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• W1983336154 abstract "Background & Aims: Defective transforming growth factor (TGF)-β1 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-β1 by enzyme-linked immunosorbent assay. The effect of oral Smad7 antisense oligonucleotide on mucosal inflammation was assessed. Results: TGF-β1 levels were increased in the inflamed tissues of mice with colitis induced by either TNBS or oxazolone. Nevertheless, TGF-β1 did not exert a regulatory effect, probably because TGF-β1 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-β1 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-β1 levels and defective TGF-β1 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. Background & Aims: Defective transforming growth factor (TGF)-β1 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-β1 by enzyme-linked immunosorbent assay. The effect of oral Smad7 antisense oligonucleotide on mucosal inflammation was assessed. Results: TGF-β1 levels were increased in the inflamed tissues of mice with colitis induced by either TNBS or oxazolone. Nevertheless, TGF-β1 did not exert a regulatory effect, probably because TGF-β1 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-β1 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-β1 levels and defective TGF-β1 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. Crohn’s disease and ulcerative colitis, the major forms of inflammatory bowel disease (IBD) in humans, result from the interaction of genetic and environmental factors that ultimately promote an immunopathologic process leading to chronic inflammation.1Podolsky D.K. Inflammatory bowel disease.N Engl J Med. 2002; 347: 417-429Crossref PubMed Scopus (3184) Google Scholar Recent studies of experimental models of colitis suggest that this immunopathologic process consists of an aberrant local immune response to components of the bacterial microflora, either due to abnormally strong effector cell activity or to normal effector cell activity that is poorly controlled by counterregulatory mechanisms.2Bouma G. Strober W. The immunological and genetic basis of inflammatory bowel disease.Nat Rev Immunol. 2003; 3: 521-533Crossref PubMed Scopus (1507) Google Scholar One such counterregulatory mechanism involves transforming growth factor (TGF)-β1, a multifunctional cytokine capable of exerting a number of negative effects on immune cells, including inhibition of T-cell proliferation and differentiation as well as down-regulation of macrophage activation and dendritic cell maturation.3Letterio J.J. Roberts A.B. Regulation of immune responses by TGF-β.Annu Rev Immunol. 1998; 16: 137-161Crossref PubMed Scopus (1686) Google Scholar The role of TGF-β1 in immune homeostasis is dramatically evident in mice with global TGF-β1 defects, such as TGF-β1–null mice or transgenic mice expressing a dominant negative TGFβ receptor (TGF/βR) II chain that are unresponsive to TGF-β1 signaling. The former die soon after birth due to systemic inflammation, and the latter develop severe colitis and pulmonary inflammation.4Kulkarni A.B. Karlsson S. Transforming growth factor-beta 1 knockout mice A mutation in one cytokine gene causes a dramatic inflammatory disease.Am J Pathol. 1993; 143: 3-9PubMed Google Scholar, 5Goreli L. Flavell R.A. Transforming growth factor β in T cell biology.Nat Rev Immunol. 2002; 2: 46-53Crossref PubMed Scopus (709) Google Scholar These manifestations of global TGF-β1 defects are mirrored in mouse models of colitis in that the secretion of TGF-β1 is consistently associated with either the protection from the development of colitis or greatly diminished severity of colitis. This is seen both in the Th1 model of colitis induced by the haptenating reagent trinitrobenzene sulfonic acid (TNBS), which mimics Crohn’s disease, or the Th2 model of colitis induced by the haptenating agent oxazolone, which mimics ulcerative colitis.6Neurath M.F. Fuss I. Kelsall B.L. Presky D.H. Waegell W. Strober W. Experimental granulomatous colitis in mice is abrogated by induction of TGF-β-mediated oral tolerance.J Exp Med. 1996; 183: 2605-2616Crossref PubMed Scopus (384) Google Scholar, 7Boirivant M. Fuss I.J. Chu A. Strober W. Oxazolone colitis: a murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4.J Exp Med. 1998; 188: 1929-1939Crossref PubMed Scopus (443) Google Scholar In addition, it is seen in the colitis of SCID or RAG2-deficient mice receiving CD45RBhigh (naive) T cells where the protective effect of the cotransfer of CD45RBlow T (memory) cells is abolished by concomitant administration of neutralizing TGF-β1 antibody.8Powrie F. Carlino J. Leach M.W. Mauze S. Coffman R.L. A critical role for transforming growth factor-β but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells.J Exp Med. 1996; 183: 2669-2674Crossref PubMed Scopus (788) Google Scholar These and other studies quite conclusively establish that TGF-β1 plays an essential negative regulatory role in the control of experimental colitis. A somewhat different picture, however, emerges from our recent studies of the inflamed tissues of patients with IBD, in which we showed a disruption of TGF-β1 signaling marked by a block in the phosphorylation of Smad3 due to high levels of an inhibitory Smad, Smad7.9Monteleone G. Kumberova A. Croft N.M. McKenzie C. Steer H.W. MacDonald T.T. Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease.J Clin Invest. 2001; 108: 601-609Crossref PubMed Scopus (524) Google Scholar This was substantiated by the fact that down-regulation of Smad7 in vitro with a Smad7 antisense oligonucleotide led to restoration of TGF-β1 signaling.9Monteleone G. Kumberova A. Croft N.M. McKenzie C. Steer H.W. MacDonald T.T. Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease.J Clin Invest. 2001; 108: 601-609Crossref PubMed Scopus (524) Google Scholar These studies showing that TGF-β1 signaling and thus TGF-β1 regulatory function is inhibited by Smad7 in inflamed tissue of patients with IBD (blocked in humans with IBD) raise questions about how TGF-β1 regulates inflammation in mouse models of inflammation. In the present study, we therefore examined TGF-β1–associated signaling and Smad7 in experimental models of colitis and then evaluated whether administration of Smad7 antisense oligonucleotides to mice with colitis characterized by high levels of Smad7 restores endogenous TGF-β1 signaling and ameliorates the disease. Studies of hapten-induced colitis were performed in 5- to 6-week-old male SJL mice (Harlan Laboratories, S. Pietro al Natisone, UD, Italy), which were maintained in the animal facility at the Istituto Superiore di Sanità (Rome, Italy). The experiments were performed within 7 days of the arrival of the animals. All studies were approved by the Animal Care and Use Committee of Istituto Superiore di Sanità and the Italian Ministry of Health. For induction of colitis, 2.5 mg of TNBS or 6 mg of oxazolone (Sigma-Aldrich, Milan, Italy) in 50% ethanol was administered to lightly anesthetized mice through a 3.5F catheter inserted into the rectum. The catheter tip was inserted 4 cm proximal to the anal verge, and 150 μL of fluid was slowly instilled into the colon, after which the mouse was held in a vertical position for 30 seconds. Controls consisted of mice treated with 150 μL of 50% ethanol and untreated naive mice. Weight changes were recorded daily to assess the induction of colitis, and tissues were collected for histologic study and protein analysis. For histologic analysis, tissues were fixed in 10% neutral buffered formalin solution, embedded in paraffin, cut into tissue sections, and stained with H&E. For TNBS-induced colitis, stained sections were examined for evidence of colitis and assigned a colitis score by considering the presence of acute and chronic inflammatory infiltrates, elongation and/or distortion of crypts, frank ulceration, and thickening of the bowel wall, as described elsewhere.6Neurath M.F. Fuss I. Kelsall B.L. Presky D.H. Waegell W. Strober W. Experimental granulomatous colitis in mice is abrogated by induction of TGF-β-mediated oral tolerance.J Exp Med. 1996; 183: 2605-2616Crossref PubMed Scopus (384) Google Scholar For oxazolone-induced colitis, stained sections were examined and assigned a colitis score by examining the slide for the presence of hypervascularization, mononuclear cells, epithelial hyperplasia, epithelial injury, and granulocytes.7Boirivant M. Fuss I.J. Chu A. Strober W. Oxazolone colitis: a murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4.J Exp Med. 1998; 188: 1929-1939Crossref PubMed Scopus (443) Google Scholar, 10Neurath M.F. Weigmann B. Finotto S. Glickman J. Nieuwenhuis E. Iijima H. Mizoguchi A. Mizoguchi E. Mudter J. Galle P.R. Bhan A. Autschbach F. Sullivan B.M. Szabo S.J. Glimcher L.H. Blumberg R.S. The transcription factor T-bet regulates mucosal T cell activation in experimental colitis and Crohn’s disease.J Exp Med. 2002; 195: 1129-1143Crossref PubMed Scopus (520) Google Scholar, 11Boirivant M. Fuss I.J. Ferroni L. De Pascale M. Strober W. Oral administration of recombinant cholera toxin subunit B inhibits IL-12-mediated murine experimental (trinitrobenzene sulfonic acid) colitis.J Immunol. 2001; 166: 3522-3532PubMed Google Scholar Studies of relapsing hapten-induced colitis were performed in BALB/c female mice (Harlan Laboratories). In this case, colitis was induced according to the protocol described by Lawrance et al,12Lawrance I.C. Wu F. Leite A.Z. Willis J. West G.A. Fiocchi C. Chakravarti S. A murine model of chronic inflammation-induced intestinal fibrosis down-regulated by antisense NF-kappa B.Gastroenterology. 2003; 125: 1750-1761Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar with minor modifications. Accordingly, 4 escalating doses of TNBS were administered per rectum at weekly intervals. The first and second doses were 0.5 mg TNBS, whereas the third and fourth doses were 0.75 and 1 mg TNBS, respectively. Controls consisted of mice that were administered 50% ethanol in the absence of TNBS at the same time points as TNBS administration. Tissues were collected from untreated (naive), ethanol-treated, or ethanol/TNBS-treated mice 1 day after the last dose of ethanol or ethanol/TNBS for histologic analysis and measurement of Smad levels. Colitis was assessed as described elsewhere.12Lawrance I.C. Wu F. Leite A.Z. Willis J. West G.A. Fiocchi C. Chakravarti S. A murine model of chronic inflammation-induced intestinal fibrosis down-regulated by antisense NF-kappa B.Gastroenterology. 2003; 125: 1750-1761Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar Briefly, serial paraffin sections of the colon were stained with H&E, and the degree of inflammation was scored as absent, mild, moderate, or severe based on the density and extent of both the acute and chronic inflammatory infiltrate, loss of goblet cells, and bowel wall thickening. An inflammatory infiltrate of low cellularity confined to the mucosa was scored as mild inflammation, and transmural inflammation with extension into the pericolonic adipose tissue with high cellularity was scored as severe. Intermediate changes were scored as moderate inflammation. Colitis was induced in SCID mice according to the protocol described by Read and Powrie.13Read S. Powrie F. Induction of inflammatory bowel disease in immunodeficient mice by depletion of regulatory T cells.in: Coligan J.E. Kruisbeek A.M. Margulies D.H. Shevach E.M. Strober W. Current protocols in immunology. Wiley, Indianapolis, IN1999: 1-10Google Scholar Development of colitis was monitored by weekly record of the body weight and presence of loose stools. Diagnosis of colitis was made by histologic analysis of the colon. To this end, tissues were removed, fixed in 10% buffered formalin phosphate, embedded in paraffin, sectioned, and stained with H&E. The degree of inflammation on microscopic cross sections of the colon was graded semiquantitatively from 0 to 4 (0, no signs of inflammation; 1, very low level; 2, low level of leukocyte infiltration; 3, high level of leukocyte infiltration, high vascular density, thickening of the colon wall; 4, transmural infiltration, loss of goblet cells, high vascular density, thickening of the colon wall). Grading was done in a blinded fashion by 2 experienced pathologists. Because murine and human Smad7 genes are homologous, mouse Smad7 was inhibited by using a specific antisense oligonucleotide that was previously shown to be effective in inhibiting human Smad7.9Monteleone G. Kumberova A. Croft N.M. McKenzie C. Steer H.W. MacDonald T.T. Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease.J Clin Invest. 2001; 108: 601-609Crossref PubMed Scopus (524) Google Scholar Details concerning the Smad7 antisense and sense oligonucleotides used in these studies have been reported previously.9Monteleone G. Kumberova A. Croft N.M. McKenzie C. Steer H.W. MacDonald T.T. Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease.J Clin Invest. 2001; 108: 601-609Crossref PubMed Scopus (524) Google Scholar The oligonucleotides were generated on a phosphorothioate backbone to increase their stability and to minimize degradation. In addition, the cytosine preceding the guanosine was replaced with a 5-methyl-deoxycytosine to avoid activation of immune cells by CpG motifs. To evaluate the ability of Smad7 antisense DNA to inhibit Smad7, murine macrophage cell lines (RAW cells) were transfected with Smad7 antisense or sense oligonucleotides (2 μg/mL) by lipofectamine for 48 hours. The cells were then harvested and subjected to Western blot analysis of Smad7. The efficiency of the transfection was determined with a fluorescein-labeled Smad7 antisense oligonucleotide, and transfection rate was evaluated by performing flow cytometry and fluorescent microscopy 24 hours after transfection. The same fluorescein-labeled Smad7 antisense oligonucleotide was used to assess the in vivo uptake of the orally administered oligonucleotide. To this end, mice with TNBS-induced colitis were given fluorescein-labeled antisense DNA (125 μg/mouse) by oral gavage 1 day after TNBS injection and then killed 0, 4, 8, 16, 24, and 48 hours later. At each time point, stomach, small intestine, colon, liver, spleen, and kidney were collected, cut in sections, and analyzed by fluorescent microscopy. To examine the therapeutic effect of Smad7 antisense oligonucleotide on the course of ongoing intestinal inflammation, mice were treated with a single dose of Smad7 antisense or sense oligonucleotide (from 50, 125, or 250 μg/mouse) in 500 μL of bicarbonate solution (pH 9.5) by oral gavage on the day after the induction of colitis. The mice were monitored daily for weight changes and then killed 3 days (TNBS-induced colitis) and 2 days (oxazolone-induced colitis) after oligonucleotide administration. To evaluate the effect of Smad7 antisense oligonucleotide administration on relapsing TNBS-induced colitis in BALB/c mice, mice were divided into 2 groups the day after the final TNBS administration: one group was treated with Smad7 antisense oligonucleotide and the other with sense oligonucleotide (125 μg/mouse, on days 1 and 3 after the last TNBS dose). Weight changes were recorded daily, and mice were killed at day 6. To evaluate the effect of Smad7 antisense oligonucleotide administration on the SCID model of colitis, 6 weeks after cell transfer the mice were treated with oral Smad7 antisense or sense oligonucleotide (125 μg/mouse) and weight changes were recorded on days 1, 3, 7, and 10 after treatment. Lamina propria mononuclear cells (LPMCs) were isolated as described elsewhere.7Boirivant M. Fuss I.J. Chu A. Strober W. Oxazolone colitis: a murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4.J Exp Med. 1998; 188: 1929-1939Crossref PubMed Scopus (443) Google Scholar, 11Boirivant M. Fuss I.J. Ferroni L. De Pascale M. Strober W. Oral administration of recombinant cholera toxin subunit B inhibits IL-12-mediated murine experimental (trinitrobenzene sulfonic acid) colitis.J Immunol. 2001; 166: 3522-3532PubMed Google Scholar In some experiments, LPMCs were separated into CD3-positive and CD3-negative cells using mouse pan T Dynabeads (Oxoid S.p.A., Garbagnate Milanese, Italy) or by cell sorting after staining cells with anti-mouse CD3–fluorescein isothiocyanate (BD PharMingen, San Diego, CA) using a FACSAria (BD Biosciences, San Jose, CA). Purity of CD3-positive cells was confirmed by flow cytometry and was consistently >95%. Total protein extracts were prepared as previously described.9Monteleone G. Kumberova A. Croft N.M. McKenzie C. Steer H.W. MacDonald T.T. Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease.J Clin Invest. 2001; 108: 601-609Crossref PubMed Scopus (524) Google Scholar Briefly, snap-frozen mucosal samples or cells were homogenized in buffer containing 10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, and 0.2 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, supplemented with 1 mmol/L dithiothreitol, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride (all reagents were from Sigma-Aldrich [Milan, Italy]). Proteins were then analyzed for the content of total TGF-β1 (corresponding to the amount measured in samples after TGF-β1 activation by acidification) and active TGF-β1 (corresponding to the amount of TGF-β1 already activated in vivo and that was measured by directly testing the samples without previous acidification), interleukin (IL)-12/p40, IL-12/p70, IL-4, or interferon (IFN)-γ using sensitive enzyme-linked immunosorbent assay kits (TGF-β1, IL-12/p70, and IL-4: R&D Systems, Abingdon, England; IL-12/p40 and IFN-γ: Peprotech EC Ltd, London, England). Values of cytokines are expressed as picogram per microgram of total proteins. Western blotting was performed on proteins extracted from colonic specimens or cells. For the detection of p-Smad3, blots were incubated with a rabbit anti-mouse p-Smad2/3 followed by horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G monoclonal antibody. Bands were detected by chemiluminescence. After p-Smad2/3 analysis, blots were stripped and incubated with a goat anti-mouse Smad3 antibody followed by a rabbit anti-goat antibody conjugated to horseradish peroxidase. The same protein samples used for analysis of p-Smad3 were also used for analysis of Smad7. In this case, blots were incubated with a rabbit anti-mouse Smad7 antibody followed by a horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin monoclonal antibody. As an internal loading control, blots were stripped and incubated with a mouse anti-human β-actin antibody. p-STAT1 and T-bet were analyzed using rabbit anti-mouse antibodies. All antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The significance of differences between groups was determined using either the Mann–Whitney U test or the Student t test. It has been shown previously that T cells isolated from the colon of mice with TNBS-induced colitis and then cultured in the presence or absence of polyclonal stimulants produce only small amounts of TGF-β1.14Fuss I.J. Boirivant M. Lacy B. Strober W. The interrelated roles of TGF-beta and IL-10 in the regulation of experimental colitis.J Immunol. 2002; 168: 900-908PubMed Google Scholar It was nevertheless possible that other cells might be producing TGF-β1 under these circumstances. Accordingly, TGF-β1 levels were measured in extracts of inflamed or control colon mucosa. Somewhat to our surprise, levels of total TGF-β1 were significantly higher in extracts from mice with TNBS-induced colitis (median, 202.5 pg/100 μg total proteins; range, 35–743 pg/100 μg total proteins) than those from either untreated control (median, 26.1 pg/100 μg total proteins; range, 14–47 pg/100 μg total proteins) or ethanol-treated (median, 35.3 pg/100 μg total proteins; range, 16–100 pg/100 μg total proteins) mice (Figure 1A) (TNBS vs untreated controls or ethanol-treated mice: P < .02). Levels of activated TGF-β1 were also higher in extracts from mice with TNBS-induced colitis (median, 56.5 pg/100 μg total proteins; range, 44–83 pg/100 μg total proteins) than those from untreated (median, 8.2 pg/100 μg total proteins; range, <10–19 pg/100 μg total proteins; P = .03) or ethanol-treated mice (median, 19 pg/100 μg total proteins; range, <10–28 pg/100 μg total proteins; P = .04). Because activated TGF-β1 is generated in the extracellular space, this indicates that increased amounts of TGF-β1 are secreted as well as synthesized in TNBS-induced colitis. We then determined if epithelial cells contributed to the above increase in TGF-β1 levels in colonic extracts. Accordingly, we measured total TGF-β1 levels in extracts obtained from the tissues before and after epithelial depletion. We showed that after epithelial cell depletion, levels of TGF-β1 in TNBS extracts (median, 51.5 pg/100 μg total proteins; range, 49–87.5 pg/100 μg total proteins) were not different from ethanol extracts (median, 49 pg/100 μg total proteins; range, 48–51 pg/100 μg total proteins). In a complementary analysis, TGF-β1 was analyzed in extracts of epithelial cells. Colonic epithelial cells from mice treated with TNBS contained higher levels of TGF-β1 (44 ± 7 pg/100 μg total proteins) than those measured in extracts of ethanol-treated mice (35 ± 2.5 pg/100 μg total proteins), but the difference was not statistically significant. The lack of statistical significance could rely on the fact that the isolated colonic cells undergo rapid apoptosis. Taken together, these data show that TNBS-induced colitis is associated with a significant increase in TGF-β1 production that is, at least in part, derived from epithelial cells. Inasmuch as suppression of immune responses by TGF-β1 usually involves activation of Smad proteins,15Heldin C. -H, Kohei M, ten Dijke P TGF-beta signalling from cell membrane to nucleus through SMAD proteins.Nature. 1997; 390: 465-471Crossref PubMed Scopus (3358) Google Scholar we then determined whether the high TGF-β1 level was associated with activation (phosphorylation) of Smad3. Extracts of colonic specimens were analyzed for p-Smad3 by Western blotting. Samples from mice with TNBS-induced colitis exhibited reduced p-Smad3 in comparison with controls (Figure 1B). Furthermore, active (phosphorylated)/inactive Smad3 ratios were significantly decreased in mice with TNBS-induced colitis (median, 0.33; range, 0–0.8) compared with untreated (median, 0.61; range, 0.44–0.8) or ethanol-treated (median, 0.6; range, 0.4–0.88) mice (Figure 1B, right panel, P < .01). The TGF-β1/Smad signaling pathway is subject to autoinhibition by Smad7, which blocks the docking of Smad3 to the TGFβRI chain and the phosphorylation of Smad3.16Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.H. ten Dijke P. Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling.Nature. 1997; 389: 631-635Crossref PubMed Scopus (1572) Google Scholar Thus, the reduced Smad3 levels noted previously could be due to increased expression of Smad7. Accordingly, Smad7 was analyzed by Western blotting in the same specimens previously used to analyze p-Smad3. Smad7 expression quantitated by densitometry and normalized by β-actin expression was significantly increased in mice with TNBS-induced colitis (median, 1.07; range, 0.77–1.5) as compared with untreated (median, 0.22; range, 0–0.4) and ethanol-treated (median, 0.32; range, 0–0.66) mice (P < .001) (Figure 1C). High Smad7 levels were also seen in unfractionated LPMCs as well as in lamina propria CD3-positive T lymphocytes and CD3-negative LPMCs isolated from inflamed intestine of mice with TNBS-induced colitis (Figure 1D). These data suggest that despite the presence of substantial levels of TGF-β1 in the inflammatory milieu of TNBS-induced colitis, TGF-β1 fails to exert a regulatory influence because of a block in the Smad pathway. The above data prompted us to explore the possibility that in TNBS-induced colitis, a high level of local Smad7 blocks the immunosuppressive activity of the endogenous TGF-β1 and thus contributes to ongoing intestinal inflammation. We thus determined whether the administration of Smad7-specific antisense oligonucleotide could affect the course of experimental colitis. In initial studies, we examined the capacity of the oligonucleotide to suppress Smad7 in RAW cells that constitutively expresses Smad7. In these studies, RAW cells were transfected with Smad7 antisense or sense oligonucleotides and then assessed for Smad7 protein expression by Western blotting. Using a fluorescein-labeled Smad7 antisense DNA to identify transfected cells by flow cytometry, almost two thirds of the RAW cells were efficiently transfected (Figure 2A). In addition, the transfected cells exhibited a marked decrease in Smad7 expression (Figure 2A, right inset). These results led us to determine the ability of this oligonucleotide to inhibit Smad7 in vivo. Initially, we administered fluorescein-labeled Smad7 antisense oligonucleotide to mice with TNBS-induced colitis at day 1 after induction of colitis by oral gavage to determine the tissue distribution of oligonucleotide administered by this route. Administration by oral gavage was selected so as to target the mucosal tissues rather than systemic tissues and thus to minimize potential adverse effects. Fluorescein-labeled oligonucleotide was seen in both the small intestinal and colonic lamina propria and epithelial layer at 4, 8 (Figure 2B), and 16 hours after oligonucleotide administration and then disappeared at 24 and 48 hours after administration (not shown). In contrast, fluorescein signals were not seen in the stomach, liver, kidney, or spleen at any time point (not shown). Finally, we determined whether administration of Smad7 antisense oligonucleotide administered by oral gavage affected Smad7 and p-Smad3 expression in the colon of mice with TNBS-induced colitis. Administration of Smad7 antisense but not sense oligonucleotide significantly reduced Smad7 (P = .02) and enhanced p-Smad3 (P = .03) levels (Figure 2C). We first evaluated the therapeutic efficacy of Smad7 antisense oligonucleotide in mice with TNBS-induced colitis. As reported previously,6Neurath M.F. Fuss I. Kelsall B.L. Presky D.H. Waegell W. Strober W. Experimental granulomatous colitis in mice is abrogated by induction of TGF-β-mediated oral tolerance.J Exp Med. 1996; 183: 2605-2616Crossref PubMed Scopus (384) Google Scholar, 11Boirivant M. Fuss I.J. Ferroni L. De Pascale M. Strober W. Oral administration of recombinant cholera toxin subunit B inhibits IL-12-mediated murine experimental (trinitrobenzene sulfonic acid) colitis.J Immunol. 2001; 166: 3522-3532PubMed Google Scholar rectal instillation of TNBS induces a colitis marked by profound and sustained weight loss. In contrast, mice administered Smad7 antisense oligonucleotide as a single oral dose of 125 or 250 μg/mouse (but not 50 μg/mouse) on day 1 after induction of TNBS colitis exhibited an initial weight loss that rapidly stabilized so that by day 4 the weight loss in the treated group was clearly lower than in the untreated group (P < .01) (Figure 3A and B) and by day 7 it was not different from the initial weight (not shown). In addition, as shown in Figure 3C, histologic examination of colon tissue as well as blinded histologic scoring of colitis in the different groups were significantly reduced in antisense-treat" @default.
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• W1983336154 title "Inhibition of Smad7 With a Specific Antisense Oligonucleotide Facilitates TGF-β1–Mediated Suppression of Colitis" @default.
• W1983336154 cites W1504236935 @default.
• W1983336154 cites W1553444759 @default.
• W1983336154 cites W1574615823 @default.
• W1983336154 cites W1587849076 @default.
• W1983336154 cites W1663371441 @default.
• W1983336154 cites W1954734381 @default.
• W1983336154 cites W1967261754 @default.
• W1983336154 cites W2008313660 @default.
• W1983336154 cites W2017443683 @default.
• W1983336154 cites W2026058763 @default.
• W1983336154 cites W2039992202 @default.
• W1983336154 cites W2050339689 @default.
• W1983336154 cites W2118202213 @default.
• W1983336154 cites W2129379081 @default.
• W1983336154 cites W2133226723 @default.
• W1983336154 cites W2134560060 @default.
• W1983336154 cites W2144661894 @default.
• W1983336154 cites W2150605496 @default.
• W1983336154 cites W2158673542 @default.
• W1983336154 cites W2165885428 @default.
• W1983336154 cites W2168036150 @default.
• W1983336154 cites W4292198229 @default.
• W1983336154 cites W4297022993 @default.
• W1983336154 doi "https://doi.org/10.1053/j.gastro.2006.09.016" @default.
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