M2 Macrophages and Phenotypic Modulation of Intestinal Smooth Muscle Cells Characterize Inflammatory Stricture Formation in Rats
2020; Elsevier BV; Volume: 190; Issue: 9 Linguagem: Inglês
10.1016/j.ajpath.2020.05.015
ISSN1525-2191
AutoresSandra Lourenssen, Michael G. Blennerhassett,
Tópico(s)Fibroblast Growth Factor Research
ResumoThe progression of Crohn disease to intestinal stricture formation is poorly controlled, and the pathogenesis is unclear, although increased smooth muscle mass is present. A previously described rat model of trinitrobenzenesulfonic acid–induced colitis is re-examined here. Although inflammation of the mid-descending colon typically resolved, a subset showed characteristic stricturing by day 16, with an inflammatory infiltrate in the neuromuscular layers including eosinophils, CD3-positive T cells, and CD68-positive macrophages. Closer study identified CD163-positive, CD206-positive, and arginase-positive cells, indicating a M2 macrophage phenotype. Stricturing involved ongoing proliferation of intestinal smooth muscle cells (ISMC) with expression of platelet-derived growth factor receptor beta and progressive loss of phenotypic markers, and stable expression of hypoxia inducible factor 1 subunit alpha. In parallel, collagen I and III showed a selective and progressive increase over time. A culture model of the stricture phenotype of ISMC showed stable hypoxia inducible factor 1 subunit alpha expression that promoted growth and improved both survival and growth in models of experimental ischemia. This phenotype was hyperproliferative to serum and platelet-derived growth factor BB, and unresponsive to transforming growth factor beta, a prominent cytokine of M2 macrophages, compared with control ISMC. We identified a hyperplastic phenotype of ISMC, uniquely adapted to an ischemic environment to drive smooth muscle layer expansion, which may reveal new targets for treating intestinal fibrosis. The progression of Crohn disease to intestinal stricture formation is poorly controlled, and the pathogenesis is unclear, although increased smooth muscle mass is present. A previously described rat model of trinitrobenzenesulfonic acid–induced colitis is re-examined here. Although inflammation of the mid-descending colon typically resolved, a subset showed characteristic stricturing by day 16, with an inflammatory infiltrate in the neuromuscular layers including eosinophils, CD3-positive T cells, and CD68-positive macrophages. Closer study identified CD163-positive, CD206-positive, and arginase-positive cells, indicating a M2 macrophage phenotype. Stricturing involved ongoing proliferation of intestinal smooth muscle cells (ISMC) with expression of platelet-derived growth factor receptor beta and progressive loss of phenotypic markers, and stable expression of hypoxia inducible factor 1 subunit alpha. In parallel, collagen I and III showed a selective and progressive increase over time. A culture model of the stricture phenotype of ISMC showed stable hypoxia inducible factor 1 subunit alpha expression that promoted growth and improved both survival and growth in models of experimental ischemia. This phenotype was hyperproliferative to serum and platelet-derived growth factor BB, and unresponsive to transforming growth factor beta, a prominent cytokine of M2 macrophages, compared with control ISMC. We identified a hyperplastic phenotype of ISMC, uniquely adapted to an ischemic environment to drive smooth muscle layer expansion, which may reveal new targets for treating intestinal fibrosis. Crohn disease (CD) is an inflammatory bowel disease causing chronic transmural inflammation that typically affects the terminal ileum and colon.1Torres J. Mehandru S. Colombel J.F. Peyrin-Biroulet L. Crohn's disease.Lancet. 2017; 389: 1741-1755Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar By the time of diagnosis, roughly a quarter of patients already have complications such as stenosis or abscess formation, and nearly all will do so after a decade.2Li J. Mao R. Kurada S. Wang J. Lin S. Chandra J. Rieder F. Pathogenesis of fibrostenosing Crohn's disease.Transl Res. 2019; 209: 39-54Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar,3Ma C. Jairath V. Click B. Hirota S.A. Lu C. Parker C.E. Rieder F. Stenosis Therapy and Anti-Fibrotic Research (STAR) ConsortiumTargeting anti-fibrotic pathways in Crohn's disease - the final frontier?.Best Pract Res Clin Gastroenterol. 2019; 38-39: 101603Crossref PubMed Scopus (9) Google Scholar This progression to stricture formation and subsequent surgery in CD is only poorly influenced by current therapy. By contrast, the other principal form of inflammatory bowel disease, ulcerative colitis, also causes an incurable, relapsing, chronic inflammation, but this affects the colonic mucosa and only rarely affects the deeper structures to any extent and is not associated with stricture formation.4de Bruyn J.R. Meijer S.L. Wildenberg M.E. Bemelman W.A. van den Brink G.R. D'Haens G.R. Development of fibrosis in acute and longstanding ulcerative colitis.J Crohns Colitis. 2015; 9: 966-972Crossref PubMed Scopus (49) Google Scholar This implies that the pathogenesis of CD involves key alterations or participation of the deeper neuromuscular layer, which may be related to the high frequency of recurrent stricture formation following surgery.5Burke J.P. Mulsow J.J. O'Keane C. Docherty N.G. Watson R.W. O'Connell P.R. Fibrogenesis in Crohn's disease.Am J Gastroenterol. 2007; 102: 439-448Crossref PubMed Scopus (225) Google Scholar,6Dietz D.W. Laureti S. Strong S.A. Hull T.L. Church J. Remzi F.H. Lavery I.C. Fazio V.W. Safety and longterm efficacy of strictureplasty in 314 patients with obstructing small bowel Crohn's disease.J Am Coll Surg. 2001; 192: 330-337Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar Animal models have been valuable in illustrating the processes that may occur in CD, identifying inflammatory processes and allowing interventions that clarify the underlying molecular and cellular biology. For example, the widely used model of trinitrobenzenesulfonic acid (TNBS)-induced inflammation in rodents causes a TH1-dominant transmural inflammation that resembles aspects of CD.7Antoniou E. Margonis G.A. Angelou A. Pikouli A. Argiri P. Karavokyros I. Papalois A. Pikoulis E. The TNBS-induced colitis animal model: an overview.Ann Med Surg (Lond). 2016; 11: 9-15Crossref PubMed Scopus (181) Google Scholar Key findings from this model that are relevant to understanding the pathogenesis of stricture formation include the demonstration that inflammation causes proliferation of the intestinal smooth muscle cells (ISMC), which then leads to a lasting thickening of the intestinal wall.8Blennerhassett M.G. Lourenssen S.R. Parlow L.R.G. Ghasemlou N. Winterborn A.N. Analgesia and mouse strain influence neuromuscular plasticity in inflamed intestine.Neurogastroenterol Motil. 2017; 29: 1-12Crossref PubMed Scopus (10) Google Scholar, 9Lourenssen S. Wells R.W. Blennerhassett M.G. Differential responses of intrinsic and extrinsic innervation of smooth muscle cells in rat colitis.Exp Neurol. 2005; 195: 497-507Crossref PubMed Scopus (52) Google Scholar, 10Nair D.G. Han T.Y. Lourenssen S. Blennerhassett M.G. Proliferation modulates intestinal smooth muscle phenotype in vitro and in colitis in vivo.Am J Physiol Gastrointest Liver Physiol. 2011; 300: G903-G913Crossref PubMed Scopus (37) Google Scholar This process begins at the onset of inflammation, when proinflammatory cytokines induce expression of platelet-derived growth factor (PDGF)-Rβ in ISMC in vitro and in vivo that confers sensitivity to the major mesenchymal mitogen PDGF-BB.11Nair D.G. Miller K.G. Lourenssen S.R. Blennerhassett M.G. Inflammatory cytokines promote growth of intestinal smooth muscle cells by induced expression of PDGF-Rbeta.J Cell Mol Med. 2014; 18: 444-454Crossref PubMed Scopus (41) Google Scholar,12Stanzel R.D.P. Lourenssen S. Nair D.G. Blennerhassett M.G. Mitogenic factors promoting intestinal smooth muscle cell proliferation.Am J Physiol Cell Physiol. 2010; 299: C805-C817Crossref PubMed Scopus (22) Google Scholar Further, the phenotype of the ISMC remains altered even after proliferation ceases and inflammation is resolved, with decreased expression of smooth muscle marker proteins.10Nair D.G. Han T.Y. Lourenssen S. Blennerhassett M.G. Proliferation modulates intestinal smooth muscle phenotype in vitro and in colitis in vivo.Am J Physiol Gastrointest Liver Physiol. 2011; 300: G903-G913Crossref PubMed Scopus (37) Google Scholar Recent work showed that protracted proliferation of rat ISMC in vitro, as might occur in chronic disease in vivo, caused epigenetic modification of phenotype with outcomes similar to those identified in ISMC from Crohn strictures, but absent from adjacent normal regions.13Bonafiglia Q.A. Lourenssen S.R. Hurlbut D.J. Blennerhassett M.G. Epigenetic modification of intestinal smooth muscle cell phenotype during proliferation.Am J Physiol Cell Physiol. 2018; 315: C722-C733Crossref PubMed Scopus (12) Google Scholar Although intestinal strictures in CD have been characterized as fibrostenotic, implying a noncellular accumulation of collagen, there is increasing evidence that increased smooth muscle, with an altered ISMC phenotype, is the predominant component.13Bonafiglia Q.A. Lourenssen S.R. Hurlbut D.J. Blennerhassett M.G. Epigenetic modification of intestinal smooth muscle cell phenotype during proliferation.Am J Physiol Cell Physiol. 2018; 315: C722-C733Crossref PubMed Scopus (12) Google Scholar, 14Van Assche G. Geboes K. Rutgeerts P. Medical therapy for Crohn's disease strictures.Inflamm Bowel Dis. 2004; 10: 55-60Crossref PubMed Scopus (154) Google Scholar, 15Flynn R.S. Murthy K.S. Grider J.R. Kellum J.M. Kuemmerle J.F. Endogenous IGF-I and alphaVbeta3 integrin ligands regulate increased smooth muscle hyperplasia in stricturing Crohn's disease.Gastroenterology. 2010; 138: 285-293Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 16Chen W. Lu C. Hirota C. Iacucci M. Ghosh S. Gui X. Smooth muscle hyperplasia/hypertrophy is the most prominent histological change in Crohn's fibrostenosing bowel strictures: a semiquantitative analysis by using a novel histological grading scheme.J Crohns Colitis. 2017; 11: 92-104Crossref PubMed Scopus (79) Google Scholar ISMC in the Crohn stricture are typically labeled with antibodies to markers for the contractile smooth muscle phenotype, such as α-smooth muscle actin (SMA), SM-22, or CG4A, which detects cells expressing both α- and γ-smooth muscle actin. However, coexpression with vimentin was also detected in some cells, indicative of a dedifferentiated smooth muscle phenotype.17Suekane T. Ikura Y. Watanabe K. Arimoto J. Iwasa Y. Sugama Y. Kayo S. Sugioka K. Naruko T. Maeda K. Hirakawa K. Arakawa T. Ueda M. Phenotypic change and accumulation of smooth muscle cells in strictures in Crohn's disease: relevance to local angiotensin II system.J Gastroenterol. 2010; 45: 821-830Crossref PubMed Scopus (18) Google Scholar The key role of ISMC is strongly supported in a recent quantitative study of Crohn strictures, showing that expansion of the smooth muscle layers is the single major contributor to the outcome, with fibrosis (ie, inappropriate expression of extracellular matrix2Li J. Mao R. Kurada S. Wang J. Lin S. Chandra J. Rieder F. Pathogenesis of fibrostenosing Crohn's disease.Transl Res. 2019; 209: 39-54Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) serving only a minimal role.16Chen W. Lu C. Hirota C. Iacucci M. Ghosh S. Gui X. Smooth muscle hyperplasia/hypertrophy is the most prominent histological change in Crohn's fibrostenosing bowel strictures: a semiquantitative analysis by using a novel histological grading scheme.J Crohns Colitis. 2017; 11: 92-104Crossref PubMed Scopus (79) Google Scholar This directly implicates the ISMC in the development of the stricture and the resulting luminal narrowing. Although thickening of the inflamed intestinal wall is well-reported, the stricturing process has been difficult to recapitulate in animal models. For example, smooth muscle hyperplasia and hypertrophy occur in TNBS colitis in both rats and mice, but this is normally observed for a period of a few days at most, when inflammation resolves and normal intestinal function returns. However, in Sprague-Dawley rats followed for a longer period (30 to 90 days), it was found that a subset of animals developed strictures at the site of previous inflammation.18Marlow S.L. Blennerhassett M.G. Deficient innervation characterizes intestinal strictures in a rat model of colitis.Exp Mol Pathol. 2006; 80: 54-66Crossref PubMed Scopus (32) Google Scholar By 30 days post-TNBS, about 30% of animals showed stenotic intestinal tissue with marked thickening and disorganization of the smooth muscle layers, which persisted to at least 90 days post-TNBS. Microscopic analysis showed normal tissue surrounding the strictured region, which displayed aberrant mucosal architecture in all samples. The ISMC showed an altered phenotype characterized by reduced desmin and α-SMA labeling, and the onset of vimentin expression. This model mimics the sporadic stricture formation seen in CD. Because it occurred after a Crohn-like inflammation, it could offer valuable insight into this poorly understood phenomenon. Accordingly, this model was further examined to identify earlier events that might predict or define stricture pathogenesis. This study identified and characterized divergent responses among animals as the acute colitis was resolved, identifying a persistent inflammatory milieu with the presence of M2 (alternatively activated) macrophages. Proliferating ISMC displayed abundant PDGF-Rβ expression that was associated with reduced expression of contractile markers, evidence for loss of contractile phenotype. However, the authors also found prominent nuclear HIF-1α localization that correlated with increased survival during metabolic or ischemic challenge in vitro. Further, the tissue culture model of the stricture phenotype of ISMC showed insensitivity to TGFβ, a cytokine that normally strongly suppressed ISMC growth. This suggested that in some animals, ISMC undergo ongoing exposure to PDGF-BB and inflammatory cytokines, leading to a permanently dedifferentiated phenotype that drives stricture formation. These histologic and immunocytochemical changes can provide insight into the development of approaches that can minimize or reverse the damaging effects of stricturing in CD. Adult Sprague-Dawley rats were obtained from Charles River Laboratories (Montreal, QC, Canada) and housed in pairs in microfilter-isolated cages with free access to food and water, for at least 5 days prior to use. To induce inflammation, 500 μL of 200 mmol/L TNBS (Fluka; Canadian Life Science, Peterborough, ON, Canada) dissolved in 50% ethanol was instilled into the mid-descending colon of rats (200 to 250 g) under light anesthesia with inhaled isoflurane, as previously described.11Nair D.G. Miller K.G. Lourenssen S.R. Blennerhassett M.G. Inflammatory cytokines promote growth of intestinal smooth muscle cells by induced expression of PDGF-Rbeta.J Cell Mol Med. 2014; 18: 444-454Crossref PubMed Scopus (41) Google Scholar Animals received subcutaneous injections with 1 mL of lactated Ringer saline from days 1 to 4 post-TNBS. There was no animal mortality associated with this procedure, and all experimental procedures were approved by the Queen's University Animal Care Committee. At day 6 (D6), 16 (D16), 35 (D35), or 90 (D90) post-TNBS administration, rats were sacrificed by cervical dislocation under isoflurane anesthesia. The mid-descending colon was removed and fixed immediately in 4% neutral buffered formalin or opened along the mesenteric border and pinned flat in phosphate-buffered saline (PBS; pH 7.4; NaCl 8 g, KCl 0.2 g, Na2HPO4 1.44 g, KH2PO4 0.24 g, double-distilled H2O 1 L) before fixing for 24 hours. Fixed tissue was embedded in paraffin and sectioned at 4 μm. Sections were stained with hematoxylin and eosin for routine examination as described previously.18Marlow S.L. Blennerhassett M.G. Deficient innervation characterizes intestinal strictures in a rat model of colitis.Exp Mol Pathol. 2006; 80: 54-66Crossref PubMed Scopus (32) Google Scholar Consecutive sections were treated with Sirius Red/Fast Green (BDH Chemicals; VVR International, Mississauga, ON, Canada) differential stain to visualize the proportion of collagen to cellular protein as described.18Marlow S.L. Blennerhassett M.G. Deficient innervation characterizes intestinal strictures in a rat model of colitis.Exp Mol Pathol. 2006; 80: 54-66Crossref PubMed Scopus (32) Google Scholar Images of stained tissue were acquired by light microscopy (Olympus BX60; Infinity 2 Camera; Infinity Capture; Teledyne Lumenera Corp., Ottawa, ON, Canada), and the proportions of collagen (red) and noncollagen (green) protein were determined using Image Pro Plus software version 6 (Media Cybernetics, Rockville, MD); at least three nonadjacent images were analyzed for each tissue. Specific cell types and cell components were detected using immunocytochemistry. Smooth muscle cells were detected with anti–α-SM22 or anti-smoothelin antibodies (both 1:2000; Abcam, Cambridge, MA). The presence of macrophages was detected using anti-CD68 (1:150; AbD Serotec, Kidlington, UK), anti-CD163 (1:150; AbD Serotec), anti-CD206 (1:100; Santa Cruz Biotechnology, Dallas, TX), or anti-arginase (1:200; Cell Signaling Technology, Danvers, MA), and T cells were detected using anti-pan T antibodies (anti-CD3, pre-diluted; Dako Canada, Mississauga, ON, Canada). Proliferating cells were detected with antibodies to proliferating cell nuclear antigen (PCNA; 1:4000; Cell Signaling Technology). Apoptotic cells were detected with anti-cleaved caspase-3 antibodies (1:200; Cell Signaling Technology). The receptor for PDGF-BB was detected using anti-PDGFRβ antibodies (Cell Signaling Technology; 1:200), and hypoxia was detected with anti-HIF1α antibodies (1:200; Novus, Oakville, ON, Canada). Collagen was detected with anti-collagen I and anti-collagen III (both 1:150; Novus). Further details are provided in Table 1.Table 1Primary and Secondary Antibodies Used for ImmunocytochemistryAntibodySourceCatalog numberDilutionAntigen retrievalPrimary antibody Arginase-1NEB936681:200DAKO TRS CD3DAKON1580Pre-dilutedDAKO TRS CD68SerotecMCA341GA clone ED11:300DAKO TRS CD163SerotecMCA342GA clone ED21:300DAKO TRS CD206Santa Cruzsc-589861:100NA Cleaved caspase-3NEB96641:200Citrate, pH6 Collagen INovusNB600-4081:150DAKO TRS Collagen IIINovusNB600-5941:150Citrate, pH 6 HIF 1αNovusNB100-4791:200DAKO TRS iNOSNovusNB300-6051:200DAKO TRS iNOSBD Transduction6103321:500DAKO TRS Mast cell tryptaseDAKOM70521:200DAKO TRS PCNADAKO25861:4000NA PDGF-RβNEB31691:200DAKO TRS α-SM22Abcamab141061:2000NA SmoothelinAbcamab2196521:2000DAKO TRSSecondary antibody Goat anti-mouse Alexa 488Life TechnologiesA110011:1000NA Goat anti-mouse Alexa 555Life TechnologiesA214221:1000NA Goat anti-rabbit Alexa 555Life TechnologiesA214281:2000NA Goat anti-rabbit Alexa 488Life TechnologiesA110081:2000NAHIF 1α, hypoxia-inducible factor 1-alpha; iNOS, inducible nitric oxide synthase; NA, not applicable; PCNA, proliferating cell nuclear antigen; PDGF-Rβ platelet derived growth factor receptor beta. Open table in a new tab HIF 1α, hypoxia-inducible factor 1-alpha; iNOS, inducible nitric oxide synthase; NA, not applicable; PCNA, proliferating cell nuclear antigen; PDGF-Rβ platelet derived growth factor receptor beta. All antibodies were diluted in PBS with 0.2% Tween-20 (PBS-T), and where necessary, tissue was incubated with citrate buffer (pH 6) at 95°C for 20 minutes or with 1× target retrieval solution (TRS; Dako Canada) according to the manufacturer's directions. Slides were then incubated with 1% goat serum in PBS-T for 1 hour to block nonspecific binding before overnight incubation in primary antibodies at 4°C, followed by incubation in the appropriate Alexa-linked secondary antibodies (Millipore Canada, Etobicoke, ON, Canada) for 2 hours at room temperature. Hoechst 33342 (Sigma-Aldrich, St. Louis, MO; 1 μg/mL) was used to label all nuclei, and staining was visualized by fluorescence microscopy (Olympus BX51; Olympus, Tokyo, Japan). Images of three to six nonadjacent fields of were acquired using a fluorescence microscope (Olympus BX51) and Retiga2000R camera linked to Q-Capture Pro 7 (both Teledyne QImaging, Surrey, BC, Canada). Images of nonadjacent regions to the stricture were acquired in areas at least 500 μm distant from affected regions. For measurement of the intensity of smooth muscle marker labeling, images were acquired at standardized nonsaturating exposure times. Each glass slide contained both control sections (noninflamed; no prior TNBS exposure) as well as experimental sections to allow internal comparisons. The intensity of immunostaining was measured for 10 nonadjacent circular smooth muscle (CSM) cells per image (Image Pro Plus) for at least 3 nonadjacent images per section. These were averaged and expressed relative to control ISMC intensity. Primary cultures of adult rat colonic CSM cells were obtained from strips of circular muscle that were microdissected from the mid-descending colon. These were incubated in HEPES-buffered digestion solution [papain (0.5 mg/mL), bovine serum albumin (1 mg/mL), dithiothreitol (1 μmol/L), collagenase (type F, 0.25 mg/mL)] at 4°C for 2 hours, followed by 1 hour at room temperature and then 37°C for 1 hour. Individual cells were obtained by trituration and these were plated onto 60-mm culture dishes in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum (FCS; Invitrogen, Carlsbad, CA). Culture purity was confirmed by positive staining for smooth muscle markers and the lack of immunostaining for glia or interstitial cells of Cajal as described previously.10Nair D.G. Han T.Y. Lourenssen S. Blennerhassett M.G. Proliferation modulates intestinal smooth muscle phenotype in vitro and in colitis in vivo.Am J Physiol Gastrointest Liver Physiol. 2011; 300: G903-G913Crossref PubMed Scopus (37) Google Scholar Once cultures reached 80% to 95% confluency, they were passaged (1:2 ratio) 1 to 3 times to generate low-passage cells, or 10 or more times to generate high-passage cells.13Bonafiglia Q.A. Lourenssen S.R. Hurlbut D.J. Blennerhassett M.G. Epigenetic modification of intestinal smooth muscle cell phenotype during proliferation.Am J Physiol Cell Physiol. 2018; 315: C722-C733Crossref PubMed Scopus (12) Google Scholar For antibody staining of cultured ISMC, cells were plated on collagen-coated glass cover slips (Sigma-Aldrich) and incubated in DMEM containing 5% FCS for 48 hours. Cells were fixed in neutral buffered formalin and incubated with antibodies to HIF-1α, and staining was visualized as above. For experimental treatments, ISMC were plated into 96-well microplates at 7500 cells/well in DMEM with 5% FCS for 24 hours, serum-deprived for 24 hours, and exposed to the metabolic uncoupler dinitrophenol (50 to 750 μmol/L in DMEM). Alternatively, cells were incubated in a hypoxic chamber (1% O2; BioSpherix, Parish, NY) for 24 hours. The effect of inhibition or activation of HIF-1α on cell proliferation was tested using chetomin or cobalt chloride (Sigma-Aldrich). In some experiments, cells were treated with PDGF-BB (0.05 to 50 ng/mL; Sigma-Aldrich) and cotreatment or 1-hour pretreatment with TGF-β (50 ng/mL). Cell number was assessed using the WST-8 assay (Cayman, Ann Arbor, MI) according to the manufacturer's instructions. Cells were treated for 30 minutes or 18 hours with TGFβ (50 ng/mL), then lysed using 1× Laemmli sample buffer (20 mmol/L Tris/HCl, 6% glycerol, 0.5% SDS, 2% β-mercaptoethanol, 0.001% bromophenol blue). Samples were resolved using 10% SDS-PAGE, then transferred to polyvinylidene difluoride membrane using a semidry apparatus (Bio-Rad, Hercules, CA). Membranes were incubated in blocking buffer (5% skim milk in Tris-buffered saline with 0.2% Tween 20) for 60 minutes at room temperature. Blots were incubated at 4°C overnight with primary anti-pSMAD 2/3 (1:1000; Novus), then horseradish peroxidase–linked secondary antibodies for 2 hours at room temperature and visualized using a chemiluminescent substrate (Luminata Forte; Millipore Canada) with a ChemiDoc MP system (Bio-Rad). Blots were reprobed with anti–β-actin antibodies (1:5000; Sigma-Aldrich) to confirm equal loading of cell samples. Quantitative data are expressed as means ± SEM of n animals. Differences between control and treatment conditions were considered significant for P ≤ 0.05 using analysis of variance or Kruskal-Wallis with Dunnett post-test. Differences between two groups are considered significant for P ≤ 0.05 using a two-tailed t-test. The authors' earlier study of the outcome of TNBS-induced colitis in Sprague-Dawley rats showed that a subset of animals developed intestinal strictures by 35 days post-TNBS (D35), with characteristics similar to those of human strictures in CD.18Marlow S.L. Blennerhassett M.G. Deficient innervation characterizes intestinal strictures in a rat model of colitis.Exp Mol Pathol. 2006; 80: 54-66Crossref PubMed Scopus (32) Google Scholar Now, this was examined more closely to identify the events during the progression of inflammation that might correlate with subsequent stricture formation. Application of TNBS causes a transmural inflammation that is predictably situated in the mid-descending colon. The peak inflammatory response on day 6 subsequently resolves, and the majority of animals display only a thickened muscular wall by D35.9Lourenssen S. Wells R.W. Blennerhassett M.G. Differential responses of intrinsic and extrinsic innervation of smooth muscle cells in rat colitis.Exp Neurol. 2005; 195: 497-507Crossref PubMed Scopus (52) Google Scholar,10Nair D.G. Han T.Y. Lourenssen S. Blennerhassett M.G. Proliferation modulates intestinal smooth muscle phenotype in vitro and in colitis in vivo.Am J Physiol Gastrointest Liver Physiol. 2011; 300: G903-G913Crossref PubMed Scopus (37) Google Scholar Standard histology showed a typical severe transmural inflammation by D6, with no apparent differences among animals by qualitative examination (Figure 1, D and E). By D16, roughly halfway to the previous endpoint of D35, the mid-descending colon appeared similar to the control. Indeed, the histology of the mid-descending colon of most animals showed an intact mucosa and no evidence of inflammation, although the smooth muscle layers were noticeably thickened (Figure 1F) as described.9Lourenssen S. Wells R.W. Blennerhassett M.G. Differential responses of intrinsic and extrinsic innervation of smooth muscle cells in rat colitis.Exp Neurol. 2005; 195: 497-507Crossref PubMed Scopus (52) Google Scholar However, a subset of rats (25/102) displayed ongoing circumferential inflammation and obvious changes in tissue microarchitecture. There was prominent local thickening of the smooth muscle layers (Figure 1, A–C), with mucosal damage and variable cellular disorganization in the muscularis externa accompanied by an immune cell infiltrate (Figure 1G). Overall, this resembled the stricture seen at D35, where both macroscopic and microscopic changes were readily apparent (Figure 1, G–I). Examination of the course of weight loss and recovery during TNBS-induced colitis showed a similar initial weight loss among animals regardless of stricturing outcome. However, there was a significantly slower weight gain during the recovery phase for animals that went on to show stricture formation (Figure 1J) (n = 13 to 22 rats/time point). Combined with the histological outcome, this shows that progressive changes leading to stricture formation are present well before D35. In the subset of animals with persistent inflammation, there was a characteristic abundance of immune cells in the submucosa and smooth muscle of the mid-descending colon (Figure 1), and the major contributing cell types were quantified. Evaluation of sections stained with hematoxylin and eosin showed that the number of eosinophils in the smooth muscle was greatly increased in D6 animals but remained equally high in those showing continued inflammation at D16 (D16 stricture) (Figure 2A). By contrast, eosinophils became rare or absent in cohort animals that were recovering normally (D16 recovery) (Figure 2A). Similarly, eosinophils were typically present in the smooth muscle of D35 strictures, whereas they were absent from D35 recovered cohorts, and this persisted through D90 (Figure 2B). In the smooth muscle layers, immunocytochemistry with antibodies to the pan-macrophage marker CD68 or the T-cell marker CD3 (CD4/8 T cells) showed that both markers were nearly completely absent from the control intestine, but readily detected in the inflamed intestine on D6 (Figure 2C). These dropped to undetectable levels in D16 recovery animals, but remained high in D16 stricture animals, with a similar pattern evident on D35 and D90 (Figure 2, D and E). Mast cells were normally undetectable in the smooth muscle layers of the control or inflamed (D6) intestine, or at the site of prior inflammation in D16 recovery animals. However, they were reliably present in areas adjacent to stricturing (>100 to 300 μm distant from stricture margin and underlying intact mucosa), at 11 ± 2 (3) on D16, 9 ± 2 (5) on D35 and 12 ± 2 (3) on D90 [mast cells/105 μm2 ± SEM (n animals)]. This did not correlate with the presence of mast cells in the stricture, which was detected in only 2 of 5 D16 rats, 1 of 5 rats at D35, and 0 of 3 rats at D90. The macrophages in the developing stricture were then further assessed for M1 versus M2 (alternatively activated) phenotypes. In D16 developing strictures, inducible nitric oxide synthase (iNOS)-positive cells were prominently present at the mucosal verge, whereas they were absent from adjacent regions with intact mucosal structure (Figure 3A). These cells showed no overlap with CD68 (not shown) and are evidence for ongoing inflammation. Immunoreactivity for C
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