By activating endothelium histone H4 mediates oleic acid-induced acute respiratory distress syndrome

By activating endothelium histone H4 mediates oleic acid-induced acute respiratory distress syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article By activating endothelium histone H4 mediates oleic acid-induced acute respiratory distress syndrome Yanlin Zhang, Jingjin Tan, Yiran Zhao, Li Guan, Shuqiang Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3887424/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2025 Read the published version in BMC Pulmonary Medicine → Version 1 posted 12 You are reading this latest preprint version Abstract Objective: This study investigated pathogenic role and mechanism of extracellular histone H4 during oleic acid (OA)-induced acute respiratory distress syndrome (ARDS). Methods: ARDS was induced by intravenous injection of OA in mice, and evaluated by blood gas, pathological analysis, lung edema, and survival rate. Heparan sulfate (HS) degradation was evaluated using immunofluorescence and flow cytometry. The released von Willebrand factor (vWF) was measured using ELISA. P-selectin translocation and neutrophil infiltration were measured via immunohistochemical analysis. Changes in VE-cadherin were measured by western blot. Blocking antibodies against TLRs were used to investigate the signaling pathway. Results: Histone H4 in plasma and BALF increased significantly after OA injection. Histone H4 was closely correlated with the OA dose, which determined the ARDS severity. Pretreatment with histone H4 further aggravated pulmonary edema and death rate, while anti-H4 antibody exerted obvious protective effects. Histone H4 directly activated the endothelia. Endothelial activation was evidently manifested as HS degradation, release of vWF, P-selectin translocation, and VE-Cadherin reduction . The synergistic stimulus of activated endothelia was required for effective neutrophil activation by histone H4. Both TLRs and calcium mediated histone H4-induced endothelial activation. Conclusions: Histone H4 is an essential pro-inflammatory and pro-thrombotic molecule in OA-induced ARDS in mice. Acute respiratory distress syndrome Pulmonary fat embolism Oleic acid Extracellular histone H4 Endothelium Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Significance Statement As the main risk factor for Acute respiratory distress syndrome (ARDS), pulmonary fat embolism (PFE) is a common complication following long bone fractures, cardiopulmonary resuscitation, and infusion through an intraosseous catheter. This study showed that histone H4 is an essential pro-inflammatory and pro-thrombotic molecule in OA-induced ARDS. Histone H4 directly induces pulmonary endothelial activation. Endothelial activation is an indispensable synergistic stimulus for neutrophil activation induced by histone H4. TLRs and calcium are intimately involved in histone H4 mediated endothelium activation. Introduction Acute respiratory distress syndrome (ARDS) has a mortality rate of more than 40%. The hallmark of ARDS is refractory hypoxemia resulting from acute pulmonary edema [1,2]. Unchecked overwhelming inflammation triggered by injurious mediators is intimately involved with endothelial injury [3,4]. Increased endothelial permeability is viewed as the underlying pathological basis of ARDS [5]. Endothelial cells are not only damaged by inflammatory injury but also the active participants of inflammatory response. Activated endothelial cells may further aggravate the inflammatory response, resulting in a vicious circle [6,7]. Extracellular histones have been increasingly recognized as key mediators in systemic inflammatory injuries [8,9]. As the main risk factor for fatal ARDS, pulmonary fat embolism (PFE) is a common complication following long bone fractures, cardiopulmonary resuscitation, and infusion through an intraosseous catheter [10,11]. PFE has many similarities with oleic acid (OA)-induced acute lung injury, but the underlying mechanisms remain largely unknown [12,13]. Therefor the aim of this study was to investigate the pathogenic role and mechanism of extracellular histone H4 during OA-induced ARDS. Materials and Methods Reagents The reagents used in this study included: oleic acid (OA) and Histopaque (Sigma-Aldrich St. Louis, MO, USA); histone H4 (Millipore, Billerica, MA, USA); antibodies for P-selectin, sodium-potassium ATPase, CD31 (PECAM-1), and Ly6G (Abcam, Cambridge, MA, USA); an antibody for Cadherin-5 (BD Transduction Laboratories, CA, USA); an antibody for heparan sulfate (HS) (Bioss, Woburn, MA, USA); blocking antibodies against P-selectin, TLR4 (HTA125), TLR2 (TL2.1), and TLR1 (GD2.F4) (eBioscience , San Diego, CA, USA); a blocking antibody against TLR6 (TLR6.127) (Abcam, Cambridge, MA, USA); enzyme-linked immunosorbent assay (ELISA) kits for histone H4 and von Willebrand factor (vWF) (Cusabio Biotech, Wuhan, China); and 1,2-bis(2-aminophenoxy) ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM) (MedChemExpress, Monmouth Junction, NJ, USA). Following the previously described methods a blocking antibody against histone H4 (anti-H4) was purified in autoimmune mice [14]. Animal studies Eight-week-old male C57BL/6 mice weighing 20–22g were purchased from Peking University Animal Center (Beijing, China). They were housed in a climate-controlled specific pathogen free facility at 25°C. The study was conducted in accordance with ARRIVE guidelines and the Basic & Clinical Pharmacology & Toxicology policy for experimental studies [15]. All procedures followed in this study were approved by the Peking University Animal Care and Use Committee (No. LA201783). After administering anesthesia with 1.5% sodium pentobarbital, mice in the OA group were injected with OA via the lateral tail vein at the indicated dose of 100, 200, 300, or 450 μL/kg suspended in 50 μL sterile phosphate-buffered saline (PBS). An equivalent quantity of PBS was injected into control mice using similar methods. To ensure the ubiquitous distribution of OA, the total volume injected was split into three equal parts, which were injected sequentially while the mice were placed in supine, 30° right lateral, and 30° left lateral positions [16]. To minimize animal suffering, the mice were humanely sacrificed by injecting ketamine (100 mg/kg) and xylazine (6 mg/kg) as soon as the experimental procedures were completed. Blood gas analysis After the mice were anesthetized, whole blood was collected by puncturing the abdominal aorta. To analyze arterial partial oxygen pressure (PaO 2 ), whole blood (0.25 mL) was examined using a gas analyzer (Ciba Corning, Etobicoke, ON, Canada). ARDS was diagnosed using PaO 2 analysis (PaO 2 /FiO 2 ≤ 300 mmHg). Measurement of histone H4 in plasma and bronchoalveolar lavage fluid (BALF) Plasma was separated from whole blood by centrifugation at 1,000×g for 10 min at 4°C. Because bronchoalveolar lavage can interfere with the analysis of lung wet/dry mass ratio, BALF was obtained from a different group of mice rather than from the group used for wet/dry ratio analysis. The BALF was obtained by flushing the lungs with 1 mL PBS, and then centrifuged at 1,000×g for 10 min to collect the supernatant. Histone H4 in the plasma and supernatant was measured using ELISA. Pathological and immunohistochemical analyses of pulmonary tissues Lung samples obtained from the right upper lobes were fixed with 4% formalin for 48 h at 25°C. After being embedded in paraffin, the fixed tissues were cut into 5-μm-thick sections. The tissues were stained with hematoxylin and eosin (H&E) and analyzed immunohistochemically for the neutrophil specific marker Ly6G. The sections were blocked with 1% hydrogen peroxide in methanol for 25 min and with 1% BSA in 0.05% Tween-20 for 15 min. Then, the sections were incubated with the primary antibody for Ly6G (1:50) for 30 min, followed by incubation with a biotin-labeled goat anti-rabbit secondary antibody. Peroxidase substrate was used to develop the sections. For each pulmonary section, three microscopic visual fields were randomly selected. Assessment of P-selectin in pulmonary vasculature Immunohistochemical detection of P-selectin was conducted after lung sections were fixed in 4% paraformaldehyde for 90 min at 4°C. Following a previously described protocol, a venule was defined as positively stained if it had a brown reaction product on more than 50% of the circumference of its endothelium. Ten venules were analyzed for each lung section, and 18 sections were examined for each group. The percentage of positively stained venules was calculated [17, 18]. Immunofluorescence analysis of pulmonary tissues As soon as the cryosections (8 μm) of pulmonary tissues were air-dried, they were fixed in 4% formalin for 30 min. The sections were permeabilized in blocking buffer (5% goat serum + 0.5% BSA + 1% Triton X-100) and incubated with a primary antibody for HS proteoglycan (1:50) for 2 h at room temperature. An FITC-labeled goat anti-rabbit IgG secondary antibody was used to visualize the HS proteoglycan, whereas DAPI (Vector, CA, USA) was used for nuclear staining. The fluorescence images were obtained using a confocal laser scanning microscope (Carl Zeiss LSM 710, Germany). Western blotting Protein concentration was measured using a Bio-Rad Protein Assay Kit. To analyze the vascular endothelial cadherin (VE-Cadherin) in mouse lung vascular endothelial cells (MLVECs), the cytoplasm and membrane were separated using a Cell Fractionation Kit (Cell Signaling Technology, Danvers, MA, USA). Equal quantities of protein lysates (40 μg) were mixed with the loading buffer and fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene difluoride membranes. The membranes were probed using an anti-Cadherin-5 primary antibody (1:500) and an anti-sodium-potassium ATPase antibody (1:50,000) overnight at 4°C. The bands were visualized using a chemiluminescence system. MLVEC isolation and characterization MLVECs were prepared following a previously prescribed method. Pulmonary tissues were diced into 1 mm 3 sections and cultured in 60-mm culture dishes. Adherent cells were further purified using a biotin-labeled rat anti-mouse CD31 antibody, and then cultured in endothelial growth medium-2 supplemented with 10% fetal-bovine-serum at 37°C in 5% CO 2 . The MLVECs were characterized by their cobblestone morphology and positive staining for factor VIII-related antigen (Sigma-Aldrich) [19]. Before the cells were treated with histone H4 or blocking antibodies against TLR1, TLR2, TLR4, TLR6, and P-selectin, they were incubated in serum-free medium for 12 h. Measurement of P-selectin translocation P-selectin translocation in MLVECs was assessed following the previously described cell surface ELISA [20]. The cells were treated with histone H4 in the presence or absence of specific blocking antibodies against TLRs. After being fixed with 1% paraformaldehyde for 20 min, the cells were incubated with blocking solution (5% BSA) for 15 min, and then incubated with an anti-P-selectin antibody (1:100) for 90 min. Peroxidase activity was quantified with a plate reader at 450 nm. The average level of cell surface P-selectin in the control group was considered as the baseline value. Purification of neutrophils from mouse bone marrow Isolation of mouse neutrophils from bone marrow was performed following the previously described protocol. In brief, the cells were flushed out of the marrow cavity using Hanks’ salt solution, which contains 2 mM EDTA (without magnesium and calcium). Histopaque density gradient was used to separate the remaining cells after the erythrocytes were abandoned. Through centrifugation (2,000 rpm, 40 min), neutrophils were found to be mainly located at the interface of Histopaque 1077 and 1119. The cellular viability was evaluated using Trypan blue dye exclusion assay. Neutrophil purity was assessed using Wright-Giemsa staining [21]. Neutrophil adhesion assay According to the described protocol, adhesion assay of neutrophils to endothelium was evaluated using a color digital camera attached with a binocular microscope (Olympus, Japan). Purified neutrophils were incubated with MLVECs in a culture well for 15 min. Three fields of view were randomly selected for every culture well, and the number of adhered neutrophils/mm 2 was recorded. The neutrophil adhesion results were presented as the ratio of the experiment group results to the control group results (100%) to eliminate the variations in neutrophil adhesion across groups [22]. The adhesion assay of neutrophils to the endothelial cells was performed under two conditions: (1) Neutrophils were challenged with the plasma collected from OA challenged mice (OA-plasma) and then exposed to MLVECs unchallenged with histone H4. (2) Neutrophils were challenged with OA-plasma and then exposed to MLVECs challenged with histone H4. Statistical analyses The results are shown as the mean ± SD. All data were analyzed using GraphPad Prism v8.3.0 (San Diego, CA, USA). Analysis of variance was used to analyze the statistical differences among groups, and the Student–Newman–Keuls test was used to analyze the differences between groups. The log-rank (Mantel-Cox) test was applied to analyze animal survival time. A p -value < 0.05 was considered statistically significant. Results 1. Pathogenic role of histone H4 in OA induced ARDS in mice After OA challenge, plasma histone H4 and BALF histone H4 were significantly higher than those in the control group, especially when the OA dose exceeded 300 μL/kg, as shown in Figure 1A and 1B. Significant positive correlations were observed between the doses of OA (from 100 to 450 μL/kg) and histone H4 in plasma ( r = 0.9706, p = 0.006) and BALF ( r = 0.9612, p = 0.0091). Refractory hypoxemia is the hallmark of ARDS. OA challenge caused obvious hypoxemia in mice in a dose dependent manner, as shown in Figure 1C. The PaO 2 decreased to 55.17 ± 15.52 mmHg ( p < 0.01, compared to the control group) when the OA dose reached 300 μL/kg. The pathological changes of the lungs were clear, and included widespread thickened pulmonary interstitium, obvious infiltration of inflammatory cells, alveolar collapse, and diffuse hemorrhage, as shown in Figure 1D. As shown in Figure 1E, the lung wet/dry mass ratio was significantly increased after OA challenge compared to the control group ( p < 0.01). Pretreatment with histone H4 further worsened the pulmonary edema, while pretreatment with anti-H4 antibody significantly improved it. Remarkably, histone H4 infusion alone also caused severe pulmonary edema. Ten mice (10/14) died within 72 h after being challenged with a lethal dose of OA (450 μL/kg), as shown in Figure 1F. When pretreated with intravenous histone H4, nearly all mice (13/14) died within 72 h after OA challenge ( p = 0.0909, compared to the mice only challenged with OA). In contrast, five mice (5/14) died when pretreated with the anti-H4 antibody ( p = 0.0462, compared to the mice only challenged with OA). There was a statistically significant difference ( p = 0.0005) in the survival rates between the mice pretreated with histone H4 and anti-H4 antibody. 2. Pulmonary endothelial activation mediated by histone H4 After OA challenge, pulmonary endothelial activation was increased compared to the control group. HS was significantly degraded 12 h after OA challenge, as shown in Figure 2A. Pretreatment with histone H4 further promoted HS degradation, while the anti-H4 antibody significantly reduced the HS degradation. Similarly, histone H4 infusion caused significant HS degradation. As shown in Figure 2B, OA challenge was associated with greater release of vWF compared to the control group ( p < 0.01). Pulmonary veins exhibited significant P-selectin translocation, as the proportion of veins that were positively stained for P-selectin was significantly increased compared to the control group ( p < 0.01), as shown in Figure 2C. Pretreatment with histone H4 and the anti-H4 antibody showed opposite effects on both vWF release and P-selectin translocation. VE-Cadherin status is an indicator of microvascular permeability. As shown in Figure 2D, after OA challenge, the membrane VE-Cadherin was significantly decreased compared to the control group ( p < 0.01). Pretreatment with histone H4 increased the loss of VE-Cadherin, whereas pretreatment with the anti-H4 antibody showed a protective effect. 3. Neutrophil activation mediated by histone H4 Neutrophil infiltration and activation were increased in the lung tissues compared to the control group after OA challenge. As shown in Figure 3A, staining for the specific neutrophil marker Ly6G showed that there was increased neutrophil infiltration into lung tissues. The MPO activity in the lung tissues was also significantly increased compared to the control group ( p < 0.01), as shown in Figure 3B. The serine proteases proteinase 3 and elastase were abundant in neutrophils and were released upon neutrophil degranulation. As shown in Figure 3C and 3D, circulating proteinase 3 and elastase were significantly increased 12 h after OA challenge compared to the control group ( p < 0.01). Pretreatment with histone H4 aggravated both neutrophil infiltration and activation caused by OA challenge, whereas the anti-H4 antibody showed an antagonizing effect on them. Impressively, histone H4 infusion alone also caused marked neutrophil infiltration and activation. 4. Activation effect of histone H4 on the endothelium and neutrophils in vitro As shown in Figure 4A and 4B, treatment of MLVECs with the plasma collected from OA challenged mice (OA-plasma) led to endothelial HS degradation and vWF release from WPBs compared to the control group ( p < 0.01). Pretreatment with histone H4 worsened HS degradation and vWF release caused by the OA-plasma, whereas the anti-H4 antibody showed an inhibitory effect to some extent. Treatment with histone H4 alone also caused evident HS degradation and vWF release. As shown in Figure 4C, when neutrophils were treated with the OA-plasma and then exposed to MLVECs unchallenged with histone H4, the relative proportion of neutrophils adhering to MLVECs was mildly increased compared to the control group ( p = 0.1658). On the contrary, when the treated neutrophils were exposed to MLVECs challenged with histone H4, the proportion of neutrophils adhering to the MLVECs was significantly increased ( p < 0.01), as shown in Figure 4D. As shown in Figure 4E and 4F, the change in the MPO activity in the supernatant was similar to that for the neutrophil adhesion. When the neutrophils treated with the OA-plasma were exposed to unchallenged MLVECs, MPO activity was slightly increased compared to the control group ( p = 0.4886). When the treated neutrophils were exposed to MLVECs challenged with histone H4, MPO activity was markedly increased compared to the control group ( p < 0.01). Pretreatment with histone H4 increased neutrophil adhesion and MPO activity further, whereas the anti-H4 antibody showed an antagonizing effect. By inhibiting the mutual binding of the endothelium and neutrophils, the blocking anti-P-selectin antibody significantly decreased the neutrophil adhesion ( p < 0.05) and MPO activity ( p < 0.05) compared to the OA-plasma group. 5. Roles of TLRs and calcium in histone H4 mediated endothelium activation To study the signaling pathways involved in histone H4-mediated endothelium activation, blocking antibodies against TLR1, TLR2, TLR4, and TLR6 were used to investigate the role of TLRs. As shown in Figures 5A and 5B, histone H4 treatment led to the significant degradation of membrane HS and VE-Cadherin in MLVECs, as measured by flow cytometry and western blot, respectively. Pretreatment with a blocking antibody against TLR4 distinctly inhibited the degradation of endothelial HS and VE-Cadherin. A blocking antibody against TLR2 also slightly inhibited HS and VE-Cadherin degradation. However, blocking antibodies against TLR1 and TLR6 had minimal effects. As shown in Figures 5C and 5D, the release of vWF from WPBs and P-selectin translocation in MLVECs were evident after histone H4 treatment compared to the control group ( p < 0.01). Pretreatment with the blocking antibody against TLR4 significantly reduced the release of vWF and P-selectin translocation compared to the H4-treated group ( p 0.05). The effects of the blocking antibodies against TLR1 and TLR6 were almost negligible. Calcium chelation with EGTA-AM was used to study the role of calcium. Calcium chelation showed little effect on endothelial HS and VE-Cadherin degradation (data not shown). However, calcium chelation significantly inhibited the release of vWF from WPBs and P-selectin translocation in a dose-dependent manner, as shown in Figures 5E and 5F. Additionally, calcium chelation showed a synergistic effect with the blocking antibody against TLR4 upon the release of vWF and P-selectin translocation. Discussion ARDS can be induced by a variety of diseases [23]. PFE is a life-threatening condition with characteristic manifestations of acute pulmonary edema and refractory hypoxemia [24, 25]. When nuclear histones are released passively from necrotic cells or actively by cell death, these extracellular histones not only exhibit bactericidal activity by forming neutrophil extracellular traps but can also damage normal host tissues [26,27]. In this study, histone H4 in the plasma and BALF was significantly increased after OA injection, especially when the dose of OA exceeded 300 μL/kg. Extracellular histone H4 was closely related with the OA dose which determined the severity of acute lung injury. The pathogenic role of histone H4 was revealed further by the experimental intervention. Pretreatment with histone H4 further aggravated the pulmonary edema and death rate, while the anti-H4 antibody exerted clear protective effects. Endothelial and neutrophil activation is a hallmark of the ARDS pathogenesis. This study showed that significant endothelial and neutrophil activation occurred during OA-induced ARDS in mice. The endothelial activation was manifested as HS degradation, release of vWF, P-selectin translocation, and VE-Cadherin reduction. Neutrophil activation was seen through pulmonary neutrophil infiltration, elevated circulating proteinase 3 and elastase, and increased MPO activity. Pretreatment with histone H4 further worsened endothelial and neutrophil activation, but the anti-H4 antibody showed significant antagonistic effects. The activation effect of histone H4 on endothelial cells and neutrophils was verified by in vitro experiments. Extracellular histone H4 could directly activate MLVECs, as measured by HS degradation and vWF release. In contrast to the activation of endothelial cells, neutrophil activation was mild when they were only challenged with exogenous histone H4, as shown by MPO activity and neutrophil adhesion assay. However, the synergistic stimulus of activated endothelia was associated with significant activation of neutrophils induced by extracellular histone H4. When the adhesion of endothelial cells with neutrophils was blocked using an anti-P-selectin antibody, the neutrophil activation was markedly inhibited. These findings suggest that neutrophil activation requires the synergistic stimulation of activated endothelium and histone H4. Furthermore, binding of neutrophils to the endothelium is a prerequisite for neutrophil activation. Neutrophil activation begins from recruitment to lung vasculature, adhesion to pulmonary endothelium, and ends with uncontrolled activation [28,29]. Pulmonary endothelial activation is necessary for histone H4-induced neutrophil activation. The pulmonary endothelium supplies a common platform for promoting activation of the inflammatory cascade [30,31]. Pulmonary edema resulting from increased endothelial permeability is the keystone of ARDS. Pulmonary endothelial glycocalyx has been recognized as the main regulator of vascular structural integrity and endothelial permeability [32,33]. As the most abundant glycosaminoglycan in pulmonary vascular endothelial glycocalyx, HS degradation mediates pulmonary endothelial hyper-permeability and consequential pulmonary edema during ARDS [34,35]. Furthermore, multiple types of cytokines, chemokines, signaling molecules, and growth factors are reserved within HS, which are termed HS-binding proteins (HSBPs). When HS is degraded, HSBPs are released to induce the inflammatory storm [36,37]. Weibel-Palade bodies are endothelium–specific secretory organelles that contain vWF and a variety of other inflammatory mediators, such as P-selectin, interleukin-8, and angiopoietin-2 [38]. When the endothelium detects damage, vWF is rapidly released through exocytosis, and P-selectin is translocated to the endothelial surface for mediating endothelial activation [39]. vWF is not only a pro-thrombotic mediator but also an essential pro-inflammatory molecule. It can promote neutrophil diapedesis through the modulation of the endothelial integrity. Additionally, the released vWF can interact with the DNA of neutrophil extracellular traps to further aggravate inflammatory injury [40-42]. The P-selectin translocated to the cell surface can mediate leukocyte tethering, rolling, and diapedesis through the endothelial barrier [43-45]. VE-Cadherin is the central component of endothelial adherens junctions that regulate junctional integrity and endothelial permeability in vessels [46]. In response to an inflammatory challenge, VE-Cadherin is degraded and endocytosed through phosphorylation- and ubiquitination-dependent mechanisms. The disruption of adherens junctions is the fundamental reason for inflammation-induced acute pulmonary edema [47,48]. TLRs are mainly distributed in the plasma membrane and include TLR1, TLR2, TLR4, TLR5, and TLR6 [49]. Our results showed that the blocking antibody against TLR4 inhibited HS degradation, P-selectin translocation, vWF release, and VE-Cadherin reduction caused by histone H4. Previous studies indicated that calcium was closely associated with extracellular histones induced injury [50, 51]. Our results showed that calcium chelation significantly inhibited the release of vWF and P-selectin translocation induced by histone H4. Furthermore, calcium chelation showed a synergistic effect with the blocking antibody against TLR4, which suggested that functional cooperation might exist between TLRs and calcium in histone H4-induced endothelial activation. In addition to TLRs and calcium, extracellular histones may induce cell injury through other means. Extracellular histones bind directly to the phospholipids of pulmonary endothelia and cause endothelial barrier dysfunction leading to increased vascular permeability [52]. Histone H4 can mediate membrane lysis of smooth muscle cells to trigger arterial tissue damage and inflammation [53]. Conclusions In conclusion, histone H4 is an essential pro-inflammatory and pro-thrombotic molecule in OA-induced ARDS. Histone H4 directly induces pulmonary endothelial activation. Endothelial activation is an indispensable synergistic stimulus for neutrophil activation induced by histone H4. TLRs and calcium are intimately involved in histone H4-mediated endothelium activation. The novel insights provided by this study will be helpful in clarifying the pathogenesis of ARDS caused by FES. Declarations Acknowledgements We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. Statement of Ethics All procedures used in this study were approved by the Peking University Animal Care and Use Committee (No. LA201783). All the experimental procedures were conducted strictly according to the U.S. NIH Guidelines for the Care and Use of Laboratory Animals. Conflict of Interest Statement The authors have no conflicts of interest to declare. Funding Sources The National Natural Science Foundation of China (Grant No. 81773374), Beijing, People’s Republic of China; the Natural Science Foundation of Beijing Municipality (Grant No. 7182179), Beijing, People’s Republic of China; and the Start-up Fund for Academic Leader Candidates of Peking University Third Hospital, Beijing, People’s Republic of China. Data Availability Statement The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Meyer NJ, Gattinoni L, Calfee CS. Acute respiratory distress syndrome. Lancet. 2021 Aug;398 (10300):622–37. 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Endothelial glycocalyx. Crit Care Clin. 2020 Apr;36(2):217-32. Oshima K, King SI, McMurtry SA, Schmidt EP. Endothelial heparan sulfate proteoglycans in sepsis: the role of the glycocalyx. Semin Thromb Hemost. 2021 Apr;47(3):274-82. LaRivière WB, Schmidt EP. The Pulmonary endothelial glycocalyx in ARDS: a critical role for heparan sulfate. Curr Top Membr. 2018 Sep; 82:33-52. Liao YE, Liu J, Arnold K. Heparan sulfates and heparan sulfate binding proteins in sepsis. Front Mol Biosci. 2023 Feb;10:1146685. Li M, Pedersen LC, Xu D. Targeting heparan sulfate-protein interactions with oligosaccharides and monoclonal antibodies. Front Mol Biosci. 2023 May;10:1194293. Karampini E, Fogarty H, Elliott S, Morrin H, Bergin C, O'Sullivan JM, et al. Endothelial cell activation, Weibel-Palade body secretion, and enhanced angiogenesis in severe COVID-19. Res Pract Thromb Haemost. 2023 Feb;7(2):100085. Ochoa CD, Wu S, Stevens T. New developments in lung endothelial heterogeneity: Von Willebrand factor, P-selectin, and the Weibel-Palade body. Semin Thromb Hemost. 2010 Apr;36(3):301-8. Ware LB, Eisner MD, Thompson BT, Parsons PE, Matthay MA. Significance of von Willebrand factor in septic and nonseptic patients with acute lung injury. Am J Respir Crit Care Med. 2004 Oct;170(7):766-72. Petri B, Broermann A, Li H, Khandoga AG, Zarbock A, Krombach F, et al. von Willebrand factor promotes leukocyte extravasation. Blood. 2010 Nov;116(22):4712-9. Luo GP, Ni B, Yang X, Wu YZ. von Willebrand factor: more than a regulator of hemostasis and thrombosis. Acta Haematol. 2012 Aug;128(3):158-69. Lorant DE, Topham MK, Whatley RE, McEver RP, McIntyre TM, Prescott SM, et al. Inflammatory roles of P-selectin. J Clin Invest. 1993 Aug;92(2):559-70. Takada YK, Simon SI, Takada Y. The C-type lectin domain of CD62P (P-selectin) functions as an integrin ligand. Life Sci Alliance. 2023 Apr;6(7):e202201747. Nussbaum C, Bannenberg S, Keul P, Gräler MH, Gonçalves-de-Albuquerque CF, Korhonen H, et al. Sphingosine-1-phosphate receptor 3 promotes leukocyte rolling by mobilizing endothelial P-selectin. Nat Commun. 2015 Apr;6:6416. Taveau JC, Dubois M, Le Bihan O, Trépout S, Almagro S, Hewat E, et al. Structure of artificial and natural VE-cadherin-based adherens junctions. Biochem Soc Trans. 2008 Apr;36(Pt 2):189-93. Li B, Huang X, Wei J, Huang H, Liu Z, Hu J, et al. Role of moesin and its phosphorylation in VE-cadherin expression and distribution in endothelial adherens junctions. Cell Signal. 2022 Sep;100:110466. Lampugnani MG, Dejana E, Giampietro C. Vascular endothelial (VE)-cadherin, endothelial adherens junctions, and vascular disease. Cold Spring Harb Perspect Biol. 2018 Oct;10(10):a029322. Asami J, Shimizu T. Structural and functional understanding of the toll-like receptors. Protein Sci. 2021 Apr;30(4):761-72. Abrams ST, Zhang N, Manson J, Liu T, Dart C, Baluwa F, et al. Circulating histones are mediators of trauma-associated lung injury. Am J Respir Crit Care Med. 2013 Jan;187(2): 160-9. Michels A, Albánez S, Mewburn J, Nesbitt K, Gould TJ, Liaw PC, et al. Histones link inflammation and thrombosis through the induction of Weibel-Palade body exocytosis. J Thromb Haemost. 2016 Nov;14(11):2274-86. Freeman CG, Parish CR, Knox KJ, Blackmore JL, Lobov SA, King DW, et al. The accumulation of circulating histones on heparan sulphate in the capillary glycocalyx of the lungs. Biomaterials. 2013 Jul;34(22):5670-6. Silvestre-Roig C, Braster Q, Wichapong K, Lee EY, Teulon JM, Berrebeh N, et al. Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death. Nature. 2019 May;569(7755):236-40. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 06 Jan, 2025 Read the published version in BMC Pulmonary Medicine → Version 1 posted Editorial decision: Revision requested 08 Jul, 2024 Reviews received at journal 06 Jul, 2024 Reviewers agreed at journal 06 Jul, 2024 Reviewers agreed at journal 05 Jul, 2024 Reviewers agreed at journal 04 Jul, 2024 Reviews received at journal 17 May, 2024 Reviewers agreed at journal 16 May, 2024 Reviewers invited by journal 16 May, 2024 Editor assigned by journal 16 May, 2024 Editor invited by journal 11 Mar, 2024 Submission checks completed at journal 11 Mar, 2024 First submitted to journal 22 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3887424","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":278842699,"identity":"36e9aef9-ee23-451f-b5de-ca04943e9005","order_by":0,"name":"Yanlin Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYDACCSjNx8zA+JiBB8w2IE4LGzMDszGJWoBIGsrGr0V+dvOzh1/b7PLY2HmPVRfIbEtsYG/eJsFQcwenFsY5x8yNZduSi9mY+dJuz+C5ndjAc6xMguHYM5xamCUSzKQl25gT25h5zG7zgLRI5JhJMDYcxqmFTSL9G1BLPVhLMViL/Bv8WniAZkp+bDsM1sIMsYUHvxYJiZwyaYZzx0FajKWBfjFu40krtkg4hluL/Iz0bZI/yqoT+/nPGH4u7Lkt289+eOONDzW4tYCDgJcNymLsAUcQA0MCXg1AhT/+wJg/CCgdBaNgFIyCEQkAQU1KqRsw9/0AAAAASUVORK5CYII=","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yanlin","middleName":"","lastName":"Zhang","suffix":""},{"id":278842700,"identity":"4a954bcb-5e12-476a-bb40-680189cb325d","order_by":1,"name":"Jingjin Tan","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jingjin","middleName":"","lastName":"Tan","suffix":""},{"id":278842701,"identity":"7e39e4fb-1dad-4b06-be1b-f40a5ebeeb2d","order_by":2,"name":"Yiran Zhao","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yiran","middleName":"","lastName":"Zhao","suffix":""},{"id":278842702,"identity":"c33c50ca-b0b0-41e6-9f37-c745ed0bc4b6","order_by":3,"name":"Li Guan","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Guan","suffix":""},{"id":278842703,"identity":"428edd62-0a7b-44fc-b4a5-5c6b9f3b115e","order_by":4,"name":"Shuqiang Li","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shuqiang","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-01-22 08:45:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3887424/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3887424/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12890-024-03334-w","type":"published","date":"2025-01-06T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52568518,"identity":"919c15be-c7b8-4fa5-a2ed-1053c8a0a1b4","added_by":"auto","created_at":"2024-03-13 05:03:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":321008,"visible":true,"origin":"","legend":"\u003cp\u003ePathogenic role of histone H4 in OA-induced ARDS\u003c/p\u003e\n\u003cp\u003eTwelve hours after the mice were challenged with different intravenous doses of OA (100, 200, 300, or 450 μL/kg), histone H4 in the plasma (A), H4 in the BALF (B), blood gas (C), and pathological changes in lungs (D) were evaluated. Histone H4 (10 mg/kg) or anti-H4 antibody (20 mg/kg) was injected through the tail vein 30 min prior to OA challenge (300 μL/kg for Fig.1E and 450 μL/kg for Fig.1F). Then, the lung wet/dry mass ratio (E) and survival rate (F) were analyzed. Data are presented as mean ± SD (n = 14 for the groups in Fig. 1F, and n = 6 for all other groups). The H\u0026amp;E stained lung sections are representative of three similar samples. Original magnification x 200. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to the control group; #\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ##\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to the OA group\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3887424/v1/5018735acbf3d78320f98889.png"},{"id":52568515,"identity":"437471fc-5ebb-4831-977b-4a69def59f7c","added_by":"auto","created_at":"2024-03-13 05:03:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":223357,"visible":true,"origin":"","legend":"\u003cp\u003ePulmonary endothelial activation in OA-induced ARDS\u003c/p\u003e\n\u003cp\u003eThe changes in the pulmonary vascular glycocalyx were evaluated by immunofluorescence staining of endothelial HS (A) 12 h after the mice were challenged with OA (300 μL/kg) injected intravenously. Histone H4 (10 mg/kg) or anti-H4 antibody (20 mg/kg) was injected through the tail vein 30 min prior to OA challenge. The level of circulating vWF was measured by ELISA (B), and translocation of P-selectin in the venules was evaluated by immunohistochemical detection (C). The change in VE-Cadherin in the cell membrane was measured by western blot (D). Data are presented as mean ± SD (n = 6). The results for immunofluorescence staining and western blot are representative of three similar experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to the control group; #\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ##\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to the OA group\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3887424/v1/67a948562fb9cbeaafc350f4.png"},{"id":52568516,"identity":"38f63c01-be48-4eb0-a8bf-73ed9128e399","added_by":"auto","created_at":"2024-03-13 05:03:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":202834,"visible":true,"origin":"","legend":"\u003cp\u003eNeutrophil activation in OA-induced ARDS\u003c/p\u003e\n\u003cp\u003eNeutrophil infiltration in the pulmonary tissue was examined by immunohistochemical detection of the specific marker Ly6G (A), and neutrophil activation was measured by MPO activity (B) 12 h after the mice were challenged with OA (300 μL/kg). Histone H4 (10 mg/kg) or anti-H4 antibody (20 mg/kg) was injected intravenously 30 min prior to OA challenge. The levels of circulating proteinase 3 (C) and leukocyte elastase (D) were measured by ELISA. Data are presented as mean ± SD (n = 6). The immunohistochemical images are representative of three similar experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to the control group; #\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ##\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to the OA group\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3887424/v1/bbb3e6367b2b514bed25f772.png"},{"id":52568519,"identity":"6b1c46d5-bda5-4b23-97c7-5093ab2848c6","added_by":"auto","created_at":"2024-03-13 05:03:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":329774,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of histone H4 on endothelial and neutrophil activation in vitro\u003c/p\u003e\n\u003cp\u003eThe changes in endothelial HS (A) and vWF in the supernatant (B) were evaluated by immunofluorescence staining and ELISA, respectively, after the MLVECs were treated with the plasma collected from OA-challenged mice (OA-plasma) for 12 h. Neutrophils were treated with the OA-plasma (12 h) and exposed to either unchallenged MLVECs or MLVECs challenged with histone H4 (15 mg/L) for six hours. The relative percentage of neutrophil adhesion to MLVECs was determined by a cell surface adhesion assay (C, D), and MPO activity in the supernatant was measured by ELISA (E, F). Histone H4 (15 mg/L), anti-H4 antibody (20 mg/L), or anti-P-selectin antibody (10 mg/L) was added to the medium one hour prior to the OA-plasma exposure. Data are presented as mean ± SD (n = 6). The immunofluorescence images are representative of three similar experiments (400×). Arrowheads indicate the staining of HS. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to the control group; #\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ##\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to the OA-plasma group\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3887424/v1/9c84959175c0e1fd404503ba.png"},{"id":52568520,"identity":"2779c1de-13c3-4789-b309-f3b9fd739c1f","added_by":"auto","created_at":"2024-03-13 05:03:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":314479,"visible":true,"origin":"","legend":"\u003cp\u003eTLR and calcium signaling involved in histone H4-mediated endothelium activation\u003c/p\u003e\n\u003cp\u003eAfter the MLVECs were challenged with histone H4 (15 mg/L) for 12 h, the changes in endothelial HS were evaluated by flow cytometry (A) while the VE-Cadherin in the cell membrane was measured by western blot (B). The vWF in the supernatant (C, E) and P-selectin translocation (D, F) were measured through ELISA. Prior to histone H4 challenge, the cells were pretreated for one hour with a blocking antibody (10 mg/L) against TLRs (TLR1, TLR2, TLR4, or TLR6) and the calcium chelator EGTA-AM (25, 50 or 100 μM). Data are presented as mean ± SD (n = 6). The results for flow-cytometry and western blot are representative of three similar samples. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to the control group; #\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ##\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared to the H4 group\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3887424/v1/719bfd780a7335e343152907.png"},{"id":73693844,"identity":"b8274ddc-efca-4529-80ce-4f57c4afbb46","added_by":"auto","created_at":"2025-01-13 16:08:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1919418,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3887424/v1/ff32ad09-a9de-4580-861c-3f9e58c452f5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"By activating endothelium histone H4 mediates oleic acid-induced acute respiratory distress syndrome","fulltext":[{"header":"Significance Statement","content":"\u003cp\u003eAs the main risk factor for Acute respiratory distress syndrome (ARDS), pulmonary fat embolism (PFE) is a common complication following long bone fractures, cardiopulmonary resuscitation, and infusion through an intraosseous catheter. This study showed that histone H4 is an essential pro-inflammatory and pro-thrombotic molecule in OA-induced ARDS. Histone H4 directly induces pulmonary endothelial activation. Endothelial activation is an indispensable synergistic stimulus for neutrophil activation induced by histone H4. TLRs and calcium are intimately involved in histone H4 mediated endothelium activation.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAcute respiratory distress syndrome (ARDS) has a mortality rate of more than 40%. The hallmark of ARDS is refractory hypoxemia resulting from acute pulmonary edema [1,2]. Unchecked overwhelming inflammation triggered by injurious mediators is intimately involved with endothelial injury [3,4].\u003c/p\u003e\n\u003cp\u003eIncreased endothelial permeability is viewed as the underlying pathological basis of ARDS [5]. Endothelial cells are not only damaged by inflammatory injury but also the active participants of inflammatory response. Activated endothelial cells may further aggravate the inflammatory response, resulting in a vicious\u0026nbsp;circle [6,7]. Extracellular histones have been increasingly recognized as key mediators in systemic inflammatory injuries [8,9].\u003c/p\u003e\n\u003cp\u003eAs the main risk factor for fatal ARDS, pulmonary fat embolism (PFE) is a common complication following long bone fractures, cardiopulmonary resuscitation, and infusion through an intraosseous catheter [10,11]. PFE has many similarities with oleic acid (OA)-induced acute lung injury, but the underlying mechanisms remain largely unknown [12,13]. Therefor the aim of this study was to investigate the pathogenic role and mechanism of extracellular histone H4 during OA-induced ARDS.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eReagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reagents used in this study included: oleic acid (OA) and Histopaque (Sigma-Aldrich St. Louis, MO, USA); histone H4 (Millipore, Billerica, MA, USA); antibodies for P-selectin, sodium-potassium ATPase, CD31 (PECAM-1), and Ly6G (Abcam, Cambridge, MA, USA); an antibody for Cadherin-5 (BD Transduction Laboratories, CA, USA); an antibody for heparan sulfate (HS) (Bioss, Woburn, MA, USA); blocking antibodies against P-selectin, TLR4 (HTA125), TLR2 (TL2.1), and TLR1 (GD2.F4) (eBioscience , San Diego, CA, USA); a blocking antibody against TLR6 (TLR6.127) (Abcam, Cambridge, MA, USA); enzyme-linked immunosorbent assay (ELISA) kits for histone H4 and von Willebrand factor (vWF) (Cusabio Biotech, Wuhan, China); and\u0026nbsp;1,2-bis(2-aminophenoxy) ethane N,N,N\u0026apos;,N\u0026apos;-tetraacetic acid acetoxymethyl ester (EGTA-AM)\u0026nbsp;(MedChemExpress,\u0026nbsp;Monmouth Junction, NJ, USA). Following the previously described methods a blocking antibody against histone H4 (anti-H4) was purified in autoimmune mice [14].\u003c/p\u003e\n\u003cp\u003eAnimal studies\u003c/p\u003e\n\u003cp\u003eEight-week-old male C57BL/6 mice weighing 20\u0026ndash;22g were purchased from Peking University Animal Center (Beijing, China). They were housed in a climate-controlled specific pathogen free facility at 25\u0026deg;C. The study was conducted in accordance with ARRIVE guidelines and the Basic \u0026amp; Clinical Pharmacology \u0026amp; Toxicology policy for experimental studies [15]. All procedures followed in this study were approved by the Peking University Animal Care and Use Committee (No. LA201783). After administering anesthesia with 1.5% sodium pentobarbital, mice in the OA group were injected with OA via the lateral tail vein at the indicated dose of 100, 200, 300, or 450 \u0026mu;L/kg suspended in 50 \u0026mu;L sterile phosphate-buffered saline (PBS). An equivalent quantity of PBS was injected into control mice using similar methods. To ensure the ubiquitous distribution of OA, the total volume injected was split into three equal parts, which were injected sequentially while the mice were placed in supine, 30\u0026deg; right lateral, and 30\u0026deg; left lateral positions [16]. To minimize animal suffering, the mice were humanely sacrificed by injecting ketamine (100 mg/kg) and xylazine (6 mg/kg) as soon as the experimental procedures were completed.\u003c/p\u003e\n\u003cp\u003eBlood gas analysis\u003c/p\u003e\n\u003cp\u003eAfter the mice were anesthetized, whole blood was collected by puncturing the abdominal aorta. To analyze arterial partial oxygen pressure (PaO\u003csub\u003e2\u003c/sub\u003e), whole blood (0.25 mL) was examined using a gas analyzer (Ciba Corning, Etobicoke, ON, Canada). ARDS was diagnosed using PaO\u003csub\u003e2\u003c/sub\u003e analysis (PaO\u003csub\u003e2\u003c/sub\u003e/FiO\u003csub\u003e2\u003c/sub\u003e \u0026le;\u0026nbsp;300 mmHg).\u003c/p\u003e\n\u003cp\u003eMeasurement of histone H4 in plasma and bronchoalveolar lavage fluid (BALF)\u003c/p\u003e\n\u003cp\u003ePlasma was separated from whole blood by centrifugation at 1,000\u0026times;g for 10 min at 4\u0026deg;C. Because bronchoalveolar lavage can interfere with the analysis of lung wet/dry mass ratio, BALF was obtained from a different group of mice rather than from the group used for wet/dry ratio analysis. The BALF was obtained by flushing the lungs with 1 mL PBS, and then centrifuged at 1,000\u0026times;g for 10 min to collect the supernatant. Histone H4 in the plasma and supernatant was measured using ELISA.\u003c/p\u003e\n\u003cp\u003ePathological and immunohistochemical analyses of pulmonary tissues\u003c/p\u003e\n\u003cp\u003eLung samples obtained from the right upper lobes were fixed with 4% formalin for 48 h at 25\u0026deg;C. After being embedded in paraffin, the fixed tissues were cut into 5-\u0026mu;m-thick sections. The tissues were stained with hematoxylin and eosin (H\u0026amp;E) and analyzed immunohistochemically for the neutrophil specific marker Ly6G. The sections were blocked with 1% hydrogen peroxide in methanol for 25 min and with 1% BSA in 0.05% Tween-20 for 15 min. Then, the sections were incubated with the primary antibody for Ly6G (1:50) for 30 min, followed by incubation with a biotin-labeled goat anti-rabbit secondary antibody. Peroxidase substrate was used to develop the sections. For each pulmonary section, three microscopic visual fields were randomly selected.\u003c/p\u003e\n\u003cp\u003eAssessment of P-selectin in pulmonary vasculature\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunohistochemical detection of P-selectin was conducted after lung sections were fixed in 4% paraformaldehyde for 90 min at 4\u0026deg;C. Following a previously described protocol, a venule was defined as positively stained if it had a brown reaction product on more than 50% of the circumference of its endothelium. Ten venules were analyzed for each lung section, and 18 sections were examined for each group. The percentage of positively stained venules was calculated [17, 18].\u003c/p\u003e\n\u003cp\u003eImmunofluorescence analysis of pulmonary tissues\u003c/p\u003e\n\u003cp\u003eAs soon as the cryosections (8 \u0026mu;m) of pulmonary tissues were air-dried, they were fixed in 4% formalin for 30 min. The sections were permeabilized in blocking buffer (5% goat serum + 0.5% BSA + 1% Triton X-100) and incubated with a primary antibody for HS proteoglycan (1:50) for 2 h at room temperature. An FITC-labeled goat anti-rabbit IgG secondary antibody was used to visualize the HS proteoglycan, whereas DAPI (Vector, CA, USA) was used for nuclear staining. The fluorescence images were obtained using a confocal laser scanning microscope (Carl Zeiss LSM 710, Germany).\u003c/p\u003e\n\u003cp\u003eWestern blotting\u003c/p\u003e\n\u003cp\u003eProtein concentration was measured using a Bio-Rad Protein Assay Kit. To analyze the vascular endothelial cadherin (VE-Cadherin) in mouse lung vascular endothelial cells (MLVECs), the cytoplasm and membrane were separated using a Cell Fractionation Kit (Cell Signaling Technology, Danvers, MA, USA). Equal quantities of protein lysates (40 \u0026mu;g) were mixed with the loading buffer and fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene difluoride membranes. The membranes were probed using an anti-Cadherin-5 primary antibody (1:500) and an anti-sodium-potassium ATPase antibody (1:50,000) overnight at 4\u0026deg;C. The bands were visualized using a chemiluminescence system.\u003c/p\u003e\n\u003cp\u003eMLVEC isolation and characterization\u003c/p\u003e\n\u003cp\u003eMLVECs were prepared following a previously prescribed method. Pulmonary tissues were diced into 1 mm\u003csup\u003e3\u003c/sup\u003e sections and cultured in 60-mm culture dishes. Adherent cells were further purified using a biotin-labeled rat anti-mouse CD31 antibody, and then cultured in endothelial growth medium-2 supplemented with 10% fetal-bovine-serum at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. The MLVECs were characterized by their cobblestone morphology and positive staining for factor VIII-related antigen (Sigma-Aldrich) [19]. Before the cells were treated with histone H4 or blocking antibodies against TLR1, TLR2, TLR4, TLR6, and P-selectin, they were incubated in serum-free medium for 12 h.\u003c/p\u003e\n\u003cp\u003eMeasurement of P-selectin translocation\u003c/p\u003e\n\u003cp\u003eP-selectin translocation in MLVECs was assessed following the previously described cell surface ELISA [20]. The cells were treated with histone H4 in the presence or absence of specific blocking antibodies against TLRs. After being fixed with 1% paraformaldehyde for 20 min, the cells were incubated with blocking solution (5% BSA) for 15 min, and then incubated with an anti-P-selectin antibody (1:100) for 90 min. Peroxidase activity was quantified with a plate reader at 450 nm. The average level of cell surface P-selectin in the control group was considered as the baseline value.\u003c/p\u003e\n\u003cp\u003ePurification of neutrophils from mouse bone marrow\u003c/p\u003e\n\u003cp\u003eIsolation of mouse neutrophils from bone marrow was performed following the previously described protocol. In brief, the cells were flushed out of the marrow cavity using Hanks\u0026rsquo; salt solution, which contains 2 mM EDTA (without magnesium and calcium). Histopaque density gradient was used to separate the remaining cells after the erythrocytes were abandoned. Through centrifugation (2,000 rpm, 40 min), neutrophils were found to be mainly located at the interface of Histopaque 1077 and 1119. The cellular viability was evaluated using Trypan blue dye exclusion assay. Neutrophil purity was assessed using Wright-Giemsa staining [21].\u003c/p\u003e\n\u003cp\u003eNeutrophil adhesion assay\u003c/p\u003e\n\u003cp\u003eAccording to the described protocol, adhesion assay of neutrophils to endothelium was evaluated using a color digital camera attached with a binocular microscope (Olympus, Japan). Purified neutrophils were incubated with MLVECs in a culture well for 15 min. Three fields of view were randomly selected for every culture well, and the number of adhered neutrophils/mm\u003csup\u003e2\u003c/sup\u003e was recorded. The neutrophil adhesion results were presented as the ratio of the experiment group results to the control group results (100%) to eliminate the variations in neutrophil adhesion across groups [22]. The adhesion assay of neutrophils to the endothelial cells was performed under two conditions: (1) Neutrophils were challenged with the plasma collected from OA challenged mice (OA-plasma) and then exposed to MLVECs unchallenged with histone H4. (2) Neutrophils were challenged with OA-plasma and then exposed to MLVECs challenged with histone H4.\u003c/p\u003e\n\u003cp\u003eStatistical analyses\u003c/p\u003e\n\u003cp\u003eThe results are shown as the mean \u0026plusmn; SD. All data were analyzed using GraphPad Prism v8.3.0 (San Diego, CA, USA). Analysis of variance was used to analyze the statistical differences among groups, and the Student\u0026ndash;Newman\u0026ndash;Keuls test was used to analyze the differences between groups. The log-rank (Mantel-Cox) test was applied to analyze animal survival time. A \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e1. Pathogenic role of histone H4 in OA induced ARDS in mice\u003c/p\u003e\n\u003cp\u003eAfter OA challenge, plasma histone H4 and BALF histone H4 were significantly higher than those in the control group, especially when the OA dose exceeded 300 \u0026mu;L/kg, as shown in Figure 1A and 1B. Significant positive correlations were observed between the doses of OA (from 100 to 450 \u0026mu;L/kg) and histone H4 in plasma (\u003cem\u003er\u003c/em\u003e = 0.9706, \u003cem\u003ep\u003c/em\u003e = 0.006) and BALF (\u003cem\u003er\u003c/em\u003e = 0.9612, \u003cem\u003ep\u003c/em\u003e = 0.0091). Refractory hypoxemia is the hallmark of ARDS. OA challenge caused obvious hypoxemia in mice in a dose dependent manner, as shown in Figure 1C. The PaO\u003csub\u003e2\u003c/sub\u003e decreased to 55.17 \u0026plusmn; 15.52 mmHg (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, compared to the control group) when the OA dose reached 300 \u0026mu;L/kg. The pathological changes of the lungs were clear, and included widespread thickened pulmonary interstitium, obvious infiltration of inflammatory cells, alveolar collapse, and diffuse hemorrhage, as shown in Figure 1D. As shown in Figure 1E, the lung wet/dry mass ratio was significantly increased after OA challenge compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Pretreatment with histone H4 further worsened the pulmonary edema, while pretreatment with anti-H4 antibody significantly improved it. Remarkably, histone H4 infusion alone also caused severe pulmonary edema. Ten mice (10/14) died within 72 h after being challenged with a lethal dose of OA (450 \u0026mu;L/kg), as shown in Figure 1F. When pretreated with intravenous histone H4, nearly all mice (13/14) died within 72 h after OA challenge (\u003cem\u003ep\u003c/em\u003e = 0.0909, compared to the mice only challenged with OA). In contrast, five mice (5/14) died when pretreated with the anti-H4 antibody (\u003cem\u003ep\u003c/em\u003e = 0.0462, compared to the mice only challenged with OA). There was a statistically significant difference (\u003cem\u003ep\u003c/em\u003e = 0.0005) in the survival rates between the mice pretreated with histone H4 and anti-H4 antibody.\u003c/p\u003e\n\u003cp\u003e2. Pulmonary endothelial activation mediated by histone H4\u003c/p\u003e\n\u003cp\u003eAfter OA challenge, pulmonary endothelial activation was increased compared to the control group. HS was significantly degraded 12 h after OA challenge, as shown in Figure 2A. Pretreatment with histone H4 further promoted HS degradation, while the anti-H4 antibody significantly reduced the HS degradation. Similarly, histone H4 infusion caused significant HS degradation. As shown in Figure 2B, OA challenge was associated with greater release of vWF compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Pulmonary veins exhibited significant P-selectin translocation, as the proportion of veins that were positively stained for P-selectin was significantly increased compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), as shown in Figure 2C. Pretreatment with histone H4 and the anti-H4 antibody showed opposite effects on both vWF release and P-selectin translocation. VE-Cadherin status is an indicator of microvascular permeability. As shown in Figure 2D, after OA challenge, the membrane VE-Cadherin was significantly decreased compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Pretreatment with histone H4 increased the loss of VE-Cadherin, whereas pretreatment with the anti-H4 antibody showed a protective effect.\u003c/p\u003e\n\u003cp\u003e3. Neutrophil activation mediated by histone H4\u003c/p\u003e\n\u003cp\u003eNeutrophil infiltration and activation were increased in the lung tissues compared to the control group after OA challenge. As shown in Figure 3A, staining for the specific neutrophil marker Ly6G showed that there was increased neutrophil infiltration into lung tissues. The MPO activity in the lung tissues was also significantly increased compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), as shown in Figure 3B. The serine proteases proteinase 3 and elastase were abundant in neutrophils and were released upon neutrophil degranulation. As shown in Figure 3C and 3D, circulating proteinase 3 and elastase were significantly increased 12 h after OA challenge compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Pretreatment with histone H4 aggravated both neutrophil infiltration and activation caused by OA challenge, whereas the anti-H4 antibody showed an antagonizing effect on them. Impressively, histone H4 infusion alone also caused marked neutrophil infiltration and activation.\u003c/p\u003e\n\u003cp\u003e4. Activation effect of histone H4 on the endothelium and neutrophils in vitro\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 4A and 4B, treatment of MLVECs with the plasma collected from OA challenged mice (OA-plasma) led to endothelial HS degradation and vWF release from WPBs compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Pretreatment with histone H4 worsened HS degradation and vWF release caused by the OA-plasma, whereas the anti-H4 antibody showed an inhibitory effect to some extent. Treatment with histone H4 alone also caused evident HS degradation and vWF release. As shown in Figure 4C, when neutrophils were treated with the OA-plasma and then exposed to MLVECs unchallenged with histone H4, the relative proportion of neutrophils adhering to MLVECs was mildly increased compared to the control group (\u003cem\u003ep\u003c/em\u003e = 0.1658). On the contrary, when the treated neutrophils were exposed to MLVECs challenged with histone H4, the proportion of neutrophils adhering to the MLVECs was significantly increased (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), as shown in Figure 4D. As shown in Figure 4E and 4F, the change in the MPO activity in the supernatant was similar to that for the neutrophil adhesion. When the neutrophils treated with the OA-plasma were exposed to unchallenged MLVECs, MPO activity was slightly increased compared to the control group (\u003cem\u003ep\u003c/em\u003e = 0.4886). When the treated neutrophils were exposed to MLVECs challenged with histone H4, MPO activity was markedly increased compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Pretreatment with histone H4 increased neutrophil adhesion and MPO activity further, whereas the anti-H4 antibody showed an antagonizing effect. By inhibiting the mutual binding of the endothelium and neutrophils, the blocking anti-P-selectin antibody significantly decreased the neutrophil adhesion (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) and MPO activity (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) compared to the OA-plasma group.\u003c/p\u003e\n\u003cp\u003e5. Roles of TLRs and calcium in histone H4 mediated endothelium activation\u003c/p\u003e\n\u003cp\u003eTo study the signaling pathways involved in histone H4-mediated endothelium activation, blocking antibodies against TLR1, TLR2, TLR4, and TLR6 were used to investigate the role of TLRs. As shown in Figures\u0026nbsp;5A and 5B, histone H4 treatment led to the significant degradation of membrane HS and VE-Cadherin in MLVECs, as measured by flow cytometry and western blot, respectively. Pretreatment with a blocking antibody against TLR4 distinctly inhibited the degradation of endothelial HS and VE-Cadherin. A blocking antibody against TLR2 also slightly inhibited HS and VE-Cadherin degradation. However, blocking antibodies against TLR1 and TLR6 had minimal effects. As shown in Figures 5C and 5D, the release of vWF from WPBs and P-selectin translocation in MLVECs were evident after histone H4 treatment compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Pretreatment with the blocking antibody against TLR4 significantly reduced the release of vWF and P-selectin translocation compared to the H4-treated group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The blocking antibody against TLR2 also showed some inhibitory effect, but there was no statistical difference compared to the H4-treated group (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). The effects of the blocking antibodies against TLR1 and TLR6 were almost negligible. Calcium chelation with EGTA-AM was used to study the role of calcium. Calcium chelation showed little effect on endothelial HS and VE-Cadherin degradation (data not shown). However, calcium chelation significantly inhibited the release of vWF from WPBs and P-selectin translocation in a dose-dependent manner, as shown in Figures 5E and 5F. Additionally, calcium chelation showed a synergistic effect with the blocking antibody against TLR4 upon the release of vWF and P-selectin translocation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eARDS can be induced by a variety of diseases [23]. PFE is a life-threatening condition with characteristic manifestations of acute pulmonary edema and refractory hypoxemia [24, 25]. When nuclear histones are released passively from necrotic cells or actively by cell death, these extracellular histones not only exhibit bactericidal activity by forming neutrophil extracellular traps but can also damage normal host tissues [26,27].\u003c/p\u003e\n\u003cp\u003eIn this study, histone H4 in the plasma and BALF was significantly increased after OA injection, especially when the dose of OA exceeded 300 \u0026mu;L/kg. Extracellular histone H4 was closely related with the OA dose which determined the severity of acute lung injury. The pathogenic role of histone H4 was revealed further by the experimental intervention. Pretreatment with histone H4 further aggravated the pulmonary edema and death rate, while the anti-H4 antibody exerted clear protective effects.\u003c/p\u003e\n\u003cp\u003eEndothelial and neutrophil activation is a hallmark of the ARDS pathogenesis. This study showed that significant endothelial and neutrophil activation occurred during OA-induced ARDS in mice. The endothelial activation was manifested as HS degradation, release of vWF, P-selectin translocation, and VE-Cadherin reduction. Neutrophil activation was seen through pulmonary neutrophil infiltration, elevated circulating proteinase 3 and elastase, and increased MPO activity. Pretreatment with histone H4 further worsened endothelial and neutrophil activation, but the anti-H4 antibody showed significant antagonistic effects.\u003c/p\u003e\n\u003cp\u003eThe activation effect of histone H4 on endothelial cells and neutrophils was verified by in vitro experiments. Extracellular histone H4 could directly activate MLVECs, as measured by HS degradation and vWF release. In contrast to the activation of endothelial cells, neutrophil activation was mild when they were only challenged with exogenous histone H4, as shown by MPO activity and neutrophil adhesion assay. However, the synergistic stimulus of activated endothelia was associated with significant activation of neutrophils induced by extracellular histone H4. When the adhesion of endothelial cells with neutrophils was blocked using an anti-P-selectin antibody, the neutrophil activation was markedly inhibited. These findings suggest that neutrophil activation requires the synergistic stimulation of activated endothelium and histone H4. Furthermore, binding of neutrophils to the endothelium is a prerequisite for neutrophil activation.\u003c/p\u003e\n\u003cp\u003eNeutrophil activation begins from recruitment to lung vasculature, adhesion to pulmonary endothelium, and ends with uncontrolled activation [28,29]. Pulmonary endothelial activation is necessary for histone H4-induced neutrophil activation. The pulmonary endothelium supplies a common platform for promoting activation of the inflammatory cascade [30,31].\u003c/p\u003e\n\u003cp\u003ePulmonary edema resulting from increased endothelial permeability is the keystone of ARDS. Pulmonary endothelial glycocalyx has been recognized as the main regulator of vascular structural integrity and endothelial permeability [32,33]. As the most abundant glycosaminoglycan in pulmonary vascular endothelial glycocalyx, HS degradation mediates pulmonary endothelial hyper-permeability and consequential pulmonary edema during ARDS [34,35]. Furthermore, multiple types of cytokines, chemokines, signaling molecules, and growth factors are reserved within HS, which are termed HS-binding proteins (HSBPs). When HS is degraded, HSBPs are released to induce the inflammatory storm [36,37].\u003c/p\u003e\n\u003cp\u003eWeibel-Palade bodies are endothelium\u0026ndash;specific secretory organelles that contain vWF and a variety of other inflammatory mediators, such as P-selectin, interleukin-8, and angiopoietin-2 [38]. When the endothelium detects damage, vWF is rapidly released through exocytosis, and P-selectin is translocated to the endothelial surface for mediating endothelial activation [39]. vWF is not only a pro-thrombotic mediator but also an essential pro-inflammatory molecule. It can promote neutrophil diapedesis through the modulation of the endothelial integrity. Additionally, the released vWF can interact with the DNA of neutrophil extracellular traps to further aggravate inflammatory injury [40-42]. The P-selectin translocated to the cell surface can mediate leukocyte tethering, rolling, and diapedesis through the endothelial barrier [43-45].\u003c/p\u003e\n\u003cp\u003eVE-Cadherin is the central component of endothelial adherens junctions that regulate junctional integrity and endothelial permeability in vessels [46]. In response to an inflammatory challenge, VE-Cadherin is degraded and endocytosed through phosphorylation- and ubiquitination-dependent mechanisms. The disruption of adherens junctions is the fundamental reason for inflammation-induced acute pulmonary edema [47,48].\u003c/p\u003e\n\u003cp\u003eTLRs are mainly distributed in the plasma membrane and include TLR1, TLR2, TLR4, TLR5, and TLR6 [49]. Our results showed that the blocking antibody against TLR4 inhibited HS degradation, P-selectin translocation, vWF release, and VE-Cadherin reduction caused by histone H4. Previous studies indicated that calcium was closely associated with extracellular histones induced injury [50, 51]. Our results showed that calcium chelation significantly inhibited the release of vWF and P-selectin translocation induced by histone H4. Furthermore, calcium chelation showed a synergistic effect with the blocking antibody against TLR4, which suggested that functional cooperation might exist between TLRs and calcium in histone H4-induced endothelial activation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to TLRs and calcium, extracellular histones may induce cell injury through other means. Extracellular histones bind directly to the phospholipids of pulmonary endothelia and cause endothelial barrier dysfunction leading to increased vascular permeability [52]. Histone H4 can mediate membrane lysis of smooth muscle cells to trigger arterial tissue damage and inflammation [53].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, histone H4 is an essential pro-inflammatory and pro-thrombotic molecule in OA-induced ARDS. Histone H4 directly induces pulmonary endothelial activation. Endothelial activation is an indispensable synergistic stimulus for neutrophil activation induced by histone H4. TLRs and calcium are intimately involved in histone H4-mediated endothelium activation. The novel insights provided by this study will be helpful in clarifying the pathogenesis of ARDS caused by FES.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe\u0026nbsp;thank\u0026nbsp;LetPub (www.letpub.com) for its linguistic assistance\u0026nbsp;during\u0026nbsp;the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement of Ethics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures used in this study were approved by the Peking University Animal Care and Use Committee (No. LA201783). All the experimental procedures were conducted strictly according to the U.S. NIH Guidelines for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe National Natural Science Foundation of China (Grant No. 81773374), Beijing, People\u0026rsquo;s Republic of China; the Natural Science Foundation of Beijing Municipality (Grant No. 7182179), Beijing, People\u0026rsquo;s Republic of China; and the Start-up Fund for Academic Leader Candidates of Peking University Third Hospital, Beijing, People\u0026rsquo;s Republic of China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMeyer NJ, Gattinoni L, Calfee CS. 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Nature. 2019 May;569(7755):236-40.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Acute respiratory distress syndrome, Pulmonary fat embolism, Oleic acid, Extracellular histone H4, Endothelium","lastPublishedDoi":"10.21203/rs.3.rs-3887424/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3887424/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e This study investigated pathogenic role and mechanism of extracellular histone H4 during oleic acid (OA)-induced acute respiratory distress syndrome (ARDS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e ARDS was induced by intravenous injection of OA in mice, and evaluated by blood gas, pathological analysis, lung edema, and survival rate. Heparan sulfate (HS) degradation was evaluated using immunofluorescence and flow cytometry. The released von Willebrand factor (vWF) was measured using ELISA. P-selectin translocation and neutrophil infiltration were measured via immunohistochemical analysis. Changes in VE-cadherin were measured by western blot. Blocking antibodies against TLRs were used to investigate the signaling pathway.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Histone H4 in plasma and BALF increased significantly after OA injection. Histone H4 was closely correlated with the OA dose, which determined the ARDS severity. Pretreatment with histone H4 further aggravated pulmonary edema and death rate, while anti-H4 antibody exerted obvious protective effects. Histone H4 directly activated the endothelia. Endothelial activation was evidently manifested as HS degradation, release of vWF, P-selectin translocation, and VE-Cadherin \u003ca href=\"https://www.baidu.com/link?url=YNWmBq7sWmvKJcxFo1TKJCHDwXlzbxgQI38BIkiGoTfwjzBG82pZLr5uhuxO1G7gmdBZy-OLy_lh7qTyISn-7-zNbORGhsruSWEyVlY3lQa\u0026amp;wd=\u0026amp;eqid=a16645b7000dc8840000000264590a65\" target=\"_blank\"\u003ereduction\u003c/a\u003e. The synergistic stimulus of activated endothelia was required for effective neutrophil activation by histone H4. Both TLRs and calcium mediated histone H4-induced endothelial activation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eHistone H4 is an essential pro-inflammatory and pro-thrombotic molecule in OA-induced ARDS in mice.\u003c/p\u003e","manuscriptTitle":"By activating endothelium histone H4 mediates oleic acid-induced acute respiratory distress syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-13 05:03:46","doi":"10.21203/rs.3.rs-3887424/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-08T06:31:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-07T02:17:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110523295228601318707140201675207001388","date":"2024-07-07T01:32:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143531177028325787807460077544951501518","date":"2024-07-05T14:09:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160612851882668954387475416550362858066","date":"2024-07-04T08:30:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-17T23:17:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247717656091525560138583697986265592002","date":"2024-05-16T16:46:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-16T08:54:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-16T06:12:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-11T19:57:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-11T19:55:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pulmonary Medicine","date":"2024-01-22T08:32:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e189dd7c-dcb6-41a8-8771-338aa48f37bf","owner":[],"postedDate":"March 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-13T16:01:01+00:00","versionOfRecord":{"articleIdentity":"rs-3887424","link":"https://doi.org/10.1186/s12890-024-03334-w","journal":{"identity":"bmc-pulmonary-medicine","isVorOnly":false,"title":"BMC Pulmonary Medicine"},"publishedOn":"2025-01-06 15:57:23","publishedOnDateReadable":"January 6th, 2025"},"versionCreatedAt":"2024-03-13 05:03:46","video":"","vorDoi":"10.1186/s12890-024-03334-w","vorDoiUrl":"https://doi.org/10.1186/s12890-024-03334-w","workflowStages":[]},"version":"v1","identity":"rs-3887424","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3887424","identity":"rs-3887424","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}