Toll-like receptors differentially regulate CC and CXC chemokines in skeletal muscle via NF-kappaB and calcineurin

Abstract

Immunologically active molecules such as cytokines and chemokines have been implicated in skeletal muscle weakness during sepsis as well as recovery from muscle injury. In sepsis, Toll-like receptors (TLRs) act as key sentinel molecules of the innate immune system. Here we determined skeletal muscle cell responses of two prototypical CC and CXC chemokine genes (monocyte chemoattractant protein 1 [MCP-1] and KC, respectively), to stimulation with specific TLR ligands. In addition, we examined whether NF-kappaB and calcineurin signaling are involved in these responses. Differentiated myotubes and intact whole muscles expressed TLR2, TLR4, TLR5, and TLR9. Stimulation with ligands for TLR2 (peptidoglycan) or TLR4 (LPS) elicited robust and equivalent levels of MCP-1 and KC mRNA expression, whereas stimulation of TLR5 (by flagellin) required gamma interferon priming to induce similar effects. Although both TLR2 and TLR4 ligands activated the NF-kappaB pathway, NF-kappaB reporter activity was approximately 20-fold greater after TLR4 stimulation than after TLR2 stimulation. Inhibitory effects of NF-kappaB blockade on TLR-mediated chemokine gene expression, by either pharmacological (pyrrolidine dithiocarbamate) or molecular (IKKbeta dominant-negative transfection) methods, were also more pronounced during TLR4 stimulation. In contrast, inhibitory effects on TLR-mediated chemokine expression of calcineurin blockade (by FK506) were greater for TLR2 than for TLR4 stimulation. MCP-1 and KC mRNA levels also demonstrated differential responses to NF-kappaB and calcineurin blockade during stimulation with specific TLR ligands. We conclude that skeletal muscle cells differentially utilize the NF-kappaB and calcineurin pathways in a TLR-specific manner to enable complex regulation of CC and CXC chemokine gene expression.

FIG. 1.

Expression of TLRs by skeletal muscle. RT-PCR of RNA extracted from the C2C12 muscle cell line at the myoblast (Mb) and myotube (Mt) stages, as well as C57BL/10 mouse diaphragm, limb muscle (tibialis anterior), and corresponding primary muscle cell cultures (myotube stage). PCRs were also performed without addition of reverse transcriptase (−CL) and in murine monocytes (+CL).

FIG. 2.

TLR-mediated chemokine expression by skeletal muscle cells. RNase protection assays performed 4 h after incubation of C2C12 myotubes with S. aureus peptidoglycan (10 μg/ml), poly(I:C) (25 μg/ml), E. coli LPS (1 μg/ml), S. enterica serovar Typhimurium flagellin (1 μg/ml), loxoribine (1 mM), or unmethylated CpG motif oligonucleotide (1 μM), representing TLR 2, 3, 4, 5, 7, and 9 ligands, respectively. −CL, unstimulated C2C12 myotubes.

FIG. 3.

IFN-γ priming of skeletal muscle cells augments chemokine expression in response to TLR5 stimulation. (A) RNase protection assay demonstrating chemokine expression by C2C12 myotubes under the following conditions: unstimulated cells (CL), cells primed for 24 h with IFN-γ (IFN; 200 U/ml), cells stimulated for 4 h with flagellin (Fl) alone, and IFN-primed cells stimulated for 4 h with flagellin (IFN + Fl). (B and C) Quantification of chemokine mRNA levels by densitometry, expressed in arbitrary units (a.u.) and normalized to the L32 housekeeping gene (n = 4 per group). *, P < 0.05 versus IFN alone; †, P < 0.05 for comparisons between IFN + Fl 0.01 μg/ml and IFN + Fl 1.0 μg/ml.

FIG. 4.

TLR2 and TLR4 stimulation induce different levels of NF-κB luciferase reporter activity. (A) C2C12 cells were cotransfected with a NF-κB firefly luciferase reporter plasmid and a constitutively active Renilla luciferase plasmid to control for transfection efficiency. Myotubes were stimulated for 4 h with LPS (1 μg/ml) or PGN (10 μg/ml) (n = 4 per group). *, P < 0.05 versus control (CL); †, P < 0.05 for comparisons between PGN and LPS. (B) RNase protection assay demonstrating that equivalent levels of KC and MCP-1 mRNA expression were observed in response to both ligands over a 10-fold dose range.

FIG. 5.

Chemical inhibition of NF-κB signaling decreases TLR-mediated chemokine expression in a differential manner. (A) RNase protection assay demonstrating chemokine expression by C2C12 myotubes after LPS or PGN stimulation for 4 h in the presence or absence of the NF-κB inhibitor PDTC (100 μM) and the antioxidant NAC (10 mM) or catalase (2,000 units/ml). (B and C) Quantification of chemokine mRNA levels by densitometry, expressed in arbitrary units (a.u.) and normalized to the L32 housekeeping gene (n = 4 per group). *, P < 0.05 versus LPS or PGN alone.

FIG. 6.

TLR2 and TLR4 ligands induce IκBα phosphorylation and degradation in skeletal muscle cells. Immunoblot analysis of phosphorylated IκBα (P-IκB) and total IκBα (IκB) in C2C12 myotubes transfected with a dominant-negative IKKβ plasmid vector or an empty control vector containing the same plasmid backbone and stimulated with LPS (1 μg/ml) or PGN (10 μg/ml).

FIG. 7.

Dominant-negative molecular inhibition of NF-κB signaling decreases TLR-mediated chemokine expression in a differential manner. (A) RNase protection assay demonstrating chemokine mRNA responses to LPS or PGN stimulation after transfection with either dominant-negative IKKβ (IKKB DN) or control plasmid vector as described in the legend to Fig. 6. (B and C) Quantification of chemokine mRNA levels by densitometry, expressed in arbitrary units (a.u.) and normalized to the L32 housekeeping gene (n = 4 per group). *, P < 0.05 versus control vector.

FIG. 8.

The calcineurin inhibitor FK506 differentially affects MCP-1 and KC gene expression in a TLR-specific manner. (A) RNase protection assay demonstrating the effects of FK506 (100 ng/ml) on chemokine expression by C2C12 myotubes after LPS or PGN stimulation for 4 h (together with the vehicle used for FK506, 0.1 μl ethanol/ml of media). (B and C) Corresponding quantification of chemokine mRNA levels by densitometry; *, P < 0.05 versus LPS or PGN alone. (D) Quantification of increases in chemokine mRNA levels in primary myotubes derived from wild-type (WT, C57BL/6) and TLR2 null mutant mice following stimulation with either LPS or PGN for 4 h (n = 4 per group). *, P < 0.05 versus unstimulated control.