The risk of asthma exacerbation increases because of exposure to a high concentration of PM2.5.16 However, previous studies did not clearly state the relationship between PM2.5 and the mechanism of acute asthmatic attacks. Our results suggested that PM2.5 caused respiratory acidosis, increased airway resistance, lung tissue damage, inflammation, and airway epithelium dysfunction while upregulating inflammation and pyroptosis proteins. However, CC16 ameliorated these pathological damage through downregulating inflammation and pyroptosis proteins. The final key result of this study is that CC16 acts mechanistically to offer protection against lung damage caused by PM2.5. Hence, there is potential interest in using CC16-based therapeutics for preventing asthma exacerbation.
PM2.5 particles are small and can reach the distal end of the airway with respiration, causing respiratory system damage.17 More and more research evidence suggests that PM2.5 can cause various cell death, including autophagy, necrosis, apoptosis, pyroptosis and ferroptosis.18–19 We in vivo and vitro data showed pyroptosis proteins NLRP3, Caspase-1, Gasdermin D, and IL-1β in PM2.5-induced asthma. This type of pyroptosis is detected through the classical pathway through inflammasomes, recruiting and activating caspase-1, which cleaves and activates IL-18 and IL-1βfor inflammatory factors, cleave the N-terminal sequence of GSDMD, causing it to bind to the membrane and produce membrane pores, leading to cell pyroptosis.20–21 Similarly, Wang et al. and Huang et al.found that PM2.5-induced lung toxicity via suppression of NLRP3 inflammasome-mediated pyroptosis.22–23 Besides, Xiong et al. reported that deleting the pyroptosis-associated protein NLRP3 in macrophages might minimize pyroptosis and PM2.5-induced lung toxicity.24
CC16 is so richly secreted by bronchial club cells and other epithelial cells that it is one of the most abundant proteins in respiratory secretions.25 The relationship of CC16 and asthma is revealed. A plot study found circulating CC16 deficits were associated with a 20% increased odds of asthma. Furthermore, there appeared to be a dose effect, as more frequent symptoms were more strongly associated with low CC16(40% increased odds) than less frequent symptoms.26 Another research found low CC16 mRNA expression levels in bronchial epithelial cells were associated with asthma severity.27 Recently, several studies have been performed using CC16, and most of the investigations have focused on regulating inflammatory pathways in immune cells.28 Pang et al. observed that recombinant CC16 inhibits tumor necrosis factor-α (TNF-α), IL-6, and IL-8 production in LPS-treated RAW264.7 cells.29 Moreover, CC16 has been reported to inhibit NF-κB nuclear translocation and matrix metalloprotein 9 production in LPS-stimulated rat tracheal epithelial cells.30 In sepsis-induced acute lung injury, Janicova et al. noted that endogenous CC16 modulates early macrophage-driven inflammation as an intrinsic anti-inflammatory signal. In study on particle-induced inflammation, Cui et al. revealed that rCC16 significantly lowered the IL-1β, TNF-α, and IL-6 protein and mRNA levels in THP-1 macrophages. Moreover, they showed that CC16 attenuated the increase in pro-IL-1β, NLRP3, and caspase 1 levels induced by exposure to silica particles.31 In our study, CC16 repairs airway epithelial proteins by targeting signal proteins related to pyroptosis and inflammation, promoting cell proliferation. This dual role and mechanism of action suggest further exploration of CC16 in airway epithelial repair.
Our results from previous experiments demonstrated that rCC16 inhibited LPS-stimulated apoptosis by activating the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)/ERK1/2 pathway and inhibited the release of inflammatory factors by inactivating the TLR-4/NF-κB signaling pathway.11 In this study, we revealed that PM2.5-induced pyroptosis comprises the inactivation of the NLRP3/caspase 1 signaling pathway and up-regulated expression of inflammatory mediator via the inactivation of the TLR4/NF-κB/MAPK/caspase 3 signaling pathway. However, they were both simultaneously inhibited by rCC16. Especially, CC16 appears to be highly specific of HMGB1. Our cell and animal models demonstrated that CC16 could inhibit the PM2.5-induced release of HMGB1. Liu et al. documented that CC16 attenuates house dust mite-mediated airway inflammation and damage via suppression of airway epithelial cell apoptosis in an HMGB1-dependent manner.32 Furthermore, Huang et al. identified that HMGB1 could promote the dysfunction of the epithelial barrier in synergy with IL-1β.33 Nevertheless, the underlying mechanism of how CC16 inhibits HMGB1 and IL-1β needs further investigation.
There is evidence suggesting that the binding capacities of CC16 are attributed to a modifying change between an activator and a receptor component protein inside and outside the cells. This protein is a dimer with two reverse polypeptide chains, thus forming a hydrophobic pocket for binding small lipophilic molecules.34 Johnson identified that VLA-4, created by a highly conserved sequence of amino acids (leucine-valine-aspartic acid), has vital mechanistic implications as a novel receptor. VLA-4 could impact neutrophil recruitment and leukocyte adhesion during infection by binding to integrin α4β1.35 These findings emphasize the underlying mechanism of CC16 as an intrinsic anti-inflammatory signal in the case of lung injury induced by PM2.5, sepsis, and toxicity.
According to the transcriptome analysis, CC16 has been observed to impede alveolar epithelial cells pyroptosis after PM2.5 exposure. This effect could be attributed to the upregulation of genes associated with cellular proliferation and repair, and down-regulation of genes linked to adhesion and lipid metabolism. Among the up-regulated genes, ETS1 is closely related to the AKT/mTOR proliferation pathway and governs the downstream NF- κB.36
However, there are several limitations in our study. First, apparent conceptual improvements are needed, especially regarding the experimental setup of the animal model. To determine whether CC16 moderates anaphylactic inflammation in the asthma airways or influences PM2.5-mediated inflammation, it would be beneficial to use four control groups: Control + OVA,Control + OVA + CC16, Control + PM2.5, and Control + PM2.5+CC16. Dr. Wang Aili, a member of our research team, augmented the control group in accordance with the experimental design and subsequently verified that CC16 exhibits reparative properties in response to injuries induced by both PM2.5 and OVA. The forthcoming publication of her article is anticipated.. It would be appropriate to design a new research study specifically focused on investigating the relationship between asthma and PM2.5. An additional animal experiment will be conducted by adding the above group. However, too few examples support the significant difference in sRAW between asthma and the negative control. Overall, data from HE, arterial blood gas, and IL 1β in BAL support the successful construction of the asthma model.