ROS-Mediated NLRP3 Inflammasome Activity Is Essential For Burn-Induced Acute Lung Injury

Backgroud Severe burns can cause dramatic changes in the body homeostasis, leading to serious damage to the body. Traumatic stress, direct heat damage, inadequate tissue perfusion, and systemic inflammatory response, all these stimulis make damage not only limited to skin, but also to many body organs’ function, resulting in function disorders. The incidence rate of multiple organ dysfunction is 28.1% after burns and it is difficult to reverse. Since the lungs are directly connected with the external environment and vulnerable to external environmental factors, so for severely burned patients, especially those with inhalation injury or underlying lung disease, lung injury is the most common after burns. A variety of factors have been involved in the pathogenesis of acute lung injury, which is mainly divided into two aspects: cellular factors and humoral factors. The former includes activated inflammatory cells such as neutrophils and macrophages; pulmonary resident cells such as lung alveolar epithelial cells and vascular endothelial cells. The latter includes a variety of inflammatory mediators, cytokines, and oxidative stress. After severe burns, above factors are activated and result in lung damage. Moreover, after severe burns, plenty of inflammatory cells are activated, which synthesize and release a number of inflammatory mediators, and lead to strong systemic inflammatory response, which is an important cause of burn-induced lung injury.NLRP3 inflammasome is an important component of innate immunity, it is composed of proteins complexes. Inflammasome can regulate caspase-1 activity, which is turn contributes to the activation of cytokine precursor pro-IL-1β and pro-IL-18 in the course of natural immune defense. IL-1β and IL-18 are a powerful inflammatory initiation factors, which can promote inflammatory reactions. Therefore, we hypothesize that NLRP3 inflammasome plays an important role in the pathogenesis of lung injury after burns. In this study we explored the expression of NLRP3 inflammasome signaling pathway involved by in vivo and in vitro experiments, and its effects on lung injury. In addition, we further explored the mechanism of NLRP3 inflammasome activition.Methods:1. We established a 30% of total body surface area(TBSA) model of lung injury in rats following severe burns, tested NLRP3 and Caspase-1 inflammasome signal in lung tissue, and examined the levels of IL-1β and IL-18 in serum and lung at each time point, to explore the activation of inflammasome signal after severe burns and optimize the time point for subsequent experiments.2. We inhibited NLRP3 inflammasome by intraperitoneal injection of BAY11-7082 and observed the activation of NLRP3 inflammsome signal pathway in rats with severe burn; we then used biopsy of lung tissue and lung injury indicators of rats with severe burn to explore the role of NLRP3 inflamasome in the lung after severe burn injury.3. We isolated rat alveolar macrophages from bronchoalveolar lavage fluid and stimulated them with burn serum to simulate the burn lung injury microenvironment after burns, and then observed the activity of NLRP3 inflammasome signaling pathway and ROS production to explore the underlying mechanism of NLRP3 inflammasome activation after severe burn.4. By using ROS scavenger N-acetylcysteine, we observed NLRP3 inflammasome signaling activation after burn serum stimulation of alveolar macrophages, to verify the role of ROS in NLRP3 inflammasome activation after severe burns.Results:1. The m RNA and protein expression of NLRP3, Caspase-1 inflammasome signaling pathways and IL-1β, IL-18 within 48 h after 30% of the total body surface area burns in rat lung tissue gradually increased in a time-dependent manner, and reached the highest value at 24 h or 48 h.2. After intraperitoneal injection of NLRP3 inflammasome inhibitor BAY11-7082, the expression of NLRP3, Caspase-1 inflammasome signaling pathways and IL-1β, IL-18 gene and protein were significantly inhibited in severely burned lung tissue, and the inhibition of NLRP3 inflammasome activation significantly reduced severe burncaused damage in lung pathology by reduced inflammation response in lungs.3. Intracellular signaling pathways of NLRP3 inflammasome significant activate, and ROS level was significantly increased after burn serum stimulation of alveolar macrophages. ROS may regulate NLRP3 inflammasome activation after severe burns.4. After using ROS scavenger, NLRP3 inflammasome signal activation in burn serum stimulated alveolar macrophages was significantly suppressed, indicating the activation of NLRP3 inflammasome is partially ROS-dependent.Conclusions:This study demonstrates that burn injury activates the NLRP3 inflammasome in alveolar macrophages(AMs), which further promotes the release of inflammatory initiating cytokines, such as IL-1β and IL-18, and amplifies the inflammatory response and finally cause ALI. Additionally, burn injury-induced NLRP3 inflammasome activation is a partially ROS-dependent process, inhibition of either NLRP3 or ROS activity can alleviate acute lung injury(ALI).