Trained immunity induced by muscle injury: a focus on inflammatory monocytes

RATIONALE and GENERAL OBJECTIVES Trained immunity or ‘innate immune memory’ is a process in which, innate immune cells, such as inflammatory Ly6Chi monocytes, undergo metabolic and epigenetic reprogramming in response to microbial stimuli, resulting in qualitatively and/or quantitatively enhanced responses following a subsequent challenge with either the same or unrelated stimuli. Most compelling, and relevant to this project, are the recent observations that non-microbial products including cytokines (IL-1β, GM-CSF and IFN-γ) and damage associated molecular patterns (DAMPs), typically released from inflamed tissues, can also induce trained immunity in monocytes. However, little is known about this process in the context of muscle injury. Therefore, the primary aim of my PhD project is to investigate whether and how trained immunity is induced in inflammatory Ly6Chi monocytes in response to muscle injury. Furthermore, we will investigate the mechanisms underlying the induction of trained immunity in monocytes after muscle injury and assess whether targeting trained immunity in Duchenne muscular dystrophy (DMD) can ameliorate dystrophic muscle pathology. EXPERIMENTAL DESIGN AND METHODS We addressed these questions using a CTX model of acute muscle injury and the mdx mouse model of chronic muscle injury. To assess the induction of trained immunity, monocytes isolated from the spleen and bone marrow were analysed for cytokines production using multiplex ELISA assay. Real time quantitative PCR, flow cytometry analysis and Intracellular Cytokines Staining were used to investigate trained immunity mechanisms. To therapeutically target trained immunity in DMD, mice were treated with mTORi-nanobiologics via tail vein injection. At the end of the treatment, tissues were harvested for FACS, histological and molecular analysis. RESULTS During acute muscle injury, the spleen represents an important source of inflammatory Ly6Chi monocytes. In fact, as soon as 3h post CTX muscle injury, the number of splenic monocytes rapidly decrease, indicating their mobilization to injured muscle, confirmed by using a splenectomised mouse model. Fifty days post injury both splenic and bone marrow derived monocytes exhibit hallmarks of trained immunity such as an enhanced response upon secondary stimulation in vitro (e.g. LPS), characterized by increased production of both pro and anti-inflammatory cytokines and chemokines. Furthermore, to assess the functional consequences of this process in vivo, we used a model of repeated injury. While secondary injury did not lead to any quantitative changes in the number of monocytes recruited to injured muscle, it induced qualitative changes in their response, with higher production of monocytes derived cytokines and chemokines. The functional relevance of monocytes training on muscle regeneration was further evaluated in vitro by culturing satellite cells with conditioned media from trained and untrained monocytes. Satellite cells exposed to conditioned media from trained monocytes showed enhanced proliferation and increased myotubes size, suggesting a positive influence on muscle repair. Next, we investigated the changes in the spleen microenvironment following CTX injury. Notably, as soon as 3h post injury there was a significant increase in the production, within the CD3+ T cell population, of cytokines already shown to be able to induce training on monocytes in vitro, such as IFN-γ and IL-4, suggesting that the spleen may represent a potential training site for monocytes after muscle injury. Finally, the mdx mouse model of Duchenne muscular dystrophy has been used to study trained immunity in the context of chronic muscle injury. Both splenic and bone marrow monocytes display dysregulated and exaggerated responsiveness, underlying the detrimental role of trained immunity under chronic injury conditions. To therapeutically modulate this process, we targeted trained immunity in Duchenne muscular dystrophy using mTORi-nanobiologics. This treatment significantly reduced TNF- production by splenic monocytes and leads to an enhanced muscle regeneration. CONCLUSION Trained immunity is a feature of inflammatory Ly6Chi monocytes following muscle injury, likely induced within the spleen through T-cells derived cytokines. While this process is beneficial for muscle regeneration after acute injury, in chronic conditions such as Duchenne muscular dystrophy, it becomes detrimental. Therapeutic targeting of trained immunity with mTORi-nanobiologics may reduce inflammation and improves muscle regeneration.