Eosinophil Infiltration in Muscular Dystrophy: Key Characteristics and Contributions to Disease
Duchenne Muscular Dystrophy (DMD) is a genetic disorder that affects 1 in 3,500 live male births. In DMD patients, inactivating mutations in the gene encoding dystrophin prevent expression of this critical cytoskeletal protein that is essential for myofiber integrity. Mutations in the gene encoding dystrophin have also been detected in muscular dystrophy (mdx) mice, which phenocopy the disease seen in DMD patients. As a result of dystrophin deficiency, the skeletal and cardiac muscles undergo severe degeneration accompanied by infiltration with inflammatory cells. Eosinophils are prominent among the leukocytes recruited to dystrophin-deficient skeletal muscle tissues, although their contributions to disease are incompletely understood.
Eosinophils are capable of releasing cytotoxic mediators stored in cytoplasmic granules; as such, eosinophils have been viewed largely as mediators of tissue destruction. I began this work with an evaluation of the extent and impact of eosinophil infiltration on acute muscle damage in muscular dystrophy mice. Towards this end, I generated eosinophil-deficient (mdx.PHIL) and eosinophil-overabundant (mdx.IL5tg) strains of mdx mice. Despite the varied levels of eosinophil infiltration detected in skeletal muscle tissues, my studies revealed that there were remarkably similar levels of muscle damage in mdx, mdx.IL5tg and mdx.PHIL mice. This was evaluated by four key measures: histopathology scoring, distribution of myofiber sizes, proportion of centrally-nucleated myofibers and creatine kinase levels.
To evaluate the functional characteristics of eosinophils recruited to dystrophin-deficient muscle tissue, I isolated eosinophils from the quadriceps muscles of mdx, IL5tg and mdx.IL5tg mice at 4 weeks of age, extracted RNA and performed RNA-sequencing. I identified 393 transcripts that were expressed at 5-fold higher levels in the eosinophils isolated from the muscle tissues of mdx mice when compared with those from IL5tg mice. Many of these transcripts encode tissue remodeling proteins (e.g., Ctss, Ctsk, Mmp19) as well as cell surface proteins (e.g., Cd14, Slc15a3, Trem2). By contrast, I identified 238 transcripts at 5-fold higher levels in eosinophils from the muscle tissues of IL5tg mice when compared with those from mdx mice. Many of these latter transcripts encode eosinophil-specific proteins (e.g., Ear1, Epx, Prg2) or proteins involved with cell cycle regulation.
The findings that suggest differential expression of Trem2 are particularly intriguing, as TREM2 has been shown to promote the proliferation, activation and survival of myeloid cells in response to tissue damage. Hence, in order to evaluate the role of Trem2 in mediating eosinophil survival within dystrophin-deficient muscle tissues, we are currently generating mdx.Trem2-/- mice and will be in a position to evaluate eosinophil infiltration and survival in the muscle tissues of these mice. While generation of this strain is ongoing, I have determined that Trem2 is critical for full eosinophil development in both ex vivo and in vivo experimental studies.
Taken together, these findings add to the growing body of evidence that suggests that eosinophils do not universally promote tissue destruction. In addition, findings presented in this dissertation highlight the unique features of eosinophils that may be uncovered through RNA-sequencing and analysis. Among the most intriguing findings, I established that Trem2 is critical for efficient eosinophil development. Further work will elucidate the regulation and functions of TREM2 expression in eosinophils.