# Induction of Autoimmune Myocarditis in Diversity Outbred Mice

## TL;DR

This study shows that Diversity Outbred mice can develop heart inflammation similar to human autoimmune myocarditis, offering a more realistic model for research.

## Contribution

The study demonstrates that DO mice, with human-like genetic diversity, can be used to model autoimmune myocarditis.

## Key findings

- DO mice can develop mild autoimmune myocarditis after immunization with porcine cardiac myosin.
- Sex differences were observed in MHC class II expression but not in costimulatory molecule expression.
- PCM immunization induced T cell and antibody responses, with varying degrees of myocarditis in both sexes.

## Abstract

Researchers often use inbred mouse strains to study autoimmune diseases, including autoimmune myocarditis. These mice are genetically very similar to one another, which means that their responses may not reflect what happens in genetically diverse human populations. In this study, we examined whether Diversity Outbred (DO) mice, whose genetic composition more closely resembles that of humans, could be utilized for studying inflammatory processes in the heart. Our results showed that DO mice are capable of developing heart inflammation and indicate that the variations noted in our study may have translational significance.

Background. Inbred mouse models of autoimmune myocarditis are routinely used to investigate the immune mechanisms underlying dilated cardiomyopathy. However, their translational relevance is limited because observations made in a single inbred strain may not reflect those of outbred human populations. This limitation can be overcome by using Diversity Outbred (DO) mice, whose genetic variability is comparable to that of humans. Methods. To investigate the utility of DO mice, we characterized their immune cell distributions and induced myocarditis by immunization with porcine cardiac myosin (PCM) emulsified in complete Freund’s adjuvant. Antigen-specific T cell and antibody responses were evaluated using lymphocytes and serum samples, respectively, and hearts were examined histologically for inflammatory changes. Results. First, we noted no significant variations in the majority of immune cell populations, which include T cells and B cells. However, NK cells, double positive for CD49b and NK1.1, were lacking in both sexes. While we noted sex differences in the expression of major histocompatibility complex class II molecules in antigen-presenting cells, expression of costimulatory molecules was similar in both sexes. Second, upon immunization, we demonstrated that the PCM was immunogenic, and the PCM-reactive T cell responses were generated in both males and females, as measured by a proliferation assay. Third, cytokine analysis revealed marginal detection of Th1 (IFN-γ) and Th17 (IL-17 and IL-22) cytokines, mainly with three doses of immunization. Fourth, determination of PCM-reactive antibody responses revealed significant amounts of IgG1 and IgG2b isotypes. Finally, histological analysis revealed varying degrees of myocarditis in individual mice of both sexes. Conclusions. Our data suggest that mild autoimmune myocarditis can be induced in DO mice. However, to capture the heterogeneity in disease susceptibility, large sample cohorts are required.