Short-term bed rest increases TLR4 and IL-6 expression in skeletal muscle of older adults

Abstract

Bed rest induces significant loss of leg lean mass in older adults. Systemic and tissue inflammation also accelerates skeletal muscle loss, but it is unknown whether inflammation is associated to inactivity-induced muscle atrophy in healthy older adults. We determined if short-term bed rest increases toll-like receptor 4 (TLR4) signaling and pro-inflammatory markers in older adult skeletal muscle biopsy samples. Six healthy, older adults underwent seven consecutive days of bed rest. Muscle biopsies (vastus lateralis) were taken after an overnight fast before and at the end of bed rest. Serum cytokine expression was measured before and during bed rest. TLR4 signaling and cytokine mRNAs associated with pro- and anti-inflammation and anabolism were measured in muscle biopsy samples using Western blot analysis and qPCR. Participants lost ∼4% leg lean mass with bed rest. We found that after bed rest, muscle levels of TLR4 protein expression and interleukin-6 (IL-6), nuclear factor-κB1, interleukin-10, and 15 mRNA expression were increased after bed rest (P < 0.05). Additionally, the cytokines interferon-γ, and macrophage inflammatory protein-1β, were elevated in serum samples following bed rest (P < 0.05). We conclude that short-term bed rest in older adults modestly increased some pro- and anti-inflammatory cytokines in muscle samples while systemic changes in pro-inflammatory cytokines were mostly absent. Upregulation of TLR4 protein content suggests that bed rest in older adults increases the capacity to mount an exaggerated, and perhaps unnecessary, inflammatory response in the presence of specific TLR4 ligands, e.g., during acute illness.

Keywords: aging; atrophy; cytokine; hospitalization; physical inactivity.

Fig. 1.

Effect of bed rest on toll-like receptor 4 (TLR4) and heat shock protein 60 (HSP60) protein expression. Data represent protein expression for TLR4 (A) and HSP60 (B) before (light gray bar) and after (dark gray bar) 7 days of bed rest in vastus lateralis skeletal muscle biopsy samples of healthy older adults (n = 6). Insets are representative immunoblot images. Equal protein loading was confirmed with α-tubulin. Line plots within figures represent individual means for males (M1–5) and female (F1). P values are indicated on figures. Values are in arbitrary units and presented as means ± SE.

Fig. 2.

Effect of bed rest on nuclear factor-κB (NF-κB) signaling. Data represent NF-κB phosphorylation (S536) relative to NF-κB total protein expression (A) and IκBα phosphorylation (S32) relative to IκBα total protein expression (B) before (light gray bars) and after (dark gray bars) 7 days of bed rest in vastus lateralis skeletal muscle of healthy older adults (n = 6). Insets are representative immunoblot images. Line plots below figures represent individual means for males (M1–5) and female (F1). P values are indicated on figures. Values are presented as means ± SE.

Fig. 3.

Effect of bed rest on pro-inflammatory muscle mRNA markers. Data represent NF-κB1 (A), IL-6 (B), tumor necrosis factor-α (TNF-α) (C), TNFR1SA1 (D), and IL-1β mRNA expression (E) before (light gray bars) and after (dark gray bars) 7 days of bed rest in vastus lateralis skeletal muscle of healthy older adults (n = 6). Line plots below figures represent individual means for males (M1–5) and female (F1). Values are reported as fold change from baseline. P values are indicated on figures. Values are presented as means ± SE.

Fig. 4.

Effect of bed rest on the mRNA expression of anti-inflammatory and anabolic cytokines. Data represent SOCS3 (A), IL-10 (B), and IL-15 mRNA expression (C) before (light gray bars) and after (dark gray bars) 7 days of bed rest in vastus lateralis skeletal muscle of healthy older adults (n = 6). Line plots below figures represent individual means for males (M1–5) and female (F1). Values are reported as fold change from baseline. P values are indicated on figures. Values are presented as means ± SE.

Fig. 5.

Relationship between the changes in leg lean mass and TLR4 and IL-6 expression. Data represent the delta change (pre- and post-bed rest) correlation for the following dependent variables in skeletal muscle of healthy older adults (n = 6): TLR4 protein expression vs. leg (right + left) lean mass loss (A) and IL-6 mRNA expression vs. leg (right + left) lean mass loss (B).