Increased Circulating Levels of Interleukin-6 Affect the Redox Balance in Skeletal Muscle

Abstract

The extent of oxidative stress and chronic inflammation are closely related events which coexist in a muscle environment under pathologic conditions. It has been generally accepted that the inflammatory cells, as well as myofibers, are sources of reactive species which are, in turn, able to amplify the activation of proinflammatory pathways. However, the precise mechanism underlining the physiopathologic interplay between ROS generation and inflammatory response has to be fully clarified. Thus, the identification of key molecular players in the interconnected pathogenic network between the two processes might help to design more specific therapeutic approaches for degenerative diseases. Here, we investigated whether elevated circulating levels of the proinflammatory cytokine Interleukin-6 (IL-6) are sufficient to perturb the physiologic redox balance in skeletal muscle, independently of tissue damage and inflammatory response. We observed that the overexpression of circulating IL-6 enhances the generation and accumulation of free radicals in the diaphragm muscle of adult NSE/IL-6 mice, by deregulating redox-associated molecular circuits and impinging the nuclear factor erythroid 2-related factor 2- (Nrf2-) mediated antioxidant response. Our findings are coherent with a model in which uncontrolled levels of IL-6 in the bloodstream can influence the local redox homeostasis, inducing the establishment of prooxidative conditions in skeletal muscle tissue.

Figure 1

IL-6 induced the enhanced ROS production and accumulation in the diaphragm muscle. Western blot analysis (right panels show representative images) for the expression of gp91phox (a) and G6PD (e) proteins in 24-week-old NSE/IL-6 and wild-type (WT) mice. Values represent mean ± SEM; n = 5 to 6 mice per group. ^∗p < 0.05, ^∗∗p < 0.005 by Student's two-tailed unpaired t-test. Lanes were run on the same gel but were not contiguous. Original images are shown in and . (b) Quantitative analysis (left panel) of the mean intensity of fluorescence derived from DHE staining on diaphragm muscle sections from 24-week-old NSE/IL-6 and WT mice. Right panels show representative images of DHE staining on muscle sections of indicated genotypes (scale bar 100 μm). Values represent mean ± SEM; n = 3 to 5 mice per group. ^∗p < 0.05 by unpaired t-test. (c) Western blot analysis for the detection of nitrated proteins (right panels show representative images) in the diaphragm muscle of NSE/IL-6 and WT mice. Values represent mean ± SEM; n = 6 mice per group. ^∗∗p < 0.005 by unpaired t-test. Lanes were run on the same gel but were not contiguous. Original images are shown in . (d) Real-time PCR analysis of the expression of miR-1 performed on diaphragm muscle samples from NSE/IL-6 and WT mice at 24 weeks of age. Values represent mean ± SEM; n = 3 to 5 mice per genotype. ^∗p < 0.05 by unpaired t-test. DHE: dihydroethidium; G6PD: glucose 6-phosphate dehydrogenase.

Figure 2

Nrf2-dependent antioxidant genes are differentially modulated in NSE/IL-6 diaphragm muscles. Real-time PCR analysis of the expression of Nrf2 (a), SIRT1 (b), and Nrf2-dependent genes: SOD1 (c), SOD2 (d), Txnrd2 (e), CAT-1 (f), NQO1 (g), and GCL (h). Gene expression analysis was performed on diaphragm muscle samples deriving from wild-type (WT) and NSE/IL-6 mice at 24 weeks of age. Values represent mean ± SEM; n = at least 4 mice per genotype. ^∗p < 0.05 by unpaired t-test. Nrf2: nuclear factor erythroid 2-related factor 2; SIRT1: sirtuin 1; SOD: superoxide dismutase; Txnrd2: thioredoxin reductase 2; CAT-1: catalase-1; NQO1: NAD(P)H quinone dehydrogenase 1; GCL: glutamate-cysteine ligase.

Figure 3

Morphofunctional analysis of NSE/IL-6 and wild-type muscles at 24 weeks of age. (a) Representative image of Haematoxylin and Eosin staining of transverse sections of diaphragm from 24-week-old wild-type (WT) and NSE/IL-6 mice. Scale bar, 100 μm. (b) Frequency distribution of myofiber cross-sectional area (CSA) in transgenic (NSE/IL-6) and wild-type (WT) diaphragm muscle. Data are represented as medians; n = 4; ^∗∗∗∗p < 0.0001. Real-time PCR analysis for the expression of Atrogin1 (c), MURF-1 (d), CTSL (e), and LC3 (f). Values represent mean ± SEM; n ≥ 5 mice per genotype. ^∗p < 0.05 by unpaired t-test. (g) Haematoxylin and Eosin staining of transverse section of extensor digitorum longus (EDL) muscles from indicated genotypes. Scale bar, 100 μm. (h) Frequency distribution of myofiber cross-sectional area (CSA) in transgenic (NSE/IL-6) and wild-type (WT) EDL muscle. Data are represented as medians; n ≥ 3; ^∗∗∗∗p < 0.0001. Physiological properties of EDL muscles from 24-week-old wild-type (WT) and NSE/IL-6 mice: (i) Maximum force (mN) and (j) specific force (mN/mm²). Data are represented as mean ± SD of EDL muscle maximum force (i) and specific force (j); n > 31; ^∗∗p < 0.01. Atrogin1: MAFbx/Atrogin1; MURF-1: Muscle RING Finger-1; CTSL: cathepsin L; LC3: microtubule-associated protein 1 light chain 3.

Figure 4

A proposed model of the impact of elevated levels of circulating IL-6 on skeletal muscle redox balance. The reported scheme represents molecular circuits involved in the generation and neutralization of reactive species in skeletal muscle. Red lines indicate mechanisms that are potentially enhanced by elevated levels of circulating IL-6. Grey dot lines represent processes which might be impaired in NSE/IL-6 muscle. In the presence of nonphysiologic amounts of serum IL-6, NOX2 expression is enhanced in diaphragm muscle, inducing a sustained generation of superoxide (O₂⁻). The downmodulation of NQO1 in muscle tissue exposed to increased levels of serum IL-6 indicates that the NOX2-derived superoxide might be not efficiently neutralized. On the other hand, O₂⁻ is converted by SOD into hydrogen peroxide (H₂O₂), whilst its further detoxification can be impaired by the reduced expression of CAT. H₂O₂ can also be neutralized through the oxidation of glutathione. The reduced expression of the rate-limiting enzyme to produce glutathione (GSH), GCL, might reflect an impaired activity of the glutathione system. The excess of O₂⁻ can also interact with nitric oxide (NO) inducing protein modifications. Moreover, the altered regulation of the NO signalling pathway might induce a deregulated expression of glucose 6-phosphate dehydrogenase (G6PD), the enzyme responsible for the production of NADPH, further enhancing the activity of the NOX2 complex in a feed-forward circuit. NOX2: NADPH oxidase 2; NQO1: NAD(P)H quinone dehydrogenase 1; SOD: superoxide dismutase; CAT: catalase; GCL: glutamate-cysteine ligase; ONOO⁻: peroxynitrite; HDAC2: histone deacetylase 2; GSSG: oxidized glutathione.