Inflammation. Neutrophil extracellular traps license macrophages for cytokine production in atherosclerosis.

Cholesterol crystal embolism (CCE) is a systemic thromboinflammatory disorder (the intertwined activation of inflammatory pathways and coagulation cascades) characterized by the embolization of cholesterol crystals (CCs) from atherosclerotic plaques into microvasculature, leading to multiorgan dysfunction. Despite its clinical significance, CCE remains underdiagnosed due to nonspecific presentations and diagnostic challenges. This review synthesizes current evidence on the central role of CC-induced neutrophil extracellular trap (NET) formation in driving endothelial injury, thrombosis, and gastrointestinal cancer progression with a specific focus on gastrointestinal malignancies. Mechanistically, CCs activate the TLR4/9-NLRP3 inflammasome cascade, triggering IL-1β release and PAD4-mediated histone citrullination, which culminate in NETosis. These NETs exacerbate vascular damage by promoting endothelial dysfunction, platelet adhesion, and immune evasion. In gastrointestinal cancer (e.g., colorectal, hepatocellular, pancreatic), CCs remodel the tumor microenvironment (TME) by inducing NET-driven immunosuppression, extracellular matrix degradation, and metastatic niche formation. The interplay between CCs, NETs, and inflammation creates a self-perpetuating cycle that worsens atherosclerosis, CCE, and tumor metastasis. Emerging therapeutic strategies targeting this cholesterol-NET-inflammation axis show promise. DNase I and heparin disrupt NET scaffolds, while PAD4 inhibitors (e.g., GSK484) block NET generation. Colchicine demonstrates dual anti-inflammatory and NETosis-inhibitory effects, and lipid-lowering agents (statins, PCSK9 inhibitors) mitigate CC burden. Nanotherapies, such as HDL-mimetic nanoparticles, offer targeted delivery to restore immune surveillance. This review highlights the need for personalized, biomarker-guided therapies to disrupt the pathogenic CC-NET axis proposing an integrated approach to mitigate CC-mediated damage in cardiovascular and gastrointestinal oncologic disease.

Neutrophil extracellular trap (NET) formation promotes vascular damage, thrombosis, and activation of interferon-α-producing plasmacytoid dendritic cells in diseased arteries. Peptidylarginine deiminase inhibition is a strategy that can decrease in vivo NET formation. To test whether peptidylarginine deiminase inhibition, a novel approach to targeting arterial disease, can reduce vascular damage and inhibit innate immune responses in murine models of atherosclerosis. Apolipoprotein-E (Apoe)(-/-) mice demonstrated enhanced NET formation, developed autoantibodies to NETs, and expressed high levels of interferon-α in diseased arteries. Apoe(-/-) mice were treated for 11 weeks with daily injections of Cl-amidine, a peptidylarginine deiminase inhibitor. Peptidylarginine deiminase inhibition blocked NET formation, reduced atherosclerotic lesion area, and delayed time to carotid artery thrombosis in a photochemical injury model. Decreases in atherosclerosis burden were accompanied by reduced recruitment of netting neutrophils and macrophages to arteries, as well as by reduced arterial interferon-α expression. Pharmacological interventions that block NET formation can reduce atherosclerosis burden and arterial thrombosis in murine systems. These results support a role for aberrant NET formation in the pathogenesis of atherosclerosis through modulation of innate immune responses.

BackgroundThe formation and accumulation of cholesterol crystals (CC) at the lesion site is a hallmark of atherosclerosis. Although studies have shown the importance of vascular smooth muscle cells (VSMCs) in the disease atherosclerosis, little is known about the molecular mechanism behind the uptake of CC in VSMCs and their role in modulating immune response.MethodsHuman aortic smooth muscle cells were cultured and treated with CC. CC uptake and CC mediated signaling pathway and protein induction were studied using flow cytometry, confocal microscopy, western blot and Olink proteomics. Conditioned medium from CC treated VSMCs was used to study neutrophil adhesion, ROS production and phagocytosis. Neutrophil extracellular traps (NETs) formations were visualized using confocal microscopy.ResultsVSMCs and macrophages were found around CC clefts in human carotid plaques. CC uptake in VSMCs are largely through micropinocytosis and phagocytosis via PI3K–AkT dependent pathway. The uptake of CC in VSMCs induce the release inflammatory proteins, including IL-33, an alarming cytokine. Conditioned medium from CC treated VSMCs can induce neutrophil adhesion, neutrophil reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. IL-33 neutralization in conditioned medium from CC treated VSMCs inhibited neutrophil ROS production and NETs formation.ConclusionWe demonstrate that VSMCs due to its vicinity to CC clefts in human atherosclerotic lesion can modulate local immune response and we further reveal that the interaction between CC and VSMCs impart an inflammatory milieu in the atherosclerotic microenvironment by promoting IL-33 dependent neutrophil influx and NETs formation.

NET formation in noninfectious conditionsFormation of neutrophil extracellular traps (NETs), a new effector function of neutrophils, was first described in 2004 [1].Since then, the formation of NETs has widely been studied.NETs have a web-like structure mainly made of decondensed chromatin fibers, whose expulsion from the cell is aided by enzymatic citrullination of histones and which are decorated with granule proteins, such as elastase, myeloperoxidase (MPO) and histones [2,3].There is ample evidence supporting the idea that formation of NETs plays an important role in immune responses, allowing neutrophils to capture, neutralize, and degrade a variety of invading microorganisms.NETosis, the type of programmed cell death resulting in NET formation, also occurs during sterile inflammation resulting in thrombosis [4,5], autoimmunity [6], and NET-mediated cytotoxicity [7,8], and therefore can be harmful.DNA and damage-associated molecular patterns (DAMPs) embedded within NETs act as alarmins, augmenting inflammation, and exerting cytotoxic effects.The prothrombotic role of NETs has been well documented [9][10][11].This feature of NETs may become problematic, especially in cases of sterile insults.Thus, while NETs may provide evolutionary advantages of trapping and killing bacteria in infectious diseases, they appear to do more harm in sterile inflammation and thrombosis, as dysregulated NET formation leads to vascular inflammation, thrombosis, and atherogenesis, Let us provide some examples of sterile insults causing NET formation.As shown by many of the studies we discuss below, ischemia/reperfusion (IR) injury is a clinically relevant cause for NET formation.IR injury is a major sterile insult resulting from hemoragic, traumatic or septic shock, burns, and surgical procedures, including organ transplantation.The response to IR injury is comprised of a diverse network ranging from, innate to adaptive immune responses.[12].Restoration of the blood supply, paradoxically, causes cell damage and exacerbation of inflammatory responses through reactive oxygen species (ROS) and other reactive molecules, worsening organ injury [13].However, the specific pathways that link IR to NETs are only partially understood.Besides IR injury, several milder insults are known to induce NET formation.Beiter et al. showed that NET formation increases in response to exhaustive treadmill or cycling exercise in healthy individuals [14].This study proved that NETosis is the main cause for increased cell-free DNA in the circulation, as reported in an earlier study [e.g., 15].Jain and coworkers analyzed NETosis associated with dry eye disease, in which hyperosmolar stress is considered the main factor [16].They further showed that hyperosmotic stress promotes NET formation by neutrophils [17].NETs have been found in venous and arterial thrombosis, trauma-induced coagulopathy, and disseminated intravascular coagulation [18][19][20].NETs promote clot formation; in addition to the interaction of NET components with the coagulation pathway, NETs provide scaffolds for clot growth by catching platelets, fibrin, von Willebrand factor (vWF), and other cells/molecules [21].It is notable that fluid shear stress is a critical factor for rapid and intense NETosis [22].Intriguingly, in sterile occlusive clots, fibrin suppresses NET generation, and the absence of fibrin promotes NETs.Shear-induced NETosis is strongly inversely correlated with fibrin in sterile occlusive clots.

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Sepsis is a deadly inflammatory syndrome caused by an exaggerated immune response to infection. Much has been focused on host response to pathogens mediated through the interaction of pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs). PRRs are also activated by host nuclear, mitochondrial, and cytosolic proteins, known as damage-associated molecular patterns (DAMPs) that are released from cells during sepsis. Some well described members of the DAMP family are extracellular cold-inducible RNA-binding protein (eCIRP), high mobility group box 1 (HMGB1), histones, and adenosine triphosphate (ATP). DAMPs are released from the cell through inflammasome activation or passively following cell death. Similarly, neutrophil extracellular traps (NETs) are released from neutrophils during inflammation. NETs are webs of extracellular DNA decorated with histones, myeloperoxidase, and elastase. Although NETs contribute to pathogen clearance, excessive NET formation promotes inflammation and tissue damage in sepsis. Here, we review DAMPs and NETs and their crosstalk in sepsis with respect to their sources, activation, release, and function. A clear grasp of DAMPs, NETs and their interaction is crucial for the understanding of the pathophysiology of sepsis and for the development of novel sepsis therapeutics.

Damage-associated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury.

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.

Following fifteen years of research, neutrophil extracellular traps (NETs) are widely reported in a large range of inflammatory infectious and non-infectious diseases. Cumulating evidences from in vitro, in vivo and clinical diagnostics suggest that NETs may play a crucial role in inflammation and autoimmunity in a variety of autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Most likely, NETs contribute to breaking self-tolerance in autoimmune diseases in several ways. During this review, we discuss the current knowledge on how NETs could drive autoimmune responses. NETs can break self-tolerance by being a source of autoantigens for autoantibodies found in autoimmune diseases, such as anti-citrullinated protein antibodies (ACPAs) in RA, anti-dsDNA in SLE and anti-myeloperoxidase and anti-protein 3 in AAV. Moreover, NET components could accelerate the inflammatory response by mediating complement activation, acting as danger-associated molecular patterns (DAMPs) and inflammasome activators, for example. NETs also can activate other immune cells, such as B cells, antigen-presenting cells and T cells. Additionally, impaired clearance of NETs in autoimmune diseases prolongs the presence of active NETs and their components and, in this way, accelerate immune responses. NETs have not only been implicated as drivers of inflammation, but also are linked to resolution of inflammation. Therefore, NETs may be central regulators of inflammation and autoimmunity, serve as biomarkers, as well as promising targets for future therapeutics of inflammatory autoimmune diseases.

Atherosclerosis is a chronic inflammatory disease driven by dynamic interactions between lipid metabolism and immune responses. Its initiation and progression are co-regulated by innate and adaptive immunity. Innate immune cells are central to early plaque development. Neutrophils release pro-inflammatory mediators, including myeloperoxidase (MPO) and neutrophil extracellular traps (NETs), which contribute to endothelial dysfunction and tissue injury. Circulating monocytes infiltrate the arterial intima and differentiate into macrophages. These macrophages can polarize into distinct phenotypes, typically categorized as pro-inflammatory M1 or anti-inflammatory M2; an imbalance between these states exacerbates plaque inflammation. A pivotal early event is the uptake of modified lipoproteins—such as oxidized low-density lipoprotein (oxLDL)—via scavenger receptors (e.g., SR-A1 and CD36), leading to the formation of lipid-laden foam cells, a hallmark of early lesions. Adaptive immune responses also shape disease progression and plaque stability. Among CD4+ T helper subsets, Th1 cells promote atherosclerosis by secreting interferon-γ (IFN-γ), whereas Th17 cells and their signature cytokine interleukin-17 (IL-17) are often associated with disease amplification. In contrast, regulatory T cells (Tregs) exert atheroprotective effects by suppressing immune activation and producing anti-inflammatory cytokines such as interleukin-10 (IL-10). B cells also contribute: B1 cells are generally protective, in part through natural IgM production, whereas B2 cells may be pro-atherogenic. Key inflammatory pathways sustain the chronic inflammatory milieu within plaques. The NLRP3 inflammasome, activated by damage-associated molecular patterns (DAMPs) such as cholesterol crystals, induces caspase-1 activation, which cleaves pro–IL-1β and pro–IL-18 into their bioactive forms. Cytokines including IL-1β and IL-6 are major drivers of inflammation; IL-1β upregulates endothelial adhesion molecules, whereas IL-6 promotes the hepatic production of acute-phase reactants. The association between systemic inflammatory diseases (e.g., systemic lupus erythematosus and rheumatoid arthritis) and accelerated atherosclerosis further underscores the importance of shared immune pathways. This review synthesizes these interactions to provide a framework for understanding atherosclerosis pathogenesis.

BACKGROUND:Neutrophil extracellular traps (NETs) contribute to atherosclerosis progression and are linked to adverse clinical outcomes such as myocardial infarction and stroke. Although the triggers of NET formation in plaques are known, the mechanisms governing DNase-mediated NET clearance and how these are disrupted during atherosclerosis remain unclear. Moreover, the consequences of impaired NET clearance on disease progression are not known.METHODS:Low-density lipoprotein receptor knockout (Ldlr−/−) mice with hematopoietic cell–specific deletion of DNase1 and DNase1L3 were fed a Western-type diet for 16 weeks to examine the impact of loss of DNase activity and the subsequent NET accumulation on advanced atherosclerosis. The effect of NETs on macrophage efferocytosis was examined in vitro and in the mouse peritoneal cavity and atherosclerotic plaque in vivo. To identify the signaling pathway impairing the NET-induced DNase response, in vitro assays were performed using selective endoplasmic reticulum stress pathway inhibitors, and the findings were validated in murine and human atherosclerotic tissues.RESULTS:Lack of DNase secretion by macrophages led to accumulation of NETs in local tissues, including atherosclerotic plaques. Persisting NETs in turn promoted cleavage of the efferocytosis receptor MerTK (c-mer proto-oncogene tyrosine kinase), resulting in defective macrophage efferocytosis and increased atherosclerotic plaque necrosis. In vitro screening identified endoplasmic reticulum stress–induced activation of the PERK (protein kinase R–like endoplasmic reticulum kinase)–ATF (activating transcription factor) 4 signaling axis in atherogenic macrophages as a key driver of impaired DNase secretion, leading to delayed NET clearance and their pathological persistence. Treatment of human atherosclerotic plaques and Ldlr−/− mice with integrated stress response inhibitor, a selective PERK inhibitor, restored vascular DNase secretion and facilitated NET clearance.CONCLUSIONS:Macrophages play a key role in clearing NETs from tissues. Endoplasmic reticulum stress suppresses macrophage DNase secretion, leading to NET accumulation in atherosclerotic plaques, which triggers efferocytosis impairment and plaque progression. Targeting the PERK-ATF4 axis to restore DNase release and NET clearance represents a promising therapeutic strategy to promote plaque stabilization.

Cholesterol crystal embolism can be a life-threatening complication of advanced atherosclerosis. Pathophysiology and molecular targets for treatment are largely unknown. We aimed to develop a new animal model of cholesterol crystal embolism to dissect the molecular mechanisms of cholesterol crystal (CC)-driven arterial occlusion, tissue infarction, and organ failure. C57BL/6J mice were injected with CC into the left kidney artery. Primary end point was glomerular filtration rate (GFR). CC caused crystal clots occluding intrarenal arteries and a dose-dependent drop in GFR, followed by GFR recovery within 4 weeks, that is, acute kidney disease. In contrast, the extent of kidney infarction was more variable. Blocking necroptosis using mixed lineage kinase domain-like deficient mice or necrostatin-1s treatment protected from kidney infarction but not from GFR loss because arterial obstructions persisted, identifying crystal clots as a primary target to prevent organ failure. CC involved platelets, neutrophils, fibrin, and extracellular DNA. Neutrophil depletion or inhibition of the release of neutrophil extracellular traps had little effects, but platelet P2Y12 receptor antagonism with clopidogrel, fibrinolysis with urokinase, or DNA digestion with recombinant DNase I all prevented arterial occlusions, GFR loss, and kidney infarction. The window-of-opportunity was <3 hours after CC injection. However, combining Nec-1s (necrostatin-1s) prophylaxis given 1 hour before and DNase I 3 hours after CC injection completely prevented kidney failure and infarcts. In vitro, CC did not directly induce plasmatic coagulation but induced neutrophil extracellular trap formation and DNA release mainly from kidney endothelial cells, neutrophils, and few from platelets. CC induced ATP release from aggregating platelets, which increased fibrin formation in a DNase-dependent manner. CC embolism causes arterial obstructions and organ failure via the formation of crystal clots with fibrin, platelets, and extracellular DNA as critical components. Therefore, our model enables to unravel the pathogenesis of the CC embolism syndrome as a basis for both prophylaxis and targeted therapy.

Aluminum hydroxide (alum) and monophosphoryl-lipid A (MPLA) are conventional adjuvants in vaccines for allergen-specific immunotherapy (AIT). Alum triggers the release of neutrophil extracellular traps (NETs) by neutrophils. NETs contain expelled decondensed chromatin associated with granular material and may act as danger-associated molecular patterns and activate antigen-presenting cells. We investigated whether adjuvant-induced NETs contribute to innate responses to AIT-vaccines. Human neutrophils were incubated with alum, MPLA and adjuvant-containing AIT-vaccine preparations. NETs were verified by time-lapse and confocal fluorescence microscopy and quantitatively assessed by DNA and elastase release and ROS production. In contrast to MPLA, alum represented a potent trigger for NET release. Vaccine formulations containing alum resulted in less NET release than alum alone, whereas the vaccine containing MPLA induced stronger NET responses than MPLA alone. NETs and alum alone and synergistically increased the expression of molecules involved in antigen presentation, i.e., CD80, CD86 and CD83, by peripheral blood monocytes. Monocyte priming with NETs resulted in individually differing IL-1β- and IL-6-responses. Thus, NETs induced by adjuvants in AIT-vaccines can provide autonomous and cooperative effects on early innate responses. The high diversity of individual innate responses to adjuvants and AIT-vaccines may affect their therapeutic efficacy.

B-1a cells, a regulatory subset of B lymphocytes, produce natural IgM and interleukin-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 colony-forming units of Escherichia coli intraperitoneally. After 4 and 20 h, 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 intraperitoneal 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 the overall survival rate in B-1a cell-treated septic mice. To understand the mechanism, we cocultured bone marrow-derived neutrophils with peritoneal B-1a cells in a contact or noncontact condition using an insert and stimulated them with eCIRP. After 4 h, we found that eCIRP significantly increased NET formation in bone marrow-derived neutrophils. 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 the 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.

Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by synovial inflammation, joint destruction, and systemic complications, eventually leading to a high rate of disability, but its exact pathogenesis remains unclear. Neutrophil extracellular traps (NETs) are chromatin fibers released by activated neutrophils during infection/inflammation, containing histones, antimicrobial proteins, and granule components. Under physiological conditions, NETs trap pathogens and act as a pivotal anti-infective mechanism of the innate immune response. During the development of RA, NET components act as danger-associated molecular patterns (DAMPs) to activate NLRP3 inflammasomes and the complements in effector lymphocytes, amplifying inflammation; NETs promote the RA-related autoantibody production in B cells, such as anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF), fueling autoimmunity, while ACPAs further induce NETosis, creating a vicious feedback loop; NETs facilitate the release of pro-inflammatory cytokines (e.g., IL-6, IL-1β, TNF-α), exacerbating joint damage; finally, NETs activate T cells, dendritic cells, and macrophages via boosting RAGE/TLR9 pathway, thereby driving the proliferation and migration fibroblast-like synoviocytes. Notably, inhibiting NET formation (e.g., FcαRI antibody, celastrol), blocking NET-mediated inflammation (e.g., RAGE/TLR9 antagonists), and clearing NET remnants to break the pathogenic cycle (e.g., PAD enzyme inhibitors, DNase I and CD19 CAR-T trials) provide novel strategies for RA treatment. This article highlights the pathogenic role of NETs in RA, and emphasizes the potential as clinical biomarkers and therapeutic targets for RA progression. It will open avenues for novel treatments targeting NETosis or its downstream effects, potentially improving outcomes for RA and other inflammatory arthritides.