Name: mechanistic insight into the development of tnbs-mediated intestinal fibrosis and evaluating the inhibitory effects of rapamycin
Uploaded: 2019-09-12T14:45:00
Duration: 10 min 21 s
Description: Watch this Scientific Journal Video about Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin at JoVE.com

This work was supported by NIH grant R01NS093045 (Y.H.), NIH grant K08DK088950 (X.Z.), R03DK099566 (X.Z.), and The Crohn&#39;s &#38; Colitis Foundation of America research Fellowship (CCFA) 481637 (R.M.).

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
<ol>
	<li>Collect human tissue samples according to the institution&#39;s research committee protocols.</li>
	<li>Procure intestinal tissue (ileocolonic) of a CD patient diagnosed with fibrosis and control samples from patients with no history of IBDs.</li>
	<li>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.</li>
	<li>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 &#181;m thick tissue sections by using a tissue-microtome.</li>
	<li>Using a brush, gently spread out each cut section on a glass slide for staining.</li>
	<li>Stain tissue section slide with trichrome stain to detect collagen deposition, and with &#945;SMA stain to detect myofibroblasts (See section 3 for detailed information about trichrome and &#945;SMA staining). Examine the stained sections under a light microscope.</li>
	<li>Process another part of the obtained human tissue sample to make protein lysate to detect &#945;SMA via western blot (see section 7 for detailed information about western blot analysis).</li>
	<li>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.</li>
	<li>Analyze fibrotic and cytokine genes by qPCR (see section 6 for detailed information about mRNA preparation).</li>
</ol>
2. Induction of TNBS fibrosis and rapamycin treatment in mice
<ol>
	<li>Carry out animal research according to the animal research protocols approved by the institution.</li>
	<li>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.</li>
	<li>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 &#181;L enema via a 1 mL syringe attached to a 3.5 French polyurethane catheter and give control mice 100 &#181;L of 25% ethanol only.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Use veterinary ointment on eyes to prevent dryness while mice are under anesthesia.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Cut the colon into small pieces and keep at -80 &#176;C for storage for future purposes (RNA/western blot analysis). Use another piece of the colon for FACS analysis.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Immuno-histopathologic assessment of gut fibrosis
<ol>
	<li>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.</li>
	<li>Paraffin embed the fixed colon tissues, slice to a 5 &#181;m thickness, and directly put the tissues on glass slides for histology.</li>
	<li>Perform H&#38;E and Trichrome Blue staining according to manufacturer&#39;s instructions to detect collagen.
	<ol>
		<li>Briefly, first deparaffinize sections by submerging the slide containing sections in two chambers of xylene for 5 min in each chamber.</li>
		<li>Rinse slides with 100% alcohol for 1 min; repeat this step three times. Rinse again with tap water for 1 min.</li>
		<li>After that, immerse slides in preheated Bouin&#39;s solution at 56 &#176;C for 60 min. Bouin&#39;s solution is yellow in appearance; wash after taking out the slides from Bouin&#39;s solution in tap water for 3 min or until slides became colorless.</li>
		<li>Immerse slides in Modified Mayer&#39;s Hematoxylin solution for 7 min at room temperature.</li>
		<li>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.</li>
		<li>Dehydrate slides with 100% alcohol for 1 min. Repeat this procedure three times.</li>
		<li>Clear slides with xylene for 1 min and repeat the step 3x.</li>
		<li>Mount slides with mounting media (Table of Materials).</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Use immunostaining to detect &#945;SMA.
	<ol>
		<li>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.</li>
		<li>Incubate with 100 &#181;L of diluted primary antibody for &#945;SMA (Table of Materials) on the slide solution (0.2% Triton X-100 and 3% normal goat serum in 1x PBS; anti-&#945;SMA 1:200) at 4 &#176;C overnight.</li>
		<li>Wash the sections three times with 1x PBS.</li>
		<li>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.</li>
		<li>Wash the sections three times with 1x PBS.</li>
		<li>Use DAPI to counterstain the nucleus for 5 min.</li>
		<li>Wash the sections three times with 1x PBS.</li>
		<li>Mount slides with fluorescent mounting media (Table of Materials), and seal with a coverslip using nail polish.</li>
	</ol>
	</li>
	<li>Acquire images by using LSM 880 confocal microscope and process images using microscope software.</li>
	<li>Quantify images by taking an average of multiple selected areas in the same section with ImageJ.</li>
</ol>
4. Isolation of intestinal lamina propria and purification of Cx3Cr1+ mononuclear phagocytes
<ol>
	<li>Longitudinally open the colon in ice-cold Hank&#39;s Balanced Salt Solution (HBSS; Table of Materials) and wash the colon with the same buffer.</li>
	<li>Cut approximately 5 cm small pieces of colon tissues using sterile scissors in HBSS.</li>
	<li>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 &#176;C incubator11. 
	NOTE: Incubation of colonic tissue in 37 &#176;C at 100 rpm shaking condition allows for efficient collagenase tissue digestion and high yield of viable cells.</li>
	<li>Discard detached colonic epithelium by passing through a 40 &#181;m cell strainer.</li>
	<li>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 &#176;C at 100 rpm.</li>
	<li>Vortex the digested tissues for approximately 20 s and pass through a 40 &#181;m cell strainer to obtain lamina propria fractions.</li>
	<li>Centrifuge the lamina propria fractions to pellet down the cells at 700 x g in 4 &#176;C</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Wash the obtained cells by re-suspending in ice-cold HBSS and centrifuge at 500 x g, 20 &#176;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).</li>
	<li>Purify mononuclear phagocytes from the collected cells purification using magnetic beads and/or FACS sorting.
	<ol>
		<li>Magnetic purification
		<ol>
			<li>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.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>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.
		<ol>
			<li>Block lamina propria cells with anti-mouse CD16/CD32 Fc blocker (Table of Materials).</li>
			<li>Stain cells by incubating with anti-CD64, CD11c, CD11b, Cx3Cr1, Ly6C, and MHCII antibodies.</li>
			<li>Sort using a FACS flow cytometer, gating for CD11c-CD64+ CD11b+ Cx3Cr1+ Ly6C- cells to purify the CX3Cr1 (P3 + P4) population.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Lyse sorted cells for total RNA preparation and detect cytokine and fibrotic markers (see section 6 for RNA and cDNA preparation).</li>
	<li>Analyze mRNA expression of fibrotic markers and inflammatory cytokine analysis from isolated single-cell suspensions from magnetic purification.</li>
	<li>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.</li>
</ol>
5. Flow cytometry analysis
<ol>
	<li>Cell-surface staining and analysis
	<ol>
		<li>Resuspend 5-10 &#215; 105 isolated lamina propria cells in 50 mL of FACS buffer (1% BSA, 2 mM EDTA in PBS, pH 7.4).</li>
		<li>Incubate cells with anti-mouse CD16/CD32 (Table of Materials) at a 1:50 dilution to block Fc receptors on ice for 10 min.</li>
		<li>Wash cells with 500 &#181;L of ice-cold FACS buffer to remove unbound anti-CD16/CD32 prior to cell-surface staining.</li>
		<li>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.</li>
		<li>Wash labeled cells with 500 &#181;L of ice-cold FACS buffer twice to remove unbound antibodies.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Intracellular cytokine staining and analysis
	<ol>
		<li>For intracellular cytokine staining, fix and permeabilize the surface-stained cells using a Fixation/Permeabilization Solution Kit (Table of Materials); please follow the manufacturer&#39;s instructions for detailed steps.</li>
		<li>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+IL1&#946;+ to detect intracellular level of IL1&#946; cytokine.</li>
	</ol>
	</li>
	<li>&#945;SMA staining and analysis
	<ol>
		<li>Wash cells with FACS buffer twice by centrifuging at 200 x g and 4 &#176;C for 5 min to remove excess fixative buffer.</li>
		<li>To determine the &#945;SMA level, permeabilize cells with a Fixation/Permeabilization Solution Kit (Table of Materials).</li>
		<li>Incubate cells with anti-&#945;SMA-AF488 antibody (Table of Materials) using 1:1,000 dilution on ice for 30 min.</li>
		<li>Wash cells twice and then do FACS analysis. Gate for Live, FSC+SSC+ followed by detection of &#945;SMA+ cells in colonic cells.</li>
	</ol>
	</li>
</ol>
6. RNA isolation, RT-PCR, real-time PCR
<ol>
	<li>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.</li>
	<li>Add 0.5 &#181;L of RNase-free glycogen from 20 &#181;g/mL glycogen stock solution (Table of Materials) to improve the recovery of total RNA prior to precipitating RNA with isopropanol.</li>
	<li>Resuspend the RNA pellet in 50 &#181;L of RNase free water (Table of Materials) and incubate at 55 &#176;C for 5 min to dissolve the RNA palate completely.</li>
	<li>Read RNA concentrations using a spectrophotometer at 260 nm.</li>
	<li>Synthesize cDNA using 1 &#181;g of total RNA and a reverse transcription synthesis kit (Table of Materials).</li>
	<li>Analyze gene expression using 2 &#181;L 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.</li>
</ol>
7. Western blotting
<ol>
	<li>Lyse colon tissue by using RIPA lysis buffer (1% NP40).</li>
	<li>Resolve protein lysate in an 8% bis-Tris gel at a constant voltage of 80 V.</li>
	<li>Transfer gel-protein to nitrocellulose membrane(s) in cold transfer buffer running at a constant current of 50 mA for 2 h at 4 &#176;C.</li>
	<li>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.</li>
	<li>To detect &#945;SMA levels, incubate membrane(s) with &#945;SMA primary detection antibody at 4 &#176;C overnight.</li>
	<li>Wash membranes 3-4 times using TBST in 15 min intervals.</li>
	<li>Incubate with HRP-conjugated secondary antibody (1:10,000) in 5% non-fat milk in TBST.</li>
	<li>Develop membranes using an ECL developing kit.</li>
	<li>Normalize protein signal intensities with GAPDH as a loading control for densitometry analysis.</li>
</ol>
8. Statistical analysis
<ol>
	<li>Analyze the data using data analysis software of choice by comparing between wild type and KO animals.</li>
	<li>Consider P-value of &#60;0.05 to be significant (*P &#60; 0.05; **P &#60; 0.01; ***P &#60; 0.001).</li>
	<li>Use Student&#39;s t-test to test the differences between two groups and ANOVA test for analysis between more than two groups.</li>
</ol>

<ol>
	<li>Garrett, W. S., Gordon, J. I., Glimcher, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostasis+and+inflammation+in+the+intestine.">Homeostasis and inflammation in the intestine.</a> Cell. 140, 859-870 (2010).</li><li>Park, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+regulatory+cells+maintain+intestinal+homeostasis+by+suppressing+gammadelta+T+cells.">T regulatory cells maintain intestinal homeostasis by suppressing gammadelta T cells.</a> Immunity. 33, 791-803 (2010).</li><li>Speca, S., Giusti, I., Rieder, F., Latella, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+of+intestinal+fibrosis.">Cellular and molecular mechanisms of intestinal fibrosis.</a> World Journal of Gastroenterology. 18, 3635-3661 (2012).</li><li>Keubler, L. M., Buettner, M., Hager, C., Bleich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Multihit+Model:+Colitis+Lessons+from+the+Interleukin-10-deficient+Mouse.">A Multihit Model: Colitis Lessons from the Interleukin-10-deficient Mouse.</a> Inflammatory Bowel Disease. 21, 1967-1975 (2015).</li><li>Strober, W., Nakamura, K., Kitani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SAMP1/Yit+mouse:+another+step+closer+to+modeling+human+inflammatory+bowel+disease.">The SAMP1/Yit mouse: another step closer to modeling human inflammatory bowel disease.</a> Journal of Clinical Investigation. 107, 667-670 (2001).</li><li>Ostanin, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+transfer+model+of+chronic+colitis:+concepts,+considerations,+and+tricks+of+the+trade.">T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade.</a> American Journal of Physiology-Gastroenterology and Liver Physiology. 296, G135-G146 (2009).</li><li>Antoniou, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TNBS-induced+colitis+animal+model:+An+overview.">The TNBS-induced colitis animal model: An overview.</a> Annals of Medicine and Surgery. 11, 9-15 (2016).</li><li>Loeuillard, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2,4,6-trinitrobenzene+sulfonic+acid-induced+chronic+colitis+with+fibrosis+and+modulation+of+TGF-beta1+signaling.">2,4,6-trinitrobenzene sulfonic acid-induced chronic colitis with fibrosis and modulation of TGF-beta1 signaling.</a> World Journal of Gastroenterology. 20, 18207-18215 (2014).</li><li>Mathur, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+autophagy+in+Cx3cr1(+)+mononuclear+cells+limits+IL-23/IL-22+axis-mediated+intestinal+fibrosis.">Induction of autophagy in Cx3cr1(+) mononuclear cells limits IL-23/IL-22 axis-mediated intestinal fibrosis.</a> Mucosal Immunology. 12, 612-623 (2019).</li><li>Joeris, T., Muller-Luda, K., Agace, W. W., Mowat, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+functions+of+intestinal+mononuclear+phagocytes.">Diversity and functions of intestinal mononuclear phagocytes.</a> Mucosal Immunology. 10, 845-864 (2017).</li><li>Mathur, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+Salmonella+typhi+infection.">A mouse model of Salmonella typhi infection.</a> Cell. 151, 590-602 (2012).</li><li>Zhao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninflammatory+Changes+of+Microglia+Are+Sufficient+to+Cause+Epilepsy.">Noninflammatory Changes of Microglia Are Sufficient to Cause Epilepsy.</a> Cell Reports. 22, 2080-2093 (2018).</li><li>Murugaiyan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-21+promotes+Th17+differentiation+and+mediates+experimental+autoimmune+encephalomyelitis.">MicroRNA-21 promotes Th17 differentiation and mediates experimental autoimmune encephalomyelitis.</a> Journal of Clinical Investigation. 125, 1069-1080 (2015).</li><li>Hinz, B., Celetta, G., Tomasek, J. J., Gabbiani, G., Chaponnier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-smooth+muscle+actin+expression+upregulates+fibroblast+contractile+activity.">Alpha-smooth muscle actin expression upregulates fibroblast contractile activity.</a> Molecular Biology of the Cell. 12, 2730-2741 (2001).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapamycin+Inhibition+of+mTOR+Reduces+Levels+of+the+Na+/H++Exchanger+3+in+Intestines+of+Mice+and+Humans,+Leading+to+Diarrhea.">Rapamycin Inhibition of mTOR Reduces Levels of the Na+/H+ Exchanger 3 in Intestines of Mice and Humans, Leading to Diarrhea.</a> Gastroenterology. 149, 151-162 (2015).</li><li>Mutalib, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+sirolimus+(rapamycin)+in+the+management+of+refractory+inflammatory+bowel+disease+in+children.">The use of sirolimus (rapamycin) in the management of refractory inflammatory bowel disease in children.</a> Journal of Crohns and Colitis. 8, 1730-1734 (2014).</li><li>Shaw, T. J., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+at+a+glance.">Wound repair at a glance.</a> Journal of Cell Science. 122, 3209-3213 (2009).</li><li>Paul, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17-driven+intestinal+fibrosis+is+inhibited+by+Itch-mediated+ubiquitination+of+HIC-5.">IL-17-driven intestinal fibrosis is inhibited by Itch-mediated ubiquitination of HIC-5.</a> Mucosal Immunology. 11, 427-436 (2018).</li><li>Sohail, I., Ghosh, S., Mukundan, S., Zelewski, S., Khan, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Inflammatory+Risk+Factors+in+the+Pathogenesis+of+Streptococcus+pneumoniae.">Role of Inflammatory Risk Factors in the Pathogenesis of Streptococcus pneumoniae.</a> Frontiers of Immunology. 9, 2275 (2018).</li><li>Laplante, M., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTOR+signaling+in+growth+control+and+disease.">mTOR signaling in growth control and disease.</a> Cell. 149, 274-293 (2012).</li><li>Xavier, R. J., Podolsky, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unravelling+the+pathogenesis+of+inflammatory+bowel+disease.">Unravelling the pathogenesis of inflammatory bowel disease.</a> Nature. 448, 427-434 (2007).</li></ol>

Competing interests: The authors declare no competing interests.

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&#39;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 IL1&#946; 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&#39;s disease, TNBS-colitis remains a potent tool to study the immuno-pathogenesis of CD and has the potential to evaluate the Crohn&#39;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&#39;s fibrosis is and will always be warranted.

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&#39;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&#39;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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-mouse aSMA, AF488 conjugated, Clone#1A4</td>
			<td>e-bioscience</td>
			<td>53-9760-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse aSMA, purified Clone#M1/77</td>
			<td>e-bioscience</td>
			<td>149760-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b, APCCy7 conjugated, Clone#M1/70</td>
			<td>Biolegend Inc</td>
			<td>101226</td>
			<td></td>
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			<td>Anti-mouse CX3cr1 PE conjugated, Clone#SA011F11</td>
			<td>Biolegend</td>
			<td>149006</td>
			<td></td>
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			<td>Anti-mouse purified CD16/32 Fc block</td>
			<td>Biolegend Inc</td>
			<td>14-9760-80</td>
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			<td>Miltenyi</td>
			<td>130-105-639</td>
			<td></td>
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			<td>e-bioscience</td>
			<td>7074</td>
			<td></td>
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			<td>InvivoGen</td>
			<td>tlrl-isdn</td>
			<td></td>
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			<td>Roche</td>
			<td>1088866001</td>
			<td></td>
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			<td>DAPI</td>
			<td>SIGMA</td>
			<td>D9542</td>
			<td></td>
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			<td>Corning</td>
			<td>10013-CV</td>
			<td></td>
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			<td>Roche</td>
			<td>D263-5vl</td>
			<td></td>
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			<td>Corning</td>
			<td>352340</td>
			<td></td>
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			<td>BD Bioscience</td>
			<td>555028</td>
			<td></td>
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			<td>FlowJo</td>
			<td>FlowJo LLC</td>
			<td><a href="http://www.flowjo.com/">www.flowjo.com</a></td>
			<td></td>
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			<td>Glycogen</td>
			<td>Roche</td>
			<td>10901393001</td>
			<td></td>
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			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>e-bioscience</td>
			<td>5018</td>
			<td></td>
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			<td>Graphpad Prism 7</td>
			<td>GraphPad Software Inc</td>
			<td><a href="http://www.graphpad.com/">www.graphpad.com</a></td>
			<td></td>
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			<td>H&#38;E kit</td>
			<td>American Mastertech Kit</td>
			<td>HXMMHPT</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>NIH</td>
			<td><a href="http://www.imagej.nih.gov/ij/">www.imagej.nih.gov/ij/</a></td>
			<td></td>
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			<td>Mouse: C57BL/6J</td>
			<td>Jackson Laboratories</td>
			<td>664</td>
			<td></td>
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			<td>Miltenyi</td>
			<td>130-042-201</td>
			<td></td>
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			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
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			<td>PMA/Ionomycin salt</td>
			<td>Sigma</td>
			<td>P8139</td>
			<td></td>
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			<td>PowerUp SYBR Green Master Mix</td>
			<td>Thermofisher</td>
			<td>A25777</td>
			<td></td>
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			<td>Rabbit Anti mouse GAPDH, Clone# D16H11</td>
			<td>Cell signaling</td>
			<td>5174</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti mouse p70 S6 Kinase, Clone#49D7</td>
			<td>Cell signaling</td>
			<td>2708</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti mouse Phospho-p70 S6 Kinase, Clone#S371</td>
			<td>Cell signaling</td>
			<td>9208</td>
			<td></td>
		</tr>
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			<td>Rabbit Anti mouse Phospho-S6 Ribosomal Protein (Ser235/236), Clone# D57.2.2E</td>
			<td>Cell signaling</td>
			<td>4858</td>
			<td></td>
		</tr>
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			<td>Rabbit Anti mouse S6 Ribosomal Protein</td>
			<td>Cell signaling</td>
			<td>2217</td>
			<td></td>
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			<td>Rapamycin</td>
			<td>LC LABORATORIES</td>
			<td>1003799</td>
			<td></td>
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			<td>TNBS</td>
			<td>Sigma</td>
			<td>92823</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichorme staining kit</td>
			<td>American Mastertech Kit</td>
			<td>STOSTBPT</td>
			<td></td>
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			<td>TRIzol</td>
			<td>Life technologies</td>
			<td>15596018</td>
			<td></td>
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		<tr>
			<td>Verso cDNA Synthesis Kit</td>
			<td>Thermofisher</td>
			<td>AB1453B</td>
			<td></td>
		</tr>
		<tr>
			<td>Zen black 2.1</td>
			<td>Carl Zeiss</td>
			<td><a href="http://www.zeis.com/">www.zeis.com</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Zen blue lite 2.3</td>
			<td>Carl Zeiss</td>
			<td><a href="http://www.zeis.com/">www.zeis.com</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

mechanistic insight into the development of tnbs-mediated intestinal fibrosis and evaluating the inhibitory effects of rapamycin

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&#39;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&#39;s-associated intestinal fibrosis.

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 &#177; 0.5 cm in the control group to 3 &#177; 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&#39;s disease model is comparable to the human Crohn&#39;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 (&#945;SMA) 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 &#945;SMA showed a 4-6-fold increase in colonic submucosa layer positively stained with &#945;SMA (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 &#945;SMA-positive cells by FACS analysis in TNBS-treated mice colon and found a significant accumulation of &#945;SMA-positive staining (Figure 1E). Furthermore, we found substantial induction in the expression of &#945;SMA, Col-I, and Col-III measured by qPCR analysis in TNBS-treated mice (Figure 1F). Increased expression of &#945;SMA 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&#39;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 &#945;SMA-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 &#945;SMA expression in active CD samples (Figure 2B). In addition, we observed significant induction of fibrosis markers including &#945;SMA, 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 &#945;SMA and collagen level in colon histology and have shown quantitative measurement of &#945;SMA and collagen in densitometry plot (Figure 3A). Our data suggests that rapamycin reduces the &#945;SMA-positive staining in submucosal layer and lessens the collagen deposition. To further validate and quantify fibrotic responses, we determined the &#945;SMA, collagen and TGF&#946; expressions by qPCR, and &#945;SMA 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-1&#946; 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60067/60067fig1.jpg" /> 
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&#39; histological analysis stained with anti-&#945;SMA antibody for activation of myofibroblasts and trichrome blue staining for collagen; scale bar 100 &#181;m. E. FACS analysis to identify &#945;SMA-positive cells in the colon and quantification of &#945;SMA-positive cells. F. Fibrotic markers and cytokines detected by qPCR. G. western blot analysis of &#945;SMA 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. <a href="https://www.jove.com/files/ftp_upload/60067/60067fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60067/60067fig2.jpg" /> 
Figure 2: TNBS Fibrosis is comparable to Crohn&#39;s-associated Intestinal fibrosis. A. Representative images of colon biopsies of control, active CD showing a significant increase of trichrome blue staining and &#945;SMA-positive staining in submucosal layers, scale bar 50 &#181;m. B. Western blot analysis of &#945;SMA 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. <a href="https://www.jove.com/files/ftp_upload/60067/60067fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60067/60067fig3.jpg" /> 
Figure 3: Rapamycin treatment effectively ameliorates TNBS-induced fibrosis. A. Colon histological analysis - representative images of myofibroblast staining with anti-&#945;SMA antibody and collagen staining with Trichrome blue, scale bar 100 &#181;m. B. qPCR analysis of &#945;SMA, Collagen and TGF&#946; expression in Control/TNBS-treated mouse colons. C. FACS analysis of &#945;SMA 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-1&#946;. This modified figure is being reused with the permission of previous publication9 in Mucosal Immunology, 2019 by Mathur et al. <a href="https://www.jove.com/files/ftp_upload/60067/60067fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin at JoVE.com

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

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;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 ...

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Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

Development of an &lt;em&gt;in vitro&lt;/em&gt; model system for studying the interaction of &lt;em&gt;Equus&lt;/em&gt; &lt;em&gt;caballus&lt;/em&gt; IgE with its high-affinity receptor Fc&amp;#949;RI

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...