Exposure to high levels of Cl2 remains a documented public threat. The severity of Cl2-induced injury depends primarily on the concentration and duration of exposure [3]. ARDS is the most severe consequence, and the overwhelming inflammatory response triggered by the initial injury is considered as the ARDS keystone [17, 18].
In addition to the classic pathogen-associated molecular patterns (PAMPs),damage-associated molecular patterns (DAMPs) are another significant pathway of uncontrolled inflammation. Extracellular histones were recognized as DAMP molecules in 2009 [12], and are essential inflammatory mediators in a variety of diseases such as sepsis, trauma, and multiple organ dysfunction syndrome [19, 20].
In this study, it was shown that Cl2 induced concentration-dependent lung injury. The levels of circulating histone H4 were closely correlated with Cl2 concentrations (ranging from 10 to 800 ppm). The concentration of Cl2 was a major determinant for the level of circulating histone H4. The pathogenic role of histone H4 in Cl2-induced ARDS was proven by histone H4 or specific blocking anti-H4 antibody intervention. Pretreatment with histone H4 further worsened the lethality rate and PaO2, while anti-H4 antibody showed some protective effects. Compared with the control mice, both endothelial and neutrophil activation were much more distinct in mice with Cl2-induced ARDS, which was indicated by P-selectin expression, vWF release, pulmonary neutrophil infiltration, and elevated MPO activity. Pretreatment with intravenous histone H4 aggravated endothelial and neutrophil activation, while anti-H4 antibody played an antagonistic role. Thus, it can be deduced that circulating histone H4 was a critical inflammatory mediator in Cl2-induced ARDS.
Additionally, TLRs were closely involved with histone H4-mediated inflammatory injury during Cl2-induced ARDS. The Tlr4-KO mice were resistant to exposure to lethal Cl2 concentrations. Tlr4 deficiency improved lethality rate, PaO2, and pulmonary edema, and prevented the endothelial and neutrophil activation caused by Cl2 exposure. More importantly, Tlr4 gene deletion greatly diminished the effect of histone H4 or anti-H4 antibody observed in WT mice. In contrast to Tlr4 deficiency, the impact of Tlr2 on inflammatory injury was not evident. In addition to TLR4 and TLR2, the roles of TLR1 and TLR6 were also screened. The blocking antibody against TLR4 or TLR2 decreased the histone H4-mediated expression of TNFA and IL1B in human pulmonary vascular endothelial cells, while the blocking antibody against TLR1 or TLR6 showed little effect.
Histones are structural elements of nuclear chromatin that mainly regulate gene expression. When histones are released passively from necrotic cells or actively by cell death such as NETosis, extracellular histones produce toxic effects on adjacent and circulating cells [21, 22]. The predominant source of histones may be neutrophils that have been activated by C5a to form neutrophil extracellular traps (NETs) in ALI [23, 24]. In contrast, nucleosomes, composed of histones and DNA, appear to be less toxic [25, 26]. Extracellular histones are DAMP molecules that can cause systemic inflammation involved in a wide range of inflammatory conditions, such as acute lung injury, liver injury, kidney injury, myocardial injury, cerebral stroke, coagulopathy, systemic lupus erythematosus, and even hair follicle death [27-34]. Extracellular histones can induce the release of chemokines, and activate the vascular endothelium, so as to facilitate leukocyte adhesion and transmigration [35, 36]. Both endothelial and neutrophilic activation are key events during the ARDS inflammatory response [37, 38]. Elevated P-selectin expression is an important marker for endothelial activation. Additionally, P-selectin translocation is a prerequisite for neutrophil adhesion to the pulmonary vascular endothelium during inflammation [39, 40]. Along with P-selectin translocation, abundant vWF is released from the endothelia, which excessively activates the coagulation cascade and aggravates lung injury [41, 42]. In addition to the endothelia and neutrophils, Westman et al. showed that extracellular histones induced monocytes to produce chemokines such as CXCL9 and CXCL10, which triggered neutrophil recruitment [36]. Fuchs et al. showed that extracellular histones bound to platelets, inducing calcium influx,recruiting plasma adhesion proteins such as fibrinogen,and triggering microaggregation [43].
Extracellular histones serve as DAMP molecules to promote inflammation. ARDS is an overwhelming inflammatory response that is triggered by various damaging factors. The initial detrimental stimuli are sensed by pattern recognition receptors (PRRs) [44]. TLRs are responsible for sensing invading pathogens and injury stimuli outside of the cell, as well as in intracellular endosomes and lysosomes. TLR1, TLR2, TLR4, TLR5, and TLR6 are present in the plasma membrane, while TLR3, TLR7, and TLR9 are mainly present in the membrane of the endoplasmic reticulum [45]. Thus, TLR1, TLR2, TLR4, TLR5, and TLR6 may mediate the inflammation caused by extracellular histones. In accordance with the results of this study, both TLR2 and TLR4 were previously found to be involved with extracellular histones-induced liver and kidney injury [39, 46]. In addition to TLR2 and TLR4, other PRRs are also involved in extracellular histones-induced inflammation. Huang et al. demonstrated that Tlr9-KO mice were protected from histone-mediated ischemia/reperfusion-induced liver injury [47]. TLR9 is generally viewed as a receptor mediating signaling brought about by endogenous circulating DNA released from dying cells. The exogenous histones may act as a cofactor of DNA, and thus, they can amplify TLR9-mediated signaling.
Histones are unique cytotoxic DAMP molecules that elicit both PRR-dependent pro-inflammatory signaling and PRR-independent direct cytotoxicity. Rationally, the affinity of histones for phosphodiester bonds may ensure their avid binding to both DNA in the nucleus and phospholipids in the plasma membrane [48, 49]. Abrams et al. demonstrated that FITC-labeled histones directly bind to the surface of cultured EA. hy926 endothelial cells, subsequently inducing an influx of calcium, ultimately resulting in cell lysis [11]. Silvestre-Roig et al. demonstrated that when smooth muscle cells (SMCs) were exposed to histone H4, alterations in the membrane were characterized by dynamic bending and pore formation. The activated SMCs attracted neutrophils and triggered the ejection of NETs. NETs, or histone H4 could induce SMC swelling and release of ATP. Finally, extracellular histone H4 triggered inflammation and arterial tissue damage by mediating SMC membrane lysis [50].
It is well-known that histone-acetylation (open chromatin) promotes gene expression, whereas histone deacetylation (closed chromatin) represses gene expression in living cells [51, 52]. To clarify whether extracellular histones underwent acetylation modification, this study detected histone H4 in plasma samples by western blot using an anti-acetylated histone H4-antibody (K5, K8, K12, K16). As shown in Figure S2, the acetylation status of plasma histone H4 in Cl2 exposure groups (10, 50 ppm) was similar to its status in the control group. An increase in the acetylation level of plasma histone H4 in the Cl2 exposure groups (200, 800 ppm) was not evident compared with the control group. Extracellular histones are released by NETs and apoptotic/necrotic cells. The main role of histones released from NETs is to kill bacteria instead of regulating gene transcription. Thus, acetylation modification was not applicable to them [53]. For the histones released from apoptotic/necrotic cells, they might be acetylated before release and retained the acetylation modification after release. It can be deduced that the pro-inflammatory effect of extracellular histones is mainly concentration dependent.
Because extracellular histones are cytotoxic DAMP molecules, it is rational that the damaging effects can be ameliorated through the application of histone-targeted interventions [54]. Administration of specific blocking antibodies or peptides targeted to these extracellular histones could inhibit inflammatory damage and improve outcomes in several types of animal models, such as sepsis, acute lung injury, liver injury, acute pancreatitis, and multiple organ injury [10, 55-58]. The direct toxicity of extracellular histones is dependent on the electrostatic membrane’s interaction with target cells. Accordingly, histone neutralizing agents have been identified as therapeutic options in treating extracellular histone-mediated tissue injury. Heparin is a highly sulfated negatively charged proteoglycan. Heparin and its derivatives can bind histones through electrostatic interactions, and they have demonstrated the ability to inhibit ALI, vascular endothelial injury, and thrombocytopenia caused by histones released [59, 60]. In addition to heparin and its derivatives, many innate and synthetic substances have been proven to prevent histone-related toxicity, including plasma albumin, C-reactive protein, bacterial O-antigen, polyglutamic acid and polysialic acid [25, 61-64].