Immune Cells In Lamina Propria Research Articles

Macrophage LRRK2 hyperactivity impairs autophagy and induces Paneth cell dysfunction.

LRRK2 polymorphisms (G2019S/N2081D) that increase susceptibility to Parkinson's disease and Crohn's disease (CD) lead to LRRK2 kinase hyperactivity and suppress autophagy. This connection suggests that LRRK2 kinase inhibition, a therapeutic strategy being explored for Parkinson's disease, may also benefit patients with CD. Paneth cell homeostasis is tightly regulated by autophagy, and their dysfunction is a precursor to gut inflammation in CD. Here, we found that patients with CD and mice carrying hyperactive LRRK2 polymorphisms developed Paneth cell dysfunction. We also found that LRRK2 kinase can be activated in the context of interactions between genes (genetic autophagy deficiency) and the environment (cigarette smoking). Unexpectedly, lamina propria immune cells were the main intestinal cell types that express LRRK2, instead of Paneth cells as previously suggested. We showed that LRRK2-mediated pro-inflammatory cytokine release from phagocytes impaired Paneth cell function, which was rescued by LRRK2 kinase inhibition through activation of autophagy. Together, these data suggest that LRRK2 kinase inhibitors maintain Paneth cell homeostasis by restoring autophagy and may represent a therapeutic strategy for CD.

A227 EXPLORING THE ROLE OF MICROBIAL AND GENETIC FACTORS IN DEFINING THE INTESTINAL IMMUNE SYSTEM USING A HUMANIZED GNOTOBIOTIC MOUSE MODEL

Abstract Background Studies of the gut microbial ecosystem and its role in disease are often complicated by the intricate network of interactions between its microbial members and the host. Therefore, mouse models in which the microbiota diversity can be strictly controlled, and the functional potential be easily assessed, are needed. Aims Establishing a taxonomically and functionally diverse human gnotobiotic microbiota that recapitulates many microbe-host interactions of the complex human microbiome. To validate the effects of the defined bacterial gut community on the host, we will assess host physiology, intestinal immunity and the response to inflammatory stimuli. Methods Germ-free (GF) mice were inoculated with a defined bacterial community isolated from the stool of a healthy human donor. Stability of colonization across multiple generations and spatial organization of the bacterial community in the intestine were assessed using 16S rRNA gene sequencing and species-specific quantitative PCR. Immune cells were isolated from the colonic lamina propria and characterized by flow cytometry. Results Inoculated GF mice were reproducibly colonized with 8 bacterial species, collectively termed Human Defined Community 1 (HDC1). The HDC1 is vertically transmitted from mothers to offspring and the relative abundances remain stable in the colony over an extended period in a gnotobiotic isolator. We observed spatial stratification of the HDC1 microbiota between the small intestine and colon or cecum. HDC1 mice form a colonic inner mucus layer comparable in thickness to SPF mice suggesting physiological spatial organization of the bacterial community over the cross-sectional axis of the colon. Characterization of colonic lamina propria immune cells in HDC1 mice revealed myeloid and lymphoid cell abundances closely resembling those of mice harboring a complex microbiota (specific pathogen-free [SPF] mice), including normal macrophage and dendritic cell counts and normal proportions of CD4+ cells (regulatory T, Th1 and Th17 cells). Finally, we showed that HDC1 mice can be transplanted with human gut commensals and Crohn's disease (CD)-associated bacteria, demonstrating the tractability of this human defined microbiota. Conclusions In summary, HDC1 gnotobiotic mice are stably colonized with a human defined bacterial community and harbor an intestinal innate and adaptive immune system similar to SPF mice. Therefore, HDC1 mice represent a reliable and tractable tool for host-microbe interaction studies. Ultimately, we will employ the HDC1 model to decipher the role of Nod2 – the strongest genetic risk factor for CD development – in shaping both the microbiota and host immunity. Funding Agencies CIHROGS

P086 Identification of a Novel Immunometabolic Target and Agonist for PLXDC2 for Amelioration of DSS Colitis Model in Mice

Abstract Background PLXDC2 is expressed on the cell surface of macrophage, dendritic and specific mesenchymal and epithelial cells whose activation shifts cellular metabolism and reduces oxidative stress to rebalance the immune response and decrease inflammation. It’s role in IBD pathogenesis is unknown. PLXDC2 activation improves disease severity in rheumatoid arthritis models1, while loss of PLXDC2 exacerbates the severity of disease in DSS colitis2. PX-04 is a novel, first-in-class, orally active PLXDC2 agonist. Here, we report on the consequences of activating PLXDC2 using PX-04 in an acute DSS colitis model. Methods Mice were given DSS in drinking water for seven days to induce disruption of the epithelial layer. At project initiation, mice were 8 weeks of age and began once daily dosing with 20mg/kg of PX-04 24 hours after being placed on DSS. Mice were weighed and scored daily for 15 symptoms of disease (weight loss, diarrhea, rectal bleeding, rectal inflammation, pain, & overall behavior). Colons were collected and digested, and the resultant cellular suspensions were filtered to produce single cell suspensions. Colonic Lamina Propria Immune cells were isolated by Percoll gradient centrifugation. Cells were labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CD19, NK1.1, CD25, F4/80, CD11b, Gr1, CX3CR1, CD64) and intracellular (Tbet, RORγT, FOXP3, IFNγ, IL17, IL10, TNFα) antibodies in a sequential live staining in 96-well plates. Data was acquired using a FACS Celesta flow cytometer with FACSDiva software. Results Oral PX-04 treatment decreased the cumulative disease activity of mice challenged with DSS (FIG 1A). Immunologically, PX-04 affected both the adaptive and innate immune responses. First, PX-04 greatly decreased Th17 cells in the colon, while providing a slight increase to regulatory CD4+ T cells (FIG. 1C). A lower proportion of Natural Killer (NK) cells (FIG. 7D) produced IFNγ. Meanwhile, the proportion of TNF producing dendritic cells was decreased by PX-04 treatment. Conclusion Conclusion: PLXDC2 is expressed in cells relevant to IBD pathogenesis. PX-04, which binds to and activates PLXDC2, effectively decreases levels of effector T cells and myeloid cells, as well as overall disease severity in acute DSS colitis, warranting further investigation. 1Tubau-Juni et al. J Immunol 206(Supp) 2Tubau-Juni et al. Sci Rep 10(1)

Host-gut microbiota derived secondary metabolite mediated regulation of Wnt/β-catenin pathway: a potential therapeutic axis in IBD and CRC.

The intestinal tract encompasses one of the largest mucosal surfaces with a well-structured layer of intestinal epithelial cells supported by a network of underlying lamina propria immune cells maintaining barrier integrity. The commensal microflora in this environment is a major contributor to such functional outcomes due to its prominent role in the production of secondary metabolites. Of the several known metabolites of gut microbial origin, such as Short Chain Fatty Acids (SCFAs), amino acid derivatives, etc., secondary bile acids (BAs) are also shown to exhibit pleiotropic effects maintaining gut homeostasis in addition to their canonical role in dietary lipid digestion. However, dysbiosis in the intestine causes an imbalance in microbial diversity, resulting in alterations in the functionally effective concentration of these secondary metabolites, including BAs. This often leads to aberrant activation of the underlying lamina propria immune cells and associated signaling pathways, causing intestinal inflammation. Sustained activation of these signaling pathways drives unregulated cell proliferation and, when coupled with genotoxic stress, promotes tumorigenesis. Here, we aimed to discuss the role of secondary metabolites along with BAs in maintaining immune-gut homeostasis and regulation of inflammation-driven tumorigenesis with emphasis on the classical Wnt/β-Catenin signaling pathway in colon cancer.

Single-Cell RNA-Sequencing Analysis of Colonic Lamina Propria Immune Cells Reveals the Key Immune Cell-Related Genes of Ulcerative Colitis.

Ulcerative colitis (UC) is a severe threat to humans worldwide. Single-cell RNA sequencing (scRNA-seq) can be used to screen gene expression patterns of each cell in the intestine, provide new insights into the potential mechanism of UC, and analyze the development of immune cell changes. These findings can provide new ideas for the diagnosis and treatment of intestinal diseases. In this study, bioinformatics analysis combined with experiments applied in dextran sulfate sodium (DSS)-induced colitis mice was used to explore new diagnostic genes for UC and their potential relationship with immune cells. We downloaded microarray datasets (GSE75214, GSE87473, GSE92415) from the Gene Expression Omnibus and used these datasets to screen differentially expressed genes (DEGs) and conduct Weighted Gene Co-expression Network Analysis (WGCNA) after quality control. The hub genes were screened, and ROC curves were drawn to verify the reliability of the results in both training set (GSE75214, GSE87473, GSE92415) and validation cohort (GSE87466). Also, we explored the relation of diagnostic genes and immune cells by CIBERSORT algorithm and single-cell analysis. Finally, the expression of hub genes and their relation with immune cells were verified in DSS-induced colitis mice. Diagnostic genes (ANXA5, MMP7, NR1H4, CYP3A4, ABCG2) were identified. In addition, we found these five genes firmly related to immune infiltration. The DSS-induced colitis mice confirm that the expression of ANXA5 mainly increased in the intestinal macrophages and had a strong negative correlation with M2 macrophages, which indicated its possible influence on the polarization of macrophages in UC patients. We identified ANXA5, MMP7, NR1H4, CYP3A4, and ABCG2 as diagnostic genes of UC that are closely related to immune infiltration and ANXA5 maintains a negative correlation with M2 macrophages which indicated its possible influence on the polarization of macrophage in UC patients.

Modulation of intestinal IL-37 expression and its impact on the epithelial innate immune response and barrier integrity.

Intestinal epithelial cells separate the luminal flora from lamina propria immune cells and regulate innate immune responses in the gut. An imbalance of the mucosal immune response and disrupted intestinal barrier integrity contribute to the evolution of inflammatory bowel diseases. Interleukin (IL)-37 has broad anti- inflammatory activity and is expressed by the human intestinal epithelium. Mice ectopically expressing human IL-37 show reduced epithelial damage and inflammation after DSS-induced colitis. Here, we investigated the impact of IL-37 on the innate immune response and tight junction protein expression of mouse intestinal organoids and the modulation of IL37 expression in human intestinal organoids. Murine intestinal organoids were generated from IL-37tg and wildtype mice. Human ileal organoids were generated from healthy young donors. Expression of transgene IL-37 or recombinant IL-37 protein did not significantly reduce overall proinflammatory cytokine mRNA expression in murine intestinal organoids. However, higher IL37 expression correlated with a reduced proinflammatory cytokine response in murine colonic organoids. IL37 mRNA expression in human ileal organoids was modulated by proinflammatory cytokines showing an increased expression upon TNF-α-stimulation and decreased expression upon IFN-gamma stimulation. Transgene IL-37 expression did not rescue TNF-α-induced changes in morphology as well as ZO-1, occludin, claudin-2, and E-cadherin expression patterns of murine jejunal organoids. We speculate that the anti-inflammatory activity of IL-37 in the intestine is mainly mediated by lamina propria immune cells protecting intestinal epithelial integrity.

AIM2 Promotes Epithelial Tuft Cell Development During Intestinal Type 2 Immune Responses

Abstract The epithelium performs a growing list of immunologically relevant functions at mucosal sites. Specialized epithelial cells of the intestine interact with the microbiota and lamina propria immune cells to regulate host defense, yet we know little about the molecular regulators linking these interactions during different immunological insults. Here, we found that the DNA sensor Absent in Melanoma 2 (AIM2) promotes development of epithelial tuft cells, which initiate intestinal type 2 immunity. Aim2-deficient intestinal epithelial organoids displayed reduced tuft cell gene signatures and numbers during treatment with the tuft cell-promoting cytokines IL-4 and IL-13. Tuft cells respond to the microbial metabolite succinate, leading to IL-13 production by type 2 innate lymphoid cells (ILC2s) and tuft cell hyperplasia in vivo. Succinate-fed Aim2 −/−mice displayed reduced tuft cells and IL13 expression compared to similarly treated wild type mice. Inflammasome-deficient Asc −/−mice and wild type mice displayed similar succinate-induced tuft cell profiles, suggesting AIM2 promotes tuft cell-ILC2 activation independently of its inflammasome function. RNA sequencing revealed Aim2 −/−intestinal organoids displayed diminished expression of O-linked N-acetylglucosamine transferase (OGT), which is required for IL13/STAT6-induced tuft cell expansion. In line with these findings, Aim2 −/−intestinal organoids and succinate-treated mice presented reduced OGT protein expression and STAT6 phosphorylation. These combined results reveal an novel role for AIM2 during type 2 intestinal inflammation in the intestine via promotion of tuft cell expansion, potentially through maintaining OGT-STAT6 signaling. Supported by grants from NIH (K22 CA212030) and the AZ Dept. of Health Services (AZBRC New Investigator Award).

Celiac Disease Diagnosis Via Histopathological Correlation of Duodenal Biopsies in Children

Aim: The purpose of this study is to compare the diagnostic accuracy of duodenal bulb biopsies to distal duodenal biopsies in patients suspected of having celiac disease. Study Design: Cross-sectional study Place and Duration: This study was conducted at Department of Pathology, The children's hospital & The institute of child health, Multan during the period from April, 2022 to September, 2022. Methods: Total 86 children of celiac disease were presented in this study. Informed written consent provided by parents of children for the collection of detailed demographic data. Under general anaesthesia, a duodenal bulb sample and four distal duodenum samples were collected during an upper gastrointestinal endoscopy. Histological alterations indicative with CD were identified. Histological diagnosis and grading were performed using a modification of the Marsh-Oberhuber classification. The entire data set was analyzed by using SPSS 20.0. Results: Forty nine (56.97%) patients were females and 37 (43.03%) cases were males. Children’s mean age was 6.4±3.27 years with mean weight 15.9±6.19 kg. Mean height of the cases was 94.7±10.42 cm. There were 51 (59.3%) children had urban residency. According to marsh grading, 50 (58.1%) cases had grade IIIC. We found that frequency of crypt hyperplasia, influx of immune cells in lamina propria and enterocytes change in both duodenal bulb and distal part biopsies were equal while only frequency of villous atrophy and intraepithelial lymphocytes were different in both biopsies. 96.5% patients reported a high level of correlation, while only 3.5% reported no correlation. Conclusion: Both biopsy sites agree strongly. We may now suggest duodenal bulb biopsy alone. Since the endoscopic method of distal duodenum is laborious and requires more knowledge and time, obtaining biopsies from the proximal section is easier and requires less experience, therefore this strategy may be used even at facilities with junior paediatric gastroenterologists. Keywords: Celiac disease, Children, Histopathology, Duodenal bulb, Distal duodenum

Intestinal Gasdermins for regulation of inflammation and tumorigenesis.

Gasdermins (GSDMs) protein family express in intestinal epithelial cells or lamina propria immune cells, and play a nonnegligible function during gut homeostasis. With the gradually in-depth investigation of GSDMs protein family, the proteases that cleave GSDMA-E have been identified. Intestinal GSDMs-induced pyroptosis is demonstrated to play a crucial role in the removal of self-danger molecules and clearance of pathogenic organism infection by mediating inflammatory reaction and collapsing the protective niche for pathogens. Simultaneously, excessive pyroptosis leading to the release of cellular contents including inflammatory mediators into the extracellular environment, enhancing the mucosal immune response. GSDMs-driver pyroptosis also participates in a novel inflammatory cell death, PANoptosis, which makes a significant sense to the initiation and progression of gut diseases. Moreover, GSDMs are expressed in healthy intestinal tissue without obvious pyroptosis and inflammation, indicating the potential intrinsic physiological functions of GSDMs that independent of pyroptotic cell death during maintenance of intestinal homeostasis. This review provides an overview of the latest advances in the physiological and pathological properties of GSDMs, including its mediated pyroptosis, related PANoptosis, and inherent functions independent of pyroptosis, with a focus on their roles involved in intestinal inflammation and tumorigenesis.

ETS-1 facilitates Th1 cell-mediated mucosal inflammation in inflammatory bowel diseases through upregulating CIRBP

Background & aimsAs a susceptibility gene for human inflammatory bowel diseases (IBD), how avian erythroblastosis virus E26 oncogene homolog-1 (ETS-1) modulates intestinal mucosal immune response remains unclear. Here we studied the potential roles of ETS-1 in the pathogenesis of IBD. MethodsETS-1 expression was examined in IBD patients. CD45RBhighCD4+ T cell-transfer colitis, dextran sulfate sodium (DSS)-induced colitis, and azomethane (AOM)/DSS-induced colitis-associated cancer (CAC) models were constructed to probe the function of ETS-1 in vivo. RNA-sequencing of CD4+ T cells from Ets-1 transgenic (Tg) mice was performed to decipher the key differentially expressed genes. Adenovirus transduction was conducted to verify the therapeutic potentials of ETS-1 in vivo. ResultsETS-1 expression was significantly increased in CD4+ T cells from active IBD patients compared with healthy controls, which was upregulated by TNF-α but markedly suppressed by anti-TNF-α mAb therapy. More severe colitis was observed in Rag1−/− mice reconstituted with Ets-1TgCD45RBhighCD4+ T cells or in Ets-1 Tg mice after DSS exposure compared with controls, characterized by higher TNF-α and IFN-γ expression in inflamed colon. Ets-1 Tg mice were more prone to develop AOM/DSS-induced CAC, and bone marrow chimeras further proved that lamina propria immune cells but not intestinal epithelial cells contributed to the development of colitis. RNA-sequencing and luciferase analysis revealed cold-inducible RNA-binding protein (CIRBP) as a functional target of ETS-1 to promote Th1 cell-driven immune response. Consistently, intraperitoneal administration of adenovirus-m-cirbp-shRNA ameliorated trinitrobenzene sulfonic acid (TNBS)-induced colitis of Ets-1 Tg mice. ConclusionsOur data identify that ETS-1 is highly expressed in IBD patients and promotes Th1-driven mucosal inflammation through CIRBP. CIRBP may serve as a novel therapeutic target for treatment of human IBD.

Coronin 1A is Uniquely Expressed in the Human Follicle Associated Epithelium and is Required For Human M Cell Maturation and Function

IntroductionMicrofold (M) cells are specialized epithelial cells that play an integral role in immune surveillance of the gut luminal environment. They functionally act as gatekeepers within the follicle‐associated epithelium (FAE) of Peyer’s patches to regulate antigen delivery to immune cells of gut‐associated lymphoid tissue. Studies have associated damage in the FAE with chronic inflammatory conditions, such as IBD; however, studies designed to understand how human M cells contribute to disease onset and/or pathogenesis under pathogenic conditions remain incomplete. OBJECTIVE: Since M cells are differentiated from Lgr5+ intestinal stem cells, we proposed to utilize human enteroids to determine the factors that regulate human M cell differentiation and function. Quantitative proteomics analysis of human enteroids differentiated to express M cells revealed enriched expression of coronin 1A, an actin‐remodeling protein involved in phagocytosis and expressed immune cells. Since M cells engulf luminal gut antigens via phagocytosis, we hypothesized that coronin 1A is uniquely expressed in the human FAE and plays a role in gut antigen uptake and transcytosis in human M cells.MethodsHuman ileal enteroids derived from biopsies obtained from healthy donors were grown as monolayers and differentiated (no Wnt3a, R‐spondin‐1) for at least 5 days. To express M cells, cultures were exposed to RANKL and TNF during differentiation. Human expression of coronin 1A was validated by immunoblot and confocal microscopy of ileal biopsies obtained from healthy donors. Stable knockdown (KD) of coronin 1A in human enteroids was achieved by shRNA lentivirus and puromycin selection. M cell monolayers, expressing or lacking coronin 1A, were exposed apically to commensal (HS) and pathogenic strains of E. coli (EPEC, AIEC) to determine the contribution of gut microbes to stimulate M cell transcytosis, as measured by uptake of fluorescent polystyrene latex beads and immunoglobulins.ResultsCoronin 1A was expressed in human enteroid monolayers differentiated to express M cells, but not in monolayers differentiated to mimic the villus epithelium. Similar results were obtained in biopsy sections of the human ileum with coronin 1A detected in the FAE, but not in crypt or villus epithelia. This staining pattern was unique for human, as coronin 1A expression in the mouse ileum was confined to lamina propria and lymphoid follicle immune cells. Coronin 1A KD resulted in decreased expression of glycoprotein 2, a marker of mature M cells, while other markers of the FAE and M cells, including CD14, ICAM1, Spi‐B, SOX8, and FcRN remained unchanged. Coronin 1A KD also resulted in impaired uptake/transcytosis of IgG (via FcRN) as well as pathogen‐stimulated uptake/transcytosis of polystyrene beads by human M cells.ConclusionsThe studies show: (1) human enteroids can be differentiated to model coronin 1A expression in the intact human FAE; (2) coronin 1A expression is necessary for terminal differentiation and function of human M cells; and (3) coronin 1A is required for pathogen‐stimulated uptake and transcytosis of various types of luminal gut cargo. We conclude that M cell‐expressing enteroid monolayers can be used as a relevant human model to further interrogate tropism for host‐pathogen interactions, mechanisms of luminal surveillance, and response to oral vaccine therapies.

XIAP restrains TNF-driven intestinal inflammation and dysbiosis by promoting innate immune responses of Paneth and dendritic cells.

Deficiency in X-linked inhibitor of apoptosis protein (XIAP) is the cause for X-linked lymphoproliferative syndrome 2 (XLP2). About one-third of these patients suffer from severe and therapy-refractory inflammatory bowel disease (IBD), but the exact cause of this pathogenesis remains undefined. Here, we used XIAP-deficient mice to characterize the mechanisms underlying intestinal inflammation. In Xiap−/− mice, we observed spontaneous terminal ileitis and microbial dysbiosis characterized by a reduction of Clostridia species. We showed that in inflamed mice, both TNF receptor 1 and 2 (TNFR1/2) cooperated in promoting ileitis by targeting TLR5-expressing Paneth cells (PCs) or dendritic cells (DCs). Using intestinal organoids and in vivo modeling, we demonstrated that TLR5 signaling triggered TNF production, which induced PC dysfunction mediated by TNFR1. TNFR2 acted upon lamina propria immune cells. scRNA-seq identified a DC population expressing TLR5, in which Tnfr2 expression was also elevated. Thus, the combined activity of TLR5 and TNFR2 signaling may be responsible for DC loss in lamina propria of Xiap−/− mice. Consequently, both Tnfr1−/−Xiap−/− and Tnfr2−/−Xiap−/− mice were rescued from dysbiosis and intestinal inflammation. Furthermore, RNA-seq of ileal crypts revealed that in inflamed Xiap−/− mice, TLR5 signaling was abrogated, linking aberrant TNF responses with the development of a dysbiosis. Evidence for TNFR2 signaling driving intestinal inflammation was detected in XLP2 patient samples. Together, these data point toward a key role of XIAP in mediating resilience of TLR5-expressing PCs and intestinal DCs, allowing them to maintain tissue integrity and microbiota homeostasis.

CCL11 exacerbates colitis and inflammation-associated colon tumorigenesis.

CCL11, also known as eotaxin-1, is described as an eosinophil chemoattractant, which has been implicated in allergic and Th2 inflammatory diseases. We have reported that CCL11 is significantly increased in the serum of inflammatory bowel disease (IBD) patients, colonic eosinophils are increased and correlate with tissue CCL11 levels in ulcerative colitis patients, and CCL11 is increased in dextran sulfate sodium (DSS)-induced murine colitis. Here, we show that CCL11 is involved in the pathogenesis of DSS-induced colitis and in colon tumorigenesis in the azoxymethane (AOM)-DSS model of colitis-associated carcinogenesis (CAC). Ccl11-/- mice exposed to DSS then allowed to recover had significantly less body weight loss and a decrease in histologic injury versus wild-type (WT) mice. In the AOM-DSS model, Ccl11-/- mice exhibited decreased colonic tumor number and burden, histologic injury, and colonic eosinophil infiltration versus WT mice. Ccl11 is expressed by both colonic epithelial and lamina propria immune cells. Studies in bone marrow chimera mice revealed that hematopoietic- and epithelial-cell-derived CCL11 were both important for tumorigenesis in the AOM-DSS model. These findings indicate that CCL11 is important in the regulation of colitis and associated carcinogenesis and thus anti-CCL11 antibodies may be useful for treatment and cancer chemoprevention in IBD.

Back Cover: Glycine Attenuates Citrobacter rodentium‐Induced Colitis by Regulating ATF6‐Mediated Endoplasmic Reticulum Stress in Mice

Mol. Nutr. Food Res. 2021, 65, 2001065 DOI: 10.1002/mnfr.202001065 Citrobacter rodentium (C. rodentium) is a well described murine pathogen that closely resembles human diarrheal pathogens enteropathogenic Escherichia coli (E. coli.) and enterohaemorrhagic E. coli. Adherent C. rodentium adapt to the gastrointestinal environment and undergo a virulence switch that facilitates colonization of the distal colon. The virulence proteins produced by C. rodentium lead to breakdown of the intestinal tight junction proteins and apoptosis of the intestinal epithelial cells, which enables permeation of C. rodentium into the lamina propria. Recognition of C. rodentium by the lamina propria immune cells initiates the mucosal inflammatory responses, including of secretion of pro-inflammatory cytokines and infiltration of immune cells themselves in the colonic epithelium. Supplementation with glycine restores the integrity of the colonic epithelium by upregulating tight junction protein expression and inhibiting of intestinal epithelial cell apoptosis, thus suppressing the overactivated inflammatory responses. Supplementation with glycine is a nutritional strategy to alleviate C. rodentium-induced colitis in mice. This is reported by Zhenlong Wu and co-workes in article number 2001065.

Macrophages Compensate for Loss of Protein Tyrosine Phosphatase N2 in Dendritic Cells to Protect from Elevated Colitis.

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) plays a critical role in the pathogenesis of inflammatory bowel diseases (IBD). Mice lacking PTPN2 in dendritic cells (DCs) develop skin and liver inflammation by the age of 22 weeks due to a generalized loss of tolerance leading to uncontrolled immune responses. The effect of DC-specific PTPN2 loss on intestinal health, however, is unknown. The aim of this study was to investigate the DC-specific role of PTPN2 in the intestine during colitis development. PTPN2fl/flxCD11cCre mice were subjected to acute and chronic DSS colitis as well as T cell transfer colitis. Lamina propria immune cell populations were analyzed using flow cytometry. DC-specific PTPN2 deletion promoted infiltration of B and T lymphocytes, macrophages, and DCs into the lamina propria of unchallenged mice and elevated Th1 abundance during acute DSS colitis, suggesting an important role for PTPN2 in DCs in maintaining intestinal immune cell homeostasis. Surprisingly, those immune cell alterations did not translate into increased colitis susceptibility in acute and chronic DSS-induced colitis or T cell transfer colitis models. However, macrophage depletion by clodronate caused enhanced colitis severity in mice with a DC-specific loss of PTPN2. Loss of PTPN2 in DCs affects the composition of lamina propria lymphocytes, resulting in increased infiltration of innate and adaptive immune cells. However, this did not result in an elevated colitis phenotype, likely because increased infiltration of macrophages in the intestine upon loss of PTPN2 loss in DCs can compensate for the inflammatory effect of PTPN2-deficient DCs.

Antibiotic-associated dysbiosis affects the ability of the gut microbiota to control intestinal inflammation upon fecal microbiota transplantation in experimental colitis models

BackgroundThe gut microbiota plays a central role in host physiology and in several pathological mechanisms in humans. Antibiotics compromise the composition and functions of the gut microbiota inducing long-lasting detrimental effects on the host. Recent studies suggest that the efficacy of different clinical therapies depends on the action of the gut microbiota. Here, we investigated how different antibiotic treatments affect the ability of the gut microbiota to control intestinal inflammation upon fecal microbiota transplantation in an experimental colitis model and in ex vivo experiments with human intestinal biopsies.ResultsMurine fecal donors were pre-treated with different antibiotics, i.e., vancomycin, streptomycin, and metronidazole before FMT administration to colitic animals. The analysis of the gut microbiome, fecal metabolome, and the immunophenotyping of colonic lamina propria immune cells revealed that antibiotic pre-treatment significantly influences the capability of the microbiota to control intestinal inflammation. Streptomycin and vancomycin-treated microbiota failed to control intestinal inflammation and were characterized by the blooming of pathobionts previously associated with IBD as well as with metabolites related to the presence of oxidative stress and metabolism of simple sugars. On the contrary, the metronidazole-treated microbiota retained its ability to control inflammation co-occurring with the enrichment of Lactobacillus and of innate immune responses involving iNKT cells. Furthermore, ex vivo cultures of human intestinal lamina propria mononuclear cells and iNKT cell clones from IBD patients with vancomycin pre-treated sterile fecal water showed a Th1/Th17 skewing in CD4+ T-cell populations; metronidazole, on the other hand, induced the polarization of iNKT cells toward the production of IL10.ConclusionsDiverse antibiotic regimens affect the ability of the gut microbiota to control intestinal inflammation in experimental colitis by altering the microbial community structure and microbiota-derived metabolites.6E6mfZzU_A62fWsXVW3UYFVideo

Lithium Carbonate Treatment Alleviates Gut Inflammation Through Activating Treg Cell Responses in a Microbiota-Dependent Manner

Background: Recent evidence suggests that antidepressant and anti-anxiety treatments may also have a place in resolving inflammation through the gut-brain axis. Lithium carbonate (LC) is one of the most commonly used drugs for bipolar disorder. However, little is known about the effects of LC on gut inflammation. In this study, we explore the protective effect of LC on colitis, and clarify its underlying mechanism of intestinal microbial dependence. Methods: Colitis mice were established by oral administration of 2.5% dextran sulfate sodium (DSS). Colon inflammation severity in mouse model was measured by body weight change, mortality, colon length, disease activity index (DAI) score and H&E staining. Gut microbiota alteration were analyzed through high-throughput 16S rRNA gene sequencing. Enzyme-linked immunosorbent assay (ELISA) were conducted to measure the colon cytokines profile. The frequency of immune cells in lamina propria (LP) were phenotyped by flow cytometry. Findings: LC treatment significantly alleviated colon inflammation in DSS-induced colitis models. It is worth noting that when antibiotics (ABX) are used to deplete the intestinal microbiota, the protective effect of LC on colon inflammation is not significant. LC treatment downregulated the levels of pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and IL-17A in colon tissue, and upregulated the levels of immunosuppressive cytokine IL-10. The 16S rRNA sequencing results showed that LC administration significantly increased gut microbial diversity and altered flora composition, especially accumulated the abundance of potential probiotics Akkermansia muciniphila (A. muciniphila). Moreover, LC treatment selectively activated the anti-inflammatory responses of colonic regulatory T (Treg) cells and maintained intestinal homeostasis. Fecal microbiota transplantation (FMT), and Akkermansia muciniphila (A. muciniphila) single bacteria gavage experiments confirmed this intestinal flora-dependent mechanism. Interpretation: Accordingly, our results demonstrate the role of LC as a potential regulator of intestinal flora in the prevention and treatment of gut inflammation. Therefore, LC treatment or targeting specific microbiota, such as A. muciniphila, provides a new strategy for the treatment of ulcerative colitis or IBD. Funding Statement: This work was supported by the National Natural Science Foundation of China (Grant NO.82030020). Declaration of Interests: None declared. Ethics Approval Statement: All animal experimental protocols were performed following the guidelines of the National Institutes of Health guide for the care and use of Laboratory Animals, approved by the Third Military Medical University Institutional Animal Care and Use Committee.

Endoplasmic Reticulum Stress and Intestinal Inflammation: A Perilous Union.

The intestinal tract encompasses the largest mucosal surface fortified with a fine layer of intestinal epithelial cells along with highly sophisticated network of the lamina propria immune cells that are indispensable to sustain gut homeostasis. However, it can be challenging to uphold homeostasis when these cells in the intestine are perpetually exposed to insults of both endogenous and exogenous origin. The complex networking and dynamic microenvironment in the intestine demand highly functional cells ultimately burdening the endoplasmic reticulum (ER) leading to ER stress. Unresolved ER stress is one of the primary contributors to the pathogenesis of inflammatory bowel diseases (IBD). Studies also suggest that ER stress can be the primary cause of inflammation and/or the consequence of inflammation. Therefore, understanding the patterns of expression of ER stress regulators and deciphering the intricate interplay between ER stress and inflammatory pathways in intestinal epithelial cells in association with lamina propria immune cells contribute toward the development of novel therapies to tackle IBD. This review provides imperative insights into the molecular markers involved in the pathogenesis of IBD by potentiating ER stress and inflammation and briefly describes the potential pharmacological intervention strategies to mitigate ER stress and IBD. In addition, genetic mutations in the biomarkers contributing to abnormalities in the ER stress signaling pathways further emphasizes the relevance of biomarkers in potential treatment for IBD.

16 CCL11 EXACERBATES COLITIS VIA MODULATION OF EPITHELIAL WOUND REPAIR

Abstract Background We have shown that CCL11 (eotaxin-1), an eosinophil chemoattractant, is significantly increased in the serum of ulcerative colitis and Crohn’s disease patients vs controls and is also increased in dextran sulfate sodium (DSS)-induced murine colitis. In response to azoxymethane (AOM)-DSS, Ccl11–/– mice have significantly decreased colonic tumor number and burden, histologic injury, and colonic eosinophil infiltration vs wild-type (WT) mice. Ccl11 is expressed by isolated colonic epithelial and lamina propria immune cells. Studies in bone marrow chimera mice revealed that both hematopoietic- and epithelial-cell derived CCL11 were important for protection in the AOM-DSS model. Our aim was to assess the role of CCL11 in an injury and recovery colitis model and in an epithelial wound restitution model. Methods C57BL/6 WT and Ccl11–/– mice were exposed to 4% DSS in the drinking water for 5 days followed by 5 days of water for an injury and recovery model. Body weights were assessed daily. Histologic injury was assessed by a validated scoring system including depth and percent involvement of inflammation and crypt damage. Colonic eosinophil and neutrophil infiltration was determined by immunohistochemistry for major basic protein (MBP) and myeloperoxidase (MPO), respectively. The number of MBP- and MPO-positive cells per high-powered field (HPF) were quantified in a blinded manner. Young Adult Mouse Colon (YAMC) epithelial cells were stained with antibodies to the CCL11 receptors CCR2, CCR3, and CCR5 for assessment by flow cytometry. YAMC cell monolayers were wounded and followed for 24 h in normal media or with the addition of 100ng/mL recombinant CCL11 (rCCL11). The wound area was measured at 0 and 24 h to determine percent restitution. RNA was isolated and assessed for Ccl11 mRNA expression. Results In the injury and recovery model, Ccl11–/– mice exhibited decreased body weight loss (p<0.05), histologic injury (20.6 ± 1.5 vs 12.4 ± 1.6, p<0.001), and fewer MBP-positive cells/HPF (23.5 ± 1.4 vs 2.4 ± 0.2, p<0.001) vs WT mice. While colonic MPO-positive cells were increased with DSS exposure, there was no difference in Ccl11–/– mice. Naïve/unstimulated YAMC cells expressed both CCR3 and CCR5. Wounding the YAMC monolayer alone did not lead to increased Ccl11 mRNA expression. However, in the presence of rCCL11, there was a decrease in wound restitution at 24 h (48.0% ± 1.7 vs 31.3% ± 2.1, p<0.001). Conclusions Loss of CCL11 leads to clinical and histologic improvement in an injury and recovery colitis model. Colonic epithelial cells express both CCR3 and CCR5, and epithelial wound restitution is decreased in the presence of rCCL11. These data suggest that CCL11 has a deleterious effect on epithelial restitution leading to an exacerbation of colitis. Therefore, treatment with anti-CCL11 antibodies may be a therapeutic strategy in IBD patients.

16 CCL11 EXACERBATES COLITIS VIA MODULATION OF EPITHELIAL WOUND REPAIR

We have shown that CCL11 (eotaxin-1), an eosinophil chemoattractant, is significantly increased in the serum of ulcerative colitis and Crohn’s disease patients vs controls and is also increased in dextran sulfate sodium (DSS)-induced murine colitis. In response to azoxymethane (AOM)-DSS, Ccl11–/– mice have significantly decreased colonic tumor number and burden, histologic injury, and colonic eosinophil infiltration vs wild-type (WT) mice. Ccl11 is expressed by isolated colonic epithelial and lamina propria immune cells. Studies in bone marrow chimera mice revealed that both hematopoietic- and epithelial-cell derived CCL11 were important for protection in the AOM-DSS model. Our aim was to assess the role of CCL11 in an injury and recovery colitis model and in an epithelial wound restitution model.