Cold-inducible RNA-binding Protein Research Articles

Unraveling the intricacies of cold-inducible RNA-binding protein: A comprehensive review.

Cold-inducible RNA-binding protein (CIRP) is a versatile RNA-binding protein, pivotal in modulating cellular responses to diverse stress stimuli including cold shock, ultraviolet radiation, hypoxia, and infections, with a principal emphasis on cold stress. The temperature range of 32-34°C is most suitable for CIRP expression. The human CIRP is an 18-21kDa polypeptide containing 172 amino acids coded by a gene located on chromosome 19p13.3. CIRP has an RNA-recognition motif (RRM) and an arginine-rich motif (RGG), both of which have roles in coordinating numerous cellular activities. CIRP itself also undergoes conformational changes in response to diverse environmental stress. Transcription factors such as hypoxia-inducible factor 1 alpha and nuclear factor-kappa B have been implicated in coordinating CIRP transcription in response to specific stimuli. The potential of CIRP to relocate from the nucleus to the cytoplasm upon exposure to different stimuli enhances its varied functional roles across different cellular compartments. The different functions include decreasing nutritional demand, apoptosis suppression, modulation of translation, and preservation of cytoskeletal integrity at lower temperatures. This review explores the diverse functions and regulatory mechanisms of CIRP, shedding light on its involvement in various cellular processes and its implications for human health and disease.

Cold inducible RNA binding protein-regulated mitochondria associated endoplasmic reticulum membranes-mediated Ca2+ transport play a critical role in hypothermia cerebral resuscitation

Cardiac arrest is a global health issue causing more deaths than many other diseases. Hypothermia therapy is commonly used to treat secondary brain injury resulting from cardiac arrest. Previous studies have shown that CIRP is induced in specific brain regions during hypothermia and inhibits mitochondrial apoptotic factors. However, the specific mechanisms by which hypothermia-induced CIRP exerts its anti-apoptotic effect are still unknown. This study aims to investigate the role of Cold-inducible RNA-binding protein (CIRP) in mitochondrial-associated endoplasmic reticulum membrane (MAM)-mediated Ca2+ transport during hypothermic brain resuscitation.We constructed a rat model of cardiac arrest and resuscitation and hippocampal neuron oxygen-glucose deprivation/reoxygenation model. We utilized shRNA transfection to interfere the expression of CIRP and observe the effect of CIRP on the structure and function of MAM.Hypothermia induced CIRP can reduce the apoptosis of hippocampal neurons, and improve the survival rate of rats. Hypothermia induced CIRP can reduce the expressions of calcium transporters IP3R and VDAC1 in MAM, reduce the concentration of calcium in mitochondria, decrease the expression of ROS, and stabilize the mitochondrial membrane potential. Immunofluorescence and immunocoprecipitation showed that CIRP could directly interact with IP3R-VDAC1 complex, thereby changing the structure of MAM, inhibiting calcium transportation and improving mitochondrial function in vivo and vitro.Both in vivo and in vitro experiments have confirmed that hypothermia induced CIRP can act on the calcium channel IP3R-VDAC1 in MAM, reduce the calcium overload in mitochondria, improve the energy metabolism of mitochondria, and thus play a role in neuron resuscitation. This study contributes to understanding hypothermia therapy and identifies potential targets for brain injury treatment.

Radiation-induced eCIRP impairs macrophage bacterial phagocytosis.

Macrophages are essential immune cells for host defense against bacterial pathogens after radiation injury. However, the role of macrophage phagocytosis in infection following radiation injury remains poorly examined. Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern that dysregulates host immune system responses such as phagocytosis. We hypothesized that radiation-induced eCIRP release impairs macrophage phagocytosis of bacteria. Adult healthy mice were exposed to 6.5-Gy total body irradiation (TBI). Primary peritoneal macrophages isolated from adult healthy mice were exposed to 6.5-Gy radiation. eCIRP-neutralizing monoclonal antibody (mAb) was added to the cell culture prior to irradiation. Bacterial phagocytosis by peritoneal macrophages was assessed using pHrodo Green-labeled E. coli 7 days after irradiation ex vivo and in vitro. Bacterial phagocytosis was also assessed after treatment with recombinant murine CIRP (rmCIRP). Rac1 and ARP2 protein expression in cell lysates and eCIRP levels in the peritoneal lavage were assessed by Western blotting. Bacterial phagocytosis by peritoneal macrophages was significantly decreased after irradiation compared to controls ex vivo and in vitro. Rac1 and ARP2 expression in the peritoneal macrophages were downregulated after TBI. TBI significantly increased eCIRP levels in the peritoneal cavity. rmCIRP significantly decreased bacterial phagocytosis in a dose-dependent manner. eCIRP mAb restored bacterial phagocytosis by peritoneal macrophages after irradiation. Ionizing radiation exposure impairs bacterial phagocytosis by macrophages after irradiation. Neutralization of eCIRP restores the phagocytic ability of macrophages after irradiation. Our findings elucidate a novel mechanism of immune dysfunction and provide a potential new therapeutic approach for limiting infection after radiation injury.

Inhibition of CIRBP represses the proliferation and migration of vascular smooth muscle cells via inhibiting Rheb/mTORC1 axis

The excessive migration and proliferation of vascular smooth muscle cells (VSMCs) plays a vital role in vascular intimal hyperplasia. CIRBP is involved in the proliferation of various cancer cells. This study was aimed to explore the role of CIRBP in the proliferation and migration of VSMCs. Adenovirus was used to interfere with cold-inducible RNA-binding protein (CIRBP) expression, while lentivirus was used to overexpress Ras homolog enriched in brain (Rheb). Western blotting and qRT-PCR were used to evaluate the expression of CIRBP, Rheb, and mechanistic target of rapamycin complex 1 (mTORC1) activity. The cell proliferation was determined by Ki67 immunofluorescence staining and CCK-8 assay. The wound healing assay was performed to assess cell migration. Additionally, immunohistochemistry was conducted to explore the role of CIRBP in intimal hyperplasia after vascular injury. We found that silencing CIRBP inhibited the proliferation and migration of VSMCs, decreased the expression of Rheb and mTORC1 activity. Restoration of mTORC1 activity via insulin or overexpression of Rheb via lentiviral transfection both attenuated the inhibitory effects of silencing CIRBP on the proliferation and migration of VSMCs. Moreover, Rheb overexpression abolished the inhibitory effect of silencing CIRBP on mTORC1 activity in VSMCs. CIRBP was upregulated in the injured carotid artery. Silencing CIRBP ameliorated intimal hyperplasia after vascular injury. In the summary, silencing CIRBP attenuates mTORC1 activity via reducing Rheb expression, thereby supressing the proliferation and migration of VSMCs and intimal hyperplasia after vascular injury.

Cold-inducible RNA-binding protein as a context-specific functional mediator of breast cancer metastasis.

3136 Background: Cold-inducible RNA-binding protein (CIRBP) is a stress-induced mRNA-binding protein associated with clinical outcomes in a variety of human disease states. CIRBP’s role as a prognostic biomarker in breast cancer (BC) has yet to be established. Methods: We describe a clinically annotated tissue micro-array cohort of 1406 hormone receptor positive (HR+) and 281 triple negative primary breast cancers (TNBC) stained by immunohistochemistry (IHC) for CIRBP. Statistical analyses were performed with the Kaplan-Meier estimator, as well as univariate and multivariate Cox proportional-hazards models. Multivariate models incorporated tumor size, lymph node status, grade and CIRBP expression levels. Co-primary endpoints were overall survival (OS) and progression-free survival (PFS). MDA-MB-231 TNBC cells and MDA-MB-361 HR+ cells were used for CIRBP knockout (KO) by CRISPR/Cas9, and subsequent mammary fat pad injections were performed in immunocompromised mice. Results: In N=281 primary TNBCs, high levels of CIRBP expression by IHC was associated with poor prognosis in multivariate analysis (OS: adjusted hazard ratio (aHR) 2.05, 95% confidence interval (CI) 1.24-3.41, P=0.005. PFS: aHR 2.46, 95% CI 1.33-4.57, P=0.004). However, in N=1406 HR+ primary BC, CIRBP expression was correlated with favorable prognosis (OS: aHR 0.927, 95% CI 0.88-0.98, P=0.05. PFS: aHR 0.904, 95% CI 0.85-0.96, P=0.002). CIRBP KO had minimal impact on mammary fat pad primary tumor growth in both MDA-MB-231 and MDA-MB-361 cell models. However, CIRBP KO resulted in the inhibition of efficient spontaneous metastasis to the liver and lungs in TNBC MDA-MB-231 cells but not HR+ MDA-MB-361 cells. Conclusions: CIRBP expression is associated with poor prognosis in TNBC but not HR+ BC patients, a finding validated with CRISPR/Cas9 KO in representative model systems. This finding highlights the prognostic significance of CIRBP in TNBC and suggests differential underlying mRNA targets bound and modulated by CIRBP in TNBC and HR+ BC, respectively.

Associations of cold-inducible RNA-binding protein with bacterial load, proinflammatory cytokines and mortality from pneumonia.

Cold-inducible RNA-binding protein (CIRP) is a damage-associated molecular pattern that plays a critical role in triggering inflammatory responses. It remains unknown whether CIRP is strongly associated with bacterial load, inflammatory response, and mortality in sepsis model. Pneumonia was induced in specific pathogen-free 8-9-week old male rats by injecting bacteria via puncture of the tracheal cartilage. The expressions of CIRP and proinflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β] in lung tissues, alveolar macrophages (AMs), plasma, and bronchoalveolar lavage fluid (BALF) were determined by reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. The numbers of bacteria recovered from the lungs were correlated with the bacterial loads injected and mortality. The expressions of CIRP increased sharply as the bacterial loads increased in the lung tissues and AMs. The amounts of TNF-α, IL-6 and IL-1β proteins synthesized were dependent on the bacterial load in the lung tissues. Releases of CIRP, TNF-α, IL-6, and IL-1β increased with the bacterial load in the blood plasma. The proteins confirmed similar patterns in the BALF. CIRP was strongly associated with the releases of TNF-α, IL-6, and IL-1β in the lung tissues, blood plasma, and BALF, and showed a close correlation with mortality. CIRP demonstrated a strong association with bacterial load, which is new evidence, and close correlations with proinflammatory cytokines and mortality of pneumonia in rats, suggesting that it might be an interesting pneumonic biomarker for monitoring host response and predicting mortality, and a promising target for immunotherapy.

Harnessing eCIRP by PS-OMe miR130: A promising shield against hemorrhage-induced lung injury.

Hemorrhagic shock (HS) poses a life-threatening condition with the lungs being one of the most susceptible organs to its deleterious effects. Extracellular cold-inducible RNA binding protein (eCIRP) has emerged as a pivotal mediator of inflammation, and its release has been observed as a case of HS-induced tissue injury. Previous studies unveiled a promising engineered microRNA, designated PS-OMe miR130, which inhibits eCIRP, thereby safeguarding vital organs. In this study, we hypothesized that PS-OMe miR130 serves as a protective shield against HS-induced lung injury by curtailing the overzealous inflammatory immune response. Hemorrhagic shock was induced in male C57BL6 mice by withdrawing blood via a femoral artery cannula to a mean arterial pressure of 30 mm Hg for 90 min. The mice were resuscitated with twice the shed blood volume with Ringer's Lactate solution. They were then treated intravenously with either PBS (vehicle) or 62.5 nmol PS-OMe miR130. At 4 h later, blood and lungs were harvested. Following PS-OMe miR130 treatment in HS mice, a substantial decrease was observed in serum injury markers including aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and blood urea nitrogen (BUN). Serum IL-6 exhibited a similar reduction. In lung tissues, PS-OMe miR130 led to a significant decrease in the mRNA expressions of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α), chemokines (KC and MIP-2), and an endothelial injury marker, E-selectin. PS-OMe miR130 also produced substantial inhibition of lung MPO activity and resulted in a marked reduction in lung injury as evidenced by histological evaluation. This was further confirmed by the observation that PS-OMe miR130 significantly reduced the presence of Ly6G-positive neutrophils and TUNEL-positive apoptotic cells. PS-OMe miR130 emerges as a potent safeguard against HS-induced lung injury by effectively inhibiting proinflammation and injuries, offering a promising therapeutic strategy in such critical clinical condition.

Extracellular CIRP co-stimulated T cells through IL6R/STAT3 in pediatric IgA vasculitis

Immunoglobulin A vasculitis (IgAV) is the most common vasculitis of childhood. Disordered immune responses play important roles in its pathogenesis, but the comprehensive immune profile of the disease and the underlying mechanisms are still largely unknown. Here we found a potential disease biomarker cold inducible RNA binding protein (CIRP) in our pediatric IgAV cohort. Serum CIRP level in these patients were elevated and positively correlated with the increased early memory (CD45RA+CD62L+CD95+) T cells revealed using multicolor flow cytometry. Immune phenotyping of the patients showed they had more activated T cells with higher IL6Ra expression. T cell culture experiment showed CIRP further activated both human CD4+ and CD8+ T cells as indicated by increased perforin secretion and phosphorylation of STAT3. Blockade of IL6Rα attenuated CIRP-induced T cell toxicity in vitro. RNA-sequencing data further supported CIRP stimulation promoted human T cell activation and migration, fueled inflammation through the JAK-STAT signaling pathway. Therefore, IL6Ra-mediated T cell activation by extracellular CIRP may contribute to pathogenesis of IgAV in children, both CIRP and IL6Ra could be new therapeutic targets for IgAV.

Tat-CIRP Peptide Facilitates Frozen Wound Healing by Ameliorating Inflammation and Promoting Angiogenesis.

Frostbite is a chemia resulting from cold-induced skin damage. The process of frostbite is often accompanied by inflammation, and the therapeutic strategies focusing on anti-inflammation are the main direction to data. Tat-CIRP is a 15 amino acid peptide containing HIV protein and cold-inducible RNA-binding protein (CIRP), which is believed to compete with endogenous CIRP for myeloid differentiation 2 (MD2) binding. This study aims to investigate the efficacy of Tat-CIRP in the treatment of frostbite. A mouse model of frostbite was established, and on the first day after frostbite occurrence, Tat-CIRP peptide was administered intravenously via the tail with a dosage interval of one day for a total of three doses. Frozen mouse skin sections were subjected to histological analysis, including hematoxylin-eosin (HE) staining, Masson staining, and immunohistochemical examination. Western blotting was performed to detect the expression level of Ki-67 in mouse skin tissue. One day after frostbite, mice exhibited skin swelling and a solid appearance. From day 1 to 5 after frostbite, MD2 expression was significantly upregulated, while CIRP expression was downregulated. Compared to the frostbite group, mice treated with Tat-CIRP showed accelerated frostbite recovery, reduced levels of inflammatory factors and MD2. Furthermore, the expression of cell proliferation-associated protein Ki-67 and angiogenesis-related protein CD31 was upregulated. Tat-CIRP promotes frozen wound healing via inhibiting inflammation and promoting angiogenesis in frostbitten mice.

Upregulation of CIRP by its agonist prevents the development of heart failure in myocardial infarction rats

BackgroundDownregulated expression of cold-inducible RNA binding protein (CIRP), a stress-response protein, has been demonstrated in the hearts of patients with heart failure (HF). However, whether CIRP plays a critical role in the pathogenesis of HF remains unknown. Zr17-2 is a recently identified CIRP agonist, which can enhance the expression of CIRP in hearts. Herein, we evaluated the effects of zr17-2 on the development of HF in a rat model of myocardial infarction (MI).MethodsMale SD rats were pretreated with CIRP agonist zr17-2 or vehicle saline for 6 consecutive days, followed by MI induction. 1-week post-MI, cardiac function, and structural and molecular changes were determined by echocardiography and molecular biology methods.ResultsExcitingly, we found that pretreatment with zr17-2 significantly attenuated MI-induced cardiac dysfunction and dilation, coupled with reduced infarction size and cardiac remodeling. In addition, increased inflammatory response in the peri-infarcted heart including macrophage infiltration and the expression of inflammatory genes were all significantly decreased by zr17-2 pretreatment, suggesting an anti-inflammatory effect of zr17-2. Moreover, zr17-2 pretreatment also upregulated the antioxidant genes (e.g. NQO-1, Nrf2, and HO-1) level in the hearts. In isolated cultured cardiomyocytes, pretreatment with zr17-2 markedly attenuated cell injury and apoptosis induced by oxidative injury, along with elevation of Nrf2-related antioxidant genes and CIRP. However, silencing CIRP abolished zr17-2’s antioxidant effects against oxidative injury, confirming that zr17-2’s role is dependent on CIRP.ConclusionCollectively, our study suggests CIRP plays a crucial role in the development of HF and a beneficial effect of CIRP agonist in preventing MI-induced HF, possibly via anti-inflammatory and anti-oxidant pathways.

CIRP increases Foxp3+ regulatory T cells and inhibits development of Th17 cells by enhancing TLR4-IL-2 signaling in the late phase of sepsis

BackgroundT helper (Th) cell imbalances have been associated with the pathophysiology of sepsis, including the Th1/Th2 and Th17/T regulatory cells (Treg) paradigms. Cold-inducible RNA-binding protein (CIRP), a novel damage-associated molecular pattern (DAMP) was reported that could induce T cell activation, and skew CD4+ T cells towards a Th1 profile. However, the effect and underlying mechanisms of CIRP on Th17/Treg differentiation in sepsis still remains unknown. MethodsA prospective exploratory study including patients with sepsis was conducted. Blood samples were collected from patients on days 0, 3 and 7 on admission. The serum CIRP and peripheral blood Treg/Th17 percentage was determined by ELISA and flow cytometry. CD4+ T cells from the spleen and lymph nodes of mice with experimental sepsis were collected after treatment with normal saline (NS), recombinant murine CIRP (rmCIRP) and C23 (an antagonist for CIRP-TLR4) at late stage of sepsis. RNA-seq was conducted to reveal the pivotal molecular mechanism of CIRP on Treg/Th17 differentiation. Naïve CD4+ T cell was isolated from the Tlr4 null and wildtype mice in the presence or absence rmCIRP and C23 to confirmed above findings. ResultsA total of 19 patients with sepsis finally completed the study. Serum CIRP levels remained high in the majority of patients up to 1 week after admittance was closely associated with high Treg/Th17 ratio of peripheral blood and poor outcome. A univariate logistic analysis demonstrated that higher CIRP concentration at Day 7 is an independent risk factor for Treg/Th17 ratio increasing. CIRP promotes Treg development and suppresses Th17 differentiation was found both in vivo and in vitro. Pretreated with C23 not only alleviated the majority of negative effect of CIRP on Th17 differentiation, but also inhibited Treg differentiation, to some extent. Tlr4 deficiency could abolish almost all downstream effects of rmCIRP. Furthermore, IL-2 is proved a key downstream molecules of the effect CIRP, which also could amplify the activated CD4+ T lymphocytes. ConclusionsPersistent high circulating CIRP level may lead to Treg/Th17 ratio elevated through TLR4 and subsequent active IL-2 signaling which contribute to immunosuppression during late phases of sepsis.

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

IntroductionNeonatal 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. MethodsC57BL6 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. ResultsMOP3 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). ConclusionDeriving 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 EvidenceLevel 1.

B-1a Cells Scavenge NETs to Attenuate Sepsis.

B-1a cells, a regulatory subset of B lymphocytes, produce natural IgM and IL-10. Neutrophil extracellular traps (NETs) play a crucial role in pathogen defense, but their excessive formation during sepsis can cause further inflammation and tissue damage. In sepsis, extracellular cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released to induce NET formation. We hypothesize that B-1a cells clear NETs to prevent sepsis-induced injury. Sepsis in mice was induced by injecting 1 × 107 and 5 × 107 CFU E. coli intraperitoneally (i.p.). After 4 and 20 hours, we assessed the number of B-1a cells in the peritoneal cavity using flow cytometry. Our results showed that the number of peritoneal B-1a cells was significantly decreased in E. coli-sepsis mice. Importantly, replenishing B-1a cells via i.p. injection in sepsis mice significantly decreased NETs in peritoneal neutrophils. We also observed a decrease in serum inflammation and injury markers and a significant increase in overall survival rate in B-1a cell-treated septic mice. To understand the mechanism, we co-cultured bone marrow-derived neutrophils (BMDNs) with peritoneal B-1a cells in a contact or non-contact condition using an insert and stimulated them with eCIRP. After 4 hours, we found that eCIRP significantly increased NET formation in BMDNs. Interestingly, we observed that B-1a cells inhibited NETs by 67% in a contact-dependent manner. Surprisingly, when B-1a cells were cultured in inserts, there was no significant decrease in NET formation, suggesting that direct cell-to-cell contact is crucial for this inhibitory effect. We further determined that B-1a cells promoted NET phagocytosis and this was mediated through natural IgM, as blocking IgM receptor attenuated the engulfment of NETs by B-1a cells. Finally, we identified that following their engulfment, NETs were localized into the lysosomal compartment for lysis. Thus, our study suggests that B-1a cells decrease NET content in eCIRP-treated neutrophils and E. coli-sepsis mice.

Cirbp suppression compromises DHODH-mediated ferroptosis defense and attenuates hypothermic cardioprotection in an aged donor transplantation model.

Hypothermia is commonly used to protect donor hearts during transplantation. However, patients transplanted with aged donor hearts still have severe myocardial injury and decreased survival rates, but the underlying mechanism remains unknown. Because aged hearts are not considered suitable for donation, the number of patients awaiting heart transplants is increasing. In this study, we examined whether hypothermic cardioprotection was attenuated in aged donor hearts during transplantation and evaluated potential therapeutic targets. Using a rat heart transplantation model, we found that hypothermic cardioprotection was impaired in aged donor hearts but preserved in young donor hearts. RNA-Seq showed that cold-inducible RNA-binding protein (Cirbp) expression was decreased in aged donor hearts, and these hearts showed severe ferroptosis after transplantation. The young donor hearts from Cirbp-KO rats exhibited attenuated hypothermic cardioprotection, but Cirbp overexpression in aged donor hearts ameliorated hypothermic cardioprotection. Cardiac proteomes revealed that dihydroorotate dehydrogenase (DHODH) expression was significantly decreased in Cirbp-KO donor hearts during transplantation. Consequently, DHODH-mediated ubiquinone reduction was compromised, thereby exacerbating cardiac lipid peroxidation and triggering ferroptosis after transplantation. A cardioplegic solution supplemented with CIRBP agonists improved hypothermic cardioprotection in aged donor hearts, indicating that this method has the potential to broaden the indications for using aged donor hearts in transplantation.

Cold-inducible RNA binding protein alleviates iron overload-induced neural ferroptosis under perinatal hypoxia insult

Cold-inducible RNA binding protein (CIRBP), a stress response protein, protects cells from mild hypothermia or hypoxia by stabilizing specific mRNAs and promoting their translation. Neurons subjected to hypobaric hypoxia insult trigger various cell death programs. One of these is ferroptosis, a novel non-apoptotic form of programmed cell death, which is characterized by excessive iron ion accumulation and lipid peroxidation. Here, we establish that CIRBP can regulate neuronal ferroptosis both in vivo and in vitro. We observe that hypoxia leads to neuronal death via intracellular ferrous iron overload and impaired antioxidant systems, accompanied by suppressed CIRBP expression. Genetic enrichment of CIRBP in hippocampal neurons CIRBPTg mice bred with Emx1-Cre mice attenuates hypoxia-induced cognitive deficits and neuronal degeneration. Mechanistically, CIRBP alleviates neuronal ferroptosis and intracellular ferrous ion accumulation by binding to the mitochondrial ferritin (FTMT) 3’UTR to stabilize mRNA and promote its translation. Our novel study shows the critical role of CIRBP in the progression of ferroptosis, and provides promising therapeutic target for hypoxia-induced neurological diseases.

Cold-Inducible RNA Binding Protein Impedes Breast Tumor Growth in the PyMT Murine Model for Breast Cancer.

RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by associating with regulatory sequences in the untranslated regions of mRNAs. Cold-inducible RBP (CIRP) is a stress-induced RBP that was recently shown to modulate inflammation in response to cellular stress, where it increases or decreases pro-tumorigenic (proinflammatory) cytokines in different contexts. CIRP expression is altered in several cancers, including breast cancer, but the effects of CIRP on inflammation in breast cancer is not known. Here, we investigate if CIRP alters growth and the inflammatory profile of breast tumors. Transgenic mice overexpressing CIRP in the mammary epithelium were crossed with the PyMT mouse model of breast cancer, and the effects on both early and late tumorigenesis and inflammation were assessed. The effects of CIRP knockdown were also assessed in Py2T cell grafts. Overexpression of CIRP led to decreased tumorigenesis in the PyMT mouse model. Conversely, the knockdown of CIRP in Py2T cell grafts led to increased tumor growth. Luminex cytokine assays assessed the effects on the inflammatory environment. CIRP/PyMT mammary glands/mammary tumors and serum had decreased cytokines that promote inflammation, angiogenesis, and metastasis compared to PyMT mammary glands and serum, documenting a shift towards an environment less supportive of tumorigenesis. CIRP overexpression also decreased CD4+ helper T cells and increased CD8+ cytotoxic T cells in mammary tumors. Overall, these data support a role for CIRP as a potent antitumor molecule that suppresses both local and systemic pro-tumorigenic inflammation.

Extracellular CIRP induces CD4CD8αα intraepithelial lymphocyte cytotoxicity in sepsis

BackgroundIn sepsis, intestinal barrier dysfunction is often caused by the uncontrolled death of intestinal epithelial cells (IECs). CD4CD8αα intraepithelial lymphocytes (IELs), a subtype of CD4+ T cells residing within the intestinal epithelium, exert cytotoxicity by producing granzyme B (GrB) and perforin (Prf). Extracellular cold-inducible RNA-binding protein (eCIRP) is a recently identified alarmin which stimulates TLR4 on immune cells to induce proinflammatory responses. Here, we hypothesized that eCIRP enhances CD4CD8αα IEL cytotoxicity and induces IEC death in sepsis.MethodsWe subjected wild-type (WT) and CIRP−/− mice to sepsis by cecal ligation and puncture (CLP) and collected the small intestines to isolate IELs. The expression of GrB and Prf in CD4CD8αα IELs was assessed by flow cytometry. IELs isolated from WT and TLR4−/− mice were challenged with recombinant mouse CIRP (eCIRP) and assessed the expression of GrB and Prf in CD4CD8αα by flow cytometry. Organoid-derived IECs were co-cultured with eCIRP-treated CD4CD8αα cells in the presence/absence of GrB and Prf inhibitors and assessed IEC death by flow cytometry.ResultsWe found a significant increase in the expression of GrB and Prf in CD4CD8αα IELs of septic mice compared to sham mice. We found that GrB and Prf levels in CD4CD8αα IELs were increased in the small intestines of WT septic mice, while CD4CD8αα IELs of CIRP−/− mice did not show an increase in those cytotoxic granules after sepsis. We found that eCIRP upregulated GrB and Prf in CD4CD8αα IELs isolated from WT mice but not from TLR4−/− mice. Furthermore, we also revealed that eCIRP-treated CD4CD8αα cells induced organoid-derived IEC death, which was mitigated by GrB and Prf inhibitors. Finally, histological analysis of septic mice revealed that CIRP−/− mice were protected from tissue injury and cell death in the small intestines compared to WT mice.ConclusionIn sepsis, the cytotoxicity initiated by the eCIRP/TLR4 axis in CD4CD8αα IELs is associated with intestinal epithelial cell (IEC) death, which could lead to gut injury.

Extracellular CIRP induces abnormal activation of fibroblast-like synoviocytes from patients with RA via the TLR4-mediated HDAC3 pathways

The development of rheumatoid arthritis (RA) is closely related to the excessive activation of fibroblast-like synoviocytes (FLSs), which are regulated by a variety of endogenous proinflammatory molecules. Extracellular cold-inducible RNA-binding protein (CIRP), as a novel endogenous proinflammatory molecule, plays an important role in inflammatory diseases. More importantly, the synovial concentration of CIRP in patients with RA was significantly higher than that in patients with osteoarthritis (OA). Thus, this study aimed to investigate the role of extracellular CIRP in the abnormal activation of RA-FLSs and its related mechanisms. Our study showed that extracellular CIRP induced proliferation, migration and invasion of RA-FLSs, increased the expression of N-cadherin and MMP-3, and promoted the release of IL-1β and IL-33. However, blocking of extracellular CIRP with C23 inhibited CIRP-induced abnormal activation of RA-FLSs and alleviated the arthritis severity in AA rats. Accumulating evidence suggests that the activity and proinflammatory effects of CIRP are mediated through Toll-like receptor 4 (TLR4). Further studies demonstrated that the TLR4 knockdown inhibited CIRP-induced abnormal activation, and histone deacetylase 3 (HDAC3) expression in RA-FLSs. In addition, we found that HDAC3 knockdown and the specific inhibitor RGFP966 significantly suppressed CIRP-induced abnormal activation of RA-FLSs. We further found that treatment with HDAC3 specific inhibitor effectively alleviated the severity of arthritis in AA rats. Taken together, these findings indicate that extracellular CIRP induces abnormal activation of RA-FLSs via the TLR4-mediated HDAC3 pathways.

A NOVEL OLIGONUCLEOTIDE MRNA MIMIC ATTENUATES HEMORRHAGE-INDUCED ACUTE LUNG INJURY.

Hemorrhagic shock (HS) is accompanied by a pronounced activation of the inflammatory response in which acute lung injury (ALI) is one of the most frequent consequences. Among the pivotal orchestrators of this inflammatory cascade, extracellular cold-inducible RNA-binding protein (eCIRP) emerges as a noteworthy focal point, rendering it as a promising target for the management of inflammation and tissue injury. Recently, we have reported that oligonucleotide poly(A) mRNA mimic termed A 12 selectively binds to the RNA binding region of eCIRP and inhibits eCIRP binding to its receptor TLR4. Furthermore, in vivo administration of eCIRP induces lung injury in healthy mice and that mouse deficient in CIRP showed protection from inflammation-associated lung injury. We hypothesize that A 12 inhibits systemic inflammation and ALI in HS. To test the impacts of A 12 on systemic and lung inflammation, extent of inflammatory cellular infiltration and resultant lung damage were evaluated in a mouse model of HS. Male mice were subjected to controlled hemorrhage with a mean arterial pressure of 30 mm Hg for 90 min and then resuscitated with Ringer's lactate solution containing phosphate-buffered saline (vehicle) or A 12 at a dose of 4 nmol/g body weight (treatment). The infusion volume was twice that of the shed blood. At 4 h after resuscitation, mice were euthanized, and blood and lung tissues were harvested. Blood and tissue markers of inflammation and injury were evaluated. Serum markers of injury (lactate dehydrogenase, alanine transaminase, and blood urea nitrogen) and inflammation (TNF-α, IL-6) were increased after HS and A 12 treatment significantly decreased their levels. A 12 treatment also decreased lung levels of TNF-α, MIP-2, and KC mRNA expressions. Lung histological injury score, neutrophil infiltration (Ly6G staining and myeloperoxidase activity), and lung apoptosis were significantly attenuated after A 12 treatment. Our study suggests that the capacity of A 12 in attenuating HS-induced ALI and may provide novel perspectives in developing efficacious pharmaceutics for improving hemorrhage prognosis.

Neuron Derived Cold-Inducible RNA-Binding Protein Promotes NETs Formation to Exacerbate Brain Endothelial Barrier Disruption after Ischemic Stroke.

In ischemic stroke, neutrophils are the first-line peripheral immune cells infiltrating the brain tissue to form neutrophil extracellular traps (NETs). The present study aimed to investigate the role of neuronal cold-inducible RNA-binding protein (CIRP) in promoting NETs-induced brain endothelial barrier destruction and cerebral edema after ischemic stroke. We found that the expression of NETs and neuronal CIRP in the penumbra increased at 6 hours after transient middle cerebral artery occlusion (tMCAO) and increased significantly at 24 hours, reaching a peak at 3 days. NETs degradation or CIRP inhibition can alleviate the leakage of brain endothelial barrier and reverse the decreased expression of tight junction proteins (zonula occludens-1, claudin-5 and occludin) in tMCAO mice. Oxygen-glucose deprivation/reperfusion treated primary neurons or recombinant CIRP could induce NETs formation via TLR4/p38 signaling pathway in vitro. Transcription factor specificity protein 1 (sp1) was responsible for the increased neuronal CIRP expression and the inhibition of sp1 could suppress the increased CIRP expression, reduce NETs formation, and diminish brain endothelial barrier leakage in tMCAO mice. We also found the upregulated CIRP level was associated with severe cerebral edema in patients with acute ischemic stroke. In conclusion, the increased expression of transcription factor sp1 after ischemic stroke can lead to elevated CIRP expression and release from the neurons, which subsequently interacts with neutrophils and promotes NETs formation, resulting in brain endothelial barrier destruction and cerebral edema.