Targeting the Toll-Like Receptor 4 Ameliorates Heart Failure in Aged Mice by Inhibiting the Formation of Neutrophil Extracellular Traps.

Heart failure (HF) is a prevalent cardiovascular condition among the elderly population, with an incidence rate that continues to rise annually, highlighting the urgent need for effective therapeutic interventions. Sustained activation of Toll-like receptor 4 (TLR4) may contribute to left ventricular dysfunction and adverse cardiac remodeling through the induction of myocardial inflammation and oxidative stress – pathological processes that closely align with the hallmark features of HF. Preclinical studies in animal models have demonstrated that TLR4 deficiency improves cardiac function in aged mice; however, the precise role and underlying mechanisms of TLR4 in human HF remain poorly understood. This study aims to test the central hypothesis that TLR4 serves as a critical molecular link between chronic inflammation and the pathophysiology of HF. HF was induced in 18-month-old male C57BL/6J mice via continuous subcutaneous infusion of isoproterenol (ISO, 30 mg/kg/day) over a period of 3 weeks. Thereafter, mice received daily intraperitoneal injections of the TLR4 inhibitor TAK-242 (2 mg/kg), deoxyribonuclease I (DNase I, 5 mg/kg), or the peptidylarginine deiminase 4 (PAD4) inhibitor GSK484 (4 mg/kg) for 7 consecutive days. Cardiac function was assessed using a ultrasound imaging system. HE staining and Masson staining were employed to evaluate myocardial pathological changes and collagen deposition. ELISA was performed to measure serum levels of myeloperoxidase-DNA (MPO-DNA), neutrophil elastase-DNA (NE-DNA), cTnI, NT-proBNP, IL-1β, IL-6 and TNF-α. Immunofluorescence staining was performed to detect the co-localization levels of Ly6G with myeloperoxidase (MPO) and citrullinated histone H3 (cit-H3) in myocardial tissue, in order to assess the formation level of neutrophil extracellular traps (NETs). Western blot were utilized to determine the expression level of TLR4 protein. The expression of TLR4 was significantly upregulated in the myocardial tissue of aged HF mice. Inhibition of TLR4 not only markedly improved cardiac function but also alleviated pathological damage to myocardial tissue and reduced collagen fiber deposition. Concurrently, it also decreased the serum levels of MPO-DNA, NE-DNA, NT-proBNP, cTnI, and inflammatory factors. Moreover, the colocalization levels of Ly6G with MPO or cit-H3 in myocardial tissue was also diminished. These findings were consistent with the effects observed following DNase I and GSK484 interventions. Targeting TLR4 can mitigate inflammatory responses and enhance cardiac function in HF mice by inhibiting NETs formation.

The initiation of liver ischemia/reperfusion (I/R) injury results in the release of damage associated molecular patterns (DAMPs) such as HMGB1 and histones, which trigger innate immune and inflammatory cascade via Toll-like receptor 4 (TLR4) or TLR9. Infiltrated neutrophils contribute to the organ damage, innate immune and inflammatory responses after liver I/R. Formation of neutrophil extracellular trap (NET) has been recently found in response to various stimuli. However, the role of NETs during liver I/R remains unknown. We show that NETs form in the sinusoids of ischemic liver lobes in vivo, associated with increased serum level of myeloperoxidase (MPO)-DNA complexes and tissue level of citrullinated-histone H3 (NET markers) compared to control mice. Treatment with peptidyl-arginine-deiminase (PAD) 4 inhibitor or DNase I conferred significant protection after liver I/R evidenced by inhibition of NET formation, indicating the pathophysiological role of NETs in liver I/R injury. DAMPs, such as HMGB1 and histones stimulate NET formation through TLR4 and TLR9-MyD88 signaling pathways. After neutrophil depletion in mice, the adoptive transfer of TLR4 knockout (KO) or TLR9 KO neutrophils confers significant protection from liver I/R injury with significant decrease in NET formation. Conclusion: DAMPs released during liver I/R promotes NET formation through TLRs signaling pathway. NETs subsequently exacerbates organ damage and initiates inflammatory responses during liver I/R.

It has been known for many years that LPS, neutrophils and platelets all participate in the pathogenesis of severe sepsis, but the inter-relationship between these players is completely unknown. Using flow chambers in vitro we suggest a novel innate immune response leading to enhanced trapping of bacteria in blood vessels. The mechanism involved platelet TLR4 detecting LPS in blood and inducing a unique response, specifically platelet binding to adherent neutrophils, but not platelet aggregation or P-selectin expression. Subsequently, the platelets stimulated very robust neutrophil activation leading to formation of NETs. These NETs retained their integrity under flow conditions and functioned to ensnare bacteria within the vasculature. Plasma from severely septic patients also induced TLR4-dependent platelet-neutrophil interactions leading to the production of NETs. We propose that this novel bacterial trapping mechanism would only occur under extreme conditions such as severe sepsis and platelet TLR4 (not leukocyte TLR4) functioned as the threshold switch for this innate immune response to occur. With the advent of antibiotics perhaps reducing the need for NET formation, we would propose that inhibiting platelet activation with TLR4 inhibitors may inhibit NET formation and reduce inadvertent tissue injury. This work was funded by: CIHR and AHFMR

Ischemic stroke prompts a strong inflammatory response which is associated with exacerbated stroke outcomes. However, classic anti-inflammatory strategies have been unsuccessful in stroke patients implying other unknown mechanisms contribute to injurious inflammation in stroke. Increasing evidence suggests immunothrombosis, a process involving coagulation, neutrophil and platelet activation, and neutrophil extracellular trap (NET) formation, is an important contributor to cardiovascular diseases. However, mechanistic regulators of immunothrombosis and their role in ischemic stroke remain unclear. We examined markers of immunothrombosis in ischemic stroke patients and matched healthy donors. Stroke patients had significantly increased levels of D-Dimers, platelet factor 4, neutrophil calprotectin, citrullinated histone H3 (H3cit) and MPO-DNA complexes, markers of NET formation. In particular, H3cit and MPO-DNA complexes positively correlated with long-term stroke outcomes. Mechanistically, we observed increased plasma and platelet HMGB1 in stroke patients, which significantly correlated with plasma NETs, indicating a role for platelet HMGB1 in NET formation. To directly examine the role of platelet HMGB1, we employed a transient ischemic stroke mouse model. Depleting platelets significantly reduced plasma HMGB1 levels, inhibited NET formation and improved stroke outcomes. Correspondingly, administrating a HMGB1 inhibitor reduced NET formation and improved stroke outcomes, implying a causative role for platelet HMGB1 in mediating NET formation after stroke. As NETs appeared detrimental in ischemic stroke, we investigated the therapeutic potential of an endogenous NET inhibitory factor (NIF), recently discovered in neonates. Mice pretreated with NIF had reduced brain injury, improved neurological and motor function and enhanced survival after stroke. Importantly, NIF specifically blocked NET formation after stroke without affecting brain neutrophil recruitment. Critically, NIF still improved stroke outcomes when administered after stroke onset. These results support a pathological role for NETs in stroke brain injury and indicate the use of NIF as a therapeutic strategy to target immunothrombosis in stroke.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMNs), extrude molecular lattices of decondensed chromatin studded with histones, granule enzymes, and antimicrobial peptides that are referred to as neutrophil extracellular traps (NETs). NETs capture and contain bacteria, viruses, and other pathogens. Nevertheless, experimental evidence indicates that NETs also cause inflammatory vascular and tissue damage, suggesting that identifying pathways that inhibit NET formation may have therapeutic implications. Here, we determined that neonatal NET-inhibitory factor (nNIF) is an inhibitor of NET formation in umbilical cord blood. In human neonatal and adult neutrophils, nNIF inhibits key terminal events in NET formation, including peptidyl arginine deiminase 4 (PAD4) activity, neutrophil nuclear histone citrullination, and nuclear decondensation. We also identified additional nNIF-related peptides (NRPs) that inhibit NET formation. nNIFs and NRPs blocked NET formation induced by pathogens, microbial toxins, and pharmacologic agonists in vitro and in mouse models of infection and systemic inflammation, and they improved mortality in murine models of systemic inflammation, which are associated with NET-induced collateral tissue injury. The identification of NRPs as neutrophil modulators that selectively interrupt NET generation at critical steps suggests their potential as therapeutic agents. Furthermore, our results indicate that nNIF may be an important regulator of NET formation in fetal and neonatal inflammation.

BackgroundObesity is a global health challenge linked to chronic non-communicable diseases. Low-grade inflammation and altered immune responses, including neutrophil extracellular trap (NET) formation, contribute to metabolic complications. Peptidyl arginine deiminase 4 (PAD4) is critical for NET formation, and its inhibition shows therapeutic potential. However, the sex-specific effects of PAD4 deficiency in diet-induced obesity remain unexplored.MethodsThis study investigated the impact of PAD4 deficiency on obesity-related metabolic pathologies in male and female C57BL/6 wild-type (WT) and Pad4(-/-) mice (n=5-6/group) fed a 22-week obesogenic cafeteria (CAF) diet. We hypothesized that PAD4 deficiency would ameliorate obesity-related metabolic and behavioral complications for both sexes. Body weight and composition, glucose and lipid metabolism, liver damage markers, and behavior were assessed. NETs were quantified via flow cytometry.ResultsPad4(-/-) males on CAF diet exhibited delayed obesity onset, lower body weight gain, and improved dyslipidemia than WT CAF males. This was associated with enhanced metabolic adaptation, indicated by higher brown adipose tissue temperature in Pad4(-/-) males. Conversely, Pad4(-/-) females on the CAF diet showed comparable weight gain to WT CAF females, similar or worsened dyslipidemia, impaired glucose metabolism, and higher liver lipid accumulation. While WT CAF females showed increased brown adipose tissue temperature, Pad4(-/-) CAF females did not.ConclusionPAD4 deficiency exerts sex-specific effects on obesity-related metabolic complications in mice. Inhibition of NET formation appears protective in males but not females on an obesogenic diet. These findings underscore the importance of considering sex as a biological variable in obesity research and developing sex-specific therapies.

Mesenchymal stem cell transplantation alleviated TBI-induced lung injury by inhibiting PAD4-dependent NET formation.

OP0054 Neuroimmunomodulation of Neutrophils as A Novel Mechanism of Levamisole-Induced Vasculitis

Introduction Histone hypercitrullination by the enzyme peptidylarginine deiminase 4 (PAD4) leads to nuclear chromatin decondensation that is needed for neutrophil extracellular trap (NET) formation. NETs consist of chromatin and granule proteins that are released into the extracellular environment. NETs were shown to be involved in thrombosis by promoting coagulation and platelet adhesion and were identified in the thrombus scaffold in animal models of deep vein thrombosis (DVT). Objective Whether NETs are involved in the pathogenesis of DVT or whether they are merely a consequence of neutrophil recruitment to the thrombus is unknown. We hypothesized that NET formation would be impaired in PAD4-deficient mice during deep vein thrombosis and that this may affect thrombus formation and/or stability. Methods PAD4-deficient mice are incapable of citrullinating histones and therefore fail to decondense chromatin during NETosis. We performed the inferior vena cava stenosis model of DVT in wild-type or PAD4-/- mice. Intravital microscopy was done to assess leukocyte vessel wall interaction in PAD4 deficiency. Results We induced NET formation in isolated peripheral blood mouse neutrophils with ionomycin and found that PAD4-/- neutrophils had a complete inability to produce NETs (WT, 20.65±2.61% NETs; PAD4-/-, not detected. n=4). Leukocyte-endothelial interactions in PAD4-/- mice were not impaired upon induction of systemic Weibel-Palade body release (WT, 55.2±11.8; PAD4-/-, 62.0±17.5 cells/min, n=5-6). In the DVT model, while a majority (9/10) of wild-type mice formed a thrombus 48 hours after stenosis, only 1 of 11 PAD4-/- mice formed a thrombus. Thrombus formation could be rescued by infusions of isolated WT bone marrow neutrophils into PAD4-/- mice, and extracellular H3Cit+ areas were seen within these thrombi. This data suggests that neutrophil PAD4 was essential for thrombus formation in deep veins. Conclusion NETs comprise a crucial part of the pathologic thrombus scaffold, and here we report that the lack of NETs inhibits pathological thrombosis. Chromatin decondensation initiated by PAD4 in neutrophils is a key player in the formation of deep vein thrombi and targeting neutrophil histone modification could be a new way to prevent DVT.

Background: We have previously demonstrated that neutrophil extracellular traps (NETs) in myocardial tissue are associated with cardiac dysfunction and adverse outcomes in patients with heart failure with reduced ejection fraction. Here, we aimed to elucidate the underlying mechanisms and to clarify whether inhibiting NETs could rescue the cardiac phenotype using ex vivo and in vivo models. Methods and Results: Given that peptidyl arginine deiminase 4 (PAD4) is essential for the formation of NETs, PAD4 knockout mice were used for this study. First, we assessed the mitochondrial function of cardiomyocytes by conducting real-time measurements of the oxygen consumption rate in ex vivo. Extracellular flux analysis revealed that NETs-containing conditioned medium from wild-type neutrophils or purified NET components led to impaired mitochondrial oxygen consumption of adult mouse cardiomyocytes, while these effects were abolished when PAD4 in neutrophils was genetically ablated (Figure 1). In a murine model of pressure overload-induced heart failure, transverse aortic constriction (TAC) operation was performed on wild-type and PAD4 knockout mice. Immunohistological analysis demonstrated that TAC induced the formation of NETs in the myocardium, with the greatest number of NETs occurring at 1 day, persisting into the failing stage at 4 weeks in wild-type mice, while PAD4-deficient hearts displayed no presence of NETs in the heart tissue (Figure 2). Four weeks after TAC, left ventricular ejection fraction was reduced in wild-type mice, whereas PAD4 knockout mice displayed preserved left ventricular ejection fraction (Figure 2). Finally, we investigated the bioenergetics of cardiomyocytes isolated from PAD4 knockout mice after TAC. Maximal mitochondrial oxygen consumption rate in cardiomyocytes isolated from TAC-operated PAD4 knockout mice was significantly higher than in those from TAC-operated wild-type mice (Figure 3). Conclusion: Our data suggest that NETs directly derived impairment of mitochondrial function in cardiomyocytes. Inhibiting NETs represents a potential therapeutic approach for heart failure, preventing mitochondrial dysfunction of cardiomyocytes.

Inhibition of NET formation by polydatin protects against collagen-induced arthritis

Early intravenous administration of tranexamic acid ameliorates intestinal barrier injury induced by neutrophil extracellular traps in a rat model of trauma/hemorrhagic shock

Background Neutrophil extracellular traps (NETs) are critical mediators of thromboinflammation during acute myocardial infarction (AMI). However, triggers and signalling pathways of NETosis in AMI remain incompletely understood. Levels of extracellular vesicles (EV) carrying oxidation-specific epitopes (OSE) originating from lipid peroxidation are increased at the culprit site in AMI. Importantly, natural IgM antibodies with specificity for OSE, such as malondialdehyde (MDA), have been shown to modulate functional effects of EV, and are associated with reduced cardiovascular risk. Purpose We investigated the stimulatory capacity of EV on neutrophil effector functions and specifically targeted key mediators of NET formation using pharmacological inhibitors and atheroprotective natural IgM to inhibit this process. Methods Patients were included after diagnosis of ST-segment elevation myocardial infarction and blood was aspirated from the culprit site and peripheral arterial site (n=28) during primary percutaneous coronary intervention (pPCI). Myocardial function was documented by cardiac magnetic resonance imaging 4±2 days and 195±15 days after pPCI. EV were isolated from cell culture supernatants and culprit site plasma for neutrophil stimulation in vitro. Pharmacological inhibitors were used to map NET signalling pathways of EV. Isolated EV were used for neutrophil stimulation in a murine injection model in the presence of the MDA-specific IgM LR04 or an isotype control. NET formation and other neutrophil functions were assessed by flow cytometry, ELISA and fluorescence microscopy. Results Levels of NET surrogate markers and CD45+ MDA+ EV were higher at the culprit site than in the peripheral circulation. EV generated by LPS-activated THP-1 monocytic cells induced in vitro NET formation, release of neutrophil elastase and IL-8, and degranulation of MPO and NGAL in primary human neutrophils, but not reactive oxygen species. Toll-like receptor 4 (TLR4) and peptidyl-arginine deiminase 4 (PAD4) were identified as key mediators of NET formation induced by in vitro-generated EV and EV isolated from the culprit site of AMI patients. Functionally, the MDA-specific monoclonal IgM antibody LR04, but not an isotype control, inhibited the ability of patient-derived and in vitro-generated EV to trigger release of NETs in primary human neutrophils and in mice. Titres of MDA-specific IgM antibodies were inversely associated with the NET marker citH3 in blood of AMI patients. Finally, we found that the concentration of CD45+ MDA+ EV per protective MDA-specific IgM at the culprit site showed an inverse association with left ventricular ejection fraction 72 hours and 6 months after AMI. Conclusions We show that EV can trigger several neutrophil effector functions. EV-induced NET formation is TLR4- and PAD4-dependent and can be inhibited by OSE-specific natural IgM potentially influencing cardiovascular outcomes after an acute event.

Neutrophil extracellular traps (NETs) occur when chromatin is decondensed and extruded from the cell, generating a web-like structure. NETs have been implicated in the pathogenesis of several sterile disease states and thus are a potential therapeutic target. Various pathways have been shown to induce NETs, including autophagy, with several key enzymes being activated like peptidyl arginine deiminase 4 (PAD4), an enzyme responsible for citrullination of histones, allowing for DNA unwinding and subsequent release from the cell. Pre-clinical studies have already demonstrated that chloroquine (CQ) and hydroxychloroquine (HCQ) are able to reduce NETs and slow disease progression. The exact mechanism as to how these drugs reduce NETs has yet to be elucidated. CQ and HCQ decrease NET formation from various NET activators, independent of their autophagy inhibitory function. CQ and HCQ were found to inhibit PAD4 exclusively, in a dose-dependent manner, confirmed with reduced CitH3+ NETs after CQ or HCQ treatment. Circulating CitH3 levels were reduced in pancreatic cancer patients after HCQ treatment. In silico screening of PAD4 protein structure identified a likely binding site interaction at Arg639 for CQ and Trp347, Ser468, and Glu580 for HCQ. SPR analysis confirmed the binding of HCQ and CQ with PAD4 with KD values of 54.1 µM (CQ) and 88.1 µM (HCQ). This data provide evidence of direct PAD4 inhibition as a mechanism for CQ/HCQ inhibition of NETs. We propose that these drugs likely reduce NET formation through multiple mechanisms; the previously established TLR9 and autophagy inhibitory mechanism and the novel PAD4 inhibitory mechanism.

There are no specific treatments for Sepsis-associated acute kidney injury (SAKI). We previously reported that Il-17a-knockout mice had dramatically improved survival after cecal ligation and puncture (CLP). Neutrophil extracellular traps (NETs) induce IL-17A, which causes harm in some diseases, but this pathway is poorly understood in sepsis. We found that knockout of Pad4 (Peptidyl Arginine Deiminase 4), an enzyme essential for NET formation, improved survival and AKI, and suppressed neutrophil infiltration into remote organs, involving a peritoneal IL-17A/distant organ CXCL-1/CXCL-2 pathway after CLP. NETs were detected in the peritoneal cavity, and not in plasma or distant organs. Adoptive transfer of peritoneal NETs restored the IL-17A/CXCL-1/CXCL-2 pathway in Pad4KO mice, leading to neutrophil infiltration and damge to remote organs. These results revealed a pathway from peritoneal NET formation to remote organ injury/inflammation via production of IL-17A at the infectious site and distant organ CXCL-1/CXCL-2. While NETs promoted intraperitoneal IL-17A production, we also showed that conversely, peritoneal IL-17A or CXCL-1/CXCL-2 promoted intraperitoneal NET formation after CLP. This peritoneal vicious cycle that includes NET formation, IL-17A, CXCL-1/CXCL-2 that may amplify organ injury in sepsis. Breaking this vicious cycle by inhibiting NET formation and/or IL-17A might be a promising therapeutic target for sepsis treatment.