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Background and Aim Bromodomain-containing protein 4 [BRD4], one of the components of the bromodomain and extraterminal domain [BET] family, is a transcriptional and epigenetic regulator of cellular proliferation and cytokine production. In this study, we assessed whether BRD4 regulates the cytokine response in inflammatory bowel diseases [IBD]. Materials and Methods BRD4 expression was analysed in intestinal mucosal samples of patients with ulcerative colitis [UC], patients with Crohn's disease [CD], normal controls [CTRs], and mice with chemically-induced colitis, by real-time polymerase chain reaction [PCR], western blotting, and confocal microscopy. Cytokine production was evaluated in lamina propria mononuclear cells [LPMCs] of IBD patients and mucosal tissues of colitic mice treated with BRD4 inhibitors. Finally, we evaluated the effect of JQ1, an inhibitor of the BRD4 signalling pathway, on the course of murine colitis. Results BRD4 RNA and protein expression was up-regulated in the inflamed mucosa of patients with UC and patients with CD as compared with the uninvolved areas of the same patients and CTRs, and in the inflamed colon of colitic mice. Knockdown of BRD4 with a specific antisense oligonucleotide in IBD LPMCs led to reduced expression of TNF-α, IL-6, IFN-γ, and IL-17A. Administration of JQ1 to colitic mice inhibited the inflammatory cytokine response and attenuated the ongoing colitis. Conclusions This is the first study showing the up-regulation of BRD4 in IBD and suggesting the role of such a protein in the positive control of the inflammatory cytokine response in the gut. [ABSTRACT FROM AUTHOR]

In the colorectal cancer (CRC) niche, the transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor‐κB (NF‐κB) are hyperactivated in both malignant cells and tumor‐infiltrating leukocytes (TILs) and cooperate to maintain cancer cell proliferation/survival and drive protumor inflammation. Through drug repositioning studies, the anthelmintic drug rafoxanide has recently emerged as a potent and selective antitumor molecule for different types of cancer, including CRC. Here, we investigate whether rafoxanide could negatively modulate STAT3/NF‐κB and inflammation‐associated CRC. The antineoplastic effect of rafoxanide was explored in a murine model of CRC resembling colitis‐associated disease. Cell proliferation and/or STAT3/NF‐κB activation were evaluated in colon tissues taken from mice with colitis‐associated CRC, human CRC cells, and CRC patient‐derived explants and organoids after treatment with rafoxanide. The STAT3/NF‐κB activation and cytokine production/secretion were assessed in TILs isolated from CRC specimens and treated with rafoxanide. Finally, we investigated the effects of TIL‐derived supernatants cultured with or without rafoxanide on CRC cell proliferation and STAT3/NF‐κB activation. The results showed that rafoxanide restrains STAT3/NF‐κB activation and inflammation‐associated colon tumorigenesis in vivo without apparent effects on normal intestinal cells. Rafoxanide markedly reduces STAT3/NF‐κB activation in cultured CRC cells, CRC‐derived explants/organoids, and TILs. Finally, rafoxanide treatment impairs the ability of TILs to produce protumor cytokines and promote CRC cell proliferation. We report the novel observation that rafoxanide negatively affects STAT3/NF‐κB oncogenic activity at multiple levels in the CRC microenvironment. Our data suggest that rafoxanide could potentially be deployed as an anticancer drug in inflammation‐associated CRC. [ABSTRACT FROM AUTHOR]

Background and Aims Intestinal barrier dysfunction is a hallmark of inflammatory bowel diseases [IBD], but the mechanisms that lead to such a defect are not fully understood. This study was aimed at characterising the factors involved in the defective barrier function in IBD. Methods Transcriptome analysis was performed on colon samples taken from healthy controls [CTR] and IBD patients. Expression of GATA-binding factor 6 [GATA6], a transcription factor involved in intestinal epithelial cell differentiation, was evaluated in colon samples taken from CTR and IBD patients by real-time polymerase chain reaction [PCR] and immunohistochemistry. Intestinal sections of wild-type and Gata6del mice, which exhibit a conditional Gata6 deletion in intestinal epithelial cells and which are either left untreated or receive subcutaneous indomethacin or rectal trinitrobenzene sulphonic acid, were stained with haematoxylin and eosin. In parallel, some Gata6del mice received antibiotics to deplete intestinal flora. Mucosal inflammatory cell infiltration and cytokine production were evaluated by flow cytometry and real-time PCR, respectively, and tight junction proteins were examined by immunofluorescence. Intestinal barrier integrity was assessed by fluorescein isothiocyanate [FITC]-dextran assay. Results Multiple genes involved in cell commitment/proliferation and wound healing were differentially expressed in IBD compared with CTR. Among these, GATA6 was significantly decreased in the IBD epithelium compared with CTR. In mice, conditional deletion of GATA6 in the intestinal epithelium induced primarily epithelial damage, diminished zonula occludens-1 expression, and enhanced intestinal permeability, ultimately resulting in bacteria-driven local immune response and enhanced susceptibility to gut inflammation. Conclusions Reduced expression of GATA6 promotes intestinal barrier dysfunction, thus amplifying intestinal inflammatory pathology. [ABSTRACT FROM AUTHOR]

Background and Aims The inflammatory bowel disease [IBD]-associated immune response is marked by excessive production of a variety of inflammatory cytokines, which are supposed to sustain and amplify the pathological process. OTUD5 is a deubiquitinating enzyme, which regulates cytokine production by both innate and adaptive immune cells. Here, we investigated the expression and role of OTUD5 in IBD. Methods OTUD5 expression was evaluated in mucosal samples of patients with Crohn's disease [CD], patients with ulcerative colitis [UC], and controls, as well as in mice with trinitrobenzene-sulphonic acid [TNBS]-induced colitis by real-time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. Moreover, OTUD5 was assessed in lamina propria mononuclear cells [LPMC] stimulated with inflammatory cytokines. TNF-α, IL-6, and IL-10 were evaluated in LPMCs of IBD patients and in colitic mice transfected with a specific OTUD5 antisense oligonucleotide [AS]. Results OTUD5 protein, but not RNA, expression was increased in inflamed ileal and colonic mucosal samples of patients with CD and patients with UC as compared with controls. In IBD, OTUD5-expressing cells were abundant in both epithelial and lamina propria compartments, and non-CD3+, HLA-DR+ LPMC were one of the major sources of the protein. OTUD5 expression was enhanced by IFN-γ through a p38/MAPK-dependent mechanism, and the AS-induced knockdown of OTUD5 in LPMCs of IBD patients and colitic mice reduced TNF-α. Conclusions Our data show that OTUD5 is overexpressed in both CD and UC and suggest the involvement of such a protein in the amplification of the aberrant cytokine response in IBD. [ABSTRACT FROM AUTHOR]

Background and Aims: The inflammatory bowel disease [IBD]-associated immune response is marked by excessive production of a variety of inflammatory cytokines, which are supposed to sustain and amplify the pathological process. OTUD5 is a deubiquitinating enzyme, which regulates cytokine production by both innate and adaptive immune cells. Here, we investigated the expression and role of OTUD5 in IBD., Methods: OTUD5 expression was evaluated in mucosal samples of patients with Crohn's disease [CD], patients with ulcerative colitis [UC], and controls, as well as in mice with trinitrobenzene-sulphonic acid [TNBS]-induced colitis by real-time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. Moreover, OTUD5 was assessed in lamina propria mononuclear cells [LPMC] stimulated with inflammatory cytokines. TNF-α, IL-6, and IL-10 were evaluated in LPMCs of IBD patients and in colitic mice transfected with a specific OTUD5 antisense oligonucleotide [AS]., Results: OTUD5 protein, but not RNA, expression was increased in inflamed ileal and colonic mucosal samples of patients with CD and patients with UC as compared with controls. In IBD, OTUD5-expressing cells were abundant in both epithelial and lamina propria compartments, and non-CD3+, HLA-DR+ LPMC were one of the major sources of the protein. OTUD5 expression was enhanced by IFN-γ through a p38/MAPK-dependent mechanism, and the AS-induced knockdown of OTUD5 in LPMCs of IBD patients and colitic mice reduced TNF-α., Conclusions: Our data show that OTUD5 is overexpressed in both CD and UC and suggest the involvement of such a protein in the amplification of the aberrant cytokine response in IBD., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)