Damage-associated molecular patterns (DAMPs) are endogenous molecules which foment inflammation and are associated with disorders in sepsis and cancer. Thus, therapeutically targeting DAMPs has potential to provide novel and effective treatments. When establishing anti-DAMP strategies, it is important not only to focus on the DAMPs as inflammatory mediators but also to take into account the underlying mechanisms of their release from cells and tissues. DAMPs can be released passively by membrane rupture due to necrosis/necroptosis, although the mechanisms of release appear to differ between the DAMPs. Other types of cell death, such as apoptosis, pyroptosis, ferroptosis and NETosis, can also contribute to DAMP release. In addition, some DAMPs can be exported actively from live cells by exocytosis of secretory lysosomes or exosomes, ectosomes, and activation of cell membrane channel pores. Here we review the shared and DAMP-specific mechanisms reported in the literature for high mobility group box 1, ATP, extracellular cold-inducible RNA-binding protein, histones, heat shock proteins, extracellular RNAs and cell-free DNA.

PW is an inventor of patent applications (WO/2010/120726 and 61/881.798) covering the fundamental concept of targeting cold-inducible RNAbinding protein for the treatment of inflammatory diseases, licensed by TheraSource LLC. PW is a cofounder of TheraSource LLC.

Sepsis is a severe state of infection with high mortality. Pathogen-associated molecular patterns and damage-associated molecular patterns (DAMPs) initiate dysregulated systemic inflammation upon binding to pattern recognition receptors. Exosomes are endosome-derived vesicles, which carry proteins, lipids and nucleic acids, and facilitate intercellular communications. Studies have shown altered contents and function of exosomes during sepsis. In sepsis, exosomes carry increased levels of cytokines and DAMPs to induce inflammation. Exosomal DAMPs include, but are not limited to, high mobility group box 1, heat shock proteins, histones, adenosine triphosphate, and extracellular RNA. Exosomes released during sepsis have impact on multiple organs, including the lungs, kidneys, liver, cardiovascular system, and central nervous system. Here, we review the mechanisms of inflammation caused by exosomes, and their contribution to multiple organ dysfunction in sepsis.

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Extracellular cold-inducible RNA-binding protein (eCIRP) is an important damage-associated molecular pattern (DAMP). Despite our understanding of the potentially harmful effects of eCIRP in sepsis, how eCIRP is released from cells remains elusive. Exosomes are endosome-derived extracellular vesicles, which carry proteins, lipids, and nucleic acids to facilitate intercellular communication and several extracellular functions. We hypothesized that eCIRP is released via exosomes to induce inflammation in sepsis. Exosomes isolated from the supernatants of LPS-treated macrophage culture and serum of endotoxemia and polymicrobial sepsis mice showed high purity, as revealed by their unique median sizes ranging between 70 and 126 nm in diameter. eCIRP levels of the exosomes were significantly increased after LPS treatment in the supernatants of macrophage culture, mouse serum, and cecal ligation and puncture (CLP)-induced sepsis mouse serum. Protease protection assay demonstrated the majority of eCIRP was present on the surface of exosomes. Treatment of WT macrophages and mice with exosomes isolated from LPS-treated WT mice serum increased TNFα and IL-6 production. However, treatment with CIRP−/- mice serum exosomes significantly decreased these levels compared with WT exosome-treated conditions. CIRP−/- mice serum exosomes significantly decreased neutrophil migration in vitro compared with WT exosomes. Treatment of mice with serum exosomes isolated from CIRP−/- mice significantly reduced neutrophil infiltration into the peritoneal cavity. Our data suggest that eCIRP can be released via exosomes to induce cytokine production and neutrophil migration. Thus, exosomal eCIRP could be a potential target to inhibit inflammation.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern. Neutrophils present in the mononuclear cell fraction of Ficoll gradient separation are called low-density neutrophils (LDNs). Here we report the novel role of eCIRP on LDNs' heterogeneity in sepsis. Sepsis was induced in male C57BL/6 wild-type (WT) and CIRP −/− mice by cecal ligation and puncture (CLP). At 20 h after CLP, LDNs in the blood were isolated by Ficoll gradient separation, followed by staining the cells with anti-Ly6G and anti-CD11b Abs and detection by flow cytometry. Sepsis or recombinant murine CIRP (rmCIRP) injection in mice resulted in significant increase in the frequency (%) and number of Ly6G + CD11b hi and Ly6G + CD11b lo LDNs in the blood compared to sham-or vehicle-treated mice. At 20 h of CLP, CIRP −/− mice had significantly lower frequency and number of Ly6G + CD11b hi and Ly6G + CD11b lo LDNs in the blood compared to WT mice. In sepsis mice or rmCIRP-injected mice, compared to Ly6G + CD11b lo LDNs, the expression of CXCR4, ICAM-1, and iNOS and formation of reactive oxygen species, and neutrophil extracellular traps in Ly6G + CD11b hi LDNs in the blood were significantly increased. Treatment of WT bone marrow-derived neutrophils (BMDNs) with rmCIRP increased Ly6G + CD11b hi LDN frequency, whereas treatment of TLR4 −/− BMDNs with rmCIRP significantly decreased the frequency of Ly6G + CD11b hi LDNs. BMDNs' stimulation with rmCIRP increased the expression of transcription factors in LDNs. eCIRP induces the formation of a proinflammatory phenotype Ly6G + CD11b hi of LDNs through TLR4. Targeting eCIRP may provide beneficial outcomes in sepsis by decreasing proinflammatory Ly6G + CD11b hi LDNs.

Phagocytic clearance of apoptotic cells by the macrophages (efferocytosis) is impaired in sepsis, but its mechanism is poorly understood. Extracellular cold-inducible RNA-binding protein (eCIRP) is a novel damage-associated molecular pattern that fuels inflammation. We identify that eCIRP-induced neutrophil extracellular traps (NETs) impair efferocytosis through a novel mechanism. Coculture of macrophages and apoptotic thymocytes in the presence of recombinant murine CIRP (rmCIRP)–induced NETs significantly inhibited efferocytosis. Efferocytosis was significantly inhibited in the presence of rmCIRP-treated wild-type (WT), but not PAD4−/− neutrophils. Efferocytosis in the peritoneal cavity of rmCIRP-injected PAD4−/− mice was higher than WT mice. Milk fat globule–EGF–factor VIII (MFG-E8), an opsonin, increased macrophage efferocytosis, whereas the inhibition of efferocytosis by NETs was not rescued upon addition of MFG-E8, indicating disruption of MFG-E8’s receptor(s) αvβ3 or αvβ5 integrin by the NETs. We identified neutrophil elastase in the NETs significantly inhibited efferocytosis by cleaving macrophage surface integrins αvβ3 and αvβ5. Using a preclinical model of sepsis, we found that CIRP−/− mice exhibited significantly increased rate of efferocytosis in the peritoneal cavity compared with WT mice. We discovered a novel role of eCIRP-induced NETs to inhibit efferocytosis by the neutrophil elastase–dependent decrease of αvβ3/αvβ5 integrins in macrophages. Targeting eCIRP ameliorates sepsis by enhancing efferocytosis.

In sepsis, macrophage bacterial phagocytosis is impaired, but the mechanism is not well elucidated. Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern that causes inflammation. However, whether eCIRP regulates macrophage bacterial phagocytosis is unknown. Here, we reported that the bacterial loads in the blood and peritoneal fluid were decreased in CIRP−/− mice and anti-eCIRP Ab-treated mice after sepsis. Increased eCIRP levels were correlated with decreased bacterial clearance in septic mice. CIRP−/− mice showed a marked increase in survival after sepsis. Recombinant murine CIRP (rmCIRP) significantly decreased the phagocytosis of bacteria by macrophages in vivo and in vitro. rmCIRP decreased the protein expression of actin-binding proteins, ARP2, and p-cofilin in macrophages. rmCIRP significantly downregulated the protein expression of βPIX, a Rac1 activator. We further demonstrated that STAT3 and βPIX formed a complex following rmCIRP treatment, preventing βPIX from activating Rac1. We also found that eCIRP-induced STAT3 phosphorylation was required for eCIRP’s action in actin remodeling. Inhibition of STAT3 phosphorylation prevented the formation of the STAT3-βPIX complex, restoring ARP2 and p-cofilin expression and membrane protrusion in rmCIRP-treated macrophages. The STAT3 inhibitor stattic rescued the macrophage phagocytic dysfunction induced by rmCIRP. Thus, we identified a novel mechanism of macrophage phagocytic dysfunction caused by eCIRP, which provides a new therapeutic target to ameliorate sepsis.