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 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 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 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–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.
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.
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.
3. General protocol notes
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 analysis of induced CD4+CD25+FoxP3+ Tregs obtained on day 14. Figure 4 shows representative hematoxylin and eosin stained slides demonstrating the evolution of hepatitis on day 21 6. Figure 5 shows representative hematoxylin and eosin stained slides demonstrating more severe hepatitis in female BALB/c mice when compared to males on day 21 in addition to the comparative cellular content in these livers10. Figure 6 shows representative confocal microscopy slides demonstrating the absence of co-localization of mouse IgG with mitochondria. Figure 7 shows representative confocal microscopy demonstrating co-localization of JHDN5 IgG with mitochondria.
Figure 1: Immunization of mice to induce hepatitis. DIH can be induced in female BALB/c mice (as an example) by immunization with TFA-JHDN5 (100 µg) emulsified in complete Freund’s adjuvant (CFA) subcutaneously (s.c.) at the base of the neck and 50 ng of pertussis toxin intramuscularly (i.m.) in the hind leg on day 0 (Step 1). On day 7, BALB/c mice can then be immunized with TFA-JHDN5 (100µg) emulsified in CFA (s.c.) at the bae of the tail. Please click here to view a larger version of this figure.
Figure 2: Determination of CD4+ T cell immune responses to whole self-proteins, epitopes of self-proteins or the TFA hapten using flow cytometry. Single cell suspensions of splenocytes from 6 – 8 week-old BALB/c mice isolated day 14 after the initial immunization, labeled with CFSE, stimulated with TFA-OVA, CYP2E1 or JHDN5 (10 µg/mL) for 72 h at 37 °C in 5% CO2, 95% air (humidified), stained with CD4-APC and analyzed by flow cytometry. Wells without antigen (media) were used as controls. BALB/c mice developed proliferation in response to OVA-TFA and CYP2E1 and not JHDN5, when compared to media. Please click here to view a larger version of this figure.
Figure 3: Gating strategy for the identification of CD4+CD25+FoxP3+ induced Tregs using the method described in 2.3.7. The first gate identifies live cells. To detect immune cells, CD45+ cells were initially gated. Next, CD4+ T cells were identified and gated, followed by identification of CD25+FoxP3+ cells within the CD4+ T cell population. Please click here to view a larger version of this figure.
Figure 4: Histological analysis of liver tissues for hepatitis. CFA-immunized mice (top panel) were used as vehicle controls. S100-immunized mice (middle panel) were evaluated following immunizations on the same schedule. On day 21, mice were euthanized, and the liver fixed in formalin. Sections 5 μm thick were made and stained with hematoxylin and eosin (H&E). Minimal hepatic inflammation (blue cells) is demonstrated following CFA (top) and S100 (middle) immunizations and large amounts of inflammation following immunizations with TFA-S100. (H&E, magnification 64X). This figure is used with permission6. Please click here to view a larger version of this figure.
Figure 5: Female BALB/c mice develop more DIH when compared to male BALB/c mice. (A) Female mice (n = 8) had significantly more severe hepatitis 3 weeks after TFA-S100/CFA immunizations than did males (n = 7/group). (B) Representative liver sections from female and male mice (H&E, magnification 64X). (C) Numbers of hepatic CD4+, CD8+, NK+, and NKT+ cells were significantly higher in females than in males. Mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. This figure is used with permission10. Please click here to view a larger version of this figure.
Figure 6: Mouse IgG does not co-localize with mitochondria. Confocal image of terminally differentiated hepatic cells stained with green fluorescent (488nm) -labeled mouse IgG (1:100) (green) in addition to red fluorescent (594)–-labeled Mitotracker Red (1:100). Green fluorescent (488nm)–labeled Mouse IgG does not co-localize with Mitotracker Red (63X magnification). Please click here to view a larger version of this figure.
Figure 7: JHDN5 IgG co-localizes with mitochondria. Confocal image of terminally differentiated hepatic progenitor cells stained with green fluorescent (488)–- labeled JHDN-5 IgG (1:100) in addition to red fluorescent (594)–-labeled Mitotracker Red (1:100). Green fluorescent (488nm)–labeled JHDN-5 IgG co-localizes with Mito-tracker Red, demonstrated by the yellow hue on the representative image (63X Magnification). Please click here to view a larger version of this figure.
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 have been demonstrated to be trifluoroacetylated upon halogenated anesthetic exposure. Most likely due to epitope spreading, some of these proteins are also target of autoantibodies in their native, non-trifluoroacetylated state. As an example, the E2 subunit of the pyruvate dehydrogenase complex (PDH-E2) carries epitopes (the lipoic acid prosthetic group) with a structural similarity to the TFA-moiety in TFA-adducts. Antibodies generated in patients with halothane hepatitis have been demonstrated to be cross-reactive to TFA-proteins and PDH-E2 20,21.
Our DIH model requires two immunizations that are emulsified in CFA along the back of the mice. We know that footpad injections with CFA have been associated with pain and distress in the mouse. Hence, the development of the experimental DIH model included several experimental trials. When we evaluated the model using one immunization using CFA, with the second immunization using incomplete Freund’s adjuvant (IFA) or IFA in both injections with our immunogen, hepatic inflammation did not develop. In sharp contrast, when we immunized the mice with the immunogen using two immunizations with CFA, significant hepatic inflammation was present. The original description of another model of autoimmune hepatitis included studies similar to ours and required two immunizations with CFA in order to demonstrate significant hepatic inflammation8. Even so, we continuously re-evaluate adjuvants using literature searches to attempt to optimize inflammation without CFA. As an example, there is a well-known adjuvant Titer Max that would augment B cell responses; however, although antibodies are a component of DIH, we and others have demonstrated a critical role for T cell responses in our model14.
The development of reliable models facilitates the investigations of the pathogenesis of DIH. We demonstrate that liver histology and immune cells can be reliably evaluated at various stages in this model. We demonstrate identification of antigen-specific T cells, serum antibodies and tissue cytokines that can be utilized to study the development of DIH. We demonstrate the methods for isolating cells from the inflamed liver and suggest the use of heparin. However, we have performed this technique without heparin and the results were indistinguishable. Additionally, current methods of tissue disruption may also release cells from the liver.
Recently, we have utilized modern tools such next gen sequencing and quantitative PCR and have experienced reproducible results. We have published results regarding DIH using IL-4, IL-4 receptor, IL-6, IL-6 receptor IL-33 and ST2 deficient mice that were all derived on a BALB/c background so that we can utilize the BALB/c mouse as our control mouse. Future applications of this model of DIH will include development of knock in mice in addition to the utilization of CRISPR technology in order uncover previously unrecognized mechanisms responsible for the pathogenesis of DIH.
The authors have nothing to disclose.
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.
0.1% 2,4,6-trinitrobenzene sulfonic acid (TNBS) | ThermoFisher | 28997 | |
AKP Substrate Kit | BioRad | 172-1063 | |
BALB/c mice | Jackson | ||
CellTrace™ CFSE Cell Proliferation Kit | ThermoFisher | C34554 | |
CFA H37Ra | Becton Dickinson (Difco Bacto) | 231131 | |
FcR Blocking reagent | Milteyi | 130-092-575 | |
General supplement | ThermoFisher | HPRG770 | |
HepaRG™ cells cryopreserved | ThermoFisher | HPR GC10 | |
Live/Dead Fixable Aqua Dead Cell stain kit | ThermoFisher | L34965 | |
NaHC03 | Millipore Sigma | S5761 | |
Percoll® | Millipore Sigma | P1644-1L | |
Pertussis Toxin | List Biologicals | 180 | |
Phosphate Buffered Saline pH 7.4 | Various | ||
Pierce™ Protease Inhibitor Mini Tablets, EDTA Free | ThermoFisher | 88666 | |
Potassium Hydroxide | JT Baker | 3140-01 | |
S-ethyltrifluorothioacetate (S-ETFA) | Millipore Sigma | 177474 | |
Slide-a-lyzer dialysis cassettes (10 K, 12 ml) | ThermoFisher | 66810 | |
UltraPure™ SDS Solution, 10% | ThermoFisher | 24730020 | |
Williams Media E, no phenol red | ThermoFisher | A1217601 |