Cold-inducible RNA-binding protein mediates neuroinflammation in cerebral ischemia

Abstract

Background: Neuroinflammation is a key cascade after cerebral ischemia. Excessive production of proinflammatory mediators in ischemia exacerbates brain injury. Cold-inducible RNA-binding protein (CIRP) is a newly discovered proinflammatory mediator that can be released into the circulation during hemorrhage or septic shock. Here, we examine the involvement of CIRP in brain injury during ischemic stroke.

Methods: Stroke was induced by middle cerebral artery occlusion (MCAO). In vitro hypoxia was conducted in a hypoxia chamber containing 1% oxygen. CIRP and tumor necrosis factor-α (TNF-α) levels were assessed by RT-PCR and Western blot analysis.

Results: CIRP is elevated along with an upregulation of TNF-α expression in mouse brain after MCAO. In CIRP-deficient mice, the brain infarct volume, induction of TNF-α, and activation of microglia are markedly reduced after MCAO. Using microglial BV2 cells, we demonstrate that hypoxia induces the expression, translocation, and release of CIRP, which is associated with an increase of TNF-α levels. Addition of recombinant murine (rm) CIRP directly induces TNF-α release from BV2 cells and such induction is inhibited by neutralizing antisera to CIRP. Moreover, rmCIRP activates the NF-κB signaling pathway in BV2 cells. The conditioned medium from BV2 cells exposed to hypoxia triggers the apoptotic cascade by increasing caspase activity and decreasing Bcl-2 expression in neural SH-SY5Y cells, which is inhibited by antisera to CIRP.

Conclusion: Extracellular CIRP is a detrimental factor in stimulating inflammation to cause neuronal damage in cerebral ischemia.

General significance: Development of an anti-CIRP therapy may benefit patients with brain ischemia.

Keywords: Cerebral ischemia; Cold shock protein; Hypoxia; Inflammation; Microglia; Neural cell.

Fig. 1

Induction of CIRP and TNF-α in the mouse brain after MCAO. The brain tissues from wild type mice were harvested at 30 h and 48 h after MCAO. Real-time PCR analysis of gene expression of (A) CIRP and (C) TNF-α. Western blotting measurement of (B) CIRP and (D) TNF-α from lysate of ischemic portion of the brain. Representative blots of CIRP (19 kDa), TNF-α (26 kDa) and β-Actin (43 kDa) are shown. Data are expressed as means ± SE (n = 4/group) and compared by one-way ANOVA and SNK test. *P < 0.05 vs. sham; Sham values were normalized as 1.0.

Fig. 2

Effect of CIRP deficiency on infarct volume after MCAO. The brain tissues from wild type and Cirbp^−/− mice were harvested at 30 h after MCAO. Serial coronal slices of fresh brain tissue were stained with triphenyl tetrazolium chloride. Infarcted area of the brain appears as pale staining on the slice and viable brain area shows plum red color. The representative images are shown. The infarcted area was quantified using NIH image J and infarct volume was calculated. Data are expressed by mean ± SE (n = 6–8/group) and compared by Student’s t-test. *P < 0.05 vs. wild type.

Fig. 3

Effect of CIRP deficiency on activation of microglia after MCAO. The brain tissues from wild type and Cirbp^−/− mice were harvested at 30 h after MCAO. Brain sections were immunostained with allograft inflammatory factor 1 (AIF1), a microglia activation marker (in brown). Resting microglia indicated with white arrows and activated microglia indicated with black arrows. Representative microphotographs of sham and MCAO brain sections are shown. Original magnification, 400×.

Fig. 4

Induction of CIRP production and translocation in microglia after exposure to hypoxia. Microglial BV2 cells were cultured under normoxia or hypoxia for 20 h or 30 h. (A) Real-time PCR analysis of CIRP gene expression from total cell lysate. Western blotting of CIRP from (B) total cell lysate and (C) cultured conditioned medium. Representative blots of CIRP (19 kDa) and β-Actin (43 kDa) are shown. Data are expressed as means ± SE (n = 4–6/group) and compared by one-way ANOVA and SNK test. *P < 0.05 vs. normoxia; ^#P < 0.05 vs. hypoxia at 20 h. Normoxia values were normalized as 1.0. (D) Images of BV2 cells expressing GFP-CIRP (green) after exposure to normoxia or hypoxia for 24 h. The cells were viewed under fluorescence and light microscope. Original magnification, 400×.

Fig. 5

Induction of TNF-α production in microglia after exposure to hypoxia. Microglial BV2 cells were cultured in normoxia or hypoxia for 20 h or 30 h. (A) Real-time PCR analysis of TNF-α gene expression from total cell lysate. (B) Western blotting of TNF-α from total cell lysate. Representative blots of TNF-α (26 kDa) and β-Actin (43 kDa) are shown. (C) The levels of TNF-α in cultured conditioned medium as measured by ELISA. Data are expressed as means ± SE (n = 4–6/group) and compared by one-way ANOVA and SNK test. *P < 0.05 vs. normoxia; ^#P < 0.05 vs. hypoxia at 20 h. Normoxia values were normalized as 1.0 in A and B.

Fig. 6

Effect of CIRP on TNF-α release and NF-κB activation in microglia. (A) TNF-α levels in the cultured medium from BV2 cells incubated with rmCIRP (0, 0.1, or 1.0 μg/ml) or co-administered with neutralizing antisera to CIRP (Anti-CIRP, 10 μg/ml) for 20 h, were measured by ELISA. Data are expressed as means ± SE (n = 4) and compared by one-way ANOVA and SNK test. *P < 0.05 vs. no rmCIRP; ^#P < 0.05 vs. rmCIRP at 1.0 μg/ml alone. (B) The luciferase activity of BV2 cells transfected with a plasmid containing NF-κB binding elements and a luciferase as reporter gene, followed by incubation with rmCIRP (1 μg/ml) or LPS (40 ng/ml) for 20 h. Data are expressed as means ± SE (n = 3). ND, non-detectable; RLU, relative light unit.

Fig. 7

Effect of the conditioned medium from microglia exposed to hypoxia on neuron injury. The conditioned medium (CM) from BV2 cells exposed to normoxia or hypoxia in the presence of antisera to CIRP (Anti-CIRP, 10 μg/ml) or rabbit control IgG (10 μg/ml) for 30 h were added to differentiated SH-SY5Y cells for another 30 h. (A) Caspase activity in SH-SY5Y cells as determined by a fluorimetric assay kit. (B) Western blotting of Bcl-2 protein in SH-SY5Y cells. Representative blots of Bcl-2 (26 kDa) and β-Actin (43 kDa) are shown. (C) Caspase activity in differentiated SH-SY5Y cells incubated with rmCIRP (1 μg/ml) or rmTNF-α (5 ng/ml) for 30 h. Data are expressed as means ± SE (n = 4) and compared by one-way ANOVA and SNK test. *P < 0.05 vs. normoxia or untreated; ^#P < 0.05 vs. hypoxia; ⁺P < 0.05 vs. hypoxia plus Anti-CIRP. Normoxia or untreated values were normalized as 1.0.