CIRP increases ICAM-1+ phenotype of neutrophils exhibiting elevated iNOS and NETs in sepsis

Abstract

Sepsis represents uncontrolled inflammation due to an infection. Cold-inducible RNA-binding protein (CIRP) is a stress-induced damage-associated molecular pattern (DAMP). A subset of neutrophils expressing ICAM-1 neutrophils was previously shown to produce high levels of reactive oxygen species. The role of CIRP for the development and function of ICAM-1 neutrophils during sepsis is unknown. We hypothesize that CIRP induces ICAM-1 expression in neutrophils causing injury to the lungs during sepsis. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis, we found increased expression of CIRP and higher frequencies and numbers of ICAM-1 neutrophils in the lungs. Conversely, the CIRP mice showed significant inhibition in the frequencies and numbers of ICAM-1 neutrophils in the lungs compared to wild-type (WT) mice in sepsis. In vitro treatment of bone marrow-derived neutrophils (BMDN) with recombinant murine CIRP (rmCIRP) significantly increased ICAM-1 phenotype in a time- and dose-dependent manner. The effect of rmCIRP on increasing frequencies of ICAM-1 neutrophils was significantly attenuated in BMDN treated with anti-TLR4 Ab or NF-κB inhibitor compared, respectively, with BMDN treated with isotype IgG or DMSO. The frequencies of iNOS producing and neutrophil extracellular traps (NETs) forming phenotypes in rmCIRP-treated ICAM-1 BMDN were significantly higher than those in ICAM-1 BMDN. Following sepsis the ICAM-1 neutrophils in the lungs showed significantly higher levels of iNOS and NETs compared to ICAM-1 neutrophils. We further revealed that ICAM-1 and NETs were co-localized in the neutrophils treated with rmCIRP. CIRP mice showed significant improvement in their survival outcome (78% survival) over that of WT mice (48% survival) in sepsis. Thus, CIRP could be a novel therapeutic target for regulating iNOS producing and NETs forming ICAM-1 neutrophils in the lungs during sepsis.

Extracellular CIRP (eCIRP) and inflammation

“…In addition to homologues and orthologues, CIRP has at least two paralogues: RNA‐binding motif protein 3 (RBM3)—an RNA‐binding protein up‐regulated in response to low temperature and aggrecan promoter‐binding protein‐1 (APBP‐1)—a 19‐kDa DNA/RNA‐binding protein . CIRP is constitutively and ubiquitously expressed at low levels, with studies showing its presence in the brain, lungs, heart, liver, kidneys, and endometrium, as well as in macrophages and neutrophils . Recently, both CIRP expression and release have been shown to be induced in cellular stress and inflammatory conditions .…”

“…eCIRP promotes inflammation by binding to the TLR4‐MD2 receptor complex, leading to the activation and nuclear translocation of the transcription factor NF‐κB . Accordingly, CIRP −/− mice are protected and exhibit attenuated inflammation and organ injury in preclinical models of inflammatory diseases . These observations suggested that targeting eCIRP could be an effective therapeutic strategy for the treatment of inflammatory diseases.…”

“…eCIRP then acts as a DAMP increasing macrophage production of proinflammatory cytokines . Since its original discovery as a DAMP, eCIRP's emerging proinflammatory role has been progressively extended to include additional target cells, such as neutrophils, lymphocytes, epithelial cells, and endothelial cells . eCIRP promotes inflammation by binding to the TLR4‐MD2 receptor complex, leading to the activation and nuclear translocation of the transcription factor NF‐κB .…”

“…Since its original discovery as a DAMP, eCIRP's emerging proinflammatory role has been progressively extended to include additional target cells, such as neutrophils, lymphocytes, epithelial cells, and endothelial cells . eCIRP promotes inflammation by binding to the TLR4‐MD2 receptor complex, leading to the activation and nuclear translocation of the transcription factor NF‐κB . Accordingly, CIRP −/− mice are protected and exhibit attenuated inflammation and organ injury in preclinical models of inflammatory diseases .…”

Extracellular CIRP as a Novel Endogenous TREM-1 Ligand to Fuel Inflammation

“…Similar to other DAMPs, eCIRP recognizes Toll-like receptor 4 (TLR4) 5,8 expressed in macrophages, lymphocytes, and neutrophils to increase cytokine production 5 , T cell polarization 9 , and neutrophil activation 10 . Healthy mice injected with recombinant murine (rm) CIRP developed acute lung injury (ALI) via activation of macrophages, neutrophils, and endothelial cells, and displayed increased vascular permeability in the lungs 11,12 . Conversely, CIRP -/mice were protected from sepsis and ALI 5,6 .…”