Smad7 induces plasticity in tumor-infiltrating Th17 cells and enables TNF-alpha-mediated killing of colorectal cancer cells.

Transforming growth factor-beta (TGF-ß) is deeply involved in colorectal cancer development and the disruption of the TGF-ß signaling in dysplastic cells is required for tumor to grow. Nevertheless, tumor cells express TGF-ß to escape the immune surveillance mediated by T cells. T-cell expression of Smad7, an intracellular inhibitor of the TGF-ß signaling, protects against colitis-associated colorectal cancer. However, whether Smad7 in T cells might influence colorectal cancer growth independently of chronic inflammation and which T-cell subset is involved in this process is unknown. To address this issue, T-cell-specific Smad7 transgenic mice and wild-type (WT) littermates were subcutaneously transplanted with syngenic MC38 colon carcinoma cells. Smad7Tg mice were resistant to tumor development compared with WT mice and protection was dependent on CD4(+) T cells. Smad7 expression in T cells increased the number of tumor-infiltrating Tbet/ROR-γ-t double-positive CD4 T cells characterized by the expression of tumor necrosis factor-alpha (TNF-α) and interferon-gamma but lower IL17A. The low expression of IL17A caused by the Smad7 expression in tumor-infiltrating CD4(+) T cells enabled the TNF-α-mediated killing of cancer cells both in vitro and in vivo, thus indicating that the Smad7-mediated plastic effect on T-cell phenotype induces protection against colorectal cancer.

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.

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.

Aryl hydrocarbon receptor-induced signals up-regulate IL-22 production and inhibit inflammation in the gastrointestinal tract.

BACKGROUND & AIMS: The pathogenesis of inflammatory bowel disease (IBD) is believed to involve an altered balance between effector and regulatory T cells. Aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor that mediates the toxicity of dioxins, controls T-cell responses. We investigated the role of AhR in inflammation and pathogenesis of IBD in humans and mouse models. METHODS: AhR expression was evaluated in intestinal tissue samples from patients with IBD and controls by real-time polymerase chain reaction (PCR) and flow cytometry. Intestinal lamina propria mononuclear cells (LPMCs) were activated in the presence or absence of the AhR agonist 6-formylindolo(3, 2-b)carbazole (Ficz). Colitis was induced in mice using trinitrobenzene sulfonic acid (TNBS), dextran sulfate sodium (DSS), or T-cell transfer. Mice were given injections of Ficz or the AhR antagonist 2-metyl-2H-pyrazole-3-carboxylic acid; some mice first received injections of a blocking antibody against interleukin (IL)-22. Cytokines were quantified by real-time PCR and flow cytometry. RESULTS: Intestine tissue from patients with IBD expressed significantly less AhR than controls. In LPMCs from patients with IBD, incubation with Ficz reduced levels of interferon gamma (IFN)-γ and up-regulated IL-22. Mice injected with Ficz were protected against TNBS-, DSS-, and T-cell transfer-induced colitis; they had marked down-regulation of inflammatory cytokines and induction of IL-22. Mice given AhR antagonist produced more inflammatory cytokines and less IL-22 and developed a severe colitis. Neutralization of endogenous IL-22 disrupted the protective effect of Ficz on TNBS-induced colitis. CONCLUSIONS: AhR is down-regulated in intestinal tissue of patients with IBD; AhR signaling, via IL-22, inhibits inflammation and colitis in the gastrointestinal tract of mice. AhR-related compounds might be developed to treat patients with IBDs.

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.

Inhibition of monocyte-derived inflammatory cytokines by IL-25 occurs via p38 Map kinase-dependent induction of Socs-3.

IL-25, a member of the IL-17 cytokine family, is known to enhance Th2-like responses associated with increased serum levels of IgE, IgG1, IgA, blood eosinophilia, and eosinophilic infiltrates in various tissues. However, IL-25 also abrogates inflammatory responses driven by Th17 cells. However, the cell types that respond to IL-25 and the mechanisms by which IL-25 differentially regulates immune reactions are not well explored. To identify potential targets of IL-25, we initially examined IL-25 receptor (IL-25R) in human peripheral blood cells. IL-25R was predominantly expressed by CD14(+) cells. We next assessed the functional role of IL-25 in modulating the response of CD14(+) cells to various inflammatory signals. CD14(+) cells responded to IL-25 by down-regulating the synthesis of inflammatory cytokines induced by toll-like receptor (TLR) ligands and inflammatory cytokines. Inhibition of cytokine response by IL-25 occurred via a p38 Map kinase-driven Socs-3-dependent mechanism. In vivo, IL-25 inhibited monocyte-derived cytokines and protected against LPS-induced lethal endotoxemia in mice. These data indicate that IL-25 is a negative regulator of monocyte proinflammatory cytokine responses, which may have therapeutic implications.

Interleukin-25 inhibits interleukin-12 production and Th1 cell-driven inflammation in the gut.

BACKGROUND & AIMS: During the pathogenesis of Crohn's disease (CD), interleukin (IL)-12, a cytokine produced by mucosal CD14+ monocyte-like cells, promotes tissue-damaging T helper cell (Th) 1-mediated inflammation through mechanisms that are not fully understood. IL-25 promotes Th2 cell responses by activating major histocompatibility complex class II-positive non-T and non-B cells. Because Th1 and Th2 cells, and the cytokines they release, are often mutually antagonistic, we examined whether IL-25 affects IL-12 production or Th1 cell-mediated inflammation in the gut. METHODS: Studies were performed using colonic samples from patients and mice with peptidoglycan (PGN)-, 2,4,6-trinitrobenzenesulphonic acid (TNBS)-, or oxazolone-induced colitis. IL-25 receptor (IL-25R) levels were evaluated in intestinal lamina propria mononuclear cells by flow cytometry, and IL-25 levels were measured by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. Mucosal CD14+ cells from patients with CD were incubated with IL-25 and/or lipopolysaccharide or PGN. Mice were injected with IL-25, and some mice first received injections of an IL-13 blocking antibody. Cytokines were quantified by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: CD14+ cells from the mucosa of CD patients expressed IL-25R and responded to IL-25 by decreasing the synthesis of IL-12 and IL-23. IL-25 prevented PGN-induced colitis in mice. IL-25 induced IL-13 production in the colon, but IL-13 was not required for suppression of PGN colitis. IL-25 ameliorated TNBS- and oxazolone-colitis. Patients with CD or ulcerative colitis produced significantly less IL-25 compared with controls. CONCLUSIONS: IL-25 inhibits CD14+ cell-derived cytokines and experimental colitis. IL-25 could be a useful treatment of CD and ulcerative colitis.

Smad7 controls resistance of colitogenic T cells to regulatory T cell-mediated suppression.

BACKGROUND & AIMS: Foxp3-expressing regulatory T cells (Tregs) play a key role in the maintenance of the gut immune homeostasis, and an intact transforming growth factor (TGF)-beta signaling is required for their function. In inflammatory bowel disease (IBD), the TGF-beta signaling is impaired because of high expression of the inhibitory molecule Smad7. Although no intrinsic defects in Tregs function have been shown in IBD, it is still unknown whether colitogenic T cells are susceptible to Treg-mediated suppression. In this study, we have investigated whether IBD mucosal CD4+ T cells are resistant to Tregs and whether Smad7 is involved in this process. METHODS: IBD lamina propria mononuclear cells (LPMC) were cultured with or without Tregs, and proliferation was assessed by flow cytometry. Proliferation of IBD LPMC was also evaluated after Smad7 antisense oligonuclotide treatment. Treg-mediated suppression of T-cell proliferation and proinflammatory cytokine expression was investigated in murine Smad7 transgenic cells. In vivo, the Smad7-dependent resistance of colitogenic naïve T cells to Tregs was studied in the adoptive transfer model of colitis. RESULTS: IBD LPMC were resistant to Treg-mediated suppression, and this phenomenon was reverted by Smad7 antisense treatment. Consistently, CD4+ T cells isolated from Smad7 transgenic mice showed high proliferation, produced considerable amount of inflammatory cytokines following activation, and induced a severe colitis when transferred in immunodeficient RAG1 knockout mice even in the presence of wild-type Tregs. CONCLUSIONS: Smad7 makes CD4+ T cells resistant to Tregs-mediated suppression thus fine-tuning their proinflammatory potential.

Cadherin-11 Is a Regulator of Intestinal Fibrosis.

BACKGROUND AND AIMS: Although 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. METHODS: CDH-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-ß 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. RESULTS: CDH-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-1ß enhanced CDH-11 expression. Knockdown of CDH-11 in FS CD fibroblasts impaired RhoA/ROCK/TGF-ß signalling and reduced collagen synthesis, whereas activation of CDH-11 increased collagen secretion. CDH-11 KO mice were largely protected from intestinal fibrosis. CONCLUSIONS: Data 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.

Regulation of gut inflammation and th17 cell response by interleukin-21.

BACKGROUND & AIMS: Interleukin (IL)-21, a T-cell-derived cytokine, is overproduced in inflammatory bowel diseases (IBD), but its role in the pathogenesis of gut inflammation remains unknown. We here examined whether IL-21 is necessary for the initiation and progress of experimental colitis and whether it regulates specific pathways of inflammation. METHODS: Both dextran sulfate sodium colitis and trinitrobenzene sulfonic acid-relapsing colitis were induced in wild-type and IL-21-deficient mice. CD4(+)CD25(-) T cells from wild-type and IL-21-deficient mice were differentiated in T helper cell (Th)17-polarizing conditions, with or without IL-21 or an antagonistic IL-21R/Fc. We also examined whether blockade of IL-21 by anti-IL-21 antibody reduced IL-17 in cultures of IBD lamina propria CD3(+) T lymphocytes. Cytokines were evaluated by real-time polymerase chain reaction and/or enzyme-linked immunosorbent assay. RESULTS: High IL-21 was seen in wild-type mice with dextran sulfate sodium- and trinitrobenzene sulfonic acid-relapsing colitis. IL-21-deficient mice were largely protected against both colitides and were unable to up-regulate Th17-associated molecules during gut inflammation, thus suggesting a role for IL-21 in controlling Th17 cell responses. Indeed, naïve T cells from IL-21-deficient mice failed to differentiate into Th17 cells. Treatment of developing Th17 cells from wild-type mice with IL-21R/Fc reduced IL-17 production. Moreover, in the presence of transforming growth factor-beta1, exogenous IL-21 substituted for IL-6 in driving IL-17 induction. Neutralization of IL-21 reduced IL-17 secretion by IBD lamina propria lymphocytes. CONCLUSIONS: These results indicate that IL-21 is a critical regulator of inflammation and Th17 cell responses in the gut.

Tbet Expression in Regulatory T Cells Is Required to Initiate Th1-Mediated Colitis.

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 IFNγ, 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 IFNγ, 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 IFNγ 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 IFNγ 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.

Mesalazine negatively regulates CDC25A protein expression and promotes accumulation of colon cancer cells in S phase.

Regular consumption of mesalazine has been associated with a reduced risk of colorectal cancer (CRC) in patients with inflammatory bowel disease. The molecular mechanisms underlying the antineoplastic effect of 5-aminosalicylic acid remain, however, poorly characterized. In this study, we examined whether mesalazine affects cell cycle progression and analyzed specific checkpoint pathways in experimental models of CRC. Mesalazine inhibited the growth of HCT-116 and HT-29 cells, two CRC cell lines that express either a wild-type or mutated p53. Cell cycle analysis revealed that mesalazine induced cells to accumulate in S phase. This effect was associated with a sustained phosphorylation of the cyclin-dependent kinase (CDK)2 at threonine 14 and tyrosine 15 residues, an event that inactivates the CDK2-cyclin complex and blocks S-G(2) phase cell cycle transition. Consistently, mesalazine reduced the protein content of CDC25A, a phosphatase that regulates CDK2 phosphorylation status. Analysis of upstream kinases that negatively control CDC25A expression showed that mesalazine enhanced the activation of CHK1 and CHK2. However, silencing of CHK1 and CHK2 did not prevent the mesalazine-induced CDC25A protein downregulation. In contrast, CDC25A protein ubiquitination and degradation and accumulation of cells in S phase following mesalazine exposure were reverted by proteasome inhibitors. Notably, mesalazine also inhibited CDC25A in human CRC explants. Finally, we showed that mesalazine downregulated CDC25A in CT26, a murine CRC cell line, and prevented the formation of CT26-derived tumors in mice. Data show that mesalazine negatively regulates CDC25A protein expression, thus delaying CRC cell progression.

Cyclooxygenase-2-dependent and -independent inhibition of proliferation of colon cancer cells by 5-aminosalicylic acid.

The cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway may have a pathogenic role in colorectal cancer (CRC). Recent studies suggest that 5-aminosalicylic acid (5-ASA) reduces the risk of inflammatory bowel disease-related CRC, but the mechanism by which 5-ASA interferes with CRC cell growth remains unknown. In this study, we have examined whether the negative effect of 5-ASA on CRC cells is dependent on COX-2/PGE2 axis inhibition. We show that 5-ASA down-regulates both constitutive and TNF-alpha or IL-1beta-induced COX-2 in HT-115 and HT-29 cells. Inhibition of COX-2 by 5-ASA occurs at the RNA and protein level, and is associated with a significant decrease in PGE2 synthesis, arrest of growth and enhanced death of CRC cells. However, exogenous PGE2 does not revert the 5-ASA-mediated CRC cell proliferation block. 5-ASA also inhibits the growth of DLD-1, a COX-deficient CRC cell line, thus suggesting that the anti-proliferative effect of 5-ASA on CRC cells is not strictly dependent on the inhibition of COX-2/PGE2. Taken together our data indicate that 5-ASA causes both a COX-2-dependent and -independent inhibition of CRC cell growth.

Reciprocal Regulation Between Smad7 and Sirt1 in the Gut.

In inflammatory bowel disease (IBD) mucosa, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine transforming growth factor-ß1, 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.

Knockdown of Smad7 With a Specific Antisense Oligonucleotide Attenuates Colitis and Colitis-Driven Colonic Fibrosis in Mice.

Background: In Crohn's disease (CD), the pathogenic immune response is associated with high Smad7, an inhibitor of TGF-ß1 signaling. Smad7 knockdown with Mongersen, a specific antisense oligonucleotide-containing compound, restores TGF-ß1 activity leading to inhibition of inflammatory signals and associates with clinical benefit in CD patients. As TGF-ß1 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-ß1 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-ß1 and reduced p-Smad3 protein expression. Conclusion: Attenuation of colitis with Smad7 antisense oligonucleotide limits development of colonic fibrosis.

RORγt-Expressing Tregs Drive the Growth of Colitis-Associated Colorectal Cancer by Controlling IL6 in Dendritic Cells.

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.

Interleukin-21 sustains inflammatory signals that contribute to sporadic colon tumorigenesis.

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.

Inhibition of colitis by IL-25 associates with induction of alternatively activated macrophages.

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-ß1) 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 naïve 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-ß1, 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-ß1 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.

Intestinal inflammation and colorectal cancer: a double-edged sword?

Chronic inflammation is thought to be the leading cause of many human cancers including colorectal cancer (CRC). Accordingly, epidemiologic and clinical studies indicate that patients affected by ulcerative colitis and Crohn's disease, the two major forms of inflammatory bowel disease, have an increased risk of developing CRC. In recent years, the role of immune cells and their products have been shown to be pivotal in initiation and progression of colitis-associated CRC. On the other hand, activation of the immune system has been shown to cause dysplastic cell elimination and cancer suppression in other settings. Clinical and experimental data herein reviewed, while confirming chronic inflammation as a risk factor for colon carcinogenesis, do not completely rule out the possibility that under certain conditions the chronic activation of the mucosal immune system might protect from colonic dysplasia.

Interleukin-25 negatively controls pathogenic responses in the gut.

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.