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Fine Particulate Matter Induces Childhood Asthma Attacks via Extracellular Vesicle‐Packaged Let‐7i‐5p‐Mediated Modulation of the MAPK Signaling Pathway

Abstract Fine particulate matter less than 2.5 µm in diameter (PM2.5) is a major risk factor for acute asthma attacks in children. However, the biological mechanism underlying this association remains unclear. In the present study, PM2.5‐treated HBE cells‐secreted extracellular vesicles (PM2.5‐EVs) caused cytotoxicity in “horizontal” HBE cells and increased the contractility of “longitudinal” sensitive human bronchial smooth muscle cells (HBSMCs). RNA sequencing showed that let‐7i‐5p is significantly overexpressed in PM2.5‐EVs and asthmatic plasma; additionally, its level is correlated with PM2.5 exposure in children with asthma. The combination of EV‐packaged let‐7i‐5p and the traditional clinical biomarker IgE exhibits the best diagnostic performance (area under the curve [AUC] = 0.855, 95% CI = 0.786–0.923). Mechanistically, let‐7i‐5p is packaged into PM2.5‐EVs by interacting with ELAVL1 and internalized by both “horizontal” recipient HBE cells and “longitudinal” recipient‐sensitive HBSMCs, with subsequent activation of the MAPK signaling pathway via suppression of its target DUSP1. Furthermore, an injection of EV‐packaged let‐7i‐5p into PM2.5‐treated juvenile mice aggravated asthma symptoms. This comprehensive study deciphered the remodeling of the extracellular environment mediated by the secretion of let‐7i‐5p‐enriched EVs during PM2.5‐induced asthma attacks and identified plasma EV‐packaged let‐7i‐5p as a novel predictor of childhood asthma.


Introduction
Asthma is a common chronic respiratory disease characterized by airway hyperresponsiveness, airway inflammation and reversible airway remodeling. [1,2] Globally, asthma remains a serious health problem that affects 358 million individuals, especially among children. [3,4] Clinically, asthma attacks in children lead to reversible and repeated attacks of coughing, wheezing, shortness of breath, and chest tightness, thereby impairing quality of life and imposing a heavy burden on the family and community. [5] As environmental pollution is predictable and preventable, airborne fine particulate matter <2.5 μm in diameter (PM 2.5 ) has been considered a crucial type of allergen causing acute asthma attacks in children. [5][6][7] Here, two airway cell types ultimately responsible for asthma pathogenesis are considered: human bronchial epithelial (HBE) cells, which form the first barrier between the external environment and the internal bronchus and are the major

EVs Secreted by PM 2.5 Stimulation "Horizontally" Induce Cytotoxicity in HBE Cells
The flowchart of the evaluation strategy for EVs is presented in Figure 1A. First, we performed a direct PM 2.5 exposure assay and found that PM 2.5 dose-dependently decreased the viability of HBE cells after exposure for different times (Table S1, Supporting Information). Compared with the PM 2.5 group exposed for 12 h, PM 2.5 concentrations greater than or equal to 250 μg mL −1 exhibited a significant difference in their ability to reduce the number of HBE cells after treatment for 24 or 48 h ( Figure 1B). Because the concentration of PM 2.5 may be up to 100-200 μg m −3 in winter, combined with children's daily inhalation of 10 m 3 of air and the average volume of 6.2 mL of fluid in the lungs and airway, the corresponding PM 2.5 concentration was calculated to be 161-323 μg mL −1 . [22] We selected 250 μg mL −1 PM 2.5 and an exposure time of 24 h as the PM 2.5 treatment conditions in this study to account for children exposed to high levels of PM 2.5 . As shown in Figure 1C,D, PM 2.5 significantly induced apoptosis and G1/G2 arrest in normal HBE cells. Thus, PM 2.5 exposure is cytotoxic to normal HBE cells.
Next, after coculture of normal and PM 2.5 /NC-treated HBE cells in a Transwell plate for 24 h, the culture medium of PM 2.5 -treated HBE cells exerted stronger cytotoxic effects than those observed on normal HBE cells exposed to NC-treated HBE cells ( Figure S1, Supporting Information), suggesting that PM 2.5 -treated HBE cells may secrete EVs to induce cytotoxicity in HBE cells. Using visualization approaches, we found that EVs purified from the culture medium of PM 2.5 /NC-treated HBE cells (PM 2.5 /NC-EVs) exhibited a typical cup-shaped morphology, were ≈100 nm in size, expressed the typical EV markers CD63, TSG101 and Alix, and lacked cellular contaminants such as calnexin and Grp94 ( Figure 1E-G and Figure S2, Supporting Information). Fluorescence microscopy showed PKH67 green fluorescent dye in the cytoplasm of HBE cells, while no PKH67 green fluorescent dye was observed in the non-EVs group, indicating that PM 2.5 /NC-EVs were taken up by normal HBE cells (Figure 1H). However, this ability was abolished by inhibiting endocytosis with cytochalasin D ( Figure S3, Supporting Information), indicating the efficient transportability of the secreted EVs. Subsequently, direct incubation with PM 2.5 -EVs for 24 h markedly decreased HBE cell viability, promoted apoptosis, and stimulated S phase arrest ( Figure 1I-L). Moreover, these aberrant cellular activities were rescued by the pharmacological inhibition of EV secretion with GW4869 ( Figure 1I and Figure S4, Supporting Information). Taken together, these findings indicate that EVs secreted by PM 2.5 -stimulated HBE cells are "horizontally" transported into normal HBE cells, with subsequent induction of cytotoxicity. The effect of PM 2.5 -EVs on cytotoxicity in HBE cells. PM 2.5 -treated HBE cells were designated PM 2.5 -HBE cells. EVs isolated from PM 2.5 -treated HBE cells and NC-treated HBE cells were designated PM 2.5 -EVs and NC-EVs, respectively. A) Flowchart of the strategy used to evaluate the effect of PM 2.5 -EVs on cytotoxicity in HBE cells and the contractility of sensitive HBSMCs. B) The viability of HBE cells was evaluated using a CCK-8 assay after PM 2.5 treatment. C) The apoptosis of HBE cells was assessed using flow cytometry after PM 2.5 treatment. D) The cell cycle of HBE cells was analyzed using flow cytometry after PM 2.5 treatment. E) Purified PM 2.5 /NC-EVs were identified using TEM. Scale bar, 100 nm. F) The EV-specific markers (CD63, TSG101 and Alix) and negative controls (calnexin and Grp94) were evaluated using Western blotting. G) The size distributions of PM 2.5 /NC-EVs were confirmed using NanoFCM. H) Representative fluorescence images of HBE cells after an incubation with PM 2.5 /NC-EVs labelled with PKH67 (green) or non-EVs. Scale bar, 25 μm. I) Schematic illustration of HBE cells incubated with PM 2.5 -EVs or PM 2.5 -EVs pretreated with DMSO/GW4869. J) The viability of HBE cells was evaluated using a CCK-8 assay after an incubation with PM 2.5 /NC-EVs. K) The apoptosis of HBE cells was assessed using flow cytometry after an incubation with PM 2.5 /NC-EVs. L) The cell cycle of HBE cells was analyzed using flow cytometry after an incubation with PM 2.5 /NC-EVs. Statistical significance was assessed using two-tailed Student's t-test. Values represent means ± SD. * p < 0.05. the expression of three identified vital proteins that regulate HB-SMC contractility: -smooth muscle actin ( -SMA), smooth muscle myosin heavy chain 11 (SM-MHC) and RhoA ( Figure 1A). No differences in the expression of contractile proteins were observed in normal HBSMCs after coculture with PM 2.5 -treated HBE cells (Figure 2A and Figure S5A, Supporting Information). Although PKH67-labelled PM 2.5 /NC-EVs were transferred into normal HBSMCs ( Figure 2B and Figure S6, Supporting Information), PM 2.5 -EVs did not regulate the expression of contractile proteins ( Figure 2C,D). Based on these data, PM 2.5 exposure may function as an inducing factor rather than an initiating factor of asthma by causing airway hyperresponsiveness and subsequently inducing asthma attacks. To simulate the condition of HBSMCs during asthma remission, we established a sensitive HBSMC model by using 100 ng mL −1 IL-13 and found that sensitive HBSMCs exhibited increased eotaxin expression ( Figure 2E). Compared with NC-treated HBE cells, sensitive HBSMCs cocultured with PM 2.5 -treated HBE cells showed higher expression levels of contractile proteins ( Figure 2F and Figure S5B, Supporting Information). Similarly, "longitudinal" recipient-sensitive HB-SMCs displayed efficient uptake of PKH67-labelled PM 2.5 /NC-EVs ( Figure 2G, and Figure S7, Supporting Information) and exhibited increased expression of contractile proteins after treatment with PM 2.5 -EVs ( Figure 2H and Figure S5C, Supporting Information). In contrast, GW4869 treatment of PM 2.5 -treated HBE cells reduced the expression levels of contractile proteins in "longitudinal" recipient-sensitive HBSMCs ( Figure 2I and Figure S5D, Supporting Information). Collectively, these results suggest that PM 2.5 -EVs are also "longitudinally" delivered to sensitive HBSMCs, enhancing their contractility.

EV-Packaged Let-7i-5p Correlates with PM 2.5 Exposure in Children with Asthma
Aiming to explore the epigenetic regulation of PM 2.5 -induced childhood asthma, we verified EVs isolated from plasma in children ( Figure 3A and Figure S8, Supporting Information) and performed RNA sequencing on EV-packaged miRNAs in plasma samples from children and PM 2.5 /NC-treated HBE cells (Figure 3B). As shown in Figure 3C, 262 EV-packaged miRNAs were differentially expressed in PM 2.5 -EVs compared with NC-EVs (118 upregulated and 144 downregulated; |fold change| >2, P adj < 0.05), and 173 EV-packaged miRNAs (113 upregulated and 60 downregulated; |fold change| >2, P adj < 0.05) were differentially expressed in the comparison of plasma from children with asthma and healthy controls. Furthermore, we identified 11 overlapping upregulated EV-packaged miRNAs in the cell models and plasma from children ( Figure 3D), among which the most abundant EV-packaged miRNA, let-7i-5p, was selected as the candidate miRNA for further verification ( Figure 3E). Consistent with the RNA sequencing results, the level of EV-packaged let-7i-5p was approximately two-fold higher in PM 2.5 -EVs than in NC-EVs ( Figure S9, Supporting Information). We confirmed the existing pattern of extracellular let-7i-5p by treating cells with RNaseA /Triton X-100 and found that the level of let-7i-5p in the culture medium of PM 2.5 -treated HBE cells did not change after RNaseA treatment but decreased markedly when RNaseA and Triton X-100 were added simultaneously ( Figure 3F). However, the let-7i-5p level was reduced in the culture medium of NC-treated HBE cells after treatment with RNaseA. Additional EV inhibition experiments verified that the level of let-7i-5p in the culture medium of PM 2.5 -treated HBE cells was significantly reduced after treatment with GW4869 ( Figure 3G). Based on these results, extracellular let-7i-5p is mainly encapsulated in EVs instead of directly secreted.
Next, we sought to examine the association of EV-packaged let-7i-5p with childhood asthma attacks and PM 2.5 exposure based on 55 patients and 55 healthy controls ( Figure 3H). The EVpackaged let-7i-5p level remained stable after different freezethaw cycles ( Figure S10A, Supporting Information) and room temperature incubation periods ( Figure S10B, Supporting Information). Compared with the healthy controls (Table S2, Supporting Information), children with asthma had significantly higher plasma total IgE levels and peripheral blood eosinophil percentages (p < 0.001) and were exposed to higher levels of PM 2.5 . We further detected let-7i-5p levels in plasma EVs from children and found that the expression of EV-packaged let-7i-5p was significantly increased in children with asthma ( Figure 3I). Moreover, EV-packaged let-7i-5p exhibited a high capacity to discriminate children with asthma from healthy controls, with an area under the curve (AUC) of 0.785 (95% confidence interval [CI] = 0.701-0.869; Figure 3J). Notably, the AUC of the combination of IgE and EV-packaged let-7i-5p was increased to 0.855 (95% CI = 0.786-0.923), accompanied by increases in the sensitivity and specificity to 70.90% and 85.20%, respectively ( Figure 3J). Correlation analysis revealed that the EV-packaged let-7i-5p level exhibited a significant positive correlation with PM 2.5 exposure in children with asthma but not in healthy controls ( Figure 3K and Figure S11, Supporting Information). These observations imply that plasma levels of EV-packaged let-7i-5p may be a biomarker for PM 2.5induced asthma in children.

EV-Packaged Let-7i-5p Affects Cytotoxicity in HBE Cells and the Contractility of Sensitive HBSMCs
We first transfected the let-7i-5p mimic into recipient cells to further determine the biological significance of EV-packaged let-7i-5p in cytotoxicity in HBE cells and the contractility of sensitive HBSMCs and found that let-7i-5p overexpression reduced HBE cell growth, induced an increase in the apoptosis rate of HBE cells, and promoted S phase arrest ( Figure S12A-D, Supporting Information). Moreover, let-7i-5p overexpression markedly increased the expression of contractile proteins in sensitive HB-SMCs ( Figure S12E,F, Supporting Information). Next, we cocultured HBE cells transfected with Cy3-labelled let-7i-5p mimic with recipient cells and observed the red fluorescence of the labelled let-7i-5p in both "horizontal" recipient HBE cells and "longitudinal" recipient-sensitive HBSMCs, indicating that EVpackaged let-7i-5p was taken up by recipient cells ( Figure S13A, Supporting Information). Subsequently, we evaluated the level of let-7i-5p after incubating the recipient cells with EVs and found that the cellular let-7i-5p levels were increased upon incubation with PM 2.5 -EVs but not upon incubation with NC-EVs or direct exposure to PM 2.5 ( Figure S13B,C, Supporting Information). These findings indicate that the enriched let-7i-5p in recipient cells is mainly transported by PM 2.5 -EVs. We further examined the  Figure 3. The role of EV-packaged let-7i-5p in children with asthma and the phenotype of recipient cells. HBE cells were transfected with NC-or let-7i-5p inhibitor, exposed to NC/PM 2.5 and then isolated the corresponding EVs, namely, NC-EVs, (NC+PM 2.5 )-EVs, and (Anti-let-7i-5p+PM 2.5 )-EVs, respectively. A) EV-specific markers (CD63, TSG101 and Alix) and negative controls (APOA1 and albumin) were evaluated using Western blotting. B) Schematic model showing the generation of EV-packaged miRNA expression profiles. C) Volcano plots of different EV-packaged miRNAs in PM 2.5 -treated HBE cell models and children with asthma. The red and blue plots indicate the differentially expressed EV-packaged miRNAs. D) Venn diagram showing the overlapping upregulated EV-packaged miRNAs. E) Hierarchical clustering analysis of the 11 overlapping upregulated EV-packaged miRNAs identified from the Venn diagram. F) The level of let-7i-5p in the culture medium of PM 2.5 /NC-treated HBE cells incubated with 2 μg mL −1 RNaseA alone or in combination with 0.1% Triton X-100 was determined using RT-qPCR. G) The level of let-7i-5p in the culture medium of PM 2.5 -treated HBE cells exposed to DMSO or GW4869 was determined using RT-qPCR. H) Locations of the individuals' residences and air quality monitoring stations. I) The level of plasma EV-packaged let-7i-5p was determined using RT-qPCR. J) ROC curve analysis of plasma levels of EV-packaged let-7i-5p and IgE. K) Correlation analysis was performed between plasma EV-packaged let-7i-5p and PM 2.5 exposure levels in children with asthma. L) The viability of HBE cells incubated with NC-EVs, (NC+PM 2.5 )-EVs or (Anti-let-7i-5p+PM 2.5 )-EVs was evaluated using a CCK-8 assay. M) The apoptosis of HBE cells incubated with NC-EVs, (NC+PM 2.5 )-EVs or (Anti-let-7i-5p+PM 2.5 )-EVs was assessed using flow cytometry. N) The cell cycle of HBE cells incubated with NC-EVs, (NC+PM 2.5 )-EVs, or (Anti-let-7i-5p+PM 2.5 )-EVs was analyzed using flow cytometry. O) The levels of contractile proteins in sensitive HBSMCs incubated with NC-EVs, (NC+PM 2.5 )-EVs, or (Anti-let-7i-5p+PM 2.5 )-EVs were measured using Western blotting. Statistical significance was assessed using two-tailed Student's t-test. Values represent means ± SD. * p < 0.05. www.advancedsciencenews.com www.advancedscience.com biological effect of EV-packaged let-7i-5p on recipient HBE cells and sensitive HBSMCs. As shown in Figure 3L-O and Figure  S14, Supporting Information, the effect of PM 2.5 -EVs on enhancing cytotoxicity in HBE cells or increasing the contractility of sensitive HBSMCs was blocked when let-7i-5p within PM 2.5 -treated HBE cells was antagonized by a let-7i-5p inhibitor. Moreover, the effects of PM 2.5 -EVs on recipient cells were enhanced when let-7i-5p was overexpressed in recipient cells transfected with the let-7i-5p mimic ( Figure S15, Supporting Information). Collectively, these data indicate that EV-packaged let-7i-5p released by PM 2.5 -treated HBE cells induces cytotoxicity in "horizontal" recipient HBE cells and increases the contractility of "longitudinal" recipient-sensitive HBSMCs.

Let-7i-5p Is Packaged into EVs via ELAVL1 and Induces Asthma by Activating the MAPK Signaling Pathway
To investigate the mechanism by which let-7i-5p is loaded into EVs, we used the database of RNA-binding protein specificities (RBPDB) to predict the specific interaction between let-7i-5p and the RNA-binding proteins (RBPs) binding motif. As shown in Figure 4A, three RBPs, including ELAV-like protein 1 (ELAVL1), a well-defined RBP for sorting miRNAs into EVs, [23,24] was predicted to specifically bind let-7i-5p (threshold 0.7). Notably, let-7i-5p FISH and ELAVL1 immunostaining indicated the colocalization of let-7i-5p and ELAVL1 in the cytoplasm of HBE cells (Figure 4B). Consistent with these findings, RNA binding protein immunoprecipitation (RIP) assays confirmed a more obvious interaction between let-7i-5p and ELAVL1 in PM 2.5 -treated HBE cells than in NC-treated HBE cells ( Figure 4C), indicating that let-7i-5p is more likely to interact with ELAVL1 following exposure to PM 2.5 . Moreover, ELAVL1 was significantly upregulated in PM 2.5treated HBE cells ( Figure 4D), and the EV-packaged let-7i-5p level was significantly reduced in cells with ELAVL1 knockdown (Figure 4E and Figure S16, Supporting Information). These findings suggest that ELAVL1 is responsible for the specific loading of let-7i-5p into EVs.
Next, we used three target prediction software programs and performed an overlap analysis to identify 169 putative targets of let-7i-5p ( Figure 4F and Figure S17, Supporting Information). The subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed consistent biological pathways involved in cellular activities ( Figure 4G). Notably, the mitogen-activated protein kinase (MAPK) signaling pathway was previously found to be functionally related to asthma, and among 8 candidate targets, dual-specificity protein phosphatase 1 (DUSP1), a key inhibitor, directly inactivates Jun amino-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 via dephosphorylation. [25] Thus, we preferentially selected DUSP1 as a putative downstream let-7i-5p target gene. As shown in Figure 4H and Figure S18, Supporting Information, let-7i-5p overexpression significantly increased the phosphorylation of JNK, ERK and p38 in recipient cells, similar to treatment with EVpackaged let-7i-5p derived from PM 2.5 -treated HBE cells. Further dual-luciferase reporter assays showed that let-7i-5p significantly reduced the luciferase activity of the wild-type DUSP1 3′-UTR reporter ( Figure 4I). Moreover, let-7i-5p overexpression or treatment with EV-packaged let-7i-5p released by PM 2.5 -treated HBE cells dramatically inhibited DUSP1 expression in both normal HBE cells and sensitive HBSMCs ( Figure 4J and Figure S19, Supporting Information). Furthermore, DUSP1 knockdown in recipient cells led to potent activation of the MAPK signaling pathway ( Figure 4K and Figure S20, Supporting Information), induced cytotoxicity in HBE cells and increased the contractility of sensitive HBSMCs ( Figure S21, Supporting Information). These findings imply that EV-packaged let-7i-5p derived from PM 2.5 -treated HBE cells may inhibit DUSP1 expression to increase the phosphorylation of JNK, ERK and p38, thereby activating the MAPK signaling pathway and inducing asthma attacks through both "horizontal" and "longitudinal" mechanisms.

EV-Packaged Let-7i-5p Mediates PM 2.5 Exposure-Induced Asthma In Vivo
We established a model of ovalbumin (OVA)-induced asthma in juvenile mice and subjected the mice to the indicated treatments to assess the role of EV-packaged let-7i-5p in PM 2.5 -induced asthma in vivo ( Figure 5A). Consistent with the in vitro observations, treatment with PM 2.5 or PM 2.5 +let-7i-5p EVs significantly increased bronchoconstriction ( Figure 5B), increased the levels of three contractile proteins ( Figure S22, Supporting Information), upregulated EV-packaged let-7i-5p in bronchoalveolar lavage fluid (BALF) (Figure 5C), reduced the DUSP1 mRNA level in BALF ( Figure 5D), and damaged lung tissues ( Figure 5E); these effects were accompanied by DUSP1 downregulation (Figure 5F) and an elevated let-7i-5p level in lung tissues ( Figure 5G). Compared with the OVA or OVA+PM 2.5 +NC-EVs group, the levels of phosphorylated JNK, ERK and p38 were increased in the OVA+PM 2.5 or OVA+PM 2.5 +let-7i-5p EVs group ( Figure S23, Supporting Information), indicating that EV-packaged let-7i-5p stimulated PM 2.5 -induced asthma by activating the MAPK signaling pathway. Furthermore, the IL-6 concentration in BALF was significantly increased by the same treatment, further confirming the establishment of the mouse model of OVA-induced asthma ( Figure 5H). The observations described the above highlight that EV-packaged let-7i-5p promotes PM 2.5 -induced airway contraction and cytotoxicity by inhibiting its target gene DUSP1.

Discussion
In the current study, we first conducted RNA sequencing of EVpackaged miRNAs in plasma from children with asthma and healthy controls and PM 2.5 /NC-treated HBE cell models, and we identified a novel miRNA, let-7i-5p, expressed at high levels in PM 2.5 -EVs and plasma from children with asthma. Let-7i-5p was packaged into EVs through an interaction with ELAVL1 and then taken up by "horizontal" recipient normal HBE cells and "longitudinal" recipient-sensitive HBSMCs. Functionally, EV-packaged let-7i-5p activated the MAPK signaling pathway by inhibiting its target gene DUSP1, resulting in cytotoxicity in "horizontal" recipient HBE cells and increased contractility of "longitudinal" recipient-sensitive HBSMCs ( Figure 5I).
Because the composition of PM 2.5 is easily affected by the geographical location and meteorological conditions, [26,27] we used National Institute of Standards and Technology (NIST) reference materials that have a well-characterized chemical content of . Let-7i-5p is packaged into PM 2.5 -EVs in an ELAVL1-dependent manner and regulates the DUSP1 expression level in recipient cells. A) The specific interaction between the let-7i-5p sequence and RNA-binding protein motifs was predicted by RBPDB (threshold value = 0.7). B) Immunofluorescence assessment of let-7i-5p (red) and ELAVL1 (green) colocalization in HBE cells. Scale bar, 20 μm. C) Upper panel: image of gel electrophoresis of PCR products from the RIP assay. Lower panel: an RIP assay with an anti-ELAVL1 antibody or control IgG was performed on PM 2.5 /NC-treated HBE cells. The level of let-7i-5p in the immunoprecipitates was determined using RT-qPCR. D) The level of the ELAVL1 mRNA in PM 2.5 /NC-treated HBE cells was determined using RT-qPCR. E) The level of let-7i-5p in EVs derived from HBE cells transfected with si-ELAVL1 was determined using RT-qPCR. F) The potential target genes containing the let-7i-5p seed sequence were predicted using the Diana-MicroT, MiRwalk and TargetScan databases. G) KEGG pathway enrichment analysis of the 169 target genes. H) The protein levels of MAPK pathway-related signaling molecules in NC mimic-, let-7i-5p mimic-, NC-EVs-, or PM 2.5 -EVs-treated recipient cells (HBE cells and sensitive HBSMCs) were determined using Western blotting. I) The binding affinity of let-7i-5p for DUSP1 was assessed using a dual-luciferase reporter assay. J) The expression of the DUSP1 protein in NC mimic-, let-7i-5p mimic-, NC-EVs, or PM 2.5 -EVs-treated HBE cells (left panel) and sensitive HBSMCs (right panel) was detected using Western blotting. K) The protein levels of MAPK pathway-related signaling molecules in NC-or si-DUPS1-transfected HBE cells (left panel) and sensitive HBSMCs (right panel) were determined using Western blotting. Statistical significance was assessed using a two-tailed Student's t-test. Values represent means ± SD. * p < 0.05. Figure 5. EV-packaged let-7i-5p induces asthma in vivo. Thirty-two female BALB/c mice were randomly assigned to the OVA group (group 1), OVA+PM 2.5 group (group 2), OVA+PM 2.5 +NC-EVs group (group 3), or OVA+PM 2.5 +let-7i-5p-EVs group (group 4). A) Schematic showing the process used to establish the mouse model. The green arrows indicate that each mouse was injected with 28.8 μL of the OVA suspension containing 3.6 μg of OVA and 144 μg of Al(OH) 3 . The yellow arrows indicate that mice were exposed to 2% OVA for 20 min. The grey arrows indicate that mice were exposed to 1.57 mg kg −1 PM 2.5 . The blue arrows indicate that mice were challenged by an endotracheal instillation of 2 μg of NC-EVs. The red arrows indicate that mice were challenged by an endotracheal instillation of 2 μg of let-7i-5p-EVs. B) Detection of bronchoconstriction using myography. C) The level of EV-packaged let-7i-5p in BALF was determined using RT-qPCR. D) The level of DUSP1 in BALF was determined using RT-qPCR. E) Representative images of H&E staining in lung tissues and total lung injury scores for each group. Scale bar, 50 μm. F) Representative photographs and quantification of DUSP1 immunostaining in lung tissues. Scale bar, 50 μm. G) The level of let-7i-5p in lung tissues was determined using RT-qPCR. H) The concentration of IL-6 in BALF was measured using ELISA. I) In the proposed model, EV-packaged let-7i-5p secreted by PM 2.5 -treated HBE cells induce asthma by regulating the expression of its target gene DUSP1 and activating the MAPK signaling pathway in recipient cells. MVB, multivesicular bodies. Statistical significance was assessed using two-tailed Student's t-test. Values represent means ± SD. * p < 0.05. PM 2.5 and represent urban PM 2.5 levels. Our results revealed that PM 2.5 decreased the viability of normal HBE cells, consistent with our previous studies. [28] We then hypothesized that PM 2.5 -treated HBE cells might secrete EVs to regulate the biological function of neighboring or distant cells. Our coculture experiments indicated that EVs released by PM 2.5 -treated HBE cells may induce cytotoxicity in normal HBE cells. As expected, PM 2.5 -EVs were efficiently transferred into recipient HBE cells [29,30] and then "horizontally" induce cytotoxicity in HBE cells; however, this effect was abolished by pretreatment of recipient HBE cells with the EV secretion inhibitor GW4869. The biological effects of PM 2.5 and PM 2.5 -EVs on the cell cycle of HBE cells were not consistent because PM 2.5 -EVs represent the effect of aberrantly expressed intracellular cargos carried by PM 2.5 -EVs. Similarly, PM 2.5 -EVs were taken up by recipient-sensitive HBSMCs and subsequently promoted sensitive HBSMC contractility by directly increasing the expression of -SMA, SM-MHC and RhoA. Thus, PM 2.5 -EVs play a key role in PM 2.5 -induced asthma through both "horizontal" and "longitudinal" mechanisms. The air-liquid interface (ALI) model is becoming an efficient and realistic tool for bronchial epithelial toxicity testing following exposure to air pollutants. [31] A better understanding of the biological function of EV-packaged let-7i-5p in PM 2.5 -induced asthma based on the ALI model is needed.
Based on RNA sequencing, we determined that let-7i-5p was overexpressed in PM 2.5 -EVs and plasma-derived EVs from children with asthma and was mainly encapsulated in EVs instead of being freely secreted. Let-7i-5p, a member of the lethal-7 (let-7) miRNA family, has been detected in various tissues and cell types (i.e., lung tissue, smooth muscle, epithelial cells, and macrophages). [15,32] To date, only one study has reported that let-7i-5p was differentially expressed in bronchial cells from steroidnaive subjects with asthma and was downregulated in patients with asthma, [25] which is inconsistent with our results. The main explanation for the inconsistency might be the difference in the ages and ethnicity of the subjects. Various studies have indicated that EVs are ideal biomarkers of many diseases; in addition to being stable in body fluids, EVs carry disease-specific functional molecules. [33] The advantages of EV-packaged let-7i-5p are based on three aspects. First, we determined that plasma EV-packaged let-7i-5p levels were positively correlated with PM 2.5 exposure in children with asthma, indicating that let-7i-5p is a specific predictor of PM 2.5 -induced asthma in children. Second, we observed a significant increase in EV-packaged let-7i-5p levels produced by PM 2.5 -stimulated HBE cells, and EV-packaged let-7i-5p performs crucial functions in effector cells of asthma (HBE and sensitive HBSMCs), which is helpful for both explaining the biological mechanism of PM 2.5 -induced asthma and further investigating therapeutic approaches. Third, EV-packaged let-7i-5p was stable in plasma and had the ability to discriminate children with asthma from healthy controls with acceptable sensitivity and specificity. These observations imply that EV-packaged let-7i-5p has the potential to serve as a specific biomarker for PM 2.5 -induced childhood asthma compared with a series of classical predictors. Furthermore, further investigations are needed to determine whether EV-packaged let-7i-5p is related to asthma induced by other allergens.
Interestingly, we revealed that EV-packaged let-7i-5p was delivered into recipient cells and then "horizontally" induced cy-totoxicity in HBE cells and "longitudinally" promoted the contractility of sensitive HBSMCs. This conclusion was based on the findings listed below. 1) Let-7i-5p overexpression caused cytotoxicity in HBE cells and increased the expression of contractile proteins in sensitive HBSMCs; 2) HBE cells transfected with Cy3labelled let-7i-5p mimic were cocultured with recipient cells [34] to show that EV-packaged let-7i-5p produced by HBE cells was directly transferred into either "horizontal" or "longitudinal" recipient cells; 3) Let-7i-5p overexpression in recipient cells was caused mainly by treatment with PM 2.5 -EVs rather than by direct exposure to PM 2.5 ; 4) Incubation with (Anti-let-7i-5p+PM 2.5 )-EVs significantly decreased the capacity of PM 2.5 -EVs to inhibit cytotoxicity in "horizontal" recipient HBE cells and decrease -SMA, SM-MHC and RhoA expression levels in "longitudinal" recipient-sensitive HBSMCs; and 5) Transfection of the let-7i-5p mimic into recipient cells significantly increased the capacity of PM 2.5 -EVs to induce asthma. Asthma is driven by a complex cellular communication network composed of multiple cell types. [10,35] The cell-cell communication of EV-packaged let-7i-5p with other cell types in the cell microenvironment of asthma must be investigated in further studies. Moreover, PM 2.5 accelerated the production of EV-packaged let-7i-5p by HBE cells by upregulating levels of the EV packaging-related protein ELAVL1 rather than increasing cellular let-7i-5p expression levels. Notably, previous studies have reported that EV-packaged miRNAs are selectively packaged into EVs by several RBPs, including hn-RNPA2B1, hnRNPQ and ELAVL1. [23,36,37] Consistent with these results, ELAVL1 (also called HuR) specifically bound let-7i-5p through a specific motif (GUUU) and directed the sorting of let-7i-5p into EVs in the present study, which might suggest unique strategies to eliminate EV-packaged let-7i-5p during PM 2.5 exposure. In addition to RBPs, Ago2 and Y-box protein 1 have been reported to participate in the EV packaged export of miRNA. [38,39] Additionally, lncRNA-miRNA interactions have recently been described as a new regulatory mechanism for EV-packaged miRNA export. [40] Thus, we need to fully elucidate the regulatory mechanism in further studies.
The MAPK signaling pathway is involved in almost all aspects of asthma pathophysiology, and its activity is self-adjusting between phosphorylation and dephosphorylation. [41,42] Extracellular stimulation (by environmental exposure, growth factors or proinflammatory cytokines) leads mainly to the activation of the three main MAPK signaling pathway modules: JNK, ERK and p38. [43] Here, we found that EV-packaged let-7i-5p secreted by PM 2.5 -treated HBE cells increased the levels of phosphorylated JNK, ERK and p38 by suppressing the expression of the let-7i-5p target gene DUSP1 in both HBE cells and sensitive HB-SMCs, thereby activating the MAPK signaling pathway to induce asthma. Consistent with our in vitro results, juvenile mice treated with EV-packaged let-7i-5p exhibited more severe bronchial contraction, higher levels of EV-packaged let-7i-5p in BALF and lower DUSP1 protein levels in lung tissues compared to those in control mice (OVA group or OVA+PM 2.5 +NC-EVs group). Because all the functions we investigated were based on OVA-induced asthmatic mouse models treated with PM 2.5 , OVA and PM 2.5 could be used as internal controls for the OVA+PM 2.5 +NC/let-7i-5p EVs group, thereby eliminating the effect of OVA or PM 2.5 on target molecules and other indices. Interestingly, EV-packaged let-7i-5p regulated the MAPK signaling pathway through the target gene DUSP1 and increased IL-6 expression levels. These results indicate that EV-packaged let-7i-5p may regulate the release of inflammatory factors. Thus, studies exploring the function of let-7i-5p in inflammatory cells are urgently needed. Due to the lower amount of plasma in the juvenile mouse model, we were unable to detect plasma EV-packaged let-7i-5p in vivo. We therefore isolated BALF samples in vivo to measure the expression of EV-packaged let-7i-5p. Overexpression and knockout of let-7i-5p in mice exposed to OVA and PM 2.5 are necessary controls to further verify the biological effects of EV-packaged let-7i-5p on PM 2.5 -induced asthma. Moreover, the CRISPR/Cas9 system will be used to generate let-7i-5p knockout cell lines, thereby further validating the effect of EV-packaged let-7i-5p on PM 2.5 -induced asthma in vivo and in vitro. House dust mites (HDMs), important natural allergens, have been shown to more closely mimic human asthma than OVA when constructing an asthmatic mouse model. [44] Therefore, we will use the HDM-induced asthmatic mouse model to further validate our results.
In summary, our findings provide evidence of a mechanism underlying PM 2.5 -induced childhood asthma in which EVpackaged let-7i-5p "horizontally" induces cytotoxicity in HBE cells and "longitudinally" promotes the contractility of sensitive HBSMCs by inhibiting the expression of its target gene DUSP1. In addition, EV-packaged let-7i-5p was upregulated in the plasma of children with asthma, and the EV-packaged let-7i-5p level was positively correlated with PM 2.5 exposure. Our results not only revealed a crucial mechanism of EV-packaged miRNA-regulated cell-cell communication from PM 2.5 -treated HBE cells to recipient cells to induce asthma, but also opened a new avenue for a diagnostic strategy and therapeutic approach for asthma in children exposed to high levels of PM 2.5.

Experimental Section
Study Subjects and Clinical Samples: Beginning in July 2017, the authors recruited 110 children from Children's Hospital of Nanjing Medical University (Nanjing, China) and collected their plasma samples. Children with asthma were diagnosed according to the guidelines for the diagnosis, prevention and treatment of childhood asthma (2016 edition) formulated by the Chinese Medical Association. During the same period, non-asthmatic children who were age-and sex-matched by the frequency with the cases were selected as healthy controls. Written informed consent forms were signed by the children and their parents, and the study protocol was approved by the Ethics Committee of Nanjing Medical University (2017-392). PM 2.5 Exposure Assessment: The PM 2.5 concentration distribution was obtained from China National Environmental Monitoring Center (CNEMC) data provided by Qingyue Data (https://data.epmap.org). The PM 2.5 concentration at the site 30 days before recruitment of the subjects into this study was recorded as the average concentration in the casecontrol study. XGeocoding software was utilized to obtain the latitude and longitude of the subjects' residence and monitoring sites. Inverse distance weighted interpolation (IDW) is an important spatial interpolation method in ArcGIS software (version 10.2), that was employed to evaluate the individual PM 2.5 exposure concentrations. [45,46] The details are explained as follows: in Equation (1), Z (u) denotes the PM 2.5 exposure concentration of subjects, z(u i ) is the observed value at the n measured sites, and d a represents the inverse distance weighted interpolation (IDW) weight distance.
Treatment of Recipient Cells with PM 2.5 : Standard reference material 2786 (SRM2786) from the NIST was used as the PM 2.5 reference in this study. [28] The PM 2.5 standard was suspended in culture medium at a concentration of 2 mg mL −1 . When recipient cells reached 50-60% confluence at the bottom of the petri dish, recipient cells were treated with different concentrations (0, 31.25, 62.5, 125, 250, 500, 1000, or 1500 μg mL −1 ) of the stock PM 2.5 standard suspension for 12, 24 or 48 h. [47] EV Isolation, Characterization, and Internalization: EVs were isolated from the culture medium of HBE cells with an ExoQuick TC Kit (SBI, USA), and EVs were purified from the plasma of children using an Exo-Quick Plasma Prep with Thrombin Kit (SBI, USA). The shape and size of EVs were assessed using transmission electron microscopy (TEM) (Tecnai G2, FEI, USA). The size distribution of EVs was quantified using nanoflow cytometry (NanoFCM) with a U30 Flow NanoAnalyzer (NanoFCM, Inc., China) and technical assistance provided by KeyGEN Biotech Co. Ltd (Jiangsu Province, China). Western blotting was utilized to detect the EV protein markers CD63 (antibody: ab134045, Abcam, USA), TSG101 (antibody: ab125011, Abcam, USA) and Alix (antibody: 92880, CST, USA), and the negative controls calnexin (antibody: ab133615, Abcam, USA), Grp94 (antibody: ab108606, Abcam, USA), APOA1 (antibody: ab52945, Abcam, USA), and albumin (antibody: ab207327, Abcam, USA) to confirm that active substances isolated from culture medium or plasma were EVs. The 2 μg of PM 2.5 /NC-EVs diluted with 200 μL of PBS were extracted (100 μL) and then labelled with 4 μL of PKH67 (green fluorescence; Sigma, USA) and 1 mL Diluent C (Sigma, USA). After an incubation for 4 min at room temperature, PM 2.5 /NC-EV labelling was terminated by the addition of 2 mL of 0.5% bovine serum albumin (BSA). Dyed EVs were isolated from the above staining liquid using an ExoQuick TC Kit to remove unbound dye from the PKH67-labelled PM 2.5 /NC-EVs. The dyed EVs were added to recipient cells and incubated for 3 h. Fluorescence microscopy (Zeiss, Germany) was used to visualize PKH67-labelled EVs in recipient cells. The details of this protocol are described in the authors' previous study. [30] EV Treatment: The concentration of EVs was determined with a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, China). For in vitro treatment, 2 μg of EVs (equivalent to ≈1 × 10 7 of the indicated HBE cells) were incubated with 2 × 10 5 recipient cells for 24 h. EV secretion was inhibited by pretreating normal HBE cells with 20 μM GW4869 (Sigma, USA) or 0.005% DMSO (Sigma, USA) as a control for 6 h and then exposing cells to the PM 2.5 standard for 24 h. The culture medium of these cells was prepared for EV isolation or RNA extraction. The indicated HBE cells were treated with 2 μg mL −1 RNaseA (Beyotime Institute of Biotechnology, China) alone or in combination with 0.1% Triton X-100 (Beyotime Institute of Biotechnology, China) for 30 min, and the expression of miRNAs in the culture medium of HBE cells was detected using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) to investigate the expression pattern of extracellular miRNAs.
Analysis of the EV-Packaged miRNA Expression Profile: EV-packaged RNA was isolated from the plasma of children with asthma or nonasthmatic children, from which two pooled plasma samples of 4.8 mL each (4.8 mL = 8 samples × 600 μL/sample) were obtained (Table S3, Supporting Information). Moreover, EV-packaged RNA was isolated from the culture medium of PM 2.5 /NC-treated HBE cells. The EV-packaged miRNA expression profiles of plasma from children and culture medium from the cell models were generated with the Illumina HiSeq 2500 platform (Novogene Co., Ltd., China).
Statistical Analysis: Significant differences between the two groups were analyzed using Student's t-test or Pearson's 2 test. The ROC curve was analyzed to obtain the AUC values and evaluate the capability for discriminating cases from healthy controls. Spearman's correlation analysis was utilized to determine the relationship between PM 2.5 exposure and the plasma level of EV-packaged let-7i-5p. Three target prediction software programs, Diana-MicroT, MiRwalk and TargetScan, were used to predict putative targets of let-7i-5p. KEGG pathway analyses were performed with Database for Annotation, Visualization and Integrated Discovery (DAVID) 6.8 software (https://david-d.ncifcrf.gov/) to identify the main biological pathways of the differentially expressed genes. RBPDB(http://rbpdb.ccbr. utoronto.ca/) was used to predict the interaction between motifs of RBPs and let-7i-5p . [48] p < 0.05 was considered statistically significant. All experiments were replicated at least three times. Quantitative data are presented as the means ± standard deviations (SD). Statistical analyses were performed with SAS 9.1.3 software (SAS Institute, Inc., USA). Additional experimental methods are described in the Supporting Information Materials and Methods.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.