Neutrophil- and Myeloid-Derived Suppressor Cell-Conditioned Medias Cause Myotube Atrophy and Alter the Myotube Transcriptome

Cancer-related inflammation and altered hematopoiesis leads to increased neutrophil and myeloid-derived suppressor cell (MDSC) production and reduced lymphoid and erythroid cell production. Recently, we found cells bearing a surface antigen belonging to both MDSCs and neutrophils accumulate in skeletal muscle and contribute to atrophy that occurs in cancer cachexia. It is unknown if these cells cause skeletal muscle atrophy directly, or if MDSCs and neutrophils impact atrophy differently. The PURPOSE of this study was to explore the direct effects of neutrophil- and MDSC-conditioned media relative to control medium on myotube atrophy and transcriptome.METHODS: Neutrophils were isolated from the blood of a healthy male donor using magnetic activated cell sorting and cultured in one of two 24-hour conditions. 1) control neutrophils were cultured in standard medium. 2) Treated neutrophils were cultured in the same medium with the addition of two cytokines shown to induce the MDSC phenotype (10ng/ml of IL-6 and10ng/ml GM-CSF). As an additional control, an equal volume of medium containing the cytokines was placed in a flask with no cells. Conditioned medias were added to fully differentiated C2C12 myotubes. After 24 hours, the myotubes were imaged and myotube diameter was measured. RNA was collected after 4 hours for RNA sequencing and bioinformatics. RESULTS: The average diameter of myotubes exposed to Neutrophil- (26.9 ± 9.6 um) and MDSC-conditioned medium (26.8 ± 13.6 um) were similarly and significantly smaller than myotubes grown in fresh medium (32 ± 15.4 um). Myotubes exposed to the control medium (containing cytokines, but not conditioned by cells) were not smaller than control myotubes (31.6 ± 11.8 um). RNA sequencing revealed significant upregulation of several histone H2B proteins and downregulation of Mybpc2 , Mef2c, and Timp3. CONCLUSION: These results show that neutrophils, whether untreated or exposed to a MDSC-inducing stimulus (IL-6 and GM-CSF), secrete muscle atrophy-producing factors. RNA sequencing showed downregulation of key thick filament protein, and activators for many muscle specific genes. This finding may help in understanding the mechanism of muscle atrophy that occurs in cancer cachexia. Further research is needed to determine specific gene impacts..