Peyer's patches (PPs) play a central role in supporting B cell responses against intestinal antigens, yet the factors controlling B cell passage through these mucosal lymphoid tissues are incompletely understood. We report that, in mixed chimeras, CXCR4-deficient B cells accumulate in PPs compared with their representation in other lymphoid tissues. CXCR4-deficient B cells egress from PPs more slowly than wild-type cells, whereas CXCR5-deficient cells egress more rapidly. The CXCR4 ligand, CXCL12, is expressed by cells adjacent to lymphatic endothelial cells in a zone that abuts but minimally overlaps with the CXCL13(+) follicle. CXCR4-deficient B cells show reduced localization to these CXCL12(+) perilymphatic zones, whereas CXCR5-deficient B cells preferentially localize in these regions. By photoconverting KikGR-expressing cells within surgically exposed PPs, we provide evidence that naive B cells transit PPs with an approximate residency half-life of 10 h. When CXCR4 is lacking, KikGR(+) B cells show a delay in PP egress. In summary, we identify a CXCL12(hi) perilymphatic zone in PPs that plays a role in overcoming CXCL13-mediated retention to promote B cell egress from these gut-associated lymphoid tissues.

Peyer's patches (PP), a key component of the gut-associated lymphoid tissue, serve as the primary inductive sites for intestinal immunity. In the present study, we addressed the hypothesis that the morphological features of PP innervation are consistent with an immunomodulatory role for the enteric nervous system. Laser scanning confocal microscopy was used to collect images through large tissue volumes, yielding a three-dimensional perspective of the neuronal network superimposed on PP follicles from porcine jejunum and human ileum. Peptidergic nerve fibers were found in close apposition to immunocytes within PP subepithelial domes and the adjacent villi. The results suggest that nerve fibers in PP may participate in neuroimmune cross-talk within individual antigen-sampling sites as well as integrate information across multiple antigen-sampling sites.

Peyer's patches are nodules that play a central role in intestinal immunity. Few studies demonstrate the relationship between the number of Peyer's patches and intestinal polyps. Here we identify a statistically significant inverse correlation between the quantity of Peyer's patches and of the development of intestinal polyps in Apc (Min/+) mice, which are a useful model to clarify the role of Peyer's patches in intestinal tumorigenesis. Using this model, we increased the number of Peyer's patches using 0.1% and 1% corn husk arabinoxylan through feed. Intestinal polyp formation significantly decreased, concomitant with an increase in Peyer's patches development (n = 12/group). In Aly (-/-) Apc (Min/+) mice (negative control; no Peyer's patches) there was no change in the amount of intestinal polyps (n = 10/group). Immune reaction following corn husk arabinoxylan treatment was measured by cytokine array. Increasing the number of Peyer's patches decreased interleukin-17 production, which showed a dose dependent correlation with transcription factor/lymphoid enhancer-binding factor. This study identified a relationship between levels of Peyer's patches and intestinal polyp formation, partly explained by the involvement of interleukin-17 production and β-catenin signaling in Apc (Min/+) mice.

CARD15/NOD2 mutations are associated with susceptibility to Crohn's Disease (CD) and Graft Versus Host Disease (GVHD). CD and GVHD are suspected to be related with the dysfunction of Peyer's patches (PP) and isolated lymphoid follicles (LFs). Using a new mouse model invalidated for Card15/Nod2 (KO), we thus analysed the impact of the gene in these lymphoid formations together with the development of experimental colitis.

Jejunal Peyer's patches (JPP) are innervated sites of immune induction and enteropathogen infection. We investigated the role of enteric nerves in modulating pathogen entry into porcine JPP. Presumptive norepinephrine (NE)-containing nerve fibers were localized in JPP domes and follicle-associated villi by secondary immunofluorescence histochemistry. NE or the neuronal conduction blocker saxitoxin increased intracellular internalization of pathogenic Salmonella choleraesuis and Escherichia coli O157:H7, but not nonpathogenic E. coli, into isolated JPP mucosa. NE action was prevented by the alpha-adrenergic antagonist phentolamine. Withdrawal of enteric neural activity or NE administration appears to modulate JPP interactions with pathogenic bacteria.

B cell entry to lymph nodes and Peyer's patches depends on chemokine receptor signaling, but the principal chemokine involved has not been defined. Here we show that the homing of CXCR4-/- B cells is suppressed in CCL19 (ELC)- and CCL21 (SLC)-deficient paucity of lymph node T cells mice, but not in wild-type mice. We also find that CXCR4 can contribute to T cell homing. Using intravital microscopy, we find that B cell adhesion to high endothelial venules (HEVs) is disrupted when CCR7 and CXCR4 are predesensitized. In Peyer's patches, B cell entry is dependent on CXCR5 in addition to CCR7/CXCR4. CXCL12 (SDF1) is displayed broadly on HEVs, whereas CXCL13 (BLC) is found selectively on Peyer's patch follicular HEVs. These findings establish the principal chemokine and chemokine receptor requirements for B cell entry to lymph nodes and Peyer's patches.

Fibroblastic reticular cells (FRCs) determine the organization of lymphoid organs and control immune cell interactions. While the cellular and molecular mechanisms underlying FRC differentiation in lymph nodes and the splenic white pulp have been elaborated to some extent, in Peyer's patches (PPs) they remain elusive. Using a combination of single-cell transcriptomics and cell fate mapping in advanced mouse models, we found that PP formation in the mouse embryo is initiated by an expansion of perivascular FRC precursors, followed by FRC differentiation from subepithelial progenitors. Single-cell transcriptomics and cell fate mapping confirmed the convergence of perivascular and subepithelial FRC lineages. Furthermore, lineage-specific loss- and gain-of-function approaches revealed that the two FRC lineages synergistically direct PP organization, maintain intestinal microbiome homeostasis and control anticoronavirus immune responses in the gut. Collectively, this study reveals a distinct mosaic patterning program that generates key stromal cell infrastructures for the control of intestinal immunity.

Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens whose survival and virulence in the human digestive tract remain unclear owing to paucity of relevant models. EHEC interact with the follicle-associated epithelium of Peyer's patches of the distal ileum and translocate across the intestinal epithelium via M-cells, but the underlying molecular mechanisms are still unknown. Here, we investigated the involvement of Long polar fimbriae (Lpf) in EHEC pathogenesis. Of the 236 strains tested, a significant association was observed between the presence of lpf operons and pathogenicity. In sophisticated in vitro models of the human gastro-intestinal tract, lpf expression was induced during transit through the simulated stomach and small intestine, but not in the colonic compartment. To investigate the involvement of Lpf in EHEC pathogenesis, lpf isogenic mutants and their relative trans-complemented strains were generated. Translocation across M-cells, interactions with murine ileal biopsies containing Peyer's patches and the number of hemorrhagic lesions were significantly reduced with the lpf mutants compared to the wild-type strain. Complementation of lpf mutants fully restored the wild-type phenotypes. Our results indicate that (i) EHEC might colonize the terminal ileum at the early stages of infection, (ii) Lpf are an important player in the interactions with Peyer's patches and M-cells, and could contribute to intestinal colonization.

Daikenchuto (TU-100), a Japanese herbal medicine, is widely used for various gastrointestinal diseases. We have previously reported that TU-100 suppresses CPT-11-induced bacterial translocation (BT) by maintaining the diversity of the microbiome. In this study we show that TU-100 modulates the immune response during BT by inducing PD-1 expression in Peyer's patches.

Intestinal allergic diseases are initiated by aberrant Th2-type immune responses, including elevation of IgE antibodies (Abs) and infiltration of eosinophils. However, little is known about the role of Peyer's patches (PP) in the control of allergic diseases. Using a mouse model for food allergy, we here show that mice lacking PP are more susceptible to disease development and show higher levels of antigen-specific IgE Abs than do PP-intact mice. In our study, we noted that high numbers of eosinophils infiltrated into the small intestine of PP-null mice. In contrast, the PP of intact mice contained regulatory CD4+CD25+ Foxp3+ T cells (Treg) that are known to produce high levels of IL-10, and inhibited the development of allergic diarrhea. PP-intact mice thus developed allergic diarrhea when treated with anti-CD25 or anti-IL-10 monoclonal antibody (mAb) in vivo. These studies demonstrate that PP, as the site where IL-10-producing Treg cells are created, mediate the mucosal regulatory network for the control of undesired allergic responses in the intestine.

In many species such as sheep and pig, there are two types of Peyer's patches (PP): several discrete patches in the jejunum and a long and continuous patch in the ileum. Most of the immunoglobulin A in the gut is generated by B-cells in the PP germinal centers. Moreover, swine like ovine ileal PP might be important for antigen independent B-cell repertoire diversification. We examined, by quantitative real-time PCR, the expression of 36 transcripts of antimicrobial peptides, chemokines, interleukines, Toll-like receptors and transcription factors from both PP and we highlighted the differences by a principal component analysis. Ileal PP was characterized by a higher mRNA expression of CCL28, IL5, IL10, TLR2 and TLR4 while jejunal PP showed higher mRNA expression of antimicrobial peptides, CCL25, FOXP3, IL4, T-Bet, TSLP and SOCS2. Then, we analyzed some VDJ rearrangements to assess immunoglobulin repertoire diversity in jejunal and ileal PP from weaned piglets. The IgA and IgM repertoires were more diverse in ileal than in jejunal piglet PP. All these results could be related to the rarefaction of interfollicular T-cell zone and the presence in ileal versus jejunal lumen of a more diversified microflora. These findings shed a light on the functional differences between both PP.

Peyer's patches (PPs) are specialized gut-associated lymphoid tissues that initiate follicular helper T (Tfh)-mediated immunoglobulin A (IgA) response to luminal antigens derived from commensal symbionts, pathobionts, and dietary sources. IgA-producing B cells migrate from PPs to the small intestinal lamina propria and secrete IgA across the epithelium, modulating the ecological balance of the commensal microbiota and neutralizing pathogenic microorganisms. α-glucosidase inhibitors (α-GIs) are antidiabetic drugs that inhibit carbohydrate digestion in the small intestinal epithelium, leading to alterations in the commensal microbiota composition and metabolic activity. The commensal microbiota and IgA responses exhibit bidirectional interactions that modulate intestinal homeostasis and immunity. However, the effect of α-GIs on the intestinal IgA response remains unclear. We investigated whether α-GIs affect IgA responses by administering voglibose and acarbose to mice via drinking water. We analyzed Tfh cells, germinal center (GC) B cells, and IgA-producing B cells in PPs by flow cytometry. We also assessed pathogen-specific IgA responses. We discovered that voglibose and acarbose induced Tfh cells, GCB cells, and IgA-producing B cells in the PPs of the proximal small intestine in mice. This effect was attributed to the modification of the microbiota rather than a shortage of monosaccharides. Furthermore, voglibose enhanced secretory IgA (S-IgA) production against attenuated Salmonella Typhimurium. Our findings reveal a novel mechanism by which α-GIs augment antigen-specific IgA responses by stimulating Tfh-GCB responses in PPs, and suggest a potential therapeutic application as an adjuvant for augmenting mucosal vaccines.

Peyer's patches (PPs) are collections of lymphoid follicles in the small intestine, responsible for scanning the intestinal content for foreign antigens such as soluble molecules, particulate matter as well as intact bacteria and viruses. The immune cells of the patch are separated from the intestinal lumen by a single layer of epithelial cells, the follicle-associated epithelium (FAE). This epithelium covers the dome of the follicle and contains enterocyte-like cells and M cells, which are particularly specialized in taking up antigens from the gut. However, the presence and number of goblet cells as well as the presence of mucus on top of the FAE is controversial. When mouse ileal PPs were mounted in a horizontal Ussing-type chamber, we could observe a continuous mucus layer at mounting and new, easily removable mucus was released from the villi on the patch upon stimulation. Confocal imaging using fluorescent beads revealed a penetrable mucus layer covering the domes. Furthermore, immunostaining of FAE from mice, rats and humans with a specific antibody against the main component of intestinal mucus, the MUC2 mucin, clearly identify mucin-containing goblet cells. Transmission electron micrographs further support the identification of mucus releasing goblet cells on the domes of PPs in these species.

The regional specialization of intestinal immune cells is affected by the longitudinal heterogeneity of environmental factors. Although the distribution of group 3 innate lymphoid cells (ILC3s) is well characterized in the lamina propria, it is poorly defined in Peyer's patches (PPs) along the intestine. Given that PP ILC3s are closely associated with mucosal immune regulation, it is important to characterize the regulatory mechanism of ILC3s. Here, we found that terminal ileal PPs of specific pathogen-free (SPF) mice have fewer NKp46+ ILC3s than jejunal PPs, while there was no difference in NKp46+ ILC3 numbers between terminal ileal and jejunal PPs in antibiotics (ABX)-treated mice. We also found that butyrate levels in the terminal ileal PPs of SPF mice were higher than those in the jejunal PPs of SPF mice and terminal ileal PPs of ABX-treated mice. The reduced number of NKp46+ ILC3s in terminal ileal PPs resulted in a decrease in Csf2 expression and, in turn, resulted in reduced regulatory T cells and enhanced antigen-specific T-cell proliferation. Thus, we suggest that NKp46+ ILC3s are negatively regulated by microbiota-derived butyrate in terminal ileal PPs and the reduced ILC3 frequency is closely associated with antigen-specific immune induction in terminal ileal PPs.

Peyer's patches (PPs) can be considered as the immune site of the intestine. Within PPs, Dendritic cells (DCs) can uptake antigens from the gut lumen by extending dendrites into epithelium, and process it and then present to lymphocytes, which effectively antigen produces an immune response. Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), an acute and highly contagious enteric viral disease. The interaction between inactivated porcine epidemic diarrhea virus and porcine monocyte-derived dendritic cells (Mo-DCs) has been reported. However, little is known about the interaction between inactivated PEDV and DCs in porcine PPs.

The primary induction sites for intestinal IgA are the gut-associated lymphoid tissues (GALT), such as Peyer's patches (PPs) and isolated lymphoid follicles (ILFs). The commensal microbiota is known to contribute to IgA production in the gut; however, the role of dietary antigens in IgA production is poorly understood. To understand the effect of dietary antigens on IgA production, post-weaning mice were maintained on an elemental diet without any large immunogenic molecules. We found that dietary antigens contribute to IgA production in PPs through induction of follicular helper T cells and germinal center B cells. The role of dietary antigens in the PP responses was further confirmed by adding bovine serum albumin (BSA) into the elemental diet. Although dietary antigens are important for PP responses, they have fewer effects than the microbiota on the development and maturation of ILFs. Furthermore, we demonstrated that dietary antigens are essential for a normal antigen-specific IgA response to Salmonella typhi serovar Typhimurium infection. These results provide new insights into the role of dietary antigens in the regulation of mucosal immune responses.

High mobility group box-1 protein (HMGB1) is an alarmin that, once released, promotes inflammatory responses, alone and as a complex with the chemokine CXCL12. Here, we report that the HMGB1-CXCL12 complex plays an essential role also in homeostasis by controlling the migration of B lymphocytes. We show that extracellular HMGB1 is critical for the CXCL12-dependent egress of B cells from the Peyer's patches (PP). This promigratory function of the complex was restricted to the PPs, since HMGB1 was not required for B-cell migratory processes in other locations. Accordingly, we detected higher constitutive levels of the HMGB1-CXCL12 complex in PPs than in other lymphoid organs. HMGB1-CXCL12 in vivo inhibition was associated with a reduced basal IgA production in the gut. Collectively, our results demonstrate a role for the HMGB1-CXCL12 complex in orchestrating B-cell trafficking in homeostasis, and provide a novel target to control lymphocyte migration in mucosal immunity.

The intestinal mucosa is suggested to support extrathymic T cell development, particularly for T cell receptor (TCR)-gammadelta intraepithelial lymphocytes (IELs). TCR-gammadelta cell development requires interleukin (IL)-7; IL-7(-/)- or IL-7 receptor(-/)- mice lack TCR-gammadelta cells. Using the intestinal fatty acid binding protein (iFABP) promoter, we reinstated expression of IL-7 to mature enterocytes of IL-7(-/)- mice (iFABP-IL7). In iFABP-IL7 mice, TCR-gammadelta IELs were restored, as were cryptopatches and Peyer's patches. TCR-gammadelta cells remained absent from all other tissues. Likewise, T cell development in thymus and B cell maturation in the bone marrow and spleen retained the IL-7(-/)- phenotype. Thus, IL-7 expression by enterocytes was sufficient for extrathymic development of TCR-gammadelta cells in situ within the intestinal epithelium and was crucial for organization of mucosal lymphoid tissue.

Intestinal microfold cells (M cells) in Peyer's patches are a special subset of epithelial cells that initiate mucosal immune responses through uptake of luminal antigens. Although the cytokine receptor activator of nuclear factor-κB ligand (RANKL) expressed on mesenchymal cells triggers differentiation into M cells, other environmental cues remain unknown. Here, we show that the metastasis-promoting protein S100A4 is required for development of mature M cells. S100A4-producing cells are a heterogenous cell population including lysozyme-expressing dendritic cells and group 3 innate lymphoid cells. We found that in the absence of DOCK8, a Cdc42 activator critical for interstitial leukocyte migration, S100A4-producing cells are reduced in the subepithelial dome, resulting in a maturation defect of M cells. While S100A4 promotes differentiation into mature M cells in organoid culture, genetic inactivation of S100a4 prevents the development of mature M cells in mice. Thus, S100A4 is a key environmental cue that regulates M cell differentiation in collaboration with RANKL.

The mechanism by which indigenous bacteria on the follicle-associated epithelium (FAE) of lymphatic follicles (LFs) accelerate the differentiation of microvillous columnar epithelial cells (MV) into M-cells was immunohistochemically investigated in rat Peyer's patches. The results showed that the number of Toll-like receptor (TLR) -4+ M-cells was greater in the FAE with expansion of bacterial colonies (LFs with bacterial colonies on the FAE: b-LF) than the FAE without expansion of bacterial colonies (nb-LF). TLR-4 was also expressed in the striated borders of MV upstream next to M-cells in the FAE of the b-LF. TLR-4+ vesicles were frequently detected in the cytoplasms of MV with TLR-4+ striated borders upstream next to TLR-4+ M-cells in the FAE of b-LF. These findings suggest that TLR-4+ MV take up TLR-4 ligands and differentiate into M-cells in the b-LF. Neither the distribution of RANK nor that of RANKL was coincident with that of M-cells in the b-LF. Moreover, RANK, but not RANKL, was expressed in intestinal villi, whereas cleaved caspase-3 was immunonegative in the MV and M-cells of the FAE, unlike in villous epithelial cells. Therefore, RANK/RANKL signaling in the LF might contribute to the down-regulation of epithelial apoptosis to facilitate the differentiation of MV into M-cells in rat Peyer's patches.