Characterizing the Effects of Vaccine Adjuvants on Skeletal Muscle Myotubes and Macrophages

Vaccines train the immune system to recognize and defend against pathogens. Currently, six types of vaccines are in use and include live-attenuated, inactivated, viral vector, protein subunit, toxoid, and messenger RNA (mRNA), the latter of which was recently approved for humans during the COVID-19 pandemic. To increase the longevity and magnitude of immune responses, some vaccines are combined with adjuvants. Mouse models have shown that adjuvants in combination with antigens can elicit a pro-inflammatory immune system response that is required for proper development of protective immunity. There has been recent appreciation for the immunomodulatory functions of skeletal muscle, yet their contribution to the immunology of vaccination remains incompletely understood. Considering most vaccines are administered intramuscularly, we utilized C2C12 mouse myotubes and J774 macrophages to explore the cytokine response that skeletal muscle cells and macrophages evoke in response to several types of vaccine adjuvants in absence of the antigen. C2C12 myotubes and J774 macrophages were treated with 7 commonly used adjuvants or appropriate controls and collected at 6 hours and 24 hours. Cytokine secretion, cytotoxicity, and effects on myotube diameter were analyzed. Most adjuvants, except for the positive controls (LPS and PAM3CSK4), CpG 1826, and Quil-A, did not induce a pro-inflammatory response in C2C12 myotubes or J774 macrophages. Interestingly, LPS, PAM3CSK4, MF59, Quil-A, AS03, and CFA, led to increases in C2C12 myotube diameter indicating an activation of hypertrophy. The lack of pro-inflammatory effects indicates that most adjuvants need antigens or additional cell-cell interactions at the injection site to produce a pro-inflammatory cytokine response