Plasma Histone Research Articles

Association of corticosteroid therapy with reduced acute kidney injury and lower NET markers in severe COVID-19: an observational study

BackgroundAcute kidney injury (AKI) is common in critical cases of coronavirus disease 2019 (COVID-19) and associated with worse outcome. Dysregulated neutrophil extracellular trap (NET) formation is one of several suggested pathophysiological mechanisms involved in the development of COVID-19 associated AKI. The corticosteroid dexamethasone was implemented as a standard treatment for severe COVID-19 as of June 2020. A sub-analysis of a prospective observational single center study was performed to evaluate the effect of corticosteroid treatment on AKI development and NET markers in critical cases of COVID-19.ResultsTwo hundred and ten adult patients admitted to intensive care at a tertiary level hospital due to respiratory failure or shock secondary to SARS-CoV-2-infection between March 13th 2020 and January 14th 2021 were included in the study. Ninety-seven of those did not receive corticosteroids. One hundred and thirteen patients were treated with corticosteroids [dexamethasone (n = 98) or equivalent treatment (n = 15)], but the incidence of AKI was assessed only in patients that received corticosteroids before any registered renal dysfunction (n = 63). Corticosteroids were associated with a lower incidence of AKI (19% vs 55.8%, p < 0.001). Fewer patients demonstrated detectable concentrations of extracellular histones in plasma when treated with corticosteroids (8.7% vs 43.1%; p < 0.001). Extracellular histones and in particular non-proteolyzed histones were observed more frequently with increasing AKI severity (p < 0.001). MPO-DNA was found in lower concentrations in patients that received corticosteroids before established renal dysfunction (p = 0.03) and was found in higher concentrations in patients with AKI stage 3 (p = 0.03). Corticosteroids did not ameliorate established AKI during the first week of treatment.ConclusionCorticosteroid treatment in severe COVID-19 is associated with a lower incidence of AKI and reduced concentrations of NET markers in plasma.

Histone-modifying enzymes and gastric cancer: Search for potential biomarkers and therapeutic targets based on Mendelian randomization

Gastric cancer, a common and severe malignancy, is associated with unfavorable outcomes and limited therapeutic options. The exploration of the potential link between plasma histone-modifying enzymes and gastric cancer through Mendelian randomization (MR) analysis offers an opportunity to identify new therapeutic targets and biomarkers. In this study, data on plasma histone-modifying enzymes were obtained from the International Working Unit Open genome-wide association studies project, and summary statistics of gastric cancer from the FinnGen study were analyzed. Forward and inverse MR were performed to determine the causal relationship between plasma histone-modifying enzymes and gastric cancer. The principal methodology for MR is the inverse variance weighted (IVW) method. Additionally, a sensitivity analysis was performed to determine the robustness of the findings. Finally, bioinformatics was used for the preliminary functional analysis. Our forward MR analysis revealed that the plasma Set1/Ash2 histone methyltransferase complex subunit ASH2 (ASH2L) was positively associated with gastric cancer risk, and the histone-lysine N-methyltransferase SETMAR (SETMAR) was negatively associated. Inverse MR analysis revealed that gastric cancer incidence was negatively correlated with the expression of plasma histone acetyltransferase KAT6A (KAT6A). These findings were consistent across different statistical methods and were deemed unlikely to have been distorted by horizontal pleiotropy. Furthermore, bioinformatics analysis indicated that ASH2L, SETMAR, and KAT6A are differentially expressed in various tumors and are significantly correlated with both the prognosis of gastric cancer and the infiltration of various immune cells. Thus, plasma histone-modifying enzymes may be causally linked to gastric cancer, and ASH2L, SETMAR, and KAT6A could play crucial roles as biomarkers and therapeutic targets in managing gastric cancer.

Blood Clot Dynamics and Fibrinolysis Impairment in Cancer: The Role of Plasma Histones and DNA.

Blood viscoelasticity and plasma protein levels can play an important role in the diagnosis and prognosis of cancer. However, the role of histones and DNA in modulating blood clot properties remains to be investigated. This study investigates the differences in blood viscoelasticity and plasma protein levels among cancer patients, individuals with other diseases, and healthy individuals. Blood samples were collected from 101 participants, including 45 cancer patients, 22 healthy individuals, and 34 individuals with other diseases. Rheological properties of clots formed in vitro by reconstituted elements of fibrinogen or plasma were analyzed with an Anton Paar Rheometer, USA. Plasma protein levels of D-dimer, TPA, EPCR, fibrinogen, and histone H3 were measured through ELISA. Blood clots were formed with or without DNA and histones (H3) by adding thrombin and calcium to plasma samples, and were evaluated for viscoelasticity, permeability, and degradation. Cancer patients show higher blood viscoelasticity and plasma D-dimer levels compared to healthy individuals and individuals with other diseases. Our in vitro analysis showed that the addition of histone to the plasma results in a significant decrease in viscoelasticity and mean fiber thickness of the clot formed thereafter. In parallel studies, using plasma from patients, DNA and histones were detected in fibrin clots and were associated with less degradation by t-PA. Moreover, our results show that the presence of DNA and histones not only increases clots' permeability, but also makes them more prone to degradation. Plasma histones and DNA affect the structure of the clot formed and induce defective fibrinolysis. Moreover, the increased viscoelastic properties of plasma from cancer patients can be used as potential biomarkers in cancer prognosis.

Morinda Citrifolia (Noni) attenuated transcriptional and degenerative markers of cardiac toxicity in bisphenol A treated male wistar rats

Bisphenol A (BPA) is an endocrine disruptor that causes metabolic disruption. It is widely used in the manufacturing industry as an internal protective lining for food and beverage cans. Morinda citrifolia (Noni) is a known ethno-medicinal plant of Pantropical origin. The aim of this study was to investigate the effect of Noni aqueous extract (NAE) on transcriptional and degenerative markers of cardiac toxicity in Bisphenol A treated male Wistar rats. Twenty-four male Wistar rats were randomly assigned into four groups as: Control (Ctr: normal chow + vehicle: Olive oil), BPA (7 g/kg, orally), (NAE:500 mg/kg; orally), and NAE: (500 mg/kg + BPA;7g/kg orally). After six weeks of administration, animals were anesthetized with 0.8 mg Phenobarbitone intraperitoneally and blood was collected by cardiac puncture. Plasma and cardiac homogenate were sampled and data were analyzed using a one-way ANOVA followed by Bonferroni’s Post hoc test. Results are expressed as mean ± standard error of mean (SEM) and p < 0.05 were accepted as significant. Bisphenol A reduced significantly plasma and cardiac histone deacetylate (HDAC), Nitric oxide (NO) and adenosine and increased triglycerides (TG), lipoprotein (a) or Lp(a), tumor necrotic factor alpha (TNF-α), uric acid (UA), and (malondialdehyde) MDA when compared with control and noni-treated group. NAE increased HDAC and NO significantly compared to the control while reducing significantly TNF-α, catalase, MDA, TG, and Lp(a). In conclusion, NAE reduced Bisphenol A worsening cardiovascular risks through a significant positive epigenetic HDAC expression and increased neovascularization activities.

C-TYPE LECTIN-2D RECEPTOR CONTRIBUTES TO HISTONE-INDUCED VASCULAR BARRIER DYSFUNCTION DURING BURN INJURY.

Severe burns are associated with massive tissue destruction and cell death where nucleus histones and other damage-associated molecular patterns are released into the circulation and contribute to the pathogenesis of multiple-organ dysfunction. Currently, there is limited information regarding the pathophysiology of extracellular histones after burns, and the mechanisms underlying histone-induced vascular injury are not fully understood. In this study, by comparing the blood samples from healthy donors and burn patients, we confirmed that burn injury promoted the release of extracellular histones into the circulation, evidenced by increased plasma levels of histones correlating with injury severity. The direct effects of extracellular histones on human endothelial monolayers were examined, and the results showed that histones caused cell-cell adherens junction discontinuity and barrier dysfunction in a dose-related manner. Like burn patients, mice subjected to a scald burn covering 25% total body surface area also displayed significantly increased plasma histones. Intravital microscopic analysis of mouse mesenteric microcirculation indicated that treatment with a histone antibody greatly attenuated burn-induced plasma leakage in postcapillary venules, supporting the pathogenic role of extracellular histones in the development of microvascular barrier dysfunction during burns. At the molecular level, intrigued by the recent discovery of C-type lectin domain family 2 member D (Clec2d) as a novel receptor of histones, we tested its potential involvement in the histone interaction with endothelial cells. Indeed, we identified abundant expression of Clec2d in vascular endothelial cells. Further proximity ligation assay demonstrated a close association between extracellular histones and endothelial expressing Clec2d. Functionally, in vivo administration of an anti-Clec2d antibody attenuated burn-induced plasma leakage across mesenteric microvessels. Consistently, Clec2d knockdown in endothelial cells partially inhibited histone-induced endothelial barrier dysfunction. Together, our data suggest that burn injury-induced increases in circulating histones contribute to microvascular leakage and endothelial barrier dysfunction via a mechanism involving the endothelial Clec2d receptor.

Apolipoprotein M bound sphingosine 1-phosphate suppresses NETosis through activating S1P1 and S1P4

The pleiotropic effects of high-density lipoprotein (HDL), including its protective properties against sepsis, are attributed to the sphingosine 1-phosphate and apolipoprotein M (ApoM) that are carried on the lipoproteins. In this study, we attempted to elucidate the possible mechanisms underlying the sepsis coagulopathic state by considering the modulation of NETosis. Our results revealed that in a lipopolysaccharide-induced sepsis mouse model, the levels of NETosis markers, such as plasma DNA and histone, were elevated in ApoM-knockout (KO) mice and attenuated in ApoM-overexpressing mice. In ApoM-KO mice, the survival rate decreased and the occurrence rates of coagulopathy and organ injury increased following the administration of histone. Treatment with a conditioned medium of ApoM-overexpressing cells attenuated the observed NETosis in HL-60S cells that differentiated into neutrophils and were inhibited through the suppression of S1P1 or S1P4. The attenuation of PKCδ and PKCα/β by S1P1 and S1P4 activation may also be involved. In ApoM-overexpressing mice, coagulopathy and organ injuries were attenuated following an injection of histone; these effects were partially inhibited by S1P1, 3, S1P4, or S1P1 antagonists. Furthermore, the exogenous administration of ApoM protected ApoM-KO mice that were challenged with histone from developing NETosis. In conclusion, the ApoM/S1P axis protects against NETosis through the attenuation of PKC activation by S1P1 and S1P4. The development of drugs targeting the ApoM/S1P axis may be beneficial for the treatment of pathological conditions involving uncontrolled NETosis, such as sepsis.

Omega 6/Omega 3 Ratio Is High in Individuals with Increased Sperm DNA fragmentation.

An imbalance between omega-6 and omega-3 fatty acids in sperm has been linked with lipid peroxidation and DNA damage in sperm, indicating a possible correlation to fertility potential. This cross-sectional study involved 56 infertile men (aged 25-45), and assessed the relationship between the omega-6 to omega-3 fatty acid ratio in sperm and seminal plasma with sperm DNA fragmentation. Individuals were categorized based on high or low levels of sperm DNA fragmentation according to two tests (TUNEL and SCSA assay less or greater than 10 and 30%, respectively), and their fatty acid composition, as well as sperm functional tests, were analyzed. Results showed that men with high DNA fragmentation exhibited higher percentages of total saturated, monounsaturated, and omega-6 to omega-3 fatty acid ratios in both sperm (P < 0.001) and seminal plasma (P < 0.001) compared to men with low DNA fragmentation. The percentage of sperm lipid peroxidation, and residual histone (P < 0.05) were higher, while the percentage of sperm motility (P < 0.001) was lower in the former compared to the latter group. Moreover, Pearson's correlation revealed positive associations between the omega-6 to omega-3 fatty acid ratio with sperm lipid peroxidation, DNA fragmentation, and residual histones in both sperm and seminal plasma. Overall, these observations suggest that consumption of omega-3 fatty acids may be related to male fertility potential, as it appears that individuals with a high percentage of omega-3 fatty acids have better sperm quality compared to men with a lower omega-3 fatty acid.

Plasma Histone Deacetylase activities, Lipid Profile, and Glycated Haemoglobin levels as indicator of Glycemic control in Type 2 Diabetes Mellitus

Background: The number of people with Diabetes mellitus (DM) is steadily rising globally and its socio-economic effects extend beyond the individual to affect their families and whole societies.Aim: This study aimed at determining the Plasma Lipid Profile, Glycated Haemoglobin (HbA1C) and Histone Deacetylase (HDAC) enzyme activities as indicator of Glycaemic control in Type 2 Diabetes mellitus.Methods: A total of 100 newly diagnosed DM patients and 50 control subjects were recruited. The weight, height, body mass index (BMI), waist circumference (WC), and blood pressure of each participant were determined. Also, fasting plasma glucose (FPG), triglycerides, total Cholesterol (TC), high density lipoprotein (HDLC) and HDAC activities were measured using standard methods. Low density lipoprotein (LDLC) was computed.Results: A significant increase in mean age, FPG, systolic and diastolic blood pressure, HbA1c total cholesterol, triglyceride, and low density lipoprotein, and HDAC activities; and a decrease in plasma HDLC were observed among the diabetics when compared with the controls. A direct but significant correlation existed between HDAC activities and FPG, WC, and duration of diabetes; and between HbA1C and FBG, TC, LDLC, HDLC, and duration of diabetes; a direct but non-significant correlation existed between HDAC and HbA1C, HDLC; an inverse and significant correlation existed between BMI and TC, Triglyceride and LDLC.Conclusion: It could be inferred from thia study that plasma HDAC activities is directly related to FPGÂ as it was found to increase with increasing FPG, indicating its likely role in metabolism.

First Report of Alternaria alternata Inciting Leaf Spot of Dangshen (Codonopsis pilosula) in China.

As a commonly traditional Chinese medicine, the perennial herb Dangshen (Codonopsis pilosula) has superior curative effects including regulating immunity, strengthening the spleen, and tonifying lungs (Bai et al. 2020). To imitate natural ecological conditions, plants were grown on hillside fields with stems prostrate on the ground, tangle-up with each other. In August 2020, leaf spots were observed on C. pilosula in Wutai county, Shanxi province, China, and indicated a high disease incidence (70%-80%) in investigated fields (6.67 ha). Small brown necrotic spots, occasionally enclosed by chlorotic halos, were observed on leaves, stems, and sepals. For identification of the pathogen, 15 small pieces (5×5 mm) of symptomatic tissues from 5 randomly-collected diseased plants were surface sterilized, placed on potato dextrose agar plates, and incubated for 4 d in darkness at 25 °C to obtain the colonies. Cultures were purified by single spore isolation from these colonies. A total of 15 isolates named as Dcp-3, and Dcp-5~Dcp-18 were recovered. They produced ovoid or obclavate spores with 15.9-57.5×9.1-20.1 μm in size, 1-6 transverse septa, and 0-4 longitudinal septa. The conidial chains with 4 to 6 spores had numerous secondary and occasionally tertiary chains on potato carrot agar plates. Because all isolates had identical morphological traits, five genes from the representative isolate Dcp-3, actin (ACT), Alternaria major allergen (Alt a1), plasma membrane ATPase (ATP), histone 3 (H3), and rDNA ITS, were amplified with primer pairs ACTDF1/ACTDR1, Alt-for/Alt-rev, ATPDF1/ATPDR1, H3-1a/H3-1b, and ITS1/ITS4, respectively (Hong et al. 2005; Lawrence et al. 2013; Ma et al. 2020). BLASTn searches indicated species of Dcp-3 could not be accurately confirmed by rDNA ITS, ATP, ACT, and Alt a1 (GenBank accession nos. OM334894, OM362504, OM326344, OM362500). Phylogenetic analysis showed it was most closely related to Alternaria alternata, A. arborescens, and A. tenuissima based on concatenated sequences of above four genes. The H3 sequence (OM362508) shared 100% homology with that of A. alternata (MN481948). The phylogenetic tree using H3 also confirmed Dcp-3 as A. alternata. Heathly, two-year-old C. pilosula were transplanted to a greenhouse. A surface-sterilized leaf was sprayed with 50 μL spore suspension (106 conidia/mL) of Dcp-3. A leaf sprayed with isometric sterile water was used as controls. Each treatment used six plants (five leaves per plant). Plants were covered with sterilized plastic bags and incubated at 22 ℃. The test was repeated twice. A week later, control leaves were healthy, but brown necrotic spots similar to field symptoms emerged on treated leaves. The A. alternata isolates were re-isolated from the border of lesions, and confirmed by morphological and molecular characteristics mentioned above, fulfilling Koch's postulates. Leaf spot of C. pilosula caused by Septoria codonopsidis has been reported in China (Wang et al. 2011). However, to our knowledge, this is the first report of A. alternata inciting leaf spot of C. pilosula in China. Our report would promote growers to enhance the field management and consider associated strategies on controlling Alternaria leaf spot of C. pilosula.

Prognostic value of plasma high mobility group box 1 protein and histone H3 levels in patients with disseminated intravascular coagulation: a multicenter prospective cohort study

BackgroundWe compared the prognostic value of plasma high mobility group box 1 protein (HMGB1) and histone H3 levels with the International Society on Thrombosis and Haemostasis (ISTH) disseminated intravascular coagulation (DIC) scores for 28-day in-hospital mortality in patients with DIC caused by various underlying diseases.MethodsWe conducted a multicenter prospective cohort study including two hematology departments, four emergency departments, and one general medicine department in Japan, between August 2017 and July 2021. We included patients diagnosed with DIC by the ISTH DIC scoring system.ResultsOverall, 104 patients were included: 50 with hematopoietic disorders, 41 with infections, and 13 with the other diseases. The 28-day in-hospital mortality rate was 21%. The receiver operator characteristic (ROC) curve showed that a DIC score of 6 points, plasma HMGB1 level of 8 ng/mL, and plasma histone H3 level of 2 ng/mL were the optimal cutoff points. The odds ratios of more than these optimal cutoff points of the DIC score, plasma HMGB1, and histone H3 levels were 1.58 (95% confidence interval [CI]: 0.60 to 4.17, p = 0.36), 5.47 (95% CI: 1.70 to 17.6, p = 0.004), and 9.07 (95% CI: 2.00 to 41.3, p = 0.004), respectively. The area under the ROC curve of HMGB1 (0.74, 95% CI: 0.63 to 0.85) was better than that of the ISTH DIC scores (0.55, 95% CI: 0.43 to 0.67, p = 0.03), whereas that of histone H3 was not (0.71, 95% CI: 0.60 to 0.82, p = 0.07). Calibration and net reclassification plots of HMGB1 identified some high-risk patients, whereas the ISTH DIC scores and histone H3 did not. The category-free net reclassification improvement of HMGB1 was 0.45 (95% CI: 0.01 to 0.90, p = 0.04) and that of histone H3 was 0.37 (95% CI: − 0.05 to 0.78, p = 0.08).ConclusionsPlasma HMGB1 levels have a prognostic value for mortality in patients with DIC. This finding may help physicians develop treatment strategies.

MO283: A Novel Hemoperfusion Adsorbent for Selective Removal of Circulating Histones in Septic Blood

Abstract BACKGROUND AND AIMS Circulating histones play a crucial role in the development and progression of sepsis and is associated with high mortality. Hemadsorption with the CytoSorb® filter has been shown to non-specifically eliminate systemic circulating histones in plasma from injured patients ex vivo and in vitro. The aim of this study was to fabricate another novel hemoperfusion adsorbent for selective removal of histones in the blood of septic patients. METHOD Chitosan-cellulose composite hydrogels (CSCE) were first prepared using a liquid-liquid inversion method. Then, negatively charged heparin was grafted onto the surfaces of the CSCE hydrogels by EDC chemistry to obtain the histone sorbents (CSCEHEP hydrogels). The chemical structure of CSCEHEP hydrogels was characterized by Fourier transform infrared spectroscopy (FTIR) and its hemocompatibility was evaluated. The adsorption capacity and adsorption selectivity of CSCEHEP hydrogels for histone were then analysed by BCA protein quantitative method. Besides, the antagonistic effect of CSCEHEP hydrogels on histone-induced cytotoxicity was verified by in vitro cell experiments using HK-2 cell lines. RESULTS The obtained CSCEHEP hydrogels showed characteristic adsorption peaks of the sulfonic group at 1217 and 940 cm-1 in their FTIR spectrum, suggesting the successful immobilization of heparin onto the surface of the sorbent. The clotting time of plasma after a 30-min incubation with CSCEHEP hydrogels was significantly prolonged by 3 times while no obvious hemolysis, platelet activation, complement activation and other blood-material adverse reactions occurred during this incubation. With a heparin grafting, the adsorption capacity of CSCEHEP hydrogels towards histones (from calf thymus) increased significantly to 314.3 μg/g, with a clearance rate of 62.8%, compared to that of pristine CSCE, which is dependent on the initial histone concentrations in testing solutions. On the contrary, the adsorption capacity of bovine serum albumin by CSCEHEP hydrogels was significantly lower than that of histones, suggesting that the adsorption process of histone by CSCEHEP in plasma was selective. Meanwhile, CSCEHEP hydrogels significantly antagonized histone-induced cytotoxicity by binding histones, and the cell viability increased significantly from 47.1% (model group) to 72.0% (CSCEHEP-treat group). CONCLUSION CSCEHEP hydrogels are of good hemocompatibility and can effectively adsorb histones in PBS solution and antagonize histone-induced cytotoxicity. It is believed that the use of such a histone sorbent for extracorporeal blood purification may provide a novel insight into the modulation of aberrant immune responses in patients with sepsis.

Role of Complement and Histones in Sepsis.

The wide use of the mouse model of polymicrobial sepsis has provided important evidence for events occurring in infectious sepsis involving septic mice and septic humans. Nearly 100 clinical trials in humans with sepsis have been completed, yet there is no FDA-approved drug. Our studies of polymicrobial sepsis have highlighted the role of complement activation products (especially C5a anaphylatoxin and its receptors C5aR1 and C5aR2) in adverse effects of sepsis. During sepsis, the appearance of these complement products is followed by appearance of extracellular histones in plasma, which have powerful proinflammatory and prothrombotic activities that cause cell injury and multiorgan dysfunction in septic mice. Similar responses occur in septic humans. Histone appearance in plasma is related to complement activation and appearance of C5a and its interaction with its receptors. Development of the cardiomyopathy of sepsis also depends on C5a, C5a receptors and histones. Neutralization of C5a with antibody or absence of C5aR1 blocks appearance of extracellular histones and cell and organ failure in sepsis. Survival rates in septic mice are greatly improved after blockade of C5a with antibody. We also review the various strategies in sepsis that greatly reduce the development of life-threatening events of sepsis.

The study on the role of extracellular histones in the pathogenesis of coal worker's pneumoconiosis

Objective: To explore the role of extracellular histones in the pathogenesis of coal worker's pneumoconiosis (CWP) , the relationship of extracellular histones in plasma with pulmonary fibrosis caused by coal mine dust was analyzed, and the stimulating effect of extracellular histones on fibroblast proliferation was studied. Methods: In May 2019, a total of 220 coal mine dust exposure workers (including coal miners and CWP patients) who visited the occupational disease outpatient department of Peking University Third Hospital from 2012 to 2015 were enrolled in the study. According to the classification of small opacity profusion (SOP) in chest radiograph for pneumoconiosis diagnosis (category 0, 1, 2, 3) , 61 coal miners were in category 0 SOP, 65 coal miners were in category 1 SOP, 56 coal miners were in category 2 SOP and 38 coal miners were in category 3 SOP. The plasma levels of extracellular histone H4 and platelet-derived growth factor (PDGF) were measured by enzyme-linked immunosorbent assay (ELISA) kit. The stimulating effects of CWP patients' plasma and calf thymus histones (CTHs) on fibroblast and the antagonizing effect of anti-H4 antibody were investigated by fibroblast proliferation experiment in vitro. Results: Among the study subjects, there were 195 males (88.6%, 195/220) and 25 females (11.4%, 25/220) , age (55.1±7.2) years, coal mine dust exposure time (16.3±4.4) years. The plasma concentrations of histone H4 in the coal miners with category 0, 1, 2 and 3 SOP were (3.92±1.75) 、(9.84±4.17) 、(14.35±5.52) and (17.83±7.69) μg/ml, respectively. There were significant differences among the four groups (P<0.01) . The plasma level of histone H4 was positively correlated with the plasma level of PDGF in the coal miners (r=0.769, P<0.01) . Compared with healthy control plasma group, the cell proliferation percentages of patients' plasma group (272%±87%) and CTH group (283%±84%) were significantly increased (P<0.05) . Compared with patients' plasma group, the cell proliferation percentage of patients' plasma+anti-H4 antibody group (185%±66%) was significantly decreased (P<0.05) . Compared with CTH group, the cell proliferation percentage of CTH+anti-H4 antibody group (167%±59%) was significantly decreased (P<0.05) . Conclusion: Extracellular histones in plasma are associated with pulmonary fibrosis in patients with CWP. Studies in vitro have shown that extracellular histones can promote proliferation of pulmonary fibroblasts. It is suggested that extracellular histones can be important biomarkers for pulmonary fibrosis caused by coal mine dust.

Requirement of Complement C6 for Intact Innate Immune Responses in Mice.

Over the first days of polymicrobial sepsis, there is robust activation of the innate immune system, causing the appearance of proinflammatory cytokines and chemokines, along with the appearance of extracellular histones, which are highly proinflammatory and prothrombotic. In the current study, we studied different innate immune responses in mice with knockout (KO) of complement protein 6 (C6). Polymorphonuclear neutrophils (PMNs) from these KO mice had defective innate immune responses, including defective expression of surface adhesion molecules, generation of superoxide anion, and appearance of reactive oxygen species and histone release after activation of PMNs, along with defective phagocytosis. In addition, in C6-/- mice, the NLRP3 inflammasome was defective both in PMNs and in macrophages. When these KO mice were subjected to polymicrobial sepsis, their survival was improved, associated with reduced levels in the plasma of proinflammatory cytokines and chemokines and lower levels of histones in plasma. In addition, sepsis-induced cardiac dysfunction was attenuated in these KO mice. In a model of acute lung injury induced by LPS, C6-/- mice showed reduced PMN buildup and less lung epithelial/endothelial cell dysfunction (edema and hemorrhage). These data indicate that C6-/- mice have reduced innate immune responses that result in less organ injury and improved survival after polymicrobial sepsis.

1818-P: High-Sucrose Diet Causes Increased Hepatic Histone Acetylation Due to Increased Bacterial Acetate Production and Reduced Lipogenesis in ChREPB-Deficient Mice

ChREBP is known to play a critical role in the development of obesity and diabetes by regulating hepatic lipogenesis. High-sucrose diet (HSD) feeding is known to cause irritable bowel syndrome-like phenotype in ChREBP knockout mice (KO) with some alterations in gut microbiota. However, effects of the gut microbiota alteration on whole-body metabolism in KO remains unknown. Twelve-week old wild type mice (WT) and KO, both fed on HSD for 7 days, were analyzed. KO had 3-fold higher cecum contents compared to WT (WT, 71±24.4; and KO, 205.7±46.0 g). KO had a remarkable increase in acetate-producing bacteria (e.g., Bifidobacterium and Bacteroides) in the cecum. Consistently, KO showed 2-fold higher acetate levels in the cecum (WT, 27.5±6.0; and KO, 55.6±22.3 μmoles/g stool). Interestingly, KO also showed higher hepatic acetate contents (WT, 0.81±0.18; and 1.08±0.14 μmoles/g liver). Since acetate is converted into acetyl-CoA; and used for lipogenesis, ketogenesis and protein acetylation, we investigated hepatic triglyceride contents, plasma 3OHBA levels and histone acetylation. While hepatic acetyl-CoA contents and plasma 3-OHBA levels did not differ between the two groups, hepatic triglyceride contents were much lower in KO (WT, 11.45±5.47; and KO, 4,11±0.44 mg/g liver), consistent with reduced expression of hepatic lipogenic genes. In contrast, histone acetylation on selected genes (e.g., Fasn, Chrebp and Pklr) was significantly enhanced in KO. These results together indicated that increased supply of acetyl-CoA in the liver, possibly due to enhanced acetate-production in the gut and reduced hepatic lipogenesis, leads to enhancement of histone acetylation in KO, while physiological relevance awaits further investigation. Disclosure K. Iizuka: Speaker’s Bureau; Self; Kowa Company, Ltd., Novo Nordisk Inc. K. Takao: None. Y. Liu: None. K. Nonomura: None. T. Kato: None. M. Mizuno: None. Y. Horikawa: None. D. Yabe: Research Support; Self; ARKRAY, Astellas Pharma Inc., AstraZeneca K.K., Eli Lilly Japan K.K., Japan Agency for Medical Research and Development, Japan Society for the Promotion of Science, Kowa Company, Ltd., Nippon Boehringer Ingelheim Co. Ltd., Sanofi K.K., Sanwa Kagaku Kenkyusho, Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker’s Bureau; Self; Abbott, ARKRAY, Astellas Pharma Inc., AstraZeneca K.K., Bayer Yakuhin, Ltd., Daiichi Sankyo, Eli Lilly Japan K.K., Fukuda Denshi, Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., LifeScan, Inc., Medtronic, Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Inc., Novo Nordisk Inc., Sanofi K.K., Sanwa Kagaku Kenkyusho, Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited. Other Relationship; Self; Japan Diabetes Society. Funding Grant-in-Aid for Scientific Research (17K00850)

Association of Prenatal Exposure to Ambient Air Pollution With Circulating Histone Levels in Maternal Cord Blood

Exposure to ambient air pollution has been associated with the risk of carcinogenesis in later life. Changes in histone modifications might have long-term adverse health effects. To investigate the association of prenatal exposure to ambient air pollution with levels of circulating total histone H3 and specific trimethylation marks (ie, H3 lysine 4, H3 lysine 36) in maternal cord blood. The Environmental Influence on Aging (ENVIRONAGE) birth cohort study included 609 mothers and their newborns. Participants were recruited when mothers entered the Hospital East Limburg (Genk, Belgium) for delivery between February 2010 and January 2017. The inclusion criteria were singleton pregnancies and the ability to fill out questionnaires in Dutch. Data analysis was conducted from March to August 2019. Exposure to particulate matter with a diameter less than 2.5 μm (PM2.5), black carbon, and nitrogen dioxide during pregnancy was modeled with a high-resolution air pollution model on the basis of maternal address for each trimester of pregnancy as well as for the entire pregnancy. Circulating total histone H3 levels and specific trimethylation marks (ie, trimethylated H3 lysine 4 and trimethylated H3 lysine 36) in cord blood. A total of 609 mother-newborn pairs were included in the study. Mean (SD) maternal age was 29.3 (4.6) years, 391 mothers (64.2%) never smoked, and 314 (51.3%) had a high education level. Overall, 322 newborns (52.4%) were boys, and mean (SD) birth weight was 3414 (485) g. Participants experienced mean (SD) exposure to PM2.5, black carbon, and nitrogen dioxide of 13.4 (2.6) μg/m3, 1.29 (0.31) μg/m3, and 17.98 (4.57) μg/m3, respectively, during their entire pregnancies. Trimethylated H3 lysine 4 and total histone H3 were positively associated with gestational PM2.5 exposure, with a 74.4% increment (95% CI, 26.7% to 140.2%, P < .001) and a 40.2% increment (95% CI, 24.1% to 58.3%, P < .001), respectively, observed for each 5-μg/m3 increase in PM2.5 exposure during the entire pregnancy. For the same exposure window, trimethylated H3 lysine 36 levels were inversely associated with PM2.5 exposure (-34.4%; 95% CI, -50.1% to -13.7%; P = .003). Exposure to black carbon during the entire pregnancy was positively associated with trimethylated H3 lysine 4 (38.4%; 95% CI, 6.2% to 80.3%; P = .003). Associations of ambient air pollution with cord plasma histone H3 modifications during early life might indicate that circulating histones are a risk factor in the development of air pollution-associated disease later in life. Additional study is required to correctly estimate the long-term consequences of our findings.

Elevated plasma histone H4 level predicts increased risk of mortality in patients with sepsis.

Histones play a vital role in the pathogenesis of sepsis. However, studies on histones and the prognosis of sepsis patients are scarce. This study aims to investigate the relationship between histones and other biomarkers of sepsis. Furthermore, we aim to determine the role histones play in the prognosis of sepsis patients to explore the possibility of using them as a potential biomarker of sepsis. We performed a prospective observational study on 136 patients. One hundred twenty-six of them had sepsis, and 10 were enrolled as healthy controls. Baseline blood samples were collected for plasma histone H4, cardiac troponin I (TnI), N-terminal pro-b-type natriuretic peptide (NT-proBNP), procalcitonin (PCT), and lactate. The site of infection, the use of vasopressor, and assessment scores of sequential organ failure were documented within 24 hours of admission. The duration of ICU stay and mortality was also recorded. The mean plasma histone levels of the patients were significantly higher than the healthy controls (P<0.001). Compared with the 89 survivors, the 37 patients who died had a higher rate of sequential organ failure assessment (SOFA) scores (P=0.002), more frequent use of vasopressors (P=0.033), and higher levels of histone H4 (P<0.001). Binary logistic regression analysis showed that high plasma histone H4 levels were independent risk factors for predicting mortality. The area under the receiver operating characteristic curve (0.731) verified that high plasma histone H4 level significantly predicted mortality. Plasma histone H4 levels positively correlated with the SOFA score, and plasma cardiac TnI. For patients with sepsis in the ICU, an elevated level of plasma histone H4 could be a risk factor associated with an increased mortality rate. Therefore, plasma histone H4 may be a useful biomarker for determining the prognosis of these patients.

Plasma Histone H4 and Its Further Implications in the Setting of Sepsis-related Myocardial Dysfunctıon

Plasma Histone H4 and Its Further Implications in the Setting of Sepsis-related Myocardial Dysfunctıon

Elevated plasma histone is an independent risk factor for the development of septic cardiomyopathy

To investigate the epidemiology and independent risk factors of septic cardiomyopathy. A prospective study was conducted. Patients with sepsis in intensive care unit (ICU) of Subei People's Hospital of Jiangsu Province, Yangzhou University, Fuxing Hospital, Capital Medical University and Beijing Electric Power Hospital from May 2016 to August 2019 were enrolled. All patients received standardized treatments according to the Surviving Sepsis Campaign (SSC) guidelines. Blood were collected within 24 hours of admission to ICU, and plasma histone H4, cardiac troponin I (cTnI) and N-terminal pro-brain natriuretic peptide (NT-proBNP) were detected by enzyme linked immunosorbent assay (ELISA). Transthoracic echocardiography was performed to record the ultrasonic parameters within 24 hours after admission. Sequential organ failure assessment (SOFA) score, usage of vasopressor drugs, and the prognosis of ICU were recorded. Patients were divided into two groups according to whether cardiomyopathy occurred or not, and the differences of each index between the two groups were compared. The correlation between plasma histone H4 and SOFA score, cTnI, NT-proBNP were investigated. Multivariate binary Logistic regression was used to determine the risk factors for septic cardiomyopathy. The predictive value of histone H4 in septic cardiomyopathy was shown by the receiver operating characteristic (ROC) curve. 121 patients were included in this study, and there were 60 patients (49.6%) with septic cardiomyopathy. Thirty-six patients died, with an ICU mortality of 29.8%. (1) Correlation analysis showed that plasma histone H4 in patients with septic cardiomyopathy was positively correlated with cTnI, SOFA score and NT-proBNP (r value was 0.512, 0.403 and 0.274, respectively, all P < 0.01). (2) Compared with the non-cardiomyopathy group, the plasma histone H4, cTnI, usage of vasopressor drugs, SOFA score and ICU mortality in the cardiomyopathy group were significantly increased [histone H4 (mg/L): 0.26 (0.23, 0.30) vs. 0.22 (0.17, 0.27), cTnI (μg/L): 0.21 (0.17, 0.30) vs. 0.18 (0.14, 0.22), usage of vasopressor drugs: 83.3% (50/60) vs. 65.6% (40/61), SOFA score: 11 (9, 12) vs. 9 (8, 10), ICU mortality: 40.0% (24/60) vs. 19.7% (12/61), all P < 0.05]. Multivariate binary Logistic regression analysis showed that high histone H4 level [odds ratio (OR) = 6.502, 95% confidence interval (95%CI) was 1.203-78.231, P = 0.044] and usage of vasopressor drugs (OR = 2.622, 95%CI was 1.034-6.849, P = 0.042) were independent risk factors for septic cardiomyopathy. (4) ROC curve analysis showed the cut-off of histones H4 for predicting septic cardiomyopathy was 0.24 mg/L, the area under the curve was 0.684 (P < 0.01), with the sensitivity of 65.2%, and specificity of 68.9%. Septic cardiomyopathy had a high incidence. Higher plasma histone H4 and the usage of vasopressor drugs were independent risk factors for septic cardiomyopathy.

Extracellular histones aggravate acute respiratory distress syndrome by inducing peripheral blood mononuclear cells pyroptosis

To explore whether extracellular histones aggravate acute respiratory distress syndrome (ARDS) by inducing peripheral blood mononuclear cell (PBMC) pyroptosis. Twenty patients with ARDS admitted to Shanghai Pulmonary Hospital, Tongji University School of Medicine from April to September in 2019 were enrolled, and 20 healthy volunteers were enrolled as controls. In vivo experiment: peripheral blood samples of patients with ARDS within 24 hours after diagnosis and healthy volunteers were collected, and the levels of plasma extracellular histone, interleukins (IL-1β and IL-18) and lactic dehydrogenase (LDH) were determined by enzyme-linked immunosorbent assay (ELISA). PBMC were harvested, the expression levels of the pyroptosis associated N terminal-gasdermin-D (GSDMD-N) protein were determined by Western Blot. In vitro experiment: PBMC isolated from healthy volunteers were divided into four groups. Blank control group without any treatment; lipopolysaccharide (LPS) group was treated with 1 mg/L LPS for 4 hours; LPS+histones group was treated with 100 mg/L exogenous histones for 24 hours after LPS treatment; LPS+histone+heparin group was treated with 200 U heparin for 24 hours after LPS and exogenous histones treatment. The GSDMD-N protein expression was determined by Western Blot, and the levels of IL-1β, IL-18 and LDH in cell supernatant were determined by ELISA. Spearman test was used to test the correlation among the parameters. In vivo experiment results: compared with healthy control group, the GSDMD-N protein expression in PBMC of patients with ARDS was significantly increased [GSDMD-N/GAPDH: 0.136 (0.062, 0.246) vs. 0.026 (0.018, 0.036), P < 0.01], as well as the plasma levels of IL-1β, IL-18, LDH and extracellular histones [IL-1β (ng/L): 120.0 (94.2, 213.0) vs. 88.5 (82.3, 105.3), IL-18 (ng/L): 164.5 (70.8, 236.3) vs. 60.5 (52.0, 89.0), LDH (U/L): 30.9 (24.7, 39.5) vs. 19.8 (17.2, 21.5), extracellular histones (mg/L): 73.0 (42.8, 112.9) vs. 12.2 (9.6, 16.9), all P < 0.01], indicating that the PBMC of ARDS patients had significant pyroptosis and release of a large number of inflammatory factors. The oxygenation index (PaO2/FiO2) of ARDS patients was 135.5 (94.5, 196.0) mmHg (1 mmHg = 0.133 kPa). Correlation analysis showed that the expression of GSDMD-N protein in patients with ARDS was negatively correlated with PaO2/FiO2 (r = -0.935, P < 0.01) and positively correlated with IL-1β, IL-18, LDH and extracellular histones (r value was 0.844, 0.843, 0.887, 0.899, respectively, all P < 0.01). In vitro experiment results: compared with blank control group, the expression of GSDMD-N protein in PBMC and the levels of inflammatory mediators in the supernatant of the LPS group were significantly increased [GSDMD-N/GAPDH: 0.035±0.006 vs. 0.028±0.006, IL-1β (ng/L): 39.8±5.5 vs. 22.6±4.7, IL-18 (ng/L): 31.2±4.4 vs. 20.0±2.2, LDH (U/L): 51.2±7.3 vs. 36.6±7.6, all P < 0.05], indicating that LPS stimulation could increase PBMC pyroptosis and the release of inflammatory mediators. Compared with LPS group, the expression of GSDMD-N protein and the levels of inflammatory mediators of the LPS+histones group were further increased [GSDMD-N/GAPDH: 0.114±0.009 vs. 0.035±0.006, IL-1β (ng/L): 119.0±18.7 vs. 39.8±5.5, IL-18 (ng/L): 49.2±8.5 vs. 31.2±4.4, LDH (U/L): 127.8±19.8 vs. 51.2±7.3, all P < 0.01], indicating that the stimulation of LPS on PBMC could be significantly amplified by exogenous histone treatment, GSDMD-N protein expression could be up-regulated and inflammatory factor release could be promoted to further induce PBMC pyroptosis. These adverse effects of exogenous histones on PBMC could be abrogated by heparin, the expression of GSDMD-N protein and the levels of inflammatory mediators were significantly lower than those of LPS+histones group [GSDMD-N/GAPDH: 0.063±0.004 vs. 0.114±0.009, IL-1β (ng/L): 46.8±8.6 vs. 119.0±18.7, IL-18 (ng/L): 33.0±5.1 vs. 49.2±8.5, LDH (U/L): 65.4±11.0 vs. 127.8±19.8, all P < 0.05]. Extracellular histones in plasma may aggravate ARDS by mediating PBMC pyroptosis.