Sepsis remains a leading cause of death. Reverse migrated neutrophils, characterized as ICAM1hiCXCR1lo, have been recognized as a key driver of systemic inflammation and organ injury in sepsis. We discovered a distinct delta-like ligand 4-positive (DLL4+) subset of neutrophils that accumulate in the lungs, contributing to lung injury; however, the underlying mechanism is less understood. In sepsis, ICAM1hiCXCR1lo neutrophils, being hyperactive, are detrimental. Here, we investigated how DLL4+ neutrophils activate alveolar macrophages (AMs) to cause endothelial cell barrier disruption and promote neutrophil reverse migration. AMs were treated with DLL4+ neutrophils or recombinant mouse DLL4, and a disintegrin and metalloprotease (ADAM17) generated by AMs was assessed at both mRNA and protein levels. Conditioned medium was subsequently applied to pulmonary vascular endothelial cells (pVECs); junctional adhesion molecule-C (JAM-C) protein was detected by Western blot assays; and ICAM1hiCXCR1lo neutrophils were detected by flow cytometry. We demonstrate that during sepsis induced by cecal ligation and puncture (CLP), DLL4+ neutrophils interact with AMs via the Notch1 pathway, leading to increase of ADAM17 expression. ADAM17 decreased JAM-C on pVECs, causing endothelial barrier disruption and ICAM1hiCXCR1lo neutrophil generation. A small-molecule inhibitor of ADAM17 effectively preserved pulmonary endothelial barrier integrity and reduced ICAM1hiCXCR1lo neutrophil accumulation. Importantly, we have developed a novel DLL4-Notch1 inhibitory peptide (NDI) that effectively suppresses ADAM17 expression, restores JAM-C, and reduces ICAM1hiCXCR1lo neutrophil accumulation in sepsis. These findings identify DLL4+ neutrophils as critical inflammatory mediators that exacerbate systemic inflammation and worsen sepsis, and highlight the DLL4-Notch1-ADAM17 axis as a promising therapeutic target.

Exposure of healthy tissue to ionizing radiation (IR) occurs due to nuclear accidents and terrorism, as well as radiotherapy. The vascular endothelium is a key target of IR, and microvascular endothelial cells (ECs) are particularly vulnerable to radiation. IR induces EC activation leading to endothelial cell injury. Human ghrelin is a stomach-derived peptide with pleiotropic effects, including protection against inflammation. We hypothesize that human ghrelin improves survival in total body irradiation (TBI) and that ghrelin’s protective effect could be mediated by attenuating endothelial cell injury. To test this, mice were exposed to TBI and after 24 h were treated subcutaneously with human ghrelin once daily for 4 days and monitored for 30 days. The survival rate of the human ghrelin-treated group was significantly higher than that of the vehicle group. Subsequently, human ghrelin treatment showed an effective dose modification factor of 1.0681. On day 4 after TBI, human ghrelin significantly attenuated EC permeability in the lungs and improved tight junction protein ZO-1 expression. Human ghrelin also improved ZO-1 and Claudin5 expression in primary mouse lung vascular endothelial cells. Taken together, these results indicate that human ghrelin improves survival after TBI, and its survival benefit is in part due to the attenuation of EC permeability and microvascular barrier dysfunction.

The gut has a profound influence on the liver through their anatomical connection via the portal vein. During acute inflammation, gut tissue injury leads to increased barrier permeability, allowing the translocation of external contents that can affect hepatic function. Gut–liver crosstalk contributes to the pathophysiology of acute inflammatory disorders, such as sepsis, intestinal ischaemia–reperfusion, hepatitis and drug-induced liver injury. This organ-to-organ crosstalk is mediated by the microbiome, pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs) and various proinflammatory mediators. Different types of gut and liver resident cells as well as circulating cells also facilitate inflammatory gut–liver crosstalk. These cell types include intestinal epithelial and myeloid cells, Kupffer cells, sinusoidal endothelial cells, hepatic stellate cells, hepatocytes, lymphocytes and neutrophils. PAMPs and DAMPs activate pattern recognition receptors, such as Toll-like receptors, on various cells, leading to proinflammatory signal transduction, including NFκB activation, cytokine and chemokine production, and NETosis. Collectively, these soluble and cellular factors exacerbate acute inflammation and tissue injury via the gut–liver axis, leading to poor outcomes in critically ill patients. Potential therapeutic interventions for this deadly clinical condition include modulation of the microbiome and pharmacological inhibition of proinflammatory mediators and cellular interactions. In this article we review the pathophysiology of inflammatory gut–liver crosstalk and potential therapeutic interventions. The gut–liver axis plays a crucial role in maintaining homeostasis and is implicated in various diseases. This review explores the complex interactions between the gut and liver, particularly during acute inflammation. The authors highlight the role of gut microorganisms, microenvironment and immune cells in mediating this crosstalk. They describe how gut-derived pathogen-associated molecular patterns and damage-associated molecular patterns activate gut-resident immune cells, which subsequently interact with and activate hepatic cells, thereby exacerbating liver injury. Key findings include the identification of gut-primed neutrophils that migrate to the liver, activating Kupffer cells and causing hepatic damage. This understanding is significant as it underscores the potential of targeting the gut–liver axis therapeutically. Future directions may involve developing strategies to modulate the microbiome and inhibit proinflammatory pathways to mitigate acute liver injury during systemic inflammation. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

Sepsis and rheumatoid arthritis (RA) are distinct yet mechanistically related conditions commonly driven by dysregulated inflammatory responses. Here, we explored the counterintuitive hypothesis that an epitope from a deleterious anti-tetranectin (TN) antibody (mAb9) could hold unforeseen therapeutic potential. By mapping mAb9’s epitope to P2 (residues 55–70), a region crucial for TN’s protective functions, we developed P2-1, a water-soluble derivative as a targeted therapy. We then employed animal models of sepsis (cecal ligation and puncture) and arthritis (collagen antibody-induced arthritis) to evaluate the therapeutic effects of P2, P2-1, and a procathepsin L (pCTS-L)-neutralizing antibody by assessing septic survival, arthritis severity, pain sensitivity, and joint tissue histology. In parallel, we utilized a surface plasmon resonance (SPR) assay and computational modeling to examine the P2-1/high mobility group box 1 (HMGB1) interaction. Finally, we elucidate the effect of P2-1 on the HMGB1-induced release of pCTS-L and other cytokines and chemokines using primary human peripheral blood mononuclear cells (PBMCs). P2-1 significantly improved survival and reduced systemic inflammation in a sepsis model, and attenuated arthritis severity and pain sensitivity in an RA model, even with therapeutic administration after disease onset. Mechanistically, P2-1 exhibited high-affinity binding to HMGB1 and selectively suppressed HMGB1-induced cathepsin L (Ctsl) mRNA upregulation and pCTS-L secretion from human immune cells, crucially without perturbing other HMGB1-induced cytokines and chemokines. We further validated pCTS-L as a therapeutic target by demonstrating that a neutralizing antibody conferred potent antiarthritic effects, reducing joint inflammation, pain, and structural damage. Our findings introduce a paradigm-shifting drug discovery strategy that transforms insights from harmful antibody action into targeted therapeutics for the HMGB1-pCTS-L axis. This approach not only delivers P2-1 as a potent therapy but also establishes pCTS-L as a crucial mediator in inflammatory diseases such as sepsis and RA.

Acute kidney injury (AKI) from renal ischemia–reperfusion (IR) injury is a major cause of acute organ dysfunction with significant morbidity and mortality. Critically, up to 50

Background: Radiation-induced sepsis resulting from intestinal inflammation and injury is a severe complication of high-dose radiation exposure. High-dose radiation compromises the integrity of the intestinal lining, causing bacterial translocation, systemic inflammation, and sepsis. Extracellular cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, plays a pivotal role in the pathogenesis of inflammatory diseases and contributes to organ injury. C23, a small molecular peptide antagonist of eCIRP, has demonstrated anti-inflammatory and organ-protective effects during organ-injury indications. However, its role in radiation-induced inflammation and injury is not known. The objective of this study is to evaluate whether C23 mitigates inflammation and injury, leading to improved survival in a murine model of partial-body irradiation (PBI) combined with sepsis. Methods: Mice were exposed to 10 Gy PBI, and at 48 h, they were subjected to cecal ligation and puncture (CLP), a well-established model of sepsis. C23 (8 mg/kg body weight [BW]) or vehicle (saline) was administered subcutaneously at 24 h and 48 h after PBI. Blood and intestinal tissue samples were collected 20 h after CLP (i.e., 68 h post-PBI) for various analyses. In another set of mice after PBI–sepsis, intestinal permeability was also assessed. In an additional cohort of mice subjected to the same experimental procedure, 10-day survival was monitored. Results: Our findings demonstrate that administration of C23 attenuated systemic inflammatory responses, intestinal permeability, intestinal injury, and apoptosis; increased crypt cell proliferation and improved survival after PBI–sepsis. Conclusions: These results reveal a novel protective role of the eCIRP antagonist, C23, in mitigating PBI–sepsis-induced inflammation and injury and represents a promising therapeutic candidate for the treatment of radiation combined injury with sepsis.

Cranial radiotherapy is associated with neuroinflammation, cognitive dysfunction, and dementia. Tau pathology plays a key role in Alzheimer’s disease (AD) but has not been studied in the context of radiation injury. Extracellular cold-inducible RNA-binding protein (eCIRP) is a novel neuroinflammatory mediator that activates neuronal IL6Rα/p25/Cdk5, leading to tau hyperphosphorylation. We hypothesized that radiation promotes pathological tau phosphorylation via the eCIRP/IL6Rα/p25/Cdk5 pathway, which can be mitigated by the eCIRP inhibitor C23. Adult C57BL/6 and CIRP−/− mice were exposed to a single dose of 10-Gy X-rays at a dose-rate of 1 Gy/min. Hippocampal tissue lysates were analyzed for p25, phosphorylated tau, and CIRP using Western blotting. Cdk5 activity was assessed utilizing a modified Cdk5/p35 kinase enzyme system. Serum and cerebrospinal fluid (CSF) eCIRP was quantified using ELISA. eCIRP (1 µg) was injected intracerebroventricularly (icv) and C23 (8 mg/kg BW) retro-orbitally. N2a and primary neurons were pre-treated with 3 μg/ml IL-6Rα neutralizing Abs or IgG control, or 25 µg/mL C23 and stimulated with up to 2.5 μg/mL eCIRP for 48 h. Irradiation significantly increased hippocampal p25 expression, Cdk5 activity, and tau phosphorylation [AT-8 (Ser202/Thr205/Ser208), and combined Ser199/Ser202 and Ser396 p-tau antibodies] at 48 h post-irradiation. eCIRP increased by 1.2-fold in the serum and 2.6-fold in the CSF, and CIRP increased by 65

Objective To investigate the effects of kangaroo mother care combined with massage therapy on heart rate, oxygen saturation, perfusion index (PI), systolic blood pressure in extremely premature infants, and the impact on parents’ satisfaction with care. Methods A total of 74 extremely premature infants admitted to the Neonatal Intensive Care Unit (NICU) of Xinxiang Central Hospital from January 2024 to January 2025 were selected as the study subjects and randomly divided into the control group and the intervention group. The control group received routine care, while the intervention group received kangaroo mother care combined with massage therapy. Kangaroo mother care was performed once daily for 2 hours, lasting for 4 weeks, and massage therapy was conducted during kangaroo mother care, lasting 10 minutes for 2 times. The heart rate, oxygen saturation, PI value, and systolic blood pressure of the two groups of premature infants were compared on the 1st, 7th, 14th, 21st, and 28th days of intervention, and the parents’ satisfaction with care at discharge was compared between the two groups. Results 1. On the 7th, 14th, 21st, and 28th days of intervention, the PI value and oxygen saturation of the premature infants in the intervention group were significantly higher than those in the control group. 2. At discharge, the parents’ satisfaction with care in the intervention group was significantly higher than that in the control group. Conclusion Kangaroo mother care combined with massage therapy can significantly increase the oxygen saturation in extremely premature infants, improve peripheral circulation perfusion, and enhance parents’ satisfaction with care.

Abstract Introduction Sepsis is a severe systemic inflammatory condition. Over half of the septic patients develop acute lung injury (ALI), worsening mortality. Inflammasome activation triggers procaspase-1 cleavage to active Caspase-1 (p10/p20 tetramer), which then cleaves GSDMD, forming membrane pores for the release of DAMPs. We hypothesize that Caspase-1 is released via GSDMD pores, and extracellular Caspase-1 (eCasp-1) exacerbates ALI in sepsis. Methods Plasma from surgical intensive care unit patients and healthy controls was analyzed for eCasp-1 by ELISA. Sepsis was induced in mice by cecal ligation and puncture, and their plasma was assessed for eCasp-1. Murine peritoneal macrophages were treated with LPS+Nigerecin ± GSDMD inhibitor disulfiram, and eCasp-1 levels were assessed. The eCasp-1/TLR4 interaction was assessed by BIAcore, and the TLR4-mediated effects of eCasp-1 were evaluated using TLR4-/- mice. A small peptide, C16, was developed to interrupt eCasp-1/TLR4 interaction, and its therapeutic effects on ALI were evaluated in septic mice. Results We discovered that the p20 subunit of Caspase-1 (termed eCasp-1) significantly increased in the blood of patients and septic mice but is undetectable in healthy controls or sham mice. We determined that eCasp-1 is released via GSDMD pores. We identified a strong affinity between eCasp-1 and TLR4, demonstrating that eCasp-1-induced inflammation occurs via TLR4. eCasp-1 administration (i.p.) significantly increased systemic inflammatory and organ injury markers in WT mice. These effects were significantly reduced in TLR4-/- mice. In septic mice, C16 treatment significantly decreased blood injury and inflammatory markers, reduced lung chemokine/cytokine expression, and decreased histologic lung injury. C16 treatment significantly improved the survival rate in sepsis from 40% to 80%. Conclusion eCasp-1 is a novel DAMP that exacerbates ALI in sepsis. Targeting eCasp-1 with C16 attenuates ALI and improves survival, suggesting a novel therapeutic approach for sepsis. Funding Source National Institutes of Health (NIH) grants R35GM118337, and R01HL076179 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Gut ischemia/reperfusion (I/R) injury releases damage-associated molecular patterns (DAMPs), such as extracellular cold-inducible RNA-binding protein (eCIRP). Milk fat globule–epidermal growth factor VIII-derived oligopeptide 3 (MOP3) is a novel peptide enabling macrophage uptake of eCIRP via αvβ3-integrin. MOP3 reduces inflammation in gut I/R, but its mechanisms are not completely understood. We hypothesized MOP3 promotes macrophage polarization toward an anti-inflammatory, M2-like phenotype in gut I/R. We induced gut I/R in mice through 60 min of superior mesenteric artery occlusion followed by 4 h of reperfusion. Intestines were evaluated for macrophage polarization by flow cytometry and immunofluorescence histology. Peritoneal cavity macrophages were isolated from mice and treated with eCIRP, MOP3, αvβ3-antibody, and/or naïve IgG for 4 or 24 h. Polarity was assessed by flow cytometry, qPCR, and ELISA. Compared to the sham, the M2 proportion after gut I/R decreased by 22.7%, and the M1 proportion increased by 241%. MOP3 treatment increased the M2 proportion by 64.3%, and the M1 proportion decreased by 22.7%. In eCIRP-stimulated macrophages, MOP3 treatment increased M2-like and reduced M1-like cell-surface markers, gene expression, and cytokine levels. αvβ3 antibody dramatically reduced MOP3′s effects. MOP3 promotes M2 polarization through αvβ3 integrin-mediated clearance of eCIRP, a novel mechanism whereby MOP3 reduces gut I/R injury.

Introduction:The mechanism by which extracellular cold-inducible RNA-binding protein (eCIRP) aggravates renal ischemia/reperfusion (RIR) injury leading to acute kidney injury (AKI) is poorly understood. The natural killer group 2D (NKG2D) receptor and its ligand MULT-1 are key immunoregulatory mechanisms promoting responses to damaged and inflamed cells. Methods:We subjected wild-type and CIRP-/- mice to RIR. We then used immunohistochemistry (IHC), flow cytometry, and Western blotting to assess NKG2D and MULT-1 in kidney tissues, macrophages, and renal tubular epithelial cells (RTECs), and ELISA to assess TNFa and IL-6. Results:The expression levels of NKG2D and its ligand MULT-1 were significantly elevated in wild-type mice subjected to RIR compared with sham. In contrast, CIRP-/- mice exhibited markedly reduced expression of both NKG2D and MULT-1 after RIR compared to wild-type mice. In vitro, eCIRP stimulated the expression of NKG2D in peritoneal macrophages and of MULT-1 in RTECs. Treatment of eCIRP-stimulated peritoneal macrophage and RTEC co-cultures with an NKG2D-neutralizing antibody significantly and markedly downregulated supernatant levels of TNFa and IL-6. Discussion:In conclusion, eCIRP induces NKG2D+ macrophages and MULT-1+ RTECs, and their interaction further increases the inflammatory response. Targeting the NKG2D/MULT-1 may reduce RIR-induced inflammation and thus attenuate AKI.

Sepsis is characterized by dysregulated immune responses induced by damage-associated molecular patterns, such as extracellular cold-inducible RNA-binding protein (eCIRP), that frequently lead to acute lung injury (ALI) and high mortality. Recently, a subset of CD4+ T cells possessing both T helper 1 (Th1) and regulatory T cell (Treg) phenotypes, termed Th1-Treg cells, has been identified; however, their function in sepsis remains unknown. In this study, we investigated the dynamics, induction mechanisms, and functional roles of Th1-Treg cells in the development of sepsis-induced ALI. Polymicrobial sepsis was induced in mice using cecal ligation and puncture. In vivo, Th1-Treg cell accumulation in the lungs was analyzed in WT and CIRP-/- mice following sepsis. In vitro, isolated CD4+ T cells from WT and TLR4-/- mice were treated with eCIRP to evaluate Th1-Treg cell differentiation and downstream signaling pathways. STAT1 and STAT5 activation were evaluated, and pharmacological inhibitors were used to assess their involvement. Adoptive transfer of Th1-Treg cells was conducted to determine their functional impact on ALI and mortality in septic mice. We observed a significant accumulation of Th1-Treg cells in the lungs of WT septic mice compared to sham mice. eCIRP drove the induction of Th1-Treg cells in vitro, and CIRP-/- mice exhibited decreased Th1-Treg cell accumulation in the lungs compared to WT mice after sepsis. In parallel to Th1-Treg cell induction, eCIRP activated signal transducer and activator of transcription, STAT1 and STAT5. Both the induction of Th1-Treg cells and the activation of STAT1/5 proteins were significantly attenuated in TLR4-/- mice. Furthermore, pharmacological inhibition of STAT1/5 signaling significantly reduced eCIRP-induced Th1-Treg cell differentiation. Intriguingly, adoptive transfer of Th1-Treg cells significantly exacerbated ALI, resulting in increased mortality in sepsis. Our findings indicate Th1-Treg cells induced by the eCIRP-TLR4-STAT1/5 axis aggravate ALI, worsening mortality in sepsis. Targeting these pathogenic cells potentially alleviates sepsis-induced ALI.

Gastrointestinal acute radiation syndrome (GI-ARS) is a deadly consequence of radiation exposure. We hypothesized that the peptide ghrelin is enteroprotective after radiation injury, and that ghrelin promotes intestinal stem cell regeneration via the vagus nerve. We subjected mice to 12-Gy partial body irradiation (PBI) with 5% bone marrow sparing. Some mice were vagotomized prior to PBI. We then injected the mice with human ghrelin (6 nmol/mouse) or vehicle at 24, 48, and 72 h post-irradiation, and collected blood and tissues at 96 h. PBI caused an 80% reduction in plasma citrulline, 32% shorter villi, 59% fewer crypts, a 14-fold increase in TUNEL+ cells, a 9-fold increase in intestinal permeability (FD4), and 4- to 30-fold increases in bacterial translocation (16S rRNA) to the liver and mesentery. Ghrelin significantly improved all these parameters. Remarkably, vagotomy attenuated ghrelin's protective effects by 22-58%. Mechanistically, ghrelin increased proliferating crypt cells by 2.3-fold, Lgr5+ active stem cells by 2.6-fold, Clu+ revival stem cells by 3.3-fold (immunofluorescence), and Clu mRNA by 1.6-fold compared to PBI alone, and all these effects were significantly diminished by vagotomy. Thus, ghrelin mitigates GI-ARS through vagus nerve-dependent activation of Clu+ revival stem cells and Lgr5+ stem cells, identifying a novel vagal-dependent neuroenteric pathway that regulates intestinal crypt regeneration after radiation injury.

Efferocytosis, the phagocytic clearance of dying cells, by microglia is crucial for limiting neuroinflammation and promoting resolution in ischemic stroke. Extracellular cold-inducible RNA-binding protein (eCIRP) is an inflammatory mediator that impairs macrophage bacterial phagocytosis in sepsis and radiation injury, but its role in microglial efferocytosis in ischemic stroke has not yet been studied. Using a transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke, this study demonstrated that eCIRP is released into the cerebrospinal fluid and microglial expression of the crucial efferocytic receptor MerTK decreases in tMCAO mice. CIRP deficiency significantly improved MerTK expression and microglial efferocytosis in tMCAO mice, reducing brain infarction, inflammation, neurological deficit, and survival in acute stroke. eCIRP induces pro-inflammatory micro-RNA 155 (miR-155) via TLR4, which suppresses its target pro-efferocytic transcription factor MAF bZIP (MafB), downregulating MerTK and the downstream cytoskeletal regulators, to impair microglial efferocytosis. Pharmacological blockade of eCIRP-TLR4 interaction using small peptide C23 attenuates miR-155 induction, restores MerTK expression, rescues microglial efferocytosis, and improves outcomes in tMCAO mice. This study uncovers a previously unknown pathway through which eCIRP signaling impairs neuroprotective efferocytic microglial function in ischemic stroke, suggesting that targeting eCIRP may promote functional recovery after stroke.

Gut-liver crosstalk exacerbates hepatic injury in gut ischemia/reperfusion (I/R), but its mechanism and therapeutic intervention remain elusive. Given the important roles of neutrophils and gut-resident intraepithelial lymphocytes (IELs) during acute inflammation, we hypothesized that the interaction between these two cell types worsens liver injury during gut I/R. Gut I/R was induced in mice by occluding superior mesenteric artery (SMA) for 60 min, followed by 4 h resuscitation. Blood, intestine and liver tissues were collected for various analysis. Neutrophil extracellular traps (NETs) were determined by microscopy. Gut I/R mice were injected (i.p.) with DPX2 (1 µg/g BW) at the time of reperfusion. After 4 h, blood and liver tissues were collected for various analysis. We discovered that neutrophils in contact with IELs in gut I/R mice had increased ability to form NETs. As such, NETs+ neutrophils were increased in the portal vein blood of gut I/R mice compared to sham mice and the systemic blood, accompanied by increased NETs in the liver, indicating the migration of the activated neutrophils from the gut to the liver. Adoptive transfer of IEL-primed neutrophils into gut I/R mice exacerbated liver inflammation as indicated by increased liver tissue levels of inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), keratinocyte-derived chemokine (KC), and C-X-C motif chemokine ligand 2 (CXCL2). The interaction of neutrophils and IELs is known to be mediated via neutrophil CD112. Increased numbers of CD112+ neutrophils were observed in the gut epithelium after gut I/R. We have discovered a CD112-derived peptide, named DPX2, which inhibits the interaction between CD112 on neutrophils and its proinflammatory ligand CD226 on IELs. In vitro, DPX2 attenuated IEL-induced NETosis under inflammatory conditions. In vivo, the administration of DPX2 significantly decreased NET-forming neutrophils in the portal vein of gut I/R mice. In parallel to NETs inhibition, DPX2 administration significantly mitigated the gene expression of iNOS, IL-6, IL-1β, KC, and CXCL2 in the liver. Furthermore, the administration of DPX2 significantly attenuated liver injury as indicated by decreased serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), tissue injury score, and liver cell death. Neutrophil-gut IEL interaction mediates proinflammatory gut-liver crosstalk and the novel CD112-derived peptide DPX2 targeting this interaction has the potential to mitigate hepatic injury.

BACKGROUND:Intestinal ischemia/reperfusion triggers systemic inflammatory responses that contribute to acute lung injury. Enolase-1 is a glycolytic enzyme increasingly implicated in its proinflammatory functions and in tissue injury. We hypothesized that an Enolase-1 inhibitor, ENOblock treatment would attenuate acute lung injury and improve survival following intestinal ischemia/reperfusion. METHODS:Adult C57BL/6 mice were subjected to superior mesenteric artery occlusion for 1 hour followed by 4 hours of reperfusion. Mice received either vehicle or ENOblock (5 mg/kg body weight) via retro-orbital injection at reperfusion. Mice were harvested for blood, intestines, and lungs after 4 hours of reperfusion or euthanized at 36 hours for survival. Enolase-1 messenger RNA was measured by quantitative polymerase chain reaction, and enolase-1 protein was measured by Western blotting. Plasma cytokines and injury markers were quantified with enzyme-linked immunosorbent assay or colorimetric assays. Lung tissues were analyzed by quantitative polymerase chain reaction, histology, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assays. Neutrophil infiltration was assessed by myeloperoxidase activity. RESULTS:Intestinal ischemia/reperfusion significantly increased lung enolase-1 messenger RNA and protein expression. ENOblock attenuated intestinal injury and reduced epithelial cell death. ENOblock treatment also significantly reduced circulating inflammatory and injury markers, as evidenced by the decreased plasma levels of interleukin-6, lactate dehydrogenase, and alanine aminotransferase. In the lungs, ENOblock significantly reduced intestinal ischemia/reperfusion-induced messenger RNA expression of interleukin-6, keratinocyte-derived chemokine, and macrophage inflammatory protein-2, and decreased pulmonary myeloperoxidase activity. Histology showed reduced alveolar injury and decreased pulmonary terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling-positive cells in ENOblock-treated mice. ENOblock was found to improve survival after intestinal ischemia/reperfusion. CONCLUSION:Enolase-1 is upregulated in the lungs following intestinal ischemia/reperfusion. ENOblock administered at reperfusion attenuates intestinal and lung injury and improves survival, supporting it as a potential therapeutic strategy for mitigating ischemia/reperfusion-induced acute lung injury and mortality.

Rationale: Liver regeneration is regulated by both metabolic processes and immune responses. Nonetheless, there is limited comprehension of the mechanisms involved. PINK1/Parkin-mediated mitophagy has been well documented, the role and underlying alternative mechanism of PINK1/Parkin in regulating mitochondrial metabolism during liver regeneration remains unclear. Methods: Liver tissues from mice undergoing hepatectomy were utilized to evaluate the expression levels of PINK1/Parkin. Hepatocyte-specific PINK1 knockout and transgenic mouse models were generated to investigate the impact of PINK1 on regeneration. Mass spectrometry, co-immunoprecipitation, and ubiquitination assays were performed to explore the underlying molecular mechanisms. Results: We observed PINK1/Parkin expression was markedly upregulated in hepatic tissue following liver resection. PINK1 depletion in hepatocytes caused impaired liver regeneration. Moreover, mitochondrial calcium overload was found be responsible for restricted TCA by inhibiting succinate dehydrogenase activity in PINK1 deficient hepatocytes. Interestingly, PINK1 deficiency leads to succinate accumulation and release from hepatocytes, which impairs liver regeneration by restricting macrophage pro-repair phenotypes. This effect was further confirmed by enhanced regeneration in myeloid SUCNR1 knockout mice. Mechanistically, Sigma-1 is a molecular chaperone of the endoplasmic reticulum calcium channel IP3R, which helps maintain its normal functional conformation. Parkin was able to bind Sigma-1 through its UBL domain, facilitating its k48-linked ubiquitination, which promotes Sigma-1 degradation and subsequently suppressing calcium transfer from the ER to mitochondria at the mitochondrial-associated ER membrane. Conclusions: Collectively, PINK1/Parkin signaling regulates hepatocellular mitochondrial ATP and succinate production by modulating ER-mitochondria calcium transfer to promote liver regeneration, revealing a promising therapeutic target for liver regeneration.

Traumatic brain injury (TBI) frequently precipitates chronic psychological disorder, particularly anxiety, depression, post-traumatic stress disorder (PTSD), and increased suicide risk, that worsen with injury severity yet remain understudied relative to sensorimotor and cognitive deficits. As TBI severity increases, conventional interventions (pharmacotherapy, cognitive-behavioral therapy, exercise) lose efficacy, revealing an urgent need for novel treatments targeting affective and stress-related disorders. Neuromodulatory approaches such as transcranial magnetic stimulation (TMS), transcranial direct current stimulation (tDCS), deep brain stimulation (DBS), photobiomodulation (PBM), vagus nerve stimulation (VNS) and trigeminal nerve stimulation (TNS), offer a mechanism-based strategy to recalibrate dysfunctional neural circuits across the full spectrum of TBI severity. In this review, we present the first comprehensive review of 45 preclinical and clinical studies examining all six modalities in TBI. We delineate intervention-specific mechanisms encompassing enhancement of synaptic plasticity, restoration of large-scale network connectivity, normalization of neurotransmitter homeostasis, augmentation of cerebral perfusion, and attenuation of neuroinflammatory cascades, correlating these neurobiological changes with quantifiable improvements in affective symptoms and stress adaptation. Through rigorous mechanistic-to-clinical mapping, this analysis establishes an evidence-based framework for developing precision neuromodulation protocols and mechanism-targeted pharmacological interventions targeting TBI-associated psychological disorders.

Caspase-1 is known to function intracellularly. However, whether caspase-1 can be released extracellularly and if so, the mechanisms of its release and action remain unknown. Here, we identify that cleaved caspase-1 (p20), which we named extracellular caspase-1 (eCasp-1), is released from immune cells in association with gasdermin D (GSDMD) pores formation. We identified significantly elevated eCasp-1 levels in the blood of critically ill surgical ICU patients, and in the blood and peritoneal fluid of gut ischemia-reperfusion (I/R) injury mice. In vitro, hypoxia-reoxygenation promoted GSDMD-dependent eCasp-1 release, and pharmacological inhibition of GSDMD reduced this release, supporting a contributing role of GSDMD-mediated membrane permeabilization in eCasp-1 release. Gut I/R demonstrated robust GSDMD-dependent release of eCasp-1. Functionally, eCasp-1 engaged TLR4 on macrophages, eliciting robust inflammatory cytokine release and organ injury. Importantly, we developed a novel peptide, C16, designed to specifically inhibit the eCasp-1-TLR4 interaction. In gut I/R, C16 administration exerted therapeutic benefits, markedly reducing systemic inflammation, attenuating acute lung injury (ALI), and significantly improving the survival. Our findings identify eCasp-1 as a new alarmin, that contributes to ALI through TLR4 signaling, with GSDMD-dependent processes contributing to its extracellular release. Targeting eCasp-1 with C16 offers a promising therapeutic strategy against ALI in acute inflammation.

Macrophages are key innate immune cells in the host defense against pathogens. Ionizing radiation can impair macrophage functions such as phagocytosis and activate them, potentially exacerbating tissue injury. Macrophage extracellular traps (METs) are formed upon stimulation of macrophages with PAMPs or DAMPs. We hypothesized that macrophages exposed to ionizing radiation can release extracellular traps. Peritoneal macrophages were collected from C57BL/6 mice and subjected to 5 Gy radiation. We performed assays to detect METs, including the immunofluorescence of citrullination of histone H3 and cell-free DNA measurement in cell culture medium as well as cell death. The exposure of ionizing radiation killed a significant number of mouse peritoneal macrophages through pyroptosis, which was mediated by Gasdermin D (GSDMD). The onset of pyroptosis eventually caused METs by suicidal METosis via pyroptosis and vital METosis occurring in the cells surviving after exposure to radiation. We found that exposure of peritoneal macrophages to 5 Gy radiation significantly increased METosis, as revealed by increased levels of citrullinated histone H3 and an increased surface area of extracellular DNA surrounding the cells. We discovered that peptidyl arginine deiminase (PAD) 2 and 4 are required for peritoneal macrophages to generate extracellular traps in response to radiation exposure. Our data demonstrate that the ionizing radiation induces METs via the activation of GSDMD, and we confirmed the requirement of PADs for METosis after exposure to the ionizing radiation. Targeting METs may direct a new therapeutic strategy for mitigating radiation-induced tissue injury.