Multiple Toll-like receptor ligands induce an IL-6 transcriptional response in skeletal myocytes

Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania Submitted 7 July 2005 ; accepted in final form 24 October 2005 Toll-like receptors (TLRs) comprise a critical sentinel that monitors body compartments for the presence of pathogens. Skeletal muscle expresses TLRs and responds to pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), by mounting an innate immune response. In the present study, we used C 2 C 12 myocytes as a model system for skeletal muscle during infection. C 2 C 12 cells responded to LPS in a time frame and with a pattern of gene expression that faithfully mimicked the response of skeletal muscle to LPS in vivo. LPS from a variety of Escherichia coli serotypes stimulated IL-6 synthesis. C 2 C 12 cells expressed TLR1–7, but not TLR8 or TLR9, mRNA by RT-PCR. A synthetic tripalmitoylated cysteine-, serine-, and lysine-containing peptide (Pam) and LPS from Porphyromonas gingivalis , two TLR2 ligands, also stimulated IL-6 expression. LPS and Pam stimulated luciferase activity driven from NF- B and IL-6 promoter-containing plasmids, and this response was blunted when the NF- B binding site was mutated. LPS- and Pam-stimulated IL-6 expression was inhibited by the proteasome inhibitor MG-132 and the I B kinase-2 (IKK2) inhibitor 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1). Pam-stimulated NF- B and IL-6 promoter activities were disrupted by a dominant-negative form of TLR2, but not TLR4. Local injection of LPS or Pam into the gastrocnemius muscle stimulated IL-6 mRNA expression in the injected, but not the contralateral, muscle. The LPS- but not Pam-stimulated expression of IL-6 mRNA was blunted in skeletal muscle of mice carrying an inactivating mutation in TLR4. The data suggest that skeletal muscle and muscle cells recognize pathogen-associated molecules with specific TLRs to initiate an IL-6 transcriptional response. skeletal muscle; muscle cells; immune response; exercise Address for reprint requests and other correspondence: R. A. Frost, Dept. of Cellular and Molecular Physiology (H166), Pennsylvania State Univ. College of Medicine, 500 University Dr., Hershey, PA 17033 (e-mail: rfrost{at}psu.edu )