Abstract
Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern promoting inflammation and tissue injury. During bacterial or viral infection, macrophages release DNA decorated with nuclear and cytoplasmic proteins known as macrophage extracellular traps (METs). Gasdermin D (GSDMD) is a pore-forming protein that has been involved in extracellular trap formation in neutrophils. We hypothesized that eCIRP induces MET formation by activating GSDMD. Human monocytic cell line THP-1 cells were differentiated with phorbol 12-myristate 13-acetate (PMA) and treated with recombinant murine (rm) CIRP. The MET formation was detected by three methods: time-lapse fluorescence microscopy (video imaging), colorimetry, and ELISA. Cleaved forms of GSDMD, and caspase-1 were detected by Western blotting. Treatment of THP-1 cells with rmCIRP increased MET formation as revealed by SYTOX Orange Staining assay in a time- and dose-dependent manner. METs formed by rmCIRP stimulation were further confirmed by extracellular DNA, citrullinated histone H3, and myeloperoxidase. Treatment of THP-1 cells with rmCIRP significantly increased the cleaved forms of caspase-1 and GSDMD compared to PBS-treated cells. Treatment of macrophages with caspase-1, and GSDMD inhibitors z-VAD-fmk, and disulfiram, separately, significantly decreased rmCIRP-induced MET formation. We also confirmed rmCIRP-induced MET formation using primary cells murine peritoneal macrophages. These data clearly show that eCIRP serves as a novel inducer of MET formation through the activation of GSDMD and caspase-1.
Highlights
Cold-inducible RNA-binding protein (CIRP) is an 18-kDa nuclear protein [1]
We found that the THP-1 cells pre-treated with anti-Tolllike receptor 4 (TLR4) Abs significantly decreased rmCIRP-induced macrophage extracellular traps (METs)
We found that the treatment of the cells with only disulfiram at a dose of 40 μM, the highest dose of it used to inhibit Extracellular CIRP (eCIRP)-induced gasdermin D (GSDMD) activation and MET formation (Figures 5F, G), showed no adverse effects on the cell viability as the results were compared with the DMSO-treated control (Figure 5H)
Summary
Trauma, hemorrhage, and organ ischemia–reperfusion injury, nuclear CIRP is translocated to the cytoplasm and released outside the cells to serve as a damage-associated molecular pattern (DAMP), worsening inflammation and tissue injury [1, 2]. Extracellular CIRP (eCIRP) activates macrophages, neutrophils, and lymphocytes by binding to Tolllike receptor 4 (TLR4) and/or triggering receptor expressed on myeloid cells-1 (TREM-1) [2, 3]. While the innate immune cells, i.e., neutrophils and macrophages, serve as the first line of defense against the pathogen, their exaggerated activation and effector functions lead to severe inflammation and tissue injury in sepsis [4–6]. ECIRP induces NET formation by activating peptidyl arginine deiminase 4 (PAD4), which causes citrullination of histone H3 [9]. Studies have demonstrated the generation of METs by several macrophage subsets and cell lines in response to a wide range of microbes and their products [13, 14]. In human macrophages, the release of METs and their formation mechanism are not thoroughly studied
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