DAMPs and radiation injury.

The heightened risk of ionizing radiation exposure, stemming from radiation accidents and potential acts of terrorism, has spurred growing interests in devising effective countermeasures against radiation injury. High-dose ionizing radiation exposure triggers acute radiation syndrome (ARS), manifesting as hematopoietic, gastrointestinal, and neurovascular ARS. Hematopoietic ARS typically presents with neutropenia and thrombocytopenia, while gastrointestinal ARS results in intestinal mucosal injury, often culminating in lethal sepsis and gastrointestinal bleeding. This deleterious impact can be attributed to radiation-induced DNA damage and oxidative stress, leading to various forms of cell death, such as apoptosis, necrosis and ferroptosis. Damage-associated molecular patterns (DAMPs) are intrinsic molecules released by cells undergoing injury or in the process of dying, either through passive or active pathways. These molecules then interact with pattern recognition receptors, triggering inflammatory responses. Such a cascade of events ultimately results in further tissue and organ damage, contributing to the elevated mortality rate. Notably, infection and sepsis often develop in ARS cases, further increasing the release of DAMPs. Given that lethal sepsis stands as a major contributor to the mortality in ARS, DAMPs hold the potential to function as mediators, exacerbating radiation-induced organ injury and consequently worsening overall survival. This review describes the intricate mechanisms underlying radiation-induced release of DAMPs. Furthermore, it discusses the detrimental effects of DAMPs on the immune system and explores potential DAMP-targeting therapeutic strategies to alleviate radiation-induced injury.

Human cell-expressed tag-free rhMFG-E8 as an effective radiation mitigator.

Human milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. E. coli-expressed histidine-tagged recombinant human MFG-E8 (rhMFG-E8) is protective in various disease conditions. However, due to improper recombinant protein glycosylation, misfolding and the possibility of antigenicity, E. coli-expressed histidine-tagged rhMFG-E8 is unsuitable for human therapy. Therefore, we hypothesize that human cell-expressed, tag-free rhMFG-E8 will have suitable structural and functional properties to be developed as a safe and effective novel biologic to treat inflammatory diseases including radiation injury. We produced a new tag-free rhMFG-E8 protein by cloning the human MFG-E8 full-length coding sequence without any fusion tag into a mammalian vector and expressed it in HEK293-derived cells. The construct includes the leader sequence of cystatin S to maximize secretion of rhMFG-E8 into the culture medium. After purification and confirmation of the protein identity, we first evaluated its biological activity in vitro. We then determined its efficacy in vivo utilizing an experimental rodent model of radiation injury, i.e., partial body irradiation (PBI). HEK293 cell supernatant containing tag-free rhMFG-E8 protein was concentrated, purified, and rhMFG-E8 was verified by SDS-PAGE with the standard human MFG-E8 loaded as control and, mass spectrometry followed by analysis using MASCOT for peptide mass fingerprint. The biological activity of human cell-expressed tag-free rhMFG-E8 was superior to that of E. coli-expressed His-tagged rhMFG-E8. Toxicity, stability, and pharmacokinetic studies indicate that tag-free rhMFG-E8 is safe, highly stable after lyophilization and long-term storage, and with a terminal elimination half-life in circulation of at least 1.45 h. In the 15 Gy PBI model, a dose-dependent improvement of the 30-day survival rate was observed after tag-free rhMFG-E8 treatment with a 30-day survival of 89%, which was significantly higher than the 25% survival in the vehicle group. The dose modification factor (DMF) of tag-free rhMFG-E8 calculated using probit analysis was 1.058. Tag-free rhMFG-E8 also attenuated gastrointestinal damage after PBI suggesting it as a potential therapeutic candidate for a medical countermeasure for radiation injury. Our new human cell-expressed tag-free rhMFG-E8 has proper structural and functional properties to be further developed as a safe and effective therapy to treat victims of severe acute radiation injury.

Antigen-presenting aged neutrophils induce CD4+ T cells to exacerbate inflammation in sepsis.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a key mediator of severity and mortality in sepsis. We found that stimulation of mouse bone marrow-derived neutrophils (BMDNs) with eCIRP generated a distinct neutrophil subpopulation, characterized by cell surface markers of both antigen-presenting cells and aged neutrophils as well as expression of IL-12, which we named antigen-presenting aged neutrophils (APANs). The frequency of APANs was significantly increased in the blood, spleen, and lungs of WT mice subjected to cecal ligation and puncture-induced sepsis but not in CIRP-/- mice. Patients with sepsis had a significant increase in circulating APAN counts compared with healthy individuals. Compared with non-APAN-transfered mice, APAN-transferred septic mice had increased serum levels of injury and inflammatory markers, exacerbated acute lung injury (ALI), and worsened survival. APANs and CD4+ T cells colocalized in the spleen, suggesting an immune interaction between these cells. APANs cocultured with CD4+ T cells significantly induced the release of IFN-γ via IL-12. BMDNs stimulated with eCIRP and IFN-γ underwent hyper-NETosis. Stimulating human peripheral blood neutrophils with eCIRP also induced APANs, and stimulating human neutrophils with eCIRP and IFN-γ caused hyper-NETosis. Thus, eCIRP released during sepsis induced APANs to aggravate ALI and worsen the survival of septic animals via CD4+ T cell activation, Th1 polarization, and IFN-γ-mediated hyper-NETosis.

Development of human cell-expressed tag-free rhMFG-E8 as a radiation mitigator and a therapeutic for acute kidney injury.

Background: Human milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. E. coli-expressed histidine-tagged recombinant human MFG-E8 (rhMFG-E8) is protective in various disease conditions. However, due to improper recombinant protein glycosylation, misfolding and possible antigenicity, E. coli-expressed histidine-tagged rhMFG-E8 is unsuitable for human therapy. Therefore, we hypothesize that human cell-expressed, tag-free rhMFG-E8 can be developed as a safe and effective novel biologic to treat inflammatory diseases such as radiation injury and acute kidney injury (AKI). Methods: We produced a new tag-free rhMFG-E8 protein by cloning the human MFG-E8 full-length coding sequence without any fusion tag into a mammalian vector and expressed it in HEK293-derived cells. The construct includes the leader sequence of cystatin S to maximize secretion of rhMFG-E8 into the culture medium. After purification and confirmation of the protein identity, we first evaluated its biological activity in vitro. We then determined its efficacy in vivo utilizing two experimental rodent models of organ injury: partial body irradiation (PBI) and ischemia/reperfusion-induced AKI. Results: HEK293 cell supernatant containing tag-free rhMFG-E8 protein was concentrated, purified, and rhMFG-E8 was verified by SDS-PAGE analysis and mass spectrometry. The biological activity of human cell-expressed tag-free rhMFG-E8 was superior to that of E. coli-expressed His-tagged rhMFG-E8. Toxicity, stability, and pharmacokinetic studies indicate that tag-free rhMFG-E8 is safe, highly stable after lyophilization and long-term storage, and with an adequate half-life for therapeutic applications. In the PBI model, a dose-dependent improvement of the 30-day survival rate was observed after tag-free rhMFG-E8 treatment with a 30-day survival of 89%, which was significantly higher than the 25% survival in the vehicle group. The dose modification factor (DMF) of tag-free rhMFG-E8 was 1.073. Tag-free rhMFG-E8 also attenuated gastrointestinal damage after PBI. In the model of AKI, tag-free rhMFG-E8 treatment significantly attenuated kidney injury and inflammation, and improved the 10-day survival. Conclusion: Our new human cell-expressed tag-free rhMFG-E8 can be further developed as a safe and effective therapy to treat victims of severe acute radiation injury and patients with acute kidney injury.

Radiation upregulates macrophage TREM-1 expression to exacerbate injury in mice.

Introduction: Exposure to high-dose ionizing radiation causes tissue injury, infections and even death due to immune dysfunction. The triggering receptor expressed on myeloid cells-1 (TREM-1) has been demonstrated to critically amplify and dysregulate immune responses. However, the role of TREM-1 in radiation injury remains unknown. Extracellular cold-inducible RNA-binding protein (eCIRP), a new damage-associated molecular pattern, is released from activated or stressed cells during inflammation. We hypothesized that ionizing radiation upregulates TREM-1 expression via eCIRP release to worsen survival. Methods: RAW264.7 cells and peritoneal macrophages collected from C57BL/6 wild-type (WT) mice were exposed to 5- and 10-Gray (Gy) radiation. C57BL/6 WT and CIRP-/- mice underwent 10-Gy total body irradiation (TBI). TREM-1 expression on RAW264.7 cells and peritoneal macrophages in vitro and in vivo were evaluated by flow cytometry. eCIRP levels in cell culture supernatants and in peritoneal lavage isolated from irradiated mice were evaluated by Western blotting. We also evaluated 30-day survival in C57BL/6 WT, CIRP-/- and TREM-1-/- mice after 6.5-Gy TBI. Results: The surface protein and mRNA levels of TREM-1 in RAW264.7 cells were significantly increased at 24 h after 5- and 10-Gy radiation exposure. TREM-1 expression on peritoneal macrophages was significantly increased after radiation exposure in vitro and in vivo. eCIRP levels were significantly increased after radiation exposure in cell culture supernatants of peritoneal macrophages in vitro and in peritoneal lavage in vivo. Moreover, CIRP-/- mice exhibited increased survival after 6.5-Gy TBI compared to WT mice. Interestingly, TREM-1 expression on peritoneal macrophages in CIRP-/- mice was significantly decreased compared to that in WT mice at 24 h after 10-Gy TBI. Furthermore, 30-day survival in TREM-1-/- mice was significantly increased to 64% compared to 20% in WT mice after 6.5-Gy TBI. Conclusion: Our data indicate that ionizing radiation increases TREM-1 expression in macrophages via the release of eCIRP, and TREM-1 contributes to worse survival after total body irradiation. Thus, targeting TREM-1 could have the potential to be developed as a novel medical countermeasure for radiation injury.

The protective effect of H151, a novel STING inhibitor, in renal ischemia-reperfusion-induced acute kidney injury.

Renal ischemia-reperfusion (RIR)-induced acute kidney injury (AKI) is a common renal functional disorder with high morbidity and mortality. Stimulator of interferon (IFN) genes (STING) is the cytosolic DNA-activated signaling pathway that mediates inflammation and injury. Our recent study showed that extracellular cold-inducible RNA-binding protein (eCIRP), a newly identified damage-associated molecular pattern, activates STING and exacerbates hemorrhagic shock. H151 is a small molecule that selectively binds to STING and inhibits STING-mediated activity. We hypothesized that H151 attenuates eCIRP-induced STING activation in vitro and inhibits RIR-induced AKI in vivo. In vitro, renal tubular epithelial cells incubated with eCIRP showed increased levels of IFN-ß, STING pathway downstream cytokine, IL-6, tumor necrosis factor-α, and neutrophil gelatinase-associated lipocalin, whereas coincubation with eCIRP and H151 diminished those increases in a dose-dependent manner. In vivo, 24 h after bilateral renal ischemia-reperfusion, glomerular filtration rate was decreased in RIR-vehicle-treated mice, whereas glomerular filtration rate was unchanged in RIR-H151-treated mice. In contrast to sham, serum blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin were increased in RIR-vehicle, but in RIR-H151, these levels were significantly decreased from RIR-vehicle. In contrast to sham, kidney IFN-ß mRNA, histological injury score, and TUNEL staining were also increased in RIR-vehicle, but in RIR-H151, these levels were significantly decreased from RIR-vehicle. Importantly, in contrast to sham, in a 10-day survival study, survival decreased to 25% in RIR-vehicle, but RIR-H151 had a survival of 63%. In conclusion, H151 inhibits eCIRP-induced STING activation in renal tubular epithelial cells. Therefore, STING inhibition by H151 can be a promising therapeutic intervention for RIR-induced AKI.NEW & NOTEWORTHY Renal ischemia-reperfusion (RIR)-induced acute kidney injury (AKI) is a common renal functional disorder with a high morbidity and mortality rate. Stimulator of interferon genes (STING) is the cytosolic DNA-activated signaling pathway responsible for mediating inflammation and injury. Extracellular cold-inducible RNA-binding protein (eCIRP) activates STING and exacerbates hemorrhagic shock. H151, a novel STING inhibitor, attenuated eCIRP-induced STING activation in vitro and inhibited RIR-induced AKI. H151 shows promise as a therapeutic intervention for RIR-induced AKI.

Extracellular CIRP Induces Calpain Activation in Neurons via PLC-IP3-Dependent Calcium Pathway.

Abnormal calcium homeostasis, activation of protease calpain, generation of p25 and hyperactivation of cyclin-dependent kinase 5 (Cdk5) have all been implicated in the pathogenesis of neurogenerative diseases including Alzheimer's disease. We have recently shown that extracellular cold-inducible RNA-binding protein (eCIRP) induces Cdk5 activation via p25. However, the precise molecular mechanism by which eCIRP regulates calcium signaling and calpain remains to be addressed. We hypothesized that eCIRP regulates p25 via Ca2+-dependent calpain activation. eCIRP increased calpain activity and decreased the endogenous calpain inhibitor calpastatin in Neuro 2a (N2a) cells. Calpain inhibition with calpeptin attenuated eCIRP-induced calpain activity and p25. eCIRP specifically upregulated cytosolic calpain 1, and calpain 1 silencing attenuated the eCIRP-induced increase in p25. eCIRP stimulation increased cytosolic free Ca2+, especially in hippocampal neuronal HT22 cells, which was attenuated by the eCIRP inhibitor Compound 23 (C23). Endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor (IP3R) inhibition using 2-aminoethoxy-diphenyl-borate or xestospongin-C (X-C), interleukin-6 receptor alpha (IL-6Rα)-neutralization, and phospholipase C (PLC) inhibition with U73122 attenuated eCIRP-induced Ca2+ increase, while Ca2+ influx across the plasma membrane remained unaffected by eCIRP. Finally, C23, IL-6Rα antibody, U73122 and X-C attenuated eCIRP-induced p25 in HT-22 cells. In conclusion, the current study uncovers eCIRP-triggered Ca2+ release from ER stores in an IL-6Rα/PLC/IP3-dependent manner as a novel molecular mechanism underlying eCIRP's induction of Cdk5 activity and potential involvement in neurodegeneration.

Extracellular CIRP induces acute kidney injury via endothelial TREM-1.

Introduction: Acute kidney injury is associated with elevated serum levels of extracellular cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern released during ischemia/reperfusion injury, hemorrhagic shock, and sepsis. It is unknown if circulating eCIRP and eCIRP-induced activation of receptor triggering receptor expressed on myeloid cells-1 (TREM-1), expressed on endothelial cells, play an important role in the pathogenesis of AKI. Methods: Male B6 wild-type (WT) and TREM-1-/- mice were subjected to intravenous injection of recombinant murine (rm) CIRP. Serum, urine, and renal tissue were collected 6 h later for analysis. Additionally, primary human renal glomerular endothelial cells (HRGEC) were stimulated in vitro with rmCIRP after pretreatment with M3, a novel inhibitory peptide of TREM-1, or vehicle. Supernatants and cells were collected 20 h after stimulation. Results: After injection with rmCIRP, WT mice had a significant increase in serum levels of BUN, creatinine, and NGAL compared to control. Additionally, NGAL was significantly increased in the urine of rmCIRP-injected mice, suggesting that circulating eCIRP can directly induce AKI. The levels of TREM-1 mRNA in the kidneys, as well as soluble (s) TREM-1 released into the serum and urine, were significantly increased in rmCIRP-injected mice. TREM-1-/- mice injected with rmCIRP had attenuated AKI, indicated by significantly decreased serum BUN, creatinine, and NGAL, and renal mRNA expression of NGAL and KIM-1 compared to WT mice. TREM-1-/- mice also had attenuated endothelial activation, with decreased mRNA and protein expression of ICAM-1 in renal tissue. HRGEC stimulated with rmCIRP in vitro had significant increases in cytokine production and sTREM-1 release, which was attenuated in cells treated with M3. Conclusion: Activation of renal TREM-1 with circulating eCIRP is sufficient to cause AKI. Elevated levels of eCIRP may be critical for the development of AKI under conditions such as ischemia/reperfusion injury, hemorrhagic shock, and sepsis. Mice deficient in the TREM-1 receptor have attenuated AKI and reduced endothelial cell activation after injection of rmCIRP. TREM-1 inhibition with M3 attenuates HRGEC activation after eCIRP stimulation. Targeting eCIRP activation of TREM-1 may provide a novel and effective treatment for AKI.

TREM-1 Modulation Strategies for Sepsis.

The triggering receptor expressed on myeloid cells-1 (TREM-1) is a pattern recognition receptor, which can be upregulated in inflammatory diseases as an amplifier of immune responses. Once activated, TREM-1 induces the production and release of pro-inflammatory cytokines and chemokines, in addition to increasing its own expression and circulating levels of the cleaved soluble extracellular portion of TREM-1 (sTREM-1). This amplification of the inflammatory response by TREM-1 has now been considered as a critical contributor to the dysregulated immune responses in sepsis. Studies have shown that in septic patients there is an elevated expression of TREM-1 on immune cells and increased circulating levels of sTREM-1, associated with increased mortality. As a result, a considerable effort has been made towards identifying endogenous ligands of TREM-1 and developing TREM-1 inhibitory peptides to attenuate the exacerbated inflammatory response in sepsis. TREM-1 modulation has proven a promising strategy for the development of therapeutic agents to treat sepsis. Therefore, this review encompasses the ligands investigated as activators of TREM-1 thus far and highlights the development and efficacy of novel inhibitors for the treatment of sepsis and septic shock.

Active Release of eCIRP via Gasdermin D Channels to Induce Inflammation in Sepsis.

Extracellular cold-inducible RNA binding protein (eCIRP) is an inflammatory mediator that causes inflammation and tissue injury in sepsis. Gasdermin D (GSDMD) is a protein that, when cleaved, forms pores in the cell membrane, releasing intracellular contents into the extracellular milieu to exacerbate inflammation. We hypothesize that eCIRP is released actively from viable macrophages via GSDMD pores. We found that LPS induced eCIRP secretion from macrophages into the extracellular space. LPS significantly increased the expression of caspase-11 and cleavage of the GSDMD, as evidenced by increased N-terminal GSDMD expression in RAW 264.7 cells and mouse primary peritoneal macrophages. GSDMD inhibitor disulfiram decreased eCIRP release in vitro. Treatment with glycine to prevent pyroptosis-induced cell lysis did not significantly decrease eCIRP release from LPS-treated macrophages, indicating that eCIRP was actively released and was independent of pyroptosis. Downregulation of GSDMD gene expression by siRNA transfection suppressed eCIRP release in vitro after LPS stimulation. Moreover, GSDMD-/- peritoneal macrophages and mice had decreased levels of eCIRP in the culture supernatants and in blood treated with LPS in vitro and in vivo, respectively. GSDMD inhibitor disulfiram inhibited serum levels of eCIRP in endotoxemia and cecal ligation and puncture-induced sepsis. We conclude that eCIRP release from living macrophages is mediated through GSDMD pores, suggesting that targeting GSDMD could be a novel and potential therapeutic approach to inhibit eCIRP-mediated inflammation in sepsis.

The role of eCIRP in bleomycin-induced pulmonary fibrosis in mice.

OBJECTIVE AND DESIGN: We examined the role of eCIRP in the pathogenesis of bleomycin-induced pulmonary fibrosis (PF). MATERIAL AND METHODS: Publicly available gene expression omnibus datasets were analyzed for the expression of CIRP in lung samples from patients with PF. Wild type (WT) or CIRP-/- mice received daily injections of 10 µg/g bleomycin for 10 days. A subset of bleomycin-injected WT mice was treated with the eCIRP antagonist C23 (8 µg/g/day) from day 10 to day 19. At three weeks, transthoracic echocardiography was performed to measure the degree of pulmonary hypertension, and lung tissues were collected and analyzed for markers of fibrosis. RESULTS: Analysis of the mRNA data of human lung samples showed a significant positive correlation between CIRP and α-smooth muscle actin (α-SMA), an important marker of fibrosis. Moreover, the expression of CIRP was higher in patients with acute exacerbation of PF than in patients with stable PF. CIRP-/- mice showed attenuated induction of α-SMA and collagens (Col1a1, Col3a1), reduced hydroxyproline content, decreased histological fibrosis scores, and improved pulmonary hypertension as compared to WT mice. WT mice treated with C23 also had significant attenuation of the above endpoint measure. CONCLUSIONS: Our study demonstrates that eCIRP plays a key role in promoting the development of PF, and blocking eCIRP with C23 can significantly attenuate this process.

A novel eCIRP/TREM-1 pathway inhibitor attenuates acute kidney injury.

BACKGROUND: Extracellular cold-inducible RNA-binding protein aggravates acute kidney injury after renal ischemia/reperfusion. Although extracellular cold-inducible RNA-binding protein activates triggering receptor expressed on myeloid cells-1, how this receptor and its antagonism with a novel peptide M3 affects acute kidney injury is poorly understood. We, therefore, hypothesize that inhibiting the extracellular cold-inducible RNA-binding protein/triggering receptor expressed on myeloid cells-1 pathway with M3 attenuates acute kidney injury. METHODS: Wild-type and triggering receptor expressed on myeloid cells-1-/- mice were subjected to bilateral 30-minute renal hilum clamping followed by reperfusion or sham. After 4 hours, wild-type mice received M3 (10 mg/kg BW) or normal saline intraperitoneally. After 24 hours, renal tissue and serum were collected for analysis. Additionally, wild-type mice were subjected to bilateral renal ischemia for 34 minutes and treated with M3 at 10 mg/kg BW or vehicle at the time of reperfusion. Survival was monitored for 10 days. RESULTS: After renal ischemia/reperfusion, triggering receptor expressed on myeloid cells-1 messenger ribonucleic acid expression increased by 9-fold in wild-type mice compared to sham mice. Wild-type mice also demonstrated significant increases in serum blood urea nitrogen, creatinine, and interleukin-6 and renal tissue levels of interleukin-6 and neutrophil gelatinase-associated lipocalin after renal ischemia/reperfusion compared to sham mice. Triggering receptor expressed on myeloid cells-1-/- mice demonstrated significant reductions in serum blood urea nitrogen, creatinine, and interleukin-6 compared to wild-type mice after renal ischemia/reperfusion. Levels of renal interleukin-6 and neutrophil gelatinase-associated lipocalin were also significantly decreased in the kidneys of triggering receptor expressed on myeloid cells-1-/- mice. Furthermore, treatment with M3 in wild-type mice significantly decreased serum and renal levels of interleukin-6 after renal ischemia/reperfusion. M3 treatment demonstrated significant reductions in renal messenger ribonucleic acid and protein levels of neutrophil gelatinase-associated lipocalin, serum blood urea nitrogen and creatinine, and histologic structural damage as well as apoptosis. Treatment with M3 also increased survival from 35% to 65% in mice with acute kidney injury. CONCLUSION: Triggering receptor expressed on myeloid cells-1 mediates the deleterious effects of extracellular cold-inducible RNA-binding protein in acute kidney injury after renal ischemia/reperfusion. The novel extracellular cold-inducible RNA-binding protein/triggering receptor expressed on myeloid cells-1 pathway antagonist, M3, attenuates acute kidney injury and has the potential to be developed as a therapeutic agent for acute kidney injury.

Human Dermcidin Protects Mice Against Hepatic Ischemia-Reperfusion-Induced Local and Remote Inflammatory Injury.

Background: Hepatic ischemia and reperfusion (I/R) injury is commonly associated with surgical liver resection or transplantation, and represents a major cause of liver damage and graft failure. Currently, there are no effective therapies to prevent hepatic I/R injury other than ischemic preconditioning and some preventative strategies. Previously, we have revealed the anti-inflammatory activity of a sweat gland-derived peptide, dermcidin (DCD), in macrophage/monocyte cultures. Here, we sought to explore its therapeutic potential and protective mechanisms in a murine model of hepatic I/R. Methods: Male C57BL/6 mice were subjected to hepatic ischemia by clamping the hepatic artery and portal vein for 60 min, which was then removed to initiate reperfusion. At the beginning of reperfusion, 0.2 ml saline control or solution of DCD (0.5 mg/kg BW) or DCD-C34S analog (0.25 or 0.5 mg/kg BW) containing a Cys (C)→Ser (S) substitution at residue 34 was injected via the internal jugular vein. For survival experiments, mice were subjected to additional resection to remove non-ischemic liver lobes, and animal survival was monitored for 10 days. For mechanistic studies, blood and tissue samples were collected at 24 h after the onset of reperfusion, and subjected to measurements of various markers of inflammation and tissue injury by real-time RT-PCR, immunoassays, and histological analysis. Results: Recombinant DCD or DCD-C34S analog conferred a significant protection against lethal hepatic I/R when given intravenously at the beginning of reperfusion. This protection was associated with a significant reduction in hepatic injury, neutrophilic CXC chemokine (Mip-2) expression, neutrophil infiltration, and associated inflammation. Furthermore, the administration of DCD also resulted in a significant attenuation of remote lung inflammatory injury. Mechanistically, DCD interacted with epidermal growth factor receptor (EGFR), a key regulator of liver inflammation, and significantly inhibited hepatic I/R-induced phosphorylation of EGFR as well as a downstream signaling molecule, protein kinase B (AKT). The suppression of EGFR expression by transducing Egfr-specific shRNA plasmid into macrophages abrogated the DCD-mediated inhibition of nitric oxide (NO) production induced by a damage-associated molecular pattern (DAMP), cold-inducible RNA-binding protein, CIRP. Conclusions: The present study suggests that human DCD and its analog may be developed as novel therapeutics to attenuate hepatic I/R-induced inflammatory injury possibly by impairing EGFR signaling.

Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4.

Extracellular cold-inducible RNA-binding protein (eCIRP), a new damage-associated molecular pattern (DAMP), has been recently shown to play a critical role in promoting the development of bleomycin-induced pulmonary fibrosis. Although fibroblast activation is a critical component of the fibrotic process, the direct effects of eCIRP on fibroblasts have never been examined. We studied eCIRP's role in the induction of inflammatory phenotype in pulmonary fibroblasts and its connection to bleomycin-induced pulmonary fibrosis in mice. We found that eCIRP causes the induction of proinflammatory cytokines and differentially expression-related pathways in a TLR4-dependent manner in pulmonary fibroblasts. Our analysis further showed that the accessory pathways MD2 and Myd88 are involved in the induction of inflammatory phenotype. In order to study the connection of the enrichment of these pathways in priming the microenvironment for pulmonary fibrosis, we investigated the gene expression profile of lung tissues from mice subjected to bleomycin-induced pulmonary fibrosis collected at various time points. We found that at day 14, which corresponds to the inflammatory-to-fibrotic transition phase after bleomycin injection, TLR4, MD2, and Myd88 were induced, and the transcriptome was differentially enriched for genes in those pathways. Furthermore, we also found that inflammatory cytokines gene expressions were induced, and the cellular responses to these inflammatory cytokines were differentially enriched on day 14. Overall, our results show that eCIRP induces inflammatory phenotype in pulmonary fibroblasts in a TLR4 dependent manner. This study sheds light on the mechanism by which eCIRP induced inflammatory fibroblasts, contributing to pulmonary fibrosis.

Extracellular CIRP activates STING to exacerbate hemorrhagic shock.

Stimulator of IFN genes (STING) activates TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3) to produce type I IFNs. Extracellular cold-inducible RNA-binding protein (eCIRP) is released from cells during hemorrhagic shock (HS). We hypothesized that eCIRP activates STING to induce inflammation and acute lung injury (ALI) after HS. WT and STING-/- mice underwent controlled hemorrhage by bleeding, followed by fluid resuscitation. Blood and lungs were collected at 4 hours after resuscitation. Serum ALT, AST, LDH, IL-6, and IFN-ß were significantly decreased in STING-/- mice compared with WT mice after HS. In STING-/- mice, the levels of pTBK1 and pIRF3, and expression of TNF-α, IL-6, and IL-1ß mRNAs and proteins in the lungs, were significantly decreased compared with WT HS mice. The 10-day mortality rate in STING-/- mice was significantly reduced. I.v. injection of recombinant mouse CIRP (rmCIRP) in STING-/- mice showed a significant decrease in pTBK1 and pIRF3 and in IFN-α and IFN-ß mRNAs and proteins in the lungs compared with rmCIRP-treated WT mice. Treatment of TLR4-/-, MyD88-/-, and TRIF-/- macrophages with rmCIRP significantly decreased pTBK1 and pIRF3 levels and IFN-α and IFN-ß mRNAs and proteins compared with WT macrophages. HS increases eCIRP levels, which activate STING through TLR4/MyD88/TRIF pathways to exacerbate inflammation.

The interplay of DAMPs, TLR4, and proinflammatory cytokines in pulmonary fibrosis.

Pulmonary fibrosis is a chronic debilitating condition characterized by progressive deposition of connective tissue, leading to a steady restriction of lung elasticity, a decline in lung function, and a median survival of 4.5 years. The leading causes of pulmonary fibrosis are inhalation of foreign particles (such as silicosis and pneumoconiosis), infections (such as post COVID-19), autoimmune diseases (such as systemic autoimmune diseases of the connective tissue), and idiopathic pulmonary fibrosis. The therapeutics currently available for pulmonary fibrosis only modestly slow the progression of the disease. This review is centered on the interplay of damage-associated molecular pattern (DAMP) molecules, Toll-like receptor 4 (TLR4), and inflammatory cytokines (such as TNF-α, IL-1ß, and IL-17) as they contribute to the pathogenesis of pulmonary fibrosis, and the possible avenues to develop effective therapeutics that disrupt this interplay.

Extracellular CIRP Activates the IL-6Rα/STAT3/Cdk5 Pathway in Neurons.

Extracellular cold-inducible RNA-binding protein (eCIRP) stimulates microglial inflammation causing neuronal damage during ischemic stroke and is a critical mediator of alcohol-induced cognitive impairment. However, the precise role of eCIRP in mediating neuroinflammation remains unknown. In this study, we report that eCIRP activates neurotoxic cyclin-dependent kinase-5 (Cdk5)/p25 through the induction of IL-6Rα/STAT3 pathway in neurons. Amyloid ß (Aß)-mediated neuronal stress, which is associated with Alzheimer's disease, increased the levels of eCIRP released from BV2 microglial cells. The released eCIRP levels from BV2 cells increased 3.2-fold upon stimulation with conditioned medium from Neuro-2a (N2a) cells containing Aß compared to control N2a supernatant in a time-dependent manner. Stimulation of N2a cells and primary neurons with eCIRP upregulated the neuronal Cdk5 activator p25 expression in a dose- and time-dependent manner. eCIRP directly induced neuronal STAT3 phosphorylation and p25 increase via its novel receptor IL-6Rα. Next, we showed using surface plasmon resonance that eCIRP-derived peptide C23 inhibited the binding of eCIRP to IL-6Rα at 25 µM, with a 40-fold increase in equilibrium dissociation constant (Kd) value (from 8.08 × 10-8 M to 3.43 × 10-6 M), and completely abrogated the binding at 50 µM. Finally, C23 reversed the eCIRP-induced increase in neuronal STAT3 phosphorylation and p25 levels. In conclusion, the current study demonstrates that the upregulation of neuronal IL-6Rα/STAT3/Cdk5 pathway is a key mechanism of eCIRP's role in neuroinflammation and that C23 as a potent inhibitor of this pathway has translational potential in neurodegenerative pathologies controlled by eCIRP.

Release mechanisms of major DAMPs.

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.

A Machine Learning Prediction Model of Respiratory Failure Within 48 Hours of Patient Admission for COVID-19: Model Development and Validation.

BACKGROUND: Predicting early respiratory failure due to COVID-19 can help triage patients to higher levels of care, allocate scarce resources, and reduce morbidity and mortality by appropriately monitoring and treating the patients at greatest risk for deterioration. Given the complexity of COVID-19, machine learning approaches may support clinical decision making for patients with this disease. OBJECTIVE: Our objective is to derive a machine learning model that predicts respiratory failure within 48 hours of admission based on data from the emergency department. METHODS: Data were collected from patients with COVID-19 who were admitted to Northwell Health acute care hospitals and were discharged, died, or spent a minimum of 48 hours in the hospital between March 1 and May 11, 2020. Of 11,525 patients, 933 (8.1%) were placed on invasive mechanical ventilation within 48 hours of admission. Variables used by the models included clinical and laboratory data commonly collected in the emergency department. We trained and validated three predictive models (two based on XGBoost and one that used logistic regression) using cross-hospital validation. We compared model performance among all three models as well as an established early warning score (Modified Early Warning Score) using receiver operating characteristic curves, precision-recall curves, and other metrics. RESULTS: The XGBoost model had the highest mean accuracy (0.919; area under the curve=0.77), outperforming the other two models as well as the Modified Early Warning Score. Important predictor variables included the type of oxygen delivery used in the emergency department, patient age, Emergency Severity Index level, respiratory rate, serum lactate, and demographic characteristics. CONCLUSIONS: The XGBoost model had high predictive accuracy, outperforming other early warning scores. The clinical plausibility and predictive ability of XGBoost suggest that the model could be used to predict 48-hour respiratory failure in admitted patients with COVID-19.

MFG-E8-derived peptide attenuates inflammation and injury after renal ischemia-reperfusion in mice.

BACKGROUND: Renal ischemia-reperfusion (renal I/R) injury may lead to acute kidney injury (AKI). After renal I/R, proinflammatory mediators cause immune cell infiltration and further injury. Milk fat globule-epidermal growth factor-factor 8 (MFG-E8) is a protein involved in cell-cell and cell-matrix interactions. MSP68 is an MFG-E8-derived peptide that inhibits neutrophil adhesion and migration. Here, we evaluated whether MSP68 attenuates renal I/R injury. MATERIALS AND METHODS: Adult C57BL/6 mice were subjected to bilateral renal ischemia for 30 min followed by reperfusion and intraperitoneal administration of saline (vehicle) or MSP68 (5 mg/kg). Sham animals underwent laparotomy without renal I/R. The blood collected and studied for BUN, creatinine, and LDH by colorimetry. The kidneys were analyzed for IL-6 and TNFα by qPCR, ELISA, histological injury, and apoptosis by TUNEL. RESULTS: At 24 h after surgery, serum levels of BUN, creatinine, and LDH were markedly higher in vehicle-treated renal I/R mice than in sham mice, but significantly lower in MSP68-treated renal I/R mice. Similarly, compared to sham, renal levels of IL-6 mRNA and protein and TNFα protein were markedly higher in vehicle-treated renal I/R mice, but significantly lower in MSP68-treated renal I/R mice. Vehicle-treated renal I/R mice also had severe renal tubular histological injury, which was significantly lower in MSP68-treated renal I/R mice. Additionally, the kidneys of vehicle-treated renal I/R mice had a 93-fold increase in TUNEL-positive cells, which were reduced by 35% in mice treated with MSP68. CONCLUSION: MSP68 has the potential to be developed as novel therapeutic agent for patients with AKI.