Acute respiratory distress syndrome (ARDS), characterized by bilateral chest radiographically opacities and severe hypoxemia (Force and V Marco Ranieri GDRB, 2012), remains a major challenge with its high morbidity and mortality. In an observational study across 50 countries and involving 459 intensive care units (ICU), ARDS was responsible for 10% of ICU admissions and 23% of mechanically ventilated patients; mortality was 35% and could be as high as 46% in severe ARDS (Bellani et al., 2016). Currently, only therapies that have been shown to be effective are lung-protective ventilatory strategies and supportive treatment including prophylactic or therapeutic application of antibiotics and restriction of fluid accumulation (Yanhui et al., 2023). There are no pharmaceutical therapies aimed at the underlying pathology have been shown to be effective (Confalonieri et al., 2017).

Neutrophils constitute the major components of the innate immunity. They are the first to respond against a large range of pathogens, and are then recruited to the inflamed tissue, acting by phagocytosis, release of reactive oxygen species (ROS), degranulation (Segal, 2005) and decondensed chromatin fibers coated with antimicrobial proteins, such as histones and neutrophil elastase, which form neutrophil extracellular traps (NETs) to trap and kill bacteria (Brinkmann et al., 2004). However, NETs may be double-edged swords (Kaplan and Radic, 2012), with excessive formation playing an important role in the pathogenesis of ARDS (Porto and Stein, 2016): NETs expand into the pulmonary alveoli, causing lung injury, and induce epithelial and endothelial cell death (Yago et al., 2018). NETs also provoke the formation of immuno-thrombosis (Yago et al., 2018), which is associated with worse ARDS outcomes. There is therefore a sound rationale for strategies that target histones and decrease NET formation in the treatment of ARDS.

Recent studies showed that extracellular histones significantly increased the development of ARDS. In an observational study that included 96 patients with ARDS and 30 healthy volunteers, extracellular histone levels from plasma and bronchoalveolar fluid (BALF) were highly correlated to the severity of ARDS and were significantly higher in non-survivors (Lv et al., 2017). In 52 patients with non-thoracic trauma, high circulating histone levels were associated with an increased incidence of acute lung injury (ALI) and higher Sequential Organ Failure Assessment (SOFA) scores (Abrams et al., 2013). Interestingly, anti-histone antibody therapy protected mice from histone-induced lethality (Lv et al., 2017). Targeting extracellular histones may thus represent a promising therapeutic option for ARDS (Karki et al., 2020).

A polyanion molecule of β-O-methyl cellobiose sulfate sodium salt (mCBS.Na), STC3141, has been identified as able to neutralize histones and decrease histone-induced red blood cell (RBC) and platelet aggregation in vitro (Meara et al., 2020). Its use was associated with a survival benefit in a rat cecal ligation and puncture model (Meara et al., 2020). This molecule has also been shown to decrease infarct size in a rat cardiac ischemia/reperfusion model (Shah et al., 2021). Beneficial effects in ARDS are therefore anticipated.

Our hypothesis was that STC3141 administration would improve outcomes in a rat model of ARDS. Dexamethasone administration has been shown to increase ventilator-free days and decrease 60-day mortality in patients with ARDS (Villar et al., 2020), so we decided to use dexamethasone as a control for treatment in this study. The reason we chosen this rat model is that it closely reproduces the acute phase of human ARDS (Chimenti et al., 2020).

The main findings of the current study are: 1) that a double-hit of LPS induced a model of ALI in rats as shown by the presence of inflammation, decreased oxygenation, lung edema, and histological changes, representing clinical ARDS findings; 2) neutralization of extracellular histone with low, medium and high dose STC3141 significantly decreasing circulating and BALF WBC and neutrophil counts, improving lung oxygenation and decreasing lung inflammation; 3) high dose STC3141 seems to have a similarly beneficial effect on oxygenation and lung edema formation to dexamethasone administration in this ALI model.

The pathological study of ARDS showed that there were a large number of proteins and inflammatory cells in edema fluid accumulated in alveoli and interstitial lung, among which neutrophils (Grommes and Soehnlein, 2011) were dominant. Activation and recruitment of neutrophils are considered to play a key role in the development of ARDS. Neutrophils are the first cells to be recruited to inflammatory sites. They have a strong antibacterial mechanism included oxidizing agent, protease and cationic peptide. However, under pathological conditions, uncontrolled release of these antibacterial substances outside the cell will destroy the host tissue (Grommes and Soehnlein, 2011). With the deepening of research on neutrophils, more and more researchers have found that neutrophil extracellular traps (NETs) are closely related to the inflammatory injury of ARDS. Excessive neutrophils played a pathogenic role in acute lung injury of influenza pneumonia, and NETs formed by neutrophils could cause alveolar-capillary injury (Narasaraju et al., 2011). Since then, animal experiments and clinical trials have reported that NETs can cause lung tissue damage and promote ARDS. NETs (Lefrançais et al., 2018; Li et al., 2018; Mikacenic et al., 2018) can be detected in alveoli and plasma of ARDS patients. The level of NETs is related to the poor prognosis of patients (Zhu et al., 2018). These results indicate that NETs is involved in the inflammatory reaction in the pathogenesis of ARDS.

Since NETs was first proposed by Brinkmann et al., in 2004 (Brinkmann et al., 2004), the related research has made new progress continuously. At first, NETs was only regarded as a complex function of innate immune system to fight against invading microorganisms. Later, people clearly realized that NETs had a far-reaching role. NETs is involved in a series of pathophysiological mechanisms from inflammation to thrombosis, When the generation or elimination of NETs is not fully controlled, they will have a serious impact on the organism. NETs is composed of filamentous DNA and a large number of protein particles attached to it, among which histone is dominant, accounting for about 70% of NETs-related proteins (Urban et al., 2009), besides neutrophil elastase and myeloperoxidase. When DNA as the backbone of NETs is degraded by DNAse, Histones separated from DNA will exist in free form. More and more studies have shown that histone, which plays a bactericidal role in NETs, is an important factor (Jamison and Grailer, 2014) leading to lung injury in ARDS. Free histones and extracellular histones contained in NETs are highly cytotoxic to alveolar epithelial cells and endothelial cells, which can directly lead to the death of these cells (Saffarzadeh et al., 2012). It can continue to induce neutrophils to produce NETs (Nakazawa et al., 2017; Shrestha et al., 2019), so that a large number of neutrophils will be recruited into the lungs, thus forming the amplification effect of inflammatory waterfall, further aggravating the degree and expanding the scope of lung injury. After these necrotic cells disintegrate, histones in the nucleus will also disperse outside the cells, damaging surrounding cells and tissues, and further expanding the damage (Jamison and Grailer, 2014; Nakazawa et al., 2017). Therefore, studying NETs, especially extracellular histone, as a target may provide an effective method for ARDS treatment.

At present, many studies have tried to treat ARDS by targeting NETs. DNA enzyme can destroy the three-dimensional structure of NETs by degrading DNA in NETs, and it has shown good efficacy in some ARDS animal models (Caudrillier et al., 2012). Activated protease C (APC), which was once listed as a serum protease to degrade extracellular histone, also showed good results in animal experiments. However, in human body, because APC has anticoagulant effect at the same time, when its concentration has not reached the ideal concentration for removing extracellular histone, However, it has shown obvious anticoagulant effect, which leads to the increase of bleeding risk, and it is difficult to balance the efficacy and safety risk, which eventually leads to the withdrawal of APC from the market. In addition, some studies have shown that negatively charged drugs, including heparin and albumin, can combine with extracellular histone through electric charge, thus reducing the toxic effect of histone (Lam et al., 2013; Wildhagen et al., 2014). Treat with histone antibody, It can also effectively neutralize the toxicity of extracellular histone (Xu et al., 2009; Wildhagen et al., 2014; Kusano et al., 2015).

LPS exposure may lead to lung inflammation. An LPS stimulated rat model is a standard experimental ALI and ARDS model and widely used for new drug development for this condition (Martin and Silverman, 1992; Uhlig and Kuebler, 2018; Liang et al., 2020). ALI and ARDS could be induced in rats by intratracheal, intraperitoneal administration of LPS or by an LPS double-hit method. Intraperitoneal injection with LPS can induce indirect ALI while intra-tracheal nebulized LPS is used to induce direct ALI. Recent studies have reported that double-hit model is more stable and reliable, closer to the diagnostic criteria of ARDS in humans and better for mimicking the pathological process of ARDS (Chen et al., 2010). Moreover, intra-tracheal nebulized LPS causes robust neutrophilic alveolitis and is used to evaluate both the early and resolution phases of ALI (Yanhui et al., 2023). In our study, LPS animals exhibited decreased oxygenation, lung inflammation (intra-alveolar leukocytes, histological injury), and higher histone concentrations than sham animals. Meanwhile, targeting extracellular histones using STC3141 significantly decreased WBC and neutrophil counts not only in the circulation, but also in the BALF, which suggests it may interrupt NET formation, thereby ameliorating the impaired microcirculation and improving lung function. However, low and medium dose STC3141 treatment did not decrease circulating histone levels, but high dose STC3141 and dexamethasone did. This dose-dependent effect is reflected by the findings of similar lung dry/wet ratio in the high dose and dexamethasone groups: improved oxygenation and decreased lung inflammation and edema formation. STC3141 administration significantly decreased histone-induced RBC and platelet aggregation in vitro, and histone-induced tissue injury, thrombocytopenia and anemia in mice (Meara et al., 2020). These preclinical findings could help to explain the observations in ARDS patients, with a significant increase in plasma histones in mild, moderate, and severe ARDS and significantly higher plasma histones in non-survivors than in survivors (Lv et al., 2017). Furthermore, in ARDS patients with a good prognosis, plasma histone levels decreased after admission whereas they increased in those with a poor prognosis (Lv et al., 2017). All these findings support the concept of histone neutralization therapy in ARDS.

The study has several limitations. First, LPS is well known to induce inflammation rather than infection, and the model therefore does not fully mimic the disease kinetics in ARDS (Xu et al., 2009). Second, the young healthy animals used in the current study are different from the typical ARDS patient with comorbidities. Third, the shortage of direct evidence immunohistochemistry results of NETs from lung with STC3141 due to limited label technology.

In the current LPS double-hit rat model of ALI, high dose STC3141 showed similar effects to dexamethasone therapy, including significantly decreased circulating histone concentrations, improved oxygenation, and decreased lung edema formation. Clinical trials are necessary to identify the effects of histone neutralization in ARDS patients.