Targeting CIRP and IL-6R-mediated microglial inflammation to improve outcomes in intracerebral hemorrhage

This study illustrates that targeting Cold-inducible RNA-binding protein (CIRP) and IL-6 receptor can significantly attenuate neuroinflammation and enhance recovery following ICH. ① Intracerebral hemorrhage (ICH) occurs, ② leading to neuronal injury. ③ The damaged neurons release cold-inducible RNA-binding protein (CIRP), ④ which stimulates the activation of microglia. ⑤ CIRP can also bind to interleukin-6 receptor (IL-6R) on the microglial cell membrane, ⑥–⑦ resulting in the phosphorylation of STAT3 within the microglial nucleus. ⑧ This interaction promotes the polarization of microglia toward a pro-inflammatory phenotype, ⑨ thereby amplifying the microglial inflammatory response. ⑩ The excessive inflammation, in turn, exacerbates neuronal damage, forming a vicious cycle. It demonstrates that the CIRP-targeting peptide Tat-CIRP CMA (TCC) effectively reduces inflammatory responses by inhibiting the IL-6R/JAK/STAT3 pathway, modulating microglial polarization, and enhancing neuronal protection. • Single-cell RNA-seq identified key inflammatory genes in microglia after ICH. • CIRP promotes neuroinflammation after ICH via microglial IL-6R/STAT3 signaling. • Targeting CIRP-IL-6R interaction reduced CIRP release and microglial activation. • IL-6Rα deletion in microglia replicated anti-inflammatory and protective effects. • CIRP levels in blood were positively correlated with infarct volume in ICH patients. Cold-inducible RNA-binding protein (CIRP) is an emerging inflammatory mediator implicated in neuronal injury following intracerebral hemorrhage (ICH). However, the underlying mechanisms by which CIRP contributes to neuroinflammation remain unclear. To investigate the role of neuron-derived CIRP in activating microglial inflammation via IL-6 receptor (IL-6R) signaling after ICH and to evaluate the therapeutic potential of targeting CIRP and IL-6R using a designed peptide inhibitor. Single-cell RNA sequencing was performed to identify key genes and pathways involved in microglial responses post-ICH. A peptide inhibitor, Tat-CIRP-CMA (TCC), was designed to target CIRP and IL-6R and tested in both in vitro (oxygen-glucose deprivation model) and in vivo (rat ICH model) systems. The effects of TCC on neuronal CIRP release, microglial activation, inflammation, and phagocytic function were assessed. A microglia-specific IL-6Rα knockout mouse model was used to further validate the functional relevance of the IL-6R/ signal transducer and activator of transcription 3 (STAT3) pathway. Clinical analysis of human ICH patients evaluated the correlation between peripheral CIRP expression and infarct volume. Neuron-derived CIRP activates microglia through IL-6R and downstream STAT3 signaling, driving pro-inflammatory responses in ICH. TCC treatment significantly reduced neuronal CIRP release, suppressed microglial activation, and decreased inflammatory cytokine levels. TCC inhibited the IL-6R /STAT3 pathway and enhanced microglial phagocytosis of red blood cells. Microglia-specific IL-6Rα deletion mirrored the anti-inflammatory and neuroprotective effects observed with TCC, reducing hematoma volume and improving sensory and behavioral outcomes. In human ICH patients, elevated peripheral CIRP expression positively correlated with infarct volume, supporting its value as a biomarker for ICH severity and prognosis. CIRP plays a pivotal role in post-ICH neuroinflammation by acting on IL-6R in microglia. The TCC peptide inhibitor effectively reduces CIRP-IL-6R mediated inflammation and supports neuroprotection. CIRP represents a promising therapeutic target and biomarker for ICH.