DSS-induced Chronic Colitis Research Articles

Metabolomics reveals the potential metabolic mechanism of infliximab against DSS-induced acute and chronic ulcerative colitis.

Inflammatory bowel disease (IBD) is often accompanied by metabolic imbalance, and infliximab (IFX) can alleviate IBD symptoms, but its metabolic mechanisms remain unclear. To investigate the relationship between IBD, metabolism, and IFX, an acute and chronic ulcerative colitis (UC) model induced by dextran sulfate sodium (DSS) was established. Plasma samples were analyzed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, followed by multivariate statistical analysis. The results showed that IFX could alleviate colonic shortening and reduce colonic pathological damage in acute and chronic mouse colitis, improve acute and chronic UC, and ameliorate metabolic disturbances. Among the 104 elevated metabolites and 170 decreased metabolites, these metabolites mainly belonged to amino acids, glucose, and purines. The changes in these metabolites were mainly associated with drug metabolism-other enzymes, riboflavin metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phosphonate and phosphinate metabolism, and phenylalanine metabolism. In summary, this study provides a valuable approach to explore the metabolic mechanisms of IFX in treating acute and chronic UC from a metabolomics perspective.

Anti-Inflammatory Effect of Dietary Pentadecanoic Fatty Acid Supplementation on Inflammatory Bowel Disease in SAMP1/YitFc Mice.

Dietary fats have been linked to the increasing incidence of chronic diseases, including inflammatory bowel diseases (IBD), namely, Crohn's disease (CD). This study investigated the impact of pentadecanoic acid (C15:0), a type of an odd-numbered chain saturated fatty acid, for its potential anti-inflammatory properties in different mouse models of experimental IBD using the SAMP1/YitFc (SAMP) mouse line (14- or 24-week-old), including chronic ileitis and DSS-induced colitis. To quantitively assess the effect of C:15, we tested two dosages of C:15 in selected experiments in comparison to control mice. Intestinal inflammation and intestinal permeability were used as primary outcomes. In ileitis, C:15 supplementation showed an anti-inflammatory effect in SAMP mice (e.g., a reduction in ileitis severity vs. control p < 0.0043), which was reproducible when mice were tested in the DSS model of colitis (e.g., reduced permeability vs. control p < 0.0006). Of relevance, even the short-term C:15 therapy prevented colitis in mice by maintaining body weight, decreasing inflammation, preserving gut integrity, and alleviating colitis signs. Collectively, the findings from both ileitis and colitis in SAMP mice indicate that C:15 may have therapeutic effects in the treatment of IBD (colitis in the short term). This promising effect has major translational potential for the alleviation of IBD in humans.

Diminished Immune Response and Elevated Abundance in Gut Microbe Dubosiella in Mouse Models of Chronic Colitis with GBP5 Deficiency.

Guanylate binding protein 5 (GBP5) is an emerging immune component that has been increasingly recognized for its involvement in autoimmune diseases, particularly inflammatory bowel disease (IBD). IBD is a complex disease involving inflammation of the gastrointestinal tract. Here, we explored the functional significance of GBP5 using Gbp5 knockout mice and wildtype mice exposed to dextran sulfate sodium (DSS) to generate chronic colitis model. We found that Gbp5 deficiency protected mice from DSS-induced chronic colitis. Transcriptome analysis of colon tissues showed reduced immune responses in Gbp5 knockout mice compared to those in corresponding wildtype mice. We further observed that after repeated DSS exposure, the gut microbiota was altered, both in wildtype mice and Gbp5 knockout mice; however, the gut microbiome health index was higher in the Gbp5 knockout mice. Notably, a probiotic murine commensal bacterium, Dubosiella, was predominantly enriched in these knockout mice. Our findings suggest that GBP5 plays an important role in promoting inflammation and dysbiosis in the intestine, the prevention of which might therefore be worth exploring in regards to IBD treatment.

Glycoprotein Nonmetastatic Melanoma Protein B: A Potential Therapeutic Target in Chronic Intestinal Fibrosis Induced by Dextran Sulfate Sodium

Abstract Background: Intestinal fibrosis is a complication of inflammatory bowel disease (IBD). Currently, there are no effective preventive measures or medical therapies for intestinal fibrosis. Surgery remains the only available strategy in the management of fibro stenotic enteropathies. However, more than 50% of patients undergoing surgery experience recurrence of stenosis. We assessed effects of glycoprotein nonmetastatic melanoma protein B (Gpnmb) on chronic colonic fibrosis induced by dextran sulfate sodium (DSS) in mice. Materials and Methods: GSE42768 mRNA microarray dataset was selected to carry out GEO2R bioinformatics analysis to predict differentially expressed genes. Chronic colonic inflammation-associated fibrosis was induced by DSS in mice. Twenty-four healthy male BALB/c were assigned to four groups: Control, model, T1: Intragastric administration of Thalidomide (Thal) 100 mg/kg.day beginning at day 18, T2: Intragastric administration of Thal (100 mg/kg.day) beginning at day 0 (n = 6 in each group). The colon was removed after modeling and assessed by pathological staining, Western blot, and reverse-transcription polymerase chain reaction. Col1α2, Gpnmb, Wnt1, and β-catenin antibodies were used. Results: The degree of chronic colitis and fibrosis was highest in the model group, and lowest in the control group. Thal treatment significantly alleviated DSS-induced chronic colitis and intestinal fibrosis, decreasing Gpnmb at both mRNA and Western blot levels. Expressions of Col1α2, Wnt1, and β-catenin got the same results. Conclusions: From bioinformatic analysis and fundamental experiment, we have illustrated that Gpnmb may stimulate the occurrence of intestinal fibrosis via Wnt1/β-catenin pathway. It may be a new therapeutic target for IBD-related intestinal fibrosis.

Hydroethanolic Extract of Lepidium apetalum Willdenow Alleviates Dextran Sulfate Sodium-Induced Colitis by Enhancing Intestinal Barrier Integrity and Inhibiting Oxidative Stress and Inflammasome Activation.

The prevalence of ulcerative colitis (UC) has surged in Asian nations recently. The limitations of traditional drug treatments, including biologics, have spurred interest in herbal medicines for managing UC. This study aimed to elucidate the protective mechanisms of hydroethanolic extract from Lepidium apetalum Willdenow (LWE) on intestinal integrity and inflammation in a dextran sodium sulfate (DSS)-induced colitis model of inflammatory bowel disease (IBD). Using UPLC-MS/MS analysis, eleven phytochemicals were identified in LWE, including catechin, vicenin-2, and quercetin. LWE restored transepithelial electrical resistance (TEER) and reduced paracellular permeability in IL-6-stimulated Caco-2 cells, increasing the expression of the tight junction proteins ZO-1 and occludin. LWE treatment alleviated DSS-induced colitis symptoms in mice, reducing body weight loss, disease activity index values, and spleen size, while improving colon length and reducing serum FITC-dextran levels, indicating enhanced intestinal barrier function. LWE suppressed NLRP3 inflammasome activation, reducing protein levels of pro-caspase-1, cleaved-caspase-1, ASC, and NLRP3, as well as mRNA levels of IL-1β, IL-6, and TNF-α. LWE treatment upregulated activity and mRNA levels of the antioxidant enzymes SOD1 and NQO1. Additionally, LWE modulated the Nrf2/Keap1 pathway, increasing p-Nrf2 levels and decreasing Keap1 levels. LWE also restored goblet cell numbers and reduced fibrosis in DSS-induced chronic colitis mice, increasing gene and protein expressions of ZO-1 and occludin. In summary, LWE shows promise as a therapeutic intervention for reducing tissue damage and inflammation by enhancing intestinal barrier function and inhibiting colonic oxidative stress-induced inflammasome activation.

Prevotella histicola ameliorates DSS-induced colitis by inhibiting IRE1α-JNK pathway of ER stress and NF-κB signaling

Inflammatory bowel disease (IBD) is a chronic and recurrent gastrointestinal inflammation regulated by intricate mechanisms. Recently, prebiotics is considered as promising nutritional strategy for the prevention and treatment of IBD. Prevotella histicola (P. histicola), an emerging probiotic, possesses apparently anti-inflammatory bioactivity. However, the role and underlying mechanism of P. histicola on IBD remain unclear. Hence, we probe into the effect of P. histicola on dextran sulfate sodium (DSS)-induced colitis and clarified the potential mechanism. Our results revealed that DSS-induced colonic inflammatory response and damaged epithelial barrier in mice were attenuated by oral administration of P. histicola. Moreover, supplementary P. histicola significantly enriched short-chain fatty acid (SCFA)-producing bacteria (Lactobacillus, and Bacillus) and reduced pathogenic bacteria (Erysipelotrichaceae, Clostridium, Bacteroides) in DSS-induced colitis. Notably, In DSS-treated mice, endoplasmic reticulum stress (ERS) was persistently activated in colonic tissue. Conversely, P. histicola gavage suppressed expansion of endoplasmic reticulum, downregulated PERK-ATF4-CHOP and IRE1α-JNK pathway. In vitro, the P. histicola supernatant eliminated LPS-induced higher production of pro-inflammatory cytokines regulated by NF-κB and impairment of epithelial barrier by inhibiting IRE1α-JNK signaling in Caco-2 cell. In summary, our study indicated that P. histicola mitigated DSS-induced chronic colitis via inhibiting IRE1α-JNK pathway and NF-κB signaling. These findings provide the new insights into the promotion of gut homeostasis and the application potential of P. histicola as a prebiotic for IBD in the future.

CCR2/CCR5 antagonist cenicriviroc reduces colonic inflammation and fibrosis in experimental colitis.

Cenicriviroc (CVC) is a CCR2/CCR5 antagonist that has been shown to be effective in the treatment of inflammatory and fibrotic diseases. Our study evaluated its efficacy in colitis. Mouse models of DSS-induced acute and chronic colitis were established. The efficacy of CVC in colitis was assessed by disease activity index (DAI) scores, histological assessment of inflammation and fibrosis, and expression assays of key molecules. In in vitro experiments, HT29 cell line was exposed to TNFα to study inflammatory signaling in intestinal epithelial cells. CCD-18Co colonic myofibroblasts and human primary colonic fibroblasts were activated by TGFβ1 to mimic fibroblast activation. In HT29 cells, CVC significantly reduced mRNA expression of CCL5 (P<0.01) but had no effect on CCL2. Furthermore, CVC reduced downstream CX3CL1 (P<0.01) and TNFα (P<0.05) expression, thereby inhibiting inflammatory progression. In acute colitis mice, CVC significantly reduced DAI scores and serum TNFα levels (P<0.05) and attenuated colonic inflammation as shown by HE staining. Meanwhile, CVC had no adverse effects on the liver, heart, and kidney of mice. On the other hand, in cellular models of chronic colitis, CVC decreased the expression of fibrosis markers, including FN, CTGF, α-SMA, and MMP9, and inhibited TGFβ1-induced fibrotic activation (P<0.01). In addition, CVC attenuated colonic fibrosis in chronic colitis mice. Moreover, CVC significantly promoted autophagy, which contributed to its regulation of inflammation. CVC significantly inhibited inflammation through CCL5/CCR5 signaling without damaging vital organs and suppressed fibrotic activation in chronic colitis, suggesting its great potential to relieve colonic inflammation and fibrosis.

Methionine-choline deficient diet deteriorates DSS-induced murine colitis through disturbance of gut microbes and infiltration of macrophages.

Ulcerative colitis (UC) is associated with changed dietary habits and mainly linked with the gut microbiota dysbiosis, necroptosis of epithelial cells, and mucosal ulcerations. Liver dysfunction and abnormal level of liver metabolism indices were identified in UC patients, suggesting a close interaction between gut and liver disorders. Methionine-choline deficient diet (MCD) has been shown to induce persistent alterations of gut microbiota and metabolome during hepatitis. In this study we further explored the disease phenotypes in UC patients and investigated whether MCD functioned as a trigger for UC susceptibility. After assessing 88 serum specimens from UC patients, we found significant liver dysfunction and dyslipidemia including abnormal ALT, AST, TG, TC, LDL-c and HDL-c. Liver dysfunction and dyslipidemia were confirmed in DSS-induced colitis mice. We fed mice with MCD for 14 days to cause mild liver damage, and then treated with DSS for 7 days. We found that MCD intake significantly exacerbated the pathogenesis of mucosal inflammation in DSS-induced acute, progressive, and chronic colitis, referring to promotion of mucosal ulcers, colon shortening, diarrhea, inflammatory immune cell infiltration, cytokines release, and abnormal activation of inflammatory macrophages in colon and liver specimens. Intraperitoneal injection of clodronate liposomes to globally delete macrophages dramatically compromised the pathogenesis of MCD-triggering colitis. In addition, MCD intake markedly changed the production pattern of short-chain fatty acids (SCFAs) in murine stools, colons, and livers. We demonstrated that MCD-induced colitis pathogenesis largely depended on the gut microbes and the disease phenotypes could be transmissible through fecal microbiota transplantation (FMT). In conclusion, this study supports the concept that intake of MCD predisposes to experimental colitis and enhances its pathogenesis via modulating gut microbes and macrophages in mice.

Tectochrysin ameliorates dextran sulfate sodium-induced chronic colitis by regulating the intestinal flora and inflammatory responses

Tectochrysin (TEC) is a natural flavonoid with anti-inflammatory, antioxidant, antitumor, and wound-healing activity. However, little is known about the therapeutic effects of TEC on inflammatory bowel disease (IBD). This study investigated the anti-inflammatory effect of TEC on IBD. Mice with dextran sulfate sodium (DSS)-induced chronic colitis served as an in vivo model of IBD. Lipopolysaccharide (LPS)-stimulated J774A.1 macrophages and mouse bone marrow-derived macrophages (BMDMs) served as in vitro models of inflammation. In vivo, 0.5% (w/w) TEC ameliorated DSS-induced colitis in mice by decreasing lipocalin-2 levels, disease activity index, colon shorten, weight loss, histological scores, and the apoptosis of colonic mucosal cells; it improved intestinal barrier function by increasing the expression of ZO-1 and occludin, decreasing the Th17/Treg ratio, and reducing the protein expression of NLRP3 and caspase-1 in the colon. Moreover, TEC decreased inflammation by reducing the serum levels of pro-inflammatory cytokines IFN-γ, IL-1β, G-CSF, IL-6, IL-8, iNOS, and TNF-α and increasing the levels of anti-inflammatory cytokines Bcl-2 and IL-10. TEC increased the relative abundance of the beneficial bacteria norank_f_Muribaculaceae in the mouse intestine. In vitro, 500 ng/mL TEC reduced oxidative stress by decreasing LPS-induced mitochondrial ROS production in BMDMs. TEC alleviated the increased levels of IL-1β, IL-6, and TNF-α and increased IL-10 levels in the culture supernatant of LPS-stimulated J774A.1 macrophages and BMDMs. These results indicate that TEC has a protective effect on DSS-induced chronic colitis by inhibiting inflammation, improving intestinal barrier function, and regulating intestinal microbial composition. Therefore, TEC has a high potential to treat IBD.

Didymin ameliorates ulcerative colitis-associated secondary liver damage by facilitating Notch1 degradation

BackgroundDidymin is a dietary flavonoid originally discovered by our group as a potent anti-ulcerative colitis (UC) agent. However, whether didymin plays a protective role in UC-associated inflammatory liver injury is still unclear. PurposeThis study aimed to evaluate the therapeutic potential of didymin on UC-associated inflammatory liver injury and explore the underlying mechanism. Study design and methodsColitis model was established in C57BL/6 mice by exposure to DSS, and didymin was administrated intragastrically for consecutive 10 days. The inflammatory liver injury was assessed by levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) in serum and histopathological damage in the liver. In vitro Kupffer cells and RAW264.7 cells challenged with lipopolysaccharides (LPS) were used to explore the modulatory activity of didymin on pro-inflammatory cytokines secretion and Notch1 signaling pathway activation. ResultsDidymin significantly mitigated liver coefficiency, ALT and AST levels in serum, and the hepatic histopathological damage caused by DSS-induced acute and chronic colitis. The mRNA expressions of pro-inflammatory factors including Tnf, Il1, and Il6 in liver tissues, Kupffer cells, and RAW264.7 cells stimulated by the influx of LPS was significantly deprived after didymin treatment. Mechanistically, didymin obstructed the protein expression, nuclear translocation of notch intracellular domain 1 (Notch1-ICD) and mRNA expression of hairy and enhancer of split 1 (Hes1). Further, the inhibitory mechanism of the Notch1-Hes1 pathway was dependent on c-Cbl-mediated Notch1-ICD lysosomal degradation. ConclusionOur study verified for the first time that didymin could prevent UC-associated diseases, such as inflammatory liver injury, and the mechanism was related to facilitating Notch1 lysosomal degradation rather than proteasome degradation via promoting protein expression of c-Cbl in macrophages. Our findings that the inhibition of Notch1 signaling transduction helps to alleviate UC-associated liver injury provides possible therapeutics for the treatment of colitis and also furnishes a research paradigm for the study of flavonoids with similar structures.

Dual RNA sequencing of Helicobacter pylori and host cell transcriptomes reveals ontologically distinct host-pathogen interaction.

Simultaneous profiling of the dynamic interaction between Helicobacter pylori and the human gastric epithelium represents a novel strategy for identifying regulatory responses that drive pathogenesis. This study presents the first dual-transcriptome analysis of H. pylori when cocultured with gastric epithelial cells, revealing a bacterial adaptation strategy and a general repression of electron transportation-associated genes, both of which were modulated by cytotoxin-associated gene A (cagA). Temporal profiling of host mRNA signatures dissected the aberrant pre-mRNA splicing of functional genes involved in the cell cycle process in response to H. pylori infection. We demonstrated a protective effect of gastric H. pylori colonization against chronic DSS-induced colitis through both in vitro and in vivo experiments. These findings significantly enhance our understanding of how H. pylori promotes infection and pathogenesis in the human gastric epithelium and provide evidence to identify targets for antimicrobial therapies.

Selenium Yeast Alleviates Dextran Sulfate Sodium-Induced Chronic Colitis in Mice by Reducing Proinflammatory Cytokines and Regulating the Gut Microbiota and Their Metabolites.

Inflammatory bowel disease (IBD) is a chronic recurrent gastrointestinal inflammatory disease. Selenium has been reported to have therapeutic potential in IBD. Selenium yeast is a common selenium supplement that is convenient to access. This study explored the effect of selenium yeast on dextran sulfate sodium- (DSS-)induced chronic colitis in mice. Mice were randomly divided into four groups: the control group, selenium yeast group, chronic colitis group, and chronic colitis+selenium yeast group (n=6). Mice were killed on the 26th day. The disease activity index (DAI) score and histological damage score were calculated. Cytokines, serum selenium, colonic tissue selenium, gut microbiota and their metabolites short-chain fatty acids (SCFAs) were evaluated. Selenium yeast lowered IL-1β, IL-6, TNF-α, IL-17A, IL-22 and IFN-γ (P<0.05). In addition, selenium yeast significantly elevated Turicibacter, Bifidobacterium, Allobaculum, Prevotella, Halomonas, Adlercreutzia (P<0.05), and butyric acid (P<0.05). Selenium yeast could improve DSS-induced chronic colitis in mice by regulating cytokines, gut microbiota and their metabolites.

Wogonin improves colitis by activating the AhR pathway to regulate the plasticity of ILC3/ILC1

BackgroundIntestinal barrier dysfunction caused by the disrupted balance of group 3 innate lymphoid cells (ILC3)/group 1 innate lymphoid cells (ILC1) is a significant feature in the pathogenesis of inflammatory bowel disease (IBD). Activation of aryl hydrocarbon receptor (AhR) signaling contributes to the maintenance of ILC3/ILC1 balance. Wogonin, a natural flavonoid from Scutellaria baicalensis Georgi, can repair intestinal mucosal damage of IBD. However, it remains unclear if wogonin can exert a therapeutic effect by activating the AhR pathway to regulate the plasticity of ILC3/ILC1. PurposeIn this study, we investigated the immunomodulatory effects of wogonin on IBD and its potential mechanisms in vitro and in vivo. Study design and MethodsChronic colitis was induced by four cycles of 2 % DSS treatment in mice. 20 mg kg-1/day wogonin was administrated by oral gavage and mice were treated intraperitoneally with 10 mg kg-1/2 days CH223191 to block the AhR pathway. Colon tissues were processed for histopathological examination and evaluation of the epithelial barrier function by immunohistochemistry. The activation of the AhR pathway and the plasticity of ILC3/ILC1 were determined by western blot and flow cytometry. Then, we also detected the intestinal microflora and their metabolites by 16 s sequencing and non-targeted Metabolomics analysis. Furthermore, an in vitro culture system consisting of MNK3 cells and NCM460 cells, and a CETSA assay were performed to confirm the molecular mechanism. ResultsWogonin ameliorated histological severity of the colon, decreased the secretion of inflammatory factors, and increased tight junction proteins in colitis mice. These effects are associated with the tendency of conversion from ILC3 to ILC1 prevented by wogonin, which was offset by AhR antagonist CH223191. In addition, wogonin exerted the curative effect by altering gut microbiota to produce metabolites such as Kynurenic acid, and 1H-Indole-3-carboxaldehyde as AhR endogenous ligands. In vitro data further verified that wogonin as an exogenous ligand directly binds to the structural domain of AhR by CETSA. Also, the supernatant of MNK-3 cells stimulated with wogonin enhanced expression of Occludin and Claudin1 in NCM460 cells induced by LPS. ConclusionCumulatively, our study illustrated that wogonin improved the outcomes of DSS-induced chronic colitis via regulating the plasticity of ILC3/ILC1. Its specific mechanism is to binding to AhR directly, and to activate the AhR pathway indirectly by altering the tryptophan metabolisms of gut microbiota.

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.

DOP90 Amphiregulin promotes colitis-associated intestinal fibrosis through activation of PI3K/AKT signaling in Intestinal fibroblasts

Abstract Background Intestinal fibrosis is one of the most threatening complications of Crohn’s disease (CD). Amphiregulin (AREG) expression is increased in patients with CD with fibrosis. Methods The role of AREG in activating intestinal fibroblasts and driving fibrogenesis is largely unexplored. In this study, fibrotic and nonfibrotic tissues were obtained from CD patients undergoing surgical resection while normal intestinal tissues were obtained from patients with diverticulum of small intestine. Fibrosis index score for fibrosis were assessed by Masson staining. AREG, collagen Ⅰ and α-SMA expression were determined in intestinal tissues of patients with CD with fibrosis or without fibrosis by qRT-PCR. Wild-type (WT) and Areg knockout (Areg-/-) mice were induced into intestinal fibrosis by dextran sulfate sodium (DSS). We induced intestinal fibrosis in WT mice using a chronic DSS-induced colitis model with or without AREG intraperitoneal injection. The fibrotic indicators in colons were evaluated. Fibroblasts were isolated from fibrotic intestinal surgical resections of patients with CD. The effect of AREG on activation and proliferation in human intestinal fibroblasts was determined. RNA-sequencing of human intestinal fibroblasts treated with or without AREG was performed. Results AREG expression was increased in intestinal fibrotic sites compared with nonfibrotic sites from patients with CD. Mice with Areg deficiency represented with severer intestinal inflammation but alleviated fibrosis in colons. Although additional given AREG attenuated intestinal inflammation in mice with DSS-induced chronic colitis, it promoted more severe intestinal fibrosis. AREG promoted human intestinal fibroblast activation and proliferation by activating PI3K/AKT signaling. PI3K inhibitor suppressed AREG-induced fibroblast activation and proliferation, thus lightened intestinal fibrosis in mice. Besides, AREG promoted secretion of LPA (Lysophosphatidic acid) from intestinal fibroblast, which activated fibroblast through LPAR3/PI3K/AKT pathway. Conclusion These findings reveal that AREG promotes intestinal fibrotic responses in experimental colitis and human patients with CD by promoting fibroblast activation and proliferation through PI3K/AKT pathway. Moreover, LPA secreted from intestinal fibroblast induced by Areg is involved in formation of CD intestinal fibrosis. Thereby, PI3K, AKT, LPA might serve as potential therapeutic targets for fibrosis in CD.

P171 Rescuing Intestinal Stem Cells via TMEM219 inhibition promotes mucosal healing in Crohn’s disease

Abstract Background Intestinal mucosa regeneration is disrupted in inflammatory bowel disease as Chron’s disease, but whether this relies on a defect in intestinal stem cells remains to be established. Methods Transcriptome and receptome profile in Crohn’s disease bioptic samples demonstrated a defect in intestinal stem cells (ISCs). The TMEM219 knockout mouse, in which TMEM219 expression is abrogated on ISCs was tested in dextran sulfate sodium (DSS) acute colitis model. DSS-induced chronic colitis was also used to confirm in vivo relevance for TMEM219 blockade in mucosal recovery. Results An increased intestinal stem cells death and dysfunction in colonic samples obtained from patients with Crohn’s disease was observed, which is controlled through the death factor TMEM219. Based on a receptome analysis, we documented large alterations in the expression of the death receptor TMEM219 in patients with Crohn’s disease, particularly in those with refractory disease and/or non responders to conventional therapy, which were paralleled by altered peripheral levels of the TMEM219 ligand, insulin-like growth factor binding protein 3 (IGFBP3). Pharmacological blockade of the IGFBP3/TMEM219 axis restored the self-renewal abilities of mini-guts generated from patients with Crohn’s disease in vitro, ameliorated DSS-induced colitis in vivo and favored mucosal healing. Genetic deletion of TMEM219 in intestinal stem cells in newly generated TMEM219flflLGR5cre mice restored mucosal regenerative abilities both in vitro and in vivo. Conclusion Our findings demonstrate that genetic and pharmacological TMEM219 blockade re-establishes intestinal self-renewal properties and offers a therapeutic opportunity for mucosal healing in inflammatory bowel disease.

P061 The macrophage-specific role of PTPN23 in Acute and Chronic Colitis

Abstract Background Single nucleotide polymorphisms within the gene locus encoding PTPN2, are associated with Crohn’s disease and ulcerative colitis. We have previously shown that, deletion of PTPN2 results in PTPN23 upregulation in intestinal epithelial cells (IEC) in vivo. PTPN23 is known to be involved in endosomal sorting and responsible for epidermal growth factor receptor (EGFR) internalization from the cell surface and thus negatively controls EGFR signalling in epithelial cells. Though PTPN23 is highly expressed in IEC and intestinal macrophages, nothing is known about its function in myeloid cells and its role in intestinal inflammation. Methods Experimental colitis was induced in PTPN23fl/flLysMCre+/- (KO) and PTPN23fl/flLysMCre-/- (WT) littermates by administration of 2% DSS in drinking water for 7 days (acute colitis) or for 4 cycles of 1.5% DSS for 7 days followed by 10 days recovery, each (chronic colitis). Immune cells were isolated from spleen and colon lamina propria from KO and WT mice and PTPN23 expression was detected by qPCR of RNA isolated after cell sorting with flow cytometry. Results The mRNA expression of PTPN23 in untreated WT mice was significantly higher in macrophages isolated from the lamina propria of the colon than in those isolated from spleen (p=0.0279). PTPN23fl/flLysMCre+/- (KO) mice were protected from DSS-induced acute colitis when compared to their WT littermates. This was apparent by a lack of DSS-induced weight loss (p=0.02); longer colon length (p=0.01), reduced endoscopic colitis scores (p=0.0016) and lower spleen weights (p=0.0063) in KO mice. In HE staining of colon tissue, the loss of crypts was less pronounced in KO mice. On mRNA level a lower expression of the pro-inflammatory cytokines interleukin 6 (IL-6) (p=0.0117) and tumour necrosis factor α (TNFα) (p=0.0024) was observed in KO compared to WT mice, supporting a reduced inflammatory response. In chronic colitis the same trends was observed, however the effects were less pronounced. WT mice lost more weight during the DSS cycles compared to KO mice, whereas no difference in weight development was detected between the genotypes in water control groups. Higher MEICSs scores were observed in WT compared to KO mice (p&amp;lt;0.0001) and loss of colonic crypts was more pronounced in HE stained colon samples in WT mice than KO mice. Conclusion PTPN23 deletion in myeloid cells results in protection from DSS-induced acute and chronic colitis. Additionally, a higher expression in intestinal macrophages compared to macrophages isolated from the spleen suggest a specific role in the intestinal immune system.

Activation of mucosal insulin receptor exacerbates intestinal inflammation by promoting tissue resident memory T cells differentiation through EZH2

BackgroundInflammatory Bowel Diseases (IBD), an autoimmune disease characterised by abnormal intestinal immunity, are related to vital morbidity around the world. However, therapeutic agents for IBD have not achieved desired benefit. Exploring new therapeutic targets for IBD, especially based on its abnormally intestinal immunity, could alleviate the flare-up and worsening of IBD. Tissue resident memory T cells (TRM) are core of multiple autoimmune diseases, including IBD. However, the mechanism of TRM differentiation remains to be investigated.MethodsThe alterations in mRNA and lncRNA profile of intestinal intraepithelial lymphocytes (IELs), the largest component of intestinal TRM, were analyzed in DSS-induced chronic colitis. Based on it, we examined the function of rectal insulin instillation in a dextran sodium sulfate (DSS) induced chronic colitis. Furthermore, we investigated the downstream-target of the insulin pathway—EZH2 and the crucial role of EZH2 in intestinal tissue resident memory T cell differentiation by utilizing EZH2fl/flCD4cre mice.ResultsInsulin receptor (INSR) expression was found to be significantly reduced. Activation of mucosal insulin pathway by rectal insulin instillation exacerbated colitis by disrupting IELs subgroups and up-regulating TNF-ɑ and IL-17 expression. Rectal insulin instillation promoted EZH2 expression and EZH2 inhibition alleviated chronic colitis. EZH2fl/flCD4cre mice restored the normal IEL subgroups and suppressed TNF-ɑ and IL-17 expression, exhibiting alleviated colitis. IELs from EZH2fl/flCD4cre mice exhibit significant changes in TRM related phenotype. CD4+TRM was significantly increased in chronic colitis and decreased in EZH2fl/flCD4cre mice.ConclusionInsulin receptor of intestinal mucosal T-cells could promote intestinal TRM differentiation via EZH2. Our discoveries suggest that therapies targeting colonic INSR and EZH2 could be potential treatment for IBD based on its regulatory effects on TRM. Insulin receptor inhibitors rather than insulin should be applied during colitis-active phase. In addition, EZH2 shows to be a downstream signal of the insulin pathway and EZH2 inhibitor could alleviating intestinal inflammation. However, the critical role of EZH2 in TRM differentiation restricts the anti-tumor effects of EZH2 inhibitor in vivo.

Huang Lian Jie Du decoction attenuated colitis via suppressing the macrophage Csf1r/Src pathway and modulating gut microbiota.

Ulcerative colitis, a subtype of chronic inflammatory bowel disease (IBD), is characterized by relapsing colonic inflammation and ulcers. The traditional Chinese herbal formulation Huang Lian Jie Du (HLJD) decoction is used clinically to treat diarrhea and colitis. However, the mechanisms associated with the effects of treatment remain unclear. This study aims to elucidate the molecular mechanistic effects of HLJD formulation on colitis. Chronic colitis in mice was induced by adding 1% dextran sulfate sodium (DSS) to their drinking water continuously for 8 weeks, and HLJD decoction at the doses of 2 and 4 g/kg was administered orally to mice daily from the second week until experimental endpoint. Stool consistency scores, blood stool scores, and body weights were recorded weekly. Disease activity index (DAI) was determined before necropsy, where colon tissues were collected for biochemical analyses. In addition, the fecal microbiome of treated mice was characterized using 16S rRNA amplicon sequencing. HLJD decoction at doses of 2 and 4 g/kg relieved DSS-induced chronic colitis in mice by suppressing inflammation through compromised macrophage activity in colonic tissues associated with the colony-stimulating factor 1 receptor (Csf1r)/Src pathway. Furthermore, the HLJD formula could modify the gut microbiota profile by decreasing the abundance of Bacteroides, Odoribacter, Clostridium_sensu_stricto_1, and Parasutterella. In addition, close correlations between DAI, colon length, spleen weight, and gut microbiota were identified. Our findings revealed that the HLJD formula attenuated DSS-induced chronic colitis by reducing inflammation via Csf1r/Src-mediated macrophage infiltration, as well as modulating the gut microbiota profile.

PepT1-targeted nanodrug based on co-assembly of anti-inflammatory peptide and immunosuppressant for combined treatment of acute and chronic DSS-induced ColitiS.

Inflammatory bowel disease (IBD), as a chronic and recurrent inflammatory bowel diseases with limited therapeutic outcomes, is characterized by immune disorders and intestinal barrier dysfunction. Currently, the most medications used to cure IBD in clinic just temporarily induce and maintain remission with poor response rates and limited outcomes. Therefore, it is urgently necessary to develop an appropriate therapeutic candidate with preferable efficacy and less adverse reaction for curing IBD. Five groups of mice were utilized: control that received saline, DSS group (mice received 2.5% DSS or 4% DSS), KPV group (mice received KPV), FK506 group (mice received FK506) and NPs groups (mice received NPs). The effect of NP on the inflammatory factors of macrophage was evaluated using CCK-8, quantitative polymerase chain reaction (PCR), Elisa and Western blot (WB). Immunofluorescent staining revealed the targeting relationship between NP and Petp-1. Immunohistochemistry staining showed the effect of NP on tight junction proteins. Moreover, in vivo animal experiments confirmed that NPs reduced inflammatory levels in IBD. After administering with NPs, mice with DSS-induced acute or chronic colitis exhibited significant improvement in body weight, colon length, and disease activity index, decreased the level of the factors associated with oxidative stress (MPO, NO and ROS) and the inflammatory cytokines (TNF-α, IL-1β and IL-6), which implied that NPs could ameliorate murine colitis effectively. Furthermore, treating by NPs revealed a notable reduction of the expressions of CD68 and CD3, restoring the expression levels of tight junction proteins (Claudin-5, Occludin-1, and ZO-1) were significantly restored, surpassing those observed in the KPV and FK506 groups. which indicated that NPs can reduce inflammation and enhance epithelial barrier integrity by decreasing the infiltration of macrophages and T-lymphocytes. Collectively, those results demonstrated the effectively therapeutic outcome after using NPs in both acute and chronic colitis, suggesting that the newly co-assembled of NPs can be as a potential therapeutic candidate for colitis.