A Novel Topical PPARγ Agonist Induces PPARγ Activity in Ulcerative Colitis Mucosa and Prevents and Reverses Inflammation in Induced Colitis Models

Positive Role of CCAAT/Enhancer-Binding Protein Homologous Protein, a Transcription Factor Involved in the Endoplasmic Reticulum Stress Response in the Development of Colitis

Telomere shortening occurs with human aging in many organs and tissues and is accelerated by rapid cell turnover and oxidative injury. To clarify the clinical importance of telomere shortening in colonic mucosa in ulcerative colitis (UC), we measured average telomere length using quantitative real-time PCR in non-neoplastic colonic mucosa in UC patients and assessed its relationship to various clinical subtypes. Relative telomere length in genomic DNA was measured in colonic biopsies obtained from rectal inflammatory mucosa from 86 UC patients as well as paired non-inflammatory proximal colonic mucosae from 10 patients. Data were correlated with various clinical phenotypes. In paired samples, average relative telomere length of rectal inflammatory mucosa was shortened compared to normal appearing proximal colon in eight out of ten cases (p=0.01). Telomere length shortening was significantly associated with more severe Mayo endoscopic subscore (p<0.0001) and cases needing surgery due to toxic megacolon or cancer occurrence (p=0.043). When the severe clinical phenotype was defined as having at least one of following phenotypes, more than two times of hospitalization, highest Mayo endoscopic subscore, steroid dependent, refractory, or needing operation, average relative telomere length was significantly shortened in the same phenotypes than the others (p=0.003). Telomere shortening is associated with more severe clinical phenotypes of UC, reflecting severe inflammatory state in the colonic mucosa.

Background The contribution of cytotoxic T cells to the pathogenesis and treatment outcomes of ulcerative colitis (UC) remains poorly understood. Methods We integrated in vitro functional assays, multiparameter flow cytometry, bulk transcriptomics, single-cell RNA sequencing (scRNAseq), and single-cell spatial transcriptomics analyses to characterise cytotoxic T-cell responses in UC and evaluate their association with clinical and endoscopic outcomes. Results In vitro stimulation of lamina propria mononuclear cells (LPMCs) from UC patients with agonistic anti-CD3 antibodies upregulated cytotoxic gene programs, including GZMB, indicating enhanced T-cell cytotoxicity. Bulk transcriptomics analysis using the TaMMA resource1 confirmed upregulation of these cytotoxic genes in the UC colonic mucosa compared to non-IBD controls and Crohn’s disease. Multiparameter flow cytometry of 42 UC patients and 22 controls showed increased GZMB+ LPMCs in UC. Surprisingly, CD4+ T cells were a major source of GZMB in UC patients and were significantly more abundant than in controls. These GZMB+ CD4+ T cells exhibited a “hyper-inflammatory” polyfunctional phenotype, co-expressing IFN-γ, IL-13, IL-17, IL-22, and TNFα, with cells producing ≥3 pro-inflammatory cytokines significantly increased in UC compared to controls (p = 0.004). Analysis of UC scRNAseq data2 confirmed that GZMB+ CD4+ T cells co-expressed cytotoxic genes (GZMA, PRF1) and pro-inflammatory mediators (IFNG, IL26, OSM) compared to GZMB- CD4+ T cells. A cytotoxic CD4+ T-cell gene signature derived from these data was strongly enriched in UC compared to controls in a large independent cohort (n = 525)3, increasing with endoscopic disease severity and stratifying UC patients by degree of cytotoxic enrichment (Figure 1). Independent single-cell spatial transcriptomics analysis further validated enrichment of this cytotoxic CD4+ T-cell signature in UC versus non-IBD colonic tissues. This signature was also detected in multiple murine models of colitis, with strongest upregulation observed in T-cell transfer colitis. Notably, higher cytotoxic CD4+ T-cell signature enrichment predicted poor response to ustekinumab at week 8 in the UNIFI trial4 (n = 384) for mucosal healing (OR 0.11, p-value = 0.042), even after adjusting for CRP, faecal calprotectin, Total Mayo Score, disease duration, and prior anti-TNF failure. Similarly, the signature was upregulated (p-value = 0.013) in baseline biopsies from UC patients who failed to achieve mucosal healing to infliximab compared with responders in an independent cohort. Conclusion Pro-inflammatory cytotoxic GZMB+ CD4+ T cells are increased in the colonic mucosa of UC patients, correlate with endoscopic disease severity, and predict resistance to biologic therapies.

Epidermal Growth Factor Partially Restores Colonic Ion Transport Responses in Mouse Models of Chronic Colitis

Objective: To analyze the relationship between matrix Gla protein(MGP) and clinical characteristics of patients with ulcerative colitis (UC). Methods: Fifty-one UC patients who were admitted to the gastroenterology department of Peking union medical college hospital from July 1, 2015 to May 31, 2017 were included. Twenty-seven healthy subjects in the same period were included as normal controls. The expression of MGP mRNA in the colonic mucosa was detected by real-time fluorescence quantitative PCR. Clinical data of the patients were collected during a 2-year follow-up. The public data set containing MGP gene expression profile of UC patients was downloaded from the GEO database, and was divided into four groups according to the microscopic Mayo score. The differential expression of MGP in each group was analyzed. Results: All the fifty-one UC patients were followed up. The expression of MGP mRNA in the colonic mucosa of UC patients treated with hormone and immunosuppressive agents or biological agents or surgery was higher than that of patients treated with mesalazine. MGP mRNA expression was positively correlated with C-reactive protein level. It was also higher in the colonic mucosa of UC patients with clostridium difficilis or cytomegalovirus infection than that of patients without opportunistic infection. The difference of MGP mRNA expression between groups in GEO public data set was statistically significant(P<0.01), showing an up-regulation trend with the aggravation of inflammation. The expression level of MGP was moderately correlated with the microscopic Mayo score. The relationship between MGP mRNA expression and lifestyle, lesion range, erythrocyte sedimentation rate, parenteral manifestations and recurrence frequency of UC patients was not obvious. Conclusions: MGP is associated with colonic inflammation and its abnormal expression can help to predict the disease activity of patients with UC.

Parasutterella excrementihominis (P. excrementihominis), a Betaproteobacteria species enriched in ulcerative colitis (UC) patients, is implicated in chronic inflammation. However, its mechanistic role in UC progression and colitis-associated colorectal cancer (CAC) remains unclear. This study investigates the pathogenic role of P. excrementihominis in UC and CAC, focusing on its induction of neutrophil extracellular traps (NETs) and underlying mechanisms. Clinical stool samples from UC patients and healthy controls were analysed for P. excrementihominis abundance. Murine models of dextran sulphate sodium (DSS)-induced colitis and azoxymethane/DSS-induced CAC were used to evaluate bacterial pathogenicity. RNA sequencing and metabolomic analyses were conducted on germ-free mice with monocolonisation, and in vitro cell experiments were carried out to elucidate the role of bacterial metabolites in NETosis. P. excrementihominis was significantly enriched in UC patients and exacerbated colitis and CAC in mice by expanding colonic neutrophils and NETs formation. Metabolomic profiling revealed that P. excrementihominis enhances the host's carbohydrate metabolic capacity, leading to increased production of succinic acid (Suc) and 6-hydroxyhexanoic acid (6-HHA). These metabolites activated gasdermin D (GSDMD)-dependent NETosis in lipopolysaccharide-primed neutrophils through the succinate receptor 1/G protein-coupled receptor 84 signalling pathway. Conversely, neutrophil-specific GSDMD deletion attenuated metabolite-driven tumourigenesis. Our findings identify P. excrementihominis as a critical microbial driver of UC and CAC pathogenesis. This bacterium significantly accelerates disease progression by producing specific metabolites (Suc and 6-HHA) that induce pathogenic NETosis. Targeting this bacterium or its metabolic axis offers novel therapeutic strategies for inflammation-driven colorectal carcinogenesis.

The incidence of inflammatory bowel disease (IBD) in the elderly has increased in recent years. However, the mechanisms underlying the ageing-related IBD susceptibility remain elusive. Cytokine-inducible SH2-containing protein (CISH) involved in regulating metabolism, the expansion of intestinal tuft cells and type-2 innate lymphoid cells, and ageing-related airway inflammation. Here, we investigated the role of CISH in ageing-related colitis susceptibility. CISH and phosphorylated signal transducer and activator of transcription-3 (p-STAT3) levels were evaluated in the colons of ageing mice and older ulcerative colitis (UC) patients. Mice with intestinal epithelial cell-specific knockout of Cish (CishΔIEC) and Cish-floxed mice were administered dextran sodium sulfate (DSS) or trinitrobenzene sulfonic acid (TNBS) to induce colitis. Colonic tissues were analysed in quantitative real-time polymerase chain reaction, immunoblotting, immunohistochemical, and histological staining experiments. Differentially expressed genes from colonic epithelia were analysed by RNA-sequencing. Ageing increased the severity of DSS-induced colitis and the expression of colonic epithelial CISH in mice. CishΔIEC prevented against the DSS or TNBS-induced colitis in middle-aged mice but not, in the young mice. RNA-sequencing analysis revealed that CishΔIEC significantly suppressed DSS-induced oxidative stress and proinflammatory responses. During ageing CCD841 cell model, knockdown of CISH decreased ageing-induced oxidative stress and proinflammatory responses, whereas these effects were compromised by knocking down or inhibiting STAT3. The increase in CISH expression was higher in the colonic mucosa of older patients with UC than that in healthy controls. CISH might be a proinflammatory regulator in ageing; therefore, targeted therapy against CISH may provide a novel strategy for treating ageing-related IBD.

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll-like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2-p) ameliorated DSS-induced colitis by interfering specifically with the activation of Ly6C(+) monocytes without affecting their recruitment to the colon. We report that TLR2-p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2-TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal-regulated kinases (ERK) signaling and reduced secretion of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-23, IL-12, and IL-1β. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro-inflammatory Ly6C(hi) monocytes and suggests that inhibition of this aggregation by TLR2-p might have therapeutic potential in the treatment of acute gut inflammation.

Levels of type 2 cytokines are elevated in the blood and intestinal tissues of ulcerative colitis (UC) patients in the active phase; this phenomenon indicates the participation of type 2 immune response in UC progression. The beneficial effects of melatonin in dextran sodium sulfate (DSS) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis models have been illustrated, but its role in the oxazolone (Oxa)-induced colitis model (driven by type 2 immune response) remains relatively unknown. We investigated the relationship between melatonin concentration and the severity of UC, revealing a significantly negative correlation. Subsequently, we investigated the effects of melatonin in Oxa-induced colitis mice and the potential underlying mechanisms. Administration of melatonin significantly counteracted body weight loss, colon shortening, and neutrophil infiltration in Oxa-induced colitis mice. Melatonin treatment mitigated Oxa-induced colitis by suppressing type 2 immune response. In addition, melatonin attenuated intestinal permeability by enhancing the expression of ZO-1 and occludin in colitis mice. Interestingly, the protective effect of melatonin was abolished when the mice were co-housed, indicating that the regulation of gut microbiota by melatonin was critical in alleviating Oxa-induced colitis. Subsequently, 16S rRNA sequencing was performed to explore the microbiota composition. Decreased richness and diversity of intestinal microbiota at the operational taxonomic unit (OTU) level resulted from melatonin treatment. Melatonin also elevated the abundance of Bifidobacterium, a well-known probiotic, and reduced proportions of several harmful bacterial genera, such as Desulfovibrio, Peptococcaceae, and Lachnospiraceae. Fecal microbiota transplantation (FMT) was used to explore the role of microbiota in the function of melatonin in Oxa-induced colitis. Microbiota transplantation from melatonin-treated mice alleviated Oxa-induced colitis, suggesting that the microbiome participates in the relief of Oxa-induced colitis by melatonin. Our findings demonstrate that melatonin ameliorates Oxa-induced colitis in a microbiota-dependent manner, suggesting the therapeutic potential of melatonin in treating type 2 immunity-associated UC.

To better understand the extent to which chemokines participate in the mucosal inflammatory response in patients with ulcerative colitis (UC), we assessed the expression of an array of chemokines in the colonic mucosa of UC patients and evaluated the effect of corticosteroids on their expression. Colonic mucosal biopsy specimens were obtained from 15 patients with UC and 12 normal controls. Messenger RNA levels for 10 chemokines were quantitated by reverse transcription polymerase chain reaction using synthetic standard RNAs. The biopsy specimens were also cultured in the presence or absence of tumor necrosis factor alpha (TNFalpha) and/or dexamethasone, and secreted chemokines in culture supernatants were assayed by ELISA. The messenger RNA expression of C-X-C (interleukin 8, growth-related alpha [GROalpha], GRObeta, GROgamma, epithelial cell-derived neutrophil activator 78, and interferon-gamma-inducible protein 10) and C-C (monocyte chemotactic protein 1, macrophage inflammatory protein 1beta, and RANTES [regulated on activation, normal T-cell expressed and secreted]) but not C (lymphotactin) chemokines was significantly higher in the affected mucosa of UC patients than in the unaffected mucosa of UC patients or in the normal mucosa of normal controls. The degree of increased expression was more prominent in the C-X-C than in the C-C chemokines. Further, the secretion of interleukin 8, GROalpha, epithelial cell-derived neutrophil activator 78, and monocyte chemotactic protein 1 was higher in UC patients than in normal controls, induced significantly by TNFalpha, and down-regulated by dexamethasone. Secretions of macrophage inflammatory protein 1beta and RANTES also showed a trend toward an increase in UC, induction by TNFalpha, and down-regulation by dexamethasone, but it did not reach statistical significance. The increased expression of a variety of chemokines in UC and their downregulation by dexamethasone suggest that chemokines may play an important role in the immunopathogenesis of UC.

In sepsis, circulating free DNA (cf-DNA) is increased, and is a marker of severity and prognosis of septic patients. This study aimed to evaluate cf-DNA in a dextran sodium sulfate-induced colitis mouse model, and its clinical implications. Dynamic pathology of the cecum wall in the DSS-induced colitis mouse model was analyzed using multiphoton microscopy (MPM). Plasma cf-DNA concentrations in colitis mouse were quantified using PicoGreen dsDNA Assay Kit. Plasma cf-DNA was also measured in 123 human ulcerative colitis (UC) patients [mean age: 35.9 years (3-75 years) with 20 pediatric patients] to assess its relationships with clinical severity and Matt's grade. Real-time images of cf-DNA were detected in the colitis model. The amount of labeled cf-DNA in the circulation of the colitis mice group was significantly higher compared with that in the control group (P < 0.05). In human UC blood samples, plasma cf-DNA concentrations in UC patients were significantly positively correlated with the clinical severity of UC and Matt's grade (P < 0.05, P < 0.05, respectively). Using MPM, we observed and analyzed real-time images of cf-DNA in a colitis mouse model. Plasma cf-DNA is a potential non-invasive blood marker for reflecting clinical severity and mucosal damage in UC patients.

To investigate the therapeutic effects of aqueous infusion of Cotinus coggygria Scop. leaves (CCLAI) on the dextran sulphate sodium (DSS)-induced ulcerative colitis (UC) in mice. A total of 56 C57BL16J mice were divided into 7 groups, including control, DSS model, CCLAI4, CCLAI6, DSS + CCLAI4, DSS + CCLAI6 and DSS + mesalamine groups (n=8). Induction of UC was conducted by 7-day treatment of 3% DSS in mice. CCLAI in 2 different doses (4% and 6%) or mesalamine (250 mg/kg) was administered to C57BL/6J mice orally for 7 consecutive days. The mice in the control group received only tap water. Clinical parameters, antioxidant and anti-inflammatory activities, and histopathological changes in the colon mucosa were examined. Cell proliferatipn index in colon section was determined by immunonistochemical analysis. Colonic cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) expression levels were detected by Western blot. CCLAI treatment inhibited DSS-induced colon shortening and significantly improved the Disease Activity Index score (P<0.05 or P<0.01). We also found a significant decrease in myeloperoxidase levels, oxidative stress parameters, and neutrophil infiltration index in the experimental groups receiving CCLAI compared to the control group (all P<0.01). In addition, it was observed that CCLAI administration for 7 days decreased the proinflammatory cytokines levels including interleukin (IL)-1β, IL-6, interferon-γ, and tumor necrosis factor-α in the colon tissues of mice receiving DSS (P<0.05). Further, CCLAI treatment reduced the COX-2 and iNOS levels with an accompanying decrease in prostaglandin E2 and nitric oxide levels, respectively. Additionally, CCLAI treatment markedly decreased the histological lesion score, which was consistent with the reduction of the pro-inflammatory cytokine levels. CCLAI treatment significantly improved the DSS-induced UC in mice by diminishing histopathological damage in the colonic mucosa and markedly decreased the inflammatory response through inhibiting the formation of inflammatory cytokines and markers of oxidative stress.

Background Ulcerative colitis (UC) is marked by excessive apoptosis of colonic epithelial cells, driven by intrinsic epithelial defects and impaired macrophage efferocytosis. Inefficient clearance of apoptotic cells perpetuates inflammation and obstructs tissue resolution. NR1D1, a nuclear receptor involved in cellular homeostasis and inflammation, has been implicated in immune regulation, but its role in UC pathogenesis, particularly in macrophage efferocytosis, remains poorly understood. Methods A clinical cohort of 300 individuals (200 UC patients and 100 healthy controls) was analyzed to assess the relationship between NR1D1 expression and disease severity, using the Mayo score as a measure of clinical progression. In parallel, dextran sulfate sodium (DSS) was used to induce acute colitis in wild-type and NR1D1-knockout mice. Proteomic profiling was employed to elucidate the effects of NR1D1 deficiency on intestinal inflammation. Mechanistic insights into NR1D1 regulation of CD47 expression were obtained using CUT&amp;Tag, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays in colonic epithelial cells from UC patients and healthy controls. The impact of NR1D1 deficiency on macrophage efferocytosis was explored in vitro using co-cultures of macrophages and organoids. Therapeutic potential was evaluated by administering a hydrogel loaded with SR9009, an NR1D1 agonist, to DSS-treated mice, followed by single-cell RNA sequencing (scRNA-seq) to assess cellular and molecular changes. Results NR1D1 expression was significantly reduced in UC patients and inversely correlated with disease severity, as reflected by the Mayo score. Proteomic analysis in DSS-treated mice confirmed that NR1D1 deficiency aggravated intestinal inflammation. CUT&amp;Tag and ChIP assays identified CD47 as a direct transcriptional target negatively regulated by NR1D1. Loss of NR1D1 led to elevated CD47 expression in epithelial cells, which impaired macrophage efferocytosis and disrupted the resolution of inflammation. Administration of SR9009 via a hydrogel delivery system restored NR1D1 activity, reduced CD47 expression, and improved efferocytosis. scRNA-seq of colonic tissues from treated mice revealed a shift toward anti-inflammatory cellular states, with enhanced macrophage functionality and improved epithelial integrity. Conclusion NR1D1 deficiency exacerbates epithelial apoptosis and inflammation in UC by upregulating CD47, thereby impairing macrophage efferocytosis. Restoring NR1D1 activity with SR9009 ameliorates inflammation and promotes resolution through improved apoptotic cell clearance. Targeting the NR1D1-CD47 axis represents a promising therapeutic strategy for UC.

Introduction Ulcerative colitis (UC) is an inflammatory intestine disease characterized by dysfunction of the intestinal mucosal barrier, ferroptosis, and apoptosis. Previous researches suggest that celecoxib, a nonsteroidal anti-inflammatory drug, holds promise in alleviating inflammation in UC. Therefore, this study aims to investigate the effects and mechanisms of celecoxib in UC. Methods To identify ferroptosis-related drugs and genes associated with UC, we utilized the Gene Expression Omnibus (GEO), FerrDb databases, and DGIdb database. Subsequently, we established a 2.5% DSS (Dextran sulfate sodium)-induced colitis model in mice and treated them with 10 mg/kg of celecoxib to validate the bioinformatics results. We evaluated histological pathologies, inflammatory response, intestinal barrier function, ferroptosis markers, and apoptosis regulators. Results Celecoxib treatment significantly ameliorated DSS-induced UC in mice, as evidenced by the body weight change curve, colon length change curve, disease activity index (DAI) score, and histological index score. Celecoxib treatment reduced the level of pro-inflammatory factors and promoted the expressions of intestinal tight junction proteins such as Claudin-1 and Occludin, thereby restoring the integrity of the intestinal mucosal barrier. Furthermore, celecoxib treatment reversed the ferroptosis characteristics in DSS-induced mice by increasing glutathione (GSH), decreasing malondialdehyde (MDA), and increasing the expression of GPX-4 and xCT. Additionally, apoptosis was induced in mice with UC, as evidenced by increased Caspase3, BAD, P53, BAX, Caspase9 and Aifm1 production, and decreased expression of BCL-XL and BCL2. Celecoxib treatment significantly reversed the apoptotic changes in DSS-induced mice. Conclusion Our findings suggest that celecoxib effectively treats DSS-induced UC in mice by inhibiting ferroptosis and apoptosis.

Ulcerative colitis (UC) and rheumatoid arthritis (RA) are immune-mediated inflammatory diseases (IMIDs) with similar symptoms and common genomics. However, the relationship between UC and RA has not been investigated thoroughly. Therefore, this study aimed to establish the differentially expressed genes (DEGs) and potential therapeutic targets in UC and RA. Three microarray datasets (GSE38713, GSE1919, and GSE12251) were selected from the Gene Expression Omnibus (GEO) database for analysis. We used R software to identify the DEGs and performed enrichment analyses. Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape software were used to construct the protein-protein interaction (PPI) network and identify the hub genes. A regulatory network based on the constructed PPI was generated using StarBase and PROMO databases. We identified a total of 1542 and 261 DEGs in UC and RA. There were 169 common DEGs identified in both UC and RA, including 63 upregulated genes (DEGs1) and nine downregulated genes (DEGs2). The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of DEGs1 and DEGs2 in the PPI network revealed that the genes enriched were involved in immunity. A total of 45 hub genes were selected based on high scores of correlation; three hub genes (SRGN, PLEK, and FCGR3B) were found to be upregulated in UC and RA, and downregulated in UC patients with response to infliximab treatment. The identification of novel DEGs and hub genes in the current study contributes to a novel perception for latent functional mechanisms and presents potential prognostic indicators and therapeutic targets in UC and RA.