Cells In Lamina Propria Research Articles

Allergen-encapsulating Nanoparticles Reprogram Pathogenic Allergen-Specific Th2 Cells to Suppress Food Allergy.

Food allergy is a prevalent and potentially deadly disease caused by inadvertent sensitization to benign food antigens. Pathogenic Th2 cells are a major driver for disease, and allergen-specific immunotherapies (AIT) aim to increase the allergen threshold required to elicit severe allergic symptoms. However, the majority of AIT approaches require lengthy treatments and convey transient disease suppression, likely due to insufficient targeting of pathogenic Th2 responses. Here, we investigate the ability of allergen-encapsulating nanoparticles to directly suppress pathogenic Th2 responses and reactivity in a mouse model of food allergy. NPs associate with pro-tolerogenic antigen presenting cells, provoking the accumulation of antigen-specific, functionally suppressive regulatory T cells in the small intestine lamina propria. We report that 2 intravenous doses of allergen encapsulated in poly(lactide-co-glycolide) nanoparticles (NPs) significantly reduces oral food challenge (OFC)-induced anaphylaxis. Importantly, we demonstrate that NP treatment alters the fates of pathogenic allergen-specific Th2 cells, reprogramming these cells towards CD25+FoxP3+ regulatory and CD73+FR4+ anergic phenotypes. We also demonstrate NP-mediated reductions in the frequency of effector cells in the gut and mast cell degranulation following OFC. These studies reveal mechanisms by which an allergen-encapsulating NP therapy and, more broadly, allergen-specific immunotherapies, can rapidly attenuate allergic responses by targeting pathogenic Th2 cells. This article is protected by copyright. All rights reserved.

Stroke and myocardial infarction induce neutrophil extracellular trap release disrupting lymphoid organ structure and immunoglobulin secretion

AbstractPost-injury dysfunction of humoral immunity accounts for infections and poor outcomes in cardiovascular diseases. Among immunoglobulins (Ig), IgA, the most abundant mucosal antibody, is produced by plasma B cells in intestinal Peyer’s patches (PP) and lamina propria. Here we show that patients with stroke and myocardial ischemia (MI) had strongly reduced IgA blood levels. This was phenocopied in experimental mouse models where decreased plasma and fecal IgA were accompanied by rapid loss of IgA-producing plasma cells in PP and lamina propria. Reduced plasma IgG was detectable in patients and experimental mice 3–10 d after injury. Stroke/MI triggered the release of neutrophil extracellular traps (NETs). Depletion of neutrophils, NET degradation or blockade of NET release inhibited the loss of IgA+ cells and circulating IgA in experimental stroke and MI and in patients with stroke. Our results unveil how tissue-injury-triggered systemic NET release disrupts physiological Ig secretion and how this can be inhibited in patients.

Tryptophan metabolites relieve intestinal Candida albicans infection by altering the gut microbiota to reduce IL-22 release from group 3 innate lymphoid cells of the colon lamina propria.

Invasive candidiasis may be caused by Candida albicans (C. albicans) colonization of the intestinal tract. Preventing intestinal damage caused by Candida albicans infection and protecting intestinal barrier function have become a critical issue. Integrated analyses of the microbiome with metabolome revealed a remarkable shift of the gut microbiota and tryptophan metabolites, kynurenic acid (KynA), and indolacrylic acid (IA) in mice infected with C. albicans. The transcriptome sequencing indicated that differentially expressed genes were significantly associated with innate immune responses and inflammatory responses. The results of this study suggest that KynA and IA (KI) can alleviate intestinal damage caused by Candida albicans infection in mice by reducing intestinal permeability, increasing intestinal firmness, alleviating intestinal inflammation, and reducing the secretion of interleukin-22 (IL-22) in the 3 groups of colon innate lymphoid cells (ILC3). We performed a fecal microbiota transplantation (FMT) experiment and found that the intestinal barrier function, inflammation, and IL-22 secretion of ILC3 in the colon lamina propria of the recipient mice subjected to C. albicans infection and KI treatment were consistent with the trends of the donor mice. Our results suggest that tryptophan metabolites may directly regulate colon lamina ILC3 to promote intestinal resistance to C. albicans invasion, or indirectly regulate the ILC3 secretion of IL-22 to play a protective role in the intestinal barrier by affecting intestinal microorganisms, which may become a potential target for alleviating intestine borne C. albicans infection.

444 Deciphering the role of IL-4 in post-colitis repair

OBJECTIVES/GOALS: Incomplete mucosal healingand dysbiosis prevent long-term remission after colitis. IL4 may restore colon homeostasis through its action on immune cells and the microbiome. We will demonstrate this mechanism using genetically modified mice and molecular tools. This may result in target therapies that prolong remission in patients with IBD. METHODS/STUDY POPULATION: Mice were treated with 3% dextran sulfate sodium (DSS) in drinking water for 5 days to induce colitis. Mice were monitored daily for changes in body weight, and to monitor colitis severity. At each endpoint, mice were sacrificed and colon length was measured. For disease severity assessment, mouse colons were prepared in paraffin sections by the 'swiss-rolling' method. For flow cytometry, lamina propria mononuclear cell isolation was performed and cellular populations were stained with fluorophore-conjugated antibodies. IL4-eGFP-expressing (4get) mice were used to analyze the cellular expression of IL4 after colitis. Cell-specific IL4 deletion mice were generated using the cre-lox system. RESULTS/ANTICIPATED RESULTS: IL4-deficient mice had worse colitis compared with wild-type controls. Flow cytometry of lamina propria cells from 4get mice showed that most IL4-producing cells after colitis are eosinophils (CD11b+SiglecF+). Flow cytometry of C57bl6 mice showed an influx of IL4Ra+ monocytes (CD11b+Ly6C+) and macrophages (CD11b+F480+). IL4-stimulated bone marrow-derived macrophages demonstrated an increase in HB-EGF mRNA transcription. Myeloid-specific IL4R deleted mice had no difference in colitis severity compared with controls. Neutrophil-specific IL4R-deleted mice had increased colitis severity and mortality. Co-housing of littermate mice rescued recovery after DSS in IL4 deficient mice. DISCUSSION/SIGNIFICANCE: IL4 appears to play a role in restoring homeostasis after colitis. The mechanism depends on eosinophil-derived IL4, and action through neutrophils. However, the reparative function of IL4 can be shared with deficient mice through the microbiome. I will study the cellular and microbial mechanism by which IL4 restores homeostasis after colitis.

Effects of pectin methyl-esterification on intestinal microbiota and its immunomodulatory properties in naive mice

Pectins are dietary fibers that are attributed with several beneficial immunomodulatory effects. Depending on the degree of esterification (DE), pectins can be classified as high methoxyl pectin (HMP) or low methoxyl pectin (LMP).The aim of this study was to investigate the effects of pectin methyl-esterification on intestinal microbiota and its immunomodulatory properties in naive mice.Supplementation of the diet with LMP or HMP induced changes in the composition of the intestinal microbiota in mice toward Bacteroides, which was mainly promoted by HMP. Metabolome analysis of stool samples from pectin-fed mice showed a different effect of the two types of pectin on the levels of short-chain fatty acids and bile acids, which was consistent with highly efficient in vivo fermentation of LMP. Analysis of serum antibody levels showed a significant increase in IgG and IgA levels by both pectins, while FACS analysis revealed a decrease of infiltrating inflammatory cells in the intestinal lamina propria by HMP.Our study revealed that the structural properties of the investigated pectins determine fermentability, effects on microbial composition, metabolite production, and modulation of immune responses. Consumption of HMP preferentially altered the gut microbiota and suppressed pro-inflammatory immune responses, suggesting a beneficial role in inflammatory diseases.

Human metapneumovirus respiratory infection affects both innate and adaptive intestinal immunity.

Respiratory infections are one of the leading causes of morbidity and mortality worldwide, mainly in children, immunocompromised people, and the elderly. Several respiratory viruses can induce intestinal inflammation and alterations in intestinal microbiota composition. Human metapneumovirus (HMPV) is one of the major respiratory viruses contributing to infant mortality in children under 5 years of age worldwide, and the effect of this infection at the gut level has not been studied. Here, we evaluated the distal effects of HMPV infection on intestinal microbiota and inflammation in a murine model, analyzing several post-infection times (days 1, 3, and 5). Six to eight-week-old C57BL/6 mice were infected intranasally with HMPV, and mice inoculated with a non-infectious supernatant (Mock) were used as a control group. We did not detect HMPV viral load in the intestine, but we observed significant changes in the transcription of IFN-γ in the colon, analyzed by qPCR, at day 1 post-infection as compared to the control group. Furthermore, we analyzed the frequencies of different innate and adaptive immune cells in the colonic lamina propria, using flow cytometry. The frequency of monocyte populations was altered in the colon of HMPV -infected mice at days 1 and 3, with no significant difference from control mice at day 5 post-infection. Moreover, colonic CD8+ T cells and memory precursor effector CD8+ T cells were significantly increased in HMPV-infected mice at day 5, suggesting that HMPV may also alter intestinal adaptive immunity. Additionally, we did not find alterations in antimicrobial peptide expression, the frequency of colonic IgA+ plasma cells, and levels of fecal IgA. Some minor alterations in the fecal microbiota composition of HMPV -infected mice were detected using 16s rRNA sequencing. However, no significant differences were found in β-diversity and relative abundance at the genus level. To our knowledge, this is the first report describing the alterations in intestinal immunity following respiratory infection with HMPV infection. These effects do not seem to be mediated by direct viral infection in the intestinal tract. Our results indicate that HMPV can affect colonic innate and adaptive immunity but does not significantly alter the microbiota composition, and further research is required to understand the mechanisms inducing these distal effects in the intestine.

Identification of Intestinal Lamina Propria Plasma Cells by Surface Transmembrane Activator and CAML Interactor Expression.

Plasma cells secrete an abundance of Abs and are a crucial component of our immune system. The intestinal lamina propria harbors the largest population of plasma cells, most of which produce IgA. These Abs can bind to beneficial gut bacteria to reinforce intestinal homeostasis and provide protection against enteric pathogens. Plasma cells downregulate many cell-surface proteins commonly used to identify B cells. In mice, expression of the surface marker CD138 has been widely used to identify plasma cells in lymph nodes, bone marrow, and spleen. Intestinal plasma cells require liberation via extensive tissue processing involving treatment with collagenase. We report that detection of CD138 surface expression is reduced following collagenase treatment. Using a mouse in which yellow fluorescent protein expression is controlled by the plasma cell requisite transcription factor Blimp-1, we show that surface detection of transmembrane activator and CAML interactor captures a significant proportion of Ab-secreting plasma cells in the intestinal lamina propria and gut-draining mesenteric lymph nodes. Additionally, we describe a flow cytometry panel based on the detection of surface markers to identify murine B cell subsets in the intestinal lamina propria and, as a proof of concept, combine it with a cutting-edge fate-tracking system to characterize the fate of germinal center B cells activated in early life. By identifying plasma cells and other key intestinal B subsets in a manner compatible with several downstream applications, including sorting and culturing and invitro manipulations, this efficient and powerful approach can enhance studies of mucosal immunity.

STERILE NEUTROPHILIA INDUCED VIA CXCR4 ANTAGONISM MITIGATES COLONIC INFLAMMATION BY ENHANCING IMMUNOSUPPRESSIVE REGULATORY T CELLS IN MICE

Abstract Sterile neutrophilia (SN) is commonly associated with chronic diseases including inflammatory bowel diseases (IBD), but its defined role is largely unknown. Plerixafor (AMD3100) is a CXCR4 antagonist that promotes neutrophil egress from bone marrow resulting in SN. We first confirmed that C57BL6/J mice administered Plerixafor (10 mg/kg/bw, i.p.,) increased their circulating neutrophils by 50% within 2.5 h. Further, to study the role of SN in IBD, we induced chronic colitis by neutralizing IL-10 signaling via αIL-10R mAb (1.0 mg/mouse, i.p., 4 weekly injections) with or without weekly twice Plerixafor. Control mice were given isotype control mAb. As expected, αIL-10R+Plerixafor-treated mice had increased peripheral and splenic neutrophils. Further, these mice displayed remarkably reduced epithelial damage, hyperplasia, immune cell infiltration, and disease activity index when compared to αIL-10R+vehicle treated mice, which had typical colitis symptoms. To delineate the mechanism for such protection by Plerixafor, we profiled immune cells in circulation, spleen and lamina propria. In circulation αIL-10R+Plerixafor-treated mice had elevated CD3+CD4+ helper T cells (Th) but reduced CD3+CD8+ cytotoxic T (Tc) cells and CD3+CD4+FoxP3+ cells (Tregs) and CD3+CD4+RORγt+ cells (Th17) remained unchanged. While in the spleens of αIL-10R+Plerixafor-treated mice displayed significant increased Tregs, but decreased Th and Tc cells. Interestingly, markedly elevated Tregs, RORγt+ Tregs and tolerogenic IL-10+ Tregs was observed in lamina propria when compared to αIL-10R+ vehicle given mice. Interestingly, the proinflammatory IL-17+ Th cells were decreased in αIL-10R+Plerixafor-treated mice which showed mild colitis. Moreover, to further elucidate the role of Plerixafor in intestinal barrier function in colitic mice, we noticed treatment with Plerixafor increased the expression of occludin and claudins (1, 2), which are the principal transmembrane tight junctions (TJ) proteins; however Plerixafor treatment reduced the expression of zonula occludens (ZO), a peripheral membrane TJ protein. Collectively, our results demonstrate that SN could be an adaptive and beneficial immune response during chronic intestinal inflammation.

P175 HucMSC-Exo ameliorate experimental colitis via modulating gut microbiota and metabolites

Abstract Background Gut microbiota and metabolites have been shown to influence the development of inflammatory bowel disease (IBD). Exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-Exo) have been identified as a promising biological therapy for the treatment of IBD. Previous studies have described that HucMSC-Exo could modulate gut microbiota. However, the mechanism remains unclear. Here, we explored the role of gut microbiota and metabolites in HucMSC-Exo-mediated amelioration of experimental colitis. Methods Dextran sulfate sodium (DSS) was used to induce colitis. The role of HucMSC-Exo were assessed in colitis mice. Fecal microbiota transplantation (FMT) and sterile fecal filtrate gavage were employed to evaluate the effect of gut microbiota and metabolites. Gut microbiota and metabolites were analyzed through 16S rRNA sequencing and metabolomic profiling. The proportion of CD4+ T cells were phenotyped by multicolour flow cytometry. Results HucMSC-Exo treatment alleviated colon inflammation by regulating flora composition, significantly up-regulated the levels of bacteria such as Bacteroides, Parabacteroides distasonis, and Tannerellaceae. Meanwhile, HucMSC-Exo transformed metabolite short-chain fatty acid (SCFA) profiles, particularly increased butyrate level. Additionally, HucMSC-Exo selectively up-regulated the frequencies of regulatory T (Treg) cells as well as down-regulated the ratio of T helper type 17 (Th17) cells in colonic lamina propria to maintain intestinal immune homeostasis and repaired the colonic mucus barrier. FMT and sterile fecal filtrate gavage were conducted to confirm the above mechanism. Microbiota from HucMSC-Exo-treated mice alleviated the colitis over microbiota from DSS-treated mice. Sterile fecal filtrate from HucMSC-Exo-treated mice and butyrate induced similar beneficial outcomes, such as improved the Th17/Treg balance and repaired the colonic mucus barrier. Collectively, HucMSC-Exo ameliorate colitis by directly modulating gut microbiota and metabolites. Conclusion HucMSC-Exo ameliorated experimental colitis via directly modulating gut microbiota and metabolite butyrate, which could regulate CD4+T cells homeostasis and repair colonic mucus barrier. The findings demonstrated the HucMSC-Exo as a potential gut microbiota and metabolites modulator to treat IBD.

P031 Microbiota-driven appendiceal B lymphocytes aggravate DSS-induced murine colitis through facilitating colonic CD4+ T cells polarization

Abstract Background The appendix is an immunological organ sustaining a microbiome inoculum that plays a vital role in intestinal mucosal immunity and colonic microbiome stability. Previous studies have proposed the appendix exerts effects on ulcerative colitis (UC)’s pathogenesis, but the nature and basis underlying this connection remain unclear. B lymphocytes account for the largest proportion of appendiceal immunocytes and activate in the experimental colitis model, we systematically investigated how appendiceal B lymphocytes (appB) participate in UC’s pathogenesis and provided rational evidence for appendectomy and B cell immunotherapy being used in UC treatments. Methods The chronic colitis model was established in C57BL/6J mice by three cycles of 2% dextran sodium sulfate (DSS) treatment. Bulk RNA sequencing and 16S rRNA sequencing were used to explore appB’s characteristics and the potential causes of appB activation in DSS-induced colitis. Adoptive appB transfer and intravital imaging were used to evaluate appB’s homing and functional features in DSS-induced chronic colitis. Magnetic microbead-based cell sorting and cells experiments in vitro were used to investigate the underlying mechanisms of appB’s activation and effects on DSS-induced chronic colitis. Flow cytometry was used to assess the colonic mucosal adaptive immunity alterations in colitis mice adopted appB. Results Firmicutes and Bacteroidetes are the dominant phylum in the normal appendix, while Firmicutes, Verrucomicrobia, and Proteobacteria are prominent in the colitis appendix. The appendiceal IgG+ B cells expended in colitis mice compared with the normal mice; and the DSS colitis appB (DSS_appB), compared with the normal control appB (NC_appB), had much more potential to migrate to the colon and fired the gut flare, accompanied by the increment of CD19+ IgG+ cells, CD4+ IFN-γ+ cells and CD4+ IL-17+ cells in the colonic lamina propria in colitis mice. In vitro, DSS_appB secreted more inflammatory cytokines (IFN-γ, IL-6) than NC_appB under the stimulation of Toll-like receptor agonist analogs (Pam3CSK4, LPS, and ODN1826); DSS_appB facilitated colonic CD4+ T cells to secrete IFN-γ and IL-17 by up-regulating surface marker (CD80/86) and secreting IFN-γ and IL-6. Conclusion In DSS-induced chronic colitis mice, appB activated, driven by appendiceal microbiota,migrated to the colonic lamina propria and aggravated colitis through facilitating CD4+ Tcells polarize to Th1 and Th17 by up-regulating CD80/86 and secreting IFN-γ and IL-6 (Figure 1). Our research provides evidence for the connection between the appendix and UC, and we propose that appendectomy or B cell immunotherapy is promising to treat UC.

P077 The Immunometabolic Bimodal Mechanism of NLRX1 Agonist NX-13 in a Pig Model of Ulcerative Colitis

Abstract Background In mouse models, NX-13 is an orally active, gut-restricted NLRX1 agonist that has been shown to reduce severity of disease in multiple UC models through a novel immunometabolic, bimodal mechanism. Most preclinical UC studies rely upon rodent models, the human gastrointestinal physiology, microbiome, and immune system bear a greater similarity to that of pigs. Therefore, we sought to expand and validate our preclinical NX-13 results in mice using the dextran sulfate sodium (DSS) pig model of colitis. Here, we describe the disease severity, cellular, and biomarker results, and confirm the immunometabolic MOA observed in other species. Methods Pigs were randomized by weight (n=6 per group) and were administered tablets of excipient alone (placebo), NX-13 10 mg, NX-13 50 mg, or NX-13 100mg once daily. Pigs were challenged with 1% DSS in drinking water for 6 days and monitored daily for weight gain (loss). Composite disease activity score (0-12) was taken based on the sum of three subscores (0-4) for rectal bleeding, stool consistency and overall health/activity. Necropsy was conducted on day 7. Feces were collected for fecal calprotectin (FCP) quantification. Colonic tissue was macroscopically scored and collected for analysis by flow cytometry, histopathology and gene expression. Results Oral NX-13 treatment protected against weight loss and reduced colitis development with differences as early as day 2, becoming significant between days 4 and 6 as disease worsens in placebo treated pigs (Fig1A). NX-13 yielded a dose-dependent improvement in macroscopic lesion severity and microscopic immune cell infiltration (Fig1B). Specifically, Th1 cells (Fig1C) and TNF producing myeloid cells in the colonic lamina propria were reduced by NX-13, as well as FCP levels in stool (Fig1D) and mRNA expression in the colon (Fig1E). Oral NX-13 treatment reduced inflammatory markers including TNF, monocyte and neutrophil chemo attractants, IL-6, and IL-23 (Fig3F). Additionally, NX-13 treatment affected markers specific to the bimodal immunometabolic MOA of NLRX1 activation, namely upregulation of NLRX1 and mitochondrial metabolism gene COX3 (Fig1G), and reduction of NFkB and NLRP3 (Fig1H). Conclusion NX-13 demonstrated fast and clinically meaningful improvement in disease activity, and significantly impacted on key inflammatory markers in a DSS colitis model in pigs. These results align with previous murine results and observations in the phase 1b clinical program. Therefore, they can be used to generate additional mechanistic hypotheses for translational studies in the ongoing NEXUS phase 2 trial in moderate to severe UC.

Exercise improves intestinal IgA production by T-dependent cell pathway in adults but not in aged mice.

Hypermutated high-affinity immunoglobulin A (IgA), neutralizes toxins and drives the diversification of bacteria communities to maintain intestinal homeostasis although the mechanism underlies the impact of moderate aerobic exercise (MAE) on the IgA-generation via T-dependent (TD) is not fully know. Therefore, the aim of this study was to determine the effect of long-time MAE on the production of IgA through the TD pathway in Peyer´s patches of the small intestine from aged mice. MAE protocol consisted of twenty 3-month-old (young) BALB/c mice running in an endless band at 0° inclination and a speed of 10 m/h for 5 days a week and resting 2 days on the weekend until reaching 6-month-old (adulthood, n=10) or 24-month-old (aging, n=10). Groups of young, adult, or elderly mice were included as sedentary controls (n=10/per group). At 6 or 24 months old, all were sacrificed, and small intestine samples were dissected to prepare intestinal lavages for IgA quantitation by ELISA and to obtain suspensions from Peyer´s patches (PP) and lamina propria (LP) cells for analysis of T, B, and plasma cell subpopulations by flow cytometry and mRNA analysis expression by RT-qPCR of molecular factors related to differentiation of B cells to IgA+ plasma cells, class switch recombination, and IgA-synthesis. Statistical analysis was computed with two-way ANOVA (factor A=age, factor B=group) and p<0.05 was considered for statistically significant differences. Compared to age-matched sedentary control, in exercised elderly mice, parameters were either increased (IgA concentration, IL-21, IL-10 and RDH mRNA expression), decreased (α-chain mRNA, B cells, mIgA+ B cells, mIgM+ B cells and IL-4 mRNA) or unchanged (PP mIgA+ plasmablasts and LP cyt-IgA+ plasma cells). Regarding the exercised adult mice, they showed an up-modulation of IgA-concentration, mRNA expression IL-21, IL-10, and RDH and cells (PP B and T cells, mIgM+ plasmablasts and LP cyt-IgA+plasma cells). Our findings suggest that MAE restored the IgA production in adult mice via the TD cell pathway but does not in aged mice. Other studies are necessary to know in more detail the impact of long-time MAE on the TD pathway to produce IgA in aging.

Metabolic fitness of IgA+ plasma cells in the gut requires DOCK8

Dedicator of cytokinesis 8 (DOCK8) mutations lead to a primary immunodeficiency associated with recurrent gastrointestinal infections and poor antibody responses but, paradoxically, heightened IgE to food antigens, suggesting that DOCK8 is central to immune homeostasis in the gut. Using Dock8-deficient mice, we found that DOCK8 was necessary for mucosal IgA production to multiple T cell-dependent antigens, including peanut and cholera toxin. Yet DOCK8 was not necessary in T cells for this phenotype. Instead, B cell-intrinsic DOCK8 was required for maintenance of antigen-specific IgA-secreting plasma cells (PCs) in the gut lamina propria. Unexpectedly, DOCK8 was not required for early B cell activation, migration, or IgA class switching. An unbiased interactome screen revealed novel protein partners involved in metabolism and apoptosis. Dock8-deficient IgA+ B cells had impaired cellular respiration and failed to engage glycolysis appropriately. These results demonstrate that maintenance of the IgA+ PC compartment requires DOCK8 and suggest that gut IgA+ PCs have unique metabolic requirements for long-term survival in the lamina propria.

Gasdermin D is the only Gasdermin that provides protection against acute Salmonella gut infection in mice.

Gasdermins (GSDMs) share a common functional domain structure and are best known for their capacity to form membrane pores. These pores are hallmarks of a specific form of cell death called pyroptosis and mediate the secretion of pro-inflammatory cytokines such as interleukin 1β (IL1β) and interleukin 18 (IL18). Thereby, Gasdermins have been implicated in various immune responses against cancer and infectious diseases such as acute Salmonella Typhimurium (S.Tm) gut infection. However, to date, we lack a comprehensive functional assessment of the different Gasdermins (GSDMA-E) during S.Tm infection in vivo. Here, we used epithelium-specific ablation, bone marrow chimeras, and mouse lines lacking individual Gasdermins, combinations of Gasdermins or even all Gasdermins (GSDMA1-3C1-4DE) at once and performed littermate-controlled oral S.Tm infections in streptomycin-pretreated mice to investigate the impact of all murine Gasdermins. While GSDMA, C, and E appear dispensable, we show that GSDMD i) restricts S.Tm loads in the gut tissue and systemic organs, ii) controls gut inflammation kinetics, and iii) prevents epithelium disruption by 72 h of the infection. Full protection requires GSDMD expression by both bone-marrow-derived lamina propria cells and intestinal epithelial cells (IECs). In vivo experiments as well as 3D-, 2D-, and chimeric enteroid infections further show that infected IEC extrusion proceeds also without GSDMD, but that GSDMD controls the permeabilization and morphology of the extruding IECs, affects extrusion kinetics, and promotes overall mucosal barrier capacity. As such, this work identifies a unique multipronged role of GSDMD among the Gasdermins for mucosal tissue defense against a common enteric pathogen.

Single cell transcriptome analyses reveal the roles of B cells in fructose-induced hypertension.

While the immune system plays a crucial role in the development of hypertension, the specific contributions of distinct immune cell populations remain incompletely understood. The emergence of single-cell RNA-sequencing (scRNA-seq) technology enables us to analyze the transcriptomes of individual immune cells and to assess the significance of each immune cell type in hypertension development. We aimed to investigate the hypothesis that B cells play a crucial role in the development of fructose-induced hypertension. Eight-week-old Dahl salt-sensitive (SS) male rats were divided into two groups and given either tap water (TW) or a 20% fructose solution (HFS) for 4 weeks. Systolic blood pressure was measured using the tail-cuff method. ScRNA-seq analysis was performed on lamina propria cells (LPs) and peripheral blood mononuclear cells (PBMCs) obtained from SS rats subjected to either TW or HFS. The HFS treatment induced hypertension in the SS rats. The analysis revealed 27 clusters in LPs and 28 clusters in PBMCs, allowing for the identification and characterization of various immune cell types within each cluster. Specifically, B cells and follicular helper T (Tfh) cells were prominent in LPs, while B cells and M1 macrophages dominated PBMCs in the HFS group. Moreover, the HFS treatment triggered an increase in the number of B cells in both LPs and PBMCs, accompanied by activation of the interferon pathway. The significant involvement of B cells in intestinal and PBMC responses indicates their pivotal contribution to the development of hypertension. This finding suggests that targeting B cells could be a potential strategy to mitigate high blood pressure in fructose-induced hypertension. Moreover, the simultaneous increase in follicular B cells and Tfh cells in LPs, along with the upregulation of interferon pathway genes in B cells, underscores a potential autoimmune factor contributing to the pathogenesis of fructose-induced hypertension in the intestine.

Colonic mucosal associated invariant T cells in Crohn's disease have a diverse and non-public T cell receptor beta chain repertoire.

Mucosal-Associated Invariant T (MAIT) cells are T cells with a semi-invariant T cell receptor (TCR), recognizing riboflavin precursors presented by a non-polymorphic MR1 molecule. As these precursors are produced by the gut microbiome, we characterized the frequency, phenotype and clonality of MAIT cells in human colons with and without Crohn's disease (CD). The transcriptome of MAIT cells sorted from blood and intestinal lamina propria cells from colectomy recipients were compared with other CD8+ T cells. Colon biopsies from an additional ten CD patients and ten healthy controls (HC) were analyzed by flow cytometry. TCR genes were sequenced from individual MAIT cells from these biopsies and compared with those of MAIT cells from autologous blood. MAIT cells in the blood and colon showed a transcriptome distinct from other CD8 T cells, with more expression of the IL-23 receptor. MAIT cells were enriched in the colons of CD patients, with less NKG2D in inflamed versus uninflamed segments. Regardless of disease, most MAIT cells expressed integrin α4β7 in the colon but not in the blood, where they were enriched for α4β7 expression. TCR sequencing revealed heterogeneity in the colon and blood, with few public sequences associated with cohorts. MAIT cells are enriched in the colons of CD patients and disproportionately express molecules (IL-23R, integrin α4β7) targeted by CD therapeutics, to suggest a pathogenic role for them in CD. Public TCR sequences were neither common nor sufficiently restricted to a cohort to suggest protective or pathogenic antigen-specificities.

Mmp17-deficient mice exhibit heightened goblet cell effector expression in the colon and increased resistance to chronic Trichuris muris infection.

Intestinal epithelial homeostasis is maintained by intrinsic and extrinsic signals. The extrinsic signals include those provided by mesenchymal cell populations that surround intestinal crypts and is further facilitated by the extracellular matrix (ECM), which is modulated by proteases such as matrix metalloproteinases (MMPs). Extrinsic signals ensure an appropriate balance between intestinal epithelial proliferation and differentiation. This study explores the role of MMP17, which is preferentially expressed by smooth muscle cells in the intestine, in intestinal homeostasis and during immunity to infection. Mice lacking MMP17 expressed high levels of goblet-cell associated genes and proteins, such as CLCA1 and RELM-β, which are normally associated with immune responses to infection. Nevertheless, Mmp17 KO mice did not have altered resistance during a bacterial Citrobacter rodentium infection. However, when challenged with a low dose of the helminth Trichuris muris, Mmp17 KO mice had increased resistance, without a clear role for an altered immune response during infection. Mechanistically, we did not find changes in traditional modulators of goblet cell effectors such as the NOTCH pathway or specific cytokines. We found MMP17 expression in smooth muscle cells as well as lamina propria cells such as macrophages. Together, our data suggest that MMP17 extrinsically alters goblet cell maturation which is sufficient to alter clearance in a helminth infection model.

Clinicohistological Characteristics of Patients with Oral Lichenoid Mucositis: A Retrospective Study for Dental Hospital Records.

Oral lichenoid mucositis (OLM) of the oral mucosa is a histological diagnosis mainly characterised by a band of inflammatory infiltrate in lamina propria and basal cell degeneration. These features describe oral lichen planus or oral lichenoid reaction. However, it could be seen in oral dysplasia. The study aimed to assess the demographics and clinicohistological characteristics of patients with OLM and their relevance to dysplastic changes in the oral mucosa. This was a cross-sectional and retrospective study of archived and electronic records of individuals with histological confirmation of OLM at King Saud University Medical City, Saudi Arabia. The descriptive and correlation assessments were used to describe the demographics and clinicohistological characteristics and their associations, respectively [p < 0.05]. The analysis included 140 records of patients with histological confirmation of OLM with a mean age of 47 (±13), and 57% (n = 81) were females. Notably, 40% of patients had at least one medical condition, mainly diabetes mellitus, 74% were asymptomatic, and 52% had lesions in the buccal mucosa. Dysplasia was seen in 18 (12%) of the 140 reviewed records. Regarding the associations between study variables, dysplastic changes were associated with the male gender [p = 0.024] and were of no significance with increased age [p = 0.594]. Moreover, having oral symptoms was associated with older age [p < 0.001], medical history of diabetes [p = 0.0132] and hypertension [p < 0.001]. The present study findings could help indicate the individuals with histologically confirmed OLM who suffer the most from the clinical disease and have an increased risk of dysplastic changes. Therefore, symptomatic management and long-term follow-up can be planned accordingly.

Disentangling the innate immune responses of intestinal epithelial cells and lamina propria cells to Salmonella Typhimurium infection in chickens.

Salmonella enterica serovar Typhimurium (STm) is a major foodborne pathogen and poultry are a key reservoir of human infections. To understand the host responses to early stages of Salmonella infection in poultry, we infected 2D and 3D enteroids, the latter of which contains leukocytes, neurons, and mesenchymal cells that are characteristic of the lamina propria. We infected these enteroids with wild-type (WT STm), a non-invasive mutant lacking the prgH gene (ΔprgH STm), or treated them with STm lipopolysaccharide (LPS) and analyzed the expression of innate immune related genes by qPCR at 4 and 8 h. The localization of the tight junction protein, ZO-1, expression was disrupted in WT STm infected enteroids but not ΔprgH STm or LPS treated enteroids, suggesting a loss of epithelial barrier integrity. The innate immune response to LPS was more pronounced in 2D enteroids compared to 3D enteroids and by 8 hpi, the response in 3D enteroids was almost negligible. However, when STm adhered to or invaded the enteroids, both 2D and 3D enteroids exhibited an upregulation of inflammatory responses. The presence of lamina propria cells in 3D enteroids resulted in the unique expression of genes associated with immune functions involved in regulating inflammation. Moreover, 2D and 3D enteroids showed temporal differences in response to bacterial invasion or adherence. At 8 hpi, innate responses in 3D but not 2D enteroids continued to increase after infection with WT STm, whereas the responses to the non-invasive strain decreased at 8 hpi in both 2D and 3D enteroids. In conclusion, STm infection of chicken enteroids recapitulated several observations from in vivo studies of Salmonella-infected chickens, including altered epithelial barrier integrity based on ZO-1 expression and inflammatory responses. Our findings provide evidence that Salmonella-infected enteroids serve as effective models for investigating host-pathogen interactions and exploring the molecular mechanisms of microbial virulence although the 3D model mimics the host more accurately due to the presence of a lamina propria.

Lidocaine ameliorates intestinal barrier dysfunction in irritable bowel syndrome by modulating corticotropin-releasing hormone receptor 2.

Intestinal barrier dysfunction is a prevalent pathogenic factor underlying various disorders. Currently there is no effective resolution. Previous studies have reported the potential anti-inflammatory properties of lidocaine and its ability to alleviate visceral hypersensitivity in individuals with irritable bowel syndrome (IBS). Therefore, our study will further verify the effect of lidocaine on intestinal barrier dysfunction in IBS and investigate the underlying mechanisms. In this study, we investigated the role of lidocaine by assessing visceral hypersensitivity, body weight, inflammatory factors, fluorescein isothiocyanate-dextran 4000 (FD4) flux, tight junctions (TJs) and spleen and thymus index in rats subjected to water avoidance stress (WAS) to mimic intestinal barrier dysfunction in IBS with and without lidocaine. In vitro, we investigated the role of corticotropin-releasing hormone receptor 2 (CRHR2) in lidocaine-treated Caco2 cells using small interfering RNA (siRNA) targeting CRHR2. In WAS rats, lidocaine significantly restored weight loss, damaged TJs, spleen index and thymus index and inhibited abdominal hypersensitivity as well as blood levels of markers indicating intestinal permeability, such as diamine oxidase (DAO), D-lactic acid (D-Lac) and lipopolysaccharide (LPS). Consequently, the leakage of FD4 flux from intestine was significantly attenuated in lidocaine group, and levels of intestinal inflammatory factors (IL-1β, IFN-γ, TNF-α) were reduced. Interestingly, lidocaine significantly suppressed corticotropin-releasing hormone (CRH) levels in lamina propria cells, while the CRH receptor CRHR2 was upregulated in intestinal epithelial cells. In vitro, lidocaine enhanced the expression of CRHR2 on Caco-2 intestinal epithelial cells and restored disrupted TJs and the epithelial barrier caused by LPS. Conversely, these effects were diminished by a CRHR2 antagonist and siRNA-CRHR2, suggesting that the protective effect of lidocaine depends on CRHR2. Lidocaine ameliorates intestinal barrier dysfunction in IBS by potentially modulating the expression of CRHR2 on intestinal epithelial cells.