Inhibition of Smad7 with a specific antisense oligonucleotide facilitates TGF-beta1-mediated suppression of colitis

BACKGROUND & AIMS: Defective transforming growth factor (TGF)-beta1 signaling due to high levels of Smad7 is a feature of inflammatory bowel disease (IBD). In this study, we analyzed the effect of reducing Smad7 levels with antisense oligonucleotide on mouse models of colitis. METHODS: Mucosal samples taken from colitic tissue of mice with colitis due to either haptenating reagents (trinitrobenzene sulfonic acid [TNBS] or oxazolone) or to transfer of T cells (SCID transfer colitis) were analyzed for Smad3 and/or Smad7 expression by Western blotting and, in some cases, content of TGF-beta1 by enzyme-linked immunosorbent assay. The effect of oral Smad7 antisense oligonucleotide on mucosal inflammation was assessed. RESULTS: TGF-beta1 levels were increased in the inflamed tissues of mice with colitis induced by either TNBS or oxazolone. Nevertheless, TGF-beta1 did not exert a regulatory effect, probably because TGF-beta1 signaling was blocked, as indicated by the presence of reduced Smad3 phosphorylation and high levels of Smad7. Oral administration of Smad7 antisense oligonucleotide to colitic mice restored TGF-beta1 signaling via Smad3 and ameliorated inflammation in hapten-induced colitis. In addition, Smad7 antisense oligonucleotide had a therapeutic effect on relapsing TNBS-induced colitis but not on cell-transfer colitis. CONCLUSIONS: These data suggest that colitis models associated with high endogenous TGF-beta1 levels and defective TGF-beta1 signaling due to high levels of Smad7 can be ameliorated by down-regulation of Smad7 and by oral administration of Smad7 antisense oligonucleotide. This may represent a new approach to the control of IBD, particularly during active phases when its Smad7 profile resembles that of hapten-induced colitis.     

Interleukin-21 enhances T-helper cell type I signaling and interferon-gamma production in Crohn's disease

BACKGROUND & AIMS: T-helper (Th)1 cells play a central role in the pathogenesis of tissue damage in Crohn's disease (CD). Interleukin (IL)-12/STAT4 signaling promotes Th1 cell commitment in CD, but other cytokines are needed to maintain activated Th1 cells in the mucosa. In this study, we examined the expression and role of IL-21, a T-cell-derived cytokine of the IL-2 family; in tissues and cells isolated from patients with inflammatory bowel disease. METHODS: IL-21 was examined by Western blotting in whole mucosa and lamina propria mononuclear cells (LPMCs) from patients with CD, ulcerative colitis (UC), and controls. We also examined the effects of exogenous IL-12 on IL-21 production, as well as the effects of blocking IL-21 with an IL-21-receptor Ig fusion protein. Interferon (IFN)-gamma was measured in the culture supernatants by enzyme-linked immunosorbent assay, and phosphorylated STAT4 and T-bet were examined by Western blotting. RESULTS: IL-21 was detected in all samples, but its expression was higher at the site of disease in CD in comparison with UC and controls. Enhanced IL-21 was seen in both ileal and colonic CD and in fibrostenosing and nonfibrostenosing disease. IL-12 enhanced IL-21 in normal lamina propria lymphocytes through an IFN-gamma-independent mechanism, and blocking IL-12 in CD LPMCs decreased anti-CD3-stimulated IL-21 expression. Neutralization of IL-21 in CD LPMC cultures decreased phosphorylated STAT4 and T-bet expression, thereby inhibiting IFN-gamma production. CONCLUSIONS: Our data suggest that IL-21 contributes to the ongoing Th1 mucosal response in CD.     

A functional role of flip in conferring resistance of Crohn's disease lamina propria lymphocytes to FAS-mediated apoptosis

BACKGROUND & AIMS: There is evidence that, in Crohn's disease (CD), lamina propria T lymphocytes (LPLs) are resistant to FAS-mediated apoptosis and that this defect contributes to the mucosal T-cell accumulation. In this study we examined the functional role of Flip, a Flice inhibitor protein, in the resistance of CD LPL to FAS-mediated apoptosis. METHODS: Biopsy specimens and LPLs were taken from CD and ulcerative colitis (UC) patients and normal controls and analyzed for Flip by Western blotting. We also examined whether inhibition of Flip by antisense oligonucleotide restored the susceptibility of CD LPLs to FAS-induced apoptosis. LPL apoptosis was assessed by flow cytometry. RESULTS: After FAS stimulation, the rate of apoptosis of CD3+ LPLs was higher in normal controls and patients with UC than in patients with CD. Enhanced expression of both long and short Flip isoforms was seen in biopsy specimens and purified CD3+ and CD45RO+ LPLs of CD patients in comparison with UC patients and normal controls. No increase in Flip was documented in untreated celiac disease mucosa, thus suggesting the possibility that induction of Flip in the gut does not simply rely on the ongoing inflammation. Finally, we showed that inhibition of Flip by antisense oligonucleotide reverted the resistance of CD LPLs to FAS-induced apoptosis. CONCLUSIONS: Data suggest a role for Flip in the resistance of CD LPLs to FAS-mediated apoptosis.     

Characteristics of intestinal dendritic cells in inflammatory bowel diseases

BACKGROUND & AIMS: Dendritic cells (DCs) recognize and respond to microbial structures using pattern recognition receptors, including Toll-like receptors (TLRs). In the intestine, DCs are pivotal in tolerance induction and direct the differentiation of T cells. We aimed to identify changes in intestinal DCs that may underlie the dysregulated immune response to enteric bacteria that occurs in patients with inflammatory bowel disease (IBD). METHODS: DCs were identified in freshly isolated lamina propria mononuclear cells by multicolor flow cytometry in patients with IBD and controls. Expression of TLR2, TLR4, and the activation/maturation marker CD40 was assessed by cell surface labeling. Production of cytokines (interleukin [IL]-12, IL-6, and IL-10) was assessed in the absence of exogenous stimulation by intracellular staining of permeabilized cells. RESULTS: In healthy controls, few intestinal DCs expressed TLR2 or TLR4, in contrast to blood DCs. DC expression of both TLRs was significantly enhanced in Crohn's disease and ulcerative colitis. DCs from inflamed tissue of patients with Crohn's disease expressed significantly higher levels of the maturation/activation marker CD40. Elevated levels of CD40 on DCs were decreased after treating patients with anti-tumor necrosis factor alpha. In Crohn's disease, but not ulcerative colitis, more colonic DCs produced IL-12 and IL-6. The number of IL-10-producing DCs did not differ significantly between patients with IBD and controls. CONCLUSIONS: In IBD, DCs are activated, their expression of microbial recognition receptors is up-regulated, and more DCs produce pathologically relevant cytokines. Intestinal DCs are likely to be key initiators or perpetuators of the inflammatory response that characterizes IBD.     

Transamidation of wheat flour inhibits the response to gliadin of intestinal T cells in celiac disease

BACKGROUND & AIMS: Celiac disease is characterized by activation of HLA-DQ2/DQ8-restricted intestinal gluten-specific CD4(+) T cells. In particular, gluten becomes a better T-cell antigen following deamidation catalyzed by tissue transglutaminase. To date, the only available therapy is represented by adherence to a gluten-free diet. Here, we examined a new enzyme strategy to preventively abolish gluten activity. METHODS: Enzyme modifications of the immunodominant alpha-gliadin peptide p56-68 were analyzed by mass spectrometry, and peptide binding to HLA-DQ2 was simulated by modeling studies. Wheat flour was treated with microbial transglutaminase and lysine methyl ester; gliadin was subsequently extracted, digested, and deamidated. Gliadin-specific intestinal T-cell lines (iTCLs) were generated from biopsy specimens from 12 adult patients with celiac disease and challenged in vitro with different antigen preparations. RESULTS: Tissue transglutaminase-mediated transamidation with lysine or lysine methyl ester of p56-68 or gliadin in alkaline conditions inhibited the interferon gamma expression in iTCLs; also, binding to DQ2 was reduced but not abolished, as suggested by in silico analysis. Lysine methyl ester was particularly effective in abrogating the activity of gliadin. Notably, a block in the response was observed when iTCLs were challenged with gliadin extracted from flour pretreated with microbial transglutaminase and lysine methyl ester. CONCLUSIONS: Transamidation of wheat flour with a food-grade enzyme and an appropriate amine donor can be used to block the T cell-mediated gliadin activity. Considering the crucial role of adaptive immunity in celiac disease, our findings highlight the potential of the proposed treatment to prevent cereal toxicity.     

Therapeutic treatment of experimental colitis with regulatory dendritic cells generated with vasoactive intestinal peptide

BACKGROUND & AIMS: Crohn's disease is a chronic debilitating disease characterized by severe T helper cell (Th)1-driven inflammation of the colon partially caused by a loss of immune tolerance against mucosal antigens. The use of regulatory dendritic cells (DCs) with the capacity to induce regulatory T cells has been proposed recently for the treatment of Crohn's disease in a strategy to restore immune tolerance. Vasoactive intestinal peptide is an immunomodulatory neuropeptide that induces regulatory DCs. The aim of this study was to investigate the therapeutic effect of vasoactive intestinal peptide-induced regulatory DCs (DC(VIP)) in a murine model of colitis. METHODS: We examined the therapeutic action of DC(VIP) in the colitis induced by intracolonic administration of trinitrobenzene sulfonic acid, evaluating diverse clinical signs of the disease including weight loss, diarrhea, colitis, and histopathology. We also investigated the mechanisms involved in the potential therapeutic effect of DC(VIP), such as inflammatory cytokines and chemokines, Th1-type response, and the generation of regulatory T cells. RESULTS: DC(VIP) injection significantly ameliorated the clinical and histopathologic severity of colitis, abrogating body weight loss, diarrhea, and inflammation, and increasing survival. The therapeutic effect was associated with down-regulation of both inflammatory and Th1-driven autoimmune response, by regulating a wide spectrum of inflammatory mediators directly through activated macrophages, and by generating interleukin-10-secreting regulatory T cells with suppressive capacity on autoreactive T cells. CONCLUSIONS: The possibility to generate/expand ex vivo regulatory DC(VIP) opens new therapeutic perspectives for the treatment of Crohn's disease in human beings, and may minimize the dependence on nonspecific immunosuppressive drugs used currently for autoimmune disorders.     

Proinflammatory effects of TH2 cytokines in a murine model of chronic small intestinal inflammation

BACKGROUND & AIMS: Strict T H 1 polarization is believed to underlie the pathogenesis of intestinal inflammation in Crohn's disease. In the present study we tested the hypothesis that TH2 cytokines also may participate in disease development in SAMP1/YitFc mice that spontaneously develop terminal ileitis with perianal manifestations. METHODS: Cytokine messenger RNA (mRNA) expression was studied by real-time polymerase chain reaction (PCR). Lamina propria mononuclear cells (LPMCs) were purified and stimulated cytokine secretion was analyzed. Blockade of interferon (IFN)-gamma or interleukin (IL)-4 was performed by using specific neutralizing monoclonal antibodies (MAbs). CD4+/IL-4-secreting lymphocytes were purified from SAMP1/YitFc mesenteric lymph nodes (MLNs) and their ability to induce ileitis was tested after transfer to SCID recipients. RESULTS: Initiation of ileitis in SAMP1/YitFc mice was T H 1-mediated because up-regulation of IFN-gamma and tumor necrosis factor (TNF) preceded the histologic injury, whereas IFN-gamma neutralization prevented the development of chronic inflammation (P <.005) by interfering with the expansion of lymphocytes. In contrast, the establishment of chronic ileitis coincided with significant increases in IL-5 (35x) and IL-13 (29x) mRNA expression (P <.005), as well as in T H 2 cytokine secretion by lamina propria lymphocytes (P <.05 vs. AKR controls). IL-4 blockade diminished IFN-gamma mRNA expression and significantly ameliorated the severity of established ileitis (P <.05) by decreasing the histologic indices for villous distortion and active inflammation. In addition, IL-4 augmented the in vitro IFN-gamma secretion by lymphocytes, whereas IL-4-secreting CD4+ lymphocytes were sufficient for adoptively transferring ileitis to SCID recipients. CONCLUSIONS: Our results indicate that both TH1 and TH2 pathways mediate Crohn's-like ileitis and suggest that combined TH1/TH2 manipulation may offer a therapeutic advantage for the treatment of Crohn's disease.     

Enteric flora and lymphocyte-derived cytokines determine expression of heat shock proteins in mouse colonic epithelial cells

BACKGROUND & AIMS: Inducible heat shock proteins (hsps), particularly hsp25 and hsp72, are expressed by surface colonocytes and may have a role in protecting intestinal epithelial cells against injury. This study is aimed at determining if enteric bacteria and/or immune signals regulate their physiologic expression. METHODS: Intestinal hsp25, hsp72, and constitutive hsc73 expression were studied in immunodeficient RAG-1(-/-) mice and in normal mice. Mucosal permeability was measured by mannitol flux and transepithelial resistance. Hsp expression in intestinal YAMC cells was assessed after incubation with recombinant cytokines, activated lamina propria lymphocytes (LPLs), or Bacteroides fragilis. RESULTS: Chronic metronidazole treatment decreases colonic mucosal hsp25 and hsp72 expression, an effect associated with increased susceptibility of mucosal barrier function to C. difficile toxin A. Hsp expression also was increased in YAMC cells incubated with B. fragilis, an effect mediated by lipopolysaccharide and other bacteria-derived factors. Colonic hsp72, but not hsp25 or hsc73, expression is decreased in RAG-1(-/-) mice. Recombinant IL-2 and other cytokines enhance YAMC hsp25 and/or hsp72 expression. Activated LPLs induce YAMC hsp expression, an effect blocked by IL-2 neutralizing antibody. CONCLUSIONS: Enteric flora and mucosal lymphocytes play a role in maintaining physiologic expression of colonocyte hsp25 and hsp72.     

Induction and regulation of Smad7 in the gastric mucosa of patients with Helicobacter pylori infection

BACKGROUND & AIMS: Helicobacter pylori (Hp) infection causes a chronic gastric inflammation, which can lead to peptic ulceration and cancer. The inflammatory response is multifactorial and is characterized by exaggerated Th1 cytokine production. How the Th1 response is induced and maintained in the stomach of Hp-infected patients remains unclear. Transforming growth factor (TGF)-beta 1 negatively regulates Th1 cell development, and TGF-beta 1-deficient mice spontaneously develop gastritis. Here, we examined TGF-beta 1 signaling in Hp-associated gastritis. METHODS: Gastric biopsy specimens taken from patients with or without Hp infection were analyzed for the content of activated TGF-beta1 by ELISA and Smad3 and 7 expression by Western blotting. Induction of Smad7 by interferon (IFN)-gamma was examined in normal gastric mucosal biopsy specimens, whereas the effect of Smad7 inhibition on the ongoing Th1 response was analyzed in Hp-colonized biopsy specimens. RESULTS: Activated TGF-beta 1 was abundant in the mucosa of controls and Hp-infected patients, with no significant difference between the 2 groups. Despite this, in whole biopsy specimens and isolated mucosal cells from Hp-infected patients, there was defective TGF-beta 1-associated Smad3 phosphorylation, which was associated with high expression of the inhibitor Smad7. Blocking Smad7 with antisense oligonucleotides restored TGF-beta 1 signaling in biopsy specimens from Hp-infected patients and concomitantly reduced interferon-gamma and T-bet. Smad7 was inducible in normal gastric biopsy specimens by interferon-gamma through a STAT1-dependent mechanism, and neutralization of interferon-gamma in biopsy specimens from Hp-infected patients reduced Smad7 expression. CONCLUSIONS: These data suggest that, in Hp-infected gastric mucosa, interferon-gamma induces the expression of Smad7, which then prevents endogenous TGF-beta 1 from down-regulating the ongoing tissue-damaging Th1 response.     

Activation of A2A adenosine receptor attenuates intestinal inflammation in animal models of inflammatory bowel disease

BACKGROUND & AIMS: Adenosine has been implicated as an important regulator of the inflammatory response. Four subtypes of adenosine receptors (A 1 , A 2A , A 2B , and A 3 ) have been described, of which A 2A potentially inhibits inflammation. The aim of this study was to investigate the role of A 2A in mucosal inflammation by administering a selective A 2A agonist (ATL-146e) to experimental models of inflammatory bowel disease. METHODS: The anti-inflammatory effects of ATL-146e were studied in the acute and chronic rabbit formalin-immune complex models of colitis and the SAMP1/YitFc mouse model of spontaneous ileitis. RESULTS: ATL-146e significantly reduced the acute inflammatory index and tissue necrosis compared with vehicle ( P < .01) in the acute model of rabbit immune colitis. In the chronic rabbit immune colitis model, ATL-146e significantly suppressed inflammatory cell infiltration into the colonic mucosa ( P < .05) and prevented mortality. The administration of ATL-146e significantly decreased the chronic inflammatory index ( P < .01) and villus distortion index ( P < .01) in the ileum of SAMP1/YitFc mice, and ameliorated adoptively transferred ileitis in severe combined immunodeficient mice injected with CD4 + T cells from SAMP1/Yit mice ( P < .05). Tumor necrosis factor, interferon gamma, and interleukin 4 concentrations were significantly suppressed by ATL-146e treatment in supernatants from cultures of mesenteric lymph node cells of SAMP1/YitFc mice ( P < .05 vs vehicle-treated mice). CONCLUSIONS: A 2A adenosine receptor activation by ATL-146e significantly reduced inflammation in the intestinal mucosa. This effect was associated with decreased leukocyte infiltration and inhibition of proinflammatory cytokines. Activation of A 2A by selective agonism may therefore serve as a novel therapy for the treatment of inflammatory bowel disease.     

Interleukin 15 activity in the rectal mucosa of inflammatory bowel disease

Background & Aims: Interleukin (IL)-15 has been found to share many immunoregulatory activities in lymphocytes with IL-2. The aim of this study was to investigate IL-15 activity in organ cultures, localization of IL-15 messenger RNA (mRNA), and proliferation of lamina propria mononuclear cells (LPMCs) in response to recombinant IL-15 using the mucosal tissues obtained from patients with inflammatory bowel disease (IBD). Methods: The contents of IL-15, tumor necrosis factor α, and IL-2 in the culture supernatant of the rectal mucosal tissues were determined by an enzyme-linked immunosorbent assay. Expression of IL-15 mRNA was analyzed by in situ hybridization, and proliferative response of LPMCs to recombinant IL-15 was determined by [³H]thymidine incorporation into DNA. Results: Significantly greater IL-15 activity was detected in active IBD, and this elevation was also observed in inactive ulcerative colitis. In contrast, greater tumor necrosis factor α activity was observed only in active IBD, and IL-2 was not detected in organ cultures. In situ hybridization showed IL-15 mRNA in macrophages and epithelial cells in active IBD specimens, and recombinant IL-15 induced a dose-dependent proliferative response in LPMCs. Conclusions: Mucosal IL-15 may be involved in the pathogenesis of IBD as one of the important mediators in activation of mucosal immune cells.GASTROENTEROLOGY 1998;114:1237-1243     

Hyperexpression of inducible costimulator and its contribution on lamina propria T cells in inflammatory bowel disease

BACKGROUND & AIMS: To investigate the role of inducible costimulator (ICOS), a new member of the CD28 family involved in regulation of T-cell activation and chronic intestinal inflammation, we assessed its expression and functional role in patients with inflammatory bowel disease (IBD). METHODS: Expression of ICOS, CD28, and cytotoxic T-lymphocyte antigen (CTLA) 4 on intestinal lamina propria mononuclear cells (LPMC) from patients with ulcerative colitis (UC), Crohn's disease (CD), and normal controls was determined using flow cytometry and immunohistochemistry. Expressions of the ICOS ligand, B7h, on lamina propria B cells, macrophages, and epithelial cells (EC) in the intestinal mucosa were also determined using flow cytometry. The functional costimulatory effect of ICOS on LPMC was assessed by the proliferative response and cytokine production. RESULTS: CD4(+) LPMC expressing ICOS was significantly increased in the inflamed mucosa of IBD patients but not in inflammatory or normal controls. B7h was also significantly up-regulated on B cells, macrophages, and EC in inflamed mucosa of IBD patients. Proliferative responses of anti-CD3/ICOS costimulation were significantly higher compared with those of anti-CD3 monoclonal antibody (mAb) alone. Anti-CD3/ICOS-stimulated-LPMC from UC secreted significantly increased amounts of interleukin (IL)-5 among the 3 groups. In contrast, anti-CD3/ICOS-stimulated-LPMC from CD secreted significantly increased amounts of interferon (IFN)-gamma in the presence of IL-12. CONCLUSIONS: Highly expressed ICOS in activated CD4(+) LPMC of IBD patients contributes to the dysregulated immune responses in IBD. Because ICOS hyperexpression was limited to inflammatory sites in IBD patients, ICOS would be a feasible therapeutic target for the treatment of IBD.