ARDS is a rapid non-cardiogenic bilateral lung infiltration syndrome characterized by alveolar vascular injury, neutrophil infiltration and accompanied by the release of pro-inflammatory factors . Neutrophils and macrophages play an important role in the process of lung injury, in which neutrophils are the first line of defense against the invasion of pathogens . Macrophages play a key role in the subsequent clearance of apoptotic neutrophils and promoting the regression of inflammation. This study focuses on the effect of LXA4 on the function of neutrophils in ARDS, so as to explore the anti-inflammatory mechanism of LXA4.
Neutrophils are the largest number of white blood cells in human circulation, and they play an important role in the process of immune defense against pathogenic microorganisms . As effector cells of the innate immune system, neutrophils participate in a variety of immune and inflammatory processes and play an important role in coordinating the overall immune and inflammatory response. In the last 5 years, there have been many reports on the anti-inflammatory effects of LXA4 [18–19]. As the first kind of regression medium, LXA4 has a strong anti-inflammatory and regression effect . Recently, some studies have shown that LXA4 and synthetic analogues can protect tissues from acute and chronic inflammation. Its mechanism includes down-regulation of proinflammatory cytokines and chemokines (such as IL-1β and TNF- α), inhibition of the activation of major pro-inflammatory pathways, and increase the release of proinflammatory cytokines (such as IL-10) . Therefore, LXA4 is one of the earliest endogenous lipid mediators, which is used to inhibit the aggregation of neutrophils, inhibit inflammation, and promote the resolution of inflammation .
In this study, we have identified a unique role for LXA4 related to the function of neutrophils in LPS-induced inflammatory lung injury. Our data clearly demonstrate that Exogenous administration of LXA4 can reduce the lung injury score of ARDS rats induced by LPS. Our experimental results also confirm that treatment with LXA4 inhibits plasma inflammatory cytokines, such as TNF-α and IL-1β. Decreased plasma TNF-α and IL-1β levels in LXA4-treated rats are consistent with the paradigm that LXA4 reduces the inflammatory response following ARDS, in addition to ameliorating the severity of disease. This suggests the potential of LXA4 as an effective lung protective therapeutic agent.
We used percoll density gradient centrifugation to extract neutrophils from peripheral blood of patients with ARDS. The purity of neutrophils was (97.5 ± 2.3)%, and the cell viability was more than 96% by trypan blue staining.
Neutrophils are over-activated during ARDS, which releases neutrophil respiratory burst and ROS to damage the surrounding tissues. Priming of the neutrophil ROS production is believed to be involved in many inflammatory diseases, such as acute respiratory distress syndrome (ARDS), rheumatoid arthritis (RA), atherosclerosis, ischemia-induced tissue injury, hypertension, diabetes, kidney disease, and sepsis . Our experimental results supported this conclusion. In this study, we evaluated the initiation of respiratory burst of suspended neutrophils in vitro using a superoxide dismutase (SOD)-inhibited cytochrome c reduction test, and luminol-amplified chemiluminescence to measure ROS . The results showed that IL-8, fMLP and PMA could stimulate the production of reactive oxygen species (ROS) in neutrophils, and PMA was the best. The ROS production of neutrophils in ARDS rats without stimulation was higher than that in control group. PMA could not only stimulate the ROS production of neutrophils in control group, but also in the peripheral blood of ARDS rats. However, after PMA stimulation, the ROS production of neutrophils in the peripheral blood of ARDS rats was significantly higher than that in the control group. After LXA4 treatment, the production of ROS in peripheral blood neutrophils of ARDS rats was significantly reduced. The inhibition effect of LXA4 on the production of ROS was significantly weakened by the addition of LXA4 receptor antagonist BOC-2, which indicated that LXA4 could reduce the damage of peripheral blood neutrophils to the surrounding tissues in the process of sterilization by combining with ALX receptor.
NETs is a kind of outer network structure, which is composed of complex DNA triple network structure. It contains major proteins, elastase (NE), myeloperoxidase (MPO), cathepsin G and other antibacterial proteases. This is a special bactericidal mechanism of neutrophils . In the process of inflammation, NETs is a double-edged sword. Studies have shown that a large number of extensive NETs can damage epithelial cells and endothelial cells, leading to the spread of inflammation . However, a large number of studies have shown that in the acute stage of inflammation, NETs can not only capture microorganisms, but also limit the spread of cytotoxic antibacterial proteins, reduce the damage of host tissue, and play an important role in inhibiting the spread of inflammation . In this study, we isolated neutrophils from peripheral blood of ARDS rats and stimulated neutrophils with IL-8, LPS, fMLP and PMA respectively. We found that IL-8, LPS, fMLP and PMA could induce the production of nets in peripheral blood neutrophils of ARDS rats in vitro, but PMA had the best effect. Therefore, we chose PMA as the stimulant of nets in subsequent experiments. The effect of PMA on promoting the production of neutrophil NETs is more obvious in peripheral blood neutrophils of ARDS rats, which indicates that the production of NETs helps neutrophils to play a bactericidal role in the acute phase of inflammation. The results showed that LXA4 could reduce the NETs function in ARDS rats mediated by binding to ALX receptor.
Neutrophil elastase (NE), a serine protease secreted by polymorphonuclear neutrophils (PMNs), plays an important role in many physiological and pathological processes such as the various inflammatory reactions, adult respiratory distress syndrome, and acute lung injury . When exposed to various inflammatory factors, neutrophils release NE via degranulation, which is then involved in the inflammatory response. Some studies believe that NE is a destructive elastase that attacks the extracellular matrix and modulates inଂammation and tissue remodeling. Its involvement may be direct (tissue damage) or indirect (pro-inଂammatory or pro-apoptotic) [26–28]. However, it is undeniable that NE is a necessary condition for neutrophil migration to inflammatory sites, and the proteolytic activity of NE contributes to the body's defence against infectious agents by promoting the destruction of pathogenic bacteria [29–30]. Our results show that LXA4 can reduce NE release induced by fMLP. To analyze the reasons, we consider that the time point selected in this study is 6 hours after the successful establishment of ARDS rats model induced by LPS. This is the peak of inflammation, a large number of neutrophils have already migrated to the site of inflammation and have played an effective bactericidal function. The subsequent period of inflammation subsides. So it is very important to reduce the damage of NE to surrounding tissues. At this time, LXA4 reduces the release of NE, which is beneficial to reduce NE damage to surrounding tissues.
It is confirmed that NE and MPO are involved in the generation of NETs. Neutrophil chromatin densification requires neutrophil elastase (NE), NE deficiency mice unable to form NETs and show immunodeficiency; the formation of NETs also requires myeloperoxidase (MPO) to act on histones in the transitional nucleus, and hypochlorite, a product produced by MPO, is necessary for the release of NETs, so patients without MPO activity can’t produce NETs. We treated neutrophils with NE inhibitor (NEi) and MPO inhibitor(MPO inhibitorⅠ), as shown in Fig. 4B、E, the amount of NE released by LPS group was significantly higher than that of control group after stimulation with fMLP. This indicated that neutrophils increased the release of NE, on the one hand, directly antagonized infection, on the other hand, increased the production of NETs and enhanced bactericidal function. After adding NEi, the release of NE from neutrophils was significantly reduced. And LXA4 also reduces the release of NE from neutrophils and is similar to NE inhibitors(NEi). As shown in Fig. 4C、F, the amount of MPO released by neutrophils in LPS group was significantly higher than that in control group. However, the amount of MPO released by neutrophils was significantly reduced after the addition of MPO inhibitor Ⅰ. The addition of LXA4 could reduce the release of MPO and is similar to MPO inhibitor Ⅰ. These results indicate that after the peak period of inflammation, LXA4 can reduce the production of NETs by inhibiting the release of NE and MPO from neutrophils, thereby reducing the damage to surrounding tissues caused by excessive activated neutrophils, which is conducive to the regression of inflammation.
During the inflammatory injury, neutrophils gather at the injury site and play a phagocytic role, which is an important mechanism to kill pathogenic microorganisms. Our study found that the phagocytic ability of neutrophils to S. aureus(SA) and E. coli(EC) increased with time, reaching the peak at 60 minutes. At 45 minutes and 60 minutes, the phagocytic ability of neutrophils in ARDS rats was higher than that in healthy control group, but there was no significant difference between the two groups at 30 minutes. The ability of phagocytosis of S. aureus (SA) and E. coli (EC) by neutrophils in LXA4 group was significantly higher than that in control group. The effect of BOC-2 on phagocytosis of neutrophils in LXA4 group was significantly reduced, On the other hand, it is confirmed that LXA4 can increase the bactericidal function of peripheral blood neutrophils in ARDS rats by binding with ALX receptor.
As we all know, the life span of neutrophils is very short, mainly because with the passage of time, neutrophils in the circulatory system will undergo apoptosis. The resolution of acute inflammation requires macrophages to phagocytize apoptotic neutrophils. Delayed apoptosis of activated neutrophils can lead to persistent acute pneumonia and eventually develop into ARDS. The results in Fig. 5A-D of this study suggest that: as the survival rate of normal neutrophils in vitro will gradually decrease with the extension of time, the survival rate of neutrophils in vitro will be lower at 24 hours, and LXA4 has a certain protective effect on normal neutrophils in healthy rats, which can appropriately prolong the life of neutrophils. However, most of the neutrophils isolated from ARDS rats are activated neutrophils. These neutrophils have strong bactericidal function. After 4 hours of LXA4 treatment, the survival rate of these neutrophils can be significantly reduced, and their apoptosis can be increased, so as to avoid the damage of excessive inflammatory reaction to the body. The results in Fig. 5A-D also indicate that the survival rate of neutrophils isolated from peripheral blood of ARDS rats can still reach about 25% after 24 hours of culture in vitro, which indicates that the survival time of activated neutrophils in vitro is significantly longer than that of normal neutrophils, and LXA4 treatment can reduce the survival rate of activated neutrophils and significantly promote their apoptosis, LXA4 can not only effectively promote the apoptosis of activated neutrophils, but also reduce the cell death caused by NETosis, which is helpful to the resolution of inflammation. Our results showed that that LXA4 promotes the apoptosis of neutrophils in peripheral blood of ARDS rats cultured in vitro for 24 hours, and the effect of reducing neutrophil NETosis death is mediated by ALX receptor.
Finally, we used cyclophosphamide to inhibit circulating neutrophils in rats, and made a rat model of ARDS with neutrophil deficiency. The results showed that LXA4 could not reduce the pulmonary inflammatory infiltration and lung injury score in the ARDS rats with neutrophil deficiency(Fig. 7A-B), nor could it reduce the levels of inflammatory factors TNF-α and IL-1β in lung homogenate(Fig. 7C-D). It is further confirmed that LXA4 had a protective effect on ARDS rats by affecting the function of neutropenia.
In summary, this study demonstrates that LXA4 alleviates lung injury and reduces the release of inflammatory cytokines in ARDS rats induced by LPS, but has no protective effect on ARDS rats with neutrophil deficiency. Moreover, LXA4 also down-regulated neutrophil respiratory burst and the production of ROS and NETs in neutrophils of ARDS rats, thus reducing the damage of neutrophils to the surrounding tissues. LXA4 can also up-regulate the ability of neutrophils to phagocytize bacteria, thereby enhancing the bactericidal ability of neutrophils. In addition, LXA4 can promote the apoptosis of neutrophils and accelerate the resolution of inflammation. Our findings reveal a novel mechanism for LXA4 to attenuate the inflammatory reaction and shows that LXA4 could be exploited therapeutically for ARDS.