The role of myeloid cells in lung injury‐induced muscle atrophy

Rationale Intensive care unit acquired weakness (ICUAW) occurs in nearly 50% of patients admitted to the ICU. Patients exhibiting ICUAW have increased in‐hospital and post‐ICU mortality. ICUAW, and the associated muscle atrophy specifically, cannot be completely explained by disuse and may involve a secondary trigger to enhance the breakdown of muscle fibers. Recent work has suggested that excessive systemic inflammation may contribute to this process. We hypothesized that muscle atrophy in ICUAW may, in part, be induced by myeloid cell recruitment to muscle. Methods C57Bl6/J mice received intratracheal (IT) instillation of lipopolysaccharide (LPS, 3 μg/g) or saline and were euthanized 3, 6, 12, 24, 72 hours thereafter. For antibody‐mediated myeloid cell depletion, 200 μg of αGR‐1, αLy6G, or isotype control antibodies were administered IP 24 hours and 1 hour prior to injury and mice were examined 24 hours post‐injury. Whole gastrocnemius muscle was analyzed using RT‐qPCR. For in vitro studies, C2C12 myoblasts were differentiated for 7 days and stimulated with LPS (1 μg/ml) to elicit an inflammatory response. Bone marrow derived macrophages (BMDMs) were differentiated with 10 ng/ml of macrophage‐colony stimulation factor for 7 days and seeded at a density of 2600 cells/cm 2 (4940 cells/well) onto C2C12 myotubes after 6 days of differentiation. On day 7 of C2C12 differentiation, cocultures were stimulated as previously described. Supernatants and cell lysates were analyzed by ELISA and RT‐qPCR. Myotube diameter was measured using ImageJ. Results IT LPS upregulates the atrogene muscle RING‐finger 1 (MuRF1) and induces atrophy in vivo. However, this effect of LPS is not seen in cultured C2C12 myotubes alone. Depletion of neutrophils and monocytes with αGR‐1 antibody reduced MuRF1 expression in vivo . Both C2C12 myotubes and whole muscle show rapid, transient expression of myeloid chemoattractants MCP‐1 and KC (CXCL1). Cocultures of BMDMs and C2C12 myotubes enhance the expression of IL‐6 and MCP‐1 synergistically and induce C2C12 myotubes to produce TNFα. Conclusions Our data indicate that IT LPS induces MuRF1 in vivo and our in vitro data in myotubes suggests that this effect may require myeloid cells. Further supporting this conclusion, we found that antibody depletion of myeloid cells reduced IT LPS‐induced MuRF1 expression. Moreover, expression of key myeloid cell chemoattractants are induced in muscle both in vitro and in vivo in response to LPS and addition of BMDMs to C2C12 myotubes with LPS treatment reveals synergistic increases of IL‐6 and MCP‐1 and induces C2C12 myotubes to produce TNFα. The current study implicates macrophages and neutrophils as modulators of the muscle atrophy response to following lung injury. Support or Funding Information NIH 5R01HL143452‐02NIH 2T32HL076122‐16