Cold-inducible RNA-binding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis

Abstract

A systemic inflammatory response is observed in patients undergoing hemorrhagic shock and sepsis. Here we report increased levels of cold-inducible RNA-binding protein (CIRP) in the blood of individuals admitted to the surgical intensive care unit with hemorrhagic shock. In animal models of hemorrhage and sepsis, CIRP is upregulated in the heart and liver and released into the circulation. In macrophages under hypoxic stress, CIRP translocates from the nucleus to the cytosol and is released. Recombinant CIRP stimulates the release of tumor necrosis factor-α (TNF-α) and HMGB1 from macrophages and induces inflammatory responses and causes tissue injury when injected in vivo. Hemorrhage-induced TNF-α and HMGB1 release and lethality were reduced in CIRP-deficient mice. Blockade of CIRP using antisera to CIRP attenuated inflammatory cytokine release and mortality after hemorrhage and sepsis. The activity of extracellular CIRP is mediated through the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex. Surface plasmon resonance analysis indicated that CIRP binds to the TLR4-MD2 complex, as well as to TLR4 and MD2 individually. In particular, human CIRP amino acid residues 106-125 bind to MD2 with high affinity. Thus, CIRP is a damage-associated molecular pattern molecule that promotes inflammatory responses in shock and sepsis.

Figure 1

Expression and release of CIRP after hemorrhage. (a) Western blot analysis of CIRP in the serum of healthy volunteers and surgical intense care unit (SICU) individuals with shock. (b) Western blot analysis of CIRP in the tissues of rats at the indicated times post-hemorrhage. n = 4–6 per time-point, *P < 0.05 vs. time 0, determined by one way ANOVA and Student-Newman-Keuls test. (c) qPCR analysis of CIRP mRNA in the liver and heart of rats at 240 min post-hemorrhage. n = 6 per group, *P < 0.05 vs. sham, determined by Student's t-test. (d) Western blot analysis of CIRP in the nuclear (N) and cytoplasmic (C) components of RAW 264.7 cells cultured in normoxia (NM) or exposed to hypoxia (1% O₂) for 20 h, followed by reoxygenation for 0, 2, 4, 7, or 24 h (H/R0, H/R2, H/R4, H/R7, or H/R24). Antibody to GAPDH and histone for detecting the cytoplasm and nucleus, respectively. (e) Images of RAW 264.7 cells expressing GFP-CIRP (green) and Hoechst 33245 (blue) staining of nuclei. Scale bar, 25 μm. (f) Western blot analysis of CIRP in the conditioned medium or (g) total cell lysate from RAW 264.7 cells. n = 3 per group, *P < 0.05 vs. NM, determined by one way ANOVA and Student-Newman-Keuls test. (h) Western blot analysis of CIRP in the nuclear (N) and lysosomal (L) components of RAW 264.7 cells cultured in normoxia or exposed to hypoxia (H/24R). Antibody to cathepsin D for detecting the lysosomes. Images represent three independent experiments (d–h). Data are mean ± s.e.m. (b,c,g). PS red, Ponceau S red staining. For Western blot images, small gap indicates skip lanes from the same membrane; large gap indicates separate membranes.

Figure 2

Recombinant CIRP induces cytokine release in macrophages. (a,b) TNF-α production of RAW 264.7 cells stimulated with various concentrations of rmCIRP for 4 h or rmCIRP (100 ng ml⁻¹) for various time periods. *P < 0.05 vs. no rmCIRP or time 0. (c) Western blot analysis of HMGB1 in the conditioned medium from RAW 264.7 cells stimulated with various concentrations of rmCIRP for 20 h. For the images, small gap indicates skip lanes from the same membrane. (d) TNF-α production of RAW 264.7 cells stimulated with rmCIRP (1.5 μg ml⁻¹) or LPS (10 ng ml⁻¹) for 8 h with (+) or without (–) polymyxin B (PMB; 120 U ml⁻¹) and heat (at 80 °C for 30 min). *P < 0.05 vs. rmCIRP; ^#P < 0.05 vs. LPS. (e) TNF-α production of differentiated human THP-1 cells stimulated with rhCIRP or rmCIRP at various concentrations for 4 h. *P < 0.05 vs. no CIRP. (f) TNF-α production of human PBMC stimulated with various concentrations of rhCIRP for 8 h. *P < 0.05 vs. no rhCIRP. (g) TNF-α production of THP-1 cells stimulated with rmCIRP (0.3 μg ml⁻¹), rmHMGB1 (0.3 μg ml⁻¹), or rmCIRP plus rmHMGB1 for 8 h, with (+) or without (–) 1 h pre-incubation of antisera to CIRP (Anti-CIRP; 4 μg ml⁻¹), antisera to HMGB1 (Anti-HMGB1; 4 μg ml⁻¹), and rabbit control (non-immunized) IgG (4 μg ml⁻¹). *P < 0.05 vs. rmCIRP; ^#P < 0.05 vs. rmHMGB1. All data are mean ± s.e.m. from three or four independent experiments. Statistical significance was determined by one way ANOVA and Student-Newman-Keuls test.

Figure 3

Attenuation of cytokine production and hepatic injury by antisera to CIRP after hemorrhage. (a,b) The levels of TNF-α and IL-6 in the serum and liver as well as hepatic injury markers AST, ALT, and liver myeloperoxidase (MPO) activity of rats at 4 h post-hemorrhage. Hemorrhaged rats received rabbit control (non-immunized) IgG or antisera to CIRP (Anti-CIRP; 10 mg kg⁻¹ body weight) during fluid resuscitation. n = 6 per group, *P < 0.05 vs. sham; ^#P < 0.05 vs. hemorrhage alone. (c) Survival curves of hemorrhaged rats administered normal saline (vehicle; n = 14), control IgG (10 mg kg⁻¹ body weight, n = 13), or antisera to CIRP (Anti-CIRP; 10 mg kg⁻¹ body weight, n = 13) for three consecutive days. *P < 0.05 vs. saline. (d) Survival curves of hemorrhaged wild-type (WT) and Cirp^–/– mice. n = 9 per group, *P < 0.05 vs. WT. (e,f) Serum TNF-α and HMGB1 levels of WT and Cirp^–/– mice at 4 h post-hemorrhage. n = 6 per group, *P < 0.05 vs. WT sham; ^#P < 0.05 vs. WT hemorrhage. Data are mean ± s.e.m. Statistical significance was determined by one way ANOVA and Student-Newman-Keuls test (a,b,e,f). Statistical significance in survival was compared by the log-rank test (c,d).

Figure 4

Expression and release of CIRP after sepsis. (a–c) CIRP protein and mRNA expression in the serum and liver of rats at 20 h post-CLP. Data are mean ± s.e.m. n = 4–6 per group, *P < 0.05 vs. sham, determined by Student's t-test. (d,e) CIRP mRNA and protein expression in total cell lysate or the conditioned medium from rat peritoneal macrophages either non-treated (Non) or exposed to LPS (10 ng ml⁻¹). CIRP mRNA and protein levels in the conditioned medium were determined after 6 h of LPS exposure. CIRP protein in total cell lysate was determined after 24 h of LPS exposure. Data are mean ± s.e.m. from three independent experiments. *P < 0.05 vs. Non, determined by Student's t-test. (f) Western blot analysis of CIRP in the conditioned medium of RAW 264.7 cells stimulated with (+) or without (–) rmHMGB1 (1 μg ml⁻¹), rmTNF-α (30 ng ml⁻¹) and LPS (100 ng ml⁻¹) for 24 h. Images represent three independent experiments. (g) Survival curves of septic rats administered with rabbit control (non-immunized) IgG (10 mg kg⁻¹ body weight) or antisera to CIRP (Anti-CIRP; 10 mg kg⁻¹ body weight). n = 18 per group, *P < 0.05 vs. control IgG, determined by the log-rank test. PS red, Ponceau S red staining. For Western blot images, small gap indicates skip lanes from the same membrane.

Figure 5

TLR4/MD2 complex mediates extracellular CIRP activity. (a) TNF-α production of peritoneal macrophages from wild-type (WT), Rage^–/–, Tlr2^–/–, or Tlr4^–/– mice stimulated with rmCIRP (1.5 μg ml⁻¹) for 4 h. Data are mean ± s.e.m. from three independent experiments. *P < 0.05 vs. WT. (b,c) Serum levels of TNF-α, IL-6, HMGB1, AST, and ALT in WT and Tlr4^–/– mice at 4 h after administering normal saline (vehicle) or rmCIRP (1 or 5 mg kg⁻¹ body weight). Data are mean ± s.e.m. n = 6–9 per group, *P < 0.05 vs. WT no rmCIRP; ^#P < 0.05 vs. WT with rmCIRP (5 mg kg⁻¹). (d) Binding affinity (K_D) of three oligopeptides derived from human CIRP sequence to rhMD2. Representative sensorgrams of the oligopeptide analysis are shown in Supplementary Fig. 6 from two independent experiments. Statistical significance was determined by one way ANOVA and Student-Newman-Keuls test.