were genetically from that of controls; however, the percentage of TH17 T cells deleted. It should be noted that these cells did not appear to is reduced.102,103 This promotion effect is mediated at several be limited to the gut and might present an intestinal regulation levels. First, the presence of microbiota significantly increased exerted by a systemic Treg population. the production of several cytokines; among them, the change in IL-17 levels was most profound.102–104 SFB abundance in the Gut microbiota and peripheral immune system development gut also positively regulated TH17 cell expansion.102,104–106 It In addition to their roles in lymphocyte tuning and polariza- has been reported that compared with C57BL/6 mice from tion, the gut microbiota is essential to the establishment of Jackson Laboratories, Taconic strains had much higher SFB. secondary lymphoid organs (SLO). GF animals have no Co-housing these two strains led to higher TH17 in Jackson reduction in their thymic outputs, in stark contrast with the mice, a phenomenon associated with the increase in gut SFB.104 multiple immunological deficiencies in the periphery. In GF Inoculation with SFB alone in gnotobiotic mice was sufficient mice, there is a general deficiency in SLOs, although to induce TH17 cells in LP. Strikingly, Yang et al.107 reported the lymphoid structural arrest is best studied in the gut. that most TH17 cells in the gut expressed TCRs specificfor In the intestine, both GALTs and associated MLNs are under- SFB antigens, and the specificities of these TCRs alone were a developed. Peyer’s patches are smaller, and the ILFs are arrested strong driving force to induce TH17 polarization.107 Sano at an immature growth state.56,118,119 et al.108 reported that TH17 differentiation occurred in close In the sterile embryo, LN development is independent of proximity to SFB colonization on epithelial cells. SFB drove the microbial presence. However, RA signaling is indispensable. epithelial production of serum amyloid A (SAA1/2). The latter At the distal ends of nerve fibers, the secretion of RA triggers induced enhanced gene expression of Rorc.108 At the metabolic the development of lymphoid anlagen from the primitive level, one of the main driving forces for their increase is the lymphoid sacs. This process required a specificpopulationof ATP in the large intestine. The SPF fecal content contains group 3 innate lymphoid cells, LTi (lymphoid tissue inducer) higher levels of ATP than GF mice.109,110 The effect of ATP and lymph toxin signaling.120–122 After birth, LTs were no

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345 longer present. Therefore, the conduit that links the emergence CD11b+ DCs expressing RALDH2 was driven by the micro- of gut microbes and lymphoid tissue development after birth biota and traveled to peripheral LNs, leading to local RA was not well characterized. Early on, Mazmanian et al.123 production. Injection of these cells into GF mice completely reported that PSA could trigger CD4+ T cell activation, which restored the LN development, indistinguishable from SPF may likely represent an antigen-dependent expansion of these mice.132 Arriving at LNs, they mediated the MAdCAM-1 to cells. Mebius et al.124 first described a mysterious population of PNAd (MECA79) transition and increased the HEV portal size non-T, non-B cells that appeared to be important to main- and CCL21 expression. This work for the first time directly taining the neonatal LN development. After this appearance, linked the gut microbiota to peripheral immune development. there was a steady movement of CCR7-dependent lymphocyte Three observations were particularly interesting. (1) In the first migration into SLOs, resulting in cellularity increase and two weeks, these cells induced the lymphocytes circulating volume expansion.125,126 This process was critically dependent through peripheral LNs to express gut-homing CCR9 and on gut microbiota.118 The most systematic analysis was α4β7, resulting in their further migration back to the gut. This performed for ILF maturation. After birth, numerous lymph process was slowed in adult mice. (2) These cells persisted in toxin B-dependent crypto patches dynamically form with the low frequencies in adult LNs, and this presence was essential emergence of cKit+ and B220+ cells. In the presence of for structural maintenance. (3) Gut fungi, particularly Candida microbiota, they converted to mature ILFs in a CCR7- tropicalis, were essential for these cells to leave the gut, dependent manner.112,127 Peptidoglycan from Gram-negative suggesting that eukaryotic metabolism within gut microbiota strains signaled through NOD1 and induced robust develop- was the critical force for peripheral immune development. ment of ILFs, also in a CCR7-dependent fashion,56 suggesting Therefore, this presents an interesting scheme whereby those that it is an active factor from the microbiota for ILF induction. RALDH2+ DCs come to SLOs to turn on an adult-like homing Disruption of this pathway resulted in 100-fold expansion of pattern to attract circulating lymphocytes and at the same time Clostridiales, Bacteroides and Enterobacteriaceae.56 drive these cells back to the gut to build an early defense there. Due to the proximity of MLNs and GALTs to the microbial It is not until the gut defense is fully completed that they keep a content and lymphatic drainage, it is relatively easy to imagine steady stream of incoming lymphocytes to maintain the a close exchange at work. It is, however, more difficult to peripheral lymphoid volume. Our results therefore implicate envision a similar regulation on SLOs that are distal to the a potential path linking the gut microbiota, particularly gut.128 In GF animals, peripheral challenge with Listeria commensal fungi, to peripheral immune volume expansion. monocytogenes resulted in a reduced accumulation of There are many questions to be answered. What are the lymphocytes.129 Lack of gut microbiota was also associated fungal metabolites driving these DCs to enter the peripheral with reduced responses to influenza infection.24,25 In viral LNs? Are they unique to Candida tropicalis? What are the infections alone, gut microbiota was associated with several molecular regulations that turn on PNAd expression? These are specific immune activations.130 The structural underdevelop- immediate questions for the near future. ment is certainly partially responsible for the reduced peripheral immune responses. Thus far, how the gut microbiota Prospective sustains the robust responsiveness of the distal immune organs This review focuses on several individual cell types and is elusive. A critical event in the neonatal SLO maturation is an processes regulated by gut microbiota, which are individual addressin profile switch from MAdCAM-1-mediated to PNAd steps in a multifaceted regulatory process. Even within this (MECA79, sulfated carbohydrate-decorated adhesion mole- confine, there are many other molecular details that are cules)-mediated lymphocyte homing. The arrival of DCs is unknown to us. In our view, with understanding deepening essential for this switch. Mossine et al.131 reported that in the and technologies improving, several lines of investigation will steady state, Diphtheria toxin receptor-mediated CD11c+ DC be of high priority. (1) The metabolic processes, rather than depletion caused a disappearance of MECA79+ on the HEV individual strains, should move to the center of the investiga- portal. This reduction limited the migration of peripheral tion. The most important are SCFAs, ATP, PSA and those lymphocytes entering the LNs, leading to significant structural known to play roles in lymphoid function regulations. Addi- attrition.131 There was a simultaneous increase in MAdCAM-1 tional metabolites and microbial species responsible for their at HEV, resembling a neonatal addressin profile. They further production are also important study subjects. (2) Systematic suggested that LT derived from these DCs was critical for the evaluation of the roles played by life forms other than bacteria. structural maintenance of LNs. Wendland et al.126 reported In the case of fungi, clinical diseases are commonly linked to that the migration was mediated by CCR7-expressing lympho- their dysbiosis. Fungal metabolism is similar to that of the host. cytes and relied on CCL21 expression by fibroblastic reticular Therefore, whether their signaling can be directly incorporated cells. Although these analyses demonstrated the importance of into the host immune regulation is an interesting question. DCs in supporting peripheral lymphoid volume, they did not (3) As the peripheral lymphoid system is regulated by the gut indicate any gut microbiota involvement. microbiota, how this regulation impacts all aspects of periph- Regarding the missing link to the microbiota, we recently eral immune activation should be thoroughly investigated. reported an interesting mechanism in neonatal mice up to (4) As a more distant target, it will be of great value to 4 weeks of age; specifically, a group of gut-originated CD103+ establish therapeutic protocols to see whether targeting

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