This work was supported by American Gastroenterological Association (AGA) Research Scholars Award and Crohn&#8217;s and Colitis Foundation of America (CCFA) Career Development Award (to S.Z.S.), NIH NIDDK F30 DK089692 (to E.C.S.), and University of North Carolina Center for Gastrointestinal Biology and Disease Grant P30 DK34987 (Histology Core). The UNC Flow Cytometry Core Facility is supported in part by an NCI Center Core Support Grant (P30CA016086) to the UNC Lineberger Comprehensive Cancer Center. We thank Luke B. Borst from North Carolina State University College of Veterinary Medicine for his help with histopathological analysis and immunohistochemistry.

NOTE: Ensure that all animal protocols are approved by and in compliance with Institutional Animal Care and Use Committee (IACUC) regulations and the National Research Council&#8217;s Guide for the Care and Use of Laboratory Animals. Donor mice may be either male or female, but recipient mice should be male. If female recipients are to be used, the donor mice must be female5. Maintain colonies using regular, non-sterile bedding and non-acidified water, as these may impact the intestinal microbiota, an...

<ol>
	<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 (7152), 427-434 (2007).</li><li>Cho, J. H., Brant, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+the+genetics+of+inflammatory+bowel+disease.">Recent insights into the genetics of inflammatory bowel disease.</a> Gastroenterology. 140 (6), 1704-1712 (2011).</li><li>Jostins, L., 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=Host-microbe+interactions+have+shaped+the+genetic+architecture+of+inflammatory+bowel+disease.">Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease.</a> Nature. 491 (7422), 119-124 (2012).</li><li>Powrie, F., Leach, M. W., Mauze, S., Caddle, L. B., Coffman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypically+distinct+subsets+of+CD4++T+cells+induce+or+protect+from+chronic+intestinal+inflammation+in+C.+B-17+scid+mice.">Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice.</a> Int Immunol. 5 (11), 1461-1471 (1993).</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> Am J Physiol Gastrointest Liver Physiol. 296 (2), 135-146 (2009).</li><li>Ma, B. W., 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=Routine+habitat+change:+a+source+of+unrecognized+transient+alteration+of+intestinal+microbiota+in+laboratory+mice.">Routine habitat change: a source of unrecognized transient alteration of intestinal microbiota in laboratory mice.</a> PLoS One. 7 (10), e47416 (2012).</li><li>Read, S., Powrie, F. <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+inflammatory+bowel+disease+in+immunodeficient+mice+by+depletion+of+regulatory+T+cells.">Induction of inflammatory bowel disease in immunodeficient mice by depletion of regulatory T cells.</a> Curr Protoc Immunol. Chapter 15 (Unit 15 13), (1999).</li><li>Maillard, M. H., 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+Wiskott-Aldrich+syndrome+protein+is+required+for+the+function+of+CD4(+)CD25(+)Foxp3(+)+regulatory+T+cells.">The Wiskott-Aldrich syndrome protein is required for the function of CD4(+)CD25(+)Foxp3(+) regulatory T cells.</a> J Exp Med. 204 (2), 381-391 (2007).</li><li>Hegazi, R. A., 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=Carbon+monoxide+ameliorates+chronic+murine+colitis+through+a+heme+oxygenase+1-dependent+pathway.">Carbon monoxide ameliorates chronic murine colitis through a heme oxygenase 1-dependent pathway.</a> J Exp Med. 202 (12), 1703-1713 (2005).</li><li>Kole, A., 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=Type+I+IFNs+regulate+effector+and+regulatory+T+cell+accumulation+and+anti-inflammatory+cytokine+production+during+T+cell-mediated+colitis.">Type I IFNs regulate effector and regulatory T cell accumulation and anti-inflammatory cytokine production during T cell-mediated colitis.</a> J Immunol. 191 (5), 2771-2779 (2013).</li><li>Kobayashi, T., 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=NFIL3-deficient+mice+develop+microbiota-dependent,+IL-12/23-driven+spontaneous+colitis.">NFIL3-deficient mice develop microbiota-dependent, IL-12/23-driven spontaneous colitis.</a> J Immunol. 192 (4), 1918-1927 (2014).</li><li>Steinbach, E. C., 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=Innate+PI3K+p110delta+Regulates+Th1/Th17+Development+and+Microbiota-Dependent+Colitis.">Innate PI3K p110delta Regulates Th1/Th17 Development and Microbiota-Dependent Colitis.</a> J Immunol. 192 (8), 3958-3968 (2014).</li><li>Kobayashi, T., 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=NFIL3+is+a+regulator+of+IL-12+p40+in+macrophages+and+mucosal+immunity.">NFIL3 is a regulator of IL-12 p40 in macrophages and mucosal immunity.</a> J Immunol. 186 (8), 4649-4655 (2011).</li><li>Leach, M. W., Bean, A. G., Mauze, S., Coffman, R. L., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+bowel+disease+in+C.B-17+scid+mice+reconstituted+with+the+CD45RBhigh+subset+of+CD4++T+cells.">Inflammatory bowel disease in C.B-17 scid mice reconstituted with the CD45RBhigh subset of CD4+ T cells.</a> Am J Pathol. 148 (5), 1503-1515 (1996).</li><li>Powrie, F., 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=Inhibition+of+Th1+responses+prevents+inflammatory+bowel+disease+in+scid+mice+reconstituted+with+CD45RBhi+CD4.">Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4.</a> T cells. Immunity. 1 (7), 553-562 (1994).</li><li>Read, S., Malmstrom, V., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T+lymphocyte-associated+antigen+4+plays+an+essential+role+in+the+function+of+CD25(+)CD4(+)+regulatory+cells+that+control+intestinal+inflammation.">Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation.</a> J Exp Med. 192 (2), 295-302 (2000).</li><li>Rogers, G. B., 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=Functional+divergence+in+gastrointestinal+microbiota+in+physically-separated+genetically+identical+mice.">Functional divergence in gastrointestinal microbiota in physically-separated genetically identical mice.</a> Sci Rep. 4, 5437 (2014).</li><li>Fukata, 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+myeloid+differentiation+factor+88+(MyD88)+is+required+for+CD4++T+cell+effector+function+in+a+murine+model+of+inflammatory+bowel+disease.">The myeloid differentiation factor 88 (MyD88) is required for CD4+ T cell effector function in a murine model of inflammatory bowel disease.</a> J Immunol. 180 (3), 1886-1894 (2008).</li><li>Kurtz, C. C., 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=Extracellular+adenosine+regulates+colitis+through+effects+on+lymphoid+and+nonlymphoid+cells.">Extracellular adenosine regulates colitis through effects on lymphoid and nonlymphoid cells.</a> Am J Physiol Gastrointest Liver Physiol. 307 (3), 338-346 (2014).</li><li>Naganuma, 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=Cutting+edge:+Critical+role+for+A2A+adenosine+receptors+in+the+T+cell-mediated+regulation+of+colitis.">Cutting edge: Critical role for A2A adenosine receptors in the T cell-mediated regulation of colitis.</a> J Immunol. 177 (5), 2765-2769 (2006).</li><li>Ranatunga, D. C., 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+protective+role+for+human+IL-10-expressing+CD4++T+cells+in+colitis.">A protective role for human IL-10-expressing CD4+ T cells in colitis.</a> J Immunol. 189 (3), 1243-1252 (2012).</li><li>Srikrishna, 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=Carboxylated+glycans+mediate+colitis+through+activation+of+NF-kappa.">Carboxylated glycans mediate colitis through activation of NF-kappa.</a> B. J Immunol. 175 (8), 5412-5422 (2005).</li><li>Wang, F., 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=IFN-gamma-induced+TNFR2+expression+is+required+for+TNF-dependent+intestinal+epithelial+barrier+dysfunction.">IFN-gamma-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction.</a> Gastroenterology. 131 (4), 1153-1163 (2006).</li></ol>

The authors declare that they have no competing financial interests.

Here we describe a step-by-step protocol inducing colonic inflammation in mice by adoptive transfer of CD4+CD45RB+ T cells into immunodeficient mice. We used C57BL/6 donor spleens and syngeneic Rag1-/- recipient mice, although other strains (e.g., BALB/c, 129S6/SvEv, non-obese diabetic (NOD)) and genetic models of immunodeficiency (e.g., SCID, Rag2-/-) may also be used4,14-16. It is well established that background strain affects e...

The inflammatory bowel diseases (IBD) Crohn&#8217;s disease and ulcerative colitis result from an incompletely defined and complex interaction between host immune responses, genetic susceptibility, environmental factors, and the enteric luminal contents1. Recent genome-wide association studies report associations between immune cell regulatory genes and IBD susceptibility2,3. Both innate and adaptive immune cell intrinsic genes are represented in these studies, indicating a central role for these cell populations in IBD pathogenesis.
There currently exist more than 50 animal models of human IBD. While no one model perfectly phenocopies human IBD, many are useful for studying various aspects of human disease, including disease onset and progression and the wound-healing response. In the method described here, intestinal inflammation is initiated with syngeneic splenic CD4+CD45RBhigh T cell adoptive transfer into T and B cell deficient recipient mice4. The CD4+CD45RBhigh T cell population contains mainly na&#239;ve T cells primed for activation that are capable of inducing chronic small bowel and colonic inflammation. This method allows the researcher to modify key experimental variables, including both innate and adaptive immune cell populations, to answer biologically relevant questions relating to disease pathogenesis. Additionally, this method provides precise initiation of disease onset and a well-characterized experimental time course. This permits the kinetic study of clinical features of disease progression in mice. Intestinal inflammation induced by this method shares many features with human IBD, including chronic large and small bowel transmural inflammation, pathogenesis driven by cytokines such as TNF and IL-12, and systemic symptoms such as wasting5. Thus, it is an ideal model system for studying the pathogenesis of human IBD.
The method here describes in detail the protocol for inducing experimental colitis by adoptive transfer of CD4+CD45RBhigh T cells into Rag1-/- mice. We discuss key technical steps, expected results, optimization, and trouble-shooting. We address the required elements for the successful development of this murine model of intestinal inflammation for research purposes.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Reagent/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >10x PBS</td>
			<td >Gibco</td>
			<td >14200075</td>
			<td></td>
		</tr>
		<tr >
			<td >12x75mm round-bottom tube</td>
			<td >Falcon</td>
			<td >352052</td>
			<td></td>
		</tr>
		<tr >
			<td >15 ml conical</td>
			<td >Corning</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >26g x 3/8 Needle</td>
			<td >BD Biosciences</td>
			<td >305110</td>
			<td></td>
		</tr>
		<tr >
			<td >50 ml conical</td>
			<td >Corning</td>
			<td >430828</td>
			<td></td>
		</tr>
		<tr >
			<td >70 um Cell Strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td></td>
		</tr>
		<tr >
			<td >BD IMagnet</td>
			<td >BD Biosciences</td>
			<td >552311</td>
			<td></td>
		</tr>
		<tr >
			<td >&#946;-mercaptoethanol</td>
			<td >Thermo Scientific</td>
			<td >35602</td>
			<td></td>
		</tr>
		<tr >
			<td >CD4-FITC IgG2b</td>
			<td >eBioscience</td>
			<td >11-0041</td>
			<td></td>
		</tr>
		<tr >
			<td >CD45RB-PE IgG2a</td>
			<td >BD Pharminogen</td>
			<td >553101</td>
			<td></td>
		</tr>
		<tr >
			<td >Complete Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 10% FBS, 0.0004% &#946;-ME</td>
		</tr>
		<tr >
			<td >FACS tube + strainer</td>
			<td >BD Falcon</td>
			<td >352235</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Microscope Slides</td>
			<td >Fisherbrand</td>
			<td >12550A3</td>
			<td></td>
		</tr>
		<tr >
			<td >Heat-inactivated FBS</td>
			<td >Gemini</td>
			<td >100-106</td>
			<td></td>
		</tr>
		<tr >
			<td >Labeling Buffer</td>
			<td ></td>
			<td ></td>
			<td >1x PBS, 0.5% BSA, 2 mM EDTA</td>
		</tr>
		<tr >
			<td >Lysis Buffer</td>
			<td ></td>
			<td ></td>
			<td >0.08% NH4Cl, 0.1% KHCO3, 1 mM EDTA</td>
		</tr>
		<tr >
			<td >MoFlo XDP</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse CD4 T lymphocyte Enrichment Set - DM</td>
			<td >BD Biosciences</td>
			<td >558131</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2a-PE</td>
			<td >BD Pharminogen</td>
			<td >553457</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2b-FITC</td>
			<td >eBioscience</td>
			<td >11-4732</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipet</td>
			<td >Fisherbrand</td>
			<td >13-678-20D</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin Solution, 100X</td>
			<td >Corning Cellgro</td>
			<td >30-002-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Petri Dish</td>
			<td >Fisherbrand</td>
			<td >875713</td>
			<td></td>
		</tr>
		<tr >
			<td >Pure Ethanol 200 Proof</td>
			<td >Decon Labs</td>
			<td >2705-HC</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI-1640</td>
			<td >Gibco</td>
			<td >11-875-093</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td >BD Biosciences</td>
			<td >309597</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan blue</td>
			<td >Corning Cellgro</td>
			<td >25-900-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Wash Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 0.0004% &#946;-ME</td>
		</tr>
	</tbody>
</table>

induction of murine intestinal inflammation by adoptive transfer of effector cd4+cd45rbhigh t cells into immunodeficient mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Approximately 10 x 106 CD4+CD45RBhigh T cells from 10 spleens from adult C57BL/6 donor mice are reliably isolated. This number will vary depending on the age and strain of the donor mouse and the proficiency of the researcher. When 4 x 105 C57BL/6 CD4+CD45RBhigh T cells are transferred into C57BL/6 Rag1-/- recipient mice, clinical signs of disease emerge around week 5 post-repletion or sooner if mice are genetically susceptible to more ...

Watch this Scientific Journal Video about Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice at JoVE.com

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

induction-murine-intestinal-inflammation-adoptive-transfer-effector

Research

JoVE Journal

Immunology and Infection

16.2K 조회수.  University of North Carolina at Chapel Hill. Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.