Platelets serve not only as crucial hemostatic components but also as pivotal regulators of inflammatory responses, capable of interacting with diverse cell types and secreting abundant extracellular factors. Accumulating evidence demonstrates that high mobility group box 1 (HMGB1), a DNA-binding protein and critical inflammatory mediator, plays multifaceted roles in disease progression, with platelets being one cellular source of circulating HMGB1. Under pathological conditions, platelets release HMGB1 into the extracellular matrix, establishing bidirectional communication between platelets and other immune cells. Moreover, HMGB1 reciprocally activates platelets through Toll-like receptors (TLRs) and receptor for advanced glycation end-products (RAGE), facilitating platelet activation and subsequent release of regulatory factors that drive inflammation-associated pathological thrombosis. In this review, we systematically characterize the HMGB1-platelet axis and elucidate its context-dependent roles in specific disease states. The mechanistic interplay between HMGB1 signaling and platelet pathophysiology is discussed, particularly its implications for disease progression. Furthermore, we critically evaluate therapeutic strategies targeting HMGB1 developed over the past decade, while proposing future directions for dual-target interventions that simultaneously modulate HMGB1 and platelet activity to combat inflammation-driven thrombotic disorders.

ObjectiveDeep vein thrombosis (DVT) is a global health issue caused by abnormal clotting in deep veins, which can lead to serious complications such as pulmonary embolism. This study is the first to validate the regulatory effect of miR-223-3p on the NLRP3 inflammasome in a mouse model of DVT, expanding its potential therapeutic value in venous thrombosis-associated inflammation.MethodsMicroRNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were conducted to assess miRNA expression in a DVT mouse model. The downstream target of miR-223-3p, NLRP3, was identified using miRNA target prediction databases and validated by qRT-PCR. Human umbilical vein endothelial cells (HUVECs) and a DVT mouse model were used to explore the functional relationship between miR-223-3p and Nlrp3.ResultsThe expression of miR-223-3p and Nlrp3 was significantly increased in the vein walls of mice with DVT. The tail vein injection of agomiR-223-3p reduced thrombus formation and downregulated the expression of Nlrp3, interleukin 6 (Il-6), interleukin 1 beta (IL-1beta) and Icam-1. In vitro, miR-223-3p overexpression reduced the expression of NLRP3, Il-6, IL-1beta and ICAM-1, whereas NLRP3 overexpression antagonized these effects. Additionally, miR-223-3p enhanced the viability and migration of LPS-stimulated HUVECs by reducing NLRP3 expression.ConclusionsOur findings suggest that miR-223-3p may play a role in alleviating inflammation and reducing the thrombus burden in mice with DVT by downregulating Nlrp3 expression, supporting its potential as a therapeutic target for DVT.

Sepsis, a life-threatening condition characterized by systemic inflammation and multiorgan dysfunction, is closely associated with the excessive formation of neutrophil extracellular traps (NETs) and the release of cell-free DNA. Both play a central role in sepsis progression, acting as major contributors to immunothrombosis and associated complications. Endogenous DNases play a pivotal role in degrading NETs and cell-free DNA, yet their activity is often dysregulated during thrombotic disease. Although exogenous DNase1 administration has shown potential in reducing NET burden and mitigating the detrimental effects of immunothrombosis, its therapeutic efficacy upon intravenous administration remains uncertain. The development of engineered DNase formulations and combination therapies may further enhance its therapeutic effectiveness by modifying its pharmacodynamic properties and avoiding the adverse effects associated with NET degradation, respectively. Although NETs are well-established targets of DNase1, it remains uncertain whether the positive effects of DNase1 on immunothrombosis are exclusively related to it's targeting of NETs or if other components contributing to immunothrombosis are also affected. This review examines the endogenous regulation of NETs in circulation and the therapeutic potential of DNases in immunothrombosis, underscoring the necessity for further investigation to optimize their clinical application.

Recurring lung injury, chronic inflammation, aberrant tissue repair and impaired tissue remodelling contribute to the pathogenesis of pulmonary fibrosis (PF). Neutrophil extracellular traps (NETs) are released by activated neutrophils to trap, immobilise and kill invading pathogen and is facilitated by peptidyl arginine deiminase-4 (PAD-4). Dysregulated NETs release and abnormal PAD-4 activation plays a crucial role in activating pro-fibrotic events in PF. Developmental endothelial locus-1 (Del-1), expressed by the endothelial cells of lungs and brain acts as an endogenous inhibitor of inflammation and fibrosis. We have hypothesised that PAD-4 inhibitor exerts anti-inflammatory and anti-fibrotic effects in mice model of PF. We have also hypothesised by PAD-4 regulated the transcription of Del-1 through co-repression and its inhibition potentiates anti-fibrotic effects of Del-1. In our study, the PAD-4 inhibitor chloro-amidine (CLA) demonstrated anti-NETotic and anti-inflammatory effects in vitro in differentiated HL-60 cells. In a bleomycin-induced PF mice model, CLA administration in two doses (3 mg/kg, I.P and 10 mg/kg, I.P) improved lung function, normalized bronchoalveolar lavage fluid parameters, and attenuated fibrotic events, including markers of extracellular matrix and epithelial-mesenchymal transition. Histological analyses confirmed the restoration of lung architecture and collagen deposition with CLA treatment. ELISA, IHC, IF, RT-PCR, and immunoblot analysis supported the anti-NETotic effects of CLA. Furthermore, BLM-induced PF reduced Del-1 and p53 expression, which was normalized by CLA treatment. These findings suggest that inhibition of PAD-4 results in amelioration of PF in animal model and may involve modulation of Del-1 and p53 pathways, warranting further investigation.

Immune complex (IC)-driven formation of neutrophil extracellular traps (NETs) is a major contributing factor to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Exogenous recombinant human serpin B1 (rhsB1) can regulate NET formation; however, its mechanism(s) of action is currently unknown as is its ability to regulate IC-mediated NET formation and other neutrophil effector functions. To investigate this, we engineered or post-translationally modified rhsB1 proteins that possessed specific neutrophil protease inhibitory activities and pretreated isolated neutrophils with them prior to inducing NET formation with ICs derived from patients with SLE, PMA, or the calcium ionophore A23187. Neutrophil activation and phagocytosis assays were also performed with rhsB1 pretreated and IC-activated neutrophils. rhsB1 dose-dependently inhibited NET formation by all three agents in a process dependent on its chymotrypsin-like inhibitory activity, most likely cathepsin G. Only one variant (rhsB1 C344A) increased surface levels of neutrophil adhesion/activation markers on IC-activated neutrophils and boosted intracellular ROS production. Further, rhsB1 enhanced complement-mediated neutrophil phagocytosis of opsonized bacteria but not ICs. In conclusion, we have identified a novel mechanism of action by which exogenously administered rhsB1 inhibits IC, PMA, and A2138-mediated NET formation. Cathepsin G is a well-known contributor to autoimmune disease but to our knowledge, this is the first report implicating it as a potential driver of NET formation. We identified the rhsB1 C334A variant as a candidate protein that can suppress IC-mediated NET formation, boost microbial phagocytosis, and potentially impact additional neutrophil effector functions including ROS-mediated microbial killing in phagolysosomes.

Extracellular traps formed by neutrophils (NETs) and eosinophils (EETs) have been described in coronary thrombi, contributing to thrombus stability. A key mechanism during NET formation is histone modification by the enzyme PAD4. Citrullinated histones, the product of PAD4 activity, are often attributed to neutrophils. Eosinophils also express high levels of PAD4.

We aimed to explore the contribution of PAD4 to EET formation.

We performed immunohistological analyses on thrombi, including a large, intact, and eosinophil-containing thrombus retrieved from the right coronary artery using an aspiration catheter and stroke thrombi from thrombectomy retrieval. We studied eosinophils for their capability to form PAD4-dependent EETs in response to strong ET-inducing agonists as well as activated platelets and bacteria.

Histopathology and immunofluorescence microscopy identified a coronary thrombus rich in platelets and neutrophils, with distinct areas containing von Willebrand factor and citrullinated histone H3 (H3Cit). Eosinophils were also identified in leukocyte-rich areas. The majority of the H3Cit+ signal colocalized with myeloperoxidase, but some colocalized with eosinophil peroxidase, indicating EETs. Eosinophils isolated from healthy volunteers produced H3Cit+ EETs, indicating an involvement of PAD4 activity. The selective PAD4 inhibitor GSK484 blocked this process, supporting PAD4 dependence of H3Cit+ EET release. Citrullinated histones were also present in EETs produced in response to live Staphylococci. However, limited evidence for EETs was found in mouse models of venous thrombosis or infective endocarditis.

As in NETosis, PAD4 can catalyze the formation of EETs. Inhibition of PAD4 decreases EET formation, supporting the future utility of PAD4 inhibitors as possible antithrombotic agents.

Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis.

Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies.

Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT2 PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V+ procoagulant endothelial CD62E+ (E-selectin) and neutrophil (Ly6G+) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082.

Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.

Neutrophils are a crucial component of the innate immune system and play a pivotal role in various physiological processes. From a physical perspective, hitchhiking is considered a phenomenon of efficient transportation. The combination of neutrophils and hitchhikers has given rise to effective delivery systems both in vivo and in vitro, thus neutrophils hitchhiking become a novel approach to disease treatment. This article provides an overview of the innovative and feasible application of neutrophils as drug carriers. It explores the mechanisms underlying neutrophil function, elucidates the mechanism of drug delivery mediated by neutrophil-hitchhiking, and discusses the potential applications of this strategy in the treatment of cancer, immune diseases, inflammatory diseases, and other medical conditions.

Gouty arthritis (GA) is an immune-mediated disorder characterized by severe inflammation due to the deposition of monosodium urate (MSU) crystals in the joints. The pathophysiological mechanisms of GA are not yet fully understood, and therefore, the identification of effective therapeutic targets is of paramount importance. Neutrophil extracellular traps (NETs), an intricate structure of DNA scaffold, encompassing myeloperoxidase, histones, and elastases - have gained significant attention as a prospective therapeutic target for gouty arthritis, due to their innate antimicrobial and immunomodulatory properties. Hence, exploring the therapeutic potential of NETs in gouty arthritis remains an enticing avenue for further investigation. During the process of gouty arthritis, the formation of NETs triggers the release of inflammatory cytokines, thereby contributing to the inflammatory response, while MSU crystals and cytokines are sequestered and degraded by the aggregation of NETs. Here, we provide a concise summary of the inflammatory processes underlying the initiation and resolution of gouty arthritis mediated by NETs. Furthermore, this review presents an overview of the current pharmacological approaches for treating gouty arthritis and summarizes the potential of natural and synthetic product-based inhibitors that target NET formation as novel therapeutic options, alongside elucidating the intrinsic challenges of these inhibitors in NETs research. Lastly, the limitations of HL-60 cell as a suitable substitute of neutrophils in NETs research are summarized and discussed. Series of recommendations are provided, strategically oriented towards guiding future investigations to effectively address these concerns. These findings will contribute to an enhanced comprehension of the interplay between NETs and GA, facilitating the proposition of innovative therapeutic strategies and novel approaches for the management of GA.

Graves' disease (GD) is an autoimmune disorder characterized by hyperthyroidism resulting from autoantibody-induced stimulation of the thyroid gland. Despite recent advancements in understanding GD's pathogenesis, the molecular processes driving disease progression and treatment response remain poorly understood. In this study, we aimed to identify crucial immunogenic factors associated with GD prognosis and immunotherapeutic response. To achieve this, we implemented a comprehensive screening strategy that combined computational immunogenicity-potential scoring with multi-parametric cluster analysis to assess the immunomodulatory genes in GD-related subtypes involving stromal and immune cells. Utilizing weighted gene co-expression network analysis (WGCNA), we identified co-expressed gene modules linked to cellular senescence and immune infiltration in CD4+ and CD8+ GD samples. Additionally, gene set enrichment analysis enabled the identification of hallmark pathways distinguishing high- and low-immune subtypes. Our WGCNA analysis revealed 21 gene co-expression modules comprising 1,541 genes associated with immune infiltration components in various stages of GD, including T cells, M1 and M2 macrophages, NK cells, and Tregs. These genes primarily participated in T cell proliferation through purinergic signaling pathways, particularly neuroactive ligand-receptor interactions, and DNA binding transcription factor activity. Three genes, namely PRSS1, HCRTR1, and P2RY4, exhibited robustness in GD patients across multiple stages and were involved in immune cell infiltration during the late stage of GD (p < 0.05). Importantly, HCRTR1 and P2RY4 emerged as potential prognostic signatures for predicting overall survival in high-immunocore GD patients (p < 0.05). Overall, our study provides novel insights into the molecular mechanisms driving GD progression and highlights potential key immunogens for further investigation. These findings underscore the significance of immune infiltration-related cellular senescence in GD therapy and present promising targets for the development of new immunotherapeutic strategies.

Intrahepatic infiltration of neutrophils is a character of alcoholic hepatitis (AH) and neutrophil extracellular traps (NETs) are an important strategy for neutrophils to fix and kill invading microorganisms. The gut-liver axis has been thought to play a critical role in many liver diseases also including AH. However, whether NETs appear in AH and play role in AH is still unsure.

Serum samples from AH patients were collected and LPS and MPO-DNA were detected. WT, NE KO, and TLR4 KO mice were used to build the AH model, and the intestinal bacteria were eliminated at the same time and LPS was given. Then the formation of NETs and AH-related markers were detected.

The serum MPO-DNA and LPS concentration was increased in AH patients and a correlation was revealed between these two indexes. More intrahepatic NETs formed in AH mice. NETs formation decreased with antibiotic intervention and restored with antibiotic intervention plus LPS supplement. While NETs formation failed to change with gut microbiome or combine LPS supplement in TLR4 KO mice. As we tested AH-related characters, liver injury, intrahepatic fat deposition, inflammation, and fibrosis alleviated with depletion of NE. These related marks were also attenuated with gut sterilization by antibiotics and recovered with a combined treatment with antibiotics plus LPS. But the AH-related markers did show a difference in TLR4 KO mice when they received the same treatment.

Intestinal-derived LPS promotes NETs formation in AH through the TLR4 pathway and further accelerates the AH process by NETs.

Nowadays, acute respiratory distress syndrome (ARDS) still has a high mortality rate, and the alleviation and treatment of ARDS remains a major research focus. There are various causes of ARDS, among which pneumonia and non-pulmonary sepsis are the most common. Trauma and blood transfusion can also cause ARDS. In ARDS, the aggregation and infiltration of neutrophils in the lungs have a great influence on the development of the disease. Neutrophils regulate inflammatory responses through various pathways, and the release of neutrophils through neutrophil extracellular traps (NETs) is considered to be one of the most important mechanisms. NETs are mainly composed of DNA, histones, and granuloproteins, all of which can mediate downstream signaling pathways that can activate inflammatory responses, generate immune clots, and cause damage to surrounding tissues. At the same time, the components of NETs can also promote the formation and release of NETs, thus forming a vicious cycle that continuously aggravates the progression of the disease. NETs are also associated with cytokine storms and immune balance. Since DNA is the main component of NETs, DNase I is considered a viable drug for removing NETs. Other therapeutic methods to inhibit the formation of NETs are also worthy of further exploration. This review discusses the formation and mechanism of NETs in ARDS. Understanding the association between NETs and ARDS may help to develop new perspectives on the treatment of ARDS.

During infection, neutrophil extracellular traps (NETs) are associated with severity of pulmonary diseases such as acute respiratory disease syndrome. NETs induce subsequent immune responses, are directly cytotoxic to pulmonary cells, and are highly procoagulant. Anticoagulation treatment was shown to reduce in-hospital mortality, indicating thromboinflammatory complications. However, data are sparsely available on the involvement of NETs in secondary events after virus clearance, which can lead to persistent lung damage and postacute sequelae with chronic fatigue and dyspnea.

This study focuses on late-phase events using a murine model of viral lung infection with postacute sequelae after virus resolution.

C57BL/6JRj mice were infected intranasally with the betacoronavirus murine coronavirus (MCoV, strain MHV-A95), and tissue samples were collected after 2, 4, and 10 days. For NET modulation, mice were pretreated with OM-85 or GSK484 and DNase I were administered intraperitoneally between days 2 to 5 and days 4 to 7, respectively.

Rapid, platelet-attributed thrombus formation was followed by a second, late phase of thromboinflammation. This phase was characterized by negligible virus titers but pronounced tissue damage, apoptosis, oxidative DNA damage, and presence of NETs. Inhibition of NETs during the acute phase did not impact virus burden but decreased lung cell apoptosis by 67% and oxidative stress by 94%. Prevention of neutrophil activation by immune training before virus infection reduced damage by 75%, NETs by 31%, and pulmonary thrombi by 93%.

NETs are detrimental inducers of tissue damage during respiratory virus infection but do not contribute to virus clearance.

Neutrophil extracellular traps (NETs), a key component of early defense against microbial infection, are also associated with tissue injury. NET composition has been reported to vary with some disease states, but the composition and variability of NETs across many healthy subjects provide a critical comparison that has not been well investigated. We evaluated NETs from twelve healthy subjects of varying ages isolated from multiple blood draws over a three-and-one-half-year period to delineate the variability in extracellular DNA, protein, enzymatic activities, and susceptibility to protease inhibitors. We calculated correlations for NET constituents and loss of human bronchial epithelial barrier integrity, measured by transepithelial electrical resistance, after NET exposure. We found that although there was some variability within the same subject over time, the mean NET total DNA, dsDNA, protein, LDH, neutrophil elastase (NE), and proteinase 3 (PR3) in isolated NETs were consistent across subjects. NET serine protease activity varied considerably within the same donor from day to day. The mean NET cathepsin G and MPO were significantly different across donors. IL-8 > IL-1RA > G-CSF were the most abundant cytokines in NETs. There was no significant difference in the mean concentration or variability of IL-8, IL-1RA, G-CSF, IL-1α, IL-1β, or TNF-α in different subjects' NETs. NET DNA concentration was correlated with increased NET neutrophil elastase activity and higher NET IL-1RA concentrations. The mean reduction in protease activity by protease inhibitors was significantly different across donors. NET DNA concentration correlated best with reductions in the barrier integrity of human bronchial epithelia. Defining NET concentration by DNA content correlates with other NET components and reductions in NET-driven epithelial barrier dysfunction, suggesting DNA is a reasonable surrogate measurement for these complex structures in healthy subjects.

Cells tend to disintegrate themselves or are forced to undergo such destructive processes in critical circumstances. This complex cellular function necessitates various mechanisms and molecular pathways in order to be executed. The very nature of cell death is essentially important and vital for maintaining homeostasis, thus any type of disturbing occurrence might lead to different sorts of diseases and dysfunctions. Cell death has various modalities and yet, every now and then, a new type of this elegant procedure gets to be discovered. The diversity of cell death compels the need for a universal organizing system in order to facilitate further studies, therapeutic strategies and the invention of new methods of research. Considering all that, we attempted to review most of the known cell death mechanisms and sort them all into one arranging system that operates under a simple but subtle decision-making (If \ Else) order as a sorting algorithm, in which it decides to place and sort an input data (a type of cell death) into its proper set, then a subset and finally a group of cell death. By proposing this algorithm, the authors hope it may solve the problems regarding newer and/or undiscovered types of cell death and facilitate research and therapeutic applications of cell death.

To characterize the role of neutrophil extracellular traps (NETs) in heterotopic ossification (HO) formation and progression and to use mechanical and pharmacological methods to decrease NETosis and mitigate HO formation.

Traumatic HO is the aberrant osteochondral differentiation of mesenchymal progenitor cells after traumatic injury, burns, or surgery. While the innate immune response has been shown to be necessary for HO formation, the specific immune cell phenotype and function remain unknown. Neutrophils, one of the earliest immune cells to respond after HO-inducing injuries, can extrude DNA, forming highly inflammatory NETs. We hypothesized that neutrophils and NETs would be diagnostic biomarkers and therapeutic targets for the detection and mitigation of HO.

C57BL6J mice underwent burn/tenotomy (a well-established mouse model of HO) or a non-HO-forming sham injury. These mice were either (1) ambulated ad libitum, (2) ambulated ad libitum with daily intraperitoneal hydroxychloroquine, ODN-2088 (both known to affect NETosis pathways), or control injections, or (3) had the injured hind limb immobilized. Single-cell analysis was performed to analyze neutrophils, NETosis, and downstream signaling after the HO-forming injury. Immunofluorescence microscopy was used to visualize NETosis at the HO site and neutrophils were identified using flow cytometry. Serum and cell lysates from HO sites were analyzed using enzyme-linked immunosorbent assay for myeloperoxidase-DNA and ELA2-DNA complexes to identify NETosis. Micro-computerized tomography was performed on all groups to analyze the HO volume.

Molecular and transcriptional analyses revealed the presence of NETs within the HO injury site, which peaked in the early phases after injury. These NETs were highly restricted to the HO site, with gene signatures derived from both in vitro NET induction and clinical neutrophil characterizations showing a high degree of NET "priming" at the site of injury, but not in neutrophils in the blood or bone marrow. Cell-cell communication analyses revealed that this localized NET formation coincided with high levels of toll-like receptor signaling specific to neutrophils at the injury site. Reducing the overall neutrophil abundance within the injury site, either pharmacologically through treatment with hydroxychloroquine, the toll-like receptor 9 inhibitor OPN-2088, or mechanical treatment with limb offloading, results in the mitigation of HO formation.

These data provide a further understanding of the ability of neutrophils to form NETs at the injury site, clarify the role of neutrophils in HO, and identify potential diagnostic and therapeutic targets for HO mitigation.

Ischemic thrombotic disease, characterized by the formation of obstructive blood clots within arteries or veins, is a condition associated with life-threatening events, such as stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism. The conventional therapeutic strategy relies on treatments with anticoagulants that unfortunately pose an inherent risk of bleeding complications. These anticoagulants primarily target clotting factors, often overlooking upstream events, including the release of neutrophil extracellular traps (NETs). Neutrophils are integral components of the innate immune system, traditionally known for their role in combating pathogens through NET formation. Emerging evidence has now revealed that NETs contribute to a prothrombotic milieu by promoting platelet activation, increasing thrombin generation, and providing a scaffold for clot formation. Additionally, NET components enhance clot stability and resistance to fibrinolysis. Clinical and preclinical studies have underscored the mechanistic involvement of NETs in the pathogenesis of thrombotic complications, since the clots obtained from patients and experimental models consistently exhibit the presence of NETs. Given these insights, the inhibition of NETs or NET formation is emerging as a promising therapeutic approach for ischemic thrombotic diseases. Recent investigations also implicate a role for the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome as a mediator of NETosis and thrombosis, suggesting that NLRP3 inhibition may also hold potential for mitigating thrombotic events. Therefore, future preclinical and clinical studies aimed at identifying and validating NLRP3 inhibition as a novel therapeutic intervention for thrombotic disorders are imperative.

Neutrophil Extracellular Traps (NETs), a key component of early defense against microbial infection, are also associated with tissue injury. NET composition has been reported to vary with some disease states, but the composition and variability of NETs across many healthy subjects provides a critical comparison that has not been well investigated. We evaluated NETs from twelve healthy subjects of varying ages isolated from multiple blood draws over a three and one half-year period to delineate the variability in extracellular DNA, protein, enzymatic activities, and susceptibility to protease inhibitors. We calculated correlations for NET constituents and loss of human bronchial epithelial barrier integrity, measured by transepithelial electrical resistance, after NET exposure. We found that although there was some variability within the same subject over time, the mean numbers of neutrophils, protein, LDH, serine protease activities, and cytokines IL-8, IL-1RA, and G-CSF in isolated NETs were consistent across subjects. Total DNA and double stranded DNA content in NETs were different across donors. NETs had little or no TNFα, IL-17A, or GM-CSF. NET DNA concentration correlated with increased NET neutrophil elastase activity and higher NET IL-1RA concentrations. NET serine protease activity varied considerably within the same donor from day-to-day. Mean response to protease inhibitors was significantly different across donors. NET DNA concentration correlated best with reductions in barrier integrity of human bronchial epithelia. Defining NET concentration by DNA content correlates with other NET components and reductions in NET-driven epithelial barrier dysfunction, suggesting DNA is a reasonable surrogate measurement for these complex structures in healthy subjects.

Neutrophils are a major subset of leukocytes in human circulating blood. In some circumstances, neutrophils release neutrophil extracellular traps (NETs). lnitially, NETs were considered to have a strong antibacterial capacity. However, currently, NETs have been shown to have a pivotal impact on various diseases. Different stimulators induce the production of different types of NETs, and their biological functions and modes of clearance do not appear to be the same. In this review, we will discuss several important issues related to NETs in order to better understand the relationship between NETs and diseases, as well as how to utilize the characteristics of NETs for disease treatment.

Human pluripotent stem cell (hPSC)-derived tissues can be used to model diseases in cell types that are challenging to harvest and study at-scale, such as neutrophils. Neutrophil dysregulation, specifically neutrophil extracellular trap (NET) formation, plays a critical role in the prognosis and progression of multiple diseases, including COVID-19. While hPSCs can generate limitless neutrophils (iNeutrophils) to study these processes, current differentiation protocols generate heterogeneous cultures of granulocytes and precursors. Here, we describe a method to improve iNeutrophil differentiations through the deletion of GATA1. GATA1 knockout (KO) iNeutrophils are nearly identical to primary neutrophils in form and function. Unlike wild-type iNeutrophils, GATA1 KO iNeutrophils generate NETs in response to the physiologic stimulant lipopolysaccharide, suggesting they are a more accurate model when performing NET inhibitor screens. Furthermore, through deletion of CYBB, we demonstrate that GATA1 KO iNeutrophils are a powerful tool in determining involvement of a given protein in NET formation.

Severe inflammation via innate immune system activation causes organ dysfunction. Among these, the central nervous system (CNS) is particularly affected by encephalopathies. These symptoms are associated with the activation of microglia and a potential infiltration of leukocytes. These immune cells have recently been discovered to have the ability to produce extracellular traps (ETs). While these components capture and destroy pathogens, deleterious effects occur such as reduced neuronal excitability correlated with excessive ETs production. In this study, the objectives were to determine (1) whether immune cells form ETs in the CNS during acute inflammation (2) whether ETs produce neuromuscular disorders and (3) whether an immunomodulatory treatment such as β1-adrenergic blockers limits these effects. We observed an infiltration of neutrophils in the CNS, an activation of microglia and a production of ETs following lipopolysaccharide (LPS) administration. Atenolol, a β1-adrenergic blocker, significantly decreased the production of ETs in both microglia and neutrophils. This treatment also preserved the gastrocnemius motoneuron excitability. Similar results were observed when the production of ETs was prevented by sivelestat, an inhibitor of ET formation. In conclusion, our results demonstrate that LPS administration increases neutrophils infiltration into the CNS, activates immune cells and produces ETs that directly impair neuromuscular function. Prevention of ETs formation by β1-adrenergic blockers partly restores this function and could be a good target in order to reduce adverse effects in severe inflammation such as sepsis but also in other motor related pathologies linked to ETs production.

Deep venous thrombosis (DVT) is a part of venous thromboembolism (VTE) that clinically manifests as swelling and pain in the lower limbs. The most serious clinical complication of DVT is pulmonary embolism (PE), which has a high mortality rate. To date, its underlying mechanisms are not fully understood, and patients usually present with clinical symptoms only after the formation of the thrombus. Thus, it is essential to understand the underlying mechanisms of deep vein thrombosis for an early diagnosis and treatment of DVT. In recent years, many studies have concluded that Neutrophil Extracellular Traps (NETs) are closely associated with DVT. These are released by neutrophils and, in addition to trapping pathogens, can mediate the formation of deep vein thrombi, thereby blocking blood vessels and leading to the development of disease. Therefore, this paper describes the occurrence and development of NETs and discusses the mechanism of action of NETs on deep vein thrombosis. It aims to provide a direction for improved diagnosis and treatment of deep vein thrombosis in the near future.

Neutrophil extracellular traps (NETs) as special release products of neutrophils have received extensive attention. They are composed of decondensed chromatin and coated with nucleoproteins, including histones and some granulosa proteins. NETs can form a network structure to effectively capture and eliminate pathogens and prevent their spread. Not only that, recent studies have shown that NETs also play an important role in venous thrombosis. This review provides the most important updated evidence regarding the mechanism of NETs formation and the role of NETs in the process of venous thrombosis. The potential prophylactic and therapeutic value of NETs in venous thrombotic disease will also be discussed.

Entamoeba histolytica (Eh), a microaerophilic parasite, causes deadly enteric infections that result in Amoebiasis. Every year, the count of invasive infections reaches 50 million approximately and 40,000 to 1,00,000 deaths occurring due to amoebiasis are reported globally. Profound inflammation is the hallmark of severe amoebiasis which is facilitated by immune first defenders, neutrophils. Due to size incompatibility, neutrophils are unable to phagocytose Eh and thus, came up with the miraculous antiparasitic mechanism of neutrophil extracellular traps (NETs). This review provides an in-depth analysis of NETosis induced by Eh including the antigens involved in the recognition of Eh and the biochemistry of NET formation. Additionally, it underscores its novelty by describing the dual role of NETs in amoebiasis where it acts as a double-edged sword in terms of both clearing and exacerbating amoebiasis. It also provides a comprehensive account of the virulence factors discovered to date that are implicated directly and indirectly in the pathophysiology of Eh infections through the lens of NETs and can be interesting drug targets.

Despite the perception that e-cigarettes are safer than conventional cigarettes, numerous findings demonstrated that e-cigarette aerosol (EC) exposure induced compromised immune functionality, vascular changes even after acute exposure, and lung injury. Notably, altered neutrophil functionality and platelet hemodynamics have been observed post-EC exposure. It was hypothesized that EC exposure initiates an inflammatory response resulting in altered neutrophil behavior and increased neutrophil-platelet interaction in the pulmonary microvasculature. Neutrophil and platelet responses were examined up to 48 hrs following whole-body, short-term EC exposure without flavorants or nicotine in a murine model, which most closely modeled secondhand exposure. This study is the first to investigate the impact of EC exposure through lung intravital imaging. Compared to room air-exposed mice, EC-exposed mice displayed significantly increased 1.7‒1.9-fold number of neutrophils in the pulmonary microvasculature associated with no marked change in neutrophils within whole blood or bronchoalveolar lavage fluid (BALF). Neutrophil-platelet interactions were also significantly elevated 1.9‒2.5-fold in exposed mice. Plasma concentration of myeloperoxidase was markedly reduced 1.5-fold 48 hr following exposure cessation, suggesting suppressed neutrophil antimicrobial activity. Cytokine expression exhibited changes indicating vascular damage. Effects persisted for 48 hr post-EC exposure. Data demonstrated that EC exposure repeated for 3 consecutive days in 2.5 hr intervals in the absence of flavorants or nicotine resulted in modified pulmonary vasculature hemodynamics, altered immune functionality, and a pro-inflammatory state in female BALB/cJ mice.

Neutrophils are major effectors and regulators of the immune system. They play critical roles not only in the eradication of pathogens but also in cancer initiation and progression. Conversely, the presence of cancer affects neutrophil activity, maturation, and lifespan. By promoting or repressing key neutrophil functions, cancer cells co-opt neutrophil biology to their advantage. This co-opting includes hijacking one of neutrophils' most striking pathogen defense mechanisms: the formation of neutrophil extracellular traps (NETs). NETs are web-like filamentous extracellular structures of DNA, histones, and cytotoxic granule-derived proteins. Here, we discuss the bidirectional interplay by which cancer stimulates NET formation, and NETs in turn support disease progression. We review how vascular dysfunction and thrombosis caused by neutrophils and NETs underlie an elevated risk of death from cardiovascular events in cancer patients. Finally, we propose therapeutic strategies that may be effective in targeting NETs in the clinical setting.

Neutrophils or polymorphonuclear neutrophils (PMNs) are an important component of innate host defense. These phagocytic leukocytes are recruited to infected tissues and kill invading microbes. There are several general characteristics of neutrophils that make them highly effective as antimicrobial cells. First, there is tremendous daily production and turnover of granulocytes in healthy adults-typically 1011 per day. The vast majority (~95%) of these cells are neutrophils. In addition, neutrophils are mobilized rapidly in response to chemotactic factors and are among the first leukocytes recruited to infected tissues. Most notably, neutrophils contain and/or produce an abundance of antimicrobial molecules. Many of these antimicrobial molecules are toxic to host cells and can destroy host tissues. Thus, neutrophil activation and turnover are highly regulated processes. To that end, aged neutrophils undergo apoptosis constitutively, a process that contains antimicrobial function and proinflammatory capacity. Importantly, apoptosis facilitates nonphlogistic turnover of neutrophils and removal by macrophages. This homeostatic process is altered by interaction with microbes and their products, as well as host proinflammatory molecules. Microbial pathogens can delay neutrophil apoptosis, accelerate apoptosis following phagocytosis, or cause neutrophil cytolysis. Here, we review these processes and provide perspective on recent studies that have potential to impact this paradigm.

Deep venous thrombosis (DVT) is one of the major health problems worldwide. Apolipoprotein L domain containing 1 (APOLD1) was reported to be downregulated in DVT. The present study intended to investigate whether APOLD1 affects thrombus formation in a rat model of DVT.

The rat model of DVT was established by inferior vena cava (IVC) stenosis. At 6 hr, 12 hr, 24 hr, and 48 hr after IVC stenosis, the gross IVC with thrombus was dissected and observed. Then, the rats were preinjected with the lentiviral overexpression vector, APOLD1-LVs, 1 hr before IVC stenosis, to evaluate the influence of APOLD1 on thrombosis in rats. The serum levels of D-dimer and TAT as well as the content of TF in IVC tissues were detected by enzyme-linked immunosorbent assay (ELISA).

IVC stenosis resulted in thrombus formation in rats, increased serum levels of D-dimer and TAT, and decreased APOLD1 expression. APOLD1 overexpression inhibited in vivo thrombosis, reduced serum levels of D-dimer, and downregulated tissue factor (TF) activity and level. APOLD1 overexpression also increased p-PI3K and p-Akt protein levels.

APOLD1 suppresses thrombus formation in a rat model of DVT via downregulating TF expression by activating the PI3K/Akt pathway.

Neutrophil extracellular traps (NETs) are structures consisting of decondensed chromatin with associated proteins, including histones and antimicrobial peptides, released from activated neutrophils. They are believed to be one of the body's first lines of defense against infectious agents. Despite their beneficial effect on the immune response process, some studies indicate that their excessive formation and the associated accumulation of extracellular DNA (eDNA) together with other polyelectrolytes (F-actin) plays an important role in the pathogenesis of many diseases. Thus NETs formation and removal are clinically significant. The monoclonal antibody 2C5 has strong specificity for intact nucleohistones (NS) and targets NS in NETs as we previously confirmed. Creation of a nano preparation that can specifically recognize and destroy NETs represents the aim for treatment many diseases. 2C5 antibody functionalized micelles coated with DNase I were created to achieve this aim.

A state-of-the-art lecture titled "novel mechanisms of thrombo-inflammation during infection" was presented at the ISTH Congress in 2022. Platelet, neutrophil, and endothelial cell activation coordinate the development, progression, and resolution of thrombo-inflammatory events during infection. Activated platelets and neutrophil extracellular traps (NETs) are frequently observed in patients with sepsis and COVID-19, and high levels of NET-derived damage-associated molecular patterns (DAMPs) correlate with thrombotic complications. NET-associated DAMPs induce direct and indirect platelet activation, which in return potentiates neutrophil activation and NET formation. These coordinated interactions involve multiple receptors and signaling pathways contributing to vascular and organ damage exacerbating disease severity. This state-of-the-art review describes the main mechanisms by which platelets support NETosis and the key mechanisms by which NET-derived DAMPs trigger platelet activation and the formation of procoagulant platelets leading to thrombosis. We report how these DAMPs act through multiple receptors and signaling pathways differentially regulating cell activation and disease outcome, focusing on histones and S100A8/A9 and their contribution to the pathogenesis of sepsis and COVID-19. We further discuss the complexity of platelet activation during NETosis and the potential benefit of targeting selective or multiple NET-associated DAMPs to limit thrombo-inflammation during infection. Finally, we summarize relevant new data on this topic presented during the 2022 ISTH Congress.

Deep vein thrombosis (DVT) is a common clinical problem, but its cellular and molecular mechanisms remain incompletely understood. In this study, we performed single-cell RNA sequencing on mouse inferior vena cava (IVC) 24 h after thrombus-inducing IVC ligation or sham operation. 9 cell types composed of multiple subpopulations were identified. Notable transcriptomic changes induced by DVT included a marked inflammatory response, elevated hypoxia, and globally reduced myogenesis. Analysis of individual cell populations revealed increased inflammation and reduced extracellular matrix production across smooth muscle cells and fibroblasts, juxtaposed against an early phenotypic shift in smooth muscle cell populations away from a contractile state. By characterizing the transcriptomic changes in the vein wall during acute venous thrombosis at the single-cell level, this work provides novel insights into early pathological events in the vein wall that may potentiate thrombus formation and result in long term adverse venous remodeling.

Platelets are essential for the formation of a haemostatic plug to prevent bleeding, while neutrophils are the guardians of our immune defences against invading pathogens. The interplay between platelets and innate immunity, and subsequent triggering of the activation of coagulation is part of the host system to prevent systemic spread of pathogen in the blood stream. Aberrant immunothrombosis and excessive inflammation can however, contribute to the thrombotic burden observed in many cardiovascular diseases. In this review, we highlight how platelets and neutrophils interact with each other and how their crosstalk is central to both arterial and venous thrombosis and in COVID-19. While targeting platelets and coagulation enables efficient antithrombotic treatments, they are often accompanied with a bleeding risk. We also discuss how novel approaches to reduce platelet-mediated recruitment of neutrophils could represent promising therapies to treat thrombosis without affecting haemostasis.

Prothrombotic and proinflammatory properties of neutrophil extracellular traps (NETs) contribute to brain damage after ischemic stroke. CD21 is a novel phthalide neuroprotectant against cerebral ischemia in rodents. This study investigated effects of CD21 on the platelet-NET-thrombin axis and ischemic brain injury and the underlying mechanism. CD21 exerteddose-dependent neuroprotectionin rats that were subjected to2 h middle cerebral artery occlusion,dose-dependentlyinhibited adenosine diphosphate-mediatedplatelet aggregationin rats, and dose-dependentlyexertedanti-thrombotic activityin rodents that received a collagen-epinephrine combination, ferric chloride, or an arteriovenous shunt. Equimolar CD21 doses exerted stronger efficacy than 3-N-butylphthalide (NBP, natural phthalide for the treatment of ischemic stroke). CD21 dose-dependently improved regional cerebral blood flow, neurobehavioral deficits, and infarct volume in mice that were subjected to photothrombotic stroke (PTS). CD21 (13.79 mg/kg, i.v.) significantly decreased NET components (plasma dsDNA concentrations; mRNA levels of elastase, myeloperoxidase, and neutrophil gelatinase-associated lipocalin and protein level of citrullinated histone H3 in ischemic brain tissues), mRNA and protein levels of peptidyl-arginine deiminase 4 (PDA4, NET formation enzyme), and mRNA levels of NET-related inflammatory mediators (interleukin-1β, interleukin-17A, matrix metalloproteinase 8, and matrix metalloproteinase 9) in ischemic brain tissues, despite no effect on mRNA levels of deoxyribonuclease I (NET elimination enzyme). Pretreatment with compound C (inhibitor of adenosine monophosphate-activated protein kinase [AMPK]) significantly reversed the inhibitory effects of CD21 on NETs, PDA4, and inflammatory mediators in PTS mice. These results suggest that CD21 might regulate the platelet-NET-thrombin axis and protect against ischemic brain injury partly through the induction of AMPK activation.

Neutrophil extracellular traps (NETs) promote intra-acinar trypsin activation and tissue damage. Therefore, reducing NET formation can reduce tissue damage in severe acute pancreatitis (SAP). However, NET formation pathways may differ among disease models. In this study, we evaluated the role of the myeloperoxidase-neutrophil elastase (NE) pathway in NET formation in SAP. SAP was induced by intraperitoneal injection of cerulein and LPSs in mice, and NE activity was inhibited by GW311616. Pancreatic tissues were collected for multiplex immunofluorescence, scanning electron microscopy, and western blotting to detect NET formation and the effect of NE on citrullinated histone H3, followed by analyses of serum amylase and cytokine levels. Pretreatment with GW311616 significantly reduced NET formation, pancreatic tissue damage, and systemic inflammatory responses in SAP. Network pharmacology analyses using NE as the target revealed the monomeric compound epigallocatechin-3-gallate (EGCG). Binding between EGCG and NE was validated using molecular docking, and the ability of EGCG to inhibit NE activity was verified experimentally. NET formation by PMA-stimulated neutrophils was significantly reduced in vitro when the cells were pretreated with 40 μM EGCG. Pretreatment with EGCG significantly reduced NET formation, pancreatic tissue damage, and systemic inflammatory responses in vivo. These results reveal that NET formation requires the myeloperoxidase-NE pathway, and citrullination of histone H3 is affected by NE activity in SAP. EGCG shows therapeutic potential for affecting NE activity, NET formation, and systemic inflammation in SAP.

At the frontline of the host defence response, neutrophil antimicrobial functions have adapted to combat infections and injuries of different origins and magnitude. The release of web-like DNA structures named neutrophil extracellular traps (NETs) constitutes an important mechanism by which neutrophils prevent pathogen dissemination or deal with microorganisms of a bigger size. At the same time, nuclear and granule proteins with microbicidal activity bind to these DNA structures promoting the elimination of entrapped pathogens. However, these toxic properties may produce unwanted effects in the host, when neutrophils uncontrollably release NETs upon persistent inflammation. As a consequence, NET accumulation can produce vessel occlusion, tissue damage, and prolonged inflammation associated with the progression and exacerbation of multiple pathologic conditions. This review outlines recent advances in understanding the mechanisms of NET release and functions in sterile disease. We also discuss mechanisms of physiological regulation and the importance of neutrophil heterogeneity in NET formation and composition.

Rationale: The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. Objectives: To relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling. Methods: Production of elastase, release of extracellular traps, adhesion, and migration were assessed in neutrophils from patients with pulmonary arterial hypertension and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension, and we determined whether they produce pulmonary hypertension in mice. Measurements and Main Results: Neutrophils from patients with pulmonary arterial hypertension produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated β1-integrin and vinculin identified by proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic IFN signature that we related to an increase in human endogenous retrovirus K envelope protein. Transfection of human endogenous retrovirus K envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and IFN genes, whereas vinculin is increased by human endogenous retrovirus K deoxyuridine triphosphate diphosphatase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus K envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor. Conclusions: Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension.

Neutrophil extracellular traps (NETs) may be associated with the development of thrombosis. Experimental studies have confirmed the presence of NETs in thrombi specimens and potential role of NETs in the mechanisms of thrombosis. Clinical studies also have demonstrated significant changes in the levels of serum or plasma NETs biomarkers, such as citrullinated histones, myeloperoxidase, neutrophil elastase, nucleosomes, DNA, and their complexes in patients with thrombosis. This paper aims to comprehensively review the currently available evidence regarding the change in the levels of NETs biomarkers in patients with thrombosis, summarize the role of NETs and its biomarkers in the development and prognostic assessment of venous thromboembolism, coronary artery diseases, ischemic stroke, cancer-associated thromboembolism, and coronavirus disease 2019-associated thromboembolism, explore the potential therapeutic implications of NETs, and further discuss the shortcomings of existing NETs biomarkers in serum and plasma and their detection methods.

The objective of this study was to investigate the expression levels of microRNA-141-5p(miRNA-141-5p), MAPK1 and neutrophil elastase in patients with and without preeclampsia (PE), and the relationship between miRNA-141-5p and MAPK1 with respect to the secretion of elastase by neutrophils in patients with PE.

Thirty patients with PE and 30 healthy pregnant (HP) women were recruited from The Second Hospital of Shanxi Medical University, Taiyuan, China, between February 2017 and July 2018. Neutrophils were isolated from 8 mL peripheral blood samples and cultured. We recorded neutrophil count and morphology during culture. Apoptosis was detected by flow cytometry in different groups at 0, 24, and 48 h. The expression levels of elastase were detected in neutrophils by enzyme-linked immunosorbent assay, whereas the expression levels of miRNA-141-5p in peripheral blood neutrophils were detected by real-time polymerase chain reaction. We used TargetScanHuman Release 7.2 to analyze the target genes of miRNA-141-5p. The expression of MAPK1 in peripheral blood neutrophils was detected by western blotting. Data were analyzed by SPSS version 21.0 software, and comparisons between groups were carried out with the Student t test.

There was no significant difference between the PE and HP groups (P > 0.050) with regard to age or body mass index. The weight of newborns in the PE group (2846.00 ± 600.00 g) was significantly lower than that in the HP group (3055.00 ± 230.68 g). The number of neutrophilic granulocytes(NGs) in blood samples from the PE group was significantly higher than that in the HP group (P = 0.003). There was no significant difference between the groups with regard to morphology. Apoptosis in the PE group was delayed when compared with the HP group at different time points. The P value of apoptosis in the PE and HP groups were respectively 0.790, < 0.001 and 0.030 at 0 h, 24 h and 48 h. The expression levels of miRNA-141-5p in the PE group were significantly lower than those in the HP group (P < 0.050). The expression levels of MAPK1 in neutrophils from the PE group were significantly higher than those in the HP group (P < 0.050) by western blot. The expression levels of elastase in neutrophils from the PE group were significantly higher than those in the HP group (P < 0.050). Furthermore, the number of NGs in peripheral blood from the PE group was higher than that of the HP group; however, the levels of apoptosis were lower. The expression levels of miRNA-141-5p in NGs decreased, the expression of MAPK1 increased, and the secretion of neutrophil elastase in the NG medium increased in the PE group than those in the HP group.

Collectively, our analysis suggested that miRNA-141-5p may be involved in the pathogenesis of PE by regulating the MAPK1 signaling pathway to activate neutrophils and increase the secretion of elastase.