Peyer's Patch Cells Research Articles

Garlic Bioconverted by Bacillus subtilis Stimulates the Intestinal Immune System and Modulates Gut Microbiota Composition.

This study evaluates the potential of bioconverted garlic ferments (BGFs) to stimulate the intestinal immune system and modulate cecal microbiota composition. In vitro, BGF significantly enhances Peyer's patch (PP)-mediated bone marrow cell proliferation and increases the production of interferon-gamma (IFN-γ), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-6, and immunoglobulin A (IgA) but not IL-4, IL-5, and immunoglobulin E (IgE). Oral administration of BGF to C3H/HeN mice for 4 weeks significantly increases the GM-CSF (42.1-45.8 pgmL-1) and IFN-γ (6.5-12.1 pgmL-1) levels in PP cells. BGF also significantly elevates the levels of tumor necrosis factor-alpha (TNF-α, 165.0-236.3 pgmg-1), GM-CSF (2.4-3.0ngmg-1), and IFN-γ (1.5-3.2ngmg-1) in the small intestinal fluid, and TNF-α (2.2-3.1 pgmL-1) and IFN-γ (10.3-0.21.5 pgmL-1) in the mouse serum. Cecal microbial analysis reveals that BGF increases Bacteroidota and Verrucomicrobiota and decreases Actinobacteria and Bacillota at the phylum level in mice. At the genus level, BGF significantly increases the abundance of Fusimonas (250mgkg-1BW-1day-1), Bacteroides (125 and 250mgkg-1BW-1day-1), and Akkermansia (125mgkg-1BW-1day-1) and decreases that of Bifidobacterium (62.5 and 250mgkg-1BW-1day-1) and Limosilactobacillus (125 and 250mgkg-1BW-1day-1). This study provides the first evidence of BGF's ability to modulate the intestinal immune system and gut microbiota, supporting its potential as a novel functional material to enhance gut immunity.

Effects of parenteral nutrition supplemented with beta-hydroxy-beta-methylbutyrate on gut-associated lymphoid tissue and morphology in mice.

Parenteral nutrition (PN) without enteral nutrition (EN) leads to marked atrophy of gut-associated lymphoid tissue (GALT), causing mucosal defense failure in both the gut and the extraintestinal mucosal system. We evaluated the effects of beta-hydroxy-beta-methylbutyrate (HMB) on GALT and gut morphology in PN-fed mice. Experiment 1: male Institute of Cancer Research mice were assigned to the Chow (n = 12), Control (standard PN: n = 10), or H600 and H2000 (PN containing 600 mg/kg or H2000 mg/kg body weight of Ca-HMB: n = 12 and 10, respectively) groups. After 5 days of dietary manipulation, all mice were killed and the whole small intestine was harvested. GALT lymphocyte cell numbers and phenotypes of Peyer patch (PP), intraepithelial space, and lamina propria lymphocytes were evaluated. Experiment 2: 47 mice (Chow: n = 12; Control: n = 14; H600: n = 11; and H2000: n = 10) were fed for 5 days as in experiment 1. Proliferation and apoptosis of gut immune cells and mucosa, and protein expressions (mammalian target of rapamycin [mTOR], caspase-3, and Bcl2) were evaluated in the small intestine. Compared with the Controls, the Chow and HMB groups showed significantly higher PP cell numbers, prevented gut mucosal atrophy, inhibited apoptosis of gut cells, and increased their proliferation in association with increased mTOR activity and Bcl2 expression. HMB-supplemented PN is a potentially novel method of preserving GALT mass and gut morphology in the absence of EN.

Food antigens suppress small intestinal tumorigenesis.

Food components suppressing small intestinal tumorigenesis are not well-defined partly because of the rarity of this tumor type compared to colorectal tumors. Using Apcmin/+ mice, a mouse model for intestinal tumorigenesis, and antigen-free diet, we report here that food antigens serve this function in the small intestine. By depleting Peyer's patches (PPs), immune inductive sites in the small intestine, we found that PPs have a role in the suppression of small intestinal tumors and are important for the induction of small intestinal T cells by food antigens. On the follicle-associated epithelium (FAE) of PPs, microfold (M) cells pass food antigens from lumen to the dendritic cells to induce T cells. Single-cell RNA-seq (scRNA-seq) analysis of immune cells in PPs revealed a significant impact of food antigens on the induction of the PP T cells and the antigen presentation capacity of dendritic cells. These data demonstrate the role of food antigens in the suppression of small intestinal tumorigenesis by PP-mediated immune cell induction.

Bacillus subtilis Feed Supplementation Combined with Oral E. coli Immunization in Sows as a Tool to Reduce Neonatal Diarrhea in Piglets.

To investigate the effects of B. subtilis on the specific immune response of lactating sows to E. coli and the diarrhea rate in suckling piglets, thirty large white sows with similar farrowing dates were randomly divided into two groups: a feedback feeding (i.e., feeding a homogenate of intestinal contents and tissues from E. coli-infected piglets to sows; FB) group and a feedback feeding with B. subtilis (FB + BS) group. Serum, colostrum, and intestinal tissues from sows and piglets were collected to assess the immune response and intestinal barrier function at weaning. T and B cells from Peyer's patches (PPs) and mesenteric lymph nodes (MLNs) in lactating mice (with treatments consistent with the sows') were isolated to explore the underlying mechanism. The results showed that, compared with the FB group, the reproductive performance of sows and the growth performance of their offspring were effectively improved in the FB + BS group. Moreover, the levels of IgG/IgA and those of IgG/IgA against E. coli in the serum and colostrum of sows in the FB+BS group were increased (p < 0.05). Meanwhile, the ratio of CD4+/CD8+, CD4+CXCR5+PD1+, and B220+IgA+ cells in MLNs and PPs, and the IgA levels in the mammary glands of mice, were also increased in the FB + BS group (p < 0.05). Notably, in suckling piglets in the FB + BS group, the diarrhea rate was decreased (p < 0.05), and the intestinal barrier function and intestinal flora composition at weaning were significantly improved. Overall, these results indicated that B. subtilis feed supplementation combined with feedback feeding in pregnant and lactating sows can reduce diarrhea in suckling piglets by enhancing the maternal immune response against E. coli and intestinal barrier function in their offspring, improving survival rates and pre-weaning growth.

Cistanche deserticola polysaccharide-functionalized dendritic fibrous nano-silica as oral vaccine adjuvant delivery enhancing both the mucosal and systemic immunity

Oral vaccines are a safe and convenient alternative to injected vaccines and have great potential to prevent major infectious diseases. However, the harsh gastrointestinal (GI) environment, mucus barriers, low immunogenicity, and lack of effective and safe mucosal adjuvants are the major challenges for oral vaccine delivery. In recent years, nanoparticle-based strategies have become attractive for improving oral vaccine delivery. Here, the dendritic fibrous nano-silica (DFNS) grafted with Cistanche deserticola polysaccharide (CDP) nanoparticles (CDP-DFNS) were prepared and investigated how to impact the immune responses. CDP-DFNS facilitated the antigen uptake in mouse bone marrow-derived dendritic cells (BMDCs), and induce the activation of DCs in vitro. Furthermore, in vivo experiments, the result showed that the uptake efficiency by Peyer's patches (PPs) of CDP-DFNS/BSA was the best. And CDP-DFNS/BSA then significantly activated the DCs in lamina propria (LP), and T/B cells in PPs and mesenteric lymph nodes (MLNs). Moreover, the memory T cell responses in later period of vaccination was stronger than other groups. In addition, CDP-DFNS/BSA enhanced BSA-specific antibody IgG, IgA production, and SIgA secretion, was effective at inducing a strong mixed Th1/Th2 response and mucosal antibody responses. These results indicated that CDP-DFNS deserves further consideration as an oral vaccine adjuvant delivery system.

High productivity of immunostimulatory membrane vesicles of Limosilactobacillus antri using glycine.

Nanosized membrane vesicles (MVs) released by bacteria play important roles in both bacteria-bacteria and bacteria-host interactions. Some gram-positive lactic acid bacteria produce MVs exhibiting immunoregulatory activity in the host. We found that both bacterial cells and MVs of Limosilactobacillus antri JCM 15950, isolated from the human stomach mucosa, enhance immunoglobulin A production by murine Peyer's patch cells. However, the thick cell walls of gram-positive bacteria resulted in low MV production, limiting experiments and applications using MVs. In this study, we evaluated the effects of glycine, which inhibits cell wall synthesis, on the immunostimulatory MV productivity of L. antri. Glycine inhibited bacterial growth while increasing MV production, with 20 g/L glycine increasing MV production approximately 12-fold. Glycine was most effective at increasing MV production when added in the early exponential phase, which indicated that cell division in the presence of glycine increased MV production. Finally, glycine increased MV productivity approximately 16-fold. Furthermore, glycine-induced MVs promoted interleukin-6 production by macrophage-like J774.1 cells, and the immunostimulatory activity was comparable to that of spontaneously produced MVs. Our results indicate that glycine is an effective agent for improving the production of MVs with immunostimulatory activity in gram-positive lactic acid bacteria, which can be applied as mucosal adjuvants and functional foods.

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.

Activation of the toll-like receptor 2 signaling pathway by GAPDH from bacterial strain RD055328.

We characterized the membrane vesicle fraction (RD-MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD-MVs and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected in the RD-MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patch cells following the addition of the RD-MV fraction. In the presence of the RD-MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD-MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.

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.

Identification of galacturonic acid-rich polysaccharide with intestinal immune system modulating activity via Peyer's patch from postbiotics of Phellinus linteus mycelial submerged culture

Interests in the development and exploration of industrial applications of medicinal mushrooms as postbiotics have lately increased. We recently reported the potential use of Phellinus linteus mycelial-containing whole culture extract (PLME) prepared by submerged cultivation as a postbiotic that promotes immune system activation. Here, we aimed to isolate and structurally elucidate the active ingredients in PLME by activity-guided fractionation. The intestinal immunostimulatory activity was evaluated by bone marrow (BM) cell proliferation activity and related cytokine production in C3H-HeN mouse-derived Peyer's patch (PP) cells treated with polysaccharide fractions. The initially crude polysaccharide (PLME-CP) of PLME prepared using ethanol precipitation was further fractionated into four fractions (PLME-CP-0 to -III) by anion-exchange column chromatography. BM cell proliferation and cytokine production of PLME-CP-III were significantly improved compared to those of PLME-CP. PLME-CP-III was then fractionated into PLME-CP-III-1 and PLME-CP-III-2 by gel filtration chromatography. Based on the molecular weight distribution, monosaccharide, and glycosyl linkage analyses, PLME-CP-III-1 was revealed as a novel galacturonic acid-rich acidic polysaccharide and further shown to play an important role in facilitating PP-mediated intestinal immunostimulatory activity. This is the first study demonstrating the structural characteristics of a novel intestinal immune system modulating acidic polysaccharide from P. linteus mycelium-containing whole culture broth postbiotics.

Red Ginseng Extract and γ-Aminobutyric Acid Synergistically Enhance Immunity Against Cancer Cells and Antitumor Metastasis Activity in Mice.

The effects of combined administration of red ginseng (RG) extracts and gamma-aminobutyric acid (GABA) on immunostimulatory activity and tumor metastasis inhibition were investigated in mice. For the immunostimulatory activity, splenocyte proliferation, natural killer (NK) cell activity, including the production of granzyme B (GrB) and interferon gamma (IFN-γ), and serum level of cytokine such as IFN-γ, interleukin (IL)-17, and IL-21 were assessed. Peyer's patch cells obtained from mice administered with RG+GABA were cultured, and the cytokine level in the culture supernatant and bone marrow (BM) cell proliferation activity were examined. The proliferative activity of splenocytes was significantly higher in the RG-GABA treatment group than in RG or GABA alone (P < .05). In the experimental tumor metastasis model, oral administration of RG+GABA showed a higher antitumor metastatic effect compared to that of RG or GABA alone. Oral administration of RG+GABA significantly augmented NK cell-mediated cytotoxicity against YAC-1 tumor cells. In addition, the production of GrB and IFN-γ was stimulated in the culture supernatant of NK cells and YAC-1 cells. Serum concentrations of IFN-γ, IL-17, and IL-21 in mice with RG+GABA were significantly higher compared to the corresponding blood levels in mice administered with RG or GABA alone. The RG+GABA group showed significant BM cell proliferation and increased production of IL-6 and granulocyte-macrophage colony-stimulating factor compared to that in the monotherapy groups. Therefore, RG may have a synergistic effect with GABA for enhancing the host defense system such as BM proliferation and NK cell activity in a tumor metastasis model.

Enhancement of IgA production by membrane vesicles derived from Bifidobacterium longum subsp. infantis.

Immunoglobulin A (IgA) is involved in the maintenance of gut homeostasis. Although the oral administration of bifidobacteria increases the amount of fecal IgA, the effects of bifidobacteria on intestinal immunity remain unclear. We found and characterized membrane vesicles (MVs) derived from Bifidobacterium longum subsp. infantis toward host immune cells. Bifidobacterium infantis MVs consisted of a cytoplasmic membrane, and extracellular solute-binding protein (ESBP) was specifically detected. In the presence of B. infantis MVs or recombinant ESBP, RAW264 cells produced the pro-inflammatory cytokine IL-6. IgA was produced by Peyer's patches cells following the addition of B. infantis MVs. Therefore, ESBP of B. infantis MVs is involved in the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells.

Modulation of local and systemic immune responses by fermented garlic extract

Fermented garlic (FG), prepared by fermenting garlic with <em>Bacillus subtilis</em> for 3‑15 days at 20‑40˚C has been linked to immune‑boosting properties. In the present study, FG was utilized to investigate its immunomodulatory effects using RAW264.7 cells, as well as BALB/c and C3H/HeN mice. Nitric oxide (NO) and pro‑inflammatory cytokine levels were quantified in RAW264.7 cells treated with or without FG (0.016, 0.08, 0.4, 2 and 10 mg/ml), garlic extract (GE) (0.016, 0.08, 0.4, 2 and 10 mg/ml) and lipopolysaccharide (1 µg/ml). Peritoneal macrophages and splenocytes were obtained from BALB/c mice for <em>ex vivo</em> analyses. MTT and enzyme‑linked immunosorbent assays were used to determine splenocyte proliferation, natural killer (NK) cell activity and cytokine production. The intestinal immunostimulatory <em>in vitro</em> effects of FG were also investigated. In a concentration‑dependent manner, FG treatment markedly accelerated the proliferation of RAW264.7 cells, murine peritoneal macrophages and splenocytes. Moreover, FG initiated macrophage activation by significantly increasing the NO, tumor necrosis factor‑α, interleukin (IL)‑12 and IL‑6 levels in RAW264.7 cells and peritoneal macrophages. In contrast to FG, GE promoted RAW264.7 cell proliferation at lower concentrations and toxicity at higher concentrations, while no detectable NO synthesis was observed at the concentrations tested. Likewise, the FG extract at larger concentrations fortified the intestinal immune system and improved bone marrow cell proliferation by significantly elevating IL‑6 and granulocyte‑macrophage colony‑stimulating factor production in splenocytes and Peyer's patch cells of mice. In addition, the NK cell activities of splenocytes from FG‑treated mice at lower doses against YAC‑1 cells were significantly increased, which must have been fostered and activated by interferon‑γ generated by splenocytes. On the whole, these findings provide relevant <em>in vitro</em> and <em>ex vivo</em> information that describes the mechanisms through which FG can strengthen a compromised immune system by influencing local and systemic gut immune responses directly or indirectly.

Preparation, characterization, and bioactivity evaluation of oligosaccharides from Atractylodes lancea (Thunb.) DC.

Sixteen oligosaccharide monomers with the degree of polymerization 3 to 18 (DP 3 to DP 18) and three active fractions (DP 3-9, DP 8-11, and DP 11-17) were separated from Atractylodes lancea (Thunb.) DC. by optimized fast protein liquid chromatography coupled with refractive index detector (FPLC-RID) and preparation hydrophilic interaction chromatography (Pre-HILIC). Gas chromatography-mass spectrometer (GC–MS), liquid chromatography tandem mass spectrometry (LC-MS/MS), nuclear magnetic resonance (NMR) spectroscopy, and methylation analysis showed that the oligosaccharide in A. lancea was 1-kestose [β-D-fructofuranosyl-(2 → 1)-β-D-fructofuranosyl-(2 → 1)-α-D-glucopyranoside] (inulin-type fructooligosaccharides, FOS). Particularly, DP 3-9 showed the best capacity in stimulating phagocytic, NO, and cytokines production on RAW264.7 cells than any other purified oligosaccharide monomers and active fractions. It could also activate T-cells in Peyer's patch cells and enhance the production of colony stimulation factors. Besides, FPLC-RID showed a good capacity for large-scale preparation of DP 3-9 with the recovery of more than 93%. The bioactivity of sixteen FOS monomers (DP 3 to DP 18) and three FOS fractions (DP 3-9, DP 8-11, and DP 11-17) investigated in this study are beneficial for the utilization of FOS as a functional ingredient in novel product development.

Mechanisms underlying enhanced IgA production in Peyer's patch cells by membrane vesicles derived from Lactobacillus sakei.

We analyzed the mechanisms underlying enhanced IgA production in the cells of Peyer's patch cells via membrane vesicles derived from Lactobacillus sakei subsp. sakei NBRC 15893. Depletion of CD11c+ cells from Peyer's patch cells suppressed the enhanced IgA production mediated by membrane vesicles. Meanwhile, the stimulation of bone-marrow-derived dendritic cells with membrane vesicles increased gene expression of inducible nitric oxide synthase, retinaldehyde dehydrogenase 2, and several inflammatory cytokines. The production of nitric oxide and interleukin (IL)-6 by membrane vesicle stimulation was induced via Toll-like receptor 2 on bone marrow-derived dendritic cells. Inhibition of inducible nitric oxide synthase and retinaldehyde dehydrogenase 2, as well as the neutralization of IL-6 in Peyer's patch cells, suppressed the enhanced IgA production by membrane vesicle stimulation. Hence, nitric oxide, retinoic acid, and IL-6 induced by membrane vesicles play crucial roles in the enhanced IgA production elicited by membrane vesicles in Peyer's patch cells.

Complement C5a promotes antigen cross-presentation by Peyer's patch monocyte-derived dendritic cells and drives a protective CD8+ Tcell response.

The complement fragment C5a is closely associated with adaptive immune induction in the mucosa. However, the mechanisms that control CD8+ Tcell responses by C5a have not been extensively explored. This study reveals that C5/C5a in the Peyer's patch (PP) subepithelial dome increases upon oral Listeria infection. We hypothesize that C5aR+ PP cells play an important role in the induction of antigen-specific Tcell immunity. Using single-cell RNA sequencing, we identify C5aR- and lysozyme-expressing dendritic cells (C5aR+ LysoDCs) in PP and examine their role in CD8+ Tcell immune induction. Stimulation of C5aR+ LysoDCs by C5a increases reactive oxygen species levels, leading to efficient antigen cross-presentation, which elicits an antigen-specific CD8+ Tcell response. In C5-deficient mice, oral co-administration of C5a and Listeria enhances Listeria-specific cytotoxic Tcell levels. Collectively, these findings suggest a role of the complement system in intestinal Tcell immunity.

Immune responses in mice vaccinated with a DNA vaccine expressing a new elastase from Trichinella spiralis

The elastase, which belongs to the serine protease family, hydrolyses various proteins and may be involved in the parasite invasion. In this study, complete sequence of elastase-1 (TsE) the nematode Trichinella spiralis (Owen, 1835) was cloned into the plasmid pcDNA3.1 as TsE DNA vaccine. After intramuscular vaccination, serum anti-Trichinella antibodies (IgG and subclass IgG1/IgG2a, and IgA), total and specific intestinal mucosal sIgA in mice vaccinated with pcDNA3.1/TsE were measured by ELISA. The results showed that vaccination with pcDNA3.1/TsE induced a systemic humoral immune response (high levels of serum IgG and subclass IgG1/IgG2a and IgA) and local intestinal mucosal immune responses (high levels of TsE-specific sIgA). Vaccination of mice with TsE DNA vaccine also triggered a systemic and local concomitant Th1/Th2 response, as demonstrated by significant elevation of Th1 (IFN-γ and IL-2) / Th2 (IL-4 and IL-10) cytokine levels after the spleen, mesenteric lymph node and Peyer's patch cells from vaccinated mice were stimulated with recombinant TsE (rTsE). The vaccination of mice with pcDNA3.1/TsE displayed a 17% reduction of intestinal adult worms and a 39% reduction of muscle larvae. Our results indicated that TsE DNA vaccine elicited a systemic concomitant Th1/Th2 response and an enteral local sIgA response, and produced a partial protection against infection with T. spiralis. The TsE may be regarded as a potential candidate vaccine target against Trichinella infection. The oral polyvalent vaccines should be developed to improve the protective efficacy of anti-Trichinella vaccines.

Tracking of an Oral Salmonella-Based Vaccine for Type 1 Diabetes in Non-obese Diabetic Mice.

Type 1 diabetes (T1D) arises secondary to immune-driven destruction of pancreatic β-cells and manifests as insulin-deficient hyperglycemia. We showed that oral vaccination with live attenuated Salmonella, which simultaneously delivers autoantigens and a TGFβ expression vector to immune cells in the gut mucosa, provides protection against the progression of T1D in non-obese diabetic (NOD) mice. In this study we employed the Sleeping Beauty (SB) transposon system that is composed of a transposase and transposon encoding the td-Tomato to express red fluorescent protein (RFP) to permanently mark the cells that take up the Salmonella vaccine. After animal vaccination, the transposon labeled-dendritic cells (DCs) with red fluorescence appeared throughout the secondary lymphoid tissues. Furthermore, Sleeping Beauty containing tgfβ1 gene (SB-tgfβ1) co-expressed TGFβ and RFP. The labeled DCs were detected predominantly in Peyer’s patches (PP) and mesenteric lymph nodes (MLN) and expressed CD103 surface marker. CD103+ DCs induced tolerogenic effects and gut homing. TGFβ significantly increased programmed death-ligand-1 (PDL-1 or CD274) expression in the DCs in the MLN and PP of treated mice. Also, TGFβ increased cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) levels in CD4+ cells in MLN and PP. Interestingly, DCs increased in all lymphatic organs of mice vaccinated with oral live Salmonella-based vaccine expressing preproinsulin (PPI), in combination with TGFβ, IL10, and subtherapeutic-doses of anti-CD3 mAb compared with vehicle-treated mice. These DCs are mostly tolerogenic in MLN and PP. Furthermore the DCs obtained from vaccine-treated but not vehicle-treated mice suppressed in vitro T cell proliferation. These data suggest that the MLN and the PP are a central hub for the beneficial anti-diabetic effects of an oral Salmonella-based vaccine prevention of diabetes in rodents.

Effect of Probiotics and Herbal Products on Intestinal Histomorphological and Immunological Development in Piglets.

The aim of the study was to evaluate the effect of probiotics and herbal products on the intestinal histomorphological and immunological development in piglets. Accordingly, 2-week-old piglets were allocated in 4 groups: C (basal diet), Pro (basal diet + probiotics), Pro+B (basal diet + probiotics + buckwheat bran), and H (powder of herbs). After 6 weeks of the experiment, 4 piglets from each experimental group were randomly selected and slaughtered at a slaughterhouse. Samples of tissue and digestive content from the jejunum and colon were collected for bacteriological, histological, and immunohistochemical examination. The results showed that probiotics increased the number of Lactobacillus spp. in the small (p < 0.05) and large intestines. The intestinal histomorphology was improved (p < 0.05) in all experimental groups by an increased villus height, VH : CD ration, colon crypt depth, and number of Ki-67+ epithelial cells. A higher number (p < 0.05) of goblet cells and their acidification were observed in group Pro, while the density of goblet cells was decreased by the herbs. Probiotics increased (p < 0.05) the number of intraepithelial lymphocytes (IELs), density of CD3+ cells in Peyer's patches (PPs), and lamina propria (LP). In group H, a dual effect on the CD3+ cell distribution was observed. The herbs reduced (p < 0.05) the number of IELs and CD3+ in LP but increased the distribution of CD3+ cells in PPs. In the colon, herbs increased CD3+ cells in LP as well. It suggests that probiotics and herbs had influence on the intestinal histomorphology and the ability to modulate the mucosal immune system; however, the combination of probiotics and buckwheat bran was not so convincing, probably due to the inhibitory effect of the buckwheat bran on the probiotics used.

Exopolysaccharide Produced by Lactobacillus casei Promotes the Differentiation of CD4+ T Cells into Th17 Cells in BALB/c Mouse Peyer's Patches in Vivo and in Vitro.

The immunomodulatory activity of a few Lactobacillus exopolysaccharides (EPS) has been reported. However, whether Lactobacillus EPS can promote the differentiation of CD4 T lymphocytes (CD4+T) cells into T-helper 17 cells (Th17 cells) in the Peyer's Patches (PPs) of mice has not been addressed. In this study, we found the molecular weight (Mw) of the purified EPS from L. casei ranged from 2.7 × 106 Da to 1.7 × 107 Da, and the average Mw was approximately 8.4 × 106 Da. In healthy BALB/c mice, EPS elevated the numbers of Th17 cells and levels of Th17-related cytokines. In vitro, EPS induced BMDCs to stimulate the differentiation of CD4+T cells of PPs into Th17 cells and the related cytokine secretions. Results suggest that L. casei EPS can effectively induce and promote the differentiation of CD4+T cells of PPs into Th17 cells in healthy mice and has the potential ability to improve intestinal mucosa immunity.