Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

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

1. Collect human tissue samples according to the institution's research committee protocols.
2. Procure intestinal tissue (ileocolonic) of a CD patient diagnosed with fibrosis and control samples from patients with no history of IBDs.
3. Use part of the intestinal tissue for immunohistology, another part of the tissue for analyzing mRNA, and a final part for protein expression of fibrotic and cytokine genes/markers.
4. For immunohistology analysis, first fix the human tissue sample in 4% paraformaldehyde for at least 48 h and then transfer it into a tube containing 70% ethanol for at least 16 h. Use this fixed tissue for making a paraffin-embedded block and cut 5 µm thick tissue sections by using a tissue-microtome.
5. Using a brush, gently spread out each cut section on a glass slide for staining.
6. Stain tissue section slide with trichrome stain to detect collagen deposition, and with αSMA stain to detect myofibroblasts (See section 3 for detailed information about trichrome and αSMA staining). Examine the stained sections under a light microscope.
7. Process another part of the obtained human tissue sample to make protein lysate to detect αSMA via western blot (see section 7 for detailed information about western blot analysis).
8. Obtain RNA from the final part of human tissue sample using an extraction reagent (Table of Materials) and convert mRNA into cDNA via RT-PCR.
9. Analyze fibrotic and cytokine genes by qPCR (see section 6 for detailed information about mRNA preparation).

2. Induction of TNBS fibrosis and rapamycin treatment in mice

1. Carry out animal research according to the animal research protocols approved by the institution.
2. Shave adult mice around the neck area in order to pre-sensitize to TNBS via dermal exposure. Soak TNBS in a cotton swab and then apply TNBS to the shaved area of the mouse neck.
   NOTE: Pre-sensitization is a very important step since it improves delayed hypersensitivity response to initiate TNBS-mediated inflammation.
3. Eight days post pre-sensitization, induce colitis by intra-rectal administration once a week for six weeks. Apply four mg of TNBS (in 25% ethanol) using a 100 µL enema via a 1 mL syringe attached to a 3.5 French polyurethane catheter and give control mice 100 µL of 25% ethanol only.
   1. Anesthetize mice with pentobarbital (25 mg/kg intraperitoneal) or expose them to 2.5% isoflurane along with 1 L/min of oxygen during TNBS administration. Confirm anesthetization by gently pinching toes and looking for an absence of reflex.
4. Use veterinary ointment on eyes to prevent dryness while mice are under anesthesia.
5. Inject rapamycin at 2 mg/kg/day or vehicle (5% Tween-20 and 4% ethanol) every weekday for 3-6 weeks, intraperitoneal, to both control and TNBS-treated mice.
6. Use 6-week TNBS post-treated mice intestine for analyzing the extracellular assays including histology, flow cytometry, western blotting and RNA extraction. To harvest organs, euthanize mice using CO₂ inhalation and confirm euthanasia with cervical dislocation.
   1. To harvest organs, euthanize mice using CO₂ inhalation and confirm euthanasia with cervical dislocation. After euthanasia, longitudinally open the mouse on its ventral side using surgical grade scissors and forceps. Remove the entire colon from the rectum to the terminal ilium area. Quickly transfer the colon to ice-cold 1x HBSS and wash the colon using the same buffer.
   2. Measure and compare the colon lengths of the control and TNBS-treated mice. Do this step as fast as possible to avoid drying out of the colons in order to avoid cell deaths.
   3. Cut the colon into small pieces and keep at -80 °C for storage for future purposes (RNA/western blot analysis). Use another piece of the colon for FACS analysis.
   4. Be sure to cut and collect colonic tissue samples from similar regions of the colon of both control and TNBS-treated animals.
      NOTE: A 10%-20% mortality is expected with TNBS inoculation although with experience, the mortality rate can be significantly reduced.

3. Immuno-histopathologic assessment of gut fibrosis

1. Fix human and/or mice tissues in 4% paraformaldehyde for 48 h, and then transfer to 70% ethanol for 16 h.
   NOTE: Paraformaldehyde is a neurotoxic chemical so avoid inhaling; use a face mask while using it.
2. Paraffin embed the fixed colon tissues, slice to a 5 µm thickness, and directly put the tissues on glass slides for histology.
3. Perform H&E and Trichrome Blue staining according to manufacturer's instructions to detect collagen.
   1. Briefly, first deparaffinize sections by submerging the slide containing sections in two chambers of xylene for 5 min in each chamber.
   2. Rinse slides with 100% alcohol for 1 min; repeat this step three times. Rinse again with tap water for 1 min.
   3. After that, immerse slides in preheated Bouin's solution at 56 °C for 60 min. Bouin's solution is yellow in appearance; wash after taking out the slides from Bouin's solution in tap water for 3 min or until slides became colorless.
   4. Immerse slides in Modified Mayer's Hematoxylin solution for 7 min at room temperature.
   5. Stain slides by immersing in Trichrome stain for 5-8 min. Immediately, rinse slides in running water for 5-10 s to remove excess Trichrome stain.
   6. Dehydrate slides with 100% alcohol for 1 min. Repeat this procedure three times.
   7. Clear slides with xylene for 1 min and repeat the step 3x.
   8. Mount slides with mounting media (Table of Materials).
4. Carefully hold coverslip at one end with forceps and let it cover the entire section without having any bubbles. If there are some bubbles, quickly remove bubbles by tapping the coverslip.
5. Use immunostaining to detect αSMA.
   1. Block colon sections in blocking buffer (0.2% Triton X-100 and 5% normal goat serum in 1x PBS) for 1 h at room temperature.
   2. Incubate with 100 µL of diluted primary antibody for αSMA (Table of Materials) on the slide solution (0.2% Triton X-100 and 3% normal goat serum in 1x PBS; anti-αSMA 1:200) at 4 °C overnight.
   3. Wash the sections three times with 1x PBS.
   4. Use goat anti-mouse IgG (H + L) conjugated with Alexa Fluor 488 (Table of Materials) as a secondary antibody for 2 h at room temperature.
   5. Wash the sections three times with 1x PBS.
   6. Use DAPI to counterstain the nucleus for 5 min.
   7. Wash the sections three times with 1x PBS.
   8. Mount slides with fluorescent mounting media (Table of Materials), and seal with a coverslip using nail polish.
6. Acquire images by using LSM 880 confocal microscope and process images using microscope software.
7. Quantify images by taking an average of multiple selected areas in the same section with ImageJ.

4. Isolation of intestinal lamina propria and purification of Cx3Cr1⁺ mononuclear phagocytes

1. Longitudinally open the colon in ice-cold Hank's Balanced Salt Solution (HBSS; Table of Materials) and wash the colon with the same buffer.
2. Cut approximately 5 cm small pieces of colon tissues using sterile scissors in HBSS.
3. Transfer small colon tissue pieces in a 50 mL conical tube containing 10 mL of pre-digestion buffer (1x HBSS with 5% FBS, 5 mM EDTA and 1 mM DTT), and shake for 20 min at 100 rpm in a 37 °C incubator¹¹.
   NOTE: Incubation of colonic tissue in 37 °C at 100 rpm shaking condition allows for efficient collagenase tissue digestion and high yield of viable cells.
4. Discard detached colonic epithelium by passing through a 40 µm cell strainer.
5. Collect the remaining tissue from the strainer and further digest them in digestion buffer containing Collagenase type IV and DNase I (Table of Materials) in 1x HBSS with 5% FBS for 20 min at 37 °C at 100 rpm.
6. Vortex the digested tissues for approximately 20 s and pass through a 40 µm cell strainer to obtain lamina propria fractions.
7. Centrifuge the lamina propria fractions to pellet down the cells at 700 x g in 4 °C
8. To exclude dead cells from the lamina propria use a density gradient (Table of Materials).
   NOTE: The density gradient media used here (Table of Materials) may cause loss of mononuclear cells; however, it greatly removes dead cells from the preparation, which overall increases the purity.
   1. Make 100 mL each of 30% and 70% density gradient media solutions. Resuspend the obtained cells in 10 mL of the 30% solution and overlay on top of 5 mL of the 70% solution in a 15 mL tube.
   2. Centrifuge the gradient in a brake-free condition at 1,000 x g and room temperature. Collect the white ring phase containing lamina propria lymphocytes, which will be between the 30% and 70% gradient layers.
9. Wash the obtained cells by re-suspending in ice-cold HBSS and centrifuge at 500 x g, 20 °C, for 10 min. Re-suspend cells in FACS (fluorescence-activated cell sorting) buffer (PBS, pH 7.4, with 1% BSA and 2 mM EDTA).
10. Purify mononuclear phagocytes from the collected cells purification using magnetic beads and/or FACS sorting.
    1. Magnetic purification
       1. Prior to staining with antibodies, first block cell surface of lamina propria cells by incubating with anti-mouse CD16/CD32 Fc blocker for 15 min on ice.
       2. Incubate cells with anti-Cx3Cr1-PE (phycoerythrin) antibodies along with anti-PE microbeads (Table of Materials) for 30 min on ice, to capture the bound cells. Wash cells with FACS buffer.
       3. Pass the antibody/bead bound cells through a magnetic-activated cell-sorting column in a magnetic field to remove unbound cells. Wash three times with FACS buffer. Remove the column from the magnetic field and push plunger in the column to yield bead bound cells.
    2. FACS sorting
       NOTE: FACS sorting can be used in lieu of magnetic purification. Even though magnetic purification is an easy and cost-effective method, it is limited to only purifying single cells, not for subpopulations of cells. Therefore, to isolate subpopulations of cells, the FACS sorting method is an effective method of sorting single cells as well as subpopulations of cells.
       1. Block lamina propria cells with anti-mouse CD16/CD32 Fc blocker (Table of Materials).
       2. Stain cells by incubating with anti-CD64, CD11c, CD11b, Cx3Cr1, Ly6C, and MHCII antibodies.
       3. Sort using a FACS flow cytometer, gating for CD11c-CD64⁺ CD11b⁺ Cx3Cr1⁺ Ly6C^– cells to purify the CX3Cr1 (P3 + P4) population.
11. Lyse sorted cells for total RNA preparation and detect cytokine and fibrotic markers (see section 6 for RNA and cDNA preparation).
12. Analyze mRNA expression of fibrotic markers and inflammatory cytokine analysis from isolated single-cell suspensions from magnetic purification.
13. For FACS analysis, block cells with anti-mouse CD16/CD32 Fc blocker (Table of Materials) prior to staining with antibodies against surface or intracellular markers.

5. Flow cytometry analysis

1. Cell-surface staining and analysis
   1. Resuspend 5-10 × 10⁵ isolated lamina propria cells in 50 mL of FACS buffer (1% BSA, 2 mM EDTA in PBS, pH 7.4).
   2. Incubate cells with anti-mouse CD16/CD32 (Table of Materials) at a 1:50 dilution to block Fc receptors on ice for 10 min.
   3. Wash cells with 500 µL of ice-cold FACS buffer to remove unbound anti-CD16/CD32 prior to cell-surface staining.
   4. Surface stain colonic single-cell suspensions (10⁶ cells/50 mL) with fluorescent-labelled antibody at 1:100 dilution on ice for 30 min to evaluate colonic lamina propria.
   5. Wash labeled cells with 500 µL of ice-cold FACS buffer twice to remove unbound antibodies.
   6. Analyze labeled mononuclear cells by flow cytometry. Gate for Live, then for FSC⁺SSC⁺ followed by CD11b⁺ Cx3Cr1⁺, and then analyze other macrophage activation markers including MHCII, CD80, CD86 and CD40.
2. Intracellular cytokine staining and analysis
   1. For intracellular cytokine staining, fix and permeabilize the surface-stained cells using a Fixation/Permeabilization Solution Kit (Table of Materials); please follow the manufacturer's instructions for detailed steps.
   2. Gate lamina propria cells for Live, then for FSC⁺SSC⁺ followed by CD11b⁺ Cx3Cr1⁺IL23⁺ to detect intracellular level of IL23 cytokine and CD11b⁺ Cx3Cr1⁺IL1β⁺ to detect intracellular level of IL1β cytokine.
3. αSMA staining and analysis
   1. Wash cells with FACS buffer twice by centrifuging at 200 x g and 4 °C for 5 min to remove excess fixative buffer.
   2. To determine the αSMA level, permeabilize cells with a Fixation/Permeabilization Solution Kit (Table of Materials).
   3. Incubate cells with anti-αSMA-AF488 antibody (Table of Materials) using 1:1,000 dilution on ice for 30 min.
   4. Wash cells twice and then do FACS analysis. Gate for Live, FSC⁺SSC⁺ followed by detection of αSMA⁺ cells in colonic cells.

6. RNA isolation, RT-PCR, real-time PCR

1. Isolate RNA from MACS or FACS purified cells or colon excised tissue by homogenizing them in extraction reagent (Table of Materials).
   NOTE: Use safety goggles and other lab protective gear when working with this reagent as it is a lung and skin irritant. Work safely in a hood.
2. Add 0.5 µL of RNase-free glycogen from 20 µg/mL glycogen stock solution (Table of Materials) to improve the recovery of total RNA prior to precipitating RNA with isopropanol.
3. Resuspend the RNA pellet in 50 µL of RNase free water (Table of Materials) and incubate at 55 °C for 5 min to dissolve the RNA palate completely.
4. Read RNA concentrations using a spectrophotometer at 260 nm.
5. Synthesize cDNA using 1 µg of total RNA and a reverse transcription synthesis kit (Table of Materials).
6. Analyze gene expression using 2 µL of cDNA as templates via real-time PCR. Use a 96-well PCR plate qPCR Master Mix kit (Table of Materials). Use CT values to calculate the fold change of RNA abundance after normalizing the GAPDH/HPRT values.

7. Western blotting

1. Lyse colon tissue by using RIPA lysis buffer (1% NP40).
2. Resolve protein lysate in an 8% bis-Tris gel at a constant voltage of 80 V.
3. Transfer gel-protein to nitrocellulose membrane(s) in cold transfer buffer running at a constant current of 50 mA for 2 h at 4 °C.
4. Block non-specific regions on the protein transferred membrane using 5% non-fat milk in TBST (25 mM Tris-HCl, pH 7.4, 1.5 M NaCl, 0.05% Tween-20) for at least 1 h at room temperature.
5. To detect αSMA levels, incubate membrane(s) with αSMA primary detection antibody at 4 °C overnight.
6. Wash membranes 3-4 times using TBST in 15 min intervals.
7. Incubate with HRP-conjugated secondary antibody (1:10,000) in 5% non-fat milk in TBST.
8. Develop membranes using an ECL developing kit.
9. Normalize protein signal intensities with GAPDH as a loading control for densitometry analysis.

8. Statistical analysis

1. Analyze the data using data analysis software of choice by comparing between wild type and KO animals.
2. Consider P-value of <0.05 to be significant (*P < 0.05; **P < 0.01; ***P < 0.001).
3. Use Student's t-test to test the differences between two groups and ANOVA test for analysis between more than two groups.