Name: induction of intestinal graft-versus-host disease and its mini-endoscopic assessment in live mice
Uploaded: 2019-02-11T13:00:00
Description: Watch this Scientific Journal Video about Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice at JoVE.com

This study was supported by the Collaborative Research Centers (CRC) 221 (CRC/TR221-DFG, #324392634; project B03) (to K.H.) and CRC 1181 (CRC-DFG, project B05) (to K.H.), both funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation).

The experimental methods described here have been approved by the government of Mittelfranken, Bavaria, Germany.
1. GvHD induction
<ol>
	<li>Day 0: Total body irradiation of the recipient mice
	<ol>
		<li>Use female CD45.2+ H2kd+ BALB/c mice that are at least 10 weeks old as recipients.</li>
		<li>Weigh and record the weight of the recipient mice prior to GvHD induction. 
		NOTE: Ensure that the recipient has a minimal body weight ...

<ol>
	<li>Ferrara, J. L., Levine, J. E., Reddy, P., Holler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft-versus-host+disease.">Graft-versus-host disease.</a> The Lancet. 373, 1550-1561 (2009).</li><li>Magenau, J., Runaas, L., Reddy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+understanding+the+pathogenesis+of+graft-versus-host+disease.">Advances in understanding the pathogenesis of graft-versus-host disease.</a> British Journal of Haematology. 173, 190-205 (2016).</li><li>Ball, L. M., Egeler, R. M., Party, E. P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+GvHD:+pathogenesis+and+classification.">Acute GvHD: pathogenesis and classification.</a> Bone Marrow Transplantation. 41, 58-64 (2008).</li><li>Naymagon, S., 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=Acute+graft-versus-host+disease+of+the+gut:+considerations+for+the+gastroenterologist.">Acute graft-versus-host disease of the gut: considerations for the gastroenterologist.</a> Nature Reviews. Gastroenterology &amp; Hepatology. 14, 711-726 (2017).</li><li>Zeiser, R., Blazar, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+acute+and+chronic+graft-versus-host+disease:+how+predictive+are+they+for+a+successful+clinical+translation.">Preclinical models of acute and chronic graft-versus-host disease: how predictive are they for a successful clinical translation.</a> Blood. 127, 3117-3126 (2016).</li><li>Cooke, K. 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=An+experimental+model+of+idiopathic+pneumonia+syndrome+after+bone+marrow+transplantation:+I.+The+roles+of+minor+H+antigens+and+endotoxin.">An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: I. The roles of minor H antigens and endotoxin.</a> Blood. 88, 3230-3239 (1996).</li><li>Anthony, B. A., Hadley, G. A. <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+graft-versus-host+disease+and+in+vivo+T+cell+monitoring+using+an+MHC-matched+murine+model.">Induction of graft-versus-host disease and in vivo T cell monitoring using an MHC-matched murine model.</a> Journal of Visualized Experiments. (66), e3697 (2012).</li><li>Beilhack, 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=Prevention+of+acute+graft-versus-host+disease+by+blocking+T-cell+entry+to+secondary+lymphoid+organs.">Prevention of acute graft-versus-host disease by blocking T-cell entry to secondary lymphoid organs.</a> Blood. 111, 2919-2928 (2008).</li><li>Becker, C., Fantini, M. C., Neurath, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+colonoscopy+in+live+mice.">High resolution colonoscopy in live mice.</a> Nature Protocols. 1, 2900-2904 (2006).</li><li>Buchele, 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=Targeting+Inflammatory+T+Helper+Cells+via+Retinoic+Acid-Related+Orphan+Receptor+Gamma+t+Is+Ineffective+to+Prevent+Allo-Response-Driven+Colitis.">Targeting Inflammatory T Helper Cells via Retinoic Acid-Related Orphan Receptor Gamma t Is Ineffective to Prevent Allo-Response-Driven Colitis.</a> Frontiers in Immunology. 9, 1138 (2018).</li><li>Ullrich, 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=BATF-dependent+IL-7RhiGM-CSF++T+cells+control+intestinal+graft-versus-host+disease.">BATF-dependent IL-7RhiGM-CSF+ T cells control intestinal graft-versus-host disease.</a> The Journal of Clinical Investigation. 128 (3), 916-930 (2018).</li><li>Kaplan, D. 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=Target+antigens+determine+graft-versus-host+disease+phenotype.">Target antigens determine graft-versus-host disease phenotype.</a> Journal of Immunology. 173, 5467-5475 (2004).</li></ol>

The protocol describes the methodology of induction and mini-endoscopic assessment of the colonic phenotype observed in the course of intestinal GvHD. It serves the wider purpose of enabling scientists to study intestinal GvHD longitudinally and noninvasively over the entire disease course (i.e., from the onset of colonic manifestation and progression until maximal disease activity).
However, there are some critical steps and important limitations inherent to the presented methodologies the ex...

Hematopoietic malignancies directly arising from the hematopoietic stem cell compartment and uncontrolled, rapidly progressing, and severe immune-mediated disorders are often indications to perform allo-HCT1,2. However, although accounting for the occurrence of the prognostic beneficial graft-versus-tumor response, donor lymphocytes are frequently inducing and promoting an unwanted immune-mediated attack of healthy tissue components within the allo-HCT recipient, a process that is called graft-versus-host disease3. Manifestations in the gut, the so-called intestinal GvHD, represent the most dreaded complication of acute GvHD, severe forms of which are routinely associated with a high mortality1,2,4.
Overall, murine models of allo-HCT have emerged as invaluable tools to identify and study immune-mediated mechanisms underlying the pathogenesis of GvHD5. However, kinetic assessment of, for instance, beneficial effects of novel therapeutic interventions over time in live mice is routinely based on the determination of clinical GvHD scores6. While these scores are suitable to reflect, for instance, the overall disease burden (i.e., the systemic GvHD), clinical scores lack the sensitivity to reliably mirror organ-specific manifestations (e.g., in the gut). Hence, conclusions, for instance with respect to gut-protective effects of a given therapeutic intervention, that are based on these scoring systems usually fall short.
Despite major advances through the invention of novel whole-body imaging modalities in combination with the usage of either bioluminescent or fluorescent genetic mouse models7,8, methodologies to directly and specifically assess the intestinal manifestation of GvHD in live mice are lacking. Hence, the rationale behind the protocol of the endoscopic assessment of the intestinal GvHD phenotype described in the next section is to overcome this obstacle. Furthermore, the motivation is also to reduce experimental mice numbers since, so far, a detailed assessment of the cellular, morphological, and molecular characteristics (e.g., by histopathology or molecular biology) of intestinal GvHD manifestation has ultimately required the sacrifice of the experimental mouse.
Our institution has previously reported on the methodology of a mini-endoscopic assessment of colonic manifestations in the course of syngeneic colitis models9. In the protocol presented here, we have refined and adapted the colonoscopic scoring matrix for alloresponse-driven colitis in live mice with intestinal GvHD upon transplantation of alloreactive HCT and donor lymphocytes in an MHC class I fully mismatched setting. We identified four parameters suitable to reflect intestinal GvHD-related colonic lesions. Furthermore, we established a system that allows a fine-tuned grading of any single determinant, resulting in a new score that readily informs the reader about the severity of intestinal GvHD present in a given mouse at a given time point. Histopathological analyses confirmed that an endoscopic score above a certain threshold is reliably predicting moderate-to-high grade tissue inflammation. Hence, mini-endoscopic evaluation appears to represent a working substitute for the gold standard histopathology that routinely requires the sacrifice of the experimental mice. Importantly, this protocol can be applied at virtually any given time point and can be repeatedly used during the course of the disease10,11. Furthermore, in contrast to the usage of bioluminescence-dependent approaches, no labor-intense and time-consuming measures like intercrossing genetically modified mice are required and, hence, the methodology can be applied on virtually any mouse line of interest.
Taken together, given the detrimental clinical perspective of allo-HCT patients with severe intestinal GvHD, rapid scientific progress and more insight into the molecular mechanisms underlying the immune pathogenesis are urgently needed. Similarly, important, ethical considerations demand that the gain of knowledge should be achieved with the usage of the minimal number of experimental mice. Hence, both recognized claims on the research community exploring intestinal GvHD can be advanced by implementing serial mini-endoscopic evaluations of the colon in the experimental work chain to monitor and grade intestinal GvHD in live experimental mouse models, as described and validated in the protocol presented here.

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			<td>352340</td>
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			<td height="21" style="height:21px;">Ammonium chloride (NH4Cl)</td>
			<td>Sigma-Aldrich Co. LLC.</td>
			<td style="width:152px;">11209</td>
			<td>ingredient of ACK lysing buffer</td>
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			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA)</td>
			<td>Carl Roth GmbH &#38; Co.KG</td>
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			<td>ingredient of ACK lysing buffer</td>
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			<td>Merck KGaA</td>
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			<td>ingredient of ACK lysing buffer</td>
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			<td>Miltenyi Biotec GmbH</td>
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			<td>magnet cell separation to isolate T cell-depleted bone marrow cells</td>
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			<td height="21" style="height:21px;">Pan T Cell Isolation Kit II, mouse</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-095-130</td>
			<td>magnet cell separation to isolate splenic T cells</td>
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			<td height="21" style="height:21px;">Alexa Fluor 700 anti-mouse CD45.2 Antibody (clone: 104; lot: B252126; RRID: AB_493731)</td>
			<td style="width:216px;">Biolegend</td>
			<td>109822</td>
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			<td height="21" style="height:21px;">Pacific Blue anti-mouse CD3 Antibody (clone: 17A2; lot: B227246; RRID: AB_493645)</td>
			<td style="width:216px;">Biolegend</td>
			<td>100214</td>
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			<td height="21" style="height:21px;">FITC anti-mouse CD4 Antibody (clone: GK1.5; lot: B225057; RRID: AB_312691)</td>
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			<td>100406</td>
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			<td height="21" style="height:21px;">PE/Cy7 anti-mouse CD45.1 Antibody (clone: A20; lot: B217246; RRID: AB_1134168)</td>
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			<td>110730</td>
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			<td height="21" style="height:21px;">APC/Cy7 anti-mouse CD8a Antibody (clone: 53-6.7, lot: B247008; RRID: AB_312753)</td>
			<td style="width:216px;">Biolegend</td>
			<td>100714</td>
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			<td height="21" style="height:21px;width:403px;">Filtrated bovine serum&#160;</td>
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			<td>P40-47500</td>
			<td>ingredient of FACS buffer&#160;</td>
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			<td height="21" style="height:21px;">96-well polystyrene V-bottom plates</td>
			<td style="width:216px;">Greiner Bio-One</td>
			<td align="right">651201</td>
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			<td height="21" style="height:21px;width:403px;">Polystyrene Round-Bottom Tube (5 mL)</td>
			<td style="width:216px;">Falcon</td>
			<td align="right" style="width:152px;">352052</td>
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			<td height="21" style="height:21px;">BD LSRFortessa II flow cytometer</td>
			<td>BD Bioscience Co.</td>
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			<td height="21" style="height:21px;width:403px;">Insulin syringe with sterile interior (30G)</td>
			<td style="width:216px;">BD</td>
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			<td height="21" style="height:21px;width:403px;">Oxy Vet Oxymat 3</td>
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			<td>oxygen concentrator&#160; for anesthesia</td>
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			<td height="21" style="height:21px;width:403px;">NarkoVet&#160;</td>
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			<td height="21" style="height:21px;width:403px;">Straight Forward Telescope</td>
			<td style="width:216px;">KARL STORZ SE &#38;Co KG</td>
			<td>64301 AA</td>
			<td>part of the experimental setup for colonoscopy</td>
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			<td>part of the experimental setup for colonoscopy</td>
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			<td style="width:216px;">KARL STORZ SE &#38;Co KG</td>
			<td style="width:152px;">61071 ZJ&#160;</td>
			<td>part of the experimental setup for colonoscopy</td>
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			<td height="21" style="height:21px;">&#160;175 Watt SCB XenonLight Source</td>
			<td style="width:216px;">KARL STORZ SE &#38;Co KG</td>
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			<td>part of the experimental setup for colonoscopy</td>
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			<td height="21" style="height:21px;">Fiber Optic Light Cable</td>
			<td style="width:216px;">KARL STORZ SE &#38;Co KG</td>
			<td style="width:152px;">495 NL</td>
			<td>part of the experimental setup for colonoscopy</td>
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			<td height="21" style="height:21px;">Image 1 S3 Camera Head</td>
			<td style="width:216px;">KARL STORZ SE &#38;Co KG</td>
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			<td>part of the experimental setup for colonoscopy</td>
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			<td style="width:216px;">KARL STORZ SE &#38;Co KG</td>
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			<td>part of the experimental setup for colonoscopy</td>
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			<td height="21" style="height:21px;width:403px;">LCD monitor</td>
			<td style="width:216px;">Olympus</td>
			<td>OEV181H</td>
			<td>part of the experimental setup for colonoscopy</td>
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			<td height="21" style="height:21px;">Forane / Isofluran</td>
			<td>AbbVie Inc.&#160;</td>
			<td>B506</td>
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			<td height="21" style="height:21px;">Formaldehyde solution 37 %</td>
			<td>Carl Roth GmbH &#38; Co.KG</td>
			<td style="width:152px;">7398.1</td>
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			<td height="21" style="height:21px;">5,0 mL Dispenser tip&#160;</td>
			<td style="width:216px;">Eppendorf AG</td>
			<td align="right">30089456</td>
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induction of intestinal graft-versus-host disease and its mini-endoscopic assessment in live mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

The current protocol, describing the mini-endoscopic evaluation of intestinal GvHD-associated lesions of the distal colon, has been established and validated in mice previously subjected to the systemic induction of a severe acute GvHD model. In this study, we used an MHC class I fully mismatched model system in which BALB/c mice were lethally irradiated, followed by the transplantation of T-cell-depleted allogeneic bone marrow and by the administration of GvHD-inducing alloreactive C57BL...

Watch this Scientific Journal Video about Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice at JoVE.com

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

Mini-endoscopic evaluation of the colon in live mice provides key experimental support for investigating intestinal manifestations of acute graph-versus-host disease, GvHD, upon allogenic, hematopoietic stem cell transplantation. The non-invasive assessment of acute GvHD-related colitis can function as a working substitute for gold standard histopathology, thereby rendering repeated animal sacrifices to obtain tissue specimens unnecessary. Demonstrating the procedure will be Vera Buchele, a post-doc from my laboratory.One day after total-body irradiation of the recipient BALB/c mice, place a CD-45.1 live 5B6 SJL donor mouse in the prone position on a clean working sheath, and use one 13-cm curved and serrated tip of a simkin forceps to lift the fur at one Achilles heel. Using hardened 8.5-cm fine scissors with straight tips, incise the skin between the forceps and one heel, elongating the incision cranially to facilitate removal of the skin and fur from the hind limbs. When the skin has been removed from the hind limbs, cut through each hip, ankle, and knee joint.Store the thigh and shank of every hind limb in a single 92-mm Petri dish filled with PVS on ice and carefully remove as much muscle as possible from each bone, transferring each cleaned femur and tibia into a 50-mL tube of sterile RPMI 1640 medium on wet ice. To isolate the bone marrow, transfer one bone at a time into a new 92-mL Petri dish filled with fresh RPMI 1640 medium, and use a scalpel to cut the ends off of each bone. Next, use a 1-mL syringe equipped with a 26-gauge needle to flush the bones with 1 mL of fresh medium at a time into a 50-mL collection tube containing 5 mL of medium.When all the bones have been flushed, gently pipette the crumbly bone marrow pieces up and down several times to generate a single-cell suspension before filtering the cells through a 40-micrometer mesh-screen cell strainer into a new 50-mL collection tube. Pellet the cells by centrifugation and re-suspend the cells in 5 mL of ammonium chloride potassium lysis buffer for a three-minute incubation at room temperature. At the end of the incubation, stop the reaction with 10 mL of PVS, and centrifuge the cells again.Re-suspend the pellet in 2 mL of fresh PVS for counting, and set aside a six times 10 to the sixth cell aliquot for downstream flow cytometric analysis. Next, use a commercially available cell purification kit to magnetically deplete the CD90.2-positive T cells from the bone marrow single-cell suspension, according to the manufacturer's instructions, and set aside a one times 10 to the sixth cell aliquot of the T-cell-depleted eluate for downstream flow cytometric analysis of the cell purity and composition before and after separation. Then, use a 1-mL syringe equipped with a 30-gauge needle to inject five times 10 to the fifth T-cell-depleted bone marrow cells in 100 microliters of PVS intravenously into the retrobulbar space of the venous sinus of each irradiated recipient animal.The next day, place a spleen harvested from a donor animal onto a 40-micrometer mesh strainer in a 50-mL collection tube, and use a syringe plunger to press the spleen tissue through the strainer into the tube. Wash the strainer and plunger with PVS to collect all the splenocytes, and pellet the cells by centrifugation. After red blood cell lysis is demonstrated, re-suspend the white blood cells for counting, and set aside a six times 10 to the sixth cell aliquot for downstream flow cytometric analysis.For a total CD3-positive T-cell isolation from total splenocytes, use a commercially available cell purification kit according to the manufacturer's instructions, and set aside a one times 10 to the sixth CD3-positive T-cell aliquot from the positive cell fraction for downstream flow cytometric analysis of the cell purity and composition before and after separation. Then, inject seven times 10 to the fifth alloreactive CD3-positive T cells in 100 microliters of PVS intravenously into the retrobulbar space of each recipient mouse to induce GvHD. To score GvHD-related lesions by mini-endoscopy of the distal colorectal region, lift the tail of a recipient animal just above the tail root with one hand, and use the other hand to carefully insert the endoscope into the rectum via the anus.While the air stream inflates the colorectal lumen, slowly and carefully move the endoscope forward in the aboral direction. To avoid colonic bowel injuries, keep the endoscope in the middle area of the lumen using the live video screen to aid in this positioning. Start recording the video stream and scoring the inflammation, evaluating the GvHD-related inflammation of the colon by scoring the parameters as illustrated.When a scoring spot has been located, move the endoscope gently and slightly back and forth to assess the different parameters. To assess the parameter translucency and stool consistency, position the endoscope at a wider distance in relation to the colonic wall. To assess the granularity and vascularity of the lesion, position the endoscope in the close proximity to the colonic wall, and carefully apply well-dosed tension to the colonic wall with the tip of the endoscope.Upon completion of the scoring process, stop the recording. Compared to controls, allogenic hematopoieic stem cell transplantation as demonstrated produces a reproducibly robust induction of a systemic GvHD phenotype, with progressively increasing clinical GvHD scores in recipient animals. These mini-endoscopically assessable criteria were specifically adapted from previously reported syngeneic colitis in the context of allo-response-driven colitis scoring parameters for the precise description, scoring, and grading of intestinal GvHD-disease-associated lesions in the distal colon.Indeed, the mini-endoscopically based grading system enables an easy discrimination of donor-lymphocyte-receiving mice with severe signs of intestinal inflammation from control mice that are essentially devoid of GvHD. Invalidation of the mini-endoscopically based grading system histopathological studies confirmed that the colon of mice severely affected by GvHD-related inflammation display similarly strong histopathological signs of inflammation. In contrast, colon tissues of control mice displayed no to, at most, mild histopathological signs of inflammation, in agreement with the virtual absence of mini-endoscopically detectable signs of colitis.Furthermore, correlation studies between mini-endoscopically and histopathologically assessed colitis activity and systemic GvHD scores demonstrate that mini-endoscopically determined some scores of less than or equal to three reliably predict the absence of mid-to higher-grade intestinal GvHD-associated colonic inflammation scores obtained from histopathological grading. In addition, the severity of the endoscopically assessed intestinal GvHD shows a correlation with systemic GvHD disease activity. To standardize the results of mini-endoscopic evaluation of colonic inflammation and to ensure comparability across different mice and experiments over time, a defined anatomic site should be selected for scoring.In addition to scoring colonic inflammation, the built-in working channel within the mini-endoscopic setup allows the retrieval of view-guided tissue biopsies applicable to various downstream analyses, such as standard histopathology. Due to the non-invasive nature of the mini-endoscopy, acute GvHD-related colitis can be repeatedly assessed within the same animal at virtually any time, thereby yielding fascinating insights into intestinal GvHD.

induction-intestinal-graft-versus-host-disease-its-mini-endoscopic

Research

JoVE Journal

Immunology and Infection

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

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.

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.

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

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...