Requirement of a Wnt5A-microbiota axis in the maintenance of gut B-cell repertoire and protection from infection.

We investigated the influence of a Wnt5A-gut microbiota axis on gut B-cell repertoire and protection from infection, having previously demonstrated that Wnt5A in association with gut commensals helps shape gut T-cell repertoire. Accordingly, Wnt5A heterozygous mice, which express less than wild-type level of Wnt5A, and their isolated Peyer's patches (PPs) were studied in comparison with the wild-type counterparts. The percentages of IgM- and IgA-expressing B cells were quite similar in the PP of both sets of mice. However, the PP of the Wnt5A heterozygous mice harbored significantly higher than wild-type levels of microbiota-bound B cell-secreted IgA, indicating the prevalence of a microbial population therein, which is significantly altered from that of wild-type. Additionally, the percentage of PP IgG1-expressing B cells was appreciably depressed in the Wnt5A heterozygous mice in comparison to wild-type. Wnt5A heterozygous mice, furthermore, exhibited notably higher than the wild-type levels of morbidity and mortality following infection with Salmonella typhimurium, a common gut pathogen. Differences in morbidity/mortality correlated with considerable disparity between the PP-B-cell repertoires of the Salmonella-infected Wnt5A heterozygous and wild-type mice, in which the percentage of IgG1-expressing B1b cells in the PP of heterozygous mice remains significantly low as compared to wild-type. Overall, these results suggest that a gut Wnt5A-microbiota axis is intrinsically associated with the maintenance of gut B-cell repertoire and protection from infection.IMPORTANCEAlthough it is well accepted that B cells and microbiota are required for protection from infection and preservation of gut health, a lot remains unknown about how the optimum B-cell repertoire and microbiota are maintained in the gut. The importance of this study lies in the fact that it unveils a potential role of a growth factor termed Wnt5A in the safeguarding of the gut B-cell population and microbiota, thereby protecting the gut from the deleterious effect of infections by common pathogens. Documentation of the involvement of a Wnt5A-microbiota axis in the shaping of a protective gut B-cell repertoire, furthermore, opens up new avenues of investigations for understanding gut disorders related to microbial dysbiosis and B-cell homeostasis that, till date, are considered incurable.

IL-4-Producing Vγ1+/Vδ6+ γδ T Cells Sustain Germinal Center Reactions in Peyer's Patches of Mice.

The mucosal immune system is the first line of defense against pathogens. Germinal centers (GCs) in the Peyer's patches (PPs) of the small intestine are constantly generated through stimulation of the microbiota. In this study, we investigated the role of γδ T cells in the GC reactions in PPs. Most γδ T cells in PPs localized in the GCs and expressed a TCR composed of Vγ1 and Vδ6 chains. By using mice with partial and total γδ T cell deficiencies, we found that Vγ1+/Vδ6+ T cells can produce high amounts of IL-4, which drives the proliferation of GC B cells as well as the switch of GC B cells towards IgA. Therefore, we conclude that γδ T cells play a role in sustaining gut homeostasis and symbiosis via supporting the GC reactions in PPs.

Mucosal immunity-mediated modulation of the gut microbiome by oral delivery of probiotics into Peyer's patches.

Methods capable of maintaining gut microbiota homeostasis to prevent bacterial translocation and infection under external threats are critical for multiple facets of human health but have been rarely reported. Here, we describe the elicitation of mucosal immunity to modulate the gut microbiota by oral delivery of living probiotics into Peyer's patches. Probiotics are individually camouflaged within a yeast membrane, on which the embedded ß-glucan can facilitate the phagocytosis of microfold cells that locate in the intestinal epithelium. The delivery of probiotics into lymphoid follicles after oral ingestion promotes robust mucosal immune responses and notably upgrades the production of secretory immunoglobulin A. The provoked immunity positively regulates the gut microflora, which, in turn, retains gut homeostasis and provides defense against environmental attacks. In two murine models of gut barrier impairment, oral administration with camouflaged probiotics effectively prevents the breakdown of intestinal barrier and evidences limited bacterial translocation and systemic inflammation.

Complement C5a promotes antigen cross-presentation by Peyer's patch monocyte-derived dendritic cells and drives a protective CD8+ T cell response.

The complement fragment C5a is closely associated with adaptive immune induction in the mucosa. However, the mechanisms that control CD8+ T cell 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 T cell immunity. Using single-cell RNA sequencing, we identify C5aR- and lysozyme-expressing dendritic cells (C5aR+ LysoDCs) in PP and examine their role in CD8+ T cell 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+ T cell response. In C5-deficient mice, oral co-administration of C5a and Listeria enhances Listeria-specific cytotoxic T cell levels. Collectively, these findings suggest a role of the complement system in intestinal T cell immunity.

Fibroblastic reticular cell lineage convergence in Peyer's patches governs intestinal immunity.

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.

Complete genome sequencing of Peyer's patches-derived Lactobacillus taiwanensis CLG01, a potential probiotic with antibacterial and immunomodulatory activity.

BACKGROUND: The genus Lactobacillus is an important component of the gastrointestinal tract of human and animals and commonly considered as probiotic. L. taiwanensis has long been proposed to be a probiotic whereas understanding on this species is still in its infancy. Genomic information of L. taiwanensis is fairly limited. Extensive characterization of its beneficial traits is needed. RESULTS: A new strain CLG01 of L. taiwanensis was isolated from mouse Peyer's patches. We established its probiotic profile through in vitro experiments. Complete genome of this strain was also sequenced and analyzed. L. taiwanensis CLG01 showed robust tolerance to acid and a degree of tolerance to bile salt with a promising antibacterial activity against a broad spectrum of pathogenic bacteria. In vitro treatment of mouse RAW 264.7 macrophage cells with heat-killed bacteria and bacterial supernatant of L. taiwanensis CLG01 resulted in enhancement of immune responses and upregulated expression of TNF-α and IL-6. The strain CLG01 also increased the IL-10 production of macrophages when co-treated with lipopolysaccharide (LPS). Complete genome of L. taiwanensis CLG01 contained a 1.89 Mb chromosome and two plasmids. Further genomic analysis revealed the presence of genes related to its resistance to different stresses and the beneficial effects mentioned above. Moreover, biosynthetic gene clusters (BGCs) encoding antimicrobial peptides, like bacteriocin, linear azol(in)e-containing peptide (LAP) and lanthipeptide, were also identified in the genome of L. taiwanensis CLG01. CONCLUSIONS: L. taiwanensis CLG01, isolated from mouse Peyer's patches, is the first L. taiwanensis strain with both phenotypes and genotypes systematically studied. These preliminary data confirmed the role of L. taiwanensis CLG01 as a potential probiotic candidate with antibacterial and immunomodulatory activity, which provide insight for further investigation to this species.

Coptis chinensis Franch polysaccharides provide a dynamically regulation on intestinal microenvironment, based on the intestinal flora and mucosal immunity.

ETHNOPHARMACOLOGICAL RELEVANCE: Coptis chinensis Franch is one of the most widely used traditional Chinese herbs in China and was firstly recorded in "Shennong's Classic of Materia Medica" in the Han Dynasty. The medical records in past thousands years have fully confirmed the clinical efficacies of Coptis chinensis Franch against intestinal diseases. The polysaccharides in herbal medicines can be digested by the flora and uptaken by the Peyer's patches (PPs) in intestine. It can be reasonably presumed that the polysaccharides in Coptis chinensis Franch (CCP) should be one of the critical element in the regulation of intestinal microenvironment. AIM OF THE STUDY: This study intended to explore the dynamic regulation of CCP on intestinal microenvironment from the perspective of the intestinal mucosal immunity and the intestinal flora, in order to provide a new research perspective for the pharmacological mechanism of Coptis chinensis Franch. MATERIALS AND METHODS: The absorption and distribution of CCP in intestinal tissues were observed after the perfusion of FITC labeled CCP. The influences of CCP on intestinal flora were evaluated by the 16sRNA gene illumina-miseq sequencing after gavage. The regulations of CCP on intestinal mucosal immunity were evaluated by the immunohistochemical analysis of the interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-17 (IL-17) and transforming growth factor-ß (TGF-ß) secretion in PPs and intestinal epithelial tissue. RESULTS: With the self-aggregation into particles morphology, CCP can be up-taken by PPs and promote the IFN-γ, IL-4, IL-17 and TGF-ß secretion in PPs in a dose-dependent manner. The CCP can also be utilized by the intestinal flora and dynamically regulate the diversity, composition and distribution of the intestinal flora. The temporal regulations of CCP on IFN-γ, IL-4, IL-17 and TGF-ß secretions in intestinal epithelial tissues are consistent with the variation tendency of intestinal flora. CONCLUSION: CCP can provide effective, dynamical and dose-dependent regulations on intestinal microenvironment, not only the intestinal flora but also the PPs and intestinal epithelium related immune response. These may be involved in the multiple biological activities of Coptis chinensis Franch.

Lactobacillus casei-fermented blueberry pomace augments sIgA production in high-fat diet mice by improving intestinal microbiota.

Intestinal secretory immunoglobulin A (sIgA)-improving function of Lactobacillus casei-fermented blueberry pomace (FBP) was investigated in this study. Male C57BL/6 mice were fed with control diet (CD) or high-fat diet (HFD) with or without FBP supplementation. Expressions of sIgA-associated genes/proteins were evaluated by quantitative polymerase chain reaction (qPCR), western blot and enzyme-linked immunosorbent assay (ELISA). Commensal microbiota in Peyer's patches (PPs) and caecal contents were analyzed by 16S rRNA Illumina sequencing and qPCR, respectively. FBP improved sIgA production in HFD mice at mRNA and protein levels. Akkermansia and Lactobacillus in PPs of HFD mice were statistically increased by FBP. Beneficial microbiota and short-chain fatty acids (SCFAs) in caecal contents were positively correlated with caecal immunoglobulins in HFD mice. FBP showed an ability to modulate intestinal microbiota, which improved sIgA production in HFD mice, warranting the potential use of berry by-products as functional ingredients in improving the intestinal immune barrier of HFD individuals.

Specific microbiota enhances intestinal IgA levels by inducing TGF-ß in T follicular helper cells of Peyer's patches in mice.

In humans and mice, mucosal immune responses are dominated by IgA antibodies and the cytokine TGF-ß, suppressing unwanted immune reactions but also targeting Ig class switching to IgA. It had been suggested that eosinophils promote the generation and maintenance of mucosal IgA-expressing plasma cells. Here, we demonstrate that not eosinophils, but specific bacteria determine mucosal IgA production. Co-housing of eosinophil-deficient mice with mice having high intestinal IgA levels, as well as the intentional microbiota transfer induces TGF-ß expression in intestinal T follicular helper cells, thereby promoting IgA class switching in Peyer's patches, enhancing IgA+ plasma cell numbers in the small intestinal lamina propria and levels of mucosal IgA. We show that bacteria highly enriched for the genus Anaeroplasma are sufficient to induce these changes and enhance IgA levels when adoptively transferred. Thus, specific members of the intestinal microbiota and not the microbiota as such regulate gut homeostasis, by promoting the expression of immune-regulatory TGF-ß and of mucosal IgA.

The Roles of Peyer's Patches and Microfold Cells in the Gut Immune System: Relevance to Autoimmune Diseases.

Microfold (M) cells are located in the epithelium covering mucosa-associated lymphoid tissues, such as the Peyer's patches (PPs) of the small intestine. M cells actively transport luminal antigens to the underlying lymphoid follicles to initiate an immune response. The molecular machinery of M-cell differentiation and function has been vigorously investigated over the last decade. Studies have shed light on the role of M cells in the mucosal immune system and have revealed that antigen uptake by M cells contributes to not only mucosal but also systemic immune responses. However, M-cell studies usually focus on infectious diseases; the contribution of M cells to autoimmune diseases has remained largely unexplored. Accumulating evidence suggests that dysbiosis of the intestinal microbiota is implicated in multiple systemic diseases, including autoimmune diseases. This implies that the uptake of microorganisms by M cells in PPs may play a role in the pathogenesis of autoimmune diseases. We provide an outline of the current understanding of M-cell biology and subsequently discuss the potential contribution of M cells and PPs to the induction of systemic autoimmunity, beyond the mucosal immune response.

High dietary salt-induced dendritic cell activation underlies microbial dysbiosis-associated hypertension.

Excess dietary salt contributes to inflammation and hypertension via poorly understood mechanisms. Antigen presenting cells including dendritic cells (DCs) play a key role in regulating intestinal immune homeostasis in part by surveying the gut epithelial surface for pathogens. Previously, we found that highly reactive γ-ketoaldehydes or isolevuglandins (IsoLGs) accumulate in DCs and act as neoantigens, promoting an autoimmune-like state and hypertension. We hypothesized that excess dietary salt alters the gut microbiome leading to hypertension and this is associated with increased immunogenic IsoLG-adduct formation in myeloid antigen presenting cells. To test this hypothesis, we performed fecal microbiome analysis and measured blood pressure of healthy human volunteers with salt intake above or below the American Heart Association recommendations. We also performed 16S rRNA analysis on cecal samples of mice fed normal or high salt diets. In humans and mice, high salt intake was associated with changes in the gut microbiome reflecting an increase in Firmicutes, Proteobacteria and genus Prevotella bacteria. These alterations were associated with higher blood pressure in humans and predisposed mice to vascular inflammation and hypertension in response to a sub-pressor dose of angiotensin II. Mice fed a high salt diet exhibited increased intestinal inflammation including the mesenteric arterial arcade and aorta, with a marked increase in the B7 ligand CD86 and formation of IsoLG-protein adducts in CD11c+ myeloid cells. Adoptive transfer of fecal material from conventionally housed high salt-fed mice to germ-free mice predisposed them to increased intestinal inflammation and hypertension. These findings provide novel insight into the mechanisms underlying inflammation and hypertension associated with excess dietary salt and may lead to interventions targeting the microbiome to prevent and treat this important disease.

Discovering Yersinia-Host Interactions by Tissue Dual RNA-Seq.

A detailed knowledge about virulence-relevant genes, as well as where and when they are expressed during the course of an infection is required to obtain a comprehensive understanding of the complex host-pathogen interactions. The development of unbiased probe-independent RNA sequencing (RNA-seq) approaches has dramatically changed transcriptomics. It allows simultaneous monitoring of genome-wide, infection-linked transcriptional alterations of the host tissue and colonizing pathogens. Here, we provide a detailed protocol for the preparation and analysis of lymphatic tissue infected with the mainly extracellularly growing pathogen Yersinia pseudotuberculosis. This method can be used as a powerful tool for the discovery of Yersinia-induced host responses, colonization and persistence strategies of the pathogen, and underlying regulatory processes. Furthermore, we describe computational methods with which we analyzed obtained datasets.

Effects of the expansion of bacterial colonies into the intervillous spaces on the localization of several lymphocyte lineages in the rat ileum.

The effect of bacterial colonies expanded into the intervillous spaces on the localization of several lymphocyte lineages was immunohistochemically investigated in two types of mucosa: ordinary mucosa of rat ileum, which consists of mucosa without any mucosal lymphatic tissue; and follicle-associated mucosa (FAM), which accompanies the parafollicular area under the muscularis mucosae in the rat ileal Peyer's patch. The results showed that bacterial colonies in the intervillous spaces induced increased populations of CD8+ cells in the epithelium of the intestinal villus in ordinary mucosa (IV) and intestinal villus in FAM (IV-FAM). Bacterial colonies in the intervillous spaces were also associated with increased numbers of IgA+ cells, which were mainly localized in the lamina propria of basal portions of IV and IV-FAM, and with expanded localization of IgA+ cells into the villous apex in both IV and IV-FAM. Moreover, IgA+ cells around the intestinal crypts adjacent to IV or IV-FAM were also increased in response to bacterial colonies. In the IV-FAM, but not IV, L-selectin+ cells, which were found to be immunopositive for TCRαß or CD19, were drastically increased in the lamina propria from the crypt to middle portion of IV-FAM and in the lumen of central lymph vessel of IV-FAM in response to the bacterial colonies in the intervillous spaces. These findings revealed that the expansion of bacterial colonies into the intervillous spaces accompanies the change of histological localization of the lymphocyte lineage in both the ordinary mucosa and FAM.

Microbiota Sensing by Mincle-Syk Axis in Dendritic Cells Regulates Interleukin-17 and -22 Production and Promotes Intestinal Barrier Integrity.

Production of interleukin-17 (IL-17) and IL-22 by T helper 17 (Th17) cells and group 3 innate lymphoid cells (ILC3s) in response to the gut microbiota ensures maintenance of intestinal barrier function. Here, we examined the mechanisms whereby the immune system detects microbiota in the steady state. A Syk-kinase-coupled signaling pathway in dendritic cells (DCs) was critical for commensal-dependent production of IL-17 and IL-22 by CD4+ T cells. The Syk-coupled C-type lectin receptor Mincle detected mucosal-resident commensals in the Peyer's patches (PPs), triggered IL-6 and IL-23p19 expression, and thereby regulated function of intestinal Th17- and IL-17-secreting ILCs. Mice deficient in Mincle or with selective depletion of Syk in CD11c+ cells had impaired production of intestinal RegIIIγ and IgA and increased systemic translocation of gut microbiota. Consequently, Mincle deficiency led to liver inflammation and deregulated lipid metabolism. Thus, sensing of commensals by Mincle and Syk signaling in CD11c+ cells reinforces intestinal immune barrier and promotes host-microbiota mutualism, preventing systemic inflammation.

Diabetes progression and alterations in gut bacterial translocation: prevention by diet supplementation with human milk in NOD mice.

Impaired intestinal barrier function occurs before type 1 diabetes (T1D) onset with a possible contribution of microbial translocation. Breastfeeding is associated with enhanced mucosal intestinal integrity and T1D protection. Our aim was to study the potential of human milk (HM) to prevent diabetes onset and modulate the translocation of gut bacteria susceptible to breastfeeding or associated to diabetes onset. We show that HM intake can prevent T1D in nonobese diabetic mice independently of bifidobacteria colonization. Prior to diabetes onset, HM mice harbored splenic bacterial counts and plasma lipopolysaccharides level similar to control mice but exhibited a reduced expansion of Anaerotruncus sp. in pancreas and Lactobacillus johnsonii and Barnesiella in Peyer's patches (PP). Surprisingly, pancreas and PP bacterial expansion did not correlate with their own gut localization but with ileal Escherichia coli and cecal HM-susceptible bacteria (the promoted L. murinus and Bacteroides vulgatus, and the repressed B. fragilis and E. coli), respectively. Besides, higher colonic B. vulgatus counts induced by HM intake were associated with low islet infiltration and pancreatic E. coli expansion. On another hand, splenic dendritic cells (DCs) were identified as negative covariate of PP Barnesiella, suggesting a possible HM contribution to preserving splenic DCs through the reduction of Barnesiella translocation. Fecal B. vulgatus also negatively correlated with PP Barnesiella expansion, indicating that the mouse coprophagic behavior likely added to HM effect. Our findings provide evidence that HM has a multilevel impact and cooperates with some gut bacteria for controlling bacterial translocation at the earliest stage of insulitis.

Oleoylethanolamide treatment affects gut microbiota composition and the expression of intestinal cytokines in Peyer's patches of mice.

The lipid sensor oleoylethanolamide (OEA), an endogenous high-affinity agonist of peroxisome proliferator-activated receptor-α (PPAR-α) secreted in the proximal intestine, is endowed with several distinctive homeostatic properties, such as control of appetite, anti-inflammatory activity, stimulation of lipolysis and fatty acid oxidation. When administered exogenously, OEA has beneficial effects in several cognitive paradigms; therefore, in all respects, OEA can be considered a hormone of the gut-brain axis. Here we report an unexplored modulatory effect of OEA on the intestinal microbiota and on immune response. Our study shows for the first time that sub-chronic OEA administration to mice fed a normal chow pellet diet, changes the faecal microbiota profile, shifting the Firmicutes:Bacteroidetes ratio in favour of Bacteroidetes (in particular Bacteroides genus) and decreasing Firmicutes (Lactobacillus), and reduces intestinal cytokines expression by immune cells isolated from Peyer's patches. Our results suggest that sub-chronic OEA treatment modulates gut microbiota composition towards a "lean-like phenotype", and polarises gut-specific immune responses mimicking the effect of a diet low in fat and high in polysaccharides content.

Peyer's patch myeloid cells infection by Listeria signals through gp38+ stromal cells and locks intestinal villus invasion.

The foodborne pathogen Listeria monocytogenes (Lm) crosses the intestinal villus epithelium via goblet cells (GCs) upon the interaction of Lm surface protein InlA with its receptor E-cadherin. Here, we show that Lm infection accelerates intestinal villus epithelium renewal while decreasing the number of GCs expressing luminally accessible E-cadherin, thereby locking Lm portal of entry. This novel innate immune response to an enteropathogen is triggered by the infection of Peyer's patch CX3CR1+ cells and the ensuing production of IL-23. It requires STAT3 phosphorylation in epithelial cells in response to IL-22 and IL-11 expressed by lamina propria gp38+ stromal cells. Lm-induced IFN-γ signaling and STAT1 phosphorylation in epithelial cells is also critical for Lm-associated intestinal epithelium response. GC depletion also leads to a decrease in colon mucus barrier thickness, thereby increasing host susceptibility to colitis. This study unveils a novel innate immune response to an enteropathogen, which implicates gp38+ stromal cells and locks intestinal villus invasion, but favors colitis.

Oxaliplatin-induced changes in microbiota, TLR4+ cells and enhanced HMGB1 expression in the murine colon.

Oxaliplatin is a platinum-based chemotherapeutic used for cancer treatment. Its use associates with peripheral neuropathies and chronic gastrointestinal side-effects. Oxaliplatin induces immunogenic cell death by provoking the presentation of damage associated molecular patterns. The damage associated molecular patterns high-mobility group box 1 (HMGB1) protein exerts pro-inflammatory cytokine-like activity and binds to toll-like receptors (namely TLR4). Gastrointestinal microbiota may influence chemotherapeutic efficacy and contribute to local and systemic inflammation. We studied effects of oxaliplatin treatment on 1) TLR4 and high-mobility group box 1 expression within the colon; 2) gastrointestinal microbiota composition; 3) inflammation within the colon; 4) changes in Peyer's patches and mesenteric lymph nodes immune populations in mice. TLR4+ cells displayed pseudopodia-like extensions characteristic of antigen sampling co-localised with high-mobility group box 1 -overexpressing cells in the colonic lamina propria from oxaliplatin-treated animals. Oxaliplatin treatment caused significant reduction in Parabacteroides and Prevotella1, but increase in Prevotella2 and Odoribacter bacteria at the genus level. Downregulation of pro-inflammatory cytokines and chemokines in colon samples, a reduction in macrophages and dendritic cells in mesenteric lymph nodes were found after oxaliplatin treatment. In conclusion, oxaliplatin treatment caused morphological changes in TLR4+ cells, increase in gram-negative microbiota and enhanced HMGB1 expression associated with immunosuppression in the colon.

Peyer's patch-specific Lactobacillus reuteri strains increase extracellular microbial DNA and antimicrobial peptide expression in the mouse small intestine.

Specific Lactobacillus reuteri is autochthonous Lactobacillus species in humans with potential application in food production as a probiotic. The difference in colonizing Peyer's patches (PP) might decide their health-promoting properties. We aimed to investigate the difference between PP- and lumen-specific L. reuteri on antimicrobial peptide expression in this study. L. reuteri strains were isolated from PP and the lumen of C57BL/6J mice, which were used to treat mice. PP-specific L. reuteri cells stimulate RegIIIγ mRNA expression of the crypt epithelial sample. PP-specific L. reuteri induces accumulation of extracellular DNA (eDNA) in the bottom of crypts. eDNA was extracted from the small-intestinal mucus, the yield of which was significantly increased after the PP-specific L. reuteri treatment. And it increased cytokine production in RAW264.7 murine macrophages. PP-specific L. reuteri significantly increased the relative abundance of Bacteroidetes-, Lactobacillus-, and Proteobacteria-derived eDNA. However, the levels of Strentrophomonas-derived eDNA decreased. These results provide a rationale for the screening of human derived L. reuteri with an immune-modulatory property.

Peyer's Patches as a Portal for DNA Delivery by Lactococcus lactis in Vivo.

Application of food-grade Lactococcus lactis (L. lactis) as a safe delivery tool for DNA vaccines and therapeutic proteins has been well investigated. Although some studies showed that eukaryotic expression plasmids were transferred from L. lactis to enterocytes, the precise mechanism of the DNA transfer remains unknown. In this study, we generated an invasive L. lactis strain that expresses "murinized" Internalin A, an invasin of intracellular bacteria Listeria monocytogenes with two amino acid alterations for invasion into murine cells, and confirmed that this L. lactis strain delivered DNA in an invasin-dependent manner into a monolayer of epithelial cells polarized to mimic the gastrointestinal tract environment. Although invasive L. lactis inoculated orally can deliver DNA into enterocytes in the gastrointestinal tract of mice, the efficiency of DNA transfer was similar to that of non-invasive L. lactis strain, suggesting that the in vivo DNA transfer from L. lactis occurs invasin-independently. A ligated-intestinal loop assay, a method for a short-term culturing of the whole intestine filled with materials to evaluate the interaction of the materials with intestinal cells, demonstrated that both non-invasive and invasive L. lactis strains were present in the Peyer's patches of the small intestine. On the other hand, few L. lactis was detected in the non-Peyer's patch epithelial region. Thus, our observations lead us to speculate that DNA transfer from L. lactis occurs predominantly in the Peyer's patches in an invasin-independent manner.