NOD1 priming by IFN-gamma regulates inflammatory responses and perturbs differentiation of skeletal muscle

Background Nod-like receptors (NLRs) are intracellular sensors acting as major regulators of cytokine secretion, gene expression and various signaling pathways, modulating immune responses, metabolism and cell fate. Beyond leukocytes, NLRs are also expressed in various non-immune competent cells, however, their expression and function in skeletal muscle, the largest tissue in the human body, are largely unknown. Methods We studied NLR expression in response to IFN-γ treatment in ex vivo Tibialis anterior , and in vitro C2C12 myoblast (MB) and myotube (MT) with RT-qPCR and Western blot. We focused on the assessment of gene expression changes and cytokine production in MT following IFN-γ priming and activation of NOD1, a key NLR identified, using RNA-seq, RT-qPCR and ELISA, followed by functional studies to understand its immunomodulatory roles. NOD1 function in the context of differentiation of MT was studied with scratch assay, while lipid droplet accumulation was determined with Oil Red O staining. Results We found that the expression of regulatory NLRs (NOD1, NOD2, NLRX1, NLRC3) dynamically changed during MB to MT differentiation, and identified NOD1, as a key immunomodulatory NLR in skeletal muscle. IFN-γ treatment in MT highly induced NOD1 expression, and NOD1 activation following IFN-γ priming resulted in global gene expression changes. We found that most myokines are induced in a NOD1-dependent manner, while a cluster of genes related to migration and differentiation were strongly downregulated. In particular, upon IFN-γ priming NOD1 regulates IL-6 production via NF-kB. In addition, IFN-γ-induced NOD1 strongly reduces the differentiation of MT, while enhances lipid accumulation. Conclusions Our results show that while NLRs are expressed in MB and dynamically change during MT differentiation, NOD1 has important, so far uncharacterized function in skeletal muscle. Our results suggest that NOD1 and tentatively other NLRs in the skeletal muscle are connected to diverse physiological and pathological processes, implying their potential roles in myopathies or as therapeutic targets in other disease states.