New aspects of IgA synthesis in the gut.

International Immunology International Immunology Advance Access published May 28, 2014

Journal:

INTIMM-14-0071

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Manuscript ID:

International Immunology

Manuscript Type: Date Submitted by the Author:

Complete List of Authors:

27-Apr-2014

Suzuki, Keiichiro; Kyoto University, Graduate School of Medicine Nakajima, Akira; Kyoto University, Graduate School of Medicine

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Keywords:

Invited Review

IgA, gut microbiota, homeostasis

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Downloaded from http://intimm.oxfordjournals.org/ at Polytechnic University. Bern Dibner Library of Science and Technology on September 2, 2014

New aspects of IgA synthesis in the gut

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New aspects of IgA synthesis in the gut

Keiichiro Suzuki1 and Akira Nakajima1 1


Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Yoshida Sakyo-ku, Kyoto 606-8501, Japan,

Corresponding author: Keiichiro Suzuki

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Email: [email protected] Fax: +81-75-753-9500 Tel: +81-75-753-9526

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Running title: Regulation of gut homeostasis by IgA

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Kyewords: IgA, gut microbiota, homeostasis

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The number of Figures: two Figures

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Abstract In mammals, the gastrointestinal tract is colonized by extremely dense and diverse bacterial communities that are beneficial for health. Maintenance of complexity and

when disrupted, the microbial community attacks the host’s tissues and cause inflammatory reactions. Our immune system provides the necessary mechanisms to maintain the homeostatic balance between microbial communities and the host. The

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immunoglobulin A (IgA) plays crucial roles in regulation of host-bacterial interactions in the gut. IgA is the most abundant immunoglobulin (Ig) isotype in our body, mostly produced by the IgA plasma cells residing in the lamina propria of the

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small and large intestine. Although it was well known that IgA provides protection against pathogens, only recently it became clear that IgA plays critical roles in

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regulation of bacterial communities in the gut in steady-state conditions. Here we

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summarize recent progress in our understandings for the various mechanisms of IgA synthesis in multiple anatomical sites and discuss how IgA limits bacterial access to

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the internal milieu of the host.

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proper localization and distribution of gut bacteria is of prime importance, because

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Introduction More than a half-century has passed since IgA was found as the most predominant class of antibody in mucosal secretions (1, 2). The observation that IgA but not IgG is

mucosal immune system. Pioneering works done by John Cebra and coworkers provided the cellular basis for the origin of IgA producing cells and the evidence for the involvement of gut bacteria in maturation of the immune system (3, 4). By using

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the transfer model in rabbit, they showed that Peyer’s patch (PP) cells were much more efficient than peripheral lymph node (pLN) cells to repopulate in small intestine of the transferred hosts (3). In addition, their experiments with germ free mice showed

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that germinal centers (GCs) formation in PPs and subsequent IgA secretion efficiently occurred only in response to the bacterial colonization in the intestine (4). These and

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many other subsequent studies led to the concept that the precursors of IgA plasma

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cells are generated in PP GCs by T cell-dependent mechanisms triggered by commensal bacteria. However, recent observations have revealed that the sites and

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mechanisms for the induction of IgA+ cells are multi-layered (5); IgA+ cells are also induced in isolated lymphoid follicles (ILFs) and in non-organized lamina propria

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(LP). In these anatomical sites, T cells are dispensable for the generation of IgA+ cells (5).

Although the IgAs generated by different mechanisms may play different roles in distinct contexts, the primary function of IgA is to maintain homeostasis at the mucosal surface. IgA was originally recognized as an important factor to protect the host from mucosal pathogens. More recently, we begin to appreciate that IgA plays an important role in steady-state conditions in the gut (6). Technical advances in the analysis of complex microbial community rapidly expanded our knowledge about the

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secreted across the mucosal surfaces attracted attentions for the uniqueness of


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IgA-dependent mechanisms for the maintenance of microbial community in the gut. In this review, we discuss the critical role of IgA for the maintenance of “healthy” gut microbiota and emphasize the mechanisms responsible for IgA synthesis in the gut .

The gut harbors more than 1000 species of bacteria that outnumber our own cells by a factor of 10. Since most of the normal gut bacteria are uncultured, the studies

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based on culture-dependent experiments did not give an accurate view of their composition. A more precise characterization became possible only recently with the development of culture-independent molecular approaches based on bacterial

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sequencing, (including the largely used sequencing of bacterial 16S rRNA genes) (7, 8). Such technical development allowed us to evaluate in detail the composition of

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microbial communities, and to monitor how they change with conditions of the hosts.

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One powerful driving force for shaping gut microbiota appears to be dietary habit of the hosts (9-12) (Fig1). Comparative analysis revealed that humans harbor a

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representative gut microbiota of omnivorous primates that is clearly distinct from the microbial community of carnivores and herbivores (9). Within different human

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populations, European children taking typical “western” diet have clearly distinctive bacterial community from rural African children who mainly take plant-based diet, suggesting that long-term dietary intake may have a significant effect on the composition of the gut microbiota (10). Even short-term dietary interventions can quickly change gut microbial community. For example, changing the diet from high fiber diet to high fat diet drastically altered within several days the bacterial composition and their metabolic activity such as the production of short chain fatty acids (SCFAs) in the gut (11, 12). These changes seem to deeply impact on the host’s

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1. IgA-independent regulation of gut microbiota

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immune system, including the production of pro-inflammatory cytokines such as IFNγ, IL1-β, IL-22, IL-23, and possibly the maintenance of regulatory T cells (Tregs) (1318).

inflammatory responses at bay provides another layer of microbiota regulation (Fig1). For example, NLRP6 inflammasome pathway in intestinal epithelial cells inhibits the development of colitis by IL-18 dependent regulation of some colitogenic

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commensals such as Prevotellaceae and TM7, or by clearing pathogens through mucus secretions by goblet cells (discussed by Flavell in this issue) (19, 20). Another example is that the expression of T-bet in innate immune system prevents the

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expansion of Klebsiella pneumoniae and Proteus mirabilis probably through the function of dendritic cells, thus avoiding inflammatory responses in the colon (21).

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IL-22 produced by innate lymphoid cells was also reported to be involved in

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regulation of the gut microbiota such as segmented filamentous bacteria (SFB), and avoiding the systemic dissemination of Alcaligenes species (discussed by Sonnenberg

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in this issue) (15, 22). Thus, the innate immune system by regulating the local

commensal microbiota.

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cytokine milieu contributes to modulation of “pathogenic” stimulus derived from

2. Regulation of gut microbiota by antigen specific IgA Acquired immune system has a non-redundant role in regulation of host-microbial interactions at mucosal barrier. Thus, production and secretion of IgA play an important role for the regulation of gut microbiota that cannot be achieved by the diet or the innate immune system (Fig1). The first evidence that secretory IgA play critical roles in regulating gut microbiota came from the studies of activation-induced

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In addition to diet, the innate immune system by keeping the undesirable


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cytidine deaminase (AID)-deficient mice, which lack the key enzyme required for class-switch recombination (CSR) and somatic hypermutation (SHM) of the Ig genes (23-25). In the absence of IgA, culturable bacteria in AID-/- small intestine expanded

also changed, with SFB aberrantly expanding in all segments of the small intestine (25). The expansion of SFB led to over-activation of not only the gut immune system, but also the whole body immune system. When IgA-producing cells were

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reconstituted in AID-/- mice upon parabiosis with normal mice, the amount and composition of gut microbiota were normalized (25). Although SFB expansion was reported to induce mostly differentiation of T cells into Th17 cells (26), the

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composition of Th17 cells in the gut LP of AID-/- mice was equivalent to those of normal mice, suggesting that the over-activated phenotype of AID-deficiency is not

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due to the activation of Th17 cells (our unpublished observation).

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Since AID is required both for CSR and SHM, the studies with AID-/- mice did not dissect whether the simple presence/absence or the quality of IgA is important for the

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regulation of gut bacterial communities and in general for the body homeostasis. The study with AIDG23S, a mutant from AID that has normal CSR but impaired SHM

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activity, addressed this issue (27). The knock-in mice expressing AIDG23S had the normal concentration of serum and fecal IgA (as well as other Ig isotypes), but had a limited diversity of the Ig repertoire due SHM defect. However, similar to AID-/mice, AIDG23S mice showed expansion of culturable bacteria in their small intestine, and systemic over-activation with induction of GCs in peripheral lymphoid tissues (27). These results clearly indicated that SHM and selection of IgAs are needed for specific recognition of the bacterial epitope to regulate their amount and composition of microbial community in the gut.

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more than 100 fold (24). Moreover, the composition of uncultured gut microbiota was

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The diversification of IgA repertoire by SHM occurs mostly in the PP GCs and the formation of GCs depends on the T cells help. Most of the T cells present in the GCs are CD4+ T cells generally known as follicular B helper T cells, of TFH cells. TFH cells

sustain GC reactions. Interestingly, TFH cells also express high amount of the inhibitory receptor programmed cell death-1 (PD-1), a molecule that also regulate the several aspects of GC biology. Thus, the absence of PD-1 led to considerable

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expansion of PP TFH cells with different properties (i.e producing more IFN-γ and less IL-21) that caused impaired selection of GC B cells. Thus, PD-1-/- mice had reduced affinity maturation of IgA-producing plasma cells, and less IgA binding capacity to

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gut bacteria (28). Associated with reduced IgA coating of bacteria, PD-1-/- mice had a marked reduction in the number of “healthy” gut bacteria, such as Bifidobacterium. In

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contrast, the number of Enterobacteriaceae was significantly increased in PD-1-/-

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mice. Like in AID-deficient or AIDG23S mice, the gut dysbiosis induced overactivation of the systemic immune system, which led to T and B cell hyperplasia in

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PD-1-/- mice. The systemic activation could be rescued by antibiotic treatment (28).

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These observations clearly indicate that the T cell-dependent selection of bacteriaspecific B cells in PP GCs is critical for regulating symbiotic relationships with the gut microbiota leading to homeostasis.

IgA not only coat the bacteria, but can modulate bacterial epitopes and even change the bacterial metabolism. By using mono-colonization system, it was demonstrated that Bacteroides thetaiotaomicron elicits less pro-inflammatory signals in the small intestine in the presence of a specific gut IgA that recognize capsular polysaccharide CPS4 (29). The bacteria evaded anti-CPS4 IgA response by inducing alternative expression of another capsular locus, CPS5. Thus, not only that IgA is required to

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are characterized by expression of co-stimulatory molecules and cytokines required to


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regulate the amount and composition of gut microbiota, but that the adaptive immune system drives diversification of bacterial surface structures by exposing their antigenic epitope(s) to appropriately selected IgA.

Although it is clear that the T cell-dependent selection of antigen specific B cells in PP GCs is critical to regulate gut microbiota, significant numbers of IgA+ cells are

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generated outside of GCs even in the absence of T cells (30). Many of these cells originate from B1 cells that reside in the peritoneal cavity. This was shown in lethally irradiated T cell-deficient mice, reconstituted with allotype-marked B1 cells together

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with host-derived bone marrow cells (31). Many of B1-derived IgA+ cells do not pass through T cell-dependent selection in GCs. However, IgA secreted from these cells

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can abundantly coat the surface of gut bacteria even with the germ line-encoded VH

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genes (32). Secretory IgA may naturally bind on bacteria through their heavy glycosylation on secretory components or on the heavy chains (33, 34). By coating in

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abundance the bacterial surfaces, innate IgA produced by B1-derived plasma cells may play an important role for preventing systemic dissemination of commensal

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bacteria (Fig1). Indeed, T cell-deficient mice that produce B1-derived gut IgA do not have serum IgA or IgG with the same specificities (31). By contrast, mice with IgA deficiency (IgA-/- mice and alymphoplasia aly/aly mice) have serum IgGs that are specific for commensal bacteria produced with the help of T cells (31). Coating of gut bacteria by non-selected “natural” IgA appears to protect neonatal mucosa from abrupt encounter with commensals. Pups exchanging experiments showed that SFB colonization and subsequent generation of IgA+ cells in the small intestine occurs earlier in the pups nursed by SCID females that lack milk Ig (35).

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3. Innate function of IgA

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Similarly, normal pups nursed by JH-/- mother that lack mature B cells and Ig had early induction of serum IgA (36). Milk Ig and pups-derived IgA prevented the penetration of bacteria through the intestinal epithelium even without diversification

nitrophenol-specific IgA produced in young quasi-monoclonal mice was sufficient to limit the penetration of commensal Enterobacter cloacae (36). Non-specific IgA coating on bacteria is also functionally important in adult gut. This

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is supported by the following observations. First, the invasion of Salmonella typhimurium to epithelial cells was increased in polymeric immunoglobulin receptor deficient (pIgR-/-) mice, which lack IgA secretion (37). Secretory IgA in naive mice

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inhibited this process without developing high-affinity antibodies that recognize surface epitopes of S. typhimurium. Second, pIgR-/- mice showed exacerbation of

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chemical colitis induced by exposure to dextran sodium sulfate (DSS) depend on the

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presence of intestinal microbiota (38). Third, similar to neonatal mice, the presence of secretory IgA limits the translocation of aerobic bacteria from intestinal lumen to

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mesenteric lymph nodes (mLNs) (39). Intestinal DCs carry living luminal bacteria to mLN and prevent further dissemination into the systemic immune compartment

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through MyD88-dependent Toll-like receptor (TLR) signaling. These DCs have capacity to induce IgA production in the gut (39-41). Thus, it seems that the presence of intestinal IgA and bacterial access to DCs are tightly correlated each other, and may be considered as the first and the second gateway respectively to prevent bacterial penetration into the deeper tissues (Fig1).

4. Impact of gut microbiota and other environmental factors on IgA synthesis 4-1. Generation of IgA+ cells in PP GCs

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of the antibody repertoire. This was shown by the observation that the non-mutated


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One of the most striking response to the colonization of commensal microbiota is the formation of PP GCs (4). In conventional mice, GCs are continuously found in PPs because of constant stimulation by gut bacteria. Interestingly, GCs in PPs but not in

receptors, given that B cells are activated by bacteria through a TLR signaling pathway (42). GC B cells in PPs are also unique in the sense that they undergo CSR almost

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exclusively to IgA. We found that the stimulation through bacterial and food-derived factors on stromal cells in PP GCs, i.e. follicular dendritic cells (FDCs) play an important role for the CSR to IgA (43). FDCs express TLRs and retinoic acid

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receptors (RARs) and stimulation through these receptors by bacterial and foodderived products induce large amounts of active TGF-β1 and BAFF (Fig2). Indeed,

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conditioned media from PP FDCs supports more efficiently the generation of IgA+ B

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cells and IgA plasmablasts than pLN FDCs by mechanisms that involves the TGF-β1 activation and BAFF production (43). In addition, PP FDCs produce enhanced level

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of CXCL13, by which FDCs may contribute to the augmentation of recruitment and

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motility of GC B cells in PPs. A recent report of in vivo ablation of FDCs supports this idea; FDC depletion resulted in the reduced level of Cxcl13 transcripts, disorganized the follicular structures and reduced B cell motility (44). The correct positioning of activated CXCR5-expressing T cells also requires CXCL13 production by FDCs. Priming of naive T cells by DCs at the T-B border induce upregulation of CXCR5 and downregulation of CCR7 on T cells, and those T cells are directed to Bcell follicles via the gradients of CXCL13, and mature into TFH cells (45). Interestingly, TFH cells in PPs can be generated from Foxp3+ T cells with IgA helper properties (46) (Fig2). Upon the adoptive transfer into T cell- deficient mice, Foxp3+

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pLNs can be induced in the absence of antigen-specific recognition by B cells

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T cells lose their Foxp3 expression, covert into TFH cells and efficiently induce GCs in the PPs and IgA plasma cells in the LP. The conversion of Foxp3+ cells into TFH cells is a gut-specific phenomenon, because this conversion was observed only in PPs

Thus, in PPs there seems to be a link between the Foxp3+ T cells, GCs and IgA+ cells. This is likely due to the unique microenvironment generated by the presence of bacteria and food-derived components, and also the products secreted by activated

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epithelial cells, DCs, B/T cells and stromal cells in the gut milieu (Fig2).

4-2. Generation of IgA+ cells outside of PP GCs

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Some of IgA+ cells are also generated in LP and ILFs, albeit less efficient that in the PP GCs (5). Pre-switched IgM+ B cells are found either scattered in the villi or

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forming aggregates called isolated lymphoid follicles (ILFs). The development of

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ILFs depends on the RORγt-expressing lymphoid tissue inducer (LTi) cells and their interaction via LT-LTβR with local stromal cells (47, 48). The ILF or LP B cells

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(Fig2).

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undergo CSR to IgA in situ upon their stimulation by commensal bacteria (47-49)

In LP and ILFs, B cells are directly activated by DCs that have access to luminal bacteria via extended dendrites through the tight junction of the epithelium, or through the transport of bacteria by microfold cells (M cells) (50, 51). Similar to PPs, the epithelium covering ILFs and intestinal villi differentiate into glycoprotein 2 (GP2)-expressing M cells through the stimulation of stromal-derived TNF superfamily member receptor activator of NF-κB ligand (RANKL) and transcription factor Spi-B (discussed by Ohno in this issue) (52-54).

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and partially in mLNs but not in spleen even with the repeated immunizations (46).


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Upon activation by bacterial antigens, the DCs present in the LP or ILFs are sufficient to induce CSR to IgA even in the absence of T cells. Two major subsets of DCs are present in LP: CX3CR1+ DCs and CD103+ DCs (55). CX3CR1+ DCs are

possibly IL-10 (56, 57). CD103+ DCs also help in situ CSR probably through the TLR5-dependent production of retinoic acid (58). In addition, DCs in LP or ILFs produce large amount of TNF-α, and stimulate local stromal cells to release the active

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form of TGF-β1 via the effect of matrix metalloproteinase 9 (MMP9) and MMP13 (48). Interestingly, a subset of CD11c+ DCs called Tip-DCs and also a fraction of IgA+ plasma cells were found to be crucial for IgA induction by producing iNOS and

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TNF-α in the gut (56, 59). Thus, interactions among bacteria, DCs, stromal cells and IgA-plasma cells likely generate a positive feedback loop to induce IgA+ cells in the

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5. Conclusion

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local environment (Fig2).

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It is now clear that IgA is critical for the maintenance of a diversified microbial

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community in the gut. Accumulating evidence show that IgA producing cells are generated by multiple pathways, in organized and non-organized follicular structures, by T-dependent and T-independent mechanisms. However, the functional differences between IgAs generated in different anatomical sites by different mechanisms are still unclear. Also unknown are the mechanisms by which the antigen-specific IgAs regulate the amount and composition of microbial community, or how exactly the innate IgA inhibits host-bacterial interactions in the gut. Further analyses are required to understand the complex and dynamic interactions among IgA, bacteria and immune

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resident cells that may regulate CSR to IgA through their production of BAFF and

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cells in the gut, which should pave the way to develop new strategies for the treatment of gut dysbiosis leading to inflammatory diseases or autoimmunity.

We thank to Sidonia Fagarasan for discussions and critically reading of the manuscript. This work was supported in part by the Special Coordination Funds for

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Promoting Science and Technology of the Japanese Government and in part by Astellas Pharma Inc. in the Formation of Innovation Center for Fusion of Advanced Technologies Program. Also supported in part by Grants-in-Aid for Scientific

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Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and Japan Society for the Promotion of Science to K.S.

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ACKNOWLEDGMENTS


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Figure legends Figure 1. Regulation of gut microbiota and its feed-back to the host. There are at least three

different dietary habits (i.e. high fat and high sugar “western” diet versus high fiber and low fat “traditional” diet ) results in different bacterial community. Second, innate cytokines produced by epithelial cells (IL-18), innate lymphoid cells (ILCs; IL-22)

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and dendritic cells (DCs; TNF-α) play an important role to regulate some of bacteria species that may induce diseases such as colitis. Although the detailed mechanisms how these cytokines change the bacterial composition is unclear, the regulation of

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epithelial-derived mucus and/or Paneth cells-derived anti-microbial peptides might be involved in this process. Third, secretory IgA that specifically recognize bacterial

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epitopes are crucial for regulating the bacterial composition, by inhibiting their

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expansion and facilitating diversification, thus shaping “healthy” bacterial community in the gut. Significant amounts of secretory IgA bind on bacterial surfaces even in the

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absence of specificities to bacterial antigens. These are “innate IgAs” –mostly

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generated by B1 cells- that inhibit the attachment/penetration of bacteria to the epithelial cells, and thus prevent translocation of bacteria to the systemic immune compartment. Most of bacteria-specific IgAs are generated in germinal centers (GCs) of Peyer’s patches (PPs) with the help of TFH cells.

Figure 2. T cell-dependent and T cell-independent induction of IgA+ cells in the gut. In PPs, DCs located under specialized M cells take up bacteria and then activate T cells. The activated T cells express CXCR5 and migrate into B cell follicles along with CXCL13

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major factors that contribute to shaping the commensal communities in the gut. First,


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gradient produced by FDCs. Some TFH cells are derived from Foxp3+ T cells that lose the Foxp3 expression. Upon activation and interaction with T cells, GC B cells express AID and undergo CSR and SHM. In the GC of PPs, B cells preferentially

FDCs producing TGF-β1 and BAFF upon their stimulation of TLRs and RARs. Unlike in the PPs, induction of IgA+ cells in ILFs and LP does not necessarily require help from T cells. Recruitment of IgM+ B cells from bone marrow (BM) or peritoneal

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cavity (PEC) into LP depends on the stromal cells (SCs) expressing functional LTβR. Local B cells are directly activated by DCs that take up luminal bacteria via the extended dendrites through tight junction, or via the M cells present on the epithelium

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that covers the villi and ILFs. TNF-α is abundantly produced by iNOS-expressing

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Tip DCs and a subset of IgA+ plasma cells. Upon TNF-α stimulation, SCs produce active form of TGF-β1 via the effect of matrix metalloproteinase (MMPs), which

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together with APRIL produced by ECs (and perhaps gut eosinophils) induce class switching of activated B cells from IgM to IgA(60, 61). IgA+ B cells differentiate into

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antibody-producing plasma cells upon the stimulation of IL-6, IL-10, BAFF and

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APRIL produced by SCs, DCs or epithelial cells.

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switch their isotype from IgM to IgA, due to help of TFH cells producing IL-21 and

International Immunology Page 23 of 24

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209x148mm (300 x 300 DPI)

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Page 24 of 24 International Immunology

Cellular aspects of selective IgA deficiency.

Obesity and the gut microbiome: pathophysiological aspects.

Nutritional aspects of malabsorption. Short gut adaptation.

T-cell regulation of murine IgA synthesis.

New aspects in the treatment of depression.

Secretory IgA is Concentrated in the Outer Layer of Colonic Mucus along with Gut Bacteria.

General aspects in polymer synthesis.

Development of the gut microbiota and mucosal IgA responses in twins and gnotobiotic mice.

Historical aspects of protein synthesis.

New aspects of liposomes.

Introduction to the structural and cellular aspects of the secretory IgA system.

Synthesis and characterization of a new Peptide prodrug of glucosamine with enhanced gut permeability.

The determination of IgE synthesis in vitro. Methodological aspects.

New aspects of the IF3-ribosome interaction.

New Insights into the Pathogenesis of IgA Nephropathy.

Suppression of the murine gut mucosal IgA response to cholera toxin with oral cyclosporine.

New aspects of influenza viruses.

New aspects of placental endocrinology.

[New aspects of enteral nutrition].

Biliglobulin: a new look at IgA.

[New aspects of stroke medicine].

New aspects of human chronopharmacology.

IgA synthesis by peripheral blood mononuclear cells from normal and selectively IgA deficient subjects.

Decreased Taxon-Specific IgA Response in Relation to the Changes of Gut Microbiota Composition in the Elderly.