Rafoxanide negatively modulates STAT3 and NF-κB activity and inflammation-associated colon tumorigenesis.

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.

Metformin inhibits inflammatory signals in the gut by controlling AMPK and p38 MAP kinase activation.

Metformin, a hypoglycemic drug used for treatment of type 2 diabetes, regulates inflammatory pathways. By using several models of intestinal inflammation, we examined whether metformin exerts anti-inflammatory effects and investigated the basic mechanism by which metformin blocks pathologic signals. Colitic mice given metformin exhibited less colonic inflammation and increased expression of active AMP-activated protein kinase, a mediator of the metabolic effects of metformin, in both epithelial and lamina propria compartments. Pharmacological inhibition of AMP-activated protein kinase reduced but did not prevent metformin-induced therapeutic effect as well as treatment of colitic mice with a pharmacological activator of AMP-activated protein kinase attenuated but did not resolve colitis. These data suggest that the anti-inflammatory effect of metformin relies on the control of additional pathways other than AMP-activated protein kinase. Indeed, metformin down-regulated p38 MAP kinase activation in colitic mice through an AMP-activated protein kinase-independent mechanism. Expression of active form of AMP-activated protein kinase was reduced in inflammatory bowel disease patients and treatment of mucosal cells of such patients with metformin enhanced AMP-activated protein kinase activation and reduced p38 MAP kinase activation, thereby inhibiting interleukin-6 expression. Our findings indicate that metformin is a good candidate for inhibiting pathological inflammation in the gut.

Aryl hydrocarbon receptor-driven signals inhibit collagen synthesis in the gut.

Fibrostrictures (FS) are a major complication of Crohn's disease (CD). Pathogenesis of FS is not fully understood, but activation of fibroblasts and excessive collagen deposition are crucial in the development of FS. Here, we investigated the role of aryl hydrocarbon receptor (AhR) in intestinal fibrosis. AhR RNA and protein expression were evaluated in intestinal fibroblasts of CD patients and controls. CD fibroblasts were stimulated with TGF-ß1 or TNF-α in the presence or absence of the AhR activator Ficz, an AhR antagonist CH223191, or a specific AhR-silencing RNA. In CD fibroblasts, TGF-ß1 and TNF-α increased Col1A1, Col3A1 and α-SMA transcripts and collagen secretion and this effect was reduced by Ficz and upregulated by CH22319. TGF-ß1 or TNF-α induced activation of p38 and ERK1/2 MAP kinases was decreased by Ficz and increased by CH223191. The inhibitory effect of Ficz on Map kinase activation and collagen induction was abolished by AhR silencing. To assess the role of AhR in vivo, mice with trinitrobenzene-sulfonic-acid induced colonic fibrosis were given Ficz or CH223191. Mice given either Ficz or CH223191 produced less or more collagen respectively as compared with control mice. Our results indicate that AhR is a negative regulator of profibrotic signals in the gut.

Plasma cells in the mucosa of patients with inflammatory bowel disease produce granzyme B and possess cytotoxic activities.

In both Crohn's disease (CD) and ulcerative colitis (UC), the gut is massively infiltrated with B cells and plasma cells, but the role of these cell types in the pathogenesis of gut tissue damage remains largely unknown. Human B cells express granzyme B (GrB) when cultured with IL-21, a cytokine overproduced in CD and UC mucosa. We therefore examined whether mucosal B cells express GrB and have cytotoxic activity in inflammatory bowel disease (IBD). GrB-expressing CD19(+) and IgA(+) cells were seen in the normal intestinal mucosa, but they were significantly more frequent in both CD and UC. In contrast, only a minority of CD19(+) and IgA(+) cells expressed perforin with no difference between IBD and controls. GrB-producing CD19(+) cells expressed CD27 and were CD38(high) and CD20 negative. CD19(+) B cells from IBD patients induced HCT-116 cell death. IL-21 enhanced GrB expression in control CD19(+) B cells and increased their cytotoxic activity. These data indicate that IBD-related inflammation is marked by mucosal accumulation of cytotoxic, GrB-expressing CD19(+) and IgA(+) cells, suggesting a role for these cells in IBD-associated epithelial damage.

Interleukin-34 sustains inflammatory pathways in the gut.

IBD (inflammatory bowel disease)-related tissue damage occurs in areas which are massively infiltrated with monocytes/macrophages. These cells respond to inflammatory stimuli with enhanced production of cytokines/chemokines. In the present study, we analysed the expression and role of IL (interleukin)-34, a regulator of monocyte/macrophage differentiation, survival and function, in IBD. A significant increase in IL-34 mRNA and protein expression was seen in inflamed mucosa of patients with CD (Crohn's disease) and patients with UC (ulcerative colitis) compared with the uninvolved areas of the same patients and normal controls. IL-34 was up-regulated in LPMCs (lamina propria mononuclear cells) isolated from normal colon by TNF-α (tumour necrosis factor α) and TLR (Toll-like receptor) ligands and was down-regulated in intestinal biopsies and LPMCs of IBD patients upon treatment with infliximab. Treatment of normal LPMCs with IL-34 increased TNF-α expression in an ERK1/2 (extracellular-signal-regulated kinase 1/2)-dependent fashion and neutralization of IL-34 in IBD mucosal explants reduced TNF-α and IL-6 synthesis. In conclusion, our results indicate that IL-34 is up-regulated in IBD and suggest a role for this cytokine in sustaining the inflammatory responses in this disease.

IL-25 prevents and cures fulminant hepatitis in mice through a myeloid-derived suppressor cell-dependent mechanism.

UNLABELLED: Fulminant hepatitis (FH) is a disease characterized by massive destruction of hepatocytes with severe impairment of liver function. The pathogenesis of FH is not fully understood, but hyperactivity of T cells and macrophages with excessive production of cytokines are important hallmarks of the condition. In this study, we investigated the role of interleukin (IL)-25 in FH. IL-25 expression was evaluated in patients with FH and in livers of mice with FH induced by D-galactosamine (D-Gal) and lipopolysaccharide (LPS). Mice were treated with IL-25 before D-Gal/LPS-induced FH and before or after concanavalin A (ConA)-induced FH. Mononuclear cells were isolated from livers of mice treated with or without IL-25 and analyzed for GR1(+) CD11b(+) cells. CFSE-labeled T cells were cocultured with GR1(+) CD11b(+) cells and their proliferation was evaluated by flow cytometry. Mice were also treated with a depleting anti-GR1 antibody before IL-25 and D-Gal/LPS administration. IL-25 was constitutively expressed in mouse and human liver and down-regulated during FH. IL-25 prevented D-Gal/LPS-induced FH and this effect was associated with increased infiltration of the liver with cells coexpressing GR1 and CD11b. In vitro studies showed that GR1(+) CD11b(+) cells isolated from mice given IL-25 inhibited T-cell proliferation. Consistently, in vivo depletion of GR1(+) cells abrogated the protective effect of IL-25 in experimental D-Gal/LPS-induced FH. IL-25 was both preventive and therapeutic in ConA-induced FH. CONCLUSIONS: IL-25 expression is markedly reduced during human and experimental FH. IL-25 promotes liver accumulation of GR1(+) CD11b(+) cells with immunoregulatory properties.

Bromodomain-containing 4 is a positive regulator of the inflammatory cytokine response in the gut.

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 analyzed 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 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 signaling 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 to 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.

Targeting hepcidin in colorectal cancer triggers a TNF-dependent-gasdermin E-driven immunogenic cell death response.

Interactions between colorectal cancer (CRC) cells and the noncancerous cells in the tumor microenvironment (TME) induce mechanisms for the escape of tumor cells from immune attack. Hepcidin, a peptide that controls immune cell functions, is overproduced by CRC cells. This study aimed to evaluate whether hepcidin acts as a regulator of anti-tumor immunity in CRC. Hepcidin silencing in CRC cells was followed by enhanced TNF-driven caspase-dependent cleavage of GSDM E and death. Mice engrafted with hepcidin-deficient CT26 cells developed fewer and smaller tumors than control mice as a result of the action of tumor-infiltrating CD8+ T lymphocytes and were protected from the development of tumors in a vaccination model and exhibited long-lasting tumor protection. Additionally, hepcidin deficiency enhanced the response of mice bearing CT26-derived tumors to anti-PD-1 therapy. These results suggest that targeting hepcidin in CRC cells enhances the production of TNF thereby triggering a caspase/GSDM E-driven lytic cell death with the downstream effect of boosting a robust immune response against tumor antigens.

Tissue inhibitor of metalloproteinase-3 regulates inflammation in human and mouse intestine.

BACKGROUND & AIMS: Tissue inhibitor of metalloproteinases (TIMP)-3 is an inhibitor of matrix metalloproteinases, which regulates tissue inflammation, damage, and repair. We investigated the role of TIMP-3 in intestinal inflammation in human beings and mice. METHODS: We used real-time polymerase chain reaction and flow cytometry to measure levels of TIMP-3 in intestine samples from patients with Crohn's disease (CD) and those without (controls). We also analyzed TIMP-3 levels in lamina propria mononuclear cells (LPMCs) collected from biopsy samples of individuals with or without CD (controls) and then stimulated with transforming growth factor (TGF)-ß1, as well as in biopsy samples collected from patients with CD and then incubated with a Smad7 anti-sense oligonucleotide (knock down). LPMCs and biopsy samples from patients with CD were cultured with exogenous TIMP-3 and levels of inflammatory cytokines were measured. We evaluated the susceptibility of wild-type, TIMP-3-knockout (TIMP-3-KO), and transgenic (TIMP-3-Tg) mice to induction of colitis with 2, 4, 6-trinitrobenzene-sulfonic-acid (TNBS), and the course of colitis in recombinase-activating gene-1-null mice after transfer of wild-type or TIMP-3-KO T cells. RESULTS: Levels of TIMP-3 were reduced in intestine samples from patients with CD compared with controls. Incubation of control LPMCs with TGF-ß1 up-regulated TIMP-3; knockdown of Smad7, an inhibitor of TGF-ß1, in biopsy samples from patients with CD increased levels of TIMP-3. Exogenous TIMP-3 reduced levels of inflammatory cytokines in CD LPMCs and biopsy samples. TIMP-3-KO mice developed severe colitis after administration of TNBS, whereas TIMP-3-Tg mice were resistant to TNBS-induced colitis. Reconstitution of recombinase-activating gene-1-null mice with T cells from TIMP-3-KO mice increased the severity of colitis, compared with reconstitution with wild-type T cells. CONCLUSIONS: TIMP-3 is down-regulated in inflamed intestine of patients with CD. Its expression is regulated by TGF-ß1, and knock-down of Smad7 in intestinal tissues from patient with CD up-regulates TIMP-3. Loss or reduction of TIMP-3 in mice promotes development of colitis.

Targeted Therapy of Interleukin-34 as a Promising Approach to Overcome Cancer Therapy Resistance.

Chemotherapy and immunotherapy have markedly improved the management of several malignancies. However, not all cancer patients respond primarily to such therapies, and others can become resistant during treatment. Thus, identification of the factors/mechanisms underlying cancer resistance to such treatments could help develop novel effective therapeutic compounds. Tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) are major components of the suppressive tumor microenvironment and are critical drivers of immunosuppression, creating a tumor-promoting and drug-resistant niche. In this regard, therapeutic strategies to tackle immunosuppressive cells are an interesting option to increase anti-tumor immune responses and overcome the occurrence of drug resistance. Accumulating evidence indicates that interleukin-34 (IL-34), a cytokine produced by cancer cells, and/or TAMs act as a linker between induction of a tumor-associated immunosuppressive microenvironment and drug resistance. In this article, we review the current data supporting the role of IL-34 in the differentiation/function of immune suppressive cells and, hence, in the mechanisms leading to therapeutic resistance in various cancers.

An essential role of adenosine deaminase acting on RNA 1 in coeliac disease mucosa.

Background and aim: Type I interferons (IFNs) are highly expressed in the gut mucosa of celiac disease (CD) gut mucosa and stimulates immune response prompted by gluten ingestion, but the processes that maintain the production of these inflammatory molecules are not well understood. Adenosine deaminase acting on RNA 1 (ADAR1), an RNA-editing enzyme, plays a crucial role in inhibiting self or viral RNAs from activating auto-immune mediated responses, most notably within the type-I IFN production pathway. The aim of this study was to assess whether ADAR1 could contribute to the induction and/or progression of gut inflammation in patients with celiac disease. Material and methods: ADAR1 expression was assessed by Real time PCR and Western blotting in duodenal biopsy taken from inactive and active celiac disease (CD) patients and normal controls (CTR). To analyze the role of ADAR1 in inflamed CD mucosa, lamina propria mononuclear cells (LPMC) were isolated from inactive CD and ADAR1 was silenced in with a specific antisense oligonucleotide (AS) and then incubated with a synthetic analogue of viral dsRNA (poly I:C). IFN-inducing pathways (IRF3, IRF7) in these cells were evaluated with Western blotting and inflammatory cytokines were evaluated with flow cytometry. Lastly, the role of ADAR1 was investigated in a mouse model of poly I:C-driven small intestine atrophy. Results: Reduced ADAR1 expression was seen in duodenal biopsies compared to inactive CD and normal controls. Ex vivo organ cultures of duodenal mucosal biopsies, taken from inactive CD patients, stimulated with a peptic-tryptic digest of gliadin displayed a decreased expression of ADAR1. ADAR1 silencing in LPMC stimulated with a synthetic analogue of viral dsRNA strongly boosted the activation of IRF3 and IRF7 and the production of type-I IFN, TNF-α and IFN-γ. Administration of ADAR1 antisense but not sense oligonucleotide to mice with poly I:C-induced intestinal atrophy, significantly increased gut damage and inflammatory cytokines production. Conclusions: These data show that ADAR1 is an important regulator of intestinal immune homeostasis and demonstrate that defective ADAR1 expression could provide to amplifying pathogenic responses in CD intestinal mucosa.

Corrigendum: An essential role of adenosine deaminase acting on RNA 1 in coeliac disease mucosa.

[This corrects the article DOI: 10.3389/fimmu.2023.1175348.].

2-Methoxy-5-amino-N-hydroxybenzamide, a derivative of mesalamine, inhibits colon cancer cell growth through cyclo-oxygenase-2-dependent and -independent mechanisms.

COX-2 (cyclo-oxygenase-2) and PGE2 (prostaglandin E2) play a key role in sustaining CRC (colorectal cancer) cell growth and survival. Indeed, the use of agents targeting the COX-2/PGE2 axis has been associated with a reduction in the development of CRC in both humans and murine models of colon carcinogenesis. In the present study, we investigated whether 2-methoxy-5-amino-N-hydroxybenzamide (herein termed 2-14), a derivative of mesalamine that inhibits CRC cell growth both in vitro and in vivo, negatively regulates COX-2/PGE2 expression in CRC cells and assessed whether the 2-14-mediated anti-neoplastic effect is strictly dependent on the inhibition of this pathway. Our results show that 2-14 blocks the growth and enhances the death of HT-115, a CRC cell line overexpressing COX-2, and that these effects associate with inhibition of COX-2 but not COX-1. 2-14 also down-regulates TNFα (tumour necrosis factor α)-induced COX-2 in HT-29 cells as well as COX-2/PGE2 expression in ex vivo cultures of human CRC explants. Similarly, 2-14 reduces COX-2, but not COX-1, in tumoural areas developing in a mouse model of CAC (colitis-associated colon cancer). Finally, we show that 2-14 exhibits in vitro and in vivo anti-mitogenic effects in DLD-1, a COX-deficient CRC cell line. Taken together, these results suggest that 2-14 inhibits CRC cell growth through COX-2-dependent and -independent mechanisms.

Characterization of IL-17A-producing cells in celiac disease mucosa.

Celiac disease (CD) is a gluten-sensitive enteropathy associated with a marked infiltration of the mucosa with IFN-gamma-secreting Th1 cells. Recent studies have shown that a novel subset of T cells characterized by expression of high levels of IL-17A, termed Th17 cells, may be responsible for pathogenic effects previously attributed to Th1 cells. In this study, we characterized the expression of IL-17A-producing cells in CD. By real-time PCR and ELISA, it was shown that expression of IL-17A RNA and protein is more pronounced in active CD biopsy specimens in comparison with inactive CD and normal mucosal biopsy specimens. Flow cytometry confirmed that IL-17A is overproduced in CD mucosa and that CD4(+) and CD4(+)CD8(+) cells were major sources. The majority of IL-17A-producing CD4(+) and CD4(+)CD8(+) cells coexpressed IFN-gamma but not CD161. The addition of a peptic-tryptic digest of gliadin to ex vivo organ cultures of duodenal biopsy specimens taken from inactive CD patients enhanced IL-17A production by both CD4(+) and CD4(+)CD8(+) cells. Because we previously showed that IL-21, a T cell-derived cytokine involved in the control of Th17 cell responses, is overproduced in CD, we next assessed whether IL-17A expression is regulated by IL-21. Blockade of IL-21 activity by a neutralizing IL-21 Ab reduced IL-17A expression in cultures of active CD and peptic-tryptic digest of gliadin-treated CD biopsy specimens. In conclusion, our data show that IL-17A is increased in CD and is produced by cells that also make IFN-gamma.

Interleukin-34 Mediates Cross-Talk Between Stromal Cells and Immune Cells in the Gut.

Initially known as a cytokine produced by and regulating the function of monocytes and macrophages, interleukin-34 (IL-34) can be synthesized by many cell types and interacts with receptors expressed by multiple immune and non-immune cells. IL-34 is constitutively expressed in the healthy human small intestine and colon and its production is markedly increased in damaged gut of patients with Crohn's disease and patients with ulcerative colitis, the main forms of chronic inflammatory bowel diseases (IBD) in human beings. Circumstantial evidence suggests that, in these pathologies, IL-34 plays a crucial role in mediating cross-talk between immune cells and stromal cells, thereby promoting activation of signalling pathways, which amplify the ongoing mucosal inflammation as well as production of fibrogenic molecules. In this article, we summarize the available data supporting the multiple effects of IL-34 in human IBD with particular attention to the role of the cytokine in immune and stromal cell interactions.

Hepcidin Upregulation in Colorectal Cancer Associates with Accumulation of Regulatory Macrophages and Epithelial-Mesenchymal Transition and Correlates with Progression of the Disease.

Advanced, metastatic colorectal cancer (CRC) is associated with high rate of mortality because of its poor responsiveness to chemotherapy/immunotherapy. Recent studies have shown that hepcidin, a peptide hormone produced mainly by hepatocytes, is expressed by and enhances the growth of tumor cells. We here assessed whether hepcidin expression helps identify subsets of CRC with advanced and aggressive course. By integrating results of in vitro/ex vivo studies with data of bioinformatics databases, we initially showed that hepcidin RNA and protein expression was more pronounced in tissue samples taken from the tumor area, as compared to the macroscopically unaffected, adjacent, colonic mucosa of CRC patients. The induction of hepcidin in the colonic epithelial cell line HCEC-1ct by interleukin (IL)-6, IL-21 and IL-23 occurred via a Stat3-dependent mechanism and, in primary CRC cells, hepcidin co-localized with active Stat3. In CRC tissue, hepcidin content correlated mainly with macrophage accumulation and IL-10 and CD206 expression, two markers of regulatory macrophages. Consistently, both IL-10 and CD206 were up-regulated by hepcidin in blood mononuclear cells. The highest levels of hepcidin were found in metastatic CRC and survival analysis showed that high expression of hepcidin associated with poor prognosis. Moreover, hepcidin expression correlated with markers of epithelial-to-mesenchymal transition and the silencing of hepcidin in CRC cells reduced epithelial-to-mesenchymal transition markers. These findings indicate that hepcidin is markedly induced in the advanced stages of CRC and suggest that it could serve as a prognostic biomarker in CRC.

Rafoxanide sensitizes colorectal cancer cells to TRAIL-mediated apoptosis.

Colorectal cancer (CRC) remains a leading causes of cancer-related death in the world, mainly due to the lack of effective treatment of advanced disease. TNF-related apoptosis-inducing ligand (TRAIL)-driven cell death, a crucial event in the control of tumor growth, selectively targets malignant rather than non-transformed cells. However, the fact that cancer cells, including CRC cells, are either intrinsically resistant or acquire resistance to TRAIL, represents a major hurdle to the use of TRAIL-based strategies in the clinic. Agents able to overcome CRC cell resistance to TRAIL have thus great therapeutic potential and many researchers are making efforts to identify TRAIL sensitizers. The anthelmintic drug rafoxanide has recently emerged as a potent anti-tumor molecule for different cancer types and we recently reported that rafoxanide restrained the proliferation of CRC cells, but not of normal colonic epithelial cells, both in vitro and in a preclinical model mimicking sporadic CRC. As these findings were linked with the induction of endoplasmic reticulum stress, a phenomenon involved in the regulation of various components of the TRAIL-driven apoptotic pathway, we sought to determine whether rafoxanide could restore the sensitivity of CRC cells to TRAIL. Our data show that rafoxanide acts as a selective TRAIL sensitizer in vitro and in a syngeneic experimental model of CRC, by decreasing the levels of c-FLIP and survivin, two key molecules conferring TRAIL resistance. Collectively, our data suggest that rafoxanide could potentially be deployed as an anti-cancer drug in the combinatorial approaches aimed at overcoming CRC cell resistance to TRAIL-based therapies.

GATA6 Deficiency Leads to Epithelial Barrier Dysfunction and Enhances Susceptibility to Gut Inflammation.

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.

The Deubiquitinating Enzyme OTUD5 Sustains Inflammatory Cytokine Response in Inflammatory Bowel Disease.

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.

Interleukin-25 fails to activate STAT6 and induce alternatively activated macrophages.

Interleukin-25 (IL-25), a T helper type 2 (Th2) -related factor, inhibits the production of inflammatory cytokines by monocytes/macrophages. Since Th2 cytokines antagonize classically activated monocytes/macrophages by inducing alternatively activated macrophages (AAMs), we here assessed the effect of IL-25 on the alternative activation of human monocytes/macrophages. The interleukins IL-25, IL-4 and IL-13 were effective in reducing the expression of inflammatory chemokines in monocytes. This effect was paralleled by induction of AAMs in cultures added with IL-4 or IL-13 but not with IL-25, regardless of whether cells were stimulated with lipopolysaccharide or interferon-γ. Moreover, pre-incubation of cells with IL-25 did not alter the ability of both IL-4 and IL-13 to induce AAMs. Both IL-4 and IL-13 activated signal transducer and activator of transcription 6 (STAT6), and silencing of this transcription factor markedly reduced the IL-4/IL-13-driven induction of AAMs. In contrast, IL-25 failed to trigger STAT6 activation. Among Th2 cytokines, only IL-25 and IL-10 were able to activate p38 mitogen-activated protein kinase. These results collectively indicate that IL-25 fails to induce AAMs and that Th2-type cytokines suppress inflammatory responses in human monocytes by activating different intracellular signalling pathways.