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

Journal name:
Nature Medicine
Volume:
19,
Pages:
1489–1495
Year published:
DOI:
doi:10.1038/nm.3368
Received
Accepted
Published online

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.

At a glance

Figures

  1. Increased expression and release of CIRP after hemorrhage.
    Figure 1: Increased expression and release of CIRP after hemorrhage.

    (a) Western blot analysis of CIRP in the serum of healthy volunteers and individuals admitted to the surgical ICU (SICU) with shock. PS red, Ponceau S red staining. (b) Western blot analysis of CIRP in the tissues of rats at the indicated times after hemorrhage. n = 4–6 rats per time-point. *P < 0.05 compared to time 0 determined by one-way analysis of variance (ANOVA) and Student-Newman-Keuls test. (c) Quantitative PCR analysis of CIRP mRNA in the liver and heart of rats at 240 min after hemorrhage. n = 6 rats per group. *P < 0.05 compared to sham determined by Student's t test. (d) Western blot analysis of CIRP in the nuclear (N) and cytoplasmic (C) compartments of RAW 264.7 cells cultured under normoxic (NM) or hypoxic (1% O2) conditions 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, respectively). Antibodies to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and histone were used to detect the cytoplasm and nucleus, respectively. (e) Images of RAW 264.7 cells expressing GFP-CIRP (green) and Hoechst 33245 (blue) staining of nuclei. Scale bars, 25 μm. (f,g) Western blot analysis of CIRP in conditioned medium (f) or total cell lysates (g) from RAW 264.7 cells. n = 3 independent experiments. *P < 0.05 compared to NM determined by one-way ANOVA and Student-Newman-Keuls test. (h) Western blot analysis of CIRP in the nuclear and lysosomal (L) components of RAW 264.7 cells cultured in normoxia or exposed to hypoxia/reperfusion for 24 h (H/R24). Antibody to cathepsin D was used to detect lysosomes. The images in dh represent three independent experiments. The data in b, c and g are shown as the mean ± s.e.m. For the western blot images, the small gaps indicate skipped lanes from the same membrane, and large gaps indicate separate membranes.

  2. Recombinant CIRP induces cytokine release in macrophages.
    Figure 2: Recombinant CIRP induces cytokine release in macrophages.

    (a,b) TNF-α production in RAW 264.7 cells stimulated with increasing concentrations of rmCIRP for 4 h (a) or rmCIRP (100 ng ml−1) for up to 8 h (b). *P < 0.05 compared to no rmCIRP or time 0. (c) Western blot analysis of HMGB1 in conditioned medium from RAW 264.7 cells stimulated with increasing concentrations of rmCIRP for 20 h. For the images, small gaps indicate skipped lanes from the same membrane. (d) TNF-α production in RAW 264.7 cells stimulated with rmCIRP (1.5 μg ml−1) or LPS (10 ng ml−1) for 8 h with (+) or without (−) pretreatment of polymyxin B (PMB; 120 U ml−1) and heat (80 °C for 30 min). *P < 0.05 compared to rmCIRP, #P < 0.05 compared to LPS. (e) TNF-α production in differentiated human THP-1 cells stimulated with increasing concentrations of rhCIRP or rmCIRP for 4 h. *P < 0.05 compared to no CIRP. (f) TNF-α production in human PBMCs stimulated with increasing concentrations of rhCIRP for 8 h. *P < 0.05 compared to no rhCIRP. (g) TNF-α production in 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 of preincubation with antisera to CIRP (anti-CIRP; 4 μg ml−1), antisera to HMGB1 (anti-HMGB1; 4 μg ml−1) or rabbit control (nonimmunized) IgG (4 μg ml−1). *P < 0.05 compared to rmCIRP, #P < 0.05 compared to rmHMGB1. All data throughout the figure are shown as the mean ± s.e.m. n = 3 independent experiments in all panels except for g, in which n = 4. All statistical significance was determined by one-way ANOVA and Student-Newman-Keuls test.

  3. Blockade of CIRP reduces TNF-[alpha] and IL-6 production, hepatic injury and mortality after hemorrhage.
    Figure 3: Blockade of CIRP reduces TNF-α and IL-6 production, hepatic injury and mortality after hemorrhage.

    (a,b) TNF-α and IL-6 levels in the serum and liver, as well as the activity of the hepatic injury markers AST, ALT and liver myeloperoxidase (MPO), in rats at 4 h after hemorrhage. Hemorrhaged rats received rabbit control (nonimmunized) IgG or antisera to CIRP (10 mg per kg body weight) during fluid resuscitation. n = 6 rats per group. *P < 0.05 compared to sham, #P < 0.05 compared to hemorrhage alone. (c) Survival curves of hemorrhaged rats administered normal saline (n = 14), control IgG (10 mg per kg body weight, n = 13) or antisera to CIRP (10 mg per kg body weight, n = 13) for 3 consecutive days. *P < 0.05 compared to saline. (d) Survival curves of hemorrhaged wild-type (WT) and Cirbp−/− mice. n = 9 mice per group. *P < 0.05 compared to WT. (e,f) Serum TNF-α (e) and HMGB1 (f) levels in WT and Cirbp−/− mice at 4 h after hemorrhage. n = 6 mice per group, *P < 0.05 compared to WT sham, #P < 0.05 compared to WT hemorrhage. All data throughout the figure are shown as the mean ± s.e.m. Statistical significance was determined by one-way ANOVA and Student-Newman-Keuls test (a,b,e,f) or log-rank test (c,d).

  4. Role of CIRP in CLP sepsis.
    Figure 4: Role of CIRP in CLP sepsis.

    (ac) CIRP protein and mRNA expression in the serum and liver of rats at 20 h after CLP. Data are shown as the mean ± s.e.m. n = 4–6 rats per group. *P < 0.05 compared to sham determined by Student's t test. (d,e) CIRP mRNA and protein expression in total cell lysates or conditioned medium from rat peritoneal macrophages that were either not treated (Non) or were 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 lysates was determined after 24 h of LPS exposure. Data are shown as the mean ± s.e.m. n = 3 independent experiments. *P < 0.05 compared to the untreated group 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. The images represent three independent experiments. (g) Survival curves of rats after induction of polymicrobial sepsis that were administered rabbit control (nonimmunized) IgG (10 mg per kg body weight) or antisera to CIRP 5 h after CLP (10 mg per kg body weight). n = 18 rats per group. *P < 0.05 compared to control IgG determined by log-rank test. For the western blot images, small gaps indicate skipped lanes from the same membrane.

  5. The TLR4-MD2 complex mediates extracellular CIRP activity.
    Figure 5: The TLR4-MD2 complex mediates extracellular CIRP activity.

    (a) TNF-α production in peritoneal macrophages from WT, Ager−/−, Tlr2−/− and Tlr4−/− mice stimulated with rmCIRP (1.5 μg ml−1) for 4 h. Data are shown as the mean ± s.e.m. n = 3 independent experiments. *P < 0.05 compared to WT. (b,c) Serum levels of TNF-α, IL-6, HMGB1, AST and ALT in WT and Tlr4−/− mice at 4 h after administration of normal saline (rmCIRP 0) or rmCIRP (1 or 5 mg per kg body weight). Data are shown as the mean ± s.e.m. n = 6–9 mice per group. *P < 0.05 compared to WT and no rmCIRP, #P < 0.05 compared to WT with rmCIRP (5 mg per kg body weight). (d) Binding affinities (Kd) of three oligopeptides derived from the human CIRP sequence to rhMD2. aa, amino acid. Representative sensorgrams of the oligopeptide analysis from two independent experiments are shown in Supplementary Figure 6. All statistical significance was determined by one-way ANOVA and Student-Newman-Keuls test.

Accession codes

Referenced accessions

NCBI Reference Sequence

References

  1. Holcomb, J.B. et al. Challenges to effective research in acute trauma resuscitation: consent and endpoints. Shock 35, 107113 (2011).
  2. Bulger, E.M. et al. Hypertonic resuscitation of hypovolemic shock after blunt trauma: a randomized controlled trial. Arch. Surg. 143, 139148 (2008).
  3. Kaczorowski, D.J., Mollen, K.P., Edmonds, R. & Billiar, T.R. Early events in the recognition of danger signals after tissue injury. J. Leukoc. Biol. 83, 546552 (2008).
  4. Dombrovskiy, V.Y., Martin, A.A., Sunderram, J. & Paz, H.L. Rapid increase in hospitalization and mortality rates for severe sepsis in the United States: a trend analysis from 1993 to 2003. Crit. Care Med. 35, 12441250 (2007).
  5. Ward, P.A. New approaches to the study of sepsis. EMBO Mol. Med. 4, 12341243 (2012).
  6. Medzhitov, R. Origin and physiological roles of inflammation. Nature 454, 428435 (2008).
  7. Oppenheim, J.J. & Yang, D. Alarmins: chemotactic activators of immune responses. Curr. Opin. Immunol. 17, 359365 (2005).
  8. Zhang, X. & Mosser, D.M. Macrophage activation by endogenous danger signals. J. Pathol. 214, 161178 (2008).
  9. Beutler, B.A. TLRs and innate immunity. Blood 113, 13991407 (2009).
  10. Ward, P.A. The sepsis seesaw: seeking a heart salve. Nat. Med. 15, 497498 (2009).
  11. Kawai, T. & Akira, S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11, 373384 (2010).
  12. Chen, G.Y., Tang, J., Zheng, P. & Liu, Y. CD24 and Siglec-10 selectively repress tissue damage–induced immune responses. Science 323, 17221725 (2009).
  13. Wang, H. et al. HMG-1 as a late mediator of endotoxin lethality in mice. Science 285, 248251 (1999).
  14. Tsung, A. et al. The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion. J. Exp. Med. 201, 11351143 (2005).
  15. Quintana, F.J. & Cohen, I.R. Heat shock proteins as endogenous adjuvants in sterile and septic inflammation. J. Immunol. 175, 27772782 (2005).
  16. Martinon, F., Pétrilli, V., Mayor, A., Tardivel, A. & Tschopp, J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440, 237241 (2006).
  17. Vogl, T. et al. Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock. Nat. Med. 13, 10421049 (2007).
  18. Xu, J. et al. Extracellular histones are major mediators of death in sepsis. Nat. Med. 15, 13181321 (2009).
  19. Zhang, Q. et al. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 464, 104107 (2010).
  20. Nishiyama, H. et al. Cloning and characterization of human CIRP (cold-inducible RNA-binding protein) cDNA and chromosomal assignment of the gene. Gene 204, 115120 (1997).
  21. Nishiyama, H. et al. A glycine-rich RNA-binding protein mediating cold-inducible suppression of mammalian cell growth. J. Cell Biol. 137, 899908 (1997).
  22. Xue, J.H. et al. Effects of ischemia and H2O2 on the cold stress protein CIRP expression in rat neuronal cells. Free Radic. Biol. Med. 27, 12381244 (1999).
  23. Nishiyama, H. et al. Decreased expression of cold-inducible RNA-binding protein (CIRP) in male germ cells at elevated temperature. Am. J. Pathol. 152, 289296 (1998).
  24. Nishiyama, H. et al. Diurnal change of the cold-inducible RNA-binding protein (Cirp) expression in mouse brain. Biochem. Biophys. Res. Commun. 245, 534538 (1998).
  25. Sheikh, M.S. et al. Identification of several human homologs of hamster DNA damage–inducible transcripts. Cloning and characterization of a novel UV-inducible cDNA that codes for a putative RNA-binding protein. J. Biol. Chem. 272, 2672026726 (1997).
  26. Wellmann, S. et al. Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1–independent mechanism. J. Cell Sci. 117, 17851794 (2004).
  27. Qu, Y. & Dubyak, G.R. P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways. Purinergic Signal. 5, 163173 (2009).
  28. Aida, Y. & Pabst, M.J. Removal of endotoxin from protein solutions by phase separation using Triton X-114. J. Immunol. Methods 132, 191195 (1990).
  29. Wang, Y. et al. Identification of stimulating and inhibitory epitopes within the heat shock protein 70 molecule that modulate cytokine production and maturation of dendritic cells. J. Immunol. 174, 33063316 (2005).
  30. Henderson, B. et al. Caught with their PAMPs down? The extracellular signalling actions of molecular chaperones are not due to microbial contaminants. Cell Stress Chaperones 15, 123141 (2010).
  31. Andersson, U. et al. High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J. Exp. Med. 192, 565570 (2000).
  32. Yang, H. et al. Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc. Natl. Acad. Sci. USA 101, 296301 (2004).
  33. Yang, S., Zhou, M., Chaudry, I.H. & Wang, P. Novel approach to prevent the transition from the hyperdynamic phase to the hypodynamic phase of sepsis: role of adrenomedullin and adrenomedullin binding protein-1. Ann. Surg. 236, 625633 (2002).
  34. Nagai, Y. et al. Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat. Immunol. 3, 667672 (2002).
  35. Hyakushima, N. et al. Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling. J. Immunol. 173, 69496954 (2004).
  36. Yang, C. & Carrier, F. The UV-inducible RNA-binding protein A18 (A18 hnRNP) plays a protective role in the genotoxic stress response. J. Biol. Chem. 276, 4727747284 (2001).
  37. Cammas, A., Lewis, S.M., Vagner, S. & Holcik, M. Post-transcriptional control of gene expression through subcellular relocalization of mRNA binding proteins. Biochem. Pharmacol. 76, 13951403 (2008).
  38. De Leeuw, F. et al. The cold-inducible RNA-binding protein migrates from the nucleus to cytoplasmic stress granules by a methylation-dependent mechanism and acts as a translational repressor. Exp. Cell Res. 313, 41304144 (2007).
  39. Yang, R. et al. Functional significance for a heterogenous ribonucleoprotein A18 signature RNA motif in the 3′-untranslated region of ataxia telangiectasia mutated and Rad3-related (ATR) transcript. J. Biol. Chem. 285, 88878893 (2010).
  40. Qu, Y., Franchi, L., Nunez, G. & Dubyak, G.R. Nonclassical IL-1β secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages. J. Immunol. 179, 19131925 (2007).
  41. Benhamou, Y. et al. Toll-like receptors 4 contribute to endothelial injury and inflammation in hemorrhagic shock in mice. Crit. Care Med. 37, 17241728 (2009).
  42. Wittebole, X., Castanares-Zapatero, D. & Laterre, P.F. Toll-like receptor 4 modulation as a strategy to treat sepsis. Mediators Inflamm. 2010, 568396 (2010).
  43. Park, J.S. et al. Involvement of toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 protein. J. Biol. Chem. 279, 73707377 (2004).
  44. Ohashi, K., Burkart, V., Flohé, S. & Kolb, H. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J. Immunol. 164, 558561 (2000).
  45. Termeer, C. et al. Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J. Exp. Med. 195, 99111 (2002).
  46. Okamura, Y. et al. The extra domain A of fibronectin activates Toll-like receptor 4. J. Biol. Chem. 276, 1022910233 (2001).
  47. Yang, H. et al. A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release. Proc. Natl. Acad. Sci. USA 107, 1194211947 (2010).
  48. Yang, H., Antoine, D.J., Andersson, U. & Tracey, K.J. The many faces of HMGB1: molecular structure-functional activity in inflammation, apoptosis, and chemotaxis. J. Leukoc. Biol. 93, 865873 (2013).
  49. Shin, H.J. et al. Kinetics of binding of LPS to recombinant CD14, TLR4, and MD-2 proteins. Mol. Cells 24, 119124 (2007).
  50. Scaffidi, P., Misteli, T. & Bianchi, M.E. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418, 191195 (2002).
  51. Brochu, C. et al. NF-κB–dependent role for cold-inducible RNA binding protein in regulating interleukin 1β. PLoS ONE 8, e57426 (2013).
  52. Sakurai, T. et al. Cirp protects against tumor necrosis factor-α–induced apoptosis via activation of extracellular signal-regulated kinase. Biochim. Biophys. Acta 1763, 290295 (2006).
  53. Wu, R. et al. Mechanisms responsible for vascular hyporesponsiveness to adrenomedullin after hemorrhage: the central role of adrenomedullin binding protein-1. Ann. Surg. 242, 115123 (2005).
  54. Miksa, M. et al. Immature dendritic cell–derived exosomes rescue septic animals via milk fat globule epidermal growth factor-factor VIII. J. Immunol. 183, 59835990 (2009).

Download references

Author information

  1. These authors contributed equally to this work.

    • Xiaoling Qiang &
    • Weng-Lang Yang

Affiliations

  1. Center for Translational Research, The Feinstein Institute for Medical Research, Manhasset, New York, USA.

    • Xiaoling Qiang,
    • Weng-Lang Yang,
    • Rongqian Wu,
    • Mian Zhou,
    • Asha Jacob,
    • Weifeng Dong,
    • Michael Kuncewitch,
    • Youxin Ji,
    • Jeffrey Nicastro,
    • Gene F Coppa &
    • Ping Wang
  2. Center for Biomedical Science, The Feinstein Institute for Medical Research, Manhasset, New York, USA.

    • Huan Yang,
    • Haichao Wang &
    • Kevin J Tracey
  3. Department of Clinical Molecular Biology, Kyoto University, Kyoto, Japan.

    • Jun Fujita

Contributions

X.Q. and M.Z. performed the experiments and analyzed data. W.-L.Y. conducted the translocation study, designed and coordinated SPR analysis and wrote the manuscript. R.W. designed the experiments. A.J. assisted with the design of experiments and participated in manuscript editing. W.D. and Y.J. performed animal studies. M.K. collected the serum from patients admitted to the surgical ICU and analyzed human data. J.N. and G.F.C. analyzed animal studies. H.Y. and K.J.T. assisted in the knockout mice study and SPR analysis. J.F. assisted in the CIRP knockout mice and GFP-CIRP study and revised the manuscript. H.W. assisted with the design of the study and analyzed data. P.W. designed and supervised the study and revised the manuscript.

Competing financial interests

P.W. is an inventor of the pending PCT application WO 2010/120726 A1 entitled “Treatment of inflammatory diseases by inhibiting cold-inducible RNA-binding protein (CIRP).” This patent application covers the fundamental concept of using CIRP inhibitors for the treatment of inflammatory diseases.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (599 KB)

    Supplementary Figures 1-6 and Supplementary Table 1

Additional data