Sex differences in lipopolysaccharide‐induced cytokine secretion in skeletal muscle
Authors: Cusack Kevin J. , Murray Kevin O. , Atchison Julianne M. , Clanton Thomas L. 
Published: May 2022

INTRODUCTIONPrevious work has demonstrated large sex differences in the response to sepsis when various components of immune signaling are knocked out of muscle fibers. Skeletal muscle is a heterogeneous organ that holds many phenotypes, including resident immune cells such as resident myeloid‐derived immune cells. Skeletal muscle fibers are functional contributors to the inflammatory pathway, however, the contribution of these fibers to secrete cytokines relative to resident myeloid cells remains unknown. In an effort to further characterize the homeostatic and immunologic basis by which skeletal muscle‐derived immune cells contribute to whole‐body homeostasis, we evaluated the cytokine secretory capability of solei ex vivo in response to lipopolysaccharide (LPS) exposure.METHODS75 C57BL/6 mice were divided into two groups based on genetic profile: 36 LysMCrexMyD88 knockout (K/O) mice (18 female, 23 male) and 39 wild‐type (W/T) mice (23 female, 16 male). The resident myeloid cells found within the skeletal muscle fibers of LysMCrexMyD88 K/O mice lack proper toll‐like receptor‐4 function. Following anesthetization under isoflurane, mouse hind limbs were placed in oxygenated (95% O2, 5% CO2) Krebs‐Ringer solution. Solei were promptly excised and studied ex vivo in oxygenated muscle baths containing 2 mL Krebs‐Ringer solution. Half of the female and male solei from the K/O and W/T groups were randomized to receive 1 µg/mL of LPS while the remaining half received no LPS (Vehicle). Half of the solei received 1 µg/mL of LPS while vehicles received no LPS. Three samples were obtained from the buffer over the 2‐hour time course: before treatment (T0), 60 min following treatment (T1), and 120 minutes following treatment (T2). Samples were stored at ‐80°C and later analyzed for 22 common innate immune cytokines using Luminex Multiplex Analysis.RESULTSOf the 22 cytokines assessed, 16 displayed significant secretory differences between sexes when measured across T1‐T0 and T2‐T1 overall.During T1‐T0, K/O males exhibited significant depression in secretory responsiveness with LPS treatment compared to females (IFN‐γ, p=0.03; IL‐6, p =0.009; IL‐12p70, p=0.019; IL‐15, p=0.019; IP‐10, p=0.037; KC, p=0.024; MIP‐1β, p=0.005; MIP‐2, p=0.002). During T2‐T1, K/O LPS‐treated males had significantly different secretory responses compared to females (↓G‐CSF, p=0.045; ↓Eotaxin, p=0.034; ↑IL‐1α, p=0.019; ↑IL12‐p40, p=0.038; ↑RANTES=0.018). W/T LPS‐treated males exhibited significant depression in secretory responsiveness compared to females during T1‐T0 (IL‐12p70, p=0.049; IP‐10, p=0.003; MCP‐1, p=0.021; MIP‐1α, p=0.034; TNFα, p=0.011). During T2‐T1, W/T LPS‐treated males displayed significant elevation in secretory responsiveness compared to females (MIP‐2, p=0.024; RANTES, p=0.006).CONCLUSIONSIn the initial response period (T1‐T0), both K/O and W/T LPS‐treated males secreted significantly fewer cytokines relative to K/O and W/T LPS‐treated females, respectively. In the secondary interval (T2‐T1), some cytokine responses were elevated in males compared to females. These results demonstrate significant variations in the processes by which male and female skeletal muscle fibers respond to inflammatory stimuli. This may help to elucidate recent sex differences found regarding the interference of the inflammatory signaling cascade within muscle fibers during sepsis.