Sepsis induces interleukin 6, gp130/JAK2/STAT3, and muscle wasting

Abstract

Background Sepsis and inflammation can cause intensive care unit-acquired weakness (ICUAW). Increased interleukin-6 (IL-6) plasma levels are a risk factor for ICUAW. IL-6 signalling involves the glycoprotein 130 (gp130) receptor and the JAK/STAT-pathway, but its role in sepsis-induced muscle wasting is uncertain. In a clinical observational study, we found that the IL-6 target gene, SOCS3, was increased in skeletal muscle of ICUAW patients indicative for JAK/STAT-pathway activation. We tested the hypothesis that the IL-6/gp130-pathway mediates ICUAW muscle atrophy. Methods We sequenced RNA (RNAseq) from tibialis anterior (TA) muscle of cecal ligation and puncture-operated (CLP) and sham-operated wildtype (WT) mice. The effects of the IL-6/gp130/JAK2/STAT3-pathway were investigated by analysing the atrophy phenotype, gene expression, and protein contents of C2C12 myotubes. Mice lacking Il6st, encoding gp130, in myocytes (cKO) and WT controls, as well as mice treated with the JAK2 inhibitor AG490 or vehicle were exposed to CLP or sham surgery for 24 or 96 h. Results Analyses of differentially expressed genes in RNAseq (≥2-log2-fold change, P < 0.01) revealed an activation of IL-6-signalling and JAK/STAT-signalling pathways in muscle of septic mice, which occurred after 24 h and lasted at least for 96 h during sepsis. IL-6 treatment of C2C12 myotubes induced STAT3 phosphorylation (three-fold, P < 0.01) and Socs3 mRNA expression (3.1-fold, P < 0.01) and caused myotube atrophy. Knockdown of Il6st diminished IL-6-induced STAT3 phosphorylation (À30.0%; P < 0.01), Socs3 mRNA expression, and myotube atrophy. JAK2 (À 29.0%; P < 0.01) or STAT3 inhibition (À38.7%; P < 0.05) decreased IL-6-induced Socs3 mRNA expression. Treatment with either inhibitor attenuated myotube atrophy in response to IL-6. CLP-operated septic mice showed an increased STAT3 phosphorylation and Socs3 mRNA expression in TA muscle, which was reduced in septic Il6st-cKO mice by 67.8% (P < 0.05) and 85.6% (P < 0.001), respectively. CLP caused a loss of TA muscle weight, which was attenuated in Il6st-cKO mice (WT: À22.3%, P < 0.001, cKO: À13.5%, P < 0.001; WT vs. cKO P < 0.001). While loss of Il6st resulted in a reduction of MuRF1 protein contents, Atrogin-1 remained unchanged between septic WT and cKO mice. mRNA expression of Trim63/MuRF1 and Fbxo32/Atrogin-1 were unaltered between CLP-treated WT and cKO mice. AG490 treatment reduced STAT3 phosphorylation (À22.2%, P < 0.05) and attenuated TA muscle atrophy in septic mice (29.6% relative reduction of muscle weight loss, P < 0.05). The reduction in muscle atrophy was accompanied by a reduction in Fbxo32/Atrogin-1-mRNA (À81.3%, P < 0.05) and Trim63/MuRF1-mRNA expression (À77.6%, P < 0.05) and protein content.

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“…The SuperScript ® First-Strand Synthesis System (Invitrogen™, Life Technologies Corporation, Carlsbad, CA, USA) was used to synthesize 1.5 µg RNA per sample in accordance with the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using Power SYBR ® Green PCR Master Mix (Applied Biosystems) and self-designed primers (for primer sequences see Table S15 ) on a Step-One™ Plus thermocycler (Applied Biosystems, Waltham, MA, USA) using the standard curve method as described recently [ 8 , 58 , 61 , 62 ]. The expression of individual genes was normalized to the geometric mean of the three stably expressed internal control genes mitochondrial ribosomal protein L13 ( Mrpl13 ), importin 8 ( Ipo8 ), and phosphoglycerate kinase 1 ( Pgk1 ) selected from obtained RNA sequencing data and according to previously published work [ 63 ].…”

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“…Previously we showed that MuRF1/Trim63 and atrogin-1/Fbxo32 mRNA and protein expression are increased in the vastus lateralis of critically ill human patients, especially those that were at risk to develop muscle wasting ( 27 ). Both E3 ligases were also upregulated in the tibialis anterior and gastrocnemius and plantaris of mouse models of neurogenic, inflammation- and starvation induced muscle atrophy ( 28 – 32 ), where their increased expression was associated with the atrophy phenotype and the loss of MyHC. We and others recently summarized the current knowledge about factors and signaling pathways involved in the regulation of E3 ubiquitin ligases relevant for muscle atrophy ( 6 , 33 , 34 ).…”

“…To cause muscle atrophy, these cytokines bind to their receptors at the myocyte cell surface and activate transcription factors and signaling proteins such as Nuclear Factor-κB (NF-κB), JAK/STAT, SMAD, MAPK, TFEB/TFE3, and IGF/PI3K/AKT to regulate MuRF1/Trim63 and atrogin-1/Fbxo32 expression that in turn mediate muscle atrophy ( 6 , 33 , 34 ). Importantly, in addition to the immune system the skeletal muscle itself contributes to the production and release of acute phase response proteins and proinflammatory cytokines ( 28 , 31 , 32 , 35 ), which may aggravate muscle loss especially during chronic inflammation and sepsis. Although we start to understand the role of protein degradation and its regulation in muscle atrophy only few therapeutic concepts arose from these insights.…”

“…Despite the fact that skeletal muscle atrophy due to immobilization is a prevalent and severe clinical problem in many different medical situations, the underlying mechanisms are not well understood. Of note, bed rest in humans and immobilization of animals have been shown to cause skeletal muscle atrophy mainly by an increased activity of UPS-mediated protein degradation with enhanced expression of MuRF1 and atrogin1 ( 27 , 29 – 32 , 120 ). Since skeletal muscle atrophy due to disuse leads to an upregulation of genes encoding sarcoplasmic reticulum (SR) calcium-handling proteins and considering that many of the proteins that are induced with ER stress are also calcium-handling proteins a connection between disuse atrophy and ER stress is likely.…”

“…The skeletal muscle has been shown to be affected by and to react to immunologic conditions such as proinflammatory stimuli ( 30 – 32 , 136 ). The involvement of the UPR in the pathogenesis of different myopathies such as Sporadic inclusion body myositis, Myasthenia gravis, Duchenne muscular dystrophy has been reviewed recently ( 137 ).…”