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Chronic inflammation drives colitis-associated colorectal cancer (CAC) in inflammatory bowel disease (IBD). FoxP3+ regulatory T cells (Treg) coexpressing the Th17-related transcription factor RORγt accumulate in the lamina propria of IBD patients, where they are thought to represent an intermediate stage of development toward a Th17 proinflammatory phenotype. However, the role of these cells in CAC is unknown. RORγt+FoxP3+ cells were investigated in human samples of CAC, and their phenotypic stability and function were investigated in an azoxymethane/dextran sulfate sodium model of CAC using Treg fate-mapping reporter and Treg-specific RORγt conditional knockout mice. Tumor development and the intratumoral inflammatory milieu were characterized in these mice. The functional role of CTLA-4 expressed by Tregs and FoxO3 in dendritic cells (DC) was studied in vitro and in vivo by siRNA-silencing experiments. RORγt expression identified a phenotypically stable population of tumor-infiltrating Tregs in humans and mice. Conditional RORγt knockout mice showed reduced tumor incidence, and dysplastic cells exhibited low Ki67 expression and STAT3 activation. Tumor-infiltrating DCs produced less IL6, a cytokine that triggers STAT3-dependent proliferative signals in neoplastic cells. RORγt-deficient Tregs isolated from tumors overexpressed CTLA-4 and induced DCs to have elevated expression of the transcription factor FoxO3, thus reducing IL6 expression. Finally, in vivo silencing of FoxO3 obtained by siRNA microinjection in the tumors of RORγt-deficient mice restored IL6 expression and tumor growth. These data demonstrate that RORγt expressed by tumor-infiltrating Tregs sustains tumor growth by leaving IL6 expression in DCs unchecked. Cancer Immunol Res; 6(9); 1082–92. ©2018 AACR.

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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.

Background and AimThe mechanisms underlying the formation of intestinal fibrostrictures [FS] in Crohn’s disease [CD] are not fully understood, but activation of fibroblasts and excessive collagen deposition are supposed to contribute to the development of FS. Here we investigated whether interleukin-34 [IL-34], a cytokine that is over-produced in CD, regulates collagen production by gut fibroblastsMethodsIL-34 and its receptor macrophage colony-stimulating factor receptor 1 [M-CSFR-1] were evaluated in inflammatory [I], FS CD, and control [CTR] ileal mucosal samples by real-time polymerase chain reaction [RT-PCR], western blotting, and immunohistochemistry. IL-34 and M-CSFR-1 expression was evaluated in normal and FS CD fibroblasts. Control fibroblasts were stimulated with IL-34 in the presence or absence of a MAP kinase p38 inhibitor, and FS CD fibroblasts were cultured with a specific IL-34 antisense oligonucleotide, and collagen production was evaluated by RT-PCR, western blotting, and Sircol assay. The effect of IL-34 on the wound healing capacity of fibroblasts was evaluated by scratch test.ResultsWe showed enhanced M-CSFR-1 and IL-34 RNA and protein expression in FS CD mucosal samples as compared with ICD and CTR samples. Immunohistochemical analysis showed that stromal cells were positive for M-CSFR-1 and IL-34. Enhanced M-CSFR-1 and IL-34 RNA and protein expression was seen in FS CD fibroblasts as compared with CTR. Stimulation of control fibroblasts with IL-34 enhanced COL1A1 and COL3A1 expression and secretion of collagen through a p38 MAP kinase-dependent mechanism, and wound healing. IL-34 knockdown in FS CD fibroblasts was associated with reduced collagen production and wound repair.ConclusionsData indicate a prominent role of IL-34 in the control of intestinal fibrogenesis.

Background and AimsIntestinal 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.MethodsTranscriptome 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.ResultsMultiple 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.ConclusionsReduced expression of GATA6 promotes intestinal barrier dysfunction, thus amplifying intestinal inflammatory pathology.

Background and AimsThe 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.MethodsOTUD5 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].ResultsOTUD5 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.ConclusionsOur 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.

Colorectal cancer (CRC) is a major cause of cancer-related death in the world. Emerging evidence suggests that the clinical success of conventional chemotherapy does not merely rely on cell toxicity, but also results from the restoration of tumor immune surveillance. Anti-tumor immune response can be primed by immunogenic cell death (ICD), a form of apoptosis associated with endoplasmic reticulum stress (ERS) induction and the expression/release of specific damage-associated molecular patterns (DAMPs). Unfortunately, a limited number of ICD inducers have been identified so far. The anti-helmintic drug rafoxanide has recently showed anti-tumor activity in different cancer types, including CRC. As such latter effects relied on ERS activation, we here investigated whether rafoxanide could promote ICD of CRC cells. The potential of rafoxanide to induce ICD-related DAMPs in both human and mouse CRC cells was assessed by flow-cytometry, chemiluminescent assay and ELISA. In addition, the immunogenic potential of rafoxanide was assessed in vivo using a vaccination assay. Rafoxanide induced all the main DAMPs (ecto-calreticulin exposure, adenosine triphosphate (ATP)/high mobility group box 1 (HMGB1) release) required for ICD. We observed a marked increase of tumor-free survival among immunocompetent mice immunized with rafoxanide-treated dying tumor cells as compared with sham. Altogether, our data indicate rafoxanide as a bona fide ICD inducer.

Background & Aims: Food additives, such as emulsifiers, stabilizers, or bulking agents, are present in the Western diet and their consumption is increasing. However, little is known about their potential effects on intestinal homeostasis. In this study we examined the effect of some of these food additives on gut inflammation. Methods: Mice were given drinking water containing maltodextrin (MDX), propylene glycol, or animal gelatin, and then challenged with dextran sulfate sodium or indomethacin. In parallel, mice fed a MDX-enriched diet were given the endoplasmic reticulum (ER) stress inhibitor tauroursodeoxycholic acid (TUDCA). Transcriptomic analysis, real-time polymerase chain reaction, mucin-2 expression, phosphorylated p38 mitogen-activated protein (MAP) kinase quantification, and H&E staining was performed on colonic tissues. Mucosa-associated microbiota composition was characterized by 16S ribosomal RNA sequencing. For the in vitro experiments, murine intestinal crypts and the human mucus-secreting HT29-methotrexate treated cell line were stimulated with MDX in the presence or absence of TUDCA or a p38 MAP kinase inhibitor. Results: Diets enriched in MDX, but not propylene glycol or animal gelatin, exacerbated intestinal inflammation in both models. Analysis of the mechanisms underlying the detrimental effect of MDX showed up-regulation of inositol requiring protein 1β, a sensor of ER stress, in goblet cells, and a reduction of mucin-2 expression with no significant change in mucosa-associated microbiota. Stimulation of murine intestinal crypts and HT29-methotrexate treated cell line cells with MDX induced inositol requiring protein 1β via a p38 MAP kinase–dependent mechanism. Treatment of mice with TUDCA prevented mucin-2 depletion and attenuated colitis in MDX-fed mice. Conclusions: MDX increases ER stress in gut epithelial cells with the downstream effect of reducing mucus production and enhancing colitis susceptibility. Keywords: Colitis, IBD, Unfolded Protein Response, Intestinal Epithelium

Background and AimsAlthough the mechanisms underlying the formation of intestinal fibrostrictures in Crohn’s disease [CD] are not fully understood, activation of fibroblasts and excessive collagen deposition are supposed to contribute to the development of such complications. Here, we investigated the role of cadherin-11 [CDH-11], a fibroblast-derived protein that induces collagen production in various organs, in intestinal fibrosis.MethodsCDH-11 expression was evaluated in inflammatory [I] and fibrostricturing [FS] CD mucosal samples, ulcerative colitis [UC] mucosal samples, and ileal and colonic control samples, by real-time polymerase chain reaction, western blotting, and immunohistochemistry. CDH-11 expression was evaluated in normal and in CD intestinal fibroblasts stimulated with inflammatory/fibrogenic cytokines. FS CD fibroblasts were cultured either with a specific CDH-11 antisense oligonucleotide [AS], or activating CDH-11 fusion protein and activation of RhoA/ROCK, and TGF-β pathways and collagen production were evaluated by western blotting. Finally, we assessed the susceptibility of CDH-11-knockout [KO] mice to colitis-induced intestinal fibrosis.ResultsCDH-11 RNA and protein expression were increased in both CD and UC as compared with controls. In CD, the greater expression of CDH-11 was seen in FS samples. Stimulation of fibroblasts with TNF-α, interleukin [IL]-6, IFN-γ, IL-13, and IL-1β enhanced CDH-11 expression. Knockdown of CDH-11 in FS CD fibroblasts impaired RhoA/ROCK/TGF-β signalling and reduced collagen synthesis, whereas activation of CDH-11 increased collagen secretion. CDH-11 KO mice were largely protected from intestinal fibrosis.ConclusionsData show that CDH-11 expression is up-regulated in inflammatory bowel disease [IBD] and suggest a role for this protein in the control of intestinal fibrosis.

Chronic inflammation drives colitis-associated colorectal cancer (CAC) in inflammatory bowel disease (IBD). FoxP3+ regulatory T cells (Treg) coexpressing the Th17-related transcription factor RORγt accumulate in the lamina propria of IBD patients, where they are thought to represent an intermediate stage of development toward a Th17 proinflammatory phenotype. However, the role of these cells in CAC is unknown. RORγt+FoxP3+ cells were investigated in human samples of CAC, and their phenotypic stability and function were investigated in an azoxymethane/dextran sulfate sodium model of CAC using Treg fate-mapping reporter and Treg-specific RORγt conditional knockout mice. Tumor development and the intratumoral inflammatory milieu were characterized in these mice. The functional role of CTLA-4 expressed by Tregs and FoxO3 in dendritic cells (DC) was studied in vitro and in vivo by siRNA-silencing experiments. RORγt expression identified a phenotypically stable population of tumor-infiltrating Tregs in humans and mice. Conditional RORγt knockout mice showed reduced tumor incidence, and dysplastic cells exhibited low Ki67 expression and STAT3 activation. Tumor-infiltrating DCs produced less IL6, a cytokine that triggers STAT3-dependent proliferative signals in neoplastic cells. RORγt-deficient Tregs isolated from tumors overexpressed CTLA-4 and induced DCs to have elevated expression of the transcription factor FoxO3, thus reducing IL6 expression. Finally, in vivo silencing of FoxO3 obtained by siRNA microinjection in the tumors of RORγt-deficient mice restored IL6 expression and tumor growth. These data demonstrate that RORγt expressed by tumor-infiltrating Tregs sustains tumor growth by leaving IL6 expression in DCs unchecked. Cancer Immunol Res; 6(9); 1082–92. ©2018 AACR.

Follistatin-like protein 1 (FSTL1) is a secreted glycoprotein, which controls several physiological and pathological events. FSTL1 expression is deregulated in many tumors, but its contribution to colon carcinogenesis is not fully understood. Here, we investigated the expression and functional role of FSTL1 in colorectal cancer (CRC). A significant increase of FSTL1 was seen in human CRC as compared to the surrounding non-tumor tissues and this occurred at both RNA and protein level. Knockdown of FSTL1 in CRC cells with a specific antisense oligonucleotide (AS) reduced expression of regulators of the late G1 phase, such as phosphorylated retinoblastoma protein, E2F-1, cyclin E and phospho-cyclin-dependent kinase-2, and promoted accumulation of cells in the G1 phase of the cell cycle thus resulting in diminished cell proliferation. Consistently, recombinant FSTL1 induced proliferation of normal intestinal epithelial cells through an ERK1/2-dependent mechanism. Cell cycle arrest driven by FSTL1 AS in CRC cells was accompanied by activation of caspases and subsequent induction of apoptosis. Moreover, FSTL1 knockdown made CRC cells more susceptible to oxaliplatin and irinotecan-induced death. Data indicate that FSTL1 is over-expressed in human CRC and suggest a role for this protein in favouring intestinal tumorigenesis.

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.

Background In Crohn's disease (CD), the pathogenic immune response is associated with high Smad7, an inhibitor of TGF-β1 signaling. Smad7 knockdown with Mongersen, a specific antisense oligonucleotide-containing compound, restores TGF-β1 activity leading to inhibition of inflammatory signals and associates with clinical benefit in CD patients. As TGF-β1 is pro-fibrogenic, it remains unclear whether Mongersen-induced Smad7 inhibition increases the risk of intestinal fibrosis. We assessed the impact of Smad7 inhibition on the course of colitis-driven intestinal fibrosis in mice. Methods BALB/c mice were rectally treated with increasing doses of trinitrobenzene sulfonic acid (TNBS) for 8 or 12 weeks. The effect of oral Smad7 antisense or control oligonucleotide, administered to mice starting from week 5 or week 8, respectively, on mucosal inflammation and colitis-associated colonic fibrosis was assessed. Mucosal samples were analyzed for Smad7 by immunoblotting and immunohistochemistry, TGF-β1 by enzyme-linked immunosorbent assay, and collagen by immunohistochemistry. Results TNBS-induced chronic colitis was associated with colonic deposition of collagen I and fibrosis, which were evident at week 8 and became more pronounced at week 12. TNBS treatment enhanced Smad7 in both colonic epithelial and lamina propria mononuclear cells. Colitic mice treated with Smad7 antisense oligonucleotide exhibited reduced signs of colitis, less collagen deposition, and diminished fibrosis. These findings were associated with diminished synthesis of TGF-β1 and reduced p-Smad3 protein expression. Conclusion Attenuation of colitis with Smad7 antisense oligonucleotide limits development of colonic fibrosis.

Colorectal cancer (CRC) remains one of the leading causes of mortality worldwide. Drug repositioning is a promising approach for new cancer therapies, as it provides the opportunity to rapidly advance potentially promising agents into clinical trials. The FDA-approved anti-helminthic drug rafoxanide was recently reported to antagonize the oncogenic function of the BRAF V600E mutant protein, commonly found in CRCs, as well as to inhibit the proliferation of skin cancer cells. These observations prompted us to investigate the potential anti-cancer effects of rafoxanide in CRC models. We found rafoxanide inhibited proliferation in CRC cells, but not in normal colonic epithelial cells. Rafoxanide's anti-proliferative action was associated with marked reduction in cyclin D1 protein levels and accumulation of cells in the G0/G1 phase. These effects relied on selective induction of the endoplasmic reticulum stress (ERS) response in CRC cells and were followed by caspase-dependent cell death. Systemic administration of rafoxanide to Apcmin/+ mice induced to develop CRCs caused ERS activation, proliferation inhibition and apoptosis induction in the neoplastic cells. Collectively, our data suggest rafoxanide might be repurposed as an anti-cancer drug for the treatment of CRC.

In inflammatory bowel disease (IBD) mucosa, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine transforming growth factor-β1, due to post-transcriptional mechanisms that enhance Smad7 acetylation status thus preventing ubiquitination-mediated proteosomal degradation of the protein. IBD-related inflammation is also marked by defective expression of Sirt1, a class III NAD+-dependent deacetylase, which promotes ubiquitination-mediated proteosomal degradation of various intracellular proteins and triggers anti-inflammatory signals. The aim of our study was to determine whether, in IBD, there is a reciprocal regulation between Smad7 and Sirt1. Smad7 and Sirt1 were examined in mucosal samples of IBD patients and normal controls by Western blotting and immunohistochemistry, and Sirt1 activity was assessed by a fluorimetric assay. To determine whether Smad7 is regulated by Sirt1, normal or IBD lamina propria mononuclear cells (LPMC) were cultured with either Sirt1 inhibitor (Ex527) or activator (Cay10591), respectively. To determine whether Smad7 controls Sirt1 expression, ex vivo organ cultures of IBD mucosal explants were treated with Smad7 sense or antisense oligonucleotide. Moreover, Sirt1 expression was evaluated in LPMC isolated from Smad7-transgenic mice given dextran sulfate sodium (DSS). Upregulation of Smad7 was seen in both the epithelial and lamina propria compartments of IBD patients and this associated with reduced expression and activity of Sirt1. Activation of Sirt1 in IBD LPMC with Cay10591 reduced acetylation and enhanced ubiquitination-driven proteasomal-mediated degradation of Smad7, while inhibition of Sirt1 activation in normal LPMC with Ex527 increased Smad7 expression. Knockdown of Smad7 in IBD mucosal explants enhanced Sirt1 expression, thus suggesting a negative effect of Smad7 on Sirt1 induction. Consistently, mucosal T cells of Smad7-transgenic mice contained reduced levels of Sirt1, a defect that was amplified by induction of DSS colitis. The data suggest the existence of a reciprocal regulatory mechanism between Smad7 and Sirt1, which could contribute to amplify inflammatory signals in the gut.

Background Transforming growth factor (TGF)-β1 exerts inhibitory effects on keratinocyte proliferation. Objectives To examine whether Smad7, a known inhibitor of TGF-β1 signalling, is involved in the psoriasis-associated keratinocyte hyper-proliferation. Methods Smad7 was evaluated in skin sections of psoriatic patients and healthy controls and in mice with Aldara-induced skin pathology by real-time PCR and immunohistochemistry. To assess whether Smad7 positively regulates the in vivo keratinocyte growth, mice treated with Aldara received daily cutaneous administration of Smad7 antisense oligonucleotide (AS). Keratin (K) 6 and K16, cell cycle-associated factors, cell cycle and cell proliferation were evaluated in HaCaT cells either treated with Smad7 AS or transfected with Smad7 plasmid and in mice given Smad7 AS. Results Smad7 was highly expressed in keratinocytes of patients with psoriasis and of mice treated with Aldara. In HaCaT cells, Smad7 knockdown inhibited cell growth, reduced K6 and K16 expression and promoted accumulation of cells in S-phase of cell cycle. Smad7-deficient keratinocytes exhibited reduced levels of CDC25A protein, a phosphatase that facilitates progression of cells through S phase, and hyper-phosphorylation of eukaryotic initiation factor 2 (eIF2)α, a negative regulator of CDC25 protein translation. Consistently, Smad7 overexpression in HaCaT was followed by induction of K6 and K16 and increased cell proliferation. Topical application of Smad7 AS to Aldara-treated mice reduced epidermal thickness. Conclusions Our data show that Smad7 is over-expressed in human and murine psoriasis and suggest a key role of this molecule in the control of keratinocyte proliferation. This article is protected by copyright. All rights reserved.

Interleukin-34 (IL-34), a cytokine produced by a wide range of cells, binds to the macrophage colony-stimulating factor receptor (M-CSFR-1) and receptor-type protein-tyrosine phosphatase zeta (PTP-z) and controls myeloid cell differentiation, proliferation and survival. various types of cancers over-express IL-34 but the role of the cytokine in colorectal cancer (CRC) remains unknown. We here investigated the expression and functional role of IL-34 in CRC. A more pronounced expression of IL-34 was seen in CRC samples as compared to matched normal/benign colonic samples and this occurred at both RNA and protein level. Immunohistochemical analysis of CRC tissue samples showed that both cancer cells and lamina propria mononuclear cells over-expressed IL-34. Additionally, CRC cells expressed both M-CSFR-1 and PTP-z, thus suggesting that CRC cells can be responsive to IL-34. Indeed, stimulation of DLD-1 cancer cells with IL-34, but not with MSCF1, enhanced the cell proliferation and cell invasion without affecting cell survival. Analysis of intracellular signals underlying the mitogenic effect of IL-34 revealed that the cytokine enhanced activation of ERK1/2 and pharmacologic inhibition of ERK1/2 abrogated IL-34-driven cell proliferation. Consistently, IL-34 knockdown in HT-29 cells with a specific IL-34 antisense oligonucleotide reduced ERK1/2 activation, cell proliferation and enhanced the susceptibility of cells to Oxaliplatin-induced death. This is the first study showing up-regulation of IL-34 in CRC and suggesting a role for this cytokine in colon tumorigenesis.

Refractory coeliac disease (RCD) is a form of coeliac disease (CD) resistant to gluten‐free diet and associated with elevated risk of complications. Many effector cytokines over‐produced in the gut of patients with RCD are supposed to amplify the tissue‐destructive immune response, but it remains unclear if the RCD‐associated mucosal inflammation is sustained by defects in counter‐regulatory mechanisms. The aim of the present study was to determine whether RCD‐related inflammation is marked by high Smad7, an intracellular inhibitor of transforming growth factor‐β 1 (TGF‐β 1) activity. Smad7 was evaluated in duodenal biopsy samples of patients with RCD, patients with active CD, patients with inactive CD and healthy controls by Western blotting, immunohistochemistry and real‐time PCR. In the same samples, TGF‐β 1 and phosphorylated (p)‐Smad2/3 were evaluated by ELISA and immunohistochemistry, respectively. Pro‐inflammatory cytokine expression was evaluated in RCD samples cultured with Smad7 sense or antisense oligonucleotide. Smad7 protein, but not RNA, expression was increased in RCD compared with active and inactive CD patients and healthy controls and this was associated with defective TGF‐β 1 signalling, as marked by diminished p‐Smad2/3 expression. TGF‐β 1 protein content did not differ among groups. Knockdown of Smad7 in RCD biopsy samples reduced interleukin‐6 and tumour necrosis factor‐α expression. In conclusion, in RCD, high Smad7 associates with defective TGF‐β 1 signalling and sustains inflammatory cytokine production. These results indicate a novel mechanism by which the mucosal cytokine response is amplified in RCD and suggest that targeting Smad7 can be therapeutically useful in RCD.

Upregulation of Smad7, an inhibitor of transforming growth factor-β1 (TGF-β1), occurs in sporadic colorectal cancer (CRC) and knockdown of Smad7 inhibits CRC cell growth, a phenomenon that associates with decreased expression of cell division cycle 25 homolog A and arrest of cells in the S phase of the cell cycle. These findings occur in CRC cells unresponsive to TGF-β1, thus suggesting the existence of a Smad7-mediated TGF-β1-independent mechanism that controls CRC cell behavior. Here we show that Smad7 inhibition with a specific Smad7 antisense oligonucleotide upregulates eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, a transcription factor involved in the regulation of cell cycle arrest and induction of cell death, and induces activating transcription factor 4 (ATF4) and CCAAT/enhancer binding protein homology protein (CHOP), two downstream targets of eIF2α. Among the upstream kinases that control eIF2α phosphorylation, the serine–threonine protein kinase RNA (PKR), but not general control non-derepressible 2 (GCN2) and protein kinase RNA-like endoplasmic reticulum kinase (PERK), is activated by Smad7 knockdown. PKR silencing abolishes Smad7 antisense-induced eIF2α phosphorylation and ATF4/CHOP induction, thereby preventing Smad7 antisense-driven cell death. Smad7 inhibition diminishes interaction of PKR with protein kinase inhibitor p58 (p58IPK), a cellular inhibitor of PKR, but does not change the expression and/or activity of other factors involved in the control of PKR activation. These findings delineate a novel mechanism by which Smad7 knockdown promotes CRC cell death.

Background and Aim: Production of chemokines by intestinal epithelial cells is a key step in the amplification of the destructive immune-inflammatory response in patients with inflammatory bowel diseases [IBD]. In this study, we examined whether intestinal epithelial cells express macrophage colony-stimulating factor receptor 1 [M-CSFR-1], the functional receptor of interleukin-34 [IL-34], a cytokine that is over-produced in IBD and supposed to sustain inflammatory pathways. Methods: M-CSFR-1 expression was evaluated in intestinal samples of IBD patients, controls, and colon epithelial cell lines by real-time polymerase chain reaction [PCR], immunohistochemistry, and western blotting. DLD-1 cells were stimulated with IL-34 in the presence or absence of MAP kinase inhibitors, chemokine induction was assessed by real-time PCR and enzyme-linked immunosorbent assay [ELISA], and mitogen-activated protein (MAP) kinase activation was monitored by western blotting. The effect of a neutralising IL-34 antibody on CC chemokine ligand (CCL) 20 synthesis was tested in ex vivo organ cultures of IBD mucosal explants. Results: Enhanced expression of M-CSFR-1 RNA transcripts was seen in inflamed mucosa of IBD patients as compared with controls. Immunohistochemical analysis confirmed up-regulation of M-CSFR-1 in IBD and showed that both epithelial and lamina propria mononuclear cells expressed this receptor. Stimulation of DLD-1 with IL-34 increased CCL20 production through an ERK1/2-dependent mechanism. Consistently, treatment of IBD explants with anti-IL-34 reduced CCL20 production. Conclusions: These data show that intestinal epithelial cells are a target of IL-34 and suggest that this cytokine contributes to mediating the cross-talk between epithelial cells and immune cells in IBD.

// Veronica De Simone 1 , Giulia Ronchetti 1 , Eleonora Franze 1 , Alfredo Colantoni 1 , Angela Ortenzi 1 , Massimo C. Fantini 1 , Angelamaria Rizzo 1 , Giuseppe S. Sica 2 , Pierpaolo Sileri 2 , Piero Rossi 2 , Thomas T. MacDonald 3 , Francesco Pallone 1 , Giovanni Monteleone 1 and Carmine Stolfi 1 1 Department of Systems Medicine, University of Rome “Tor Vergata”, Rome, Italy 2 Department of Surgery, University of Rome “Tor Vergata”, Rome, Italy 3 Centre for Immunology and Infectious Disease, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK Correspondence to: Carmine Stolfi, email: // Keywords : Apc min/+ mice, STAT3, COX-2/PGE2, VEGF Received : September 22, 2014 Accepted : February 17, 2015 Published : March 12, 2015 Abstract Interleukin (IL)-21 triggers inflammatory signals that contribute to the growth of neoplastic cells in mouse models of colitis-associated colorectal cancer (CRC). Because most CRCs are sporadic and arise in the absence of overt inflammation we have investigated the role of IL-21 in these tumors in mouse and man. IL-21 was highly expressed in human sporadic CRC and produced mostly by IFN-γ-expressing T-bet/RORγt double-positive CD3+CD8- cells. Stimulation of human CRC cell lines with IL-21 did not directly activate the oncogenic transcription factors STAT3 and NF-kB and did not affect CRC cell proliferation and survival. In contrast, IL-21 modulated the production of protumorigenic factors by human tumor infiltrating T cells. IL-21 was upregulated in the neoplastic areas, as compared with non-tumor mucosa, of Apc min/+ mice, and genetic ablation of IL-21 in such mice resulted in a marked decrease of both tumor incidence and size. IL-21 deficiency was associated with reduced STAT3/NF-kB activation in both immune cells and neoplastic cells, diminished synthesis of protumorigenic cytokines (that is, IL-17A, IL-22, TNF-α and IL-6), downregulation of COX-2/PGE2 pathway and decreased angiogenesis in the lesions of Apc min/+ mice. Altogether, data suggest that IL-21 promotes a protumorigenic inflammatory circuit that ultimately sustains the development of sporadic CRC.