TY - JOUR
AU - MD, Ellen M. Zimmermann,
AB - Objective Treatment of Crohn’s disease (CD) with anti-tumor necrosis factor α (TNFα) decreases intestinal inflammation, but the effect on fibrosis remains unclear. We hypothesized that treatment with rat-specific anti-TNFα will decrease the development of intestinal fibrosis in a rat model of CD. We further hypothesized that magnetization transfer magnetic resonance imaging (MT-MRI) will be sensitive in detecting these differences in collagen content. Methods Rats were injected in the distal ileum and cecum with peptidoglycan–polysaccharide (PG-PS) or human serum albumin (control) at laparotomy and then received intraperitoneal injections of rat-specific anti-TNFα or vehicle daily for 21 days after laparotomy. Rats underwent MT-MRI abdominal imaging on day 19 or 20. MT ratio was calculated in the cecal wall. Cecal tissue histologic inflammation was scored. Cecal tissue procollagen, cytokine, and growth factor messenger RNAs were measured by quantitative real-time PCR. Results PG-PS–injected rats treated with anti-TNFα had less histologic inflammation, and cecal tissue expressed lower levels of proinflammatory cytokine messenger RNAs than vehicle-treated PG-PS–injected rats (IL-1β: 5.59 ± 1.53 versus 10.41 ± 1.78, P = 0.02; IL-6: 23.23 ± 9.33 versus 45.89 ± 11.79, P = 0.07). PG-PS–injected rats treated with anti-TNFα developed less intestinal fibrosis than vehicle-treated PG-PS–injected rats by tissue procollagen I (2.87 ± 0.66 versus 9.28 ± 1.11; P = 0.00002), procollagen III (2.25 ± 0.35 versus 7.28 ± 0.76; P = 0.0000009), and MT-MRI (MT ratio: 17.79 ± 1.61 versus 27.95 ± 1.75; P = 0.0001). Insulin-like growth factor I (2.52 ± 0.44 versus 5.14 ± 0.60; P = 0.0007) and transforming growth factor β1 (2.34 ± 0.29 versus 3.45 ± 0.29; P = 0.006) were also decreased in anti-TNFα–treated PG-PS–injected rats. Conclusions Anti-TNFα prevents the development of bowel wall inflammation and fibrosis in the PG-PS rat model of CD. MT-MRI measurably demonstrates this decrease in intestinal fibrosis. inflammatory bowel disease, stricture, animal model, inflammation in IBD, animal models of IBD, fibrosis The natural history of Crohn’s disease is characterized by segmental, transmural bowel wall inflammation that waxes and wanes over years and often progresses to fibrosis, critical luminal narrowing, and obstruction. Strictures contribute to the development of fistulae and often require surgical intervention. Therapy targeted at blocking tumor necrosis factor α (TNFα) has revolutionized our approach to the care of patients with CD. Anti-TNFα therapy decreases symptoms, heals mucosa, and improves the quality of life of most of the patients with CD who are given this treatment. The profound effect that anti-TNFα therapy has on decreasing the inflammatory component of CD is well-established.1 Despite its significant effect on inflammation, the extent to which anti-TNFα therapy alters the natural history of CD remains unclear. It is often assumed that decreasing inflammation will, in turn, decrease fibrosis, ultimately decreasing the rate of stricture formation and the rate of complications, such as abscess or fistula. However, a beneficial effect of anti-TNFα therapy on intestinal fibrosis has been difficult to demonstrate in patients. In fact, early reports of increased rates of stricture development and intestinal obstruction after the introduction of infliximab raised the question as to whether anti-TNFα therapy could promote fibrotic strictures.2 More recent data demonstrate declining surgical rates in patients with CD, indirectly suggesting a beneficial effect of biologic therapy on stricture formation.3,4 Not all studies confirm this trend. Prospectively collected data from the TREAT (the Crohn’s Therapy, Resource, Evaluation, and Assessment Tool) registry do not demonstrate a change in surgical rates with anti-TNFα therapy.2 Therefore, the ability of anti-TNF therapy to decrease intestinal fibrosis and prevent the development of fibrotic strictures remains an open question. One of the limitations in our ability to understand the effect of anti-TNFα therapy on stricture development is the lack of a reliable tool for quantifying and monitoring intestinal fibrosis in vivo. Recently, we demonstrated that magnetization transfer MRI (MT-MRI) can semiquantitatively detect intestinal fibrosis in a rat model of CD.5 In this model, rats injected with peptidoglycan–polysaccharide (PG-PS) develop enterocolitis with granulomatous inflammation that leads to prominent fibrosis over time. The chronic phase of PG-PS enterocolitis mimics CD. In the present study, we hypothesized that treatment of PG-PS enterocolitis early in the course of the disease with a rat-specific anti-TNFα will decrease inflammation and prevent the development of intestinal fibrosis. We further hypothesized that MT-MRI will be sensitive to differences in intestinal wall fibrosis that occur with potent anti-TNFα therapy. Materials and Methods Animals Female Lewis strain rats raised in a specific-pathogen-free environment were obtained from Harlan (Indianapolis, IN). The animals were housed under standard specific-pathogen-free and temperature controlled conditions, and they were given unrestricted access to water and standard laboratory chow. Animals were approximately 8 to 10 weeks of age (approximately 150–165 g) at the start of the experiments. PG-PS Enterocolitis Model Anesthetized rats underwent laparotomy, and using aseptic technique, intramural injections of PG-PS (15 μg rhamnose/g body weight; PG-PS 10S from Lee Laboratories, Becton, Dickinson, and Company, Bedford, MA) were administered at 7 sites along the surgically exposed intestine (in ileal Peyer’s patches, terminal ileum, and cecum) using 33-G needles.6,–9 Control rats were injected with human serum albumin (HSA; 45 μg/g body weight; sterile solution in normal saline) in the same manner. After the surgical intervention, the animals were closely monitored, weighed 3 times per week, and allowed free access to rodent chow and water. In this model, PG-PS induces transient acute inflammation, followed by a quiescent phase, then a spontaneously reactivating chronic granulomatous inflammatory phase that begins by day 14 and is associated with prominent fibrosis.5,–9 Three separate experiments were performed as outlined in Table 1. Table 1. Rat Treatment Groups by Experiment     View Large Table 1. Rat Treatment Groups by Experiment     View Large Anti-TNFα Treatment CNTO1081, a murine monoclonal antibody raised by injecting mice with recombinant rat TNFα, followed by standard hybridoma techniques, was provided by Janssen Research & Development (Radnor, PA) at a concentration of 10 mg/mL in sterile phosphate-buffered saline (PBS) and was stored at 4°C. Aliquots were prepared aseptically in a biologically safety cabinet and were used without further dilution. The drug was administered by intraperitoneal injection at a dose of 2 to 4 mg given 2 to 3 times weekly. Control rats received intraperitoneal injections of an equal volume of sterile PBS. Treatment was started on day 1 after PG-PS or HSA injection and was continued for 3 to 4 weeks, at which time the animals were euthanized. Assay of Serum Levels of Anti-Rat TNF In experiments 2 and 3, immediately following euthanasia, blood was collected from each animal. A portion of the blood was allowed to clot at room temperature for 30 to 60 minutes and then centrifuged at ×1000g for 10 minutes. Serum was recovered and stored at −80°C. Levels of antirat TNF were measured by ELISA in high-binding 96-well plates (Nunc; Thermo Fisher, Rochester, NY). Wells were coated with rat TNFα (R&D Systems, Minneapolis, MN) at 1 µg/mL in PBS, 50 µL per well, overnight at 4°C. After washing twice with PBS containing 0.05% Tween 20, plates were blocked with 1% bovine serum albumin (BSA) in PBS at room temperature for 2 hours. Standards and serum samples, diluted in PBS containing 1% BSA and 2.5% normal rat serum (Jackson ImmunoResearch Laboratories, West Grove, PA), were added to duplicate wells and incubated at 37°C for 1.5 hours. After 3 washes, biotinylated rat anti-mouse IgG H + L (1:1000 in PBS with 1% BSA; Jackson ImmunoResearch Laboratories) was added to the wells and incubated at 37°C for 1 hour. After 3 washes, peroxidase–streptavidin (1:5000 in PBS with 1% BSA; Jackson ImmunoResearch Laboratories) was added to the wells and incubated for 1 hour at 37°C. After 4 washes, o-phenylenediaminedihydrochloride (Sigma Chemical, St Louis, MO) was added to the wells and incubated at room temperature for 10 minutes. Then, 0.2N H2SO4 was added to stop color development, and absorbance at 490 nm was measured. Hematocrit and Erythrocyte Sedimentation Rate A portion of the blood was anticoagulated with potassium EDTA. Spun hematocrits (50 μL) were performed by the Animal Diagnostic Laboratory of the University of Michigan Unit for Laboratory Animal Medicine. For erythrocyte sedimentation rate, 200 μL of anticoagulated blood was drawn into a capillary tube, the lower end of which was then sealed with clay. The capillary tube was kept upright for 1 hour, and then, the height of plasma above the settled erythrocytes was measured. Gross Abdominal Score Rats were euthanized at 21 or 29 days after PG-PS or HSA injection, and the abdominal contents were grossly evaluated by an observer (E.M.Z.) blinded to the treatments. Cecal wall thickening (based on the degree of opacity of the bowel wall and the perceived thickness at palpation), thickening and contraction of the cecal and terminal ileal mesentery, adhesions, cecal nodules, and liver nodules were each graded on a scale of 0 to 4. The composite gross abdominal score (maximum possible = 20) was calculated as the sum of the individual component scores.5,7,–9 Histologic Evaluation At the time of dissection, a 1 × 2 cm portion of the wall of the cecum was resected. One half of this piece of tissue was shaped into a Swiss roll10 and placed in 10% neutral buffered formalin; the remaining half was frozen for RNA analysis (described below). A 0.5-cm length of the cecal tip was collected and placed in formalin. After 24 hours, the tissue was removed from formalin, embedded in paraffin, and sectioned. From each tissue sample, one section was stained with hematoxylin and eosin and another with a modification of Masson’s original trichrome stain. This trichrome system stains collagen blue, nuclei purple–brown, and cytoplasm pink. Each tissue section was scored for inflammation and fibrosis by 2 gastrointestinal pathologists working independently and blinded to the treatments (see Table, Supplemental Digital Content 1, http://links.lww.com/IBD/A132 and Rahal et al9). The mean of the 2 independent scores was then calculated. Messenger RNA Analyses One half of the 1 × 2 cm portion of PG-PS–injected or HSA-injected cecum, harvested at the time of dissection, was snap frozen in liquid nitrogen and placed at −80°C for later RNA extraction. RNA was isolated from pulverized frozen cecal tissue using the RNeasy Midi Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The RNA concentration was calculated from the absorbance at 260 nm. A portion of the RNA was reverse transcribed into complementary DNA (cDNA) using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. cDNAs were used in quantitative real-time PCRs for procollagen types I and III (using primers and probes previously designed by our laboratory to span an intron11) and insulin-like growth factor I (IGF-I), transforming growth factor β1 (TGF-β1), TNFα, IL-1β, and IL-6 (using commercially available TaqMan Gene Expression Assays from Applied Biosystems). Results were normalized to glyceraldehyde 3-phosphate dehydrogenase messenger RNA (mRNA) levels (measured using commercially available TaqMan Gene Expression Assay); results were then expressed as fold the mean value for HSA-injected vehicle-treated animals. All reactions were performed in triplicate wells. Imaging Protocol MRI were obtained with a 2.0-T, 31-cm clear-bore system equipped with actively shielded gradients (Unity-Inova, Varian Inc., Palo Alto, CA). MT-MRI was performed in a standard imaging mode at 10 kHz (Msat images) and at 100 kHz off-resonance (M0 images). On each image, regions of interest (ROIs) were identified on the greater curve of the cecum. ROIs were selected in areas in which the bowel wall was clearly well-defined and visible on both the Msat and M0 images. Two to 5 ROIs were identified for each image with the use of imaging software for a Macintosh computer (Osirix version 3.8.1; Osirix, Geneva, Switzerland). The MT ratio in the cecal wall was calculated as follows: 100 × (1 − Msat/M0). The mean MT ratio of all ROIs for any given animal was used for all evaluations in this study.5 Statistical Analyses The 1-tailed Student t test was used for all bivariate comparisons of sample means. All statistical analyses were performed with software (Stata 10.1 for Mac; StataCorp, College Station, TX). A P value of <0.05 was considered significant. Ethical Considerations All animal studies were approved by the University of Michigan Committee on Use and Care of Animals. Results In all animals not receiving anti-TNFα therapy across all 3 experiments, PG-PS–injected rats (n = 21) demonstrated greater gross abdominal scores than HSA-injected control animals (n = 15) (12.6 ± 0.8 versus 0.4 ± 0.2, respectively; P < 0.00000001). Treatment with anti-TNFα resulted in a decrease in gross abdominal score (Fig. 1; low-dose regimen: 7.8 ± 1.8 versus 13.0 ± 1.8, n = 6/group, P = 0.04; high-dose regimen: 3.8 ± 0.8 versus 12.5 ± 1.0, n = 15/group, P = 0.00000006). Figure 1. View largeDownload slide Effect of anti-TNFα therapy on gross abdominal score. The gross abdominal score is a subjective assessment of intra-abdominal disease burden, with a maximum possible score of 20. In this animal model, the gross abdominal score is greater in all PG-PS–injected animals than HSA-injected animals. In PG-PS–injected rats, anti-TNFα therapy begun on day 1 after PG-PS injection resulted in a decrease in the gross abdominal score and did so in a dose-dependent manner. Results for high-dose therapy are the combined results for experiments 2 and 3. Results shown are means; error bars reflect the standard error of the mean (n = 6–15/group). Figure 1. View largeDownload slide Effect of anti-TNFα therapy on gross abdominal score. The gross abdominal score is a subjective assessment of intra-abdominal disease burden, with a maximum possible score of 20. In this animal model, the gross abdominal score is greater in all PG-PS–injected animals than HSA-injected animals. In PG-PS–injected rats, anti-TNFα therapy begun on day 1 after PG-PS injection resulted in a decrease in the gross abdominal score and did so in a dose-dependent manner. Results for high-dose therapy are the combined results for experiments 2 and 3. Results shown are means; error bars reflect the standard error of the mean (n = 6–15/group). In the initial experiment, rats were given anti-TNFα at a dose of 2 mg twice weekly. This starting dose was based on data from previous reports using the drug in rat models of immune-mediated diseases.12,13 When individual elements of the composite gross abdominal score were examined (Table 2), it appeared that the decrease in the composite score was due primarily to a decrease in liver granulomas and adhesions, whereas the effects on gut parameters (cecal thickening, cecal granulomas, mesenteric thickening) were less pronounced. Furthermore, mRNAs of inflammatory and fibrogenic cytokines and growth factors in the cecum, as well as those of procollagen types I and III, were not significantly changed in PG-PS–injected rats treated with low-dose anti-TNFα compared with those given vehicle (Fig. 2A, B). PG-PS–injected rats treated with a higher-dose regimen (anti-TNFα 4 mg, 3 times weekly) demonstrated a lower gross abdominal score than PG-PS–injected rats given vehicle (Fig. 1). Furthermore, examination of the individual components of the gross abdominal score showed reductions in fibrosis and gut parameters in animals given the higher-dose anti-TNFα treatment as compared with those given vehicle (P < 0.005; Table 2). Table 2. Mean Gross Abdominal Scores and Components Thereof     View Large Table 2. Mean Gross Abdominal Scores and Components Thereof     View Large Figure 2. View largeDownload slide Effect of anti-TNFα therapy on relative expression of mRNAs of proinflammatory cytokines and profibrotic factors. Quantitative real-time PCR was used to measure the levels of the indicated mRNAs in cecal tissue, relative to glyceraldehyde 3-phosphate dehydrogenase mRNA. Results are expressed as fold the mean value for HSA-injected vehicle-treated animals. A, Proinflammatory cytokines with the low-dose (2 mg, 2×/week) anti-TNFα regimen (experiment 1). B, Profibrotic factors in the same low-dose experiment (A and B, n = 6/group). C, Proinflammatory cytokines with the high-dose (4 mg, 3×/week) anti-TNFα regimen. D, Profibrotic factors in the same high-dose experiments (C and D show the combined results of experiments 2 and 3; n = 6–15/group). All results shown are means; error bars reflect the standard error of the mean. Proinflammatory cytokine mRNAs were variably elevated in cecal tissue of PG-PS–injected rats (A and C), but they were decreased or showed trends toward decreased levels in rats treated with high-dose anti-TNFα (C). Profibrotic factor mRNAs were variably elevated in cecal tissue of PG-PS–injected rats (B and D) but were decreased in rats treated with high-dose anti-TNFα (D), demonstrating an antifibrotic property of anti-TNFα therapy. Figure 2. View largeDownload slide Effect of anti-TNFα therapy on relative expression of mRNAs of proinflammatory cytokines and profibrotic factors. Quantitative real-time PCR was used to measure the levels of the indicated mRNAs in cecal tissue, relative to glyceraldehyde 3-phosphate dehydrogenase mRNA. Results are expressed as fold the mean value for HSA-injected vehicle-treated animals. A, Proinflammatory cytokines with the low-dose (2 mg, 2×/week) anti-TNFα regimen (experiment 1). B, Profibrotic factors in the same low-dose experiment (A and B, n = 6/group). C, Proinflammatory cytokines with the high-dose (4 mg, 3×/week) anti-TNFα regimen. D, Profibrotic factors in the same high-dose experiments (C and D show the combined results of experiments 2 and 3; n = 6–15/group). All results shown are means; error bars reflect the standard error of the mean. Proinflammatory cytokine mRNAs were variably elevated in cecal tissue of PG-PS–injected rats (A and C), but they were decreased or showed trends toward decreased levels in rats treated with high-dose anti-TNFα (C). Profibrotic factor mRNAs were variably elevated in cecal tissue of PG-PS–injected rats (B and D) but were decreased in rats treated with high-dose anti-TNFα (D), demonstrating an antifibrotic property of anti-TNFα therapy. There was a decrease in IL-1β mRNA with high-dose anti-TNFα treatment as compared with vehicle (5.59 ± 1.53 versus 10.41 ± 1.78, P = 0.02; Fig. 2C); IL-6 and TNFα mRNAs demonstrated a trend (IL-6: 23.23 ± 9.33 versus 45.89 ± 11.79, P = 0.07; TNFα: 2.40 ± 0.42 versus 3.16 ± 0.46, P = 0.12) (Fig. 2C). Levels of mRNAs for several profibrotic factors were lower in PG-PS–injected rats treated with high-dose anti-TNFα than in those that received vehicle. These included procollagen type I (2.87 ± 0.66 versus 9.28 ± 1.11; P = 0.00002), procollagen type III (2.25 ± 0.35 versus 7.28 ± 0.76; P = 0.0000009), IGF-I (2.52 ± 0.44 versus 5.14 ± 0.60; P = 0.0007), and TGF-β1 (2.34 ± 0.29 versus 3.45 ± 0.29, P = 0.006) (Fig. 2D). MT-MRI showed decreased cecal wall fibrosis in anti-TNFα–treated PG-PS–injected rats compared with vehicle controls (MT ratio: 17.79 ± 1.61 versus 27.95 ± 1.75, respectively; P = 0.0001; Fig. 3A). The decrease in cecal wall fibrosis was confirmed by histologic scoring of hematoxylin and eosin and trichrome stained sections (fibrosis score: 4.3 ± 0.7 versus 8.3 ± 0.5, respectively; P = 0.0002; Fig. 3B). Inflammation was also decreased in anti-TNFα–treated PG-PS–injected rats compared with vehicle controls, as determined by histologic scoring (inflammation score: 4.7 ± 0.8 versus 9.6 ± 0.7, respectively; P = 0.0002; Fig. 3B). Representative histologic sections are shown in Figure 4. Figure 3. View largeDownload slide A, Effect of anti-TNFα therapy on the MT ratio of the wall of the cecum. MT-MRI measures the relative amounts of large macromolecules, such as collagen in tissue, and the results are expressed as the MT ratio. MT-MRI was performed on day 19 or 20 after injection of PG-PS or HSA, in the high-dose anti-TNFα experiments only. Results shown are means and reflect the combined results of experiments 2 and 3; error bars reflect the standard error of the mean (n = 6–15/group). Anti-TNFα therapy resulted in a lower MT ratio in PG-PS–injected rats as compared with PG-PS–injected rats given vehicle, suggesting a less robust fibrotic response to PG-PS with anti-TNFα therapy. B, Effect of anti-TNFα therapy on histologic scores for fibrosis and inflammation in cecal tissue. Rats injected with PG-PS develop extensive inflammation and intense fibrosis. However, those rats treated with high-dose anti-TNFα developed less inflammation and less fibrosis in the cecum. Results shown are means and reflect the combined results of experiments 2 and 3; error bars reflect the standard error of the mean (n = 6–15/group). Figure 3. View largeDownload slide A, Effect of anti-TNFα therapy on the MT ratio of the wall of the cecum. MT-MRI measures the relative amounts of large macromolecules, such as collagen in tissue, and the results are expressed as the MT ratio. MT-MRI was performed on day 19 or 20 after injection of PG-PS or HSA, in the high-dose anti-TNFα experiments only. Results shown are means and reflect the combined results of experiments 2 and 3; error bars reflect the standard error of the mean (n = 6–15/group). Anti-TNFα therapy resulted in a lower MT ratio in PG-PS–injected rats as compared with PG-PS–injected rats given vehicle, suggesting a less robust fibrotic response to PG-PS with anti-TNFα therapy. B, Effect of anti-TNFα therapy on histologic scores for fibrosis and inflammation in cecal tissue. Rats injected with PG-PS develop extensive inflammation and intense fibrosis. However, those rats treated with high-dose anti-TNFα developed less inflammation and less fibrosis in the cecum. Results shown are means and reflect the combined results of experiments 2 and 3; error bars reflect the standard error of the mean (n = 6–15/group). Figure 4. View largeDownload slide Histology images. Microscopic appearance of representative hematoxylin and eosin–stained (left) and trichrome-stained (right) sections of cecum from HSA-injected control rats (A, B), PG-PS–injected rats (vehicle treated; C, D), and PG-PS–injected rats treated with anti-TNFα (E, F). A–F, ×40 original magnification photomicrographs of cecum. PG-PS–injected (vehicle-treated) rats have dense collagen expanding the submucosa and disrupting the muscularis externa (in brackets in C and D). PG-PS–injected (vehicle-treated) rats have granulomas in the submucosa, many of which are necrotizing (asterisk in C and D). PG-PS–injected rats treated with anti-TNFα (E, F) still have some granulomas, but less expansion of submucosa with collagen, less disruption of muscularis externa and muscularis mucosae, and no serosal expansion. Figure 4. View largeDownload slide Histology images. Microscopic appearance of representative hematoxylin and eosin–stained (left) and trichrome-stained (right) sections of cecum from HSA-injected control rats (A, B), PG-PS–injected rats (vehicle treated; C, D), and PG-PS–injected rats treated with anti-TNFα (E, F). A–F, ×40 original magnification photomicrographs of cecum. PG-PS–injected (vehicle-treated) rats have dense collagen expanding the submucosa and disrupting the muscularis externa (in brackets in C and D). PG-PS–injected (vehicle-treated) rats have granulomas in the submucosa, many of which are necrotizing (asterisk in C and D). PG-PS–injected rats treated with anti-TNFα (E, F) still have some granulomas, but less expansion of submucosa with collagen, less disruption of muscularis externa and muscularis mucosae, and no serosal expansion. PG-PS–injected rats treated with high-dose anti-TNFα had higher hematocrit (43.1% ± 0.6% versus 35.3% ± 0.9%, P = 0.00000004; Fig. 5A) and lower erythrocyte sedimentation rate (0.2 ± 0.1 versus 2.8 ± 0.6, P = 0.0002; Fig. 5B) than PG-PS–injected rats treated with vehicle. Serum levels of anti-TNFα at the time of euthanasia (2 days after the final dose) were measured in randomly selected samples from the high-dose experiments and are shown in Figure 5C. There was no difference in the drug level in PG-PS–injected versus HSA-injected animals given the drug (P = 0.27). Figure 5. View largeDownload slide Effect of anti-TNFα therapy on hematocrit and erythrocyte sedimentation rate. A, As in humans, it appears that hematocrit is a marker for inflammatory disease activity in rats. Rats injected with PG-PS (vehicle treated) become anemic, with decreased hematocrit, compared with HSA-injected control animals. PG-PS–injected rats treated with anti-TNFα are less anemic than PG-PS–injected rats given vehicle. B, Rats injected with PG-PS (vehicle treated) develop an elevated erythrocyte sedimentation rate. This effect is attenuated in PG-PS–injected rats that are treated with anti-TNFα. Results shown are means and reflect the combined results of experiments 2 and 3; error bars reflect the standard error of the mean (n = 6-15/group). C, Serum levels of anti-TNFα were measured in randomly selected animals from the high-dose experiments at the end of the experiments, 2 days after the final dose of drug (bolded horizontal lines represent the group medians, fine horizontal lines represent upper and lower quartiles; HSA-injected animals: mean 622 ± 24 µg/mL, n = 6; PG-PS–injected animals: 504 ± 123, n = 14; P = 0.27). Figure 5. View largeDownload slide Effect of anti-TNFα therapy on hematocrit and erythrocyte sedimentation rate. A, As in humans, it appears that hematocrit is a marker for inflammatory disease activity in rats. Rats injected with PG-PS (vehicle treated) become anemic, with decreased hematocrit, compared with HSA-injected control animals. PG-PS–injected rats treated with anti-TNFα are less anemic than PG-PS–injected rats given vehicle. B, Rats injected with PG-PS (vehicle treated) develop an elevated erythrocyte sedimentation rate. This effect is attenuated in PG-PS–injected rats that are treated with anti-TNFα. Results shown are means and reflect the combined results of experiments 2 and 3; error bars reflect the standard error of the mean (n = 6-15/group). C, Serum levels of anti-TNFα were measured in randomly selected animals from the high-dose experiments at the end of the experiments, 2 days after the final dose of drug (bolded horizontal lines represent the group medians, fine horizontal lines represent upper and lower quartiles; HSA-injected animals: mean 622 ± 24 µg/mL, n = 6; PG-PS–injected animals: 504 ± 123, n = 14; P = 0.27). Discussion Fibrotic intestinal strictures are the cause of abdominal pain and contribute to complications of CD, such as fistula formation and obstruction. Potent therapy with biologic agents such as anti-TNFα promotes mucosal healing, but its effects on fibrosis are largely unknown. The aim of this study was to determine if the use of anti-TNFα therapy early in the course of experimental CD would decrease intestinal fibrosis. We showed that anti-TNFα therapy administered in a disease prevention paradigm decreases the development of intestinal fibrosis in a relevant model of CD. This is consistent with accumulating data that suggest improved patient outcomes when therapy is initiated early in the disease course.14 TNFα is a well-established mediator of chronic inflammation in many organ systems including the gut, and it is a key mediator of intestinal inflammation in CD. Given the pleotropic and redundant nature of inflammatory mediators and the complexity of the intestinal immune system, it is remarkable that therapy for CD targeted at this single mediator has such profound ameliorating effects on intestinal inflammation. The clinical effects of decreasing tissue inflammation and improving patient symptoms are well-established.15,16 What is less well-accepted is whether anti-TNFα therapy decreases intestinal fibrosis and whether it can decrease stricture formation over time. At the tissue level, infliximab, the most widely used anti-TNFα therapy, downregulates basic fibroblast growth factor and vascular endothelial growth factor in patients with CD, suggesting that infliximab may decrease fibrogenesis.17 In a wound healing model, rats treated with infliximab demonstrated decreased early wound tensile strength, which may suggest decreased healing, a process that involves collagen deposition.18 Unfortunately, no direct measure of tissue collagen was made. In addition, infliximab does not itself bind or neutralize rat TNF, suggesting that infliximab may be working by a different mechanism in this model.19 In our study, anti-TNFα therapy used early in the course of PG-PS enterocolitis prevented both inflammation and fibrosis. We are unable to determine whether this is a direct effect of anti-TNFα on fibrosis versus an indirect effect on fibrosis because of the anti-inflammatory effects of the therapy. We investigated changes in IGF-I and TGF-β1 mRNAs, 2 profibrotic factors that have been associated with intestinal fibrosis in CD20,21 and in the PG-PS model of CD.7,9 Both of these factors are markedly increased in PG-PS enterocolitis and decreased with anti-TNFα therapy, consistent with a role for these factors in this model. Furthermore, as in a murine colitis model and in human CD where inflammation is associated with increased NF-kB activity and increased production of IL-1, IL-6, and TNFα,22,23 these cytokines are increased in the PG-PS model and decreased with anti-TNF therapy. Because IL-6 has well-known profibrotic effects,24 IL-6 may help mediate PG-PS–induced fibrosis, and the decrease in IL-6 mRNA with anti-TNFα therapy may play a role in abrogating the fibrosis. IL-6 in the presence of TGF-β is a strong inducer of proinflammatory IL-17+ T helper (TH-17) cells in mice, cells that are important in the pathogenesis of inflammatory bowel disease.25 Furthermore, TNFα and IGF-I have been shown to have additive effects in increasing tissue collagen, with TNFα inhibiting collagen degradation by means of increased TIMP1 expression, and IGF-I increasing collagen synthesis by increased collagen gene transcription.26 Our data are consistent with a role for IL-6, TNFα, TGF-β1, and IGF-I in PG-PS enterocolitis and consistent with the idea that changes in these mediators with early anti-TNFα therapy may be responsible for the decrease in fibrosis seen with therapy. The lack of data on the effects of anti-TNFα on fibrogenesis in patients with CD stems largely from our inability to monitor the natural history of intestinal fibrosis noninvasively over time. We previously demonstrated that MT-MRI is sensitive to the presence of fibrotic content in the bowel wall.5 In this study, we demonstrate that MT-MRI is sufficiently sensitive to detect differences in bowel wall fibrosis that occur with medical therapy. This is the first demonstration of an in vivo assessment of the effects of medical therapy on bowel wall fibrosis in a CD model. These data further support the use of this imaging technology as a noninvasive tool for monitoring fibrosis in patients with CD. Thus, once the technology is translated to humans, MT-MRI may be useful for studying the natural history of CD and for monitoring the development of fibrosis in a given individual. Our demonstration of early anti-TNFα therapy effectively preventing the development of bowel wall fibrosis in rats with experimentally induced CD supports the early use of anti-TNFα therapy in patients with CD to decrease symptoms, heal mucosa, and potentially decrease intestinal fibrosis. 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TI - Anti–Tumor Necrosis Factor α Prevents Bowel Fibrosis Assessed by Messenger RNA, Histology, and Magnetization Transfer MRI in Rats With Crohn's Disease
JO - Inflammatory Bowel Diseases
DO - 10.1097/MIB.0b013e3182802c32
DA - 2013-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/anti-tumor-necrosis-factor-prevents-bowel-fibrosis-assessed-by-zqqeJnugIA
SP - 683
EP - 690
VL - 19
IS - 4
DP - DeepDyve
ER -