Intestinal Inflammation Modulates the Epithelial Response to Butyrate in Patients With Inflammatory Bowel Disease

Background: Butyrate-producing gut bacteria are reduced in patients with active inflammatory bowel disease (IBD), supporting the hypothesis that butyrate supplementation may be beneficial in this setting. Nonetheless, earlier studies suggest that the oxidation of butyrate in IBD patients is altered. We propose that inflammation may decrease epithelial butyrate consumption. Methods: Non-IBD controls and IBD patients were recruited for the study. Stool samples were used for short-chain fatty acid and bacterial butyryl CoA:acetate CoA-transferase quantification. Colonic biopsies and ex vivo differentiated epithelial organoids (d-EpOCs) treated with bu- tyrate and/or tumor necrosis factor alpha (TNFα) were used for analyzing the expression of transporters MCT1 and ABCG2, metabolic enzyme ACADS, and butyrate receptor GPR43, and for butyrate metabolism and consumption assays. Results: We observed that lower stool content of butyrate-producing bacteria in active IBD patients did not correlate with decreased bu- tyrate concentrations. Indeed, the intestinal epithelial expression of MCT1, ABCG2, ACADS, and GPR43 was altered in active IBD patients. Nonetheless, d-EpOCs derived from IBD patients showed SLC16A1 (gene encoding for MCT1 protein), ABCG2, ACADS, and GPR43 expres- sion levels comparable to controls. Moreover, IBD- and non-IBD-derived d-EpOCs responded similarly to butyrate, as assessed by transcriptional regulation. TNFα significantly altered SLC16A1, ABCG2, and GPR43 transcription in d-EpOCs, mimicking the expression profile observed in biopsies from active IBD patients and resulting in reduced butyrate consumption. Conclusions: We provide evidence that the response to butyrate is not intrinsically altered in IBD patients. However, TNFα renders the epithe- lium less responsive to this metabolite, defeating the purpose of butyrate supplementation during active inflammation

Experimental and genetic evidence for the impact of CD5 and CD6 expression and variation in inflammatory bowel disease

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) resulting from the interaction of multiple environmental, genetic and immunological factors. CD5 and CD6 are paralogs encoding lymphocyte co-receptors involved in fine-tuning intracellular signals delivered upon antigen-specific recognition, microbial pattern recognition and cell adhesion. While CD5 and CD6 expression and variation is known to influence some immune-mediated inflammatory disorders, their role in IBD remains unclear. To this end, Cd5- and Cd6-deficient mice were subjected to dextran sulfate sodium (DSS)-induced colitis, the most widely used experimental animal model of IBD. The two mouse lines showed opposite results regarding body weight loss and disease activity index (DAI) changes following DSS-induced colitis, thus supporting Cd5 and Cd6 expression involvement in the pathophysiology of this experimental IBD model. Furthermore, DNA samples from IBD patients of the ENEIDA registry were used to test association of CD5 (rs2241002 and rs2229177) and CD6 (rs17824933, rs11230563, and rs12360861) single nucleotide polymorphisms with susceptibility and clinical parameters of CD (n=1352) and UC (n=1013). Generalized linear regression analyses showed association of CD5 variation with CD ileal location (rs2241002CC) and requirement of biological therapies (rs2241002C-rs2229177T haplotype), and with poor UC prognosis (rs2241002T-rs2229177T haplotype). Regarding CD6, association was observed with CD ileal location (rs17824933G) and poor prognosis (rs12360861G), and with left-sided or extensive UC, and absence of ankylosing spondylitis in IBD (rs17824933G). The present experimental and genetic evidence support a role for CD5 and CD6 expression and variation in IBD's clinical manifestations and therapeutic requirements, providing insight into its pathophysiology and broadening the relevance of both immunomodulatory receptors in immune-mediated disorders

An RORγt oral inhibitor modulates IL-17 responses in peripheral blood and intestinal mucosa of Crohn's disease patients

Background and Aims: Despite the negative results of blocking IL-17 in Crohn's disease (CD) patients, selective modulation of Th17-dependent responses warrants further study. Inhibition of retinoic acid-related orphan receptor gamma (RORγt), the master regulator of the Th17 signature, is currently being explored in inflammatory diseases. Our aim was to determine the effect of a novel oral RORγt antagonist (BI119) in human CD and on an experimental model of intestinal inflammation. Methods: 51 CD patients and 11 healthy subjects were included. The effects of BI119 were tested on microbial-stimulated peripheral blood mononuclear cells (PBMCs), intestinal crypts and biopsies from CD patients. The ability of BI119 to prevent colitis in vivo was assessed in the CD4+CD45RBhigh T cell transfer model. Results: In bacterial antigen-stimulated PBMCs from CD patients, BI119 inhibits Th17-related genes and proteins, while upregulating Treg and preserving Th1 and Th2 signatures. Intestinal crypts cultured with supernatants from BI119-treated commensal-specific CD4+ T cells showed decreased expression of CXCL1, CXCL8 and CCL20. BI119 significantly reduced IL17 and IL26 transcription in colonic and ileal CD biopsies and did not affect IL22. BI119 has a more profound effect in ileal CD with additional significant downregulation of IL23R, CSF2, CXCL1, CXCL8, and S100A8, and upregulation of DEFA5. BI119 significantly prevented development of clinical, macroscopic and molecular markers of colitis in the T-cell transfer model. Conclusions: BI119 modulated CD-relevant Th17 signatures, including downregulation of IL23R while preserving mucosa-associated IL-22 responses, and abrogated experimental colitis. Our results provide support to the use of RORγt antagonists as a novel therapy to CD treatment

Gastric metaplasia as a precursor of nonconventional dysplasia in inflammatory bowel disease

Gastric metaplasia in colonic mucosa with inflammatory bowel disease (IBD) develops as an adaptation mechanism. The association between gastric metaplasia and nonconventional and/or conventional dysplasia as precursors of colitis-associated colorectal cancer is unknown. To address this question, we retrospectively reviewed a series of 33 IBD colectomies to identify gastric metaplasia in 76 precursor lesions. We obtained 61 nonconventional and 15 conventional dysplasias. Among nonconventional dysplasia, 31 (50.8 %) were low-grade (LGD), 4 (6.5 %) were high-grade (HGD), 9 (14.8 %) had both LGD and HGD, and 17 (27.9 %) had no dysplasia (ND), while 14 (93 %) conventional dysplasias had LGD, and 1 (7 %) had LGD and HGD. Gastric metaplasia was assessed by concomitant immunoexpression of MUC5AC and loss of CDX2 staining. Expression of a p53-mut pattern was considered as a surrogate for gene mutation, and complete loss of MLH1 staining as presence of MLH1 hypermethylation. In nonconventional dysplasia, MUC5AC immunoexpression decreased as the degree of dysplasia increased, being 78 % in LGD and 39 % in HGD (p = 0.006). CDX2 was lost in epithelial glands with high expression of MUC5AC (p < 0.001). The p53-mut pattern was observed in 77 % HGD, 45 % LGD, and in 6 % with ND (p < 0.001). Neither nonconventional nor conventional dysplasia showed complete loss of MLH1 staining. Gastric metaplasia was also present in mucosa adjacent to nonconventional dysplasia with chronic changes or active inflammation. Our results show that gastric metaplasia appears in IBD-inflamed colon mucosa, it is the substrate of most nonconventional dysplasia and occurs prior to p53 alterations

Dissecting common and unique effects of anti-alpha4beta7 and anti-tumor necrosis factor treatment in ulcerative colitis

Background and Aims:Vedolizumab is an anti-α4β7 antibody approved for the treatment of ulcerative colitis [UC]. Although it is assumed that vedolizumab blocks intestinal homing of lymphocytes, its effects on different intestinal cell populations are not fully stablished. In order to establish the unique mechanisms of action of vedolizumab in UC patients, we compared its effects to those induced by anti-tumour necrosis factor [TNF]. Methods:patients with active UC [endoscopic Mayo score >1] starting vedolizumab [n = 33] or anti-TNF [n = 45] and controls [n = 22] were included. Colon biopsies [at weeks 0, 14 and 46] and blood samples [at weeks 0, 2, 6, 14, 30 and 46] were used for cell phenotyping, transcriptional analysis [qPCR], and to measure receptor occupancy. Results:Vedolizumab, in contrast to anti-TNF, significantly reduced the proportion of α4β7+ cells within intestinal T subsets while preserving the percentage of α4β7+ plasma cells. The marked decrease in α4β7 did not change the percentage of colonic αEβ7+ cells [at 46 weeks]. Both vedolizumab and anti-TNF significantly downregulated inflammation-related genes in the colon of responders [Mayo score < 2]. Moreover, both treatments significantly decreased the percentage of intestinal, but not blood, total lymphocytes [T and plasma cells], as well as the proportion of α4β1+ cells within intestinal T lymphocytes. Conclusions:Our data show that while vedolizumab and anti-TNF block two unrelated targets, they induce remarkably similar effects. On the other hand, vedolizumab's unique mechanism of action relies on blocking intestinal trafficking of α4β7 T cells, despite effectively binding to B and plasma cells that express α4β7

Alterations in p53, Microsatellite Stability and Lack of MUC5AC Expression as Molecular Features of Colorectal Carcinoma Associated with Inflammatory Bowel Disease

Colitis-associated colorectal carcinoma (CAC) occurs in inflammatory bowel disease (IBD) because of the "chronic inflammation-dysplasia-cancer" carcinogenesis pathway characterized by p53 alterations in the early stages. Recently, gastric metaplasia (GM) has been described as the initial event of the serrated colorectal cancer (CRC) process, resulting from chronic stress on the colon mucosa. The aim of the study is to characterize CAC analyzing p53 alterations and microsatellite instability (MSI) to explore their relationship with GM using a series of CRC and the adjacent intestinal mucosa. Immunohistochemistry was performed to assess p53 alterations, MSI and MUC5AC expression as a surrogate for GM. The p53 mut-pattern was found in more than half of the CAC, most frequently stable (MSS) and MUC5AC negative. Only six tumors were unstable (MSI-H), being with p53 wt-pattern (p = 0.010) and MUC5AC positive (p = 0.005). MUC5AC staining was more frequently observed in intestinal mucosa, inflamed or with chronic changes, than in CAC, especially in those with p53 wt-pattern and MSS. Based on our results, we conclude that, as in the serrated pathway of CRC, in IBD GM occurs in inflamed mucosa, persists in those with chronic changes and disappears with the acquisition of p53 mutations

An RORγt oral inhibitor modulates IL-17 responses in peripheral blood and intestinal mucosa of Crohn's disease patients

Background and Aims: Despite the negative results of blocking IL-17 in Crohn's disease (CD) patients, selective modulation of Th17-dependent responses warrants further study. Inhibition of retinoic acid-related orphan receptor gamma (RORγt), the master regulator of the Th17 signature, is currently being explored in inflammatory diseases. Our aim was to determine the effect of a novel oral RORγt antagonist (BI119) in human CD and on an experimental model of intestinal inflammation. Methods: 51 CD patients and 11 healthy subjects were included. The effects of BI119 were tested on microbial-stimulated peripheral blood mononuclear cells (PBMCs), intestinal crypts and biopsies from CD patients. The ability of BI119 to prevent colitis in vivo was assessed in the CD4+CD45RBhigh T cell transfer model. Results: In bacterial antigen-stimulated PBMCs from CD patients, BI119 inhibits Th17-related genes and proteins, while upregulating Treg and preserving Th1 and Th2 signatures. Intestinal crypts cultured with supernatants from BI119-treated commensal-specific CD4+ T cells showed decreased expression of CXCL1, CXCL8 and CCL20. BI119 significantly reduced IL17 and IL26 transcription in colonic and ileal CD biopsies and did not affect IL22. BI119 has a more profound effect in ileal CD with additional significant downregulation of IL23R, CSF2, CXCL1, CXCL8, and S100A8, and upregulation of DEFA5. BI119 significantly prevented development of clinical, macroscopic and molecular markers of colitis in the T-cell transfer model. Conclusions: BI119 modulated CD-relevant Th17 signatures, including downregulation of IL23R while preserving mucosa-associated IL-22 responses, and abrogated experimental colitis. Our results provide support to the use of RORγt antagonists as a novel therapy to CD treatment

An RORγt oral inhibitor modulates IL-17 responses in peripheral blood and intestinal mucosa of Crohn's disease patients

Background and Aims: Despite the negative results of blocking IL-17 in Crohn's disease (CD) patients, selective modulation of Th17-dependent responses warrants further study. Inhibition of retinoic acid-related orphan receptor gamma (RORγt), the master regulator of the Th17 signature, is currently being explored in inflammatory diseases. Our aim was to determine the effect of a novel oral RORγt antagonist (BI119) in human CD and on an experimental model of intestinal inflammation. Methods: 51 CD patients and 11 healthy subjects were included. The effects of BI119 were tested on microbial-stimulated peripheral blood mononuclear cells (PBMCs), intestinal crypts and biopsies from CD patients. The ability of BI119 to prevent colitis in vivo was assessed in the CD4+CD45RBhigh T cell transfer model. Results: In bacterial antigen-stimulated PBMCs from CD patients, BI119 inhibits Th17-related genes and proteins, while upregulating Treg and preserving Th1 and Th2 signatures. Intestinal crypts cultured with supernatants from BI119-treated commensal-specific CD4+ T cells showed decreased expression of CXCL1, CXCL8 and CCL20. BI119 significantly reduced IL17 and IL26 transcription in colonic and ileal CD biopsies and did not affect IL22. BI119 has a more profound effect in ileal CD with additional significant downregulation of IL23R, CSF2, CXCL1, CXCL8, and S100A8, and upregulation of DEFA5. BI119 significantly prevented development of clinical, macroscopic and molecular markers of colitis in the T-cell transfer model. Conclusions: BI119 modulated CD-relevant Th17 signatures, including downregulation of IL23R while preserving mucosa-associated IL-22 responses, and abrogated experimental colitis. Our results provide support to the use of RORγt antagonists as a novel therapy to CD treatment

Epigenetic landscape in the kick-and-kill therapeutic vaccine BCN02 clinical trial is associated with antiretroviral treatment interruption (ATI) outcome

Background The BCN02-trial combined therapeutic vaccination with a viral latency reversing agent (romidepsin, RMD) in HIV-1-infected individuals and included a monitored antiretroviral pause (MAP) as an efficacy read-out identifying individuals with an early or late ( 4weeks) viral-rebound. Integrated-omics analyses were applied prior treatment interruption to identify markers of virus control during MAP.& nbsp;Methods PBMC, whole-genome DNA methylation and transcriptomics were assessed in 14 BCN02 participants, including 8 Early and 4 Late viral-rebound individuals. Chromatin state, histone marks and integration analysis (his tone-3 acetylation (H3Ac), viral load, proviral levels and HIV-specific T cells responses) were included. REDUC-trial samples (n = 5) were included as a control group for RMD administration alone.& nbsp;Findings DNA methylation imprints after receiving the complete intervention discriminated Early versus Late viral rebound individuals before MAP. Also, differential chromatin accessibility and histone marks at DNA methylation level were detected. Importantly, the differential DNA methylation positions (DMPs) between Early and Late rebounders before MAP were strongly associated with viral load, proviral levels as well as the HIV-specific T-cell responses. Most of these DMPs were already present prior to the intervention and accentuated after RMD infusion.& nbsp;Interpretation This study identifies host DNA methylation profiles and epigenetic cascades that are predictive of subsequent virus control in a kick-and-kill HIV cure strategy. Copyright (C)& nbsp;2022 The Authors. Published by Elsevier B.V.& nbsp

Epigenetic landscape in the kick-and-kill therapeutic vaccine BCN02 clinical trial is associated with antiretroviral treatment interruption (ATI) outcome.

BackgroundThe BCN02-trial combined therapeutic vaccination with a viral latency reversing agent (romidepsin, RMD) in HIV-1-infected individuals and included a monitored antiretroviral pause (MAP) as an efficacy read-out identifying individuals with an early or late (&lt; or &gt; 4weeks) viral-rebound. Integrated -omics analyses were applied prior treatment interruption to identify markers of virus control during MAP.MethodsPBMC, whole-genome DNA methylation and transcriptomics were assessed in 14 BCN02 participants, including 8 Early and 4 Late viral-rebound individuals. Chromatin state, histone marks and integration analysis (histone-3 acetylation (H3Ac), viral load, proviral levels and HIV-specific T cells responses) were included. REDUC-trial samples (n&nbsp;=&nbsp;5) were included as a control group for RMD administration alone.FindingsDNA methylation imprints after receiving the complete intervention discriminated Early versus Late viral-rebound individuals before MAP. Also, differential chromatin accessibility and histone marks at DNA methylation level were detected. Importantly, the differential DNA methylation positions (DMPs) between Early and Late rebounders before MAP were strongly associated with viral load, proviral levels as well as the HIV-specific T-cell responses. Most of these DMPs were already present prior to the intervention and accentuated after RMD infusion.InterpretationThis study identifies host DNA methylation profiles and epigenetic cascades that are predictive of subsequent virus control in a kick-and-kill HIV cure strategy.FundingEuropean Union Horizon 2020 Framework Programme for Research and Innovation under Grant Agreement N°681137-EAVI2020 and N°847943-MISTRAL, the Ministerio de Ciencia e Innovación (SAF2017_89726_R), and the National Institutes of Health-National Institute of Allergy and Infectious Diseases Program Grant P01-AI131568