PM2.5‐induced pulmonary inflammation via activating of the NLRP3/caspase‐1 signaling pathway

Abstract Particulate matter 2.5 (PM2.5)‐induced pulmonary inflammation has become a public concern in recent years. In which, the activation of the NLRP3/caspase‐1 pathway was closely related to the inflammatory response of various diseases. However, the promotion effect of the NLRP3/caspase‐1 pathway on PM2.5‐induced pulmonary inflammation remains largely unclear. Here, our data showed that PM2.5 exposure caused lung injury in the mice by which inflammatory cell infiltration occurred in lung and alveolar structure disorder. Meanwhile, the exposure of human bronchial epithelial cells (16HBE) to PM2.5 resulted in suppressed cell viability, as well as elevated cell apoptosis. Moreover, a higher level of inflammatory cytokine and activation of the NLRP3/caspase‐1 pathway in PM2.5‐induced inflammation mice models and 16HBE cells. Mechanistically, pretreatment with MCC950, a NLRP3/caspase‐1 pathway inhibitor, prevented PM2.5‐induced lung injury, inflammatory response, and the number of inflammatory cells in BALFs, as well as promoted cell viability and decreased inflammatory cytokine secretion. Collectively, our findings indicated that the NLRP3/caspase‐1 pathway serves a vital role in the pathological changes of pulmonary inflammation caused by PM2.5 exposure. MCC950 was expected to be the therapeutic target of PM2.5 inhalation mediated inflammatory diseases.


| INTRODUCTION
Atmospheric fine particles, namely particulate matter 2.5 (PM2.5), refers to the suspended particles in the atmosphere in the aerodynamic diameter less than or equal to 2.5 μm, with small particle size, large surface area characteristics, propagation distance, the stagnation time is extended, easy access to the alveolar terminal, and easily dissolved in the blood and respiratory system. 1,2 Epidemiological studies found that the main components of PM2.5 are complex and have a carrier role, which can be loaded with sulfate, nitrate, ammonium salt, carbon-containing particles, heavy metals, minerals, bacteria, and viruses. 3 When PM2.5 inhaled in the lungs, it can cause an acute pulmonary inflammatory response and release a variety of inflammatory factors. At the same time, PM2.5 also contains organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and lipopolysaccharides, which can produce free radicals in the lungs, break the fast oxidation and antioxidant balance, and then cause lung function damage, resulting in adverse effects of the lung. 3 Although there are many theories on the pathogenesis of PM2.5, the mechanism and regularity of its specific harmful effects on pulmonary still need to be further explored.
Previous studies have confirmed that inflammasomes promote inflammation, and chronic inflammatory response triggered by a variety of immune cells is crucial for PM2.5-induced pulmonary inflammation. 4,5 NOD-like receptor protein 3 (NLRP3) inflammasome is an intracellular multi-protein complex containing NLRP3, apoptotic speck protein (ASC), and pro-caspase-1. 6 When stimulated, the NLRP3 inflammasome promotes caspase-1 activation and subsequently converting pro-interleukin-1β (IL-1β) and pro-IL-18 into their mature bioactive forms. Mature IL-1β and IL-18 are then released to extracellular space. 7 Interestingly, as an effector of the NLRP3 inflammasome, IL-1β is a pluripotent pro-inflammatory cytokine involved in the process of PM2.5-induced respiratory diseases and considered to play a pivotal role in pulmonary inflammation pathogenesis. 8 For example, Xu et al showed that PM2.5 exposure induced IL-1β signaling activation and resulted in pulmonary inflammation. 9 Besides, the activation of NLRP3 inflammasome accelerated pulmonary fibrosis caused by airborne fine particulate matter. 10 Another study has demonstrated that PM2.5 exposure resulted in neuronal injury via promoting NLRP3 inflammasome activation in a model of Alzheimer disease. 11 The above research findings indicated that the NLRP3 inflammasome might serve an essential role in the process of pulmonary inflammation induced by PM2.5 exposure, but its functional role is not clear.
This study aimed to examine NLRP3 inflammasome was activated in pulmonary inflammation caused by PM2.5 exposure and verify whether activation NLRP3 inflammation was involved in the harmful effect on PM2.5-induced lung injury in vivo. To do this, we used H&E to evaluate the morphological changes of the lung in the PM2.5-induced mice models. Moreover, we further to explore the possible mechanism of PM2.5-induced lung injury via detecting the levels of inflammatory cytokines in serum and lung tissues, and counting the number of inflammatory cells in bronchoalveolar lavage fluids (BALFs). Finally, we elucidated the effect of NLRP3 inflammasome activation on PM2.5-induced mice pulmonary inflammation, which provided an animal experimental theoretical basis for the clinical study of lung injury caused by air pollution (ie, PM2.5).

| Sample collection of PM2.5
The preparation and purification methods of PM2.5 were modified according to the previous study. 12 In brief, PM2.5 was collected in Shenyang Environmental Monitoring Centre (Shenyang, China) from December 10, 2017 and between December 17, 2018. After the collection, the glass fiber filter paper (1.5 cm × 1.5 cm) was applied to elute PM2.5 using ultrasonic vibration equipment (20 minutes × 5 times). And then, the detached PM2.5 suspension was put into a freeze-drying bottle and placed in an ultra-low temperature refrigerator at −80 C. Finally, the PM2.5 particles were frozen, vacuumed, and dried to dry powder and the dried PM2.5 powder was stored for standby at −20 C.

| Analysis of inflammatory cells in bronchoalveolar lavage fluid (BALF)
The preparation of BALF and inflammatory cell counting were analyzed according to the previous study. 14

| Cell culture and treatment
Human bronchial epithelial cells (16HBE cells) were from the Chines Academy of Sciences (Shanghai, China). 16HBE cells were cultured in bronchial epithelial cell medium (ScienCell Research Laboratories, Inc.) and incubated at 37 C in a humidified atmosphere containing 5% CO 2 . And then, 16HBE cells were seeded into a 6-well plate and serum deprivation for 12 hours in bronchial epithelial cell medium, PM2.5 was suspended and sonicated in sterile saline to a final concentration of 1 mg/mL, and 16HBE cells were treated with 20 μg/mL PM2.5 for 24 hours. OD490 nm value was measured. The viability of the non-treated cells (control) was defined as 100%, and the viability of cells from all other groups was calculated separately from that of the control group.

| Cell viability
2.6 | Cell apoptosis 2 × 10 5 cells were maintained in an incubator with saturated humidity for 24 hours, digested with trypsin, and washed with precooled PBS three times at 4 C. ×1 binding buffer was used to resuspend the cells and adjust the cell concentration to 1 × 10 6 cells/mL. Cells were F I G U R E 1 PM2.5 exposure triggered lung injury and enhanced the number of inflammatory cells in BALFs. A, All mice exposure to PM2.5 (120 μg/mL) by intratracheal instillation for 14 days, H&E staining was used to observe the histological changes of the lung (magnification, ×400); B-F, BALF cell numbers in leukocyte, neutrophil, macrophage, lymphocyte, and eosinophil were analyzed; G and H, ELISA assay was applied to detect the levels of TP and LDH in the serum of PM2.5 exposure to mice. * P < .05, ** P < .01, *** P < .001, compared with the PM2.5 exposure group [Color figure can be viewed at wileyonlinelibrary.com] treated with 5 μL Alexa FluorR 488 annexin V and 1 μL 100 μg/mL PI solution for 15 minutes at room temperature in dark conditions. Thereafter, 400 μL Annexin V Binding buffer was added and the cells were assessed by flow cytometry as soon as possible. The samples were analyzed with a FACScan flow cytometer (BD Biosciences, California).

| Western blot analysis
The total proteins of tissues were extracted using RIPA buffer

| Statistical analysis
All experiments were repeated at least three times. Data were presented with mean ± SD. Statistical analysis was performed by Gra-phPad Prism 8 (GraphPad Software, Inc.), and differences between groups were analyzed by one-way ANOVA followed by Tukey post hoc test. Student t test was used for comparisons between two groups. P < .05 was considered to be statistically significant.
F I G U R E 3 Inhibition of the NLRP3/caspase-1 pathway alleviated PM2.5-induced lung injury and decreased the number of inflammatory cells in BALFs. A, Mice in the PM2.5 + MCC950 group was received 2 mg/kg MCC950 for 12 hours via tail injection before PM2.5 treatment; H&E staining was used to observe the histological changes of the lung (magnification, ×200); B-F, BALF cell numbers in leukocyte, neutrophil, macrophage, lymphocyte, and eosinophil were analyzed; G and H, ELISA assay was applied to detect the expression levels of TP and LDH in the serum of mice. *P < .05, **P < .01, ***P < .001, compared with the PM2.5 exposure group [Color figure can be viewed at wileyonlinelibrary.com] 3 | RESULTS

| PM2.5 exposure induced lung damage in vivo
Previous studies have confirmed that PM2.5 exposure could cause acute airway inflammation. 15,16 In the present study, we explore the effect of PM2.5 on pulmonary inflammation in vivo. H&E staining showed that compared with the control and saline group, PM2.5 treatment (120 μg/mL) resulted in alveolar structure disorder, alveolar septal thickening, erythrocyte effusion, and infiltration of inflammatory cells (Figure 1), suggesting that PM2.5 exposure triggered to lung injury and inflammatory response. Next, we examine the BALF cell numbers in leukocytes, neutrophils, macrophages, lymphocytes, and eosinophils derived from the mice treated with PM2.5. The data revealed that PM2.5 exposure enhanced the number of leukocytes, neutrophils, macrophages, lymphocytes, and eosinophils compared with the control group and saline group ( Figure 1B-F). Moreover, previous studies have confirmed that PM2.5-induced oxidative stress appears as an excessive variation of associated-enzyme activities, oxidant accumulation and so on. 17,18 As expected, ELISA analysis results showed that the levels of TP and LDH in the PM2.5 treatment group were higher than those in the control and saline group ( Figure 1G,H).
Taken together, lung injury after chronic exposure to PM2.5 was related to the imbalance inflammatory response.

| MCC950 administration ameliorated inflammatory response in PM2.5-induced mice model
To further investigate the effect of MCC950 on pulmonary inflammation induced by PM2.5 in vivo. As shown in Figure 4A,

| DISCUSSION AND CONCLUSIONS
In recent years, air pollution is grim, and the haze occurrences frequently. And PM2.5 was one of the important constituents in the haze, plays the most important role in causing adverse health effects.
Studies have shown that most respiratory diseases are closely related to atmospheric environmental particulate matter, and atmospheric PM2.5 has a significant effect on the development of respiratory diseases. 21 In the present study, our data showed that PM2. Increasing evidence has confirmed that pulmonary inflammation caused by PM2.5 exposure was related to inflammatory cell recruitment in the alveolus and be activated. 22 Ogino et al confirmed that PM2.5-induced airway inflammation via increasing the BALFs cell numbers in total cell fractions, macrophages, neutrophils, eosinophils, and lymphocytes. 12 Zheng et al found that PM2.5 exposure significantly increased the number of neutrophilic and the levels of the Th2 cytokines in BALFs. 4 Moreover, another study has confirmed that the number of infiltrated macrophages and neutrophils in the lung of PM2.5-treated mice was enhanced, 23 as well as PM2.5-induced activated RAW264.7 macrophages inflammatory factors release. 24 In line with previous studies, our data showed that the number of inflammatory cells in BALFs were enhanced in the alveolus of mice exposure to PM2.5.
It has demonstrated that exposure to PM2.5 contributed to increasing the release of IL-1β, an inflammatory effector, which has been used as a target for the treatment of inflammatory-related diseases. 9,10,25 In the present study, our data showed that PM2.5 exposure increased the expression of inflammatory cytokines, including IL-1β, IL-6, and TNF-α in lung tissues compared with the control and saline group. Moreover, previous studies found that the expression level of IL-1β was regulated by multiple pathways in inflammation diseases, such as toll-like receptor 4 (TLR4)/nuclear factor kappa beta (NF-κB) pathway, 26,27 NLRP3 inflammasome, 28 Akt pathway. 13 For instance, Wang et al showed that PM2.5 exposure enhanced the levels of IL-1β by activating the TLR4/MyD88 pathway and NLRP3 inflammasome in murine airway inflammation. 29 We found that inhibition of the NLRP3/caspase-1 pathway by MCC950 significantly attenuated PM2.5-induced IL-1β release in lung tissues and serum of mice. Furthermore, NLRP3 inflammasome activation was associated with particulate matter induced pulmonary fibrosis, 10,30