In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn's fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.
Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn's-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn's-associated intestinal fibrosis.
Dysregulation of immune homeostasis in the gut leads to pathogenic inflammation and has been widely known to cause inflammatory bowel disease (IBD)1,2. Intestinal fibrosis is a chronic consequence of inflammatory bowel diseases (IBDs), such as Crohn's disease (CD)3. The irreversible pathophysiology of CD includes intestinal stricture or stenosis of fibrosis, which limits treatment options, and with no medications currently available, the only treatment is surgery. Ultimately, the development of effective therapies to counter inappropriate inflammation is much needed to study the mechanism of CD, and this will lead us a step closer to that.
A variety of genetic mouse models are available to study IBD including IL10 KO, SAMP/Yit and adoptive CD45+RB high cell transfer into SCID mice4,5,6. Here, we show the procedure for TNBS-mediated fibrosis in the mouse model of CD, which is comparable to the pathology of human Crohn's fibrosis. The TNBS-induced model has certain advantages. This model is technically simple; disease onset is rapid, inexpensive, and could widely be used in different animals (e.g., mouse, rat and guinea pig7). Co-administration of ethanol and TNBS (2,4,6-trinitrobenzene sulfonic acid) abruptly damages the intestinal barrier and exposes colon tissue protein to TNBS and elicit substantial immunologic responses8,9. Repeated exposure of TNBS leads to an overreactive repair process responding to inflammation and injury, and develops a fibrotic reaction in the gut. Thus, TNBS-induced fibrosis model serves to be a very compelling model to study Crohn's-associated intestinal fibrosis.
Furthermore, mononuclear phagocytes are the primary cells that arbitrate the innate immune response to pathogenesis and injury in the gut10,11,12,13. To elucidate the cellular mechanism and to establish the role of Cx3Cr1+ mononuclear phagocytes in the TNBS fibrosis model, we show the procedure of purifying the mononuclear phagocytes. Analysis of Cx3Cr1+ cells is an essential step in order to assess the inflammatory markers and determine the concomitant mechanism for intestinal fibrosis. Collectively, this detailed procedure for TNBS fibrosis will be helpful to explain the cellular mechanisms of intestinal fibrosis.
For this manuscript, all human samples were procured according to the approved protocol by Institute Review Board (IRB) and by the Committee on Human Research at Albany Medical College. All research involving animals were strictly followed according to the approved protocol by the Institutional Animal Care and Use Committee at Albany Medical College as well as the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
1. Collection of human intestinal specimens
2. Induction of TNBS fibrosis and rapamycin treatment in mice
3. Immuno-histopathologic assessment of gut fibrosis
4. Isolation of intestinal lamina propria and purification of Cx3Cr1+ mononuclear phagocytes
5. Flow cytometry analysis
6. RNA isolation, RT-PCR, real-time PCR
7. Western blotting
8. Statistical analysis
We adopted the TNBS colitis mouse model to study and elucidate the underlying mechanisms of intestinal fibrosis8. Here, we performed a detailed time course study of TNBS-mediated colitis, where TNBS was rectally administrated weekly to wild type mice for up to six weeks as represented schematically (Figure 1A). After six weeks of TNBS treatment, we noticed that colonic lengths shorten progressively over the course of the TNBS treatment, from an average of 5 ± 0.5 cm in the control group to 3 ± 0.5 cm in the TNBS group; such quantitative analysis of colon length represents a very apparent reduction in colon length (Figure 1B). To ensure that the TNBS Crohn's disease model is comparable to the human Crohn's fibrosis model and was not an artifact related to the methodology, we analyzed the fibrotic markers at multiple levels in a detailed time course study for the TNBS injection to the wild type. Accumulation of alpha-smooth muscle actin (αSMA) positive cells and collagen deposition within submucosal layers have been reported in most of the fibrosis incidences and is regarded as a hallmark for fibrotic events14. We found that the colon sections of TNBS-treated mice that were stained with αSMA showed a 4-6-fold increase in colonic submucosa layer positively stained with αSMA (Figure 1C). Besides, the Trichrome blue staining for these sections also showed a 2-4-fold increase, suggesting significant collagen deposition, which validates severe intestinal fibrosis (Figure 1D). We further assessed the activation of myofibroblasts by detecting αSMA-positive cells by FACS analysis in TNBS-treated mice colon and found a significant accumulation of αSMA-positive staining (Figure 1E). Furthermore, we found substantial induction in the expression of αSMA, Col-I, and Col-III measured by qPCR analysis in TNBS-treated mice (Figure 1F). Increased expression of αSMA protein in western blot analysis revealed increased fibrosis (Figure 1G). Overall, TNBS kinetics treatment provides an opportunity to access putative immune response in chronic conditions, which closely mimics the CD chronic phase condition and is essential to fibrosis development.
To compare TNBS fibrosis with Crohn's associated fibrosis, we analyzed the expression of fibrosis markers and cytokines in fresh tissue biopsy from the ileum of patients with active CD or under remission. Remarkably, we found marked induction of thickening of αSMA-positive layers and increased collagen deposition as detected by trichrome staining in active CD sections (Figure 2A). We also performed Western blot analysis and confirmed the induction of αSMA expression in active CD samples (Figure 2B). In addition, we observed significant induction of fibrosis markers including αSMA, Col1, as detected by qPCR analysis (Figure 2C).
Next, to determine a mechanism to limit TNBS fibrosis we evaluated the effects of rapamycin, a pharmacological inhibitor of mTOR activity15,16. Accordingly, we treated mice with both TNBS and rapamycin and analyzed αSMA and collagen level in colon histology and have shown quantitative measurement of αSMA and collagen in densitometry plot (Figure 3A). Our data suggests that rapamycin reduces the αSMA-positive staining in submucosal layer and lessens the collagen deposition. To further validate and quantify fibrotic responses, we determined the αSMA, collagen and TGFβ expressions by qPCR, and αSMA expression by flow cytometry in TNBS-treated colon (Figure 3B, 3C). We have also shown Cx3Cr1+ mononuclear phagocytes induce an inflammatory immune response to injury9. Thus, we wanted to see if administration of rapamycin in TNBS-treated mice could reverse the inflammatory and fibrotic effects. Therefore, we purified Cx3Cr1+ resident mononuclear phagocytes from colonic single cell suspensions by using magnetic microbeads (Figure 3D). We found an increased level of p-p70 and p-S6 in Cx3Cr1+ resident mononuclear phagocytes by western blot analysis from TNBS-treated mice, and this level was blocked by rapamycin (Figure 3E). Besides, we found that rapamycin treatment dampens down the IL-23 and IL-1β in the TNBS-treated group (Figure 3F). Moreover, these findings elucidate the effective mechanism involved in the induction of TNBS-fibrosis and have shown that rapamycin attenuates the induction of fibrosis.
Figure 1: Successful TNBS administration leads to developing intestinal fibrosis in mice. A. Schematic diagram representation of weekly TNBS treatment given to wild-type mice. B. Colon images and measurement of colon lengths from TNBS-treated mice, harvested on weeks 0, 2, 4, and 6 post-TNSB treatment. C-D. Colon sections' histological analysis stained with anti-αSMA antibody for activation of myofibroblasts and trichrome blue staining for collagen; scale bar 100 µm. E. FACS analysis to identify αSMA-positive cells in the colon and quantification of αSMA-positive cells. F. Fibrotic markers and cytokines detected by qPCR. G. western blot analysis of αSMA from colon lysate of TNBS-treated and control mice. This modified figure is being reused with the permission of previous publication9 in Mucosal Immunology, 2019 by Mathur et al. Please click here to view a larger version of this figure.
Figure 2: TNBS Fibrosis is comparable to Crohn's-associated Intestinal fibrosis. A. Representative images of colon biopsies of control, active CD showing a significant increase of trichrome blue staining and αSMA-positive staining in submucosal layers, scale bar 50 µm. B. Western blot analysis of αSMA expression and quantification. C. qPCR analysis of fibrotic markers and cytokines. This modified figure is being reused with the permission of previous publication9 in Mucosal Immunology, 2019 by Mathur et al. Please click here to view a larger version of this figure.
Figure 3: Rapamycin treatment effectively ameliorates TNBS-induced fibrosis. A. Colon histological analysis - representative images of myofibroblast staining with anti-αSMA antibody and collagen staining with Trichrome blue, scale bar 100 µm. B. qPCR analysis of αSMA, Collagen and TGFβ expression in Control/TNBS-treated mouse colons. C. FACS analysis of αSMA in single cell suspension from mouse colon treated with Control/TNBS. D. Schematic for purification of Cx3Cr1+ cells from colonic lamina propria fraction and FACS analysis of purified cells. E. Western blot analysis of p-p70 and p-S6 levels in purified Cx3Cr1+ mononuclear phagocytes from mice treated with TNBS and/or rapamycin. F. qPCR analysis of the expression in purified Cx3Cr1+ mononuclear phagocytes, which produces IL-23 and IL-1β. This modified figure is being reused with the permission of previous publication9 in Mucosal Immunology, 2019 by Mathur et al. Please click here to view a larger version of this figure.
Wound healing or tissue repair is a tightly regulated biological process17. During tissue injury with a chemical, mechanical and infection condition, an inflammatory response triggers the tissue repair process. However, a dysregulated and pathological inflammatory response leads to developing scarring or a fibrotic reaction, which could impair the tissue repair function9,18,19. Here, we show the procedure for the TNBS-induced fibrosis animal model, which significantly shares pathophysiology with human Crohn's disease. Successive inoculation of TNBS chemical exposure to damage the mouse epithelium causes deep ulcerations and induces fibrosis development. With a reliable, low cost, and rapid induction of disease onset, this method is widely accepted by multiple research groups involved in studies for tissue injury, transmural inflammation and the gut-brain axis.
To successfully implement the TNBS fibrosis model, there are several essential steps. For example, adequate dosage and timing of TNBS administration are very important. A 6-8-week TNBS inoculation allows deep tissue ulceration and is highly recommended for chronic fibrotic studies. Outcoming stool from mice and the retrograde reflex of inoculated TNBS are two major problems, which could result in delivery of variable doses to mice. Gentle pressure near the mice rectum may help release stools before TNBS administration. Holding the head of the animal down for a few seconds dramatically helps to stop reverse reflux of TNBS. Keeping the mice in a cage, placing the cage on a heat pad, and providing the mice with Napa nectar could also be useful strategies in preventing high mortality.
Here, we also discuss the gut inflammation during TNBS fibrosis and their impact on fibrotic response. mTOR/autophagy role is broadly implicated on intestinal homeostasis and in IBD pathogenesis20,21. We identified that mTOR/autophagy signaling is critical to modulate pro-inflammatory responses from IL-23 and IL1β cytokines from Cx3Cr1+ mononuclear phagocytes that affect pro-fibrotic IL-23/IL-22 axis (Figure 3A)9. Thereby, purifying single Cx3Cr1+ mononuclear cells for immunoprofiling and gene expression is a critical step of the model. We discuss in detail the protocol for isolating lamina propria and provide the purification procedure for Cx3Cr1+ mononuclear cells using magnetic beads.
Collectively, despite the presence of genetic and spontaneous models for Crohn's disease, TNBS-colitis remains a potent tool to study the immuno-pathogenesis of CD and has the potential to evaluate the Crohn's fibrosis treatments. The major limitation of using chemically induced fibrosis models such as TNBS is the chance of high variability from user to user and the possibility of outliers in the data. A sample size of 5-8 animals per group along with greater user experience can increase the consistency of the model. However, finding a suitable genetic model causing spontaneous Crohn's fibrosis is and will always be warranted.
This work was supported by NIH grant R01NS093045 (Y.H.), NIH grant K08DK088950 (X.Z.), R03DK099566 (X.Z.), and The Crohn's & Colitis Foundation of America research Fellowship (CCFA) 481637 (R.M.).
Name | Company | Catalog Number | Comments |
Anti-mouse aSMA, AF488 conjugated, Clone#1A4 | e-bioscience | 53-9760-82 | |
Anti-mouse aSMA, purified Clone#M1/77 | e-bioscience | 149760-80 | |
Anti-mouse CD11b, APCCy7 conjugated, Clone#M1/70 | Biolegend Inc | 101226 | |
Anti-mouse CX3cr1 PE conjugated, Clone#SA011F11 | Biolegend | 149006 | |
Anti-mouse purified CD16/32 Fc block | Biolegend Inc | 14-9760-80 | |
Anti-PE MicroBeads | Miltenyi | 130-105-639 | |
Anti-rabbit IgG, HRP-linked Antibody | e-bioscience | 7074 | |
Bovine Serum Albumin | InvivoGen | tlrl-isdn | |
Collagenase type IV | Roche | 1088866001 | |
DAPI | SIGMA | D9542 | |
DMEM | Corning | 10013-CV | |
DNase I from bovine pancreas | Roche | D263-5vl | |
Falcon® 40µm Cell Strainer | Corning | 352340 | |
Fixation/Permeabilization Solution Kit with BD GolgiPlug kit | BD Bioscience | 555028 | |
FlowJo | FlowJo LLC | www.flowjo.com | |
Glycogen | Roche | 10901393001 | |
Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate | e-bioscience | 5018 | |
Graphpad Prism 7 | GraphPad Software Inc | www.graphpad.com | |
H&E kit | American Mastertech Kit | HXMMHPT | |
Image J | NIH | www.imagej.nih.gov/ij/ | |
Mouse: C57BL/6J | Jackson Laboratories | 664 | |
MS Columns | Miltenyi | 130-042-201 | |
Percol | GE Healthcare | 17-0891-01 | |
PMA/Ionomycin salt | Sigma | P8139 | |
PowerUp SYBR Green Master Mix | Thermofisher | A25777 | |
Rabbit Anti mouse GAPDH, Clone# D16H11 | Cell signaling | 5174 | |
Rabbit Anti mouse p70 S6 Kinase, Clone#49D7 | Cell signaling | 2708 | |
Rabbit Anti mouse Phospho-p70 S6 Kinase, Clone#S371 | Cell signaling | 9208 | |
Rabbit Anti mouse Phospho-S6 Ribosomal Protein (Ser235/236), Clone# D57.2.2E | Cell signaling | 4858 | |
Rabbit Anti mouse S6 Ribosomal Protein | Cell signaling | 2217 | |
Rapamycin | LC LABORATORIES | 1003799 | |
TNBS | Sigma | 92823 | |
Trichorme staining kit | American Mastertech Kit | STOSTBPT | |
TRIzol | Life technologies | 15596018 | |
Verso cDNA Synthesis Kit | Thermofisher | AB1453B | |
Zen black 2.1 | Carl Zeiss | www.zeis.com | |
Zen blue lite 2.3 | Carl Zeiss | www.zeis.com |
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