UC Irvine

Duchenne muscular dystrophy (DMD) is a lethal, x-linked genetic neuromuscular disorder caused by mutations in the dystrophin gene. The resulting non-functional dystrophin protein renders muscle myofibers susceptible to contraction-induced injury, and normal ambulation leads to muscle degeneration and eventual death. Further contributing to disease is the activation of chronic immune responses caused by asynchronous and continuous bouts of injury. This distributes the homeostasis-promoting balance of immune responses, further causing exacerbated muscle degeneration and impaired regeneration. Importantly, different facets of the immune system, such as type I or type II immunity, are implicated as being injury-promoting or pro-regenerative, respectively. In acute injury settings, type II immune responses such as the activation of M2-like macrophages and eosinophils are critical for efficient muscle regeneration. It is suspected that a similar type II immune response is activated in dystrophic muscle, but its role in promoting regeneration is disrupted by competing pro-inflammatory responses. However, the regulation of type II immunity in dystrophic muscle is largely unknown.

Intriguingly, a recently identified subset of immune cells, group 2 innate lymphoid cells (ILC2), have been shown to be potent regulators of type II immunity and promote repair in other diseases. The focus of this dissertation is aimed at understanding the role of ILC2s in regulating skeletal muscle type II immune responses during chronic muscle disorders such as DMD (Chapter 3). We found that mdx muscle ILC2s were increased in number and expressed higher levels of type II cytokines interleukin-5 and interleukin-13 compared to wildtype controls, indicating that muscle degeneration activates ILC2s. We also sought to identify how muscle ILC2s are activated and found that fibro/adipogenic progenitors were the primary source of IL-33, an alarmin known to activate ILC2s. We found that muscle ILC2s are activated by exogenous cytokines, including IL-2/anti-IL-2 complex (IL-2c) and IL-33, which we also used to induce the expansion of ILC2s in vivo. Using additional mouse models in which ILC2s are genetically depleted, we found that ILC2s are potent regulators of skeletal muscle eosinophilia. As ILC2s have also been shown to activate M2-like macrophages in other tissues, we also evaluated this interaction in dystrophic skeletal muscle. To our surprise, ILC2s did not regulate macrophage numbers or phenotype in mdx muscle during this study.

In addition to investigating the role of ILC2s in muscle dystrophy, another aim of this work was focused on the development of software that evaluates the morphological features of skeletal muscle (Chapter 2). Muscle function is commonly assessed by evaluating and quantifying histological features of muscle cross-sections. This includes evaluating myofiber size, the expression of markers for injury and regeneration, as well as measuring centrally-located myofiber nuclei. Historically, the time-consuming and tedious nature of manually quantifying muscle samples hindered accurate evaluation, as many analyses were limited to the sampling of parts of the entire cross-section. Furthermore, morphological features are much more complex in diseased muscle compared to healthy, which further inhibited the ability to use software to automatically evaluate the tissue in a high-throughput manner. To address these issues in the field, and provide a tool that we could use to evaluate how perturbations to the immune system effect muscle pathology, we developed QuantiMus. QuantiMus is a machine learning-based software that allows the accurate detection of muscle myofibers as well as the location of nuclei (i.e., centrally-located) and intra-myofiber presence of investigator-chosen proteins stained by immunofluorescence in entire muscle cross-sections. To date, we have successfully used QuantiMus to measure myofiber regeneration, myofiber injury, centrally-nucleated fibers, and to determine the myofiber type distribution of entire muscle cross-sections. The ability to measure full cross-sections is an advancement in the field that allows investigators to avoid random sampling of tissue sections and prevents bias in the data.

All together, this study defines a role for ILC2s in regulating skeletal muscle eosinophilia during muscular dystrophy. We also developed software that allows us to evaluate muscle morphology in a high-throughput manner. Future directions will be aimed at understating what, in addition to regulating muscle eosinophilia, role ILC2s play in regulating DMD pathogenesis. In part, these future studies will encompass the power of QuantiMus to histologically evaluate how perturbations to ILC2s and eosinophils regulate pathology.