Dr. Njoku would like to acknowledge Dr. Noel R. Rose, MD PhD, for his guidance and insightful discussions that resulted in the formulation of this model.

All procedures were approved by the animal care and use committee.
1. Trifluoroacetylation of hepatic S-100 cytosolic proteins or a CYP2E1 epitope
NOTE: First, prepare the trifluoroacetylated S100 (TFA-S100) and trifluoroacetylated CYP2E1 epitope (TFA-JHDN5). Because syngeneic S100 proteins are needed for immunizations, and BALB/c mice are required to produce the immunogen. The preparation yields a large amount of immunogen; so, anticipate performing this portion around four times a year. An identical method will be used to make the TFA-JHDN5. The CYP2E1 epitope (JHDN5), GII/ FNN/ GPT/ WKD/ IRR/ FSL/ TTL, can be sequenced or purchased.
<ol>
	<li>Isolation of the S100 fraction of the liver.
	<ol>
		<li>Following sedation of 5 &#8211; 10 BALB/c mice with 40-60 mg/kg ketamine mixed with 4 - 6 mg/kg xylazine, confirm proper depth of anesthesia by observing a reduction in muscle tone and no response to painful stimuli, such as a toe pinch (withdrawal reflex), in addition to the loss of righting reflexes&#160;and the loss of palpebral reflexes.</li>
		<li>Using microsurgical scissors, expose the intra-abdominal contents using a midline incision and make a small cut in the inferior vena cava to remove the blood.</li>
		<li>Place a 24-gauge angiocatheter into the portal vein and perfuse the liver 10 mL/min with 40 mL of phosphate buffered saline (PBS) pH 7.4 in a water bath at 4 &#176;C. Remove and weigh the pooled livers and cut it into small pieces (10 &#8211; 15 mm).&#160; 
		NOTE:&#160;&#160;Euthanasia is&#160;induced&#160;by intraperitoneal injection of additional ketamine/xylazine (80 mg/kg: 8 mg/kg), followed by cervical dislocation and opening the chest cavity to collapse the lungs.&#160;This method provides the least pain and discomfort to the animals.&#160;This method is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.</li>
		<li>Add 4 times the weight of sucrose (250 mM) -TRIS (10 mM)- EDTA (1 mM) homogenization buffer (pH 7.4) supplemented with Complete Protease inhibitor Cocktail tablets (see Table of Materials) as per manufacturers recommendations. Homogenize in a 15 mL polypropylene tube, using a general laboratory tissue homogenizer on medium speed on ice until smooth. Homogenize on ice to prevent the tissue from becoming warm during homogenization.</li>
		<li>Centrifuge the liver homogenates at 1500 x g for 10 min and then pour off the supernatant. Centrifuge the supernatant for 1 h at 100,000 x g. Snap freeze the supernatant and store at -80 &#176;C. The supernatant is cytosolic S-100.</li>
	</ol>
	</li>
	<li>Trifluoroacetylation of S100 and JHDN5 
	NOTE: Trifluoroacetylation of the &#949;-amino groups of lysine residues of S-100 will be performed according to the procedure of Satoh18. All portions of this experiment with the exception of the latter days of dialysis are performed in the fume hood.
	<ol>
		<li>Determine the total protein concentration of the cytosolic S-100 using the bicinchoninic acid assay (BCA assay)7. Dilute 20 mg of BALB/c mouse S100 or JHDN5 to 10 mL with dH2O in a 50 mL Erlenmeyer flask. Adjust the pH to 10 with 1N KOH.</li>
		<li>Add 4.7 mmole of S-ethyltrifluorothioacetate (S-ETFA), to the solution. Maintain the pH between 9.9 &#8211; 10.0 with 1N KOH by administering KOH in droplet fashion for approximately 1 h. Record total volume of KOH for each reaction.</li>
		<li>Transfer the solutions into separate dialysis cassettes (Do not overfill). Dialyze the cassettes for 72 h against 4 L of dH2O with three changes per day. After dialysis, record the final volume of TFA-S100 or TFA-JHDN5 and then aliquot into labeled tubes. Snap freeze and store at &#8211;80 &#176;C.</li>
		<li>An estimated concentration is determined by dividing the initial amount of S100 or JHDN5 (in mg) by the final volume following dialysis (mL). To determine the percent modification of the native protein19, dilute 1.0 mg of each native and TFA-altered protein (if the final concentration is greater than 1.0 mg) to 1.0 mL with dH2O in separate bullet tubes and prepare a blank using 1.0 mL dH2O.&#160;If the concentration of the TFA-altered protein is less than 1.0 mg, do not dilute
		<ol>
			<li>To separate wells of a 96 well plate, add 50 &#181;L of the blank, native and altered proteins. Add 50 &#181;L of 4% NaHCO3 followed by 50 &#181;L of 0.1% 2,4,6-trinitrobenzene sulfonic acid to each well.</li>
			<li>Incubate the plate at 40 &#176;C for 2 h. Following the incubation, add 50 &#181;L of 10 % SDS to each well followed by 25 &#181;L of 1N HCl.</li>
			<li>Read at OD of 334 nm and then record absorbance of each compound from 200 &#8211; 600 nm in order to confirm the characteristic drop in absorbance at 334 nm. Calculate the percent modification of lysine residues by TFA, using the following formula: 
			<img alt="Equation 1" src="/files/ftp_upload/59174/59174eq1.jpg" /></li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Immunization of mice to induce hepatitis
NOTE: DIH is modeled in BALB/c mice by immunizations with liver cytosolic proteins that have been covalently altered by trifluoracetyl chloride (TFA), a model drug-metabolite, TFA-S1006 or an epitope of CYP2E1 covalently altered by TFA9, TFA-JHDN5 that induces hepatitis, autoreactive T cells, and CYP2E1 autoantibodies. Mice exhibit a splenic activation phase 2 weeks after the initial immunization and a hepatic phase by 3 weeks that is characterized by granulocytic inflammation. Female BALB/c mice are more susceptible than males to hepatitis in this model.
<ol>
	<li>On day 0, immunize 6&#8211;8 week-old BALB/c mice subcutaneously at the base of the neck with 200 &#956;g of TFA-S100 or 100 &#181;g of TFA-JHDN5 emulsified in equal volumes of complete Freund&#8217;s adjuvant (CFA). On day 0, immunize the mice with 50 ng of pertussis toxin, intramuscularly in one of&#160;the hind legs. On day 7, immunize the mice subcutaneously at the base of the tail with either 200 &#956;g of TFA-S100 or 100 &#181;g of TFA-JHDN5 emulsified in equal volumes of CFA to ensure that each mouse receives two injections of the same immunogen. 
	NOTE: The mice are monitored hourly for the first 6 hours following the immunization and then at least twice daily for one week. If the mice demonstrate signs of pain or distress, such as hunched posture or ruffled fur, analgesics should be administered in accordance with the local ACUC. If significant pain or distress is noted, the mice should be evaluated by a veterinarian to determine if euthanasia is necessary.</li>
	<li>Determination of CD4+ T cell immune responses to whole self-proteins, epitopes of self-proteins or the TFA hapten using flow cytometry
	<ol>
		<li>Following sedation of mice with 40-60 mg/kg ketamine mixed with 4-6 mg/kg xylazine via intraperitoneal injection, confirm the proper depth of anesthesia as described in step 1.1.1 and then identify the spleen after exposing the intra-abdominal cavity using microsurgical scissors. Cut the spleen at the pedicle and place in a Petri dish with PBS/2% fetal calf serum (FCS). 
		NOTE: Euthanasia will be induced in the mice by intraperitoneal injection of additional ketamine/xylazine (80 mg/kg: 8 mg/kg), followed by cervical dislocation and opening the chest cavity to collapse the lungs. This method provides the least pain and discomfort to the animals. This method is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.</li>
		<li>Release the cells using frosted glass slides and transfer to a 50 mL conical polypropylene tube. Wash with PBS/2% FCS by bringing the volume up to 50 mL and centrifuging at 335 x g using a benchtop refrigerated centrifuge. Pour off the supernatant and repeat this step.</li>
		<li>Remove red cells using 1 mL of ACK Lysing buffer for 1 min and bring volume up to 50 mL with PBS/2% FCS. Centrifuge at 335 x g and pour off the supernatant.</li>
		<li>Count the cells. Label the cells with CFSE for 30 min on ice in the dark, as per manufacturer&#8217;s instructions. Suspend single cell suspensions into 6 well plates of 3x106 cells/mL per well in PBS/2% FCS.</li>
		<li>Stimulate labeled cells with either CYP2E1, JHDN5, or TFA-OVA (10 &#181;g/mL) for 72 h at 37 &#176;C in 5% CO2, 95% air (humidified). After incubation, stain the cells with CD4-APC (1:100) for 30 min on ice and analyze by flow cytometry within 3 days.</li>
		<li>Utilize the following gating strategy to identify CD4+CFSE+ cells: CD4+CFSE+ cells will be identified from the gated alive cells and displayed as histograms of proliferating cells.</li>
	</ol>
	</li>
	<li>Isolation of infiltrating immune cells from immunized mice. 
	<ol>
		<li>To isolate infiltrating immune cells in the livers on day 14 or day 21, anesthetize the mice by intraperitoneal injection with 40-60 mg/kg ketamine mixed with 4-6 mg/kg xylazine and confirm the proper depth of anesthesia as described in step 1.1.1. After laparotomy using a midline incision made with micro surgical scissors as described in 1.1.2, cannulate the portal vein with a 25 gauge needle and then cut the inferior vena cava below the renal veins.</li>
		<li>Perfuse each liver at a flow rate of 10 mL/min with 40 mL of PBS in a water bath 37 &#176;C. After perfusion, using micro surgical scissors, cut the liver at the hepatic pedicle, remove the gall bladder and then cut the liver at the hilum.</li>
		<li>Disrupt the liver on a mesh stainless steel sieve using a 20 mL sterile syringe pestle and cold PBS. Filter the resulting cell suspension into 50 mL pre-sterilized centrifuge tubes using a 300 mesh screen. Bring each suspension to 50 mL using cold PBS and then wash the suspension by centrifugation for 10 min at 370 x g.</li>
		<li>Discard the supernatant and then pool each pellet by treatment into new 50 mL tubes (One tube per mouse is recommended; however, if samples are pooled, 2-4 pellets/tube is recommended). Suspend the pooled pellets in 45 mL Percoll 35% (in PBS), and 100 IU/mL heparin.</li>
		<li>Spin each tube at 500 x g for 10 min at 20 &#176;C. Discard the supernatant and suspend the pellet in 5 mL of PBS and then add 1 mL of ACK lysing buffer to each pellet for 10 min on ice.</li>
		<li>Bring each tube to 50 mL with PBS and wash by centrifugation for 10 min at 370 x g. Discard the supernatant and then wash the cells with PBS/2% FCS by centrifugation for 10 min at 370 x g. Count the cells.</li>
		<li>Analysis of cell type using flow cytometry 
		NOTE: Here is an example of how induced Foxp3+Tregs can be detected.
		<ol>
			<li>Incubate 1x106 cells with FcR blocking reagent and stain with 1:100 dilutions of CD4-FITC, CD25-PE, and CD45-PerCP for 30 min on ice. Next, stain the cells intracellularly with FoxP3-APC.</li>
			<li>Fix the cells with 250 &#181;L of fixation buffer (see Table of Materials), and store at 4 &#176;C until analysis by flow cytometry within 3 days.</li>
			<li>The following gating strategy is recommended in order to detect induced Foxp3+Tregs in single cell suspensions from liver, spleen or lymph nodes using flow cytometry: Identify live cells using Live/Dead Fixable Aqua Dead Cell stain kit. Next, gate on liver cells that are CD45+ (PerCP, clone RA3-6B2), and gate CD4+ cells (FITC, cloneGK1.5) from the CD45+ gate. From the CD4+ cells, identify the percentages of CD25+(PE, clone 7D4) and FoxP3+ (APC, clone 3G3) cells.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Histological analysis of liver tissues for hepatitis
	<ol>
		<li>On day 21, fix the liver sections (5 &#181;m thick) in 10% neutral buffered formalin and stain with Hematoxylin &#38;Eosin.</li>
		<li>Determine histology scores first at low power (40X) in an average of 2 views and confirm at 64X. Score the tissue sections as follows: Grade 0=no inflammation or necrosis; Grade 1= minor lobular inflammation with no necrosis; Grade 2= lobular inflammation involving &#60;50% of the section; Grade 3=lobular inflammation involving &#8805; 50% of the section; and Grade 4=inflammation with necrosis.</li>
	</ol>
	</li>
	<li>Determination of tissue cytokine levels in spleens and livers. 
	<ol>
		<li>On day 14 or day 21, homogenize the liver or spleen samples (1 g) from each mouse in 1 mL of RPMI/2% FCS until smooth using a general laboratory homogenizer on medium setting. Keep the sample cold on ice.</li>
		<li>Centrifuge the homogenate for 15 min at 1455 x g at 4 &#176;C using a refrigerated desktop centrifuge. Snap freeze the supernatant and store at -80 &#176;C until ready for use. Cytokine and chemokine levels can be measured with commercial ELISA kits, as per kit instructions. Standardize the levels of cytokines by converting the levels (in mL or &#181;L) to pg/g of tissue.</li>
	</ol>
	</li>
	<li>Detection of serum antibodies to CYP2E1, the CYP2E1 epitope JHDN5 and the TFA drug metabolite.
	<ol>
		<li>On days 14 or 21, sedate the mice with 40-60 mg/kg ketamine mixed with 4-6 mg/kg xylazine. Confirm proper depth of anesthesia by observing a reduction in muscle tone and no response to painful stimuli, such as a toe pinch (withdrawal reflex), in addition to the loss of righting reflexes&#160;and the loss of palpebral reflexes. Utilize a 25 G&#160;needle attached to a tuberculin syringe and approach the heart by slowly advancing the needle into the thoracic cavity under the ribs and immediately lateral to the xiphoid process. Collect blood using intracardiac puncture.</li>
		<li>Once blood is collected, allow it to clot at room temperature. Centrifuge the blood samples at 295 x g for 20 min at room temperature. Carefully remove the sera, aliquot the sera and snap freeze at -20 &#176;C.</li>
		<li>Apply 100 &#181;L of CYP2E1, JHDN5, or TFA-ovalbumin (OVA) test antigens (5 &#181;g/mL in PBS) to 96 well plates for at least 18 h at 4 &#176;C overnight. The next day, wash the plates with wash buffer (PBS/2%FCS), 2 cycles (4 washes each).</li>
		<li>Apply 100 &#181;L of mouse sera (1:100) in PBS/2% FCS in triplicate on the plates and incubate at room temperature for 2 h. After 2 h, wash the plates with wash buffer as described in step 2.6.2.</li>
		<li>Add 100 &#181;L of Alkaline phosphatase (AKP)-goat anti-mouse IgG, AKP-rat anti-mouse IgG1, or AKP-rat anti-mouse IgG2a secondary antibodies (1:1000) for 2 h followed by a wash step of 1 cycle with wash buffer and 1 cycle with PBS. Detect the antibodies using an AKP substrate kit and measure at OD 405 nm every 15 min with a spectrophotometer. TFA usually develops completely in 15 min while CYP2E1 and the CYP2E1 epitope can develop from 30 to 60 min7.</li>
	</ol>
	</li>
	<li>Studies of the development of JHDN5 IgG-induced oxidative stress in vitro.
	<ol>
		<li>Using 12 well plates, incubate 106 terminally differentiated hepatic cells per well on fibronectin-covered cover slips in 1000 &#181;L of Williams E media supplemented with glutamine and general supplement (see Table of Materials) at 37 &#176;C, 5% CO2, 95% humidity for 7 days as recommended to maximize CYP2E1 activity.</li>
		<li>Add JHDN5 IgG (1:40) or mouse IgG (1:1000) to separate wells and incubate for 2 h at 37 &#176;C, 5% CO2, 95% humidity. Hybridoma sera was very dilute. Add deep red fluorescent antibody detector (see Table of Materials) for an additional 30 min to all wells.</li>
		<li>Wash the wells 3x with 1 mL of PBS in the dark. Fix the cells in 3.7% formaldehyde for 10 min. Examine by confocal microscopy within 24 h.</li>
	</ol>
	</li>
	<li>Co-localization studies of JHDN5 IgG with intracellular organelles such as mitochondria in vitro. 
	<ol>
		<li>To demonstrate co-localization of JHDN5 IgG with mitochondria, sparsely culture (~30% confluence) terminally differentiated hepatic cells on fibronectin-covered cover slips for 7 days in dye&#8211;free Williams&#8217;s media E supplemented as described in step 2.7.</li>
		<li>After determining the correct absorption wavelengths, add green fluorescent, 488 nm -conjugated mouse IgG or JHDN-5 (1:100) and Red fluorescent, 594-conjugated Mito-tracker Red (1:100) for 2 h (37 &#176;C), 5% CO2, 95% humidity.</li>
		<li>Mount labeled fibronectin-covered cover slips with Anti-fade Reagent with DAPI, and examine by confocal microscopy.</li>
	</ol>
	</li>
</ol>
3. General protocol notes
<ol>
	<li>Utilize non-pharmaceutical grade tools when compounds are not available in a clinical use formulation. However, obtain each of these tools from reliable commercial suppliers identified in this method. Always use chemicals that conform to specifications defined by the Committee on Analytical Reagents of the American Chemical Society of at least the reagent grade level. For our methods, utilize analytical grade level reagents whenever possible.</li>
	<li>Follow strict aseptic technique for the formulation of the TFA-altered proteins in order to prevent contamination that could adversely affect animal welfare or the interpretation of data.&#160;</li>
	<li>Store and use non-pharmaceutical grade formulations at durations for which the formulation will remain potent, as per available technical information. Store CFA at room temperature, CYP2E1 and its epitopes at -20 or -80 &#176;C. Store TFA-altered proteins at -80 &#176;C and allowed to come to 4 &#176;C prior to emulsification with CFA. Store TFA altered proteins at -80 &#176;C and store in aliquots in order to prevent repeat freeze-thaw cycles.</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+free+amino+groups+in+proteins+by+trinitrobenzenesulfonic+acid.">Determination of free amino groups in proteins by trinitrobenzenesulfonic acid.</a> Analytical Biochemistry. 14 (3), 328-336 (1966).</li><li>Christen, U., Burgin, M., Gut, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halothane+metabolism:+immunochemical+evidence+for+molecular+mimicry+of+trifluoroacetylated+liver+protein+adducts+by+constitutive+polypeptides.">Halothane metabolism: immunochemical evidence for molecular mimicry of trifluoroacetylated liver protein adducts by constitutive polypeptides.</a> Molecular Pharmacology. 40 (3), 390-400 (1991).</li><li>Christen, U., Quinn, J., Yeaman, S. J., Kenna, J. G., Clarke, J. B., Gandolfi, A. 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=Identification+of+the+dihydrolipoamide+acetyltransferase+subunit+of+the+human+pyruvate+dehydrogenase+complex+as+an+autoantigen+in+halothane+hepatitis.+Molecular+mimicry+of+trifluoroacetyl-lysine+by+lipoic+acid.">Identification of the dihydrolipoamide acetyltransferase subunit of the human pyruvate dehydrogenase complex as an autoantigen in halothane hepatitis. Molecular mimicry of trifluoroacetyl-lysine by lipoic acid.</a> European Journal of Biochemistry. 223 (3), 1035-1047 (1994).</li></ol>

The authors have nothing to disclose.

The strength of this protocol rests in its reproducibility; so, it is critical to adhere to the suggested steps. Formulation of the immunogen can be a barrier for some; however, we have reproduced our model using the epitope described in our document, which removes the need to isolate the S100 fraction of the liver. It is likely that additional epitopes or proteins can be altered and induce hepatitis following immunizations; however, we describe those proteins that we have used with reliable results. Several proteins hav...

The purpose of this method is to describe a mouse model of drug-induced autoimmune hepatitis that develops in vivo and demonstrate how it can be utilized to investigate the molecular, immunologic and genetic basis of this disease. The long-term objective of our studies is to uncover mechanisms responsible for the development of chronic liver inflammation and injury by studying DIH in susceptible patients. Liver disease and cirrhosis constitute the sixth most common cause of death in adults between the ages of 25 and 64. Idiosyncratic DILI, sometimes referred to as drug-induced autoimmune hepatitis (DIH) is the third most common cause of acute liver failure in the United States. DIH is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis1. The overwhelming majority of patients with DIH are women2,3,4. A type of DIH develops in susceptible individuals following administration of halogenated volatile anesthetics such as isoflurane, sevoflurane, desflurane or halothane. These anesthetics covalently binds to liver proteins with reactive products of their metabolism, thus creating novel autoantigens capable of eliciting allergic or autoimmune responses5.
The study of pathogenic mechanisms involved in the development of anesthetic and any form of DIH has been previously hampered by the lack of an animal model that closely mimics the induction of human disease. We have developed an experimental murine model of DIH with features resembling immune-mediated DILI in patients. Hepatitis is induced by immunization with one of two autoantigens that have been covalently modified by the trifluoroacetyl chloride (TFA) metabolite that is formed following oxidative metabolism of the anesthetic by the enzyme cytochrome P450 2E1 (CYP2E1)5. One autoantigen is the hepatic cytosolic S100 liver fraction, which is a mixture of several proteins6, and the second autoantigen is an epitope of CYP2E1 that is recognized by sera from patients with anesthetic immune-mediated DILI7. By using BALB/c mice, which are relatively resistant to experimental autoimmune hepatitis, we distinguish our model from the S100-induced immunization model of autoimmune hepatitis in C57Bl/6J mice8.
Because of its diverse clinical presentations, DIH is difficult to study in patients. Translational experimental models offer the ability to evaluate the pathogenesis of disease in vivo and in vitro. At present, there are no other alternative methods for inducing DIH that fully examine in vivo or in vitro adaptive or innate immune responses without the use of animals. Moreover, since trifluoroacetylation of S-100 or the CYP2E1 epitope does not appear to produce an irritating immunogen, and we are inducing DIH by immunization with TFA-altered proteins, these animals will not receive ether, any halogenated anesthetic, barbiturate or alcohol prior to immunization or other procedures, considering that these agents may alter the parameters we are studying. Even so, we have decreased our mouse usage by utilizing computer simulation to confirm the binding preferences of our discovered CYP2E1 epitope9 and have mirrored human DIH implicating female sex by demonstrating that female BALB/c mice develop a more severe DIH10.
In spite of diverse presentations of DIH in patients and challenges in the study of clinical disease, post-translational modification of native proteins by reactive drug metabolites is an accepted key mechanism in the pathogenesis DIH that follows halogenated anesthetics11. Investigators also accept that CYP2E1 is a major autoantigen in this process12,13. The role of interleukin (IL)-4&#8211;upregulated CD4+T cells that recognize a post-translationally modified CYP2E1 and other liver proteins is an accepted initiator of anesthetic DIH by attracting neutrophils, eosinophils and mast cells into the liver14, and this mechanism has been confirmed in many forms of DIH15,16. Induced FoxP3-expressing CD4+CD25+T cells (Tregs) reduce the severity of DIH, and relative deficiencies of these cells in the spleen worsen DIH 10,7. Thus, the majority of advances in understanding DIH have been made possible by utilizing in vivo mouse models to evaluate the genetic, metabolic and immunologic mechanisms of DIH both in vivo and in vitro.
Because we and other investigators have uncovered roles for IL-4, neutrophils, and eosinophils in the initiation of DIH using different mouse models, we believe that this observation supports our contention that regardless of the DIH model utilized, hepatitis and injury are induced by IL-4. The strength of our protocol lies in the utilization of in vivo methodology, both male and female mice, and repetition of histology, CD4+ T cell proliferation assays and cytokines. The strength of our use of in vitro studies is that they reduce the numbers of mice needed while providing the methodology to isolate cellular interactions that drive DIH. We recommend the use of male and female mice because this reduces the possibility of unconscious bias in interpretation of results and strengthens the translation potential of our studies since the incidence, prevalence, and severity of DIH is higher in women17. We recommend that mice are obtained from a single vendor; however, if this is not possible, obtain litter mate controls or wild-type mice from the same vendor as the genetically altered mice.

<table><tbody><tr><td>0.1% 2,4,6-trinitrobenzene sulfonic acid (TNBS)</td><td>ThermoFisher</td><td>28997</td><td></td></tr><tr><td>AKP Substrate Kit</td><td>BioRad</td><td>172-1063</td><td></td></tr><tr><td>BALB/c mice</td><td>Jackson</td><td></td><td></td></tr><tr><td>CellTrace&amp;trade; CFSE Cell Proliferation Kit</td><td>ThermoFisher</td><td>C34554</td><td></td></tr><tr><td>CFA H37Ra</td><td>Becton Dickinson (Difco Bacto)</td><td>231131</td><td></td></tr><tr><td>FcR Blocking reagent</td><td>Milteyi</td><td>130-092-575</td><td></td></tr><tr><td>General supplement</td><td>ThermoFisher</td><td>HPRG770</td><td></td></tr><tr><td>HepaRG&amp;trade; cells cryopreserved</td><td>ThermoFisher</td><td>HPR GC10</td><td></td></tr><tr><td>Live/Dead Fixable Aqua Dead Cell stain kit&amp;nbsp;</td><td>ThermoFisher</td><td>L34965</td><td></td></tr><tr><td>NaHC03</td><td>Millipore Sigma</td><td>S5761</td><td></td></tr><tr><td>Percoll&amp;reg;</td><td>Millipore Sigma</td><td>P1644-1L</td><td></td></tr><tr><td>Pertussis Toxin</td><td>List Biologicals</td><td>180</td><td></td></tr><tr><td>Phosphate Buffered Saline pH 7.4</td><td>Various</td><td></td><td></td></tr><tr><td>Pierce&amp;trade; Protease Inhibitor Mini Tablets, EDTA Free</td><td>ThermoFisher</td><td>88666</td><td></td></tr><tr><td>Potassium Hydroxide</td><td>JT Baker</td><td>3140-01</td><td></td></tr><tr><td>S-ethyltrifluorothioacetate (S-ETFA)</td><td>Millipore Sigma</td><td>177474</td><td></td></tr><tr><td>Slide-a-lyzer dialysis cassettes (10 K, 12 ml)</td><td>ThermoFisher</td><td>66810</td><td></td></tr><tr><td>UltraPure&amp;trade; SDS Solution, 10%</td><td>ThermoFisher</td><td>24730020</td><td></td></tr><tr><td>Williams Media E, no phenol red</td><td>ThermoFisher</td><td>A1217601</td><td></td></tr></tbody></table>

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.

The immunization schedule utilized to induce DIH shown in Figure 1 represents the two immunizations required at the base of the neck (day 0) and the base of the tail (day 7). Figure 2 shows representative proliferation data obtained on day 14 using CFSE in response to CYP2E1, JHDN5, the CYP2E1 epitope and the trifluoroacetyl (TFA) metabolite of the anesthetics. Figure 3 shows the gating strategy and representative flow cytometry ana...

Watch this Scientific Journal Video about Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms at JoVE.com

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

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 ...

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Immunology and Infection

2.9K Views.  Johns Hopkins University. 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.

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

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 ...

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

Medicine

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) ...

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 ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

Induction and Evaluation of Experimental Autoimmune Encephalomyelitis in Mouse

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

Author Spotlight: Creating a Versatile Experimental Autoimmune Encephalomyelitis Model Relevant for Both Male and Female Mice 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

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

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