The Novel MFG-E8-derived Oligopeptide, MOP3, Improves Outcomes in a Preclinical Murine Model of Neonatal Sepsis.

INTRODUCTION: Neonatal sepsis is a devastating inflammatory condition that remains a leading cause of morbidity and mortality. Milk fat globule-EGF-factor VIII (MFG-E8) is a glycoprotein that reduces inflammation, whereas extracellular cold-inducible RNA binding protein (eCIRP) worsens inflammation. This study aimed to determine the therapeutic potential of a novel MFG-E8-derived oligopeptide 3 (MOP3) designed to clear eCIRP and protect against inflammation, organ injury, and mortality in neonatal sepsis. METHODS: C57BL6 mouse pups were injected intraperitoneally with cecal slurry (CS) and treated with MOP3 (20 µg/g) or vehicle. 10 h after injection, blood, lungs, and intestines were collected for analyses, and in a 7-day experiment, pups were monitored for differences in mortality. RESULTS: MOP3 treatment protected septic pups from inflammation by reducing eCIRP, IL-6, TNFα, and LDH. MOP3 reduced lung and intestinal inflammation and injury as assessed by reductions in tissue mRNA levels of inflammatory markers, histopathologic injury, and apoptosis in lung and intestines. MOP3 also significantly improved 7-day overall survival for CS-septic mouse pups compared to vehicle (75% vs. 46%, respectively). CONCLUSION: Deriving from MFG-E8 and designed to clear eCIRP, MOP3 protects against sepsis-induced inflammation, organ injury, and mortality in a preclinical model of neonatal sepsis, implicating it as an exciting potential new therapeutic. LEVEL OF EVIDENCE: Level 1.

Transcriptomic profiling of immune cells in murine polymicrobial sepsis.

Introduction: Various immune cell types play critical roles in sepsis with numerous distinct subsets exhibiting unique phenotypes even within the same cell population. Single-cell RNA sequencing (scRNA-seq) enables comprehensive transcriptome profiling and unbiased cell classification. In this study, we have unveiled the transcriptomic landscape of immune cells in sepsis through scRNA-seq analysis. Methods: We induced sepsis in mice by cecal ligation and puncture. 20 h after the surgery, the spleen and peritoneal lavage were collected. Single-cell suspensions were processed using a 10× Genomics pipeline and sequenced on an Illumina platform. Count matrices were generated using the Cell Ranger pipeline, which maps reads to the mouse reference transcriptome, GRCm38/mm10. Subsequent scRNA-seq analysis was performed using the R package Seurat. Results: After quality control, we subjected the entire data set to unsupervised classification. Four major clusters were identified as neutrophils, macrophages, B cells, and T cells according to their putative markers. Based on the differentially expressed genes, we identified activated pathways in sepsis for each cell type. In neutrophils, pathways related to inflammatory signaling, such as NF-κB and responses to pathogen-associated molecular patterns (PAMPs), cytokines, and hypoxia were activated. In macrophages, activated pathways were the ones related to cell aging, inflammatory signaling, and responses to PAMPs. In B cells, pathways related to endoplasmic reticulum stress were activated. In T cells, activated pathways were the ones related to inflammatory signaling, responses to PAMPs, and acute lung injury. Next, we further classified each cell type into subsets. Neutrophils consisted of four clusters. Some subsets were activated in inflammatory signaling or cell metabolism, whereas others possessed immunoregulatory or aging properties. Macrophages consisted of four clusters, namely, the ones with enhanced aging, lymphocyte activation, extracellular matrix organization, or cytokine activity. B cells consisted of four clusters, including the ones possessing the phenotype of cell maturation or aging. T cells consisted of six clusters, whose phenotypes include molecular translocation or cell activation. Conclusions: Transcriptomic analysis by scRNA-seq has unveiled a comprehensive spectrum of immune cell responses and distinct subsets in the context of sepsis. These findings are poised to enhance our understanding of sepsis pathophysiology, offering avenues for targeting novel molecules, cells, and pathways to combat infectious diseases.

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.

An engineered poly(A) tail attenuates gut ischemia/reperfusion-induced acute lung injury.

BACKGROUND: Gut ischemia/reperfusion causes the release of damage-associated molecular patterns, leading to acute lung injury and high mortality. Cold-inducible ribonucleic acid-binding protein is a ribonucleic acid chaperon that binds the polyadenylation tail of messenger ribonucleic acid intracellularly. Upon cell stress, cold-inducible ribonucleic acid-binding protein is released, and extracellular cold-inducible ribonucleic acid-binding protein acts as a damage-associated molecular pattern, worsening inflammation. To inhibit extracellular cold-inducible ribonucleic acid-binding protein, we have recently developed an engineered polyadenylation tail named A12. Here, we sought to investigate the therapeutic potential of A12 in gut ischemia/reperfusion-induced acute lung injury. METHODS: Male C57BL6/J mice underwent superior mesenteric artery occlusion and were treated with intraperitoneal A12 (0.5 nmol/g body weight) or vehicle at the time of reperfusion. Blood and lungs were collected 4 hours after gut ischemia/reperfusion. Systemic levels of extracellular cold-inducible ribonucleic acid-binding protein, interleukin-6, aspartate transaminase, alanine transaminase, and lactate dehydrogenase were determined. The pulmonary gene expression of cytokines (interleukin-6, interleukin-1ß) and chemokines (macrophage-inflammatory protein-2, keratinocyte-derived chemokine) was also assessed. In addition, lung myeloperoxidase, injury score, and cell death were determined. Mice were monitored for 48 hours after gut ischemia/reperfusion for survival assessment. RESULTS: Gut ischemia/reperfusion significantly increased the serum extracellular cold-inducible ribonucleic acid-binding protein levels. A12 treatment markedly reduced the elevated serum interleukin-6, alanine transaminase, aspartate transaminase, and lactate dehydrogenase by 53%, 23%, 23%, and 24%, respectively, in gut ischemia/reperfusion mice. A12 also significantly decreased cytokine and chemokine messenger ribonucleic acids and myeloperoxidase activity in the lungs of gut ischemia/reperfusion mice. Histological analysis revealed that A12 attenuated tissue injury and cell death in the lungs of gut ischemia/reperfusion mice. Finally, administration of A12 markedly improved the survival of gut ischemia/reperfusion mice. CONCLUSION: A12, a novel extracellular cold-inducible ribonucleic acid-binding protein inhibitor, diminishes inflammation and mitigates acute lung injury when employed as a treatment during gut ischemia/reperfusion. Hence, the targeted approach toward extracellular cold-inducible ribonucleic acid-binding protein emerges as a promising therapeutic strategy for alleviating gut ischemia/reperfusion-induced acute lung injury.

Targeting sting to reduce sepsis-induced acute intestinal injury.

BACKGROUND: Sepsis is a dysregulated host response to infection syndrome leading to life-threatening organ dysfunction. Sepsis-induced intestinal dysfunction is a key element in the progression to multisystem organ failure. The stimulator of interferon genes is an intracellular protein implicated in intestinal injury in sepsis. H151, a small molecule inhibitor of stimulator of interferon genes, has not yet been studied as a potential therapeutic in sepsis. We hypothesize that H151 therapeutically reduces sepsis-induced acute intestinal injury. METHODS: Male mice underwent cecal ligation and puncture and were treated with intraperitoneal H151 (10 mg/kg body weight) or vehicle. Intestines and serum were collected for analysis 20 hours after cecal ligation and puncture. Oral gavage of mice with FITC-dextran was performed 15 hours after cecal ligation and puncture. Five hours after gavage, serum was collected, and intestinal permeability was assessed. Mice were monitored for 10 days after cecal ligation and puncture to assess survival. RESULTS: Zonula occludens 1 tight junctional protein expression was reduced after cecal ligation and puncture and recovered with H151 treatment. This was associated with a 62.3% reduction in intestinal permeability as assessed by fluorimetry. After cecal ligation and puncture, treatment with H151 was associated with a 58.7% reduction in intestinal histopathologic injury (P < .05) and a 56.6% reduction in intestinal apoptosis (P < .05). Intestinal myeloperoxidase activity was decreased by 70.8% after H151 treatment (P < .05). Finally, H151 improved 10-day survival from 33% to 80% after cecal ligation and puncture (P = .011). CONCLUSION: H151, a novel stimulator of interferon genes inhibitor, reduces intestinal injury, inflammation, and permeability when administered as a treatment for cecal ligation and puncture-induced sepsis. Thus, targeting stimulator of interferon genes shows promise as a therapeutic strategy to ameliorate sepsis-induced acute intestinal 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.

A novel miRNA mimic attenuates organ injury after hepatic ischemia/reperfusion.

INTRODUCTION: Extracellular cold-inducible RNA-binding protein (eCIRP) is a novel mediator of inflammation and tissue injury. It has been shown that miRNA 130b-3p acts as an endogenous inhibitor of eCIRP. Because RNA mimics are unstable after in vivo administration, we have chemically engineered miRNA 130b-3p mimic (named PS-OMe miR130) to improve its stability by protection from nuclease activity. We hypothesize that PS-OMe miR130 reduces eCIRP-mediated injury and inflammation in a murine model of hepatic ischemia/reperfusion (I/R), a model of sterile inflammation. METHODS: Adult male mice underwent 70% hepatic ischemia for 60 minutes and 24-hour reperfusion. At the start of reperfusion, mice were treated intravenously with vehicle (phosphate-buffered saline) or PS-OMe miR130. Blood and liver tissue were collected after 24 hours for biochemical analysis. Apoptosis in the liver tissue was determined by transferase dUTP nick-end labeling assay. RESULTS: After hepatic I/R, organ injury markers including aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase significantly decreased after PS-OMe miR130 treatment. Furthermore, histological analysis of liver sections demonstrated significantly less injury in PS-OMe miR130 treatment mice versus vehicle mice. In addition, tumor necrosis factor α mRNA, interleukin-1ß mRNA, and neutrophil infiltration (myeloperoxidase activity and granulocyte receptor 1 immunohistochemistry) were significantly attenuated after PS-OMe miR130 treatment. Finally, apoptosis significantly decreased in liver tissue after treatment. CONCLUSION: PS-OMe miR130 decreases eCIRP-mediated injury and inflammation in a murine model of hepatic I/R.

Extracellular CIRP dysregulates macrophage bacterial phagocytosis in sepsis.

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 ßPIX, a Rac1 activator. We further demonstrated that STAT3 and ßPIX formed a complex following rmCIRP treatment, preventing ßPIX 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-ßPIX 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.

H151, A SMALL MOLECULE INHIBITOR OF STING AS A NOVEL THERAPEUTIC IN INTESTINAL ISCHEMIA-REPERFUSION INJURY.

BACKGROUND: Intestinal ischemia-reperfusion (I/R) injury is a severe disease associated with high mortality. Stimulator of interferon genes (STING) is an intracellular protein that is activated by cytosolic DNA and is implicated in I/R injury, resulting in transcription of type I interferons (IFN-α and IFN-ß) and other proinflammatory molecules. Extracellular cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, induces STING activation. H151 is a small molecule inhibitor of STING that has not yet been studied as a potential therapeutic. We hypothesize that H151 reduces inflammation, tissue injury, and mortality after intestinal I/R. Methods: In vitro, RAW264.7 cells were pretreated with H151 then stimulated with recombinant murine (rm) CIRP, and IFN-ß levels in the culture supernatant were measured at 24 hours after stimulation. In vivo, male C57BL/6 mice were subjected to 60-minute intestinal ischemia via superior mesenteric artery occlusion. At the time of reperfusion, mice were intraperitoneally instilled with H151 (10 mg/kg BW) or 10% Tween-80 in PBS (vehicle). Four hours after reperfusion, the small intestines, lungs, and serum were collected for analysis. Mice were monitored for 24 hours after intestinal I/R to assess survival. Results: In vitro, H151 reduced rmCIRP-induced IFN-ß levels in a dose-dependent manner. In vivo, intestinal levels of pIRF3 were increased after intestinal I/R and decreased after H151 treatment. There was an increase in serum levels of tissue injury markers (lactate dehydrogenase, aspartate aminotransferase) and cytokine levels (interleukin 1ß, interleukin 6) after intestinal I/R, and these levels were decreased after H151 treatment. Ischemia-reperfusion-induced intestinal and lung injury and inflammation were significantly reduced after H151 treatment, as evaluated by histopathologic assessment, measurement of cell death, chemokine expression, neutrophil infiltration, and myeloperoxidase activity. Finally, H151 improved the survival rate from 41% to 81% after intestinal I/R. Conclusions: H151, a novel STING inhibitor, attenuates the inflammatory response and reduces tissue injury and mortality in a murine model of intestinal I/R. H151 shows promise as a potential therapeutic in the treatment of this disease.

Necroptosis-Mediated eCIRP Release in Sepsis.

Introduction: Extracellular cold-inducible RNA-binding protein (eCIRP) is an endogenous pro-inflammatory mediator that exacerbates injury in inflammation and sepsis. The mechanisms in which eCIRP is released have yet to be fully explored. Necroptosis is a programmed cell death that is dependent on the activation of mixed lineage kinase domain-like pseudo kinase (MLKL) which causes the release of damage-associated molecular patterns. We hypothesize that eCIRP is released through necroptosis and intensifies inflammation in sepsis. Methods: RAW264.7 cells were treated with pan-caspase inhibitor z-VAD (15 µM) 1 h before stimulation with LPS (1 µg/mL). Necroptosis inhibitor, Necrostatin-1 (Nec-1) (10 µM) was added to the cells with LPS simultaneously. After 24 h of LPS stimulation, cytotoxicity was determined by LDH assay. eCIRP levels in the culture supernatants and phospho-MLKL (p-MLKL) from cell lysates were assessed by Western blot. p-MLKL interaction with the cell membrane was visualized by immunofluorescence. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Mice were treated with Nec-1 (1 mg/kg) or DMSO. 20 h post-surgery, serum and peritoneal fluid levels of eCIRP, TNF-α and IL-6 were determined by ELISA. H&E staining of lung tissue sections was performed. Results: We found that in RAW264.7 cells, LPS+z-VAD induces necroptosis as evidenced by an increase in p-MLKL levels and causes eCIRP release. Nec-1 reduces both p-MLKL activation and eCIRP release in LPS+z-VAD-treated RAW264.7 cells. Nec-1 also inhibits the release of eCIRP, TNF-α and IL-6 in the serum and peritoneal fluid in CLP-induced septic mice. We predicted a transient interaction between eCIRP and MLKL using a computational model, suggesting that eCIRP may exit the cell via the pores formed by p-MLKL. Conclusion: Necroptosis is a novel mechanism of eCIRP release in sepsis. Targeting necroptosis may ameliorate inflammation and injury in sepsis by inhibiting eCIRP release.

Neutrophil phenotypes and functions in cancer: A consensus statement.

Neutrophils are the first responders to infection and inflammation and are thus a critical component of innate immune defense. Understanding the behavior of neutrophils as they act within various inflammatory contexts has provided insights into their role in sterile and infectious diseases; however, the field of neutrophils in cancer is comparatively young. Here, we summarize key concepts and current knowledge gaps related to the diverse roles of neutrophils throughout cancer progression. We discuss sources of neutrophil heterogeneity in cancer and provide recommendations on nomenclature for neutrophil states that are distinct in maturation and activation. We address discrepancies in the literature that highlight a need for technical standards that ought to be considered between laboratories. Finally, we review emerging questions in neutrophil biology and innate immunity in cancer. Overall, we emphasize that neutrophils are a more diverse population than previously appreciated and that their role in cancer may present novel unexplored opportunities to treat cancer.

Inhibition of the Interaction of TREM-1 and eCIRP Attenuates Inflammation and Improves Survival in Hepatic Ischemia/Reperfusion.

INTRODUCTION: Triggering receptor expressed on myeloid cells-1 (TREM-1) has important implications in sepsis and inflammation and is a novel receptor for extracellular cold-inducible RNA-binding protein (eCIRP). We hypothesize that the inhibition of TREM-1 via its interaction with eCIRP by novel peptide inhibitor M3 or knockout gene will attenuate the inflammation and injury associated with severe hepatic ischemia/reperfusion (I/R). METHODS: Wild-type (WT) C57BL/6 and TREM-1-/- mice underwent 60 min of 70% hepatic ischemia, with 24 h of reperfusion. Additionally, WT mice underwent hepatic I/R and were treated with M3 (10 mg/kg body weight) or vehicle (normal saline) at the start of reperfusion. Blood and ischemic liver tissues were collected, and analysis was performed using enzymatic assays, enzyme-linked immunosorbent assay, reverse-transcription quantitative polymerase chain reaction, and pathohistology techniques. For survival surgery, mice additionally underwent resection of non-ischemic lobes of the liver and survival was monitored for 10 days. RESULTS: There was an increase in serum levels of tissue markers including aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase as well as cytokine levels (IL-6) and histological scoring of hematoxylin and eosin sections in WT I/R mice. These markers decreased substantially in TREM-1-/- mice. Additionally, neutrophil infiltration markers and markers of local inflammation (myeloperoxidase, macrophage inflammatory protein-2, cyclooxygenase-2) were attenuated in TREM-1-/- mice. Similarly, we show a significant decrease in injury and inflammation markers with M3 treatment. Additionally, we demonstrate decreased apoptosis with TREM-1 inhibition. Finally, M3 treatment improved the survival rate from 42% to 75% after hepatic I/R. CONCLUSION: TREM-1 is an important eCIRP receptor in the inflammatory response of hepatic I/R, and deficiency of TREM-1 via knockout gene or peptide inhibition attenuated liver injury and inflammation, and improved survival. Inhibition of the TREM-1 and eCIRP interaction in hepatic I/R may have important therapeutic potential.

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.

Extracellular CIRP decreases Siglec-G expression on B-1a cells skewing them towards a pro-inflammatory phenotype in sepsis.

BACKGROUND: Sepsis is a life-threatening disease syndrome caused by a dysregulated host response to infection and injury. Extracellular cold-inducible RNA-binding protein (eCIRP) acts as a damage-associated molecular pattern. Peritoneal cavity (PerC) B-1a cells attenuate inflammation and tissue injury by spontaneous releasing natural IgM and IL-10. Sialic acid-binding immunoglobulin-type lectin-G (Siglec-G) is a CD33-related receptor highly expressed in B-1a cells to serve critical immunoregulatory functions. In sepsis, B-1a cell numbers in PerC are decreased. We hypothesized that eCIRP causes the reduction of PerC B-1a cells and alters their function during sepsis. METHODS: Sepsis was induced in WT and CIRP-/- mice by cecal ligation and puncture (CLP). PerC washout cells were collected and B-1a cells and Siglec-G were assessed by flow cytometry. Mice were i.p. injected with recombinant murine (rm) CIRP and after 20 h, Siglec-G expression in PerC B-1a cells were assessed. PerC B-1a cells were treated with rmCIRP for 4 h and Siglec-G expression was assessed. PerC B-1a cells were pre-treated with anti-Siglec-G Ab and then after stimulated with rmCIRP for 24 h, IL-6 levels in the culture supernatants were assessed. RESULTS: eCIRP levels in the PerC were elevated in septic mice. In WT mice, the frequencies and numbers of total and Siglec-G+ B-1a cells in the PerC were significantly decreased in the CLP group compared to sham group, whereas in CIRP-/- mice, their frequencies and numbers in sepsis were significantly rescued compared to WT septic mice. Mice injected with rmCIRP showed decreased frequencies and numbers of total and Siglec-G+ PerC B-1a cells compared to PBS-injected mice. In vitro treatment of PerC B-1a cells with rmCIRP demonstrated significant reduction in Siglec-G mRNA and protein compared to PBS group. PerC B-1a cells treated with anti-Siglec-G Ab had significantly higher production of IL-6 in response to rmCIRP compared to IgG control. Anti-Siglec-G Ab treated B-1a cells co-cultured with macrophages produced significantly higher levels of IL-6, and TNF-α, and lower levels of IL-10 compared to IgG-treated B-1a cells and macrophage co-cultures stimulated with rmCIRP. CONCLUSION: eCIRP reduces PerC B-1a cell pool and skews them to a pro-inflammatory phenotype by downregulating Siglec-G expression. Targeting eCIRP will retain Siglec-G expressing B-1a cells in the PerC and preserve their anti-inflammatory function in sepsis.

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.

The Role of Siglec-G on Immune Cells in Sepsis.

Sepsis is a life-threatening clinical syndrome that results from an overwhelming immune response to infection. During sepsis, immune cells are activated by sensing pathogen-associated molecular patterns and damage-associated molecular patterns (DAMPs) through pattern recognizing receptors (PRRs). Regulation of the immune response is essential to preventing or managing sepsis. Sialic acid-binding immunoglobulin-type lectin-G (Siglec-G), a CD33 group of Siglec expressed in B-1a cells and other hematopoietic cells, plays an important immunoregulatory role. B-1a cells, a subtype of B lymphocytes, spontaneously produce natural IgM which confers protection against infection. B-1a cells also produce IL-10, GM-CSF, and IL-35 to control inflammation. Sialic acids are present on cell membranes, receptors, and glycoproteins. Siglec-G binds to the sialic acid residues on the B cell receptor (BCR) and controls BCR-mediated signal transduction, thereby maintaining homeostasis of Ca++ influx and NFATc1 expression. Siglec-G inhibits NF-κB activation in B-1a cells and regulates B-1a cell proliferation. In myeloid cells, Siglec-G inhibits DAMP-mediated inflammation by forming a ternary complex with DAMP and CD24. Thus, preserving Siglec-G's function could be a novel therapeutic approach in sepsis. Here, we review the immunoregulatory functions of Siglec-G in B-1a cells and myeloid cells in sepsis. A clear understanding of Siglec-G is important to developing novel therapeutics in treating sepsis.

Extracellular CIRP Induces Macrophage Extracellular Trap Formation Via Gasdermin D Activation.

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.

The vitals of NETs.

Neutrophils produce neutrophil extracellular traps (NETs) by expelling their extracellular chromatin embedded with citrullinated histone H3, myeloperoxidase, and other intracellular molecules. Since their discovery in 2004, numerous articles have demonstrated the mechanism of NET formation and their function in innate immunity and inflammation. NET components often play an antimicrobial role, but excessive NETs are deleterious and can cause inflammation and tissue damage. This review highlights recent advancements in the identification of novel pathways and mechanisms of NET formation. We also focus on the specific damaging impact of NETs in individual organs. We then discuss the progress and limitations of various NET detection assays. Collectively, these vital aspects of NETs significantly improve our understanding of the pathobiology of NETs and future diagnostics and therapeutic tools for examining and modulating NETs in inflammatory diseases.

Inhibition of Efferocytosis by Extracellular CIRP-Induced Neutrophil Extracellular Traps.

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) αvß3 or αvß5 integrin by the NETs. We identified neutrophil elastase in the NETs significantly inhibited efferocytosis by cleaving macrophage surface integrins αvß3 and αvß5 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 αvß3/αvß5 integrins in macrophages. Targeting eCIRP ameliorates sepsis by enhancing efferocytosis.

Targeting the eCIRP/TREM-1 interaction with a small molecule inhibitor improves cardiac dysfunction in neonatal sepsis.

BACKGROUND: Neonatal sepsis and the associated myocardial dysfunction remain a leading cause of infant mortality. Extracellular cold-inducible RNA-binding protein (eCIRP) acts as a ligand of triggering receptor expressed on myeloid cells-1 (TREM-1). M3 is a small CIRP-derived peptide that inhibits the eCIRP/TREM-1 interaction. We hypothesize that the eCIRP/TREM-1 interaction in cardiomyocytes contributes to sepsis-induced cardiac dysfunction in neonatal sepsis, while M3 is cardioprotective. METHODS: Serum was collected from neonates in the Neonatal Intensive Care Unit (NICU). 5-7-day old C57BL/6 mouse pups were used in this study. Primary murine neonatal cardiomyocytes were stimulated with recombinant murine (rm) CIRP with M3. TREM-1 mRNA and supernatant cytokine levels were assayed. Mitochondrial oxidative stress, ROS, and membrane potential were assayed. Neonatal mice were injected with rmCIRP and speckle-tracking echocardiography was conducted to measure cardiac strain. Sepsis was induced by i.p. cecal slurry. Mouse pups were treated with M3 or vehicle. After 16 h, echocardiography was performed followed by euthanasia for tissue analysis. A 7-day survival study was conducted. RESULTS: Serum eCIRP levels were elevated in septic human neonates. rmCIRP stimulation of cardiomyocytes increased TREM-1 gene expression. Stimulation of cardiomyocytes with rmCIRP upregulated TNF-α and IL-6 in the supernatants, while this upregulation was inhibited by M3. Stimulation of cardiomyocytes with rmCIRP resulted in a reduction in mitochondrial membrane potential (MMP) while M3 treatment returned MMP to near baseline. rmCIRP caused mitochondrial calcium overload; this was inhibited by M3. rmCIRP injection impaired longitudinal and radial cardiac strain. Sepsis resulted in cardiac dysfunction with a reduction in cardiac output and left ventricular end diastolic diameter. Both were improved by M3 treatment. Treatment with M3 attenuated serum, cardiac, and pulmonary levels of pro-inflammatory cytokines compared to vehicle-treated septic neonates. M3 dramatically increased sepsis survival. CONCLUSIONS: Inhibition of eCIRP/TREM-1 interaction with M3 is cardioprotective, decreases inflammation, and improves survival in neonatal sepsis. Trial registration Retrospectively registered.