Search

PDF file - 996K

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.

TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis is a crucial event in the control of tumor growth. However, many cancer cells, including colon cancer cells, are resistant to TRAIL-driven cell death. We have recently shown that 2-methoxy-5-amino-N-hydroxybenzamide (herein termed 2-14), a novel derivative of mesalamine, induces endoplasmic reticulum stress in colon cancer cells. Because endoplasmic reticulum stress-induced signals regulate the expression of molecules involved in TRAIL-driven apoptosis, we examined whether 2-14 makes colon cancer cells sensitive to TRAIL. Colon cancer cells were cultured with 2-14 and/or TRAIL. Death receptor (DR) 4/DR5 were analyzed by real-time PCR and flow cytometry. TRAIL pathway–associated proteins and extracellular signal–regulated kinase (ERK) were assessed by Western blotting. The in vivo capability of 2-14 to sensitize colon cancer cells to TRAIL-induced apoptosis was evaluated in a syngenic colon cancer model in which CT26-derived grafts were induced in mice. 2-14 promoted ERK-dependent induction of DR5, thereby enhancing TRAIL-mediated caspase-8 activation and apoptosis. Analysis of TRAIL-related pro- and antiapoptotic factors and functional studies revealed that survivin is involved in the protection of colon cancer cells against TRAIL-driven apoptosis. Notably, 2-14 enhanced ubiquitination and proteasome-mediated degradation of survivin. These data were confirmed in a murine model of TRAIL-resistant colon cancer in which 2-14 upregulated DR5, reduced survivin expression, and synergized with TRAIL in inhibiting tumor growth. Similarly, intraperitoneal administration of 2-14 to mice upregulated DR5 and downregulated survivin in a model of colitis-associated colon cancer. These findings indicate that 2-14 acts as a sensitizer for TRAIL-induced apoptosis and suggest that 2-14 can be useful in the therapy for TRAIL-resistant colon cancer. Mol Cancer Ther; 10(10); 1969–81. ©2011 AACR.

Suppl. figure and figure legends

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.

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.

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.

Persistent activation of Signal Transducer and Activator of Transcription (STAT)3 occurs in a high percentage of tumors, including colorectal cancer (CRC), thereby contributing to malignant cell proliferation and survival. Although STAT3 is recognized as an attractive therapeutic target in CRC, conventional approaches aimed at inhibiting its functions have met with several limitations. Moreover, the factors that sustain hyper‐activation of STAT3 in CRC are not yet fully understood. The identification of tumor‐specific STAT3 cofactors may facilitate the development of compounds that interfere exclusively with STAT3 activity in cancer cells. Here, we show that progranulin, a STAT3 cofactor, is upregulated in human CRC as compared to nontumor tissue/cells and its expression correlates with STAT3 activation. Progranulin physically interacts with STAT3 in CRC cells, and its knockdown with a specific antisense oligonucleotide (ASO) inhibits STAT3 activation and restrains the expression of STAT3‐related oncogenic proteins, thus causing cell cycle arrest and apoptosis. Moreover, progranulin knockdown reduces STAT3 phosphorylation and cell proliferation induced by tumor‐infiltrating leukocyte (TIL)‐derived supernatants in CRC cell lines and human CRC explants. These findings indicate that CRC exhibits overexpression of progranulin, and suggest a role for this protein in amplifying the STAT3 pathway in CRC.

Celiac disease (CD) is an enteropathy triggered by the ingestion of gluten proteins in genetically predisposed individuals and characterized by excessive activation of effector immune cells and enhanced production of inflammatory cytokines. However, factors/mechanisms that amplify the ongoing mucosal inflammation in CD are not fully understood. In this study, we assessed whether mammalian target of Rapamycin (mTOR), a pathway that combines intra- and extra-cellular signals and acts as a central regulator for the metabolism, growth, and function of immune and non-immune cells, sustains CD-associated immune response. Our findings indicate that expression of phosphorylated (p)/active form of mTOR is increased in protein lysates of duodenal biopsy samples taken from patients with active CD (ACD) as compared to normal controls. In ACD, activation of mTOR occurs mainly in the epithelial compartment and associates with enhanced expression of p-4EBP, a downstream target of mTOR complex (mTORC)1, while expression of p-Rictor, a component of mTORC2, is not increased. Stimulation of mucosal explants of inactive CD patients with pepsin-trypsin-digested (PT)-gliadin or IFN-γ/IL-21, two cytokines produced in CD by gluten-specific T cells, increases p-4EBP expression. Consistently, blockade of such cytokines in cultures of ACD mucosal explants reduces p-4EBP. Finally, we show that inhibition of mTORC1 with rapamycin in ACD mucosal explants reduces p-4EBP and production of IL-15, a master cytokine produced by epithelial cells in this disorder. Our data suggest that ACD inflammation is marked by activation of mTORC1 in the epithelial compartment.

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.

In normal conditions gut homeostasis is maintained by the suppressive activity of regulatory T cells (Tregs), characterized by the expression of the transcription factor FoxP3. In human inflammatory bowel disease, which is believed to be the consequence of the loss of tolerance toward antigens normally contained in the gut lumen, Tregs have been found to be increased and functionally active, thus pointing against their possible role in the pathogenesis of this immune-mediated disease. Though, in inflammatory conditions, Tregs have been shown to upregulate the T helper (Th) type 1-related transcription factor Tbet and to express the pro-inflammatory cytokine IFN gamma, thus suggesting that at a certain point of the inflammatory process, Tregs might contribute to inflammation rather than suppress it. Starting from the observation that Tregs isolated from the lamina propria of active but not inactive IBD patients or uninflamed controls express Tbet and IFN gamma, we investigated the functional role of Th1-like Tregs in the dextran sulfate model of colitis. As observed in human IBD, Th1-like Tregs were upregulated in the inflamed lamina propria of treated mice and the expression of Tbet and IFN gamma in Tregs preceded the accumulation of conventional Th1 cells. By using a Treg-specific Tbet conditional knockout, we demonstrated that Tbet expression in Tregs is required for the development of colitis. Indeed, Tbet knockout mice developed milder colitis and showed an impaired Th1 immune response. In these mice not only the Tbet deficient Tregs but also the Tbet proficient conventional T cells showed reduced IFN gamma expression. However, Tbet deficiency did not affect the Tregs suppressive capacity in vitro and in vivo in the adoptive transfer model of colitis. In conclusion here we show that Tbet expression by Tregs sustains the early phase of the Th1-mediated inflammatory response in the gut.

Background and aims In ulcerative colitis [UC], mucosal damage occurs in areas that are infiltrated with neutrophils. The antimicrobial function of neutrophils relies in part on the formation of extracellular web-like structures, named neutrophil extracellular traps [NETs]. The formation and/or clearance of aberrant NETs have been associated with several immune diseases. Here we investigated the role of NETs in UC-related inflammation. Methods The expression of NET-associated proteins was evaluated in colonic biopsies of patients with Crohn's disease [CD], UC and in normal controls [NC] by Western blotting, immunofluorescence and immunohistochemistry. Colonic biopsies of UC patients were analysed before and after anti-tumour necrosis factor α [anti-TNF-α] treatment. The capacity of neutrophils to produce NETs upon activation was tested in vitro. UC lamina propria mononuclear cells [LPMCs] were cultured with NETs in the presence or absence of an extracellular signal-regulated kinase-1/2 [ERK1/2] inhibitor and inflammatory cytokine induction was assessed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. We also characterized the contribution of NETs in dextran sodium sulfate [DSS]-induced colitis. Results NET-associated proteins were over-expressed in inflamed colon of UC patients as compared to CD patients and NC. Circulating neutrophils of UC patients produced NETs in response to TNF-α stimulation, and reduced expression of NET-related proteins and diminished NET formation were seen in patients receiving successful treatment with anti-TNF-α. Treatment of UC LPMCs with NETs activated ERK1/2, thus enhancing TNF-α and interleukin-1β [IL-1β] production. NETs were induced in mice with DSS-colitis and in vivo inhibition of NET release attenuated colitis. Conclusions Our data show that NET release occurs in UC and suggest a role for NETs in sustaining mucosal inflammation in this disorder.

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.

Colorectal cancers (CRCs) often show a dense infiltrate of cytokine-producing immune/inflammatory cells. The exact contribution of each immune cell subset and cytokine in the activation of the intracellular pathways sustaining CRC cell growth is not understood. Herein, we isolate tumor-infiltrating leukocytes (TILs) and lamina propria mononuclear cells (LPMCs) from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of patients who underwent resection for sporadic CRC and show that the culture supernatants of TILs, but not of LPMCs, potently enhance the growth of human CRC cell lines through the activation of the oncogenic transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-kB). Characterization of immune cell complexity of TILs and LPMCs reveals no differences in the percentages of T cells, natural killer T cells, natural killer (NK) cells, macrophages and B cells. However, T cells from TILs show a functional switch compared with those from LPMCs to produce large amounts of T helper type 17 (Th17)-related cytokines (that is, interleukin-17A (IL-17A), IL-17F, IL-21 and IL-22), tumor necrosis factor-alpha (TNF-alpha) and IL-6. Individual neutralization of IL-17A, IL-17F, IL-21, IL-22, TNF-alpha or IL-6 does not change TIL-derived supernatant-driven STAT3 and NF-kB activation, as well as their proproliferative effect in CRC cells. In contrast, simultaneous neutralization of both IL-17A and TNF-alpha, which abrogates NF-kB signaling, and IL-22 and IL-6, which abrogates STAT3 signaling, reduces the mitogenic effect of supernatants in CRC cells. IL-17A, IL-21, IL-22, TNF-alpha and IL-6 are also produced in excess in the early colonic lesions in a mouse model of sporadic CRC, associated with enhanced STAT3/NF-kB activation. Mice therapeutically given BP-1-102, an orally bioavailable compound targeting STAT3/NF-kB activation and cross-talk, exhibit reduced colon tumorigenesis and diminished expression of STAT3/NF-kB-activating cytokines in the neoplastic areas. These data suggest that strategies aimed at the cotargeting of STAT3/NF-kB activation and interaction between them might represent an attractive and novel approach to combat CRC.

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. (Hepatology 2013;58:1436–1450)

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: Interleukin (IL)-25, a Th2-related factor, inhibits the synthesis of inflammatory cytokines by macrophages and attenuates experimental colitis in mice. The mechanism underlying the counterregulatory effect of IL-25, however, remains unknown. Since Th2-cytokines can abrogate inflammatory pathways by inducing alternatively activated macrophages (AAMs), we evaluated whether AAMs are involved in the IL-25-mediated anticolitic effect. Methods: AAM-related markers were evaluated in peritoneal and lamina propria mononuclear cells of mice with or without 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis treated with IL-25 and/or neutralizing IL-4, IL-13, and transforming growth factor beta 1 (TGF-β1) antibodies. Peritoneal AAMs induced in vivo by injecting mice with IL-25 were transferred to mice with TNBS colitis. Finally, we assessed the in vitro effect of IL-25 on the alternative activation of peritoneal F4/80+ cells. Results: IL-25 enhanced the expression of AAM-related markers in F4/80+ cells infiltrating the peritoneum and colon of naive and colitic mice. Peritoneal F4/80+ cells isolated from IL-25-treated mice reduced the severity of TNBS colitis when injected intraperitoneally to recipient mice. Since IL-25 did not directly induce AAM in vitro and in vivo in mice, IL-25 administration enhanced the expression of IL-4, IL-13, and TGF-β1, which are known to promote AAM differentiation, we finally assessed whether such cytokines were involved in the IL-25-driven AAM induction. Blockade of IL-4, IL-13, and TGF-β1 with neutralizing antibodies in mice did not inhibit the stimulatory effect of IL-25 on AAM gene expression. Conclusions: The IL-25-mediated anticolitic effect is associated with induction of AAMs, a subset of macrophages with antiinflammatory properties. (Inflamm Bowel Dis 2012;)

TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis is a crucial event in the control of tumor growth. However, many cancer cells, including colon cancer cells, are resistant to TRAIL-driven cell death. We have recently shown that 2-methoxy-5-amino-N-hydroxybenzamide (herein termed 2-14), a novel derivative of mesalamine, induces endoplasmic reticulum stress in colon cancer cells. Because endoplasmic reticulum stress-induced signals regulate the expression of molecules involved in TRAIL-driven apoptosis, we examined whether 2-14 makes colon cancer cells sensitive to TRAIL. Colon cancer cells were cultured with 2-14 and/or TRAIL. Death receptor (DR) 4/DR5 were analyzed by real-time PCR and flow cytometry. TRAIL pathway–associated proteins and extracellular signal–regulated kinase (ERK) were assessed by Western blotting. The in vivo capability of 2-14 to sensitize colon cancer cells to TRAIL-induced apoptosis was evaluated in a syngenic colon cancer model in which CT26-derived grafts were induced in mice. 2-14 promoted ERK-dependent induction of DR5, thereby enhancing TRAIL-mediated caspase-8 activation and apoptosis. Analysis of TRAIL-related pro- and antiapoptotic factors and functional studies revealed that survivin is involved in the protection of colon cancer cells against TRAIL-driven apoptosis. Notably, 2-14 enhanced ubiquitination and proteasome-mediated degradation of survivin. These data were confirmed in a murine model of TRAIL-resistant colon cancer in which 2-14 upregulated DR5, reduced survivin expression, and synergized with TRAIL in inhibiting tumor growth. Similarly, intraperitoneal administration of 2-14 to mice upregulated DR5 and downregulated survivin in a model of colitis-associated colon cancer. These findings indicate that 2-14 acts as a sensitizer for TRAIL-induced apoptosis and suggest that 2-14 can be useful in the therapy for TRAIL-resistant colon cancer. Mol Cancer Ther; 10(10); 1969–81. ©2011 AACR.

Although interleukin-25 (IL-25) has been traditionally considered as a cytokine involved in T helper (Th) 2 cell-associated allergic diseases and host defence against helminthic parasites, recent studies have shown that IL-25 exerts negative effects on the initiation and progression of Th1/Th17-mediated pathologies. This later function of IL-25 is particularly evident at the gut level, where IL-25 could contribute to attenuate tissue-damaging immune responses. These new and exciting pre-clinical observations suggest that therapeutic interventions aimed at enhancing IL-25 activity could be useful in the management of patients with chronic gut inflammation.

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.

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-β1 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-β1 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-β1 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.

In inflammatory bowel disease (IBD), tissue damage is driven by an excessive immune response, poorly controlled by counter-regulatory mechanisms. SIRT1, a class III NAD+-dependent deacetylase, regulates negatively the expression of various proteins involved in the control of immune-inflammatory pathways, such as Stat3, Smad7, and NF-κB. Here we examined the expression, regulation, and function of SIRT1 in IBD. SIRT1 RNA and protein expression was less pronounced in whole biopsies and lamina propria mononuclear cells (LPMCs) of IBD patients in comparison with normal controls. SIRT1 expression was downregulated in control LPMC by tumor necrosis factor (TNF)-α and interleukin (IL)-21, and upregulated in IBD LPMC by neutralizing TNF-α and IL-21antibodies. Consistently, SIRT1 expression was increased in mucosal samples taken from IBD patients successfully treated with Infliximab. Treatment of IBD LPMC with Cay10591, a specific SIRT1 activator, reduced NF-κB activation and inhibited inflammatory cytokine synthesis, whereas Ex527, an inhibitor of SIRT1, increased interferon (IFN)-γ in control LPMC. SIRT1 was also reduced in mice with colitis induced by 2,4,6-trinitrobenzenesulphonic acid or oxazolone. Cay10591 prevented and cured experimental colitis whereas Ex527 exacerbated disease by modulating T cell-derived cytokine response. Data indicate that SIRT1 is downregulated in IBD patients and colitic mice and suggest that SIRT1 activation can help attenuate inflammatory signals in the gut.Mucosal Immunology advance online publication, 21 May 2014; doi:10.1038/mi.2014.35.

Monocytes/macrophages displaying different markers of activation/differentiation infiltrate the inflamed gut of patients with inflammatory bowel diseases (IBD), but the role that each monocyte/macrophage subpopulation plays in the pathogenesis of IBD is not fully understood. The hemoglobin scavenger receptor CD163, a specific marker of monocytes/macrophages, has been associated with either anti-inflammatory or inflammatory functions of macrophages in several pathologies. In this study we examined the tissue distribution and function of CD163-expressing monocytes/macrophages in IBD. CD163 RNA and protein expression was more pronounced in IBD in comparison to normal controls, with no significant difference between Crohn's disease and Ulcerative colitis. In IBD, over-expression of CD163 was restricted to areas with active inflammation and not influenced by current therapy. Immunohistochemical analysis confirmed the accumulation of CD163-expressing cells in IBD, mostly around and inside blood vessels, thus suggesting that these cells are partly recruited from the systemic circulation. Indeed, FACS analysis of circulating mononuclear cells showed that the fractions of CD163-positive monocytes were increased in IBD patients as compared to controls. Functionally, interleukin-6 up-regulated CD163 expression in lamina propria mononuclear cells and mucosal explants of normal subjects. In IBD blood and mucosal cell cultures, cross-linking of CD163 with a specific monoclonal anti-CD163 antibody enhanced tumor necrosis factor-α synthesis. These findings indicate that IBD mucosa is abundantly infiltrated with CD163-positive cells, which could contribute to amplify the inflammatory cytokine response.

IL-21 expression is increased in the gut of patients with colitis-associated colon cancer, and genetic ablation or antibody neutralization of IL-21 reduces tumor size and inflammation in mice treated with dextran sulfate sodium and azoxymethane., Chronic inflammation is a major driving force in the development of cancer in many tissues, but the array of factors involved in this neoplastic transformation are not well understood. We have investigated the role of interleukin (IL)-21 in colitis-associated colon cancer (CAC), as this cytokine is overexpressed in the gut mucosa of patients with ulcerative colitis (UC), a chronic inflammatory disease associated with colon cancer. IL-21 was increased in the gut of patients with UC-associated colon cancer, and in mice with CAC induced by azoxymethane (AOM) and dextran sulfate sodium (DSS). After AOM+DSS treatment, IL-21 KO mice showed reduced mucosal damage, reduced infiltration of T cells, and diminished production of IL-6 and IL-17A. IL-21–deficient mice also developed fewer and smaller tumors compared with wild-type (WT) mice. Absence of IL-21 reduced signal transducer and activator of transcription 3 activation in tumor and stromal cells. Administration of a neutralizing IL-21 antibody to WT mice after the last DSS cycle decreased the colonic T cell infiltrate and the production of IL-6 and IL-17A and reduced the number of tumors. These observations indicate that IL-21 amplifies an inflammatory milieu that promotes CAC, and suggest that IL-21 blockade may be useful in reducing the risk of UC-associated colon cancer.