The Role of Histones and Heparin in Sepsis: A Review

Molecular Characteristics of eHistones

All members of the histone family commonly localize in the nucleus of eukaryotic cells and are divided into core histones (H2A, H2B, H3, H4) and linker histones (H1, H5). Based upon amino acid analysis, H1, H2A, and H2B are Lys-rich macromolecules, while H3 and H4 are Arg-rich. Thus, histones have a high proportion of amino acids with alkaline side chains at physiological pH. This cationic property of all histone molecules is the component of the nucleosome that functions as the scaffold for the formation of highly ordered and well-organized chromatin structures. ¹⁵ As the structural organizers, histones play an indispensable role inside the nucleus; however, some histone family members, especially H3 and H4, have also been identified to possess pro-inflammatory functions upon being released from the nucleus into the extracellular environment. ¹⁶

eHistones are derived from dying cells. In the infectious situation, eHistones, on one hand, can function as pro-thrombotic microbicidal proteins, which locally limit the spread of infection and isolate the areas for immune cell infiltration to overturn the infection and initiate tissue regeneration and repair. On the other hand, circulating eHistones can associate with other components from the cell, such as double stranded (ds)DNA or high motility group box 1 (HMGB1), to form damage-associated molecular pattern molecules (DAMPs) that initiate and perpetuate systematic cytotoxic injuries through non-infectious inflammatory reactions. ^{17 –20} In addition, the release of eHistones contributes to the formation of neutrophil extracellular traps (NETs), which results from an expulsion of various cytosolic or chromatin-related components that possess the systematic pro-inflammatory effect of NETs. ²¹ In healthy persons, serum levels of eHistones range from 0.79-2.30 mg/L, but in persons with sepsis, inflammation, severe trauma, and other pathological processes, eHistone levels can reach up to 230 mg/L. ^17,22

The molecular functions of eHistones depend on Toll-like receptors (TLRs), which localize to the cellular membrane or to the membranes of the endoplasmic reticulum, endosome, lysosome, or endolysosome. TLRs are classified into TLR2, TLR4, and TLR9, and one of the confirmed eHistones-mediated TLR activation pathways was proposed to activate TLR2 and TLR4, ^{23 –25} which further signals MyD88/NF-kappaB-dependent transcription of various pro-inflammatory cytokines that amplify and intensify cellular injuries. ²⁶ Endogenous eHistones can activate the NOD-like receptor family through TLR9, subsequently causing the release of reactive oxygen species (ROS) to activate the pyrin domain containing 3 (NLRP3) inflammasome, producing more cytokines and recruiting more immune cells during sterile inflammation. ²⁷ eHistones can also activate complements that trigger the production of more immune cells. ²⁸ Furthermore, eHistones can insert into the phospholipid bilayer of cell membranes through affinity-interaction with phospholipid–phosphodiester bonds to alter the permeability of cell membranes, ²⁹ resulting in an influx of calcium ions and cell death. Taken together, eHistones either can enhance immune reactions through recruiting more immune cells, induce pathway-mediated cytokine production and release, or exert direct cytotoxicity to pro-inflammatory cells.

Role of eHistones in the Progression of Sepsis

Sepsis progression from the second stage to the third stage occurs when various pro-inflammatory factors or cytokines are catapulted into the bloodstream to battle infectious microorganisms. This process can result in devastating inflammatory reactions throughout the body, which can further cause a cascade of changes culminating in multiple organ failure (MOF), and sometimes even death. ³⁰ Numerous studies have investigated the pathogenicity of sepsis and sepsis shock by studying the host response, innate immunity, and coagulative abnormalities to better understand the imbalance between pro-inflammation and anti-inflammation due to the cytokine storm associated with sepsis. ^31,32 Many signaling pathways have been hypothesized in the pathogenesis of sepsis, but recent research by Xu et al and Ekaney et al, have demonstrated that eHistones are a major player in the progression of sepsis that initiate MOF through cytotoxicity, excessive inflammation, and coagulation dysfunction (Figure 1).

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Figure 1.

The proposed molecular mechanisms of eHistones in celluar injury.

Cytokine storm has been identified as the critical cellular event associated with a wide variety of diseases; however, the pathogenesis and biological consequences of cytokine overproduction are still lacking fundamental understanding. Studies found that sepsis is worsened by an uncontrolled surge of pro-inflammatory cytokines and chemokines produced by the immune system in response to infection or injury. This cytokine “tornado” creates a vicious cycle that involves the recruitment of more immune cells, which, instead of battling the initial infection, overwhelmingly produce various pro-inflammatory factors that furiously attack the body’s tissues and organs, causing organ failure and death.

In groundbreaking studies, Xu et al, found that eHistones not only possesses lethal properties, but they also promote neutrophil margination, endothelial vacuolation, alveolar hemorrhage, and vascular thrombosis in animal models. ³³ Ekaney and Xu confirmed that eHistone levels are significantly increased in septic patients and might mediate the deterioration and aggravation of sepsis-induced MOF by promoting cellular injury and inflammation via TLR4 signaling. ³⁴ Subsequent studies further demonstrated that eHistones can cause a cytokine storm by significantly up-regulating production of sepsis-associated cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and IL-1β. Moreover, Westman et al ³⁵ showed that eHistones specifically target monocytes in human blood to evoke mobilization of the chemokines CXCL9 and CXCL10 from these cells, triggering the overflow of immune cells to the site of infection (Figure 1).

Role of eHistone in the Pathogenesis of MOF

MOF is considered the most devastating consequence of severe sepsis. Studies have shown that there is a common pathway for the development of multiple system organ failure. This pathway can be initiated by diffuse endothelial injuries in the microvasculature as a result of pro-inflammatory cytokines, leukocytes, and other proteins, and continue with the formation of microvascular thrombosis, subsequently causing irreversible hypoxic injuries in multiple organs including the brain, heart, intestines, kidney, and liver. ³⁶ However, the pathogenesis of MOF is still not fully understood. The most recent observational study found that circulating histone H3 levels may initiate MOF in septic patients, and high concentrations of eHistone H3 can directly usher various organs into failure. ⁷ Many studies already addressed the possible roles of eHistones in organ damage (Figure 2). ³⁷

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Figure 2.

The putative eHistone-regulated pathogenesis of MOF.

Hypoxic-ischemic injury (HII) from sepsis-induced shock can irreversibly cause organ damage. The first identified failed organ was the lung. Sepsis-induced acute lung injury (ALI) mainly results in inefficient gas exchange, increased permeability to proteins, severe inflammation, and permanent pulmonary dysfunction due to alveoli and pulmonary endothelial cell injury. ^22,38 Sepsis-induced ALI is associated with an in-hospital mortality rate of approximately 50%. ³⁹ In vitro studies showed that administration of pure histones killed alveolar cells, and further experiments in animal models of lung injury demonstrated that NET formation in the extracellular space caused significant pulmonary edema and vascular permeability. However, pre-incubation with anti-histone antibodies significantly attenuated the NET-dependent lung cell injury, thereby suggesting that eHistones play an indispensable role in inflammatory lung damage.

The kidney is prone to ischemic injury and is the second most failed organ in septic shock. Acute kidney injury (AKI) from septic ischemia contributes to significant morbidity and mortality among septic patients. At the cellular level, repeated sepsis-induced ischemia to renal tissues triggers acute cell death that is perpetuated by the storm of cytokines and neutrophils, permanently causing renal parenchymal dysfunction. At the molecular level, one study found that, in response to acute renal ischemia, histones were released from the necrotic tubular epithelial cells into the extracellular space, resulting in dose-dependent toxicity to the vascular endothelium of renal vessels and the epithelium of renal tubules, consequently increasing vascular permeability and trans-endothelial migration of neutrophils into the renal parenchyma. ⁴⁰

The third organ that commonly undergoes failure during septic shock is the liver.

Sepsis-induced hepatic ischemia can result in acute liver failure (ALF) with the presentation of abnormal liver enzyme levels, coagulopathy, and hepatic edema due to the disturbance of the synthetic function of the liver. ⁴¹ Several studies demonstrated that eHistones associated with hepatic inflammation activate the TLR-mediated signaling pathway of Kupffer cells to generate a cytokine storm. Furthermore, in a study using an ALF mouse model, extracellular H3 release into the hepatic interstitial space was found to cause significant hepatic damage, which could be attenuated with an anti-H3 antibody, suggesting that blocking eHistone H3 can significantly reduce the risk of mortality from ALI and lower the serum levels of TNF-α and IL-6. NETosis can also be stimulated by eHistone-dependent activation of TLR4 and TLR9 on neutrophils, consequently releasing NETs to mediate repeated hepatic cytotoxicity. ²³ One study showed that co-injecting the NETosis inhibitors PAD4i or DNase1 with eHistones abolished the surge of pro-inflammatory mediators in an animal model, indicating that inhibiting the histone-mediated release of NETs confers hepatic protection.

The heart is also prone to dysfunction or failure from severe sepsis. Specifically, sepsis causes necrotic cardiac damage and the release of immunogenic intracellular components due to repeated ischemic-reperfusion injury in the cardiomyocytes. Studies have shown that extracellular histones released from necrotic cardiomyocytes immediately after ischemic myocardial injury induce further eHistone-dependent cardiomyocyte toxicity. ⁴² In addition, NETosis-derived products can activate the inflammatory signaling pathway after cardiac ischemia. Studies have shown that inhibition of toxic histone molecules can reduce neutrophil myocardial infiltration, pause further myocardial toxicity, improve ventricular remodeling, increase local cardiomyocyte survival, and enhance cardiac function. ⁴³

Brain ischemia from septic shock holds devastating consequences that frequently result in death or profound long-term neurologic disability. One study found that eHistones increase cerebral infarct size. This detrimental effect of eHistones may result from their direct toxicity to endothelial cells at the blood-brain-barrier, thereby increasing permeability, promoting leukocyte migration, and enhancing immune stimulation. In addition, eHistones were found to directly damage glial cells in a dose-dependent fashion. One recent study showed that eHistone H1 possesses a neuro-immunomodulatory property that transfigures astrocytes to an activated stellate morphology, which signals increased reactivity of the astrocytes for releasing more pro-inflammatory cytokines. These novel findings highlight the functional role of eHistones in the pathogenesis of brain injury from severe sepsis.

Taken together, the literature indicates that eHistones are toxic to host cells and elicit immune-stimulatory effects that can induce multiple organ injuries. Circulating histones have been shown to exacerbate organ injuries in various mouse models, indicating the role of eHistones in the development of MOF in the deterioration of sepsis. eHistones can induce microvascular endothelial injury, and TLR2/4-mediated inflammation leads to acute tubular necrosis in experimental AKI. eHistones can also injure endothelial cells resulting in microvascular thrombosis and hemorrhage in experimental ALI and contribute to experimental acute brain injury. Histone H4 and increased circulating NETs can activate platelets, which may cause microvascular thrombosis in sepsis. In summary, eHistones kill endothelial cells and are one of the major mediators of death in sepsis.

Molecular Characteristics and Functions of Heparin

Unfractionated heparin (UFH) is a non-synthetic mucopolysaccharide with natural anti-coagulating capacity. Jay McLean and William Henry Howell serendipitously isolated this macromolecule while conducting pro-coagulant research in 1916. UFH has been extensively applied to treat various thromboembolism diseases (VTE) since it was first introduced into clinical practice in the late 1930’s. Pharmacologically, UFH interferes with normal coagulative pathways through binding to the lysines on anti-thrombin III(ATIII), consequently inhibiting the pro-coagulative activities of thrombin and factor Xa. Studies showed that UFH binds and irreversibly changes the conformation of ATIII, freeing the arginine-reactive site on ATIII to interact with thrombin and form a ternary complex that can inhibit the pro-coagulative potential of thrombin by ∼1,000 times. Once UFH binds and accelerates the activity of one ATIII, it can dissociate from the ternary complex and interact with other AT molecules, thereby exerting an amplifying anti-coagulant effect. More interestingly, studies have shown that the release of thrombin from the coagulative cascade requires a heparin molecule consisting of 18 saccharide units in the mucopolysaccharide chain. ⁴⁴ Additionally, UFH binds to cofactor II to further attenuate the activity and availability of thrombin.

Studies suggest that only 30% of UFH polysaccharides possess the putative ATIII-activating sequence, which accounts for most of its anti-coagulative effect. Several human trials have demonstrated that heparin can exert other functions beyond just anti-coagulation, including, but not limited to, anti-inflammation, anti-complemental activation, and modulation of various proteases. ^{45 –47} UFH was shown to attenuate inflammation and prevent septic death in several animal models, ^{48 –50} and heparin has been shown to modulate immune-stimulation, platelet activation, leukocyte recruitment, lipopolysaccharide (LPS)-stimulated release of cytokines, expression of adhesion molecules, and angiogenesis. ⁵¹ Furthermore, increasing evidence suggests that, in endotoxaemic models, heparin can change the course of pulmonary hypertension by lessening neutrophil migration into the interstitial space of the lung, restricting edema, and improving hypoxemia. Heparin can also counteract chemotactic functions of neutrophils, disrupt eosinophil migration, and decrease vascular permeability.

Heparin as a Therapeutic Agent for Sepsis

Enthusiasm for researching the therapeutic benefits of heparin in the treatment of sepsis was boosted by the finding from the placebo arms of the KyberSept phase 3 AT trial, which indicated that the use of low dose prophylactic heparin might decrease mortality for patients suffering from sepsis; although the data were statistically insignificant. ⁵² At the molecular level, the very first step for the development of sepsis is neutrophil attachment to the vascular endothelium preceding their migration from the circulation into the inflamed local tissues. Growing evidence has demonstrated that heparin can interrupt neutrophil adhesion to the endothelium in sepsis. In addition, some studies found that treatment with UFH can reduce LPS-induced inflammatory responses, suggesting that the underlying mechanism for this attenuation was heparin’s ability to inhibit inflammatory mediator production. ^13,53,54 Furthermore, non-anti-coagulant heparin was shown to block circulating histones and reduce cytokine-induced inflammation in a septic animal model. ¹⁴ Therefore, heparin should be further investigated for its potential to treat sepsis, especially severe sepsis. ⁵⁵

Evidence on the Therapeutic Role of Heparin in Sepsis

The PROWESS study was the very first evidence of the life-saving potential of heparin for patients with severe sepsis, but it should be carefully referenced due to the conclusion of the study being based only on a sub-group analysis. ^{56 –58} The OPTIMIST study further confirmed that treatment with UFH alone improved the survival of patients with sepsis. However, the findings in this study should be considered with caution due to selection bias. Since randomization of patients was done after heparin was administered, heparin was used in both mildly and severely septic patients. ⁵⁹ The HETRASE trial was a prospective randomized double blind study of 319 septic patients who were randomized to receive either intravenous heparin (500 units/hour for 7 days) or placebo. The study could not establish a significant difference in the duration of hospital stay between groups and did not find that heparin significantly improved organ failure score or survival, although heparin treatment was shown to reduce the risk of bleeding. This trial suffered from selection bias due to the heterogeneity of patients with severe septic shock and the low dosage of heparin. ⁶⁰

Zarychanski et al, conducted a retrospective study of 695 patients with septic shock and found that intravenous administration of heparin in the early stage of shock was associated with a significant decrease in 28-day mortality, thereby indicating that heparin used in the early phase of the disease could be an effective treatment for patients suffering from severe sepsis. The results were further upheld by a post hoc study from a Japanese group who investigated heparin as a treatment of severe sepsis. In this multicenter retrospective cohort study, 2,663 septic patients from 42 intensive care units were stratified into different subgroups according to the Acute Physiology and Chronic Health Evaluation (APACHE) II score, systemic inflammatory response syndrome (SIRS) score, Japanese Association for Acute Medicine (JAAM) DIC score, and Sequential Organ Failure Assessment (SOFA) scores. They found that anti-coagulant treatment decreased sepsis-related mortality in subgroups of patients who were classified as having severe sepsis with sepsis-induced coagulopathy. ⁶¹ The finding from this retrospective study partly reflects the weakness in the experimental design of the HETRASE study, which did not show the benefit of heparin for treating septic patients.

Two meta-analyses that studied the therapeutic role of heparin in the treatment of severe sepsis should be thoroughly discussed here. The results from these studies suggested that treatment with low doses of heparin significantly lowered the 28-day in-hospital mortality of septic patients. The meta-analysis by Wang et al, reported a 40% reduction in septic-associated mortality after prophylactic injection of heparin. In the study with a pooled population of 1,325 patients, Zarychanski et al, concluded that the patients who received intravenous heparin had a 12% decrease in relative risk for sepsis-associated mortality, and there was a trend in survival improvement in these patients. However, this meta-analysis was under-designed, as patients were not randomized into groups. Despite the remarkable outcomes from these pooled data meta-analyses, both studies suffer from some limitations. ^62,63 In the study by Wang et al, non-randomized controlled trials accounted for most of sepsis-associated mortality and bleeding complications, while the study by Zarychanski et al, recruited various trials between 1983 and 2014 and included different definitions of sepsis, which may have influenced the results. Furthermore, 85% of the patients who underwent septic shock presented with peripheral hypoperfusion, which made the subcutaneous injection of heparin less preferable or suitable; the poor absorption via this route may have attenuated the efficacy of heparin. To have better clinical outcomes in the management of septic shock, clinicians should know the optimal dose, best route of drug delivery, and proper timing of drug administration. Neither of these 2 meta-analyses clarified these practical issues. ⁶⁴

Despite the conflicting results across various studies in the literature, the anti-septic value of heparin has been gaining more attention as a possible breakthrough in the management of severe sepsis and septic shock. However, complications, such as hemorrhaging, can occur with the clinical use of anti-coagulants, and therefore the use of heparin should be used with caution. For example, heparin-induced thrombocytopenia (HIT) is a serious complication that can further aggravate the underlying pathology and worsen outcomes in septic patients.

Taken together, the therapeutic characteristics of heparin have drawn attention in the field of critical care medicine. The findings from the current clinical trials and basic science research provide significant insight into the beneficial role of heparin for treating sepsis. With the current knowledge, there is rationale to advocate for a well-designed randomized controlled trial (RCT) to investigate the treatment benefit and safety of heparin, as well as other anti-coagulants, at different doses and routes of administration to confirm the therapeutic function of heparin. Moreover, further investigation of the pharmacological mechanism underlying heparin’s beneficial effects in treating sepsis or septic shock with MOF should improve sepsis management.

Interaction Between Heparin and Histones

eHistones have gained attention as the major initiator of cell injury in sepsis. A recent study demonstrated that non-anti-coagulant heparin can interact with eHistones to prevent histone-mediated cytotoxicity in vitro and reduce mortality from sepsis in mouse models without increasing the risk of bleeding, suggesting that administration of non-anti-coagulant heparin is a novel approach to treat patients suffering from sepsis. ^55,65 Additionally, heparin has been shown experimentally to bind to histones and exhibit a protective effect in inflammatory conditions in an in vitro study, which found that selectively de-sulfated heparin reduced histone induced inflammatory markers, such as IL-6, IL-8, tissue factor, and C3a. On the one hand, the selectively de-sulfated heparins possessed reduced anti-coagulant activities, and on the other hand, they retained a high degree of effectiveness as an anti-histone agent. These data suggest that specific structural features are required for heparin to attenuate the inflammatory action of histones. Moreover, co-injection of a low dose of heparin and a lethal dose of histones protected against organ damage and death by antagonizing circulating histones in a mouse model. Similar effects have also been observed in other septic models. Collectively, these findings indicate that a low dose of non-coagulant heparin with a specific structure might be an effective therapy to stop sepsis progression and reduce mortality.

Wildhagen et al, discovered that heparin has a strong affinity for eHistones that results from cellular destruction during severe inflammation. They further showed, using a septic mouse model, that truncating heparin’s anti-coagulative activity can block eHistone-mediated toxicity. Furthermore, Iba et al, reported that either unfractionated heparin or low molecular weight heparin can suppress the toxicity of histone H3 in vivo and in vitro, and that heparin at high concentrations prevents eHistones from binding to platelets, suggesting that histones can be established as a potential therapeutic target for heparin to attenuate the progression of sepsis (Figure 3).

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Figure 3.

The in vitro molecular effects of heparin binding eHistones.

In conclusion, sepsis is a serious disease that is common in intensive care unit patients and is associated with extremely high mortality rates. Therefore, understanding the pathogenesis and searching for an effective treatment of sepsis is critical. Histones and heparin have become a focal point of research in the field of sepsis treatment. Histones normally stay in the nucleus, but once translocated into the extracellular space, they can trigger various signaling pathways to cause overwhelming cytotoxicity in various cells and tissues. Interestingly, an increasing number of studies has suggested that heparin could be used to treat patients with sepsis, septic shock, and disseminated intravascular coagulation associated with infection. Several studies have established the hypothesis that heparin can bind histones and prevent histone-mediated cytotoxicity, subsequently reducing mortality from sterile inflammation and sepsis without increasing the risk of bleeding. However, many questions remain regarding the counter-effects of heparin on eHistones, including which part of the UFH is the functional domain responsible for the adverse mechanisms of eHistones, what is the molecular interaction and mechanism between the two, and how can the potential complications related to heparin be balanced in neutralizing eHistones? Thus, the underlying relationship between eHistones and heparin requires rigorous investigation to determine if heparin can be used to target eHistones in the treatment of severe sepsis.