(WHS-P3.05) EXCESSIVE LEVELS OF NEUTROPHILS CAUSE MACROPHAGE DYSFUNCTION IN FIBROTIC VOLUMETRIC MUSCLE LOSS (VML) INJURY
Friday, May 17, 2024
10:30 AM – 11:30 AM East Coast USA Time
Volumetric Muscle Loss (VML) is a debilitating condition defined by the rapid loss of muscle mass, leading to permanent impairment. The current standard of care is inefficient due to the poor understanding of the molecular/cellular processes governing VML repair. Macrophages are crucial for muscle repair due to its interaction with neighboring, and changes in macrophage phenotype have been observed following critical size VML injuries. The goal of this study is to uncover mechanisms driving macrophages towards a dysfunctional phenotype following VML. We employed a murine model of VML using subcritical size injuries (Regenerative), and critical size injuries (Fibrotic). We utilized flow cytometry to evaluate immune cell trafficking and macrophage phenotype in muscle over 28 days, and for 7 days in the spleen and bone marrow. Multiplex gene expression (NanoString) was used to characterize FACS-sorted macrophages and whole muscle tissue over 3 days. Systemic cytokines were quantified via multiplex cytokine analysis (Luminex) over 7 days. Finally, tissue healing was assessed via histology on Day 28. Fibrotic muscle macrophages demonstrated a higher expression of inflammatory and lower expression of reparative markers over 28 days. Noticeably, similar results were observed in the spleen over 7 days. Hierarchical clustering of muscle macrophages also uncovered hybrid macrophage phenotypes, particularly at Day 7. At the gene level, Fibrotic macrophages in muscle presented a broad gene downregulation as early as Day 1 compared to Regenerative injury. These results demonstrated that macrophage dysfunction appears soon after injury and prior to onset of fibrosis. Whole muscle and blood analyses uncovered a higher expression of neutrophil chemokines and systemic G-CSF after Fibrotic injuries, accompanied by an increase in neutrophil influx to the muscle. These results suggest an active role of neutrophils in the progression of this pathology, and perhaps in modulating macrophage phenotype. To evaluate neutrophil-macrophage crosstalk, we partially depleted neutrophils so that neutrophil accumulation in the muscle following Fibrotic injuries resembles levels observed in Regenerative group. Partial neutrophil depletion decreased macrophage expression of inflammatory markers/genes and increased reparative marker/genes expression. However, no improvement in muscle physiology was observed, suggesting that neutrophil depletion improves macrophage phenotype, but is not enough to leverage muscle healing. Here we have, for the 1st time, observed systemic changes in immune behavior following VML injury. Moreover, we uncovered neutrophils as critical macrophage modulators. These results can instruct the development of better therapeutics.