Circulating Histones Are Major Mediators of Multiple Organ Dysfunction Syndrome in Acute Critical Illnesses.

OBJECTIVES:Multiple organ dysfunction syndrome is characterized by simultaneous multiple organ failure, which is the leading cause of death in acute critically ill patients. However, what mediates multiple organ dysfunction syndrome is not fully understood. The discovery of toxic effects by extracellular histones on different individual organs strongly suggests their involvement in multiple organ dysfunction syndrome. In this study, we investigate whether circulating histones are major mediators of multiple organ dysfunction syndrome in acute critical illnesses. DESIGN:Combination of retrospective clinical studies and animal models with intervention. SETTING:ICU in a tertiary hospital and research laboratories. PATIENTS:Four hundred and twenty ICU patients, including sepsis (140), severe trauma (63), severe pancreatitis (89), and other admission diagnoses (128). LABORATORY INVESTIGATION:Cells from major organs are treated with calf thymus histones or histone-containing sera. Animal models for sepsis, trauma, and acute pancreatitis are treated with antihistone reagents. INTERVENTION:Antihistone reagents in in vitro, ex vivo, and animal models. MEASUREMENT AND MAIN RESULTS:Retrospective analysis of a prospectively recruited ICU cohort demonstrated a strong correlation between circulating histones and organ injury markers and Sequential Organ Failure Assessment scores. Ex vivo experiments showed that patient sera containing high histone levels were toxic to cultured cells from different origins, suggesting their universal toxicity to multiple organs. Animal models of sepsis, trauma, and pancreatitis further demonstrated a temporal correlation between histone levels and disease severity and multiple organ injury. Importantly, antihistone reagents, that is, antihistone single-chain variable fragment and nonanticoagulant heparin, could dramatically reduce multiple organ injury, particularly of the heart and lungs, and improve survival in mouse models. CONCLUSIONS:High levels of circulating histones are major mediators of multiple organ dysfunction syndrome. Our results indicate that monitoring upon ICU admission could inform on disease severity and developing antihistone therapy holds great potential of reducing multiple organ dysfunction syndrome and improving survival of critically ill patients

A Novel Assay for Neutrophil Extracellular Trap Formation Independently Predicts Disseminated Intravascular Coagulation and Mortality in Critically Ill Patients

Rationale: Neutrophil extracellular traps (NETs) are important in the host defense against infection, but they also promote intravascular coagulation and multiorgan failure in animal models. Their clinical significance remains unclear, and available assays for patient care lack specificity and reliability. Objectives: To establish a novel assay and test its clinical significance. Methods: A prospective cohort of 341 consecutive adult ICU patients was recruited. The NET-forming capacity of ICU admission blood samples was semiquantified by directly incubating patient plasma with isolated neutrophils ex vivo. The association of NET-forming capacity with Sequential Organ Failure Assessment scores, disseminated intravascular coagulation, and 28-day mortality was analyzed and compared with available NET assays. Measurements and Main Results: Using the novel assay, we could stratify ICU patients into four groups with absent (22.0%), mild (49.9%), moderate (14.4%), and strong (13.8%) NET formation, respectively. Strong NET formation was predominantly found in sepsis (P < 0.0001). Adjusted by Acute Physiology and Chronic Health Evaluation II score, multivariate regression showed that the degree of NET formation could independently predict disseminated intravascular coagulation and mortality, whereas other NET assays (e.g., cell-free DNA, myeloperoxidase, and myeloperoxidase–DNA complexes) could not. IL-8 concentrations were found to be strongly associated with NET formation, and inhibiting IL-8 significantly attenuated NETosis. Mitogen-activated protein kinase activation by IL-8 has been identified as a major pathway of NET formation in patients. Conclusions: This assay directly measures the NET-forming capacity in patient plasma. This could guide clinical management and enable identification of NET-inducing factors in individual patients for targeted treatment and personalized ICU medicine

Amyloid-Fibrinogen Aggregates ("Microclots") Predict Risks of Disseminated Intravascular Coagulation and Mortality.

Microclots have been associated with various conditions, including post-acute sequelae of SARS-CoV-2 infection. They have been postulated to be amyloid-fibrin(ogen) aggregates, but their role as a prognostic biomarker remains unclear. To examine for their possible clinical utility, blood samples were collected for the first 96 hours from critically ill patients (n=104) admitted to the intensive care unit (ICU). Detection was by staining platelet-poor plasma samples with Thioflavin T and visualized by fluorescent microscopy. Image J software was trained to identify and quantify microclots, which were detected in 44 [42.3%] patients on ICU admission but not in the remaining 60 [57.7%] or in 20 healthy controls [0.0%]. Microclots on admission to ICU were associated with a primary diagnosis of sepsis (microclots present in sepsis=23/44 [52.3%] vs microclots absent in sepsis=19/60 [31.7%], P=0.044). Multicolour immunofluorescence demonstrated that microclots consisted of amyloid-fibrinogen aggregates, which was supported by proteomic analysis. Patients with either a high number or larger-sized microclots had a higher likelihood of developing disseminated intravascular coagulation (DIC) (OR=51.4 [95% CI=6.3-6721.1], P<0.001) and had an increased probability of 28-day mortality (OR=5.3 [95% CI=2.0-15.6], P<0.001). This study concludes that microclots, as defined by amyloid-fibrin(ogen) aggregates, are potentially useful in identifying sepsis and predicting adverse coagulopathic and clinical outcomes

Parasite histones are toxic to brain endothelium and link blood barrier breakdown and thrombosis in cerebral malaria

Microvascular thrombosis and blood–brain barrier (BBB) breakdown are key components of cerebral malaria (CM) pathogenesis in African children and are implicated in fatal brain swelling. How Plasmodium falciparum infection causes this endothelial disruption and why this occurs, particularly in the brain, is not fully understood. In this study, we have demonstrated that circulating extracellular histones, equally of host and parasite origin, are significantly elevated in CM patients. Higher histone levels are associated with brain swelling on magnetic resonance imaging. On postmortem brain sections of CM patients, we found that histones are colocalized with P falciparum–infected erythrocytes sequestered inside small blood vessels, suggesting that histones might be expelled locally during parasite schizont rupture. Histone staining on the luminal vascular surface colocalized with thrombosis and leakage, indicating a possible link between endothelial surface accumulation of histones and coagulation activation and BBB breakdown. Supporting this, patient sera or purified P falciparum histones caused disruption of barrier function and were toxic to cultured human brain endothelial cells, which were abrogated with antihistone antibody and nonanticoagulant heparin. Overall, our data support a role for histones of parasite and host origin in thrombosis, BBB breakdown, and brain swelling in CM, processes implicated in the causal pathway to death. Neutralizing histones with agents such as nonanticoagulant heparin warrant exploration to prevent brain swelling in the development or progression of CM and thereby to improve outcomes

Extracellular Histones Inhibit Complement Activation through Interacting with Complement Component 4

Abstract Complement activation leads to membrane attack complex formation, which can lyse not only pathogens but also host cells. Histones can be released from the lysed or damaged cells and serve as a major type of damage-associated molecular pattern, but their effects on the complement system are not clear. In this study, we pulled down two major proteins from human serum using histone-conjugated beads: one was C-reactive protein and the other was C4, as identified by mass spectrometry. In surface plasmon resonance analysis, histone H3 and H4 showed stronger binding to C4 than other histones, with KD around 1 nM. The interaction did not affect C4 cleavage to C4a and C4b. Because histones bind to C4b, a component of C3 and C5 convertases, their activities were significantly inhibited in the presence of histones. Although it is not clear whether the inhibition was achieved through blocking C3 and C5 convertase assembly or just through reducing their activity, the outcome was that both classical and mannose-binding lectin pathways were dramatically inhibited. Using a high concentration of C4 protein, histone-suppressed complement activity could not be fully restored, indicating C4 is not the only target of histones in those pathways. In contrast, the alternative pathway was almost spared, but the overall complement activity activated by zymosan was inhibited by histones. Therefore, we believe that histones inhibiting complement activation is a natural feedback mechanism to prevent the excessive injury of host cells.</jats:p