Some evidences have suggested that the adverse effects on lung tissue posed by PM2.5 maybe associated with the immune and inflammatory response[11, 15, 32]. Hence, B cell receptor signaling pathway, as the important pathway responsible for immune response[33], enriched by integrated analysis was foreseeable, which was also illuminated by previous study using RNA-sequencing[25]. The transcriptional levels of the target genes involved in this pathway were also significantly up- or down-regulated by PM2.5. Among them, Cd72, type II transmembrane protein coding gene belonging to C-type lectin family[34], was elucidated play an important role in controlling the magnitude of B cell responses[35], and then responsible for the immune system homeostasis regulation[36]. The overexpression of Cd72 found in this work from PM2.5-exposed group may promote B cell survival and proliferation, and enhance release of CD23, and then activate the immune response[37–39]. In addition, anti-CD72 monoclonal antibodies (mAbs) can activate CD19 tyrosine phosphorylation[40]. The similar result was observed in this work (the up-regulation of Cd19 induced by PM2.5). It was reported that mutation of gene pik3cd was associated with the prevalence of systemic lupus erythematosus, which is a typical immune system disease[41]. Hence, the marked increase of the mRNA level of pik3cd in lung tissue from exposure group might related to the immune response induced by PM2.5. Meanwhile, the associate microRNA of Cd72 149-5p and 328-3p were significantly down-regulated by PM2.5. It has reported that the two microRNA may be responsible for the lung inflammatory and fibrotic pathology in mice[42]. The microRNA (203-5p and 7a-5p) related to Pik3cd were markedly up-regulated in mice lung from exposure group. Based on the KEGG and GO annotations, the microRNA 203-5p was involved into the B cell receptor signaling pathway (Figure. S7a). Therefore, the increase of the expression levels of gene Cd72, Cd19, and Pik3cd involved into B cell receptor signaling pathway may contribute to the lung injury including inflammation and fibrosis induced by PM2.5.
Surprisingly, we firstly found the most significantly enriched KEGG pathway of DEGs through integrated analysis was cell adhesion molecules (CAMs), which was identified to mediate the process of cell recruitment and homing, and play an important role in the inflammatory process[43]. Once the inflammatory process of pulmonary immune system was triggered by PM2.5, the up-regulation of adhesion molecules genes occurred in endothelial and immune cells to mediate leukocyte adhesion and then migrating to inflammation sites[44]. From the results of RT-qPCR quantification and transcriptomics analysis, the significant upswing of the expression levels of genes Cd22 and H2-M2 were observed from PM2.5-treated group. The gene Cd22, a immunoglobulin superfamily cell-surface molecule that serves as an adhesion receptor for sialic acid-bearing ligands[45], was elucidated to active B cells and regulate antigen receptor signaling in vitro[46]. Similarly, the associated microRNA 3110-5p for Cd22 were significantly stimulated from exposure group. Thus, our finding indicated that CAMs may be a key pathway responsible for the adverse effects posed by PM2.5 to respiratory system. According to the KEGG and GO annotations (Figure. S7b), the genes Cd22 and H2-M2 can be used as feasible biomarkers.
Furthermore, the target genes involved in the insignificant (P > 0.05) enriched KEGG pathways (i.e., antigen processing and presentation, graft-versus-host disease, primary immunodeficiency, rap1 signaling pathway, MAPK signaling pathway, ras signaling pathway, small cell lung cancer, and PI3K-Akt signaling pathway) were also significantly up- or down-regulated by PM2.5, suggesting these pathways related to immune response of lung tissue should also be concerned for the further investigation.
A number of investigations have demonstrated that long-term exposure to PAHs increased risk of developing lung cancer[47–49]. CCA was performed using concentrations of PAHs as environmental factor. As showed in Figure. S8, the top three PAHs canonically correlated to target genes and microRNA were BaP, DBP, DBA, and BaP, DBP, benzo[b]fluorene (BF), respectively, indicating the four PAHs not the one with top concentration maybe the main impact factors. Among them, BaP was traditionally used as the surrogate marker for the toxicity of PAHs mixtures[50]. DBP has been evaluated 100 times carcinogenic potency than BaP[50]. The potency value of dibenzo[a, h]anthracene (DBA), which was estimated to be associated with human cancer[51], was reported to be up to 10 times than Bap[52]. To further explore the covariation between the PAHs and genetic indicators, Pearson’s index was determined. Obvious covariable relationship was observed between the target genes including Cd72, Cd19, Pik3cd, Cd22, H2-M24, and associated microRNA, and the PAHs detected from PM2.5 (Figure. S9, p < 0.05). This result suggested the target genetic indicators we selected showed a significant covariation with environmental pollutants, and then they may be used as the biomarkers to indicate the healthy risk posed by these types of pollutants.
From the public health point of view, PM2.5 samples used in this work were collected from Baoji, the mid-scale city located at the developing area. The main components of PAHs detected from the PM2.5 were similar to the mega-city (e.g., Beijing, Nanjing, et.al), while the concentrations of them, especially for Bap, DBA, DBP were statistically higher than the developed area[53–55]. Noted the concentrations of PM2.5 in all regions in China were very high than the threshold proposed by WHO (35 µg/mL) or more permissive limits adopted by China (75 µg/mL) in spite of the general decrease trends observed recent years[56]. Meanwhile, energy structure was much different between the mid-scale city and the mega-city in China[57], which directly result in the variation of the occurrence levels of the organic pollutants (PAHs) derived from the fossil fuel combustion[58]. Combined with the obviously adverse effects of the PM2.5 collected from Baoji city on the respiratory system found in this work, the healthy risk posed by PM2.5 at the mid-scale city of China should be paid more attention.