Commensal bacterial outer membrane protein A induces interleukin-22 production.

Interleukin (IL)-22 promotes host-microbiota homeostasis. We sought to identify microbiota metabolite(s) that drive intestinal IL-22 production. We observed that exposing Peyer's patch cells (PPCs), ex vivo, to fecal supernatants (FSs) recapitulates fermentable fiber- and microbiota-dependent IL-22 production, and cellular sources thereof, thus supporting the use of this model. An interrogation of FSs generated from mice fed the fermentable fiber inulin (FS-Inu) revealed that its IL-22-inducing activity is mediated by heat-labile protein. Fractionation of FS-Inu by ion-exchange chromatography, and subsequent proteomic analysis of IL-22-inducing fractions, indicates that outer membrane protein A (OmpA) might be a microbial driver of IL-22 expression. Concomitantly, recombinant OmpA from Parabacteroides goldsteinii, which is enriched by an inulin diet, induces IL-22 production and expression of the IL-22-dependent genes REG3γ and -ß, in PPCs and mice. Thus, OmpA is one bacterial inducer of IL-22 expression, potentially linking diet, mucosal immune homeostasis, and gut health.

Peyer's Patch: Possible target for modulating the Gut-Brain-Axis through microbiota.

The gastrointestinal (GI) tract and the brain form bidirectional nervous, immune, and endocrine communications known as the gut-brain axis. Several factors can affect this axis; among them, various studies have focused on the microbiota and imply that alterations in microbiota combinations can influence both the brain and GI. Also, many studies have shown that the immune system has a vital role in varying gut microbiota combinations. In the current paper, we will review the multidirectional effects of gut microbiota, immune system, and nervous system on each other. Specifically, this review mainly focuses on the impact of Peyer's patches as a critical component of the gut immune system on the gut-brain axis through affecting the gut's microbial composition. In this way, some factors were discussed as proposed elements of missing gaps in this field.

CD200R activation on naïve T cells by B cells induces suppressive activity of T cells via IL-24.

CD200 is an anti-inflammatory protein that facilitates signal transduction through its receptor, CD200R, in cells, resulting in immune response suppression. This includes reducing M1-like macrophages, enhancing M2-like macrophages, inhibiting NK cell cytotoxicity, and downregulating CTL responses. Activation of CD200R has been found to modulate dendritic cells, leading to the induction or enhancement of Treg cells expressing Foxp3. However, the precise mechanisms behind this process are still unclear. Our previous study demonstrated that B cells in Peyer's patches can induce Treg cells, so-called Treg-of-B (P) cells, through STAT6 phosphorylation. This study aimed to investigate the role of CD200 in Treg-of-B (P) cell generation. To clarify the mechanisms, we used wild-type, STAT6 deficient, and IL-24 deficient T cells to generate Treg-of-B (P) cells, and antagonist antibodies (anti-CD200 and anti-IL-20RB), an agonist anti-CD200R antibody, CD39 inhibitors (ARL67156 and POM-1), a STAT6 inhibitor (AS1517499), and soluble IL-20RB were also applied. Our findings revealed that Peyer's patch B cells expressed CD200 to activate the CD200R on T cells and initiate the process of Treg-of-B (P) cells generation. CD200 and CD200R interaction triggers the phosphorylation of STAT6, which regulated the expression of CD200R, CD39, and IL-24 in T cells. CD39 regulated the expression of IL-24, which sustained the expression of CD223 and IL-10 and maintained the cell viability. In summary, the generation of Treg-of-B (P) cells by Peyer's patch B cells was through the CD200R-STAT6-CD39-IL-24 axis pathway.

Quercetin Improves Barrier Properties in Porcine Small Intestine but Not in Peyer's Patches.

Peyer's patches (PPs) are part of the gut-associated lymphatic tissue (GALT) and represent the first line of the intestinal immunological defense. They consist of follicles with lymphocytes and an overlying subepithelial dome with dendritic cells and macrophages, and they are covered by the follicle-associated epithelium (FAE). A sealed paracellular pathway in the FAE is crucial for the controlled uptake of luminal antigens. Quercetin is the most abundant plant flavonoid and has a barrier-strengthening effect on tight junctions (TJs), a protein complex that regulates the paracellular pathway. In this study, we aimed to analyze the effect of quercetin on porcine PPs and the surrounding villus epithelium (VE). We incubated both tissue types for 4 h in Ussing chambers, recorded the transepithelial electrical resistance (TEER), and measured the unidirectional tracer flux of [3H]-mannitol. Subsequently, we analyzed the expression, protein amount, and localization of three TJ proteins, claudin 1, claudin 2, and claudin 4. In the PPs, we could not detect an effect of quercetin after 4 h, neither on TEER nor on the [3H]-mannitol flux. In the VE, quercetin led to a higher TEER value, while the [3H]-mannitol flux was unchanged. The pore-forming claudin 2 was decreased while the barrier-forming claudin 4 was increased and the expression was upregulated. Claudin 1 was unchanged and all claudins could be located in the paracellular membrane by immunofluorescence microscopy. Our study shows the barrier-strengthening effect of quercetin in porcine VE by claudin 4 upregulation and a claudin 2 decrease. Moreover, it underlines the different barrier properties of PPs compared to the VE.

α-Glucosidase inhibitors boost gut immunity by inducing IgA responses in Peyer's patches.

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.

Antigen receptor signaling and cell death resistance controls intestinal humoral response zonation.

Immunoglobulin A (IgA) maintains commensal communities in the intestine while preventing dysbiosis. IgA generated against intestinal microbes assures the simultaneous binding to multiple, diverse commensal-derived antigens. However, the exact mechanisms by which B cells mount broadly reactive IgA to the gut microbiome remains elusive. Here, we have shown that IgA B cell receptor (BCR) is required for B cell fitness during the germinal center (GC) reaction in Peyer's patches (PPs) and for generation of gut-homing plasma cells (PCs). We demonstrate that IgA BCR drove heightened intracellular signaling in mouse and human B cells, and as a consequence, IgA+ B cells received stronger positive selection cues. Mechanistically, IgA BCR signaling offset Fas-mediated death, possibly rescuing low-affinity B cells to promote a broad humoral response to commensals. Our findings reveal an additional mechanism linking BCR signaling, B cell fate, and antibody production location, which have implications for how intestinal antigen recognition shapes humoral immunity.

Preparation of Herbal Extracts for Intestinal Immune Modulation Activity Based on In Vitro Screening and In Vivo Evaluation of Zingiber officinale Rosc. Extracts.

Ten traditional herbal extracts effective against diarrhea, infectious diseases, and bacterial activity were selected and analyzed for Peyer's patch cell-mediated intestinal immunomodulatory activity in vitro and in vivo. Among the 10 herbal extracts, Zingiber officinale Rosc. (ZO) extract induced the highest secretion of immunoglobulin A (IgA) and granulocyte macrophage colony-stimulating factor (GM-CSF) in the cells of Peyer's patches. Furthermore, animal experiments showed that IA production was enhanced with the oral administration of ZO extract (100 mg/kg and 300 mg/kg) for 10 days. In addition, 6-, 8-, 10-gingerol, and 6-, 8-, 10-shogaol, the six major index compounds of ZO extract, were analyzed using HPLC. Our study findings confirm the intestinal immunomodulatory activity of ZO extract and lay a strong foundation for future analytical studies aimed at determining the active components of ZO extracts.

Core Shell Lipid-Polymer Hybrid Nanoparticles for Oral Bioavailability Enhancement of Ibrutinib via Lymphatic Uptake.

The purpose of this research was to develop ibrutinib (IBR)-loaded lipid-polymer hybrid nanoparticles (IBR-LPHNPs) to improve oral absorption by intestinal lymphatic uptake. IBR-LPHNPs were fabricated by nanoprecipitation method using poly(lactic-co-glycolic acid), lipoid S 100, and DSPE-MPEG 2000. The IBR-LPHNPs showed particle size of 85.27±3.82 nm, entrapment efficiency of 97.70±3.85%, and zeta potential of -24.9±3.08 mV respectively. Fourier transform infrared spectroscopy and differential scanning calorimetry study revealed compatibility between IBR and excipients. X-ray diffraction study showed the conversion of IBR into amorphous form. High-resolution transmission electron microscopic image displayed spherical-shaped, discrete layered polymeric core and lipid shell structure. The drug release from IBR-LPHNPs exhibited prolong release profile up to 48 h and was best fitted to Korsmeyer-Peppas model. Higher fluorescence intensity at the end of 2 h in the intestinal tissue confirmed the uptake of LPHNPs by Peyer's patches. The oral bioavailability of IBR was improved 22.52-fold with LPHNPs as compared to free IBR. The intestinal lymphatic uptake study in rats pretreated with cycloheximide confirmed the intestinal lymphatic uptake of IBR-LPHNPs. All the results conclusively showed that LPHNPs could be a promising approach to improve oral bioavailability of IBR.

M cell maturation and cDC activation determine the onset of adaptive immune priming in the neonatal Peyer's patch.

Early-life immune development is critical to long-term host health. However, the mechanisms that determine the pace of postnatal immune maturation are not fully resolved. Here, we analyzed mononuclear phagocytes (MNPs) in small intestinal Peyer's patches (PPs), the primary inductive site of intestinal immunity. Conventional type 1 and 2 dendritic cells (cDC1 and cDC2) and RORgt+ antigen-presenting cells (RORgt+ APC) exhibited significant age-dependent changes in subset composition, tissue distribution, and reduced cell maturation, subsequently resulting in a lack in CD4+ T cell priming during the postnatal period. Microbial cues contributed but could not fully explain the discrepancies in MNP maturation. Type I interferon (IFN) accelerated MNP maturation but IFN signaling did not represent the physiological stimulus. Instead, follicle-associated epithelium (FAE) M cell differentiation was required and sufficient to drive postweaning PP MNP maturation. Together, our results highlight the role of FAE M cell differentiation and MNP maturation in postnatal immune development.

Peyer's patch-involved gut microbiota facilitates anti-HBV immunity in mice.

BACKGROUND: Gut microbiota is crucial for immune homeostasis and is associated with the prognosis of chronic hepatitis B infection. Peyer's patches (PPs), characterized by intestinal mucosa localization, are involved in the gut microbiota-mediated immune response. However, whether and how PPs orchestrate gut microbiota-modulated anti-hepatitis B virus (HBV) response remain elusive. This study aims to elucidate the role of PPs in gut microbiota-mediated anti-HBV adaptive immunity. METHODS: We investigated the effects of gut microbiota and PPs on adaptive immune responses by transcriptomic, phenotypic, and functional analyzes from an HBV mouse model with gut commensal microbiota and PP-depleting interventions. RESULTS: Depletion of gut microbiota impaired systemic adaptive immune responses, resulting in a delayed HBV antigen clearance. Differentially expressed genes analysis of PPs revealed that pathways related to adaptive immune responses were significantly downregulated in gut microbiota-deficient mice. Notably, the depletion of PPs could abolish gut microbiota-boosted intrahepatic HBV-specific T cell response, leading to a higher serum hepatitis B surface antigen level in mice. CONCLUSION: PPs orchestrate gut microbiota-mediated intrahepatic anti-HBV cellular immunity, underlining the significance of remote manipulating the "gut microbiota-PPs" axis for achieving optimum anti-HBV response.

Diurnal changes in bacterial settlement on the Peyer's patch and surrounding mucosa in the rat ileum and its effect against the intestinal immune system.

Our previous study revealed the diurnal change in the indigenous bacteria settling on the terminal region of the rat ileum. In the present study, we investigated the diurnal change in indigenous bacteria on the most distal ileal Peyer's patch (PP) and surrounding ileal mucosa and explored how stimulation from indigenous bacteria for a day affects the intestinal immune system at the beginning of the light phase. Histological measurement revealed that bacteria adjacent to the follicle-associated epithelium of PP and to the villous epithelium of the surrounding ileal mucosa are more abundant at zeitgeber time (ZT)0 and ZT18 than at ZT12. On the other hand, tissue-section 16S rRNA amplicon sequencing revealed no significant difference between ZT0 and ZT12 in the bacterial composition on the ileal tissue including the PP. One-day treatment with an antibiotic (Abx) successfully impaired the settlement of bacteria around the ileal PP. In transcriptome analysis, 1-day Abx treatment led to the downregulation of several chemokines in both PP and ordinary ileal mucosa at ZT0. Histological analysis of the 1-day Abx group revealed decreases in both CD68+ macrophages in PP and naphthol AS-D chloroacetate esterase stain-positive mast cells in the ileal villi. Together, these findings suggest that the colonies of indigenous bacteria on the distal ileal PP and surrounding mucosa expand during the dark phase, which might lead to the expression of genes to regulate the intestinal immune system and contribute to the homeostasis of at least macrophages in PP and mast cells in the ileal mucosa.

Intestinal Peyer's Patches: Structure, Function, and In Vitro Modeling.

BACKGOUND: Considering the important role of the Peyer's patches (PPs) in gut immune balance, understanding of the detailed mechanisms that control and regulate the antigens in PPs can facilitate the development of immune therapeutic strategies against the gut inflammatory diseases. METHODS: In this review, we summarize the unique structure and function of intestinal PPs and current technologies to establish in vitro intestinal PP system focusing on M cell within the follicle-associated epithelium and IgA+ B cell models for studying mucosal immune networks. Furthermore, multidisciplinary approaches to establish more physiologically relevant PP model were proposed. RESULTS: PPs are surrounded by follicle-associated epithelium containing microfold (M) cells, which serve as special gateways for luminal antigen transport across the gut epithelium. The transported antigens are processed by immune cells within PPs and then, antigen-specific mucosal immune response or mucosal tolerance is initiated, depending on the response of underlying mucosal immune cells. So far, there is no high fidelity (patho)physiological model of PPs; however, there have been several efforts to recapitulate the key steps of mucosal immunity in PPs such as antigen transport through M cells and mucosal IgA responses. CONCLUSION: Current in vitro PP models are not sufficient to recapitulate how mucosal immune system works in PPs. Advanced three-dimensional cell culture technologies would enable to recapitulate the function of PPs, and bridge the gap between animal models and human.

Efficacies of Potential Probiotic Candidates Isolated from Traditional Fermented Korean Foods in Stimulating Immunoglobulin A Secretion.

The aim of this study was to evaluate efficacies of selected lactic acid bacteria (LAB) in inducing immunoglobulin A (IgA) secretion. Twenty-five different LAB isolated from traditional fermented Korean foods were characterized for their probiotic properties and screened to identify those that could stimulate lamina propria cells (LPCs) from Peyer's patch to secret IgA in vitro. Among them, four strains (Lactiplantibacillus plantarum CJW55-10, Lactiplantibacillus pentosus CJW18-6, L. pentosus CJW56-11, and Pediococcus acidilactici CJN2696) were found to be strong IgA inducers. The number of IgA positive B cells and soluble IgA level were increased when LPCs were co-cultured with these LAB. Expression levels of toll-like receptor (TLR) such as TLR2 and TLR4 and secretion of interleuckin-6 were augmented in LPCs treated with these LAB. Further, we determined whether oral intake of these LAB enhanced IgA production in vivo. After one-week of daily oral administration, these LAB feed mice increased mucosal IgA and serum IgA. In conclusion, selected strains of LAB could induce systemic IgA secretion by activating lamina propria B cells in Peyer's patch and oral intake of selected strains of LAB can enhance systemic immunity by inducing mucosal IgA secretion.

Issues for patchy tissues: defining roles for gut-associated lymphoid tissue in neurodevelopment and disease.

Individuals diagnosed with neurodevelopmental conditions such as autism spectrum disorder (ASD; autism) often experience tissue inflammation as well as gastrointestinal dysfunction, yet their underlying causes remain poorly characterised. Notably, the largest components of the body's immune system, including gut-associated lymphoid tissue (GALT), lie within the gastrointestinal tract. A major constituent of GALT in humans comprises secretory lymphoid aggregates known as Peyer's patches that sense and combat constant exposure to pathogens and infectious agents. Essential to the functions of Peyer's patches is its communication with the enteric nervous system (ENS), an intrinsic neural network that regulates gastrointestinal function. Crosstalk between these tissues contribute to the microbiota-gut-brain axis that altogether influences mood and behaviour. Increasing evidence further points to a critical role for this signalling axis in neurodevelopmental homeostasis and disease. Notably, while the neuroimmunomodulatory functions for Peyer's patches are increasingly better understood, functions for tissues of analogous function, such as caecal patches, remain less well characterised. Here, we compare the structure, function and development of Peyer's patches, as well as caecal and appendix patches in humans and model organisms including mice to highlight the roles for these essential tissues in health and disease. We propose that perturbations to GALT function may underlie inflammatory disorders and gastrointestinal dysfunction in neurodevelopmental conditions such as autism.

Study on improvement effects and mechanism of rhein on immunoglobulin A nephropathy / 中国药房

OBJECTIVE To study the improvement effects and mechanism of rhein on immunoglobulin A nephropathy （IgAN） model rat based on signal transducer and activator of transcription 3 （STAT3） signaling pathway. METHODS Rats were randomly divided into normal control group， IgAN model group and rhein treatment group， with 10 rats in each group. IgAN model group and rhein treatment group were given combination of bovine serum albumin+lipopolysaccharide+carbon tetrachloride to induce IgAN model. Since the 7th week， rhein treatment group rats were intragastrically given relevant medicine， and normal control group and model group rats were given equal amount of normal saline intragastrically， for consecutive 4 weeks. After the last administration， the count of urine sediment erythrocyte， 24 h-urine total protein （UTP）， the levels of immunoglobulin A （IgA） in serum and secretory immunoglobulin A （sIgA） in intestinal mucosa were detected. The pathological changes of Peyer’s patch in renal cortex and intestinal mucosa and IgA deposition in renal cortex were observed. The expressions of interleukin-17 （IL-17）， IL- 6 and transforming growth factor β （TGF-β） in Peyer’s patch of intestinal mucosa in rats were detected. The expressions of STAT3 and related orphan receptor γt （RORγt） mRNA in Peyer’s patch were detected. The expressions of p-STAT3 and RORγt proteins in Peyer’s patch were detected. RESULTS Compared with normal control group， the count of urine sediment erythrocyte， 24 h-UTP， the levels of IgA in serum and sIgA in intestinal mucosa were increased significantly in IgAN model group （P＜0.01）； enlarged renal corpuscles， dilated renal sacs， obvious intratubular mesangial hyperplasia and fibrosis were observed in renal cortex； the volume and germinal center of Peyer’s patch in intestinal mucosa increased； IgA deposition of renal cortex zxyylxk20220103） was obvious； the expressions of IL-17， IL-6 and TGF-β in Peyer’s patch， mRNA expressions of STAT3 and RORγt， protein expressions of p-STAT3 and RORγt were increased significantly （P＜0.01）. Compared with IgAN model group，above indexes were decreased significantly in rhein treatment group （P＜0.01）， pathological damage of renal cortex was improved， the volume of Peyer’s patch and germinal center of intestinal mucosa were reduced， and IgA deposition in renal cortex was weakened. CONCLUSIONS Rhein can improve IgAN model rats， the mechanism of which may be associated with inhibiting STAT3 signaling pathway and regulating immune function of Peyer’s patch in intestinal mucosa.

Sheep vaccinated against paratuberculosis have increased levels of B cells infiltrating the intestinal tissue.

Systemic immunisation delivered subcutaneously is currently used to control paratuberculosis, a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). These vaccines do not provide complete protection and a small cohort of animals still succumb to clinical disease. The aim of this study was to assess mycobacterial infection site-specific variations in immune cells in vaccinated sheep that did or did not develop the disease following controlled exposure to MAP. Immunohistochemical staining of terminal ileum demonstrated that vaccination increased infiltration of CD4 + T cells and B cells. Infiltration of large numbers of CD4 + T and B cells was also seen in sheep that successfully cleared infection. Vaccination promoted the polarisation of macrophages to an M1 activation state. The presence of certain cells at the site of infection, especially CD4 + T cells, is likely to contribute to vaccine success by increasing the speed and potency of the local immune response. Systemic immunisation against MAP can alter the composition of innate and adaptive immune cell populations at the predilection site for MAP infection in the ileum one year after vaccination. This informs understanding of the impact of vaccination at the site of infection and also the duration of vaccine-elicited changes. This information may assist vaccine development and allow targeting of protective immune responses in the gut of ruminants.

Barrier Perturbation in Porcine Peyer's Patches by Tumor Necrosis Factor is Associated With a Dysregulation of Claudins.

The proinflammatory cytokine tumor necrosis factor (TNF) has been described as one of the main mediators of intestinal inflammatory diseases, affecting the composition of tight junction (TJ) proteins and leading to a disruption of the epithelial barrier. An intact intestinal barrier is mandatory, because the follicle-associated epithelium of Peyer's patches represents the first defense line of the intestinal immune system and ensures a controlled uptake of antigens from the gut lumen. In the current study, we have analyzed the detailed effects of TNF on the follicle-associated epithelium of porcine Peyer's patches by applying the Ussing chamber technique. Epithelial tissue specimens of Peyer's patches and the surrounding villus epithelium were mounted into conventional Ussing chambers and incubated with TNF for 10 h. The transepithelial resistance, representing epithelial barrier function of the tissue, was recorded. A reduction of transepithelial resistance was detected after 8 h in Peyer's patch tissue specimens, whereas the villus epithelium was not significantly affected by TNF. Subsequent molecular analysis of TJ protein expression revealed a marked decrease of claudin-1 and -4, and an increase of claudin-2. In neighboring villus epithelium, no significant changes in the expression of TJ proteins could be shown. A strong increase of TNF receptor-2 (TNFR-2) could also be detected in Peyer's patches, in agreement with the major role of this receptor in Peyer's patches. Our findings were in accordance with changes detected by confocal laser scanning immunofluorescence microscopy. The regulation of TNF effects via myosin light chain kinase (MLCK) was analyzed in blocking experiments. Our detailed analysis is the first to show that TNF affects the barrier function of the follicle-associated epithelium of porcine Peyer's patches but has no effects on the villus epithelium. These findings reveal not only the basic differences of epithelial barrier function between the two structures, but also the significance of Peyer's patches as a primary mucosal immune defense.

Three Layers of Intestinal γδ T Cells Talk Different Languages With the Microbiota.

The mucosal surfaces of our body are the main contact site where the immune system encounters non-self molecules from food-derived antigens, pathogens, and symbiotic bacteria. γδ T cells are one of the most abundant populations in the gut. Firstly, they include intestinal intraepithelial lymphocytes, which screen and maintain the intestinal barrier integrity in close contact with the epithelium. A second layer of intestinal γδ T cells is found among lamina propria lymphocytes (LPL)s. These γδ LPLs are able to produce IL-17 and likely have functional overlap with local Th17 cells and innate lymphoid cells. In addition, a third population of γδ T cells resides within the Peyer´s patches, where it is probably involved in antigen presentation and supports the mucosal humoral immunity. Current obstacles in understanding γδ T cells in the gut include the lack of information on cognate ligands of the γδ TCR and an incomplete understanding of their physiological role. In this review, we summarize and discuss what is known about different subpopulations of γδ T cells in the murine and human gut and we discuss their interactions with the gut microbiota in the context of homeostasis and pathogenic infections.

PRC2 Regulated Atoh8 Is a Regulator of Intestinal Microfold Cell (M Cell) Differentiation.

Intestinal microfold cells (M cells) are a dynamic lineage of epithelial cells that initiate mucosal immunity in the intestine. They are responsible for the uptake and transcytosis of microorganisms, pathogens, and other antigens in the gastrointestinal tract. A mature M cell expresses a receptor Gp2 which binds to pathogens and aids in the uptake. Due to the rarity of these cells in the intestine, their development and differentiation remain yet to be fully understood. We recently demonstrated that polycomb repressive complex 2 (PRC2) is an epigenetic regulator of M cell development, and 12 novel transcription factors including Atoh8 were revealed to be regulated by the PRC2. Here, we show that Atoh8 acts as a regulator of M cell differentiation; the absence of Atoh8 led to a significant increase in the number of Gp2+ mature M cells and other M cell-associated markers such as Spi-B and Sox8. In vitro organoid analysis of RankL treated organoid showed an increase of mature marker GP2 expression and other M cell-associated markers. Atoh8 null mice showed an increase in transcytosis capacity of luminal antigens. An increase in M cell population has been previously reported to be detrimental to mucosal immunity because some pathogens like orally acquired prions have been able to exploit the transcytosis capacity of M cells to infect the host; mice with an increased population of M cells are also susceptible to Salmonella infections. Our study here demonstrates that PRC2 regulated Atoh8 is one of the factors that regulate the population density of intestinal M cell in the Peyer's patch.

Gene Expression Pattern of Peyer's Patch Lymphocytes Exposed to Kagocel Suggests Pattern-Recognition Receptors Mediate Its Action.

Kagocel is a synthetic carboxymethylcellulose derivative copolymerized with gossypol. Clinical data evidence its safety and efficiency for the treatment of flu and other viral infections via enhancement of interferon production. The gut-associated lymphoid tissue seems a likely site of kagocel action. The study was aimed to investigate the molecular mechanisms of its action using murine Peyer's patches lymphocytes as a test system and the cytokines production and gene expression patterns as the primary outcomes. The Peyer's patches lymphocytes isolated from BALB/c mice were stimulated with concanavalin A, or, to mimic viral infection, with a combination of concanavalin A and TLR3 ligand poly I:C. After 24 h of stimulation the cells were treated with saline, 30, 100, or 300 µg/ml of kagocel, or, as positive controls, 300 µg/ml oats b-D-glucan or 300 µg/ml lentinan. After 24 and 72 h of incubation with these drugs cytokines production was analyzed with ELISA and gene expression pattern was investigated using nCounter Inflammation panel chips followed by bioinformatics analysis. Expression of genes involved in the inflammatory response, antiviral defense, lymphocytes survival and proliferation (C1qa, C2, C3, Ccl21a, Il11, Il1b, Il23a, Il5, Ltb4r2, Alox15, Pla2g4a, Ptger1, Mapkapk5, Hras, Ifna1, Tlr2, Mrc1, Mx2) was upregulated in kagocel-treated Peyer's patches lymphocytes. A list of plausible transcription factors (CEBPs, IRF, NFκB, RXR, Stat, Tead4, and ZSCAN) and master-regulators has been identified (cIAP, CIKS, dock9, MEKK1, FXR, IKK, IRAK, TRAF, dsRNA:TLR3:TRIF). The changes in gene expression pattern and the outcomes of bioinformatics analysis suggest that pattern recognition receptors, TLRs and dectin-1, are the key mediators of kagocel immunomodulatory action, with the possible involvement of interferon autocrine loop. The genes upregulated with kagocel include diverse components of the innate immune defense system.