Exosomal miRNA-19a and miRNA-614 Induced by Air Pollutants Promote Proinflammatory M1 Macrophage Polarization via Regulation of RORα Expression in Human Respiratory Mucosal Microenvironment

According to the World Health Organization (WHO), currently, more than 91% of the global population and 80% of city dwellers live in areas where the air pollution exceeds WHO guideline limits and particulate matter (PM) contributes to 8 million premature deaths each year (1). The WHO reports strong relationships between air pollution exposure and diseases, such as acute respiratory infections and chronic inflammatory diseases. Rhinosinusitis is a representative inflammatory disease of the nasal cavities and sinuses resulting from various causes, including exposure to viruses, bacteria, and air pollutants. According to the Sinus Allergy Health Partnership and the American Rhinologic Society, rhinosinusitis is currently defined as acute rhinosinusitis, which manifests for up to 4 wk, and chronic rhinosinusitis (CRS), which has the same symptom profile lasting for more than 12 wk (2). CRS inflammation involves a number of proinflammatory cytokines as well as cell infiltration of macrophages and lymphocytes (3). Despite many studies on rhinosinusitis over the last few decades, causative factors and mechanisms are not yet fully understood (4).

Air pollution is largely composed of urban PM that increases the risk of cancer and pulmonary and cardiovascular diseases (5, 6). PM is classified by aerodynamic diameter into coarse, fine, and ultrafine particles (7). Upon inhalation, large particles (>10 μm) can only reach the nasal epithelium, whereas smaller particles penetrate deep into the alveolar space from the nasal cavity (8). It is known that PM exposure causes inflammatory and autoimmune diseases in humans (9–12). However, the molecular mechanism through which PM drives inflammation and causes the disease is not fully known. The main targets of inhaled PM are airway epithelial cells and immune cells, such as macrophages, which are powerful producers of proinflammatory mediators that regulate the local inflammatory response of the airways (13, 14).

Numerous studies suggest microRNAs (miRNAs), which are small endogenous noncoding RNAs, as key regulators of macrophage differentiation, infiltration, and activation (15). These miRNAs regulate gene expression by reducing the translation and stability of target mRNAs through direct binding to the 3′-untranslated region (UTR). They are recently considered as regulators of many genes in inflammatory and autoimmune diseases. However, the targets of most miRNAs are unknown, and their roles responding to air pollution are not clearly understood (16).

Retinoic acid–related orphan receptors (RORs) play a crucial role in embryonic development, cellular differentiation, cancers, metabolic pathways, and inflammatory signaling pathways (17–21). In mammals, RORs have three major isoforms: α, β, and γ. Human macrophages primarily produce RORα, known as NR1F1, which regulates the polarization of human macrophages (22, 23). RORα is known to exert anti-inflammatory effects in cells by regulating inflammatory gene expression. Nevertheless, the molecular mechanism by which the expression of RORα is regulated in inflammation is not fully understood (24–27).

In this study, we report that exosomal miRNA-19a and miRNA-614 induced by PM promote proinflammatory M1 macrophage polarization via the regulation of RORα expression in the human respiratory mucosal microenvironment. Human nasal epithelial cells exposed to PM induce inflammatory miRNAs and deliver miRNAs to macrophages through exosomes. Our data strongly demonstrate that the regulation of RORα expression via exosomal miRNAs plays a crucial role in the proinflammatory mucosal microenvironment and M1/M2 macrophage polarization. Therapeutic strategies targeting these miRNAs could contribute to the treatment of proinflammatory diseases related to air pollution, including CRS.

Standard Reference Material (SRM) 1648a, which is atmospheric PM collected in an urban area, was purchased from the National Institute of Standards and Technology (Gaithersburg, MD). SRM 1648a has a 0- to 100-μm-diameter particle size distribution and a mean particle diameter of 5.85 μm. It was prepared in PBS and mixed well before use in each experiment.

Human nasal epithelial cell line RPMI 2650 (American Type Culture Collection CCL-30) and human monocyte cell line THP-1 (TIB-202) cells were purchased from the American Type Culture Collection (Rockville, MD) and maintained in Eagle’s MEM and Roswell Park Memorial Institute medium supplemented with 10% FBS and antibiotics in a humidified atmosphere with 5% CO₂ at 37°C. All cell lines were authenticated by the American Type Culture Collection and were consistent with their presumptive identity.

The primary human nasal epithelial cells (pHNEs) were harvested by scraping from the human inferior turbinate of patients during augmentation rhinoplasty and cultured in airway epithelial cell growth medium (PromoCell, Heidelberg, Germany) after being treated with dispase for 24 h at 4°C (28). We collected a total of nine pairs of normal and CRS patient samples from Korea University Hospital. Among them, four pairs were used for immunohistochemistry and immunofluorescence staining, and five pairs were used for miRNA, mRNA analysis, and Western blot. The statistical significance between the normal and CRS patients was determined by two-way ANOVA with Sidak multiple comparison test. CRS diagnosis was determined according to the current European position paper on rhinosinusitis and nasal polyps. Subjects were excluded if they used any oral or topical medication, including steroids, antihistamines, or antibiotics at least 3 mo prior to the surgery. Nasal polyp tissue was obtained from patients undergoing endoscopic sinus surgery. The CRS with nasal polyp (CRSwNP) tissue was immunohistochemically verified by H&E staining as noneosinophilic CRSwNP (NECRSwNP) tissue. Prior to obtaining the uncinate process and nasal polyp, this study protocol was approved by the Institutional Review Board for Human Studies at Korea University Hospital, College of Medicine, Seoul, Republic of Korea (No. 2018AN0061), and all participants provided written informed consent.

Patient-derived cultured human nasal epithelial cells were harvested after 100 μg/ml SRM 1648a treatment for 24 h. The TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina, San Diego, CA) was used to construct cDNA libraries. Poly A–selected RNA extraction, RNA fragmentation, random hexamer-primed reverse transcription, and 100-nt paired-end sequencing with the Illumina HiSeq2500 sequencer (Illumina) were performed. Low-quality raw reads were eliminated, and data were aligned to Homo sapiens (hg19) using HISAT v2.0.5. The sorted readings were configured to estimate their abundance as fragments per kilobase of exon per million fragments mapped values of the transcript and gene expressed in each sample using StringTie v1.3.3b (28).

The RPMI 2650 cell line was treated with 0, 25, 50, 100, 200, and 400 μg/ml SRM 1648a and incubated for 48 h. The cell viability and cytotoxicity were measured using CellTiter-Glo Luminescent Cell Viability Assay (catalog no. G7570; Promega) and CellTox Green Cytotoxicity Assay (G8741; Promega) by GloMax Discover Multimode Microplate Reader (Promega, Madison, WI).

To differentiate human THP-1 monocytes into macrophages, THP-1 cells were treated with 12-O-tetradecanoylphorbol-l3-acetate (catalog no. P1585, PMA; Sigma-Aldrich, St. Louis, MO) for 48 h in a humidified atmosphere with 5% CO₂ at 37°C. The M0 macrophages were differentiated into M1-like macrophages by administering 100 ng/ml LPS (catalog no. L2630; Sigma-Aldrich) and 20 ng/ml hIFN-ɤ (catalog no. 285-IF-100; R&D Systems, Minneapolis, MN) or M2-like macrophages by administering 20 ng/ml hIL-4 (catalog no. 204-IL-010; R&D Systems) and 20 ng/ml hIL-13 (catalog no. 213-ILB-005; R&D Systems) in complete RPMI media for 48 h (29–32).

Conditioned medium was harvested from SRM 1648a-treated cultured RPMI 2650 cells. All experiments were performed at 4°C. Cells and large debris were removed by centrifugation at 200 × g for 10 min. The supernatant fraction was subjected to sequential centrifugation at 2000 × g for 20 min and 10,000 × g for 30 min, passed through a 0.22-μM polystyrene vacuum filter (Corning, Corning, NY), and centrifuged at 100,000 × g for 70 min. The pellet containing exosomes was washed twice in PBS (pH 7.4) by centrifugation at 100,000 × g for 70 min. The washed pellet material was resuspended in 500 μl of PBS (pH 7.4) by trituration using a large-bore pipette over 30 min at 4°C (33).

miRNA was extracted using RNAzol RT (catalog no. R4533; Sigma-Aldrich), which can be used to isolate separate fractions of mRNA and miRNA. After isolation, miRNA was converted into cDNA templates for quantitative PCR (qPCR) using the MystiCq miRNA cDNA Synthesis Mix Kit (cat no. MIRRT; Sigma-Aldrich). Because of its small size, miRNA was first subjected to polyadenylation in a poly-A polymerase reaction. Then, the poly-A–tailed miRNA was converted into first-strand cDNA using reverse transcriptase and an oligo-dT adapter primer. The amplification of miRNA cDNAs was quantified in real-time SYBR Green RT-qPCR reactions according to the recommended two-step cycling procedure (preincubation/activation at 95°C for 2 min followed by 40 cycles of 95°C for 5 s, 60°C for the 30 s). MystiCq miRNA Universal PCR Primer (cat no. MIRUP; Sigma-Aldrich) and individual MystiCq microRNA qPCR Assay Primer (cat no. MIRAP00038 for hsa-miR-19a and cat no. MIRAP00599 for hsa-miR-614) were used. MiRNA RT-qPCR was performed using the Applied Biosystems 7500 real-time cyclers (Applied Biosystems, Foster City, CA). The target gene expression was quantified using a kit containing the human positive control primer, the small nucleolar RNA SNORD44.

miRNA control, mimic, and inhibitor; small interfering RNA (siRNA) duplexes against RORA (5ʹ-CUC AGA ACA ACA CCG UGU A-3ʹ, 5ʹ-UAC ACG GUG UUG UUC UGA G-3ʹ); and the control siRNA duplex serving as the negative control were purchased from Bioneer (Daejeon, Korea). Transfection of miRNAs or siRNAs was performed using the Neon Transfection System (Thermo Fisher Scientific, Waltham, MA) or Lipofectamine RNAiMAX in vitro Transfection Reagent (Thermo Fisher Scientific).

The total RNA in THP-1 cells was extracted using TRIzol reagent (cat no. 15596018; Invitrogen). cDNA was amplified and quantified using SYBR Premix Ex Taq (cat no. RR420L; Takara, Tokyo, Japan) with the following primers: TNF_α_F: 5′-GCT GCA CTT TGG AGT GAT CG-3′, TNF_α_R: 5′-TCA CTC GGG GTT CGA GAA GA-3′, IL-1B_F: 5′-CCA CCT CCA GGG ACA GGA TA-3′, IL-1B_R: 5′-TTT GGG ATC TAC ACT CTC CAG C-3′, IL-6_F: 5′-ATG TGT GAA AGC AGC AAA GAG-3′, IL-6_R: 5′-CAC CAG GCA AGT CTC CTC A-3′, DC_SIGN_F: 5′-GAA CTG GCA CGA CTC CAT CA-3′, DC_SIGN_R: 5′-GTT GGG CTC TCC TCT GTT CC-3′, RORα_F: 5′-CAC GAC GAC CTC AGT AAC TAC A-3′, RORα_R: 5′-TGG TGA ACG AAC AGT AGG GAA-3′, GAPDH_F: 5′-GTC AGT GGT GGA CCT GAC CT-3′, and GAPDH_R: 5′-AAA GGT GGA GGA GTG GGT GT-3′. Real-time PCR was performed using the Applied Biosystems 7500 real-time cyclers.

For macrophage differentiation analysis, siRNA-transfected M0 macrophages were blocked with human BD Fc Block (cat no. 564219; BD Biosciences, San Jose, CA) and stained with PE anti-human CD209 (DC-SIGN) Ab (cat no. 330105; BioLegend, San Diego, CA). The assay was performed on a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN) and analyzed using FlowJo_V10 software.

The 3′-UTR fragment of RORA (RORA_XhoI-F: 5′-TAGGCGATCG CTCGAG GAGTACCAAGCTTAGTTCAG-3′, RORA_XhoI-R: 5′-AATTCCCGGG CTCGAG TCGATCGACATTCCTTAGCC-3′) containing putative binding sites for miR-19a and miR-614 was cloned into psiCHECK-2 Vector (Promega). The sequences of the constructed plasmids were confirmed by sequencing analysis using an internal primer (5′-AAGTGGTCCAGACAAGGC-3′) and a universal primer. For luciferase reporter assays, the M0 macrophages were cotransfected with psiCHECK-2-WT or psiCHECK-2-RORA-3′-UTR reporter constructs along with miR-19a, miR-614, or negative control miRNA using Lipofectamine 2000 (Invitrogen). After 48 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) and GloMax-Multi Detection System (Promega). Renilla luciferase data were normalized to firefly luciferase data (34).

To measure human TNF-α protein levels from supernatants from the miRNA-transfected M0 macrophage cell cultures, we performed ELISA with a TNF-α Human Uncoated ELISA Kit (cat no. 88-7346; Invitrogen). The TNF-α ELISA was measured by GloMax Discover Multimode Microplate Reader (Promega) at 450 nm.

Supernatants obtained from the M0 macrophages, which were transfected with siRNAs using Lipofectamine RNAiMAX, were diluted and mixed with a biotinylated detection Ab mixture for a human cytokine array (cat no. ARY005B; R&D Systems). The luminescent images were obtained using an iBright Western blot Imaging System (Thermo Fisher Scientific). The pixel densities on the developed membrane were analyzed using iBright Analysis Software (Thermo Fisher Scientific) (35).

Immunohistochemistry assay was performed using normal samples and CRS patient samples. The frozen tissue sections were prepared using the cryotome for immunohistochemistry. After hydration and fixation, the samples were immersed in Ag retrieval solution for 15 min and blocked in 3% BSA at room temperature for 45 min. The immunoreaction was performed with anti-RORα (PA5-11224; 1:25; Invitrogen) primary Abs in the blocking solution at 4°C overnight. For the secondary Ab, HRP-labeled anti-rabbit IgG (31460; 1:1000; Cell Signaling Technology, Danvers, MA) was incubated at room temperature for 1 h. The samples were incubated with and then reacted with a 3,3′-diaminobenzidine (DAB) kit (sk-4100; Vector Labs, Burlingame, CA). The reaction was developed with DAB, nickel sulfate, and hydrogen peroxide. Cell nuclei were counterstained with hematoxylin (BioGnost, Zagreb, Croatia). The slide was scanned by Aperio AT2 (Leica, Wetzlar, Germany).

Immunofluorescence assay was performed using normal samples and CRS patient samples. The samples were blocked for 45 min with 3% BSA. After incubating overnight with anti-RORA (PA5-11224; 1:25; Invitrogen), CD 14 (46-0149-41; 1:100; Invitrogen), CD 19 (302225; 1:100; BioLegend), CD 3 (300405; 1:100; BioLegend), and CD68 (14-0688-82; 1:100; Invitrogen) at 4°C, the samples were washed three times with PBS and incubated for 1 h with Rhodamine Red-X (R6394; 1:400; Invitrogen) and Alexa Fluor 488 goat anti-mouse IgG (A11001; 1:100; Invitrogen) at room temperature. The fluorescent signals were visualized using a confocal microscope (LSM700; Carl Zeiss, Oberkochen, Germany).

All experiments in the current study were performed in triplicate. All statistical values were presented as mean ± SD. One-way ANOVA with Tukey multiple comparison test was used to determine the statistical significance of the results obtained in this study. A p value <0.05 was considered statistically significant.

Air pollutants are known to cause various inhalation-related diseases in the human respiratory mucosa. To elucidate the effects of air pollutants, we performed viability and cytotoxicity assays on the human nasal epithelial cell line RPMI 2650. As shown in Fig. 1A, morphological changes in the RPMI 2650 cells and PM accumulation were observed. PM significantly decreased RPMI 2650 cell viability measured using CellTiter-Glo Luminescent Cell Viability Assay while increasing cytotoxicity measured using CellTox Green cytotoxicity assay in a dose-dependent manner (Fig. 1B). The airway mucosal microenvironment consists of epithelial cells and various immune cells. As one of the various immune cells existing in the mucosal microenvironment, macrophages are classified into classically activated macrophages (M1) and alternatively activated macrophages (M2). It is known that M1 macrophages have a proinflammatory phenotype and produce Th1 cytokines (IL-1, IL-6, TNF-α, and IL-23). In contrast, M2 macrophages exert an anti-inflammatory phenotype and produce Th2 cytokines (IL-10). Because nasal epithelial cells are the first barrier exposed to air pollutants, we hypothesized that human nasal epithelial cells damaged by PM modulate the mucosal microenvironment. To test this hypothesis, we treated M0, M1, and M2 macrophages differentiated from human monocytes with conditioned medium from PM-treated nasal epithelial cells. The conditioned medium was generated from RPMI 2650 cell culture treated with PM for 48 h. The PM remaining in conditioned medium was removed by centrifugation prior to macrophage treatment. As shown in Fig. 1C and 1D, the conditioned medium increased the mRNA and protein expression of TNF-α, IL-1β, and IL-6 in the M1 macrophages while decreasing that of DC-SIGN in the M2 macrophages, indicating a shift in the mucosal microenvironment to the proinflammatory state. Taken together, the nasal epithelial cells release unknown factors upon PM exposure to cause inflammation.

To further investigate the molecular mechanism underlying the induction of inflammation and disease after PM exposure, we performed a whole-transcriptome analysis. We administered 100 μg/ml PM to four individual pHNE for 24 h and analyzed them using RNA sequencing. Low-quality genes were eliminated by filtering during data preprocessing and quality check. From all processed sequenced data, we identified a total of 398 differentially regulated miRNAs in pHNEs upon PM exposure. Among these 398 mature human miRNAs, significantly differential expression was validated for 58 miRNAs. Differentially expressed genes were analyzed using the fragments per kb of exon per million mapped reads considering the number of readings, transcript length, and depth of application. To compare differentially expressed genes between samples, 1.3-fold change cut-off was applied, and it was considered differentially expressed when p value was ≤ 0.05. We performed a hierarchical clustering analysis using the 58 validated miRNAs that were differentially expressed between eight samples, which revealed inverse correlations between the control and PM-treated pHNEs (Fig. 2A). The multidimensional scaling plot showed the clear separation of PM-treated samples from the control by displaying the degree of similarity among miRNA expression patterns using two components that best describe the variability of the entire data (Fig. 2B). We also identified meaningful 19 differentially expressed miRNAs with more than 3-fold changes in expression from the list of 58 validated miRNAs (Fig. 2C). Next, we predicted novel upregulated miRNAs (miR-19a, miR-614, and miR-3138) from 19 miRNAs using the TAM 2.0 program, which is a method for enrichment and depletion analysis of a miRNA category in a list of miRNAs (36). The miR-19a was the most upregulated in PM-treated samples compared with the control. The miR-614 was the second highest in the results of TAM 2.0 and had the highest number of reads. The miR-19a has been reported to be related to inflammation and allergic asthma (34, 37). To understand how miR-19a causes inflammation, we predicted the miR-19a target gene networks using the miRNet program, and the integrated platform found RORα as one of the miR-19a targets (Fig. 2D) (38).

Macrophages are one of the major components that regulate inflammation in the mucosal microenvironment. According to the results of TAM 2.0 and miRNet, we hypothesized that human nasal epithelial cells might promote proinflammatory macrophages via upregulated miR-19a and miR-614 upon PM exposure. To test this hypothesis, we first confirmed that the data were consistent in the human nasal epithelial cells, RPMI 2650, by examining PM-enhanced miR-19a and miR-614 expression. Consistent with the abovementioned data, the relative miR-19a and miR-614 expression levels increased with PM treatment compared with those of the control in RPMI 2650 cells (Fig. 3A). Valadi et al. (35) demonstrated that miRNAs could be secreted from cells through exosomes and play critical roles in cell-to-cell communication. To identify epithelial cell–to–macrophage communication, we purified exosomes released from RPMI 2650 cells with or without PM treatment and analyzed miRNAs in the exosomes according to the previous studies (39, 40). As shown in Fig. 3B and 3C, significant increases in miR-19a and miR-614 expression levels were detected in the exosomes purified from the culture supernatant of PM-treated RPMI 2650 and PM-treated pHNEs. The purified exosomes increased the mRNA expression of TNF-α, IL-1β, and IL-6 in M0 macrophages, but the exosomes treated with RNases were not able to induce mRNA expression of inflammatory molecules (Fig. 3D). The results suggest that the upregulated miRNAs can be released from the nasal epithelium exposed to PM via exosomes and travel to other mucosal components in the tissue microenvironment.

To identify the function of epithelial cell–derived miR-19a and miR-614 in macrophage differentiation, M0 macrophages were transfected with miRNAs, and differentiation to M1 or M2 macrophages was induced. The result showed that transfection with miR-19a or miR-614 increased the mRNA expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6), which are considered as M1 macrophage markers in macrophage phenotypes (Fig. 4A). Cytokine array and ELISA data showed enhanced secretion of proinflammatory cytokines (TNF-α, IL-1α, IL-1β, and IL-6) from the M0 macrophages transfected with miR-19a or miR-614 (Fig. 4B, 4C). In contrast to the enhanced M1 macrophage phenotype with miR-19a and miR-614 transfection, both qPCR and flow cytometry analyses showed decreased expression of the M2 marker (DC-SIGN) in macrophages expressing the same miRNAs (Fig. 4D). Based on these results, we concluded that the nasal epithelium could be affected by PM, thereby upregulating and releasing inflammation-related miRNAs via exosomes, and those exosomal miRNAs may alter the mucosal microenvironment through stimulating proinflammatory macrophage differentiation.

While exploring the molecular mechanism underlying how miR-19a and miR-614 stimulate the M1 macrophage phenotype, we predicted RORα as a target of these miRNAs using miRNet (Fig. 2D) and miRDB. To confirm RORα as a target molecule of miR-19a and miR-614, we first traced the changes in RORα expression after transfecting M0 macrophages with miRNA mimic or inhibitor. It was shown that RORα expression was decreased by miR-19a or miR-614 mimics, whereas it was increased by inhibitors, the complementary sequence to each miRNA neutralizing endogenous miRNAs (Fig. 5A). It is well known that miRNAs restrict gene expression by targeting the 3′-UTR of mRNAs to perform mRNA cleavage, thereby repressing translation (41). To confirm the direct regulation of RORα expression by these miRNAs, we predicted putative miR-19a and miR-614 binding sites on the RORA 3ʹ-UTR using the miRWalk program, which predicts miRNA-target interactions with a machine learning algorithm (Fig. 5B). To verify that RORα is a genuine target of miR-19a and miR-614, we cloned 2 kb of the RORα 3′-UTR fragment containing these miRNAs binding sites into the psiCheck2 dual-luciferase vector detecting changes of the expression of target sequences (Supplemental Fig. 1). The relative luciferase activity of the psiCheck2-RORA 3′-UTR reporter was significantly suppressed by cotransfection of miR-19a or miR-614 mimics (Fig. 5C), suggesting that miR-19a and miR-614 repress RORα expression by directly targeting the RORα 3′-UTR. As shown in Fig. 5D, significant decreases in RORα protein expression was detected in the macrophages when treated with the exosomes derived from PM-treated pHNE. The qPCR results showed that the downregulation of RORα increased TNF-α mRNA expression and promoted M1 phenotype polarization (Fig. 5E). Cytokine array analysis also showed that RORα depletion induced proinflammatory macrophages by increasing proinflammatory cytokines (Fig. 5F). Interestingly, the RORα-depleted macrophages could not induce TNF-α mRNA expression when treated with the exosomes derived from PM-treated pHNE (Fig. 5G). Collectively, miR-19a and miR-614 acted as a novel negative regulator of RORα through directly suppressing its mRNA, thereby promoting proinflammatory macrophages.

Because it was confirmed that the induction of miR-19a and miR-614 by PM regulates RORα expression, we next investigated the altered RORα expression in human inflammatory disease to ascertain the function of miRNA-19a and miRNA-614. The immunohistochemical results showed that RORα expression decreased in the CRS patient tissue compared with the normal tissue (Fig. 6A, Supplemental Fig. 2). As expected, miR-19a and miR-614 levels were dramatically increased in the CRS patient sample (Fig. 6B). Consistently, immunofluorescence assay showed a profound reduction in RORα expression in the CRS patient tissue, whereas enhanced expression of immune cell markers such as CD14 (monocyte), CD19 (B cell), and CD3 (T cell) was found in the CRS patient tissue (Fig. 6C, Supplemental Fig. 2). These results revealed that the reduction in RORα expression might be correlated with the enhanced immune cell infiltration and activation in human inflammatory disease compared with those of the normal. Accordingly, the mRNA and protein levels of proinflammatory cytokines were increased as RORα decreased in the CRS patient sample (Fig. 6D, 6E). Inverse correlation of RORα and CD68 (macrophage markers) expression in the human nasal tissue indicates the RORα function as a linchpin modulating the immune response (Fig. 6F). Collectively, these results indicate that miR-19a and miR-614 are inversely correlated with RORα expression and promote the inflammatory disease via downregulation of RORα. Based on these results, we concluded that RORα-mediated attenuation of proinflammatory gene expression is important in normal homeostasis in the human respiratory mucosa (Fig. 7).

Air pollution causes severe inflammation-associated respiratory and autoimmune diseases (9–12). Previous studies have shown that PM in urban air acts on nasal epithelial cells and macrophages, which are the main targets of inhaled toxicants, causing inflammatory stress that induces macrophages to release inflammatory cytokines (42, 43). Inflammatory cytokines are known to recruit and stimulate the activation of inflammatory cells and also improve the response to additional PM exposure (44). However, the molecular mechanism by which PM adversely affects the respiratory system remains unclear. Our study provides evidence that PM might drive mucosal inflammatory diseases through the regulation of miRNA expression. In this context, the current study identifies a function of miRNAs in immune cell regulation, indicating that miRNAs control the human respiratory mucosal microenvironment.

In this study, we investigated the significant differential expression of mature human miRNAs using PM-treated pHNEs. Using microarray and RT-qPCR array techniques, we found that the upregulated novel miRNAs are involved in inflammation. Furthermore, these miRNAs were also increased in exosomes released from the pHNEs. A recent study demonstrated that miRNAs could be transferred from donor cells to target cells via exosomes and influence gene regulation (45). These results suggested that miRNAs in circulating exosomes are transported to target cells, where they exert a direct effect on target genes.

Intriguingly, we found that miR-19a and miR-614 are significantly upregulated by PM and are released through exosomes from human nasal epithelial cells, leading to the differentiation of proinflammatory macrophages. Recently, miR-17-92 clusters have been reported to promote both Th1 response and Th2 cytokine production. In particular, miR-19a, a member of the miR-17-92 cluster, has been shown as associated with inflammation (34, 37). However, the molecular mechanism involved in regulating macrophage differentiation via miR-19a is still unclear. Our study provides evidence that RORα is a target gene of miR-19a and miR-614. In this study, we found that miR-19a and miR-614 directly target the 3′-UTR of RORα and subsequently inhibit the RORα-mediated transcriptional repression of proinflammatory gene expression. The downregulation of RORα promoted M1 phenotype polarization, leading to the hyperinflammatory state of the mucosal microenvironment. RORα is known to be ubiquitously expressed in various tissues (24). Our histological data showed ubiquitous RORα expression in the primary human mucosal tissue under steady-state. Based on the previous studies and our data, we expect that the exosomes released by pHNE can also target many stromal cell types as well as macrophages.

CRSwNP is subclassed as eosinophilic CRSwNP and NECRSwNP. Whereas Th2 immune responses are generally increased in eosinophilic CRSwNP, NECRSwNP shows predominant Th1/Th17 cytokine profiles (3, 46). Our results demonstrated that exposure to particles induces the Th1 inflammatory response, which is similar to the phenotype observed in NECRSwNP, indicating that air pollution might increase the incidence of the inflammatory diseases.

Collectively, this study demonstrates that PM can affect the nasal epithelium by altering inflammation-associated miRNA expression. Specifically, we revealed that miRNA-19a and miRNA-614 released from the nasal epithelial cells have a distinct regulatory role in the control of RORα expression, thereby promoting proinflammatory macrophages. In addition, the inverse correlation between the miRNAs and RORα expression was further confirmed in NECRSwNP. We believe that the molecular mechanism discovered in this study can be used to complement current therapies involving different mechanisms in proinflammatory diseases caused by air pollution.

We thank Dr. Ji Min Lee at the Department of Molecular Bioscience in Kangwon National University for useful discussions.

The authors have no financial conflicts of interest.