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Glucocorticoids are a major class of therapeutic anti-inflammatory and immunosuppressive drugs prescribed to patients with inflammatory diseases, to avoid transplant rejection, and as part of cancer chemotherapy. However, exposure to these drugs increases the risk of opportunistic infections such as with the fungus Aspergillus fumigatus, which causes mortality in >50% of infected patients. The mechanisms by which glucocorticoids increase susceptibility to A. fumigatus are poorly understood. In this article, we used a zebrafish larva Aspergillus infection model to identify innate immune mechanisms altered by glucocorticoid treatment. Infected larvae exposed to dexamethasone succumb to infection at a significantly higher rate than control larvae. However, both macrophages and neutrophils are still recruited to the site of infection, and dexamethasone treatment does not significantly affect fungal spore killing. Instead, the primary effect of dexamethasone manifests later in infection with treated larvae exhibiting increased invasive hyphal growth. In line with this, dexamethasone predominantly inhibits neutrophil function rather than macrophage function. Dexamethasone-induced mortality also depends on the glucocorticoid receptor. Dexamethasone partially suppresses NF-κB activation at the infection site by inducing the transcription of IκB via the glucocorticoid receptor. Independent CRISPR/Cas9 targeting of IKKγ to prevent NF-κB activation also increases invasive A. fumigatus growth and larval mortality. However, dexamethasone treatment of IKKγ crispant larvae further increases invasive hyphal growth and host mortality, suggesting that dexamethasone may suppress other pathways in addition to NF-κB to promote host susceptibility. Collectively, we find that dexamethasone acts through the glucocorticoid receptor to suppress NF-κB-mediated neutrophil control of A. fumigatus hyphae in zebrafish larvae., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Reactive oxygen species (ROS) scavenging of nanozymes toward acute kidney injury (AKI) is a current promising strategy, however, the glomerular filtration barrier (GFB) limits their application for treating kidney related diseases. Here, a neutrophil-mediated delivery system able to hijack neutrophil to transport nanozyme-loaded cRGD-liposomes to inflamed kidney for AKI treatment by cRGD targeting integrin αvβ1 is reported. The neutrophil-mediated nanozyme delivery system demonstrated great antioxidant and anti-apoptosis ability in HK-2 and NRK-52E cell lines. Moreover, in ischemia-reperfusion (I/R) induced AKI mice, a single dose of LM@cRGD-LPs 12 h post-ischemia significantly reduces renal function indicators, alleviates renal pathological changes, and inhibits apoptosis of renal tubular cells and the expression of renal tubular injured marker, thus remarkably reducing the damage of AKI. Mechanistically, the treatment of LM@cRGD-LPs markedly inhibits the process of Nrf2 to the nucleus and reduces the expression of the downstream HO-1, achieves a 99.51% increase in renal tissue Nrf2 levels, and an 86.31% decrease in HO-1 levels after LM@cRGD-LPs treatment. In short, the strategy of neutrophil-mediated nanozyme delivery system hold great promise as a potential therapy for AKI or other inflammatory diseases., (© 2024 Wiley‐VCH GmbH.)

Benzalkonium chloride (BAC) is a broad-spectrum antibacterial agent that possesses cleaning and bactericidal properties, but impact of BAC on wellbeing of aquatic organisms remains uncertain. Consequently, in this current study, we have examined the immunotoxic potential of BAC in zebrafish embryos, thus marking it as the pioneering effort in this field. According to the findings, zebrafish embryos exposed to BAC exhibited a decline in yolk area that varied with the concentration, along with a significant decrease in the count of neutrophils, macrophages, red blood cells, and thymus T-cells. We observed significantly up-regulated expression of immune-related signaling genes such as cxcl-c1c, il-8, tir4 and inf-γ, but expression of nf-κb was downregulated. In addition, we observed a marked reduction in the number of hematopoietic stem cells in zebrafish larvae after BAC exposure, which could be the result of oxidative stress-mediated apoptosis. We found that compared with the control group, the number of red blood cells in juvenile zebrafish in BAC-exposure group was significantly down-regulated, which could be attributed to hematopoietic stem cell defect. Astaxanthin restored immune cells and hematopoietic stem cells after BAC exposure, whereas Inhibitor of Wnt Response-1(IWR-1) restored neutrophils after BAC exposure. The research findings demonstrated that exposure to BAC displayed harmful effects on the development and immune system of zebrafish embryos. These effects might be associated with alterations in reactive oxygen species(ROS) levels and activation of the Wnt signaling pathway caused by BAC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024. Published by Elsevier Inc.)

Invasion and adhesion of neutrophils into tissues and their concomitant secretion play an important role in the development of vascular pathologies, including abdominal aortic aneurysm (AAA). Chronic administration of angiotensin II is used to initiate AAA formation in mice. The role of aldosterone in this process is being studied. We conducted for the first time a complex comparative study of the effects of angiotensin II and aldosterone on the adhesion of human neutrophils to fibronectin and the concomitant secretion of proteins, free amino acids as well as reactive oxygen (ROS) and nitrogen (NO) species. Neither angiotensin II nor aldosterone affected the attachment of neutrophils to fibronectin and the concomitant production of ROS. We showed for the first time that aldosterone stimulated the release of amino acid hydroxylysine, a product of lysyl hydroxylase, the activity of which is positively correlated with cell invasiveness. Aldosterone also initiates the secretion of matrix metalloproteinase 9 (MMP-9) and cathepsin G, which may reorganize the extracellular matrix and stimulate the recruitment and adhesion of neutrophils to the aortic walls. Angiotensin II did not affect protein secretion. It may contribute to neutrophil-induced vascular injury by inhibiting the production of NO or by increasing the secretion of isoleucine. Our results suggest that it is aldosterone-induced neutrophil secretion that may play a significant role in neutrophil-induced vascular wall destruction in angiotensin II-induced AAA or other vascular complications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Invasive fungal pathogen Candida auris has become a public health threat causing outbreaks of high mortality infections. Drug resistance often limits treatment options. For Candida albicans, subinhibitory concentrations of echinocandins unmask immunostimulatory β-glucan, augmenting immunity. Here we analyze the impact of echinocandin treatment of C. auris on β-glucan exposure and human neutrophil interactions. We show subinhibitory concentrations lead to minimal glucan unmasking and only subtle influences on neutrophil functions for the isolates belonging to circulating clades. The data suggest that echinocandin treatment will not largely alter phagocytic responses. Glucan masking pathways appear to differ between C. auris and C. albicans., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To investigate the therapeutic mechanism of Pterocarya hupehensis Skan total flavonoids (PHSTF) for rheumatoid arthritis (RA)., Methods: Twenty-five male SD rats were randomly divided into normal control group, RA model group, PHSTF treatment (45 and 90 mg/kg) groups, and Tripterygium glycosides (TPG) tablet (10 mg/kg) group ( n =5). Except for those in the normal control group, all the rats were subjected to collagen-induced arthritis (CIA) modeling using a secondary immunization method, after which PHSTF and TPG were administered via gavage once daily for 4 weeks. After the treatments, serum levels of TNF- α and IL-1β were measured using ELISA, and ankle joint pathologies were assessed with HE staining; the expression of citrullinated histone H3 (Cit-H3), a neutrophil extracellular trap (NET) marker, in the ankle joints was evaluated with immunohistochemistry. In primary cultures of rat peripheral blood neutrophils stimulated with phorbol ester (PMA), the effects of PHSTF (100 and 200 μg/mL) on the expressions of Cit-H3, peptidylarginine deiminase 4 (PADI4), neutrophil elastase (NE), and myeloperoxidase (MPO) were examined with Western blotting; immunofluorescence assay was used to observe Cit-H3 expression and NET formation in the cells., Results: In the CIA rat models, PHSTF significantly alleviated ankle swelling, decreased serum levels of TNF- α and IL-1β, improved histopathological changes in the ankle joints, and reduced Cit-H3 expression in both the serum and ankle joint cartilage. In the isolated rat neutrophils, PHSTF showed no significant effect on cell viability but strongly inhibited PMA-induced NET release., Conclusion: PHSTF can alleviate RA by inhibiting the formation of NETs.

The Panton-Valentine leukocidin (PVL) of Staphylococcus aureus is associated with necrotizing infections. After binding to complement 5a receptor (C5aR/CD88) and CD45 it causes cytolysis in polymorphonuclear neutrophils (PMNs) as well as inflammasome activation in monocytes. The objective of this study was to test if (ant)agonists of C5aR and CD45 can attenuate the effect of PVL on PMNs and monocytes. We tested the effect of various concentrations of six C5aR (ant)agonists (avacopan, BM213, DF2593A, JPE-1375, PMX205 and W-54011) and one CD45 antagonist (NQ301) to attenuate the cytotoxic effect of PVL on human PMNs and monocytes in vitro. Shifts in the half-maximal effective concentration (EC 50 ) of PVL to achieve a cytotoxic effect on PMNs and modulation of inflammatory cytokine response from monocytes were determined by flow cytometry and IL-1β detection. Pre-treatment of PMNs with avacopan, PMX205 and W-54,011 resulted in 3.6- to 4.3-fold shifts in the EC 50 for PVL and were able to suppress IL-1β secretion by human monocytes in the presence of PVL. BM213, DF2593A and NQ301 were unable to change the susceptibility of PMNs towards PVL or reduce inflammasome activation in monocytes. Avacopan, PMX205 and W-54,011 showed protection against PVL-induced cytotoxicity and suppressed IL-1β secretion by monocytes. Clinical studies are needed to prove whether these substances can be used therapeutically as repurposed drugs., (© 2024. The Author(s).)

Tumor-associated neutrophils (TANs) have been shown to promote immunosuppression and tumor progression, and a high TAN frequency predicts poor prognosis in triple-negative breast cancer (TNBC). Dysregulation of CREB-binding protein (CBP)/P300 function has been observed with multiple cancer types. The bromodomain (BRD) of CBP/P300 has been shown to regulate its activity. In this study, we found that IACS-70654, a selective CBP/P300 BRD inhibitor, reduced TANs and inhibited the growth of neutrophil-enriched TNBC models. In the bone marrow, CBP/P300 BRD inhibition reduced the tumor-driven abnormal differentiation and proliferation of neutrophil progenitors. Inhibition of CBP/P300 BRD also stimulated the immune response by inducing an IFN response and MHCI expression in tumor cells and increasing tumor-infiltrated cytotoxic T cells. Moreover, IACS-70654 improved the response of a neutrophil-enriched TNBC model to docetaxel and immune checkpoint blockade. This provides a rationale for combining a CBP/P300 BRD inhibitor with standard-of-care therapies in future clinical trials for neutrophil-enriched TNBC.

Aims: Sepsis pathophysiology is complex and identifying effective treatments for sepsis remains challenging. The study aims to identify effective drugs and targets for sepsis through transcriptomic analysis of sepsis patients, repositioning analysis of compounds, and validation by animal models., Main Methods: GSE185263 obtained from the GEO database that includes gene expression profiles of 44 healthy controls and 348 sepsis patients categorized by severity. Bioinformatic algorithms revealed the molecular, function, and immune characteristics of the sepsis, and constructed sepsis-related protein-protein interaction networks. Subsequently, Random Walk with Restart analysis was applied to identify candidate drugs for sepsis, which were tested in animal models for survival, inflammation, coagulation, and multi-organ damage., Key Findings: Our analysis found 1862 genes linked to sepsis development, enriched in functions like neutrophil extracellular trap formation (NETs) and complement/coagulation cascades. With disease progression, immune activation-associated cells were inhibited, while immune suppression-associated cells were activated. Next, the drug repositioning method identified candidate drugs, such as alpha-1 antitrypsin, that may play a therapeutic role by targeting neutrophil elastase (NE) to inhibit NETs. Animal experiments proved that alpha-1 antitrypsin treatment can improve the survival rate, reduce sepsis score, reduce the levels of inflammation markers in serum, and alleviate muti-organ morphological damage in mice with sepsis. The further results showed that α-1 antitrypsin can inhibit the NETs by suppressing the NE for the treatment of sepsis., Significance: Alpha-1 antitrypsin acted on the NE to inhibit NETs thereby protecting mice from sepsis-induced inflammation and coagulation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest in performing this study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Dendrobium officinale Kimura et Migo, known as "Tiepi Shihu" in traditional Chinese medicine, boasts an extensive history of medicinal use documented in the Chinese Pharmacopoeia. "Shen Nong Ben Cao Jing" records D. officinale as a superior herbal medicine for fortifying "Yin" and invigorating the five viscera. Erianin, a benzidine compound, emerges as a prominent active constituent derived from D. officinale, with the pharmacological efficacy of D. officinale closely linked to the anti-inflammatory properties of erianin., Aim of the Study: Acute lung injury (ALI) is a substantial threat to global public health, while P-selectin stands out as a promising novel target for treating acute inflammatory conditions. This investigation aims to explore the therapeutic potential of erianin in ALI treatment and elucidate the underlying mechanisms., Experimental Design: The effectiveness of erianin in conferring protection against ALI was investigated through comprehensive histopathological and biochemical analyses of lung tissues and bronchoalveolar lavage fluid (BALF) in an in vivo model of LPS-induced ALI in mice. The impact of erianin on fMLP-induced neutrophil chemotaxis was quantitatively assessed using the Transwell and Zigmond chamber, respectively. To determine the therapeutic target of erianin and elucidate their binding capability, a series of sophisticated assays were employed, including drug affinity responsive target stability (DARTS) assay, cellular thermal shift assay (CETSA), and molecular docking analyses., Results: Erianin demonstrated a significant alleviation of LPS-induced acute lung injury, characterized by reduced total cell and neutrophil counts and diminished total protein contents in BALF. Moreover, erianin exhibited a capacity to decrease proinflammatory cytokine production in both lung tissues and BALF. Notably, erianin effectively suppressed the activation of NF-κB signaling in the lung tissues of LPS- challenged mice; however, it did not exhibit in vitro inhibitory effects on inflammation in LPS-induced human pulmonary microvascular endothelial cells (HPMECs). Additionally, erianin blocked the adhesion and rolling of neutrophils on HPMECs. While erianin did not influence endothelial P-selectin expression or cytomembrane translocation, it significantly reduced the ligand affinity between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1)., Conclusions: Erianin inhibits P-selectin-mediated neutrophil adhesion to activated endothelium, thereby alleviating ALI. The present study highlights the potential of erianin as a promising lead for ALI treatment., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Microthrombosis is commonly seen in sepsis and COVID-19. Zixue Powder (ZXP) is a traditional Chinese herbal formula with the potential to treat microvascular and infectious diseases. However, the role and mechanism of ZXP in sepsis-associated thrombosis remain unclear., Aim of the Study: Investigating the therapeutic effectiveness and underlying mechanisms of ZXP in septic thrombosis., Materials and Methods: ZXP's compositions were examined with UPLC-QTOF-MS. The efficacy of ZXP on sepsis-induced thrombosis was assessed through various methods: liver tissue pathology was examined using hematoxylin-eosin staining, platelet count was determined by a blood cell analyzer, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum tissue factor (TF), thromboxane B2 (TXB2), D-Dimer, and plasminogen activator inhibitor-1 (PAI-1). Neutrophil extracellular traps (NETs) were localized and expressed in liver tissues by immunofluorescence, and the number of NETs in peripheral blood was evaluated by ELISA, which measured the quantity of cf-DNA and MPO-DNA in serum. Platelet P-selectin expression and platelet-neutrophil aggregation were measured by flow cytometry, and plasma P-selectin expression was measured by ELISA. Furthermore, the mechanism of the stimulator of interferon genes (STING) signaling pathway in ZXP's anti-sepsis thrombosis effect was investigated using the STING agonist, Western blot experiments, and immunoprecipitation experiments., Results: UPLC-QTOF-MS identified 40 chemical compositions of ZXP. Administration of ZXP resulted in significant improvements in liver thrombosis, platelet counts, and levels of TXB2, TF, PAI-1, and D-Dimer in septic rats. Moreover, ZXP inhibited NETs formation in both liver tissue and peripheral blood. Additionally, ZXP decreased the levels of P-selectin in both platelets and plasma, as well as the formation of platelet-neutrophil aggregates, thereby suppressing P-selectin-mediated NETs release. Immunoprecipitation and immunofluorescence staining experiments revealed that ZXP attenuated P-selectin secretion by inhibiting STING-mediated assembly of platelet soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complex, ultimately preventing inhibition of NETs formation., Conclusion: Our study showed that ZXP effectively mitigates platelet granule secretion primarily through modulation of the STING pathway, consequently impeding NET-associated thrombosis in sepsis. These findings offer valuable insights for future research on the development and application of ZXP., Competing Interests: Declaration of competing interest The author announced that they have no interest conflicts for this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Factor XII (FXII) is the zymogen of the plasma protease FXIIa that activates the intrinsic coagulation pathway and the kallikrein kinin-system. The role of FXII in inflammation has been obscure. Here, we report a single-domain antibody (nanobody, Nb) fused to the Fc region of a human immunoglobulin (Nb-Fc) that recognizes FXII in a conformation-dependent manner and interferes with FXIIa formation. Nb-Fc treatment inhibited arterial thrombosis in male mice without affecting hemostasis. In a mouse model of extracorporeal membrane oxygenation (ECMO), FXII inhibition or knockout reduced thrombus deposition on oxygenator membranes and systemic microvascular thrombi. ECMO increased circulating levels of D-dimer, alkaline phosphatase, creatinine and TNF-α and triggered microvascular neutrophil adherence, platelet aggregation and their interaction, which were substantially attenuated by FXII blockade. Both Nb-Fc treatment and FXII knockout markedly ameliorated immune complex-induced local vasculitis and anti-neutrophil cytoplasmic antibody-induced systemic vasculitis, consistent with selectively suppressed neutrophil migration. In human blood microfluidic analysis, Nb-Fc treatment prevented collagen-induced fibrin deposition and neutrophil adhesion/activation. Thus, FXII is an important mediator of inflammatory responses in vasculitis and ECMO, and Nb-Fc provides a promising approach to alleviate thrombo-inflammatory disorders., (© 2024. The Author(s).)

Background: Celecoxib, an anti-inflammatory drug, combined therapies using antimicrobials and immune modulator drugs are being studied., Objective: To assess whether Celecoxib has direct in vitro antifungal effect against the Paracoccidioides brasiliensis, the causative agent of Paracoccidioidomycosis-(PCM) and also if it improves the in vivo activity of neutrophils-(PMN) in an experimental murine subcutaneous-(air pouch) model of the disease., Methods: The antifungal activity of Celecoxib(6 mg/mL) on P. brasiliensis-(Pb18) was evaluated using the microdilution technique. Splenocytes co-cultured with Pb18 and treated with Celecoxib(6 mg/mL) were co-cultured for 24, 48 and 72-hours. Swiss mice were inoculated with Pb18 and treated with Celecoxib(6 mg/kg) in the subcutaneous air pouch. Neutrophils were collected from the air pouch. Mitochondrial activity, reactive oxygen production, catalase, peroxidase, cytokines and chemokines, nitrogen species, total protein, microbicidal activity of PMNs and viable Pb18 cells numbers were analyzed., Results: Celecoxib had no cytotoxic effect on splenocytes co-cultured with Pb18, but had a marked direct antifungal effect, inhibiting fungal growth both in vitro and in vivo. Celecoxib interaction with immune system cells in the air pouch, it leads to activation of PMNs, as confirmed by several parameters (mitochondrial activity, reactive oxygen species, peroxidase, KC and IL-6 increase, killing constant and phagocytosis). Celecoxib was able to reduce IL-4, IL-10 and IL-12 cytokine production. The number of recovered viable Pb18 decreased dramatically., Conclusions: This is the first report of the direct antifungal activity of Celecoxib against P. brasiliensis. The use of Celecoxib opens a new possibility for future treatment of PCM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Dihydromyricetin (DHM), which has various biological functions, possesses therapeutic potential for ulcerative colitis (UC). Neutrophil extracellular traps (NETs) and their components play a crucial role in several pathological processes in UC. However, whether DHM alleviates UC by regulating NETs remains unclear. Mice with dextran sulfate sodium (DSS)-induced acute colitis were treated with DHM at different concentrations, and the severity of colitis was evaluated by assessing body weight, colon length, histological scores, cytokine production, and epithelial barrier integrity. To quantify and visualize NETs, the expression of cell free-DNA (cf-DNA) in serum and Cit-H3 in colonic tissue was analyzed via western blotting and immunofluorescence analysis. HL-60 cells and mouse bone marrow-derived neutrophils (BMDNs) were used to evaluate the effects of DHM on NETs in vitro. NETs were treated with DHM at varying concentrations or DNase I and used to repair the intestinal epithelial barrier in a Caco-2/HIEC-6 cell monolayer model. Furthermore, the genes targeted by DHM through neutrophils for alleviating UC were identified by screening online databases, and the results of network pharmacological analysis were verified via western blotting and quantitative real-time polymerase chain reaction. DHM alleviated DSS-induced colitis in mice by reversing weight loss, increased DAI score, colon length shortening, enhanced spleen index, colonic pathological damage, cytokine production, and epithelial barrier loss in a dose-dependent manner. In addition, it inhibited the formation of NETs both in vivo and in vitro. Based on the results of network pharmacological analysis, DHM may target HIF-1α and VEGFA through neutrophils to alleviate UC. Treatment with PMA increased the expression of HIF-1α and VEGFA in D-HL-60 cells and BMDNs, whereas treatment with DHM or DNase I reversed this effect. Treatment with DMOG, an inhibitor of HIF prolyl hydroxylase (HIF-PH), counteracted the suppressive effects of DHM on NETs formation in D-HL-60 cells and BMDNs. Accordingly, it partially counteracted the protective effects of DHM on the intestinal epithelial barrier in Caco-2 and HIEC-6 cells. These results indicated that DHM alleviated DSS-induced UC by regulating NETs formation via the HIF-1α/VEGFA signaling pathway, suggesting that DHM is a promising therapeutic candidate for UC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background and Objective: This randomized controlled trial investigated the influence of perioperative lidocaine administration on the postoperative inflammatory response in patients undergoing robot-assisted radical prostatectomy, with the results having potential implications for postoperative recovery and cancer recurrence via neutrophil extracellular trapping (NETosis). Materials and Methods: In total, 58 patients with localized prostate cancer were randomly assigned to receive an intravenous infusion of 2% lidocaine or a saline placebo intraoperatively. Serum levels of interleukin (IL)-6, IL-10, and IL-17, tumor necrosis factor(TNF)-α, interferon(IFN)-γ, neutrophil elastase (NE), citrullinated histone3 (CitH3), and myeloperoxidase (MPO) were determined preoperatively and at 24 h postoperatively. Biochemical recurrence (BCR) was assessed over a follow-up period of 2 years. Results: The lidocaine group showed a significant change in MPO, a greater reduction in IL-10 level, and a smaller increase in the NE level compared to the placebo group, suggesting a modulatory effect of lidocaine on certain anti-inflammatory and neuroendocrine pathways. No significant difference in the BCR rate was observed between the two groups. Conclusions: Perioperative lidocaine administration selectively modulates certain inflammatory and neuroendocrine responses after robot-assisted radical prostatectomy surgery, potentially influencing recovery outcomes. These findings highlight the need for further investigations of the role of lidocaine in Enhanced Recovery After Surgery protocols, particularly in oncologic surgeries.

Objective: Antimicrobial resistance is an emerging problem and multi-drug resistant (MDR) Klebsiella pneumoniae ( K. pneumoniae ) represents an enormous risk of failing therapy in hospital-acquired pneumonia. The current study aimed to determine the immunomodulatory effect of topical flagellin in addition to antibiotic treatment during respiratory infection evoked by hypervirulent antibiotic-susceptible and antibiotic-resistant K. pneumoniae in mice., Methods: C57BL6 mice were inoculated intranasally with hypervirulent K. pneumoniae (K2:O1) which was either antibiotic-susceptible or multi-drug resistant. Six hours after infection, mice were treated with antibiotics intraperitoneally and flagellin or vehicle intranasally. Mice were sacrificed 24 hours after infection. Samples were analyzed for bacterial loads and for inflammatory and coagulation markers., Results: Flagellin therapy induced neutrophil influx in the lung during antibiotic-treated pneumonia evoked by either antibiotic-susceptible or -resistant K. pneumoniae . The pulmonary neutrophil response was matched by elevated levels of neutrophil-attracting chemokines, neutrophil degranulation products, and local coagulation activation. The combined therapy of effective antibiotics and flagellin did not impact K. pneumoniae outgrowth in the lung, but decreased bacterial counts in distant organs. Neutrophil depletion abrogated the flagellin-mediated effect on bacterial dissemination and local coagulation responses., Conclusion: Topical flagellin administration as an adjunctive to antibiotic treatment augments neutrophil responses during pneumonia evoked by MDR- K. pneumoniae , thereby reducing bacterial dissemination to distant organs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 van Linge, Kullberg, Chouchane, Roelofs, Goessens, van ‘t Veer, Sirard, de Vos and van der Poll.)

As methicillin-resistant Staphylococcus aureus (MRSA) exhibits formidable resistance to many drugs, the imperative for alternative therapeutic strategies becomes increasingly evident. At the heart of our study is the identification of a novel inhibitor through fluorescence anisotropy assays, specifically targeting the crucial multiple gene regulator A (MgrA) regulatory network in S. aureus . Isorhapontigenin (Iso), a natural compound, exhibits outstanding inhibitory efficacy, modulating bacterial virulence pathways without exerting direct bactericidal activity. This suggests a paradigm shift toward attenuating virulence instead of purely focusing on bacterial elimination. Through comprehensive in vitro and in vivo evaluations, we elucidated the complex interplay between Iso and MgrA, leading to reduced S. aureus adhesion, and overall virulence. At the cellular level, Iso offers significant protection to A549 cells infected with S. aureus , reducing cellular damage. Importantly, Iso augments the chemotaxis of neutrophils, curtailing the immune evasion capabilities of S. aureus . Furthermore, in vivo investigations highlight the notable effectiveness of Iso against MRSA-induced pneumonia and within the Galleria mellonella infection model, underscoring its pivotal role in the evolving realm of antibacterial drug discovery. Significantly, when Iso is used in combination with vancomycin, it outperforms its solo application, indicating a more pronounced therapeutic impact. This seminal research emphasizes Iso's potential as a primary defense against the surge of multidrug-resistant pathogens, heralding new prospects in antimicrobial therapy., Competing Interests: The authors declare no conflict of interest.

EGFR plays an essential role in cellular signaling pathways that regulate cell growth, proliferation, and survival and is often dysregulated in cancer. Several monoclonal IgG antibodies have been clinically tested over the years, which exert their function via blocking the ligand binding domain (thereby inhibiting downstream signaling) and inducing Fc-related effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). However, these IgG antibodies do not optimally recruit neutrophils, which are the most abundant white blood cell population in humans. Therefore, we reformatted six therapeutic EGFR antibodies (cetuximab, panitumumab, nimotuzumab, necitumumab, zalutumumab, and matuzumab) into the IgA3.0 format, which is an IgA2 isotype adapted for clinical application. Reformatting these antibodies preserved Fab-mediated functions such as EGFR binding, growth inhibition, and ligand blockade. In addition, whole leukocyte ADCC was significantly increased when using this panel of IgA3.0 antibodies compared with their respective IgG counterparts, with no major differences between IgA3.0 antibodies. In vivo, IgA3.0 matuzumab outperformed the other antibodies, resulting in the strongest suppression of tumor outgrowth in a long intraperitoneal model. We showed that neutrophils are important for the suppression of tumor outgrowth. IgA3.0 matuzumab exhibited reduced receptor internalization compared with the other antibodies, possibly accounting for its superior in vivo Fc-mediated tumor cell killing efficacy. In conclusion, reformatting EGFR antibodies into an IgA3.0 format increased Fc-mediated killing while retaining Fab-mediated functions and could therefore be a good alternative for the currently available antibody therapies., (©2024 American Association for Cancer Research.)

Peimisine has therapeutic effects on cough, asthma, acute lung injury and liver fibrosis. However, it has not been reported whether peimisine has an inhibitory effect on pulmonary fibrosis. In current study, a mouse pulmonary interstitial fibrosis model was established to investigate the efficacy of peimisine. Mice were categorized into six groups: Control, model, pirfenidone and three peimisine multi-dose groups. After the modelling, each group was given drugs for 21 days. Mice were euthanized and the histopathology changes of the lung were compared. The contents of cytokines in serum were determined. The mRNA expression levels of related genes in the lung tissue were detected. The contents of macrophages and neutrophils in the bronchoalveolar lavage fluid (BALF) were detected. The antifibrotic effect of peimisine was validated by using MRC-5 cells. The results demonstrated that peimisine could alleviate the destruction of alveolar structure and reduce the aggregation of inflammatory cells. Peimisine could reduce the protein expression levels of cytokines in serum. The mRNA levels of related genes were regulated. The contents of macrophages and neutrophils were decreased. Peimisine had a regulatory effect on the abnormal proliferation of MRC-5 cells. The mechanism was related to regulating extra cellular matrix (ECM) and epithelial-mesenchymal transition (EMT).

Inflammatory bowel disease (IBD) is a chronic non-specific intestinal inflammatory disease that affects millions of people worldwide, and current treatment methods have certain limitations. This study aimed to explore the therapeutic potential and mechanism of action of lemairamin (Wgx-50) in inflammatory bowel disease (IBD). We used dextran sulfate sodium (DSS)-treated zebrafish as an inflammatory bowel disease model, and observed the effect of Wgx-50 on DSS-induced colitis inflammation. The results of the study showed that Wgx-50 could reduce the expression of pro-inflammatory cytokines induced by DSS and inhibit the recruitment of neutrophils to the site of intestinal injury. Further experiments revealed that Wgx-50 exerted its anti-inflammatory effect by regulating the activation of the Akt pathway. These research findings indicate that Wgx-50 possesses anti-inflammatory activity.

Scope: Neutrophils play a decisive role during the immediate defense against infections. However, as observed during rheumatoid arthritis, activated neutrophils can also cause tissue damage. Previous studies indicate that zinc supplementation may alter certain neutrophil functions. However, precise underlying mechanisms and possible effects of zinc deficiency remain incompletely understood. The objective of this study is to investigate the effects of changes in zinc status on formation of neutrophil extracellular traps (NETs) and other fundamental neutrophil functions., Methods and Results: Interleukin (IL)-17 and tumor necrosis factor (TNF)-α are used to simulate the inflammatory environment observed in autoimmune diseases. The study analyzes the impact of the zinc status on NETs release, using a fluorescence plate reader, and on the expression of peptidylarginine deiminase 4 (PAD4), S100A8/A9, and certain cytokines by PCR and western blot. These results show that zinc supplementation significantly reduces NETs formation and downregulates PAD4 protein expression. Zinc supplementation results in increased protein expression of interleukin-1 receptor antagonist (IL-1RA) and IL-8 in stimulated cells., Conclusion: The results suggest that changes in extracellular zinc availability may influence the functions of neutrophils. Therefore, maintaining an appropriate zinc level is advisable for preserving innate immunity and to prevent hyper-activation of neutrophils., (© 2024 The Author(s). Molecular Nutrition & Food Research published by Wiley‐VCH GmbH.)

Intravenous thrombolysis using recombinant tissue plasminogen activator (tPA) remains the primary treatment for patients with acute ischemic stroke (AIS). However, the mechanism of tPA-related hemorrhagic transformation (HT) remains poorly understood. Elevation of histidine-rich glycoprotein (HRG) expression was detected by nano-liquid chromatography tandem mass spectrometry at 1 h following tPA infusion as compared to baseline prior to tPA infusion (discovery cohort, n = 10), which was subsequently confirmed in a validation cohort (n = 157) by ELISA. Surprisingly, no elevation of HRG was detected in individuals who subsequently developed HT. During in vitro experiments, HRG reduced neutrophil NETosis, inflammatory cytokine production, and migration across the blood-brain barrier induced by tPA. In a photothrombotic murine AIS model, HRG administration ameliorated HT with delayed thrombolysis, by inhibiting neutrophil immune infiltration and downregulating pro-inflammatory signaling pathways. Neutrophil depletion or NETosis inhibition also alleviated HT, whereas HRG siRNA treatment exacerbated HT. In conclusion, fluctuations in HRG levels may reflect tPA therapy and its associated HT. The inhibitory effect of HRG on neutrophils may counteract tPA-induced immune abnormalities and HT in patients with AIS., (© 2024. The Author(s).)

Triple-negative breast cancer (TNBC) is labeled as an aggressive type of breast cancer and still has limited therapeutic targets despite the advanced development of cancer therapy. Neutrophils, representing the conventional inflammatory response, significantly influence the malignant phenotype of tumors, supported by abundant evidence. As a vital function of neutrophils, NETs are the extracellular fibrous networks including the depolymerized chromatin DNA frames with several antimicrobial proteins. They are produced by activated neutrophils and are involved in host defence or immunological reactions. This review focuses more on the interactions between neutrophils and TNBC, focusing on how neutrophils modulate the immune response within the tumor milieu. Specifically, we delve into the role of NETs, which are involved in promoting tumor growth and metastasis, inhibiting anti-tumor immunity, and promoting tumor-associated thrombosis. Furthermore, we discuss recent advancements in therapeutic strategies aimed at targeting NETs to enhance the efficacy of TNBC treatment. The advances in the knowledge of the dynamics between neutrophils and TNBC may lead to the opportunity to devise new immunotherapeutic strategies targeted to fight this hostile type of breast cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Objective: Sex differences exist in sepsis, but the commitment of neutrophils to these differences remains unclear. Neutrophil extracellular traps (NETs) function to remove pathogens, yet excessive NETs release can contribute to organ damage. This study explores effects of the gender hormones on endotoxin-induced NETs using neutrophils from both male and female sources. Methods: Blood samples were collected from healthy volunteers. Isolated neutrophils were seeded in collagen-coated cell culture plates, and NETs were induced by lipopolysaccharide (LPS) treatment. After 15 minutes of LPS treatment, 17β-estradiol (0.03-272.4 ng/mL), testosterone enanthate (0.01-10 ng/mL), dimethyl sulfoxide, or ethanol (vehicle control) was added to the plates. These were incubated for three hours at 37°C with 5% CO 2 . Neutrophil extracellular traps formation was assessed using immunofluorescence staining. Results: Lipopolysaccharide-induced NETs formation was significantly greater in females than in males. In male-derived neutrophils, 17β-estradiol at above the blood concentrations significantly suppressed LPS-induced NETs. No effect was seen while using testosterone enanthate to NETs at any concentration. In female-derived neutrophils, 17β-estradiol, which was near to the highest concentration of non-pregnant women's blood, tended to increase NETs. Testosterone enanthate, which was near to female blood concentration, significantly promoted NETs. Conclusions: Sex differences existed in LPS-induced NETs of human neutrophil. In males, high concentrations of 17β-estradiol administration may have a suppressive effect on excessive NETs during infection. In females, endogenous gender hormones may promote NETs during infection. Sex differences in neutrophils may need to be considered in organ damage owing to NETs excess such as sepsis.

Excessive neutrophil infiltration into the tumor microenvironment (TME) is an important factor that contributes to tumor overgrowth and limited immunotherapy efficacy. Neutrophils activate various receptors involved in tumor progression, while suppressing the infiltration and activity of cytotoxic T cells and creating optimal conditions for tumor growth. Therefore, the appropriate control of neutrophil infiltration is an effective strategy for tumor treatment. In the present study, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) inhibited tumor overgrowth by suppressing excessive neutrophil infiltration, resulting in >74.97 % reduction in tumor size in a Lewis lung carcinoma (LLC-1) mouse model. All subjects in the positive control group died during the 90-day survival period, whereas only four subjects in the PLAG treatment group survived. PLAG had a significantly higher tumor growth inhibitory effect and survival rate than other neutrophil infiltration-targeting inhibitors (e.g., Navarixin, lymphocyte antigen 6 complex locus G6D antibody [aLy6G]). The ability of PLAG to regulate neutrophil infiltration and inhibit tumor growth depends on thioredoxin-interacting protein (TXNIP). In tumors lacking TXNIP expression, PLAG failed to control neutrophil infiltration and infiltration-related factor release, and the inhibitory effect of PLAG on tumor growth was reduced. PLAG-mediated inhibition of neutrophil infiltration enhances the efficacy of immune checkpoint inhibitors (ICIs), increasing the antitumor efficacy and survival rate by 30 %. In conclusion, PLAG could be a novel alternative to anti-tumor drugs that effectively targets excessive neutrophil infiltration into cancer tissues., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Leukocyte-platelet aggregates comprise a pathogenic link between hemostasis and immunity, but the prerequisites and mechanisms of their formation remain not understood., Aims: To quantify the formation, composition, and morphology of leukocyte-platelet aggregates in vitro under the influence of various cellular activators., Methods: Phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS), thrombin receptor-activating peptide (TRAP-6), and adenosine diphosphate (ADP) were used as cellular activators. Flow cytometry was utilized to identify and quantify aggregates in whole human blood and platelet-rich plasma. Cell types and cellular aggregates were identified using fluorescently labeled antibodies against the appropriate cellular markers, and cell activation was assessed by the expression of appropriate surface markers. For confocal fluorescent microscopy, cell membranes and nuclei were labeled. Neutrophil-platelet aggregates were studied using scanning electron microscopy., Results: In the presence of PMA, ADP or TRAP-6, about 17-38 % of neutrophils and 61-77 % of monocytes formed aggregates with platelets in whole blood, whereas LPS did not induce platelet aggregation with either neutrophils or monocytes due the inability to activate platelets. Similar results were obtained when isolated neutrophils were added to platelet-rich plasma. All the cell types involved in the heterotypic aggregation expressed molecular markers of activation. Fluorescent and electron microscopy of the aggregates showed that the predominant platelet/leukocyte ratios were 1:1 and 2:1., Conclusions: Formation of leukocyte-platelet aggregates depends on the nature of the cellular activator and the spectrum of its cell-activating ability. An indispensable condition for formation of leukocyte-platelet aggregates is activation of all cell types including platelets, which is the restrictive step., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A new polysaccharide, named SP40015A01, was obtained from Saposhnikoviae Radix by water extraction, isolation and purification. SP40015A01 (9.7 × 10 5  Da) is composed of Rhamnose (Rha), Galacturonic acid (GalA), Galactose (Gal), and Arabinose (Ara) with the proportion of 1.6:85.6:5.8:7.6. The backbone of SP40015A01 is composed of 3-α-GalAp, 2-α-GalAp, 2,3-β-GalAp and 2,3-β-Galp, and branched at C3 of 2,3-β-GalAp, C3 of 2,3-β-Galp. Zebrafish experiments were used to explore the immunomodulatory activity of SP40015A01. Results showed that SP40015A01 could significantly improve the neutrophils density of immunocompromised zebrafish and reduce the content of nitric oxide (NO) and interleukin-1β (IL-1β). This study demonstrated that SP40015A01 has significant immunomodulatory activity, which can improve the neutrophils density and reduce inflammatory factor content, suggesting SP40015A01 may be a potential immunomodulator in Saposhnikoviae Radix (SR) for treatment of hypoimmunity related disease. This study supplemented the research on the polysaccharide components in traditional Chinese medicine and provided a scientific explanation for the development and clinical applications of SR., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: LIU BIN reports financial support was provided by National Natural Science Foundation of China (82074283). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Modulating SYK has been demonstrated to have impacts on pathogenic neutrophil responses in COVID-19. During sepsis, neutrophils are vital in early bacterial clearance but also contribute to the dysregulated immune response and organ injury when hyperactivated. Here, we evaluated the impact of R406, the active metabolite of fostamatinib, on neutrophils stimulated by LPS. We demonstrate that R406 was able to effectively inhibit NETosis, degranulation, ROS generation, neutrophil adhesion, and the formation of CD16 low neutrophils that have been linked to detrimental outcomes in severe sepsis. Further, the neutrophils remain metabolically active, capable of releasing cytokines, perform phagocytosis, and migrate in response to IL-8. Taken together, this data provides evidence of the potential efficacy of utilizing fostamatinib in bacterial sepsis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

A balanced immune system is essential to maintain adequate host defense and effective self-tolerance. While an immune system that fails to generate appropriate response will permit infections to develop, uncontrolled activation may lead to autoinflammatory or autoimmune diseases. To identify drug candidates capable of modulating immune cell functions, we screened 1200 small molecules from the Prestwick Chemical Library for their property to inhibit innate or adaptive immune responses. Our studies focused specifically on drug interactions with T cells, B cells, and polymorphonuclear leukocytes (PMNs). Candidate drugs that were validated in vitro were examined in preclinical models to determine their immunomodulatory impact in chronic inflammatory diseases, here investigated in chronic inflammatory skin diseases. Using this approach, we identified several candidate drugs that were highly effective in preclinical models of chronic inflammatory disease. For example, we found that administration of pyrvinium pamoate, an FDA-approved over-the-counter anthelmintic drug, suppressed B cell activation in vitro and halted the progression of B cell-dependent experimental pemphigoid by reducing numbers of autoantigen-specific B cell responses. In addition, in studies performed in gene-deleted mouse strains provided additional insight into the mechanisms underlying these effects, for example, the receptor-dependent actions of tamoxifen that inhibit immune-complex-mediated activation of PMNs. Collectively, our methods and findings provide a vast resource that can be used to identify drugs that may be repurposed and used to promote or inhibit cellular immune responses., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aims: Neutrophils perform various functions in a circadian-dependent manner; therefore, we investigated here whether the effect of alpha1-antitrypsin (AAT), used as augmentation therapy, is dependent on the neutrophil circadian clock. AAT is a vital regulator of neutrophil functions, and its qualitative and/or quantitative defects have significant implications for the development of respiratory diseases., Methods: Whole blood from 12 healthy women age years, mean (SD) 29.92 (5.48) was collected twice daily, 8 h apart, and incubated for 30 min at 37 °C alone or with additions of 2 mg/ml AAT (Respreeza) and/or 5 μg/ml lipopolysaccharide (LPS) from Escherichia coli. Neutrophils were then isolated to examine gene expression, migration and phagocytosis., Results: The expression of CD14, CD16, CXCR2 and SELL (encoding CD62L) genes was significantly higher while CDKN1A lower in the afternoon than in the morning neutrophils from untreated blood. Neutrophils isolated in the afternoon had higher migratory and phagocytic activity. Morning neutrophils isolated from AAT-pretreated blood showed higher expression of CXCR2 and SELL than those from untreated morning blood. Pretreatment of blood with AAT enhanced migratory properties of morning but not afternoon neutrophils. Of all genes analysed, only CXCL8 expression was strongly upregulated in morning and afternoon neutrophils isolated from LPS-pretreated blood, whereas CXCR2 expression was downregulated in afternoon neutrophils. The addition of AAT did not reverse the effects of LPS., Significance: The circadian clock of myeloid cells may affect the effectiveness of various therapies, including AAT therapy used to treat patients with AAT deficiency, and needs further investigation., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: Increasing studies demonstrated the importance of C5a and anti-neutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation in the pathogenesis of ANCA-associated vasculitis (AAV). Sphingosine-1-phosphate (S1P) acts as a downstream effector molecule of C5a and enhances neutrophil activation induced by C5a and ANCA. The current study investigated the role of a S1P receptor modulator, FTY720, in experimental autoimmune vasculitis (EAV) and explored the immunometabolism-related mechanisms of FTY720 in modulating ANCA-induced neutrophil activation., Methods: The effects of FTY720 in EAV were evaluated by quantifying haematuria, proteinuria, crescent formation, tubulointerstitial injury and pulmonary haemorrhage. RNA sequencing of renal cortex and gene enrichment analysis were performed. The proteins of key identified pathways were analysed in neutrophils isolated from peripheral blood of patients with active AAV and normal controls. We assessed the effects of FTY720 on ANCA-induced neutrophil respiratory burst and neutrophil extracellular traps formation (NETosis)., Results: FTY720 treatment significantly attenuated renal injury and pulmonary haemorrhage in EAV. RNA sequencing analyses of renal cortex demonstrated enhanced fatty acid oxidation (FAO) and peroxisome proliferator-activated receptor (PPAR) signalling in FTY720-treated rats. Compared with normal controls, patients with active AAV showed decreased FAO in neutrophils. FTY720-treated differentiated HL-60 cells showed increased expression of carnitine palmitoyltransferase 1a (CPT1a) and PPARα. Blocking or knockdown of CPT1a or PPARα in isolated human neutrophils and HL-60 cells reversed the inhibitory effects of FTY720 on ANCA-induced neutrophil respiratory burst and NETosis., Conclusion: FTY720 attenuated renal injury in EAV through upregulating FAO via the PPARα-CPT1a pathway in neutrophils, offering potential immunometabolic targets in AAV treatment., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Recent investigation of a constitutively active ADAMTS13 variant (caADAMTS13) in murine models of acute ischaemic stroke (AIS) have revealed a potential anti-inflammatory mechanism of action contributing to its protective effect. However, it remains unclear whether these observations are a direct result of VWF proteolysis by caADAMTS13. We have implemented state of the art in vitro assays of neutrophil rolling and transmigration to quantify the impact of caADAMTS13 on these processes. Moreover, we have tested caADAMTS13 in two in vivo assays of neutrophil migration to confirm the impact of the treatment on the neutrophil response to sterile inflammation. Neutrophil rolling, over an interleukin-1β stimulated hCMEC/D3 monolayer, is directly inhibited by caADAMTS13, reducing the proportion of neutrophils rolling to 9.5 ± 3.8 % compared to 18.0 ± 4.5 % in untreated controls. Similarly, neutrophil transmigration recorded in real-time, was significantly suppressed in the presence of caADAMTS13 which reduced the number of migration events to a level like that in unstimulated controls (18.0 ± 4.5 and 15.8 ± 7.5 cells/mm 2 /h, respectively). Brain tissue from mice undergoing experimental focal cerebral ischaemia has indicated the inhibition of this process by caADAMTS13. This is supported by caADAMTS13's ability to reduce neutrophil migration into the peritoneal cavity in an ischaemia-independent model of sterile inflammation, with the VWF-dependent mechanism by which this occurs being confirmed using a second experimental stroke model. These findings will be an important consideration in the further development of caADAMTS13 as a potential therapy for AIS and other thromboinflammatory pathologies, including cardiovascular disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Ethnopharmacological Relevance: Fei-Yan-Qing-Hua decoction (FYQHD) is an empirical formula that has shown clinical success in treating community-acquired pneumonia (CAP) for two decades. Influenza viral infection is a significant trigger for severe pneumonia, yet the role of FYQHD in treating influenza remains unclear., Aim of the Study: This study aimed to assess the potential efficacy of FYQHD in treating influenza viral infection and to elucidate its underlying mechanisms., Materials and Methods: The protective effects of FYQHD against influenza were evaluated through survival assessments and pathological analyses. Transcriptomic analysis was performed to identify the genes and pathways influenced by FYQHD in influenza. The anti-inflammatory effects and molecular mechanisms of FYQHD were studied in macrophages stimulated by Toll-like receptor (TLR) 7 ligation in vitro. The key constituents of FYQHD absorbed in mouse sera were identified using untargeted metabolomics, and the anti-inflammatory activity of some of these compounds in macrophages was evaluated using ELISA., Results: Our findings demonstrate that FYQHD enhances survival and reduces lung damage in PR8-infected mice, primarily through its anti-inflammatory properties. Lung indexes and organ damage were significantly lower in the PR8 + OSV + FYQHD group compared to the PR8 + OSV group, indicating a potential complementary therapeutic effect of FYQHD and OSV in treating influenza. FYQHD effectively reduced chemokine expression, thereby decreasing the chemotaxis and infiltration of inflammatory monocytes/macrophages and neutrophils in the lungs. The anti-inflammatory effects of FYQHD in macrophages were achieved through the inhibition of NF-κB activation and p38 phosphorylation. The key constituents of FYQHD absorbed in mouse sera were identified, with some, such as wogonin, luteolin, kaempferol, and isorhamnetin, showing anti-inflammatory effects in primary macrophages., Conclusion: FYQHD demonstrates protective efficacy against influenza and shows promise as an adjuvant therapeutic agent, particularly when used in combination with antiviral drugs like OSV. The potent anti-inflammatory components within FYQHD provide a basis for further exploration in drug research and development aimed at combating influenza., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cryptococcosis, caused by infections with C. neoformans and C. gattii, presents a serious threat to global health and necessitates effective treatment strategies. Granulocyte-Macrophage Colony-Stimulating Factor, GM-CSF, is an immune-modulating cytokine that has been utilized clinically to improve host defense against infection; however, the impact of GM-CSF treatment in C. gattii infection has not been elucidated. Our current study aimed to investigate the effect of GM-CSF treatment on pulmonary immune response during C. gattii infection. In response to C. gattii infection, GM-CSF-expressing T helper cells and CD11b + myeloid were enhanced in the lungs. The intranasal administration of GM-CSF during C. gattii infection significantly reduced pulmonary cryptococcal load, promoted an increase in pulmonary Th17 cells, as well as neutrophil infiltration in the lungs. Exposure of neutrophils to C. gattii in the presence of GM-CSF resulted in an increased neutrophil phagocytosis and fungal killing capacity, generation of reactive oxygen species (ROS), and upregulation of inflammatory cytokines and anti-microbial peptides. Although GM-CSF treatment in C. neoformans-infected mice had a comparable impact on the reduction of lung fungal burden, it resulted in the enhancement of Th1-type cytokine IFN-γ and the activation of M1 macrophages. Altogether, this study demonstrated that the intranasal delivery of GM-CSF has distinct effects on promoting the protection against C. gattii and C. neoformans by activating neutrophil/type-17 immune response and stimulating M1 macrophage/type-1 immunity, respectively., (Copyright © 2024 Elsevier B.V. All rights reserved.)

NETosis happens when neutrophils are activated and neutrophil extracellular traps (NETs) are formed synchronously, which is a hallmark of psoriasis. However, the specific trigger that drives NET formation and the distinct contents and interaction with interleukin-36 receptor (IL-36R) of NETs remain to be further elucidated. This work identified NET formation driven by toll-like receptor (TLR) 3 ligand (especially polyinosinic-polycytidylic acid (Poly(I:C)) were enhanced by purinergic receptor P2X ligand-gated ion channel 7 receptor (P2X7R) ligands (especially adenosine 5'-triphosphate (ATP)). NET formation was accompanied by the secretion of inflammatory cytokines and characterized by IL-1β decoration. NET formation blockade decreased expressions of inflammatory cytokines and chemokines, which consequently improved inflammatory responses. Additionally, imiquimod (IMQ)-induced psoriasiform symptoms including neutrophilic infiltration tended to be time-sensitive. Mouse primary keratinocytes and mice deficient in Il1rl2, which encodes IL-36R, mitigated inflammatory responses and NET formation, thereby delaying the pathophysiology of psoriasis. Together, the findings provided the therapeutic potential for IL-36 targeting NET inhibitors in psoriasis treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sophora flavescens Ait. (SFA), an extensively utilized herb for the treatment of fevers, inflammatory disorders, ulcers and skin diseases related to bur, contains oxymatrine (OMT) as its principal active constituent. OMT exerts regulatory effects over inflammation, oxidative stress and apoptosis. Neutrophils, critical regulators of the inflammation response, have not been thoroughly elucidated regarding the protective properties and underlying mechanisms of OMT-mediated anti-inflammation. This study was aim to explore the protective effect of OMT on neutrophils under inflammatory conditions and delve into its potential mechanism. Leveraging the advantages of zebrafish, an animal model with a real-time dynamic observation system, we established an in vivo caudal fin wound model and a copper sulfate induced-inflammation model in zebrafish line Tg (mpx:GFP). The result revealed that OMT significantly attenuated neutrophil migration, upregulated the mRNA expression levels of JNK, casp3, mapk14a, mapkapk2a and map2k1 damaged by zebrafish caudal fin wound model, and downregulated mRNA expression levels of JNK, casp3, mapk14a, mapkapk2a and map2k1 in the copper sulfate injury model. In vitro experiments demonstrated that OMT modulated the chemotaxis response of primary neutrophils from mice, enhanced phagocytosis, reduced oxidative stress and alleviated inflammation level. We hypothesize that the OMT may exert its anti-inflammatory effects by regulating primary neutrophils through the MAPK signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lung ischemia-reperfusion (I/R) injury is the main risk factor for primary graft dysfunction and patient death after lung transplantation (LTx). It is widely accepted that the main pathological mechanism of lung I/R injury are calcium overload, oxygen free radical explosion and neutrophil-mediated damage, which leading to the lack of effective treatment options. The aim of this study was to further explore the mechanisms of lung I/R injury after LTx and to provide potential therapeutic strategies. Our bioinformatics analysis revealed that the neutrophil extracellular traps (NETs) formation was closely involved in lung I/R injury after LTx, which was accompanied by up-regulation of peptidylprolyl isomerase F (PPIF) and peptidyl arginine deiminase 4 (PADI4). We further established an orthotopic LTx mouse model to simulate lung I/R injury in vivo, and found that PPIF and PADI4 inhibitors effectively reduced neutrophil infiltration, NETs formation, inflammatory response, and lung I/R injury. In the neutrophil model induced by HL-60 cell line in vitro, we found that PPIF inhibitor cyclosporin A (Cys A) better alleviated calcium overload induced inflammatory response, reactive oxygen species content and NETs formation. Further study demonstrated that interfering with neutrophil PPIF protected mitochondrial function by alleviating store-operated calcium entry (SOCE) during calcium overload and played the above positive role. On this basis, we found that the reduction of calcium content in neutrophils was accompanied by the inhibition of calcineurin (CN) and nuclear factor of activated T cells (NFAT). In conclusion, our findings suggested that neutrophil PPIF could serve as a novel biomarker and potential therapeutic target of lung I/R injury after LTx, which provided new clues for its treatment by inhibiting calcium overload-induced NETs formation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Corneal neovascularization (CoNV) is the second leading cause of visual impairment worldwide, and current drugs have certain limitations. Inflammatory response is the core pathological process of CoNV. Neutrophil extracellular traps (NETs) are generated after neutrophil activation, which promotes neovascularization. Prior studies demonstrated that bone morphogenetic protein 4 (BMP4) could significantly reduce inflammation and CoNV formation, its exact molecular mechanism remains unclear. Therefore, we stimulated human peripheral blood neutrophils with phorbol myristate acetate (PMA) or deoxyribonuclease I (DNase I) to induce or inhibit NETs formation. By using corneal sutures and subconjunctival injections of NETs or DNase I, rat CoNV models were established. Compared with the suture group, NETs formation and inflammatory cell infiltration in the corneal stroma were significantly increased, corneal edema was aggravated, and the length, area and diameter of CoNV were significantly enhanced in the NETs group. Furthermore, by curetting the corneal epithelial apical junctional complexes (AJCs), a crucial component in preserving the function of the corneal epithelial barrier, we discovered that the damage of AJCs had a significant role in inducing CoNV formation. NETs could induce CoNV formation by injuring corneal epithelial AJCs. Finally, by comparing the aforementioned indicators after the intervention of BMP4, BMP4 inhibitor Noggin and NADPH oxidase (NOX) inhibitor, we finally demonstrated that BMP4 could inhibit NETs-induced inflammation and corneal epithelial AJC injury, repair corneal epithelial barrier function and eventually inhibit CoNV formation by blocking NOX-2-dependent NETs formation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Neutrophils, pivotal cells of innate and adaptive immune responses, employ reactive oxygen species (ROS) to combat pathogens and control gene expression. Paracetamol (acetaminophen) is widely used as an analgesic and antipyretic medication, yet its precise mechanisms of action are not yet fully understood. Here, we investigate the impact of both ingested and in-vitro paracetamol on neutrophil ROS activity, using flow cytometry and antioxidant assays. Our studies reveal that paracetamol significantly suppresses ROS activity ex-vivo in the short term. Additionally, both paracetamol and its metabolite N-acetyl-p-benzoquinone imine exhibited direct in vitro antioxidant effects, and paracetamol suppressed neutrophil extracellular trap formation ex vivo. These findings suggest a connection between paracetamol use and altered neutrophil responses, with potential implications for use in some patient groups, such as immunocompromised individuals. Further investigation into paracetamol's effects on neutrophil antimicrobial functions is warranted to elucidate possible risks, particularly when taken frequently or in conjunction with other treatments such as vaccinations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The increased production of reactive oxygen species (ROS) by human neutrophils can lead to oxidative imbalances and several diseases, such as inflammatory bowel disease (IBD). Opuntia ficus-indica (O. ficus-indica) is rich in bioactive substances with anti-inflammatory properties. This study aimed to identify the bioactive compounds present in aqueous cladodes extract (ACE) of O. ficus-indica using high-performance liquid chromatography (HPLC) and to test its effects on human neutrophil inflammatory functions and on ulcerative colitis (UC) induced by acetic acid (Aa) in rats. ROS production and degranulation by neutrophils were assessed by luminol-amplified chemiluminescence, enzymatic techniques, and western blotting. In vivo, the experiment involved seven groups of rats: a negative control group (NaCl), the acetic acid group (Aa), and groups treated with oral sulfasalazine (150 mg/kg) or various doses of ACE for 7 days. Colonic lesions were induced by an intra-rectal Aa injection, and inflammation was assessed. HPLC analysis identified gallic acid, catechin, caffeic acid, and ferulic acid as major compounds in ACE. In vitro, ACE inhibited neutrophil ROS production, including superoxide anion produced by NADPH oxidase, and significantly reduced myeloperoxidase activity and neutrophil degranulation. In vivo, ACE protected rats from Aa-induced histopathological damage of the colonic mucosa, significantly increased catalase, superoxide dismutase and reduced glutathione levels, and significantly suppressed the increases of plasma cytokines (TNF-α and IL-1β) observed in the Aa group. In conclusion, O. ficus-indica ACE has significant anti-inflammatory properties by restoring oxidative balance, indicating that it could be a potential source of therapeutic agents for inflammatory diseases, particularly UC., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Human 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes, mediators of the innate immune system that also play an important role in inflammatory diseases and cancer. In this study, we present compounds, containing a Michael-reactive cyanoacrylate moiety as potent inhibitors of 5-LO. Representatives of the tyrosine kinase inhibitor family called tyrphostins, structurally related to known 5-LO inhibitors, were screened for their 5-LO inhibitory properties using recombinant human 5-LO, intact human PMNL (polymorphonuclear leukocytes), and PMNL homogenates. Their mode of action was characterized by the addition of glutathione, using a fourfold cysteine 5-LO mutant and mass spectrometry analysis. SAR studies revealed several members of the tyrphostin family containing a Michael-reactive cyanoacrylate to efficiently inhibit 5-LO. We identified degrasyn (IC 50 0.11 µM), tyrphostin A9 (IC 50 0.8 µM), AG879 (IC 50 78 nM), and AG556 (IC 50 64 nM) as potent 5-LO inhibitors. Mass spectrometry analysis revealed that degrasyn and AG556 covalently bound to up to four cysteines, including C416 and/or C418 which surround the substrate entry site. Furthermore, the 5-LO inhibitory effect of degrasyn was remarkably impaired by the addition of glutathione or by the mutation of cysteines to serines at the surface of 5-LO. We successfully identified several tyrphostins as potent inhibitors of human 5-LO. Degrasyn and AG556 were able to covalently bind to 5-LO via their cyanoacrylate moiety. This provides a promising mechanism for targeting 5-LO by Michael acceptors, leading to new therapeutic opportunities in the field of inflammation and cancer., Competing Interests: Declarations. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s).)

Targeting neutrophil function has gained attention as a propitious therapeutic strategy for diverse inflammatory diseases. Accordingly, a series of enone-based derivatives were developed to inhibit neutrophil-mediated inflammation, showing promise for treating inflammatory diseases. These compounds fall into two clusters with distinct effects: one inhibits neutrophilic superoxide (SO) anion production and elastase release triggered by N-formyl-Met-Leu-Phe (fMLF), with compound 6a being most effective (IC 50 values of 1.23 and 1.37 μM, respectively), affecting c-Jun N-terminal kinase (JNK) and Akt phosphorylation. The second cluster suppresses formation of SO anion without affecting elastase levels, surpassed by compound 26a (IC 50 of 1.56 μM), which attenuates various mitogen-activated protein kinases (MAPKs) with minimal Akt impact. Notably, none of the tested compounds showed cytotoxicity in human neutrophils, underscoring their potential as therapeutic agents against inflammatory diseases.

Helicobacter pylori colonizes the human gastric mucosa of more than half of the human population and has a unique lipopolysaccharide (LPS) structure. LPS is the most dominant and suitable pathogen-associated molecular pattern that is detected via pattern recognition receptors. Although the priming effect of H. pylori LPS on reactive oxygen species (ROS) production of PMNs is lower than that of Escherichia coli O111:B4 LPS, LPS released from H. pylori associated with antibiotics eradication therapy may activate PMNs and increase ROS production. In addition, we describe the effects of H. pylori and E. coli O111:B4 LPSs on gene expression and the anti-inflammatory effect of lansoprazole (LPZ) in human polymorphonuclear leukocytes. LPS isolated from H. pylori and E. coli O111:B4 alters toll-like receptor 2 (TLR) and TLR4 expressions similarly. However, LPS from E. coli O111:B4 and H. pylori caused a 1.8-fold and 1.5-fold increase, respectively, in CD14 expression. All LPS subtypes upregulated TNFα and IL6 expression in a concentration-dependent manner. Although E. coli O111:B4 LPS upregulated IL8R mRNA levels, H. pylori LPS did not (≦ 100 ng/mL). Gene expression levels of ITGAM demonstrated no significant change on using both LPSs. These different effects on the gene expression in PMNs may depend on variations in LPS structural modifications related to the acquired immunomodulatory properties of H. pylori LPS. Proton pump inhibitors, i.e., LPZ, are used in combination with antibiotics for the eradication therapy of H. pylori. LPZ and its acid-activated sulphenamide form AG-2000 suppress ROS production of PMNs in a dose-dependent manner. These results suggest that LPZ combination with antibiotics for H. pylori eradication reduces gastric inflammation by suppressing ROS release from PMNs., (© 2024. The Author(s).)

The recent global resurgence of severe infections caused by the Group A streptococcus (GAS) pathogen, Streptococcus pyogenes , has focused attention on this microbial pathogen, which produces an array of virulence factors, such as the pore-forming toxin, streptolysin O (SOT). Importantly, the interactions of SOT with human neutrophils (PMN), are not well understood. The current study was designed to investigate the effects of pretreatment of isolated human PMN with purified SOT on several pro-inflammatory activities, including generation of reactive oxygen species (ROS), degranulation (elastase release), influx of extracellular calcium (Ca 2+ ) and release of extracellular DNA (NETosis), using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of PMN to SOT alone caused modest production of ROS and elastase release, while pretreatment with the toxin caused significant augmentation of chemoattractant (fMLP)-activated ROS generation and release of elastase by activated PMN. These effects of treatment of PMN with SOT were associated with both a marked and sustained elevation of cytosolic Ca 2+ concentrations and significant increases in the concentrations of extracellular DNA, indicative of NETosis. The current study has identified a potential role for SOT in augmenting the Ca 2+ -dependent pro-inflammatory interactions of PMN, which, if operative in a clinical setting, may contribute to hyper-activation of PMN and GAS-mediated tissue injury.

Context: Virtually all parts of Salvadora persica L. (Salvadoraceae) are used in traditional medicine. The twigs and leaves are used for oral health, but leaves are far less investigated., Objective: This study assesses the oral health-promoting potential of S. persica leaves with emphasis on anti-inflammatory and antiproliferative effects and provides an in depth-characterization of their metabolite profile., Materials and Methods: Hot-water and methanolic S. persica leaf extracts (1, 10, and 100 µg/mL) and their major constituents (5, 10, and 50 µM), were subjected to cellular assays on IL-8 and TNFα release in LPS-stimulated human neutrophils, NO-release in LPS/IFNγ stimulated mouse macrophages, and proliferation of HNO97 human tongue carcinoma cells. Metabolite profiling was performed by UHPLC-HRMS analysis. Major constituents were isolated and structurally elucidated., Results and Discussion: Both extracts showed pronounced anti-inflammatory activity in LPS-stimulated neutrophils. Major identified compound classes were flavonoid glycosides, the glucosinolate glucotropaeolin, phenyl- and benzylglycoside sulfates, and megastigmane glycosylsulfates, the latter ones identified for the first time in S. persica . Glucotropaeolin strongly inhibited the release of IL-8 and TNF-α (13.3 ± 2.0 and 22.7 ± 2.6% of the release of stimulated control cells at 50 µM), while some flavonoids and 3-(3'- O -sulfo-β-d-glucopyranosyloxy)-7,8-dihydro-β-ionone, a newly isolated megastigmane glycosylsulfate, were moderately active. Benzylisothiocyanate, which is likely formed from glucotropaeolin during traditional application of S. persica, showed considerable antiproliferative activity (IC 50 in HNO97 cells: 10.19 ± 0.72 µM) besides strongly inhibiting IL-8 and TNFα release., Conclusions: Glucotropaeolin and benzylisothiocyanate are likely implicated in the oral health-promoting effects of S. persica leaves. The chemistry and pharmacology of the newly identified megastigmane glycosylsulfates should be further evaluated.

Neutrophil extracellular traps (NETs) are three-dimensional reticular structures that release chromatin and cellular contents extracellularly upon neutrophil activation. As a novel effector mechanism of neutrophils, NETs possess the capacity to amplify localized inflammation and have been demonstrated to contribute to the exacerbation of various inflammatory diseases, including cardiovascular diseases and tumors. It is suggested that lysophosphatidylcholine (LPC), as the primary active component of oxidized low-density lipoprotein, represents a significant risk factor for various inflammatory diseases, such as cardiovascular diseases and neurodegenerative diseases. However, the specific mechanism of NETs formation induced by LPC remains unclear. Quercetin has garnered considerable attention due to its anti-inflammatory properties, serving as a prevalent flavonoid in daily diet. However, little is currently known about the underlying mechanisms by which quercetin inhibits NETs formation and alleviates associated diseases. In our study, we utilized LPC-treated primary rat neutrophils to establish an in vitro model of NETs formation, which was subsequently subjected to treatment with a combination of quercetin or relevant inhibitors/activators. Compared to the control group, the markers of NETs and the expression of P2X7R/P38MAPK/NOX2 pathway-associated proteins were significantly increased in cells treated with LPC alone. Quercetin intervention decreased the LPC-induced upregulation of the P2X7R/P38MAPK/NOX2 pathway and effectively reduced the expression of NETs markers. The results obtained using a P2X7R antagonist/activator and P38MAPK inhibitor/activator support these findings. In summary, quercetin reversed the upregulation of the LPC-induced P2X7R/P38MAPK/NOX2 pathway, further mitigating NETs formation. Our study investigated the potential mechanism of LPC-induced NETs formation, elucidated the inhibitory effect of quercetin on NETs formation, and offered new insights into the anti-inflammatory properties of quercetin.

Preterm birth (PTB) is a leading cause of neonatal morbidity and mortality worldwide, yet the cellular and molecular mechanisms driving this condition remain undeciphered, thus limiting discovery of new therapies. In-depth analyses of human and mouse tissues associated with PTB, in combination with cellular studies, indicated that aberrantly high-expressed neutrophil cytoplasmic factor (NCF) 1 leads to oxidative distress, recruitment, and pro-inflammatory activation of neutrophils and macrophages, while sequentially overexpressed pro-inflammatory mediators induce contractions of uterine smooth muscle cells (USMCs) as well as apoptosis of USMCs and amniotic epithelial cells, thereby causing PTB. According to these new findings, we rationally engineered an amphiphilic macromolecular conjugate LPA by covalently integrating low-molecular-weight heparin, a reactive oxygen species-responsive/scavenging component, and an anti-inflammatory peptide. This bioengineered macromolecular conjugate can self-assemble into multi-bioactive nanoparticles (LPA NP). In a mouse model of PTB, LPA NP effectively delayed PTB and inhibited adverse pregnancy outcomes, by regulating NCF1-mediated oxidative-inflammatory cascades, i.e., attenuating oxidative stress, inhibiting inflammatory cell activation, reducing local inflammation, and decreasing contraction/apoptosis of myometrial cells. Packaging LPA NP into temperature-responsive, self-healing, and bioadhesive hydrogel further potentiated its in vivo efficacies after intravaginal delivery, by prolonging retention time, sustaining nanotherapy release, and increasing bioavailability in the placenta/uterus. Importantly, both the conjugate/nanotherapy and hydrogel formulations exhibited excellent safety profiles in pregnant mice, with negligible side effects on the mother and offspring., (Copyright © 2024 Science China Press. Published by Elsevier B.V. All rights reserved.)

Cathepsin C (CatC) is an enzyme which regulates the maturation of neutrophil serine proteases (NSPs) essential for neutrophil activation. Activated neutrophils are key players in the innate immune system, and are also implicated in the etiology of various inflammatory diseases. This study aims to demonstrate a therapeutic potential for CatC inhibitors against disorders in which activated neutrophil-derived neutrophil extracellular traps (NETs) play a significant role. We demonstrate that a CatC inhibitor, MOD06051, dose-dependently suppresses the cellular activity of NSPs, including neutrophil elastase (NE), in vitro. Neutrophils derived from MOD06051-administered rats exhibit significantly lower NE activity and NET-forming ability than controls. Furthermore, MOD06051 dose-dependently ameliorates vasculitis and significantly decreases NETs when administered to a rat model of myeloperoxidase (MPO)-antineutrophil cytoplasmic antibody-associated vasculitis (AAV). These findings suggest that CatC inhibition is a promising strategy to reduce neutrophil activation and improve activated neutrophil-mediated diseases such as MPO-AAV., (© 2024. The Author(s).)

The production of superoxide anions and other reactive oxygen species (ROS) by neutrophils is necessary for host defense against microbes. However, excessive ROS production can induce cell damage that participates in the inflammatory response. Superoxide anions are produced by the phagocyte NADPH oxidase, a multicomponent enzyme system consisting of two transmembrane proteins (gp91phox/NOX2 and p22phox) and four soluble cytosolic proteins (p40phox, p47phox, p67phox and the small G proteins Rac1/2). Stimulation of neutrophils by various agonists, such as the bacterial peptide formyl-Met-Leu-Phe (fMLF), induces NADPH oxidase activation and superoxide production, a process that is enhanced by the pro-inflammatory cytokines such as GM-CSF. The pathways involved in this GM-CSF-induced up-regulation or priming are not fully understood. Here we show that GM-CSF induces the activation of the prolyl cis/trans isomerase Pin1 in human neutrophils. Juglone and PiB, two selective Pin1 inhibitors, were able to block GM-CSF-induced priming of ROS production by human neutrophils. Interestingly, GM-CSF induced Pin1 binding to phosphorylated p47phox at Ser345. Neutrophils isolated from synovial fluid of patients with rheumatoid arthritis are known to be primed. Here we show that Pin1 activity was also increased in these neutrophils and that Pin1 inhibitors effectively inhibited ROS hyperproduction by the same cells. These results suggest that the prolyl cis/trans isomerase Pin1 may control GM-CSF-induced priming of ROS production by neutrophils and priming of neutrophils in synovial fluid of rheumatoid arthritis patients. Pharmacological targeting of Pin1 may be a valuable approach to the treatment of inflammation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)