Name: experimental autoimmune uveitis: an intraocular inflammatory mouse model
Uploaded: 2022-01-12T12:30:00
Description: Watch this Scientific Journal Video about Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model at JoVE.com

JG was awarded a UCL Impact Studentship and Rosetrees Trust funding to support CB. VC was in receipt of a research collaborative grant from Akari Therapeutics Inc. We would like to thank UCL Institute of Ophthalmology, Biological Service Unit especially Ms Alison O&#39;Hara and her team for their technical support.

All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act of 1986, and institutional Animal Welfare and Ethical Review Body (AWERB) guidelines.
1. Housing C57BL/6J mice
<ol>
	<li>House mice in a specific pathogen free environment, on a 12 hour light-dark cycle and food and water available ad libitum.</li>
	<li>Perform all experiments on adult female C57BL/6J (females are preferentially chosen as there is an incidence of women to men 1.4 to 1 in uve...

<ol>
	<li>Lyu, 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=TMP778,+a+selective+inhibitor+of+RORgammat,+suppresses+experimental+autoimmune+uveitis+development,+but+affects+both+Th17+and+Th1+cell+populations.">TMP778, a selective inhibitor of RORgammat, suppresses experimental autoimmune uveitis development, but affects both Th17 and Th1 cell populations.</a> The European Journal of Immunology. 48, 1810-1816 (2018).</li><li>Klaska, I. P., Forrester, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+autoimmune+uveitis.">Mouse models of autoimmune uveitis.</a> Current Pharmaceutical Design. 21, 2453-2467 (2015).</li><li>Eskandarpour, M., Alexander, R., Adamson, P., Calder, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Inhibition+of+Bromodomain+Proteins+Suppresses+Retinal+Inflammatory+Disease+and+Downregulates+Retinal+Th17+Cells.">Pharmacological Inhibition of Bromodomain Proteins Suppresses Retinal Inflammatory Disease and Downregulates Retinal Th17 Cells.</a> The Journal of Immunology. 198, 1093-1103 (2017).</li><li>Mochizuki, 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=Preclinical+and+clinical+study+of+FK506+in+uveitis.">Preclinical and clinical study of FK506 in uveitis.</a> Current Eye Research. 11, 87-95 (1992).</li><li>Nguyen, Q. D., 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=Intravitreal+Sirolimus+for+the+Treatment+of+Noninfectious+Uveitis:+Evolution+through+Preclinical+and+Clinical+Studies.">Intravitreal Sirolimus for the Treatment of Noninfectious Uveitis: Evolution through Preclinical and Clinical Studies.</a> Ophthalmology. 125, 1984-1993 (2018).</li><li>Leal, I., 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=Anti-TNF+Drugs+for+Chronic+Uveitis+in+Adults-A+Systematic+Review+and+Meta-Analysis+of+Randomized+Controlled+Trials.">Anti-TNF Drugs for Chronic Uveitis in Adults-A Systematic Review and Meta-Analysis of Randomized Controlled Trials.</a> Frontiers in Medicine (Lausanne). 6, 104 (2019).</li><li>Chen, Y. 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=Functionally+distinct+IFN-?++IL-17A++Th+cells+in+experimental+autoimmune+uveitis:+T-cell+heterogeneity,+migration,+and+steroid+response.">Functionally distinct IFN-?+ IL-17A+ Th cells in experimental autoimmune uveitis: T-cell heterogeneity, migration, and steroid response.</a> European Journal of Immunology. 50 (12), 1941-1951 (2020).</li><li>Xu, 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=A+clinical+grading+system+for+retinal+inflammation+in+the+chronic+model+of+experimental+autoimmune+uveoretinitis+using+digital+fundus+images.">A clinical grading system for retinal inflammation in the chronic model of experimental autoimmune uveoretinitis using digital fundus images.</a> Experimental Eye Research. 87, 319-326 (2008).</li><li>Copland, D. 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=The+clinical+time-course+of+experimental+autoimmune+uveoretinitis+using+topical+endoscopic+fundal+imaging+with+histologic+and+cellular+infiltrate+correlation.">The clinical time-course of experimental autoimmune uveoretinitis using topical endoscopic fundal imaging with histologic and cellular infiltrate correlation.</a> Investigative Ophthalmology &amp; Visual Science. 49, 5458-5465 (2008).</li><li>Dick, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doyne+lecture+2016:+intraocular+health+and+the+many+faces+of+inflammation.">Doyne lecture 2016: intraocular health and the many faces of inflammation.</a> Eye (Lond). 31, 87-96 (2017).</li><li>Agarwal, R. K., Silver, P. B., Caspi, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+experimental+autoimmune+uveitis.">Rodent models of experimental autoimmune uveitis.</a> Methods in Molecular Biology. 900, 443-469 (2012).</li><li>Caspi, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+look+at+autoimmunity+and+inflammation+in+the+eye.">A look at autoimmunity and inflammation in the eye.</a> Journal of Clininical Investigation. 120, 3073-3083 (2010).</li><li>Caspi, R. 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=Mouse+models+of+experimental+autoimmune+uveitis.">Mouse models of experimental autoimmune uveitis.</a> Ophthalmic Research. 40, 169-174 (2008).</li><li>Shao, 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=Severe+chronic+experimental+autoimmune+uveitis+(EAU)+of+the+C57BL/6+mouse+induced+by+adoptive+transfer+of+IRBP1-20-specific+T+cells.">Severe chronic experimental autoimmune uveitis (EAU) of the C57BL/6 mouse induced by adoptive transfer of IRBP1-20-specific T cells.</a> Experimental Eye Research. 82, 323-331 (2006).</li><li>Horai, 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=Microbiota-Dependent+Activation+of+an+Autoreactive+T+Cell+Receptor+Provokes+Autoimmunity+in+an+Immunologically+Privileged+Site.">Microbiota-Dependent Activation of an Autoreactive T Cell Receptor Provokes Autoimmunity in an Immunologically Privileged Site.</a> Immunity. 43, 343-353 (2015).</li><li>Chen, 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=Comparative+analysis+of+induced+vs.+spontaneous+models+of+autoimmune+uveitis+targeting+the+interphotoreceptor+retinoid+binding+protein.">Comparative analysis of induced vs. spontaneous models of autoimmune uveitis targeting the interphotoreceptor retinoid binding protein.</a> PLoS One. 8, 72161 (2013).</li><li>Chen, J., Qian, H., Horai, R., Chan, C. C., Caspi, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+optical+coherence+tomography+and+electroretinography+to+evaluate+retinal+pathology+in+a+mouse+model+of+autoimmune+uveitis.">Use of optical coherence tomography and electroretinography to evaluate retinal pathology in a mouse model of autoimmune uveitis.</a> PLoS One. 8, 63904 (2013).</li><li>Harry, 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=Suppression+of+autoimmune+retinal+disease+by+lovastatin+does+not+require+Th2+cytokine+induction.">Suppression of autoimmune retinal disease by lovastatin does not require Th2 cytokine induction.</a> Journal of Immunology. 174, 2327-2335 (2005).</li></ol>

Experimental animal models are necessary tools for studying disease pathogenesis and preclinical testing of novel therapeutic paradigms. In the current protocol, we have discussed a methodology for inducing, monitoring, and scoring EAU, an experimental model of intraocular inflammatory uveitis. This EAU model has more than 95% disease incidence when all procedures are performed according to the protocol outlined herein, and results in the development of chronic, monophasic EAU. To achieve this incidence level, we stress ...

This protocol will demonstrate how to induce Experimental Autoimmune Uveitis (EAU) in the C57BL/6J mouse by a single subcutaneous injection of a retinal antigen in an emulsified adjuvant. Methods for monitoring and assessing disease progression will be detailed through fundoscopic imaging and histological examination, with measurement parameters outlined within. In addition, fluorescein angiography, a technique for examining retinal blood vessel structure and permeability will be discussed.
This EAU model recapitulates central features of non-infectious posterior uveitis in humans with regards to clinicopathologic characteristics and the basic cellular and molecular mechanisms that drive disease. EAU is mediated by Th1 and/or Th17 subsets of self-reactive CD4+T lymphocytes, as shown in adoptive transfer experiments and with IFN&#947;-depleted mice1. Much of our understanding of the potential roles for these cells in uveitis comes from studying EAU2 where both Th1 and Th17 cells are detected within the retinal tissues3. Often, EAU is used as a preclinical model to assess the utility of novel therapies in attenuating disease. Therapeutic approaches that have successfully modulated EAU disease have shown some efficacy in the clinic and reached FDA approved status. Examples of these are groups of immunoregulatory drugs such as the T cell-targeting therapies: cyclosporine, FK-506, and rapamycin4,5,6. Recently, interventions targeting novel pathways have also been explored in this model to investigate both mechanism and effect on disease outcome. These include targeting transcriptional regulation through chromatin reader Bromodomain Extra-Terminal (BET) proteins and P-TEFb inhibitors3. Moreover, more conventional approaches such as a VLA-4 inhibitor have recently demonstrated suppression in EAU via modulation of effector CD4+ T cells7. In addition, targeting Th17 cells with TMP778, a ROR&#947;t inverse agonist, has also been found to significantly suppress EAU8. Furthermore, this model offers an opportunity to study chronic autoimmune inflammation in the retina and the accompanying underlying mechanisms such as lymphocyte priming.
The primary readouts for EAU preclinical studies are clinical assessment by performing retinal fundoscopy imaging and less frequently, by assessing retinal integrity by Optical Coherence Tomography (OCT). Retinal histopathological evaluation and immunophenotyping of retinal cells by flow cytometry are then undertaken at termination. Fundoscopy is an easy-to-use live imaging system that allows for rapid and reproducible clinical assessment of the whole retina. For immunohistochemical assessments, the techniques are based on the preparation of retinal sections that allow us to study tissue architecture for the degree of inflammation and structural damage9. The assessment criteria and conventional scoring systems, for all techniques used, will be outlined within this protocol. The extent of damage recorded using fundoscopic imaging often closely correlates with histological changes. This dual approach to monitoring and assessing disease severity affords greater sensitivity and more reliable measurement outcomes.
EAU is a well-established, commonly used model for preclinical testing and investigation of immune-mediated eye disease. This model is reliable and reproducible with &#62;95% disease incidence and generates comprehensive data that can be used to validate or repudiate new therapies for the treatment of intraocular inflammatory disease that represents a major cause of working-age blindness worldwide10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>antisedan</td>
			<td>ZOETIS, USA</td>
			<td></td>
			<td>for waking up</td>
		</tr>
		<tr>
			<td>Complete Freund&#8217;s Adjuvant; CFA</td>
			<td>Sigma, UK</td>
			<td>F5881</td>
			<td>for immunisation&#160;</td>
		</tr>
		<tr>
			<td>Domitor</td>
			<td>Orion Pharma, Finland</td>
			<td></td>
			<td>for anesthesia</td>
		</tr>
		<tr>
			<td>Flourescein</td>
			<td>Sigma, UK</td>
			<td>F2456</td>
			<td>for Angiography</td>
		</tr>
		<tr>
			<td>IRBP1-20</td>
			<td>Chamberidge peptide, UK</td>
			<td></td>
			<td>peptide;antigen&#160;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Orion Pharma, Finland</td>
			<td></td>
			<td>for anesthesia</td>
		</tr>
		<tr>
			<td>Micron III</td>
			<td>Phoenix Research, USA</td>
			<td></td>
			<td>for fundoscopy</td>
		</tr>
		<tr>
			<td>Mouse Serum</td>
			<td>Sigma, UK</td>
			<td>M5905</td>
			<td>for immunisation&#160;</td>
		</tr>
		<tr>
			<td>Mycobacterium terberculosis</td>
			<td>Sigma, UK</td>
			<td>344289</td>
			<td>for immunisation&#160;</td>
		</tr>
		<tr>
			<td>Pertussis Toxin</td>
			<td>Sigma, UK</td>
			<td>P2980</td>
			<td>for immunisation&#160;</td>
		</tr>
		<tr>
			<td>phenylephrine hydrochloride 2.5%&#160;</td>
			<td>Bausch &#38; Lomb UK&#160;</td>
			<td>PHEN25</td>
			<td>for dilation&#160;</td>
		</tr>
		<tr>
			<td>Tropicamide 1%</td>
			<td>SANDOZ</td>
			<td></td>
			<td>for dilation&#160;</td>
		</tr>
		<tr>
			<td>Viscotears</td>
			<td>WELDRICKS Pharmacy, UK</td>
			<td>2082642</td>
			<td>for eye lubrication</td>
		</tr>
	</tbody>
</table>

experimental autoimmune uveitis: an intraocular inflammatory mouse model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this protocol, we describe a step-by-step method for inducing a model of experimental autoimmune uveitis (EAU) by immunizing mice with a uveitogenic retinal peptide derived from IRBP. The assessment of disease employing widely used and readily accessible approaches are covered although these are not exclusive and may be added to, or partially replaced, by other imaging techniques. The first signs of EAU in C57BL/6J mice can be detected two weeks post-immunization and peak disease reached within three weeks as illustra...

Watch this Scientific Journal Video about Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model at JoVE.com

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

This model can be used to study cellular mechanisms involving the pathogenesis of immune-mediated human posterior uveitis. The implication of this technique extend toward the translational study of human uveitis in monitoring novel drug efficacy and for providing mechanistic insight into chemical disease progression. The main advantage of using this model is the reliability of disease induction and use of non-invasive endoscopic techniques that allow us to monitor disease progression.Here we describe how to induce the disease and assess the pathology using several readouts To prepare IRBP 1-20 complete Freund's Adjuvant or CFA, resuspend a calculated amount of the peptide in a minimum volume of 100%DMSO. When the powder has completely dissolved, add small volumes of PBS to the tube until reaching to the final calculated volume of PBS according to the number of mice to be injected using gentle agitation to mix the solution. After preparing the complete Freund's Adjuvant, gently and frequently pipette the solution to form a viscus and evenly distributed emulsion while adding the DMSO PBS peptide solution dropwise at a one-to-one ratio to the suspension.To aerate the solutions, use a 1000 microliter pipette set to 700 microliters to repeatedly pipette the solution until a thick, creamy emulsion has been obtained. Before injecting the peptide suspension, place each mouse to be injected into a separate cage and use a one milliliter syringe equipped with a 23 gauge needle to intraperitoneal deliver 1.5 micrograms of Bordetella pertussis toxin in 100 microliters of RPMI 1640 medium supplemented with 1%mouse serum to each animal. When pertussis toxin solution has been injected, mouse should be held in a scruff like position and the skin pinched to form a tent like structure on the back of the neck.Thread the needle to the space between the finger and thumb, and inject 200 microliters of the IRBP emulsion to the tented skin, maintaining pressure after the injection and rotating the needle head to close the skin before retraction. To score the clinical disease, on day 21 after injection, confirm a lack of response to petal reflex in the anesthetized peptide injected mouse and restrain the animal in a scruff. Topically apply 1%tropicamide and 2%phenylephrine to the cornea of each eye immediately after injection.When the pupils have fully dilated, place the mouse onto a purpose-built microscope stage. Position the microscope for full access to the retina. Adjust the eye piece throughout the imaging process as necessary to see the optic disc and retina and acquire images of the entire retinal area, adjusting and covering all of the corners of the periphery.To measure vessel leakage, administer 100 microliters of 2%fluorescein subcutaneously at the back of the neck and reposition the mouse such that the retina is center on the middle of the live image. Set the fundoscope to a blue light excitation filter at 465 to 490 nanometers before acquiring an image of each at 1.5 and seven minutes after fluorescent injection. When all of the images have been acquired, intraperitoneally deliver an anti sedation injectable for recovery and place the mouse in a cage on a preheated mat with access to wet soaked diet with monitoring until the mouse regains consciousness.For clinical disease scoring of the images, base the clinical assessment on the severity of the optic disc inflammation, retinal vessel cuffing, retinal tissue infiltrate, and structural damage. Score each of these parameters on a scale from one to five. The collective total is representative of the clinical disease for the whole eye with a maximum score of 20 obtainable per eye.After fundus imaging for the final clinical endpoint of the study, euthanize the mouse. For histological analysis, prize apart the eyelids for access to the entire eye, then place curved forceps behind the globe to grasp the orbital connective tissue and optic nerve and enucleate the eye. Place the enucleated eye in one to five milliliters of 4%glutaraldehyde for a minimum of 15 minutes before transferring the organ to one to five milliliters of 10%formaldehyde for at least 24 hours.After histological tissue staining, according to standard protocols, assigned scores on a scale of zero to three based on the level of immune cell infiltration and the degree of retinal damage. Fundoscopic changes are classified during disease progression as inflammatory changes that include retinal tissue and vascular and optic disc inflammation and retinal structural damage in addition to histological changes based on infiltrating immune cells and structural impairments. These clinical and histopathological changes can be graded and scored to assess disease progression and to evaluate therapeutic intervention efficacy.Vascular leakage is also a pathological feature of the model and of human uveitis. As illustrated in this analysis, fluorescein can be used to visualize vascular leakage as another readout of this model. It is essential for researchers to ensure the emulsion is prepared correctly without signs of separation between the solutions.They should be mixed thoroughly and the final product should be smooth in texture. This procedure can be combined with other methods of drug administration and with for cytometry for phenotyping infiltrating retinal immune cell populations.

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Immunology and Infection

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

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 ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

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 ...

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the &#945;-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...