Genome-Wide Analysis Reveals Mucociliary Remodeling of the Nasal Airway Epithelium Induced by Urban PM2.5 Michael T

ORIGINAL RESEARCH

Genome-Wide Analysis Reveals Mucociliary Remodeling of the Nasal Airway Epithelium Induced by Urban PM2.5 Michael T. Montgomery1, Satria P. Sajuthi1, Seung-Hyun Cho2, Jamie L. Everman1, Cydney L. Rios1, Katherine C. Goldfarbmuren1, Nathan D. Jackson1, Benjamin Saef1, Meghan Cromie1, Celeste Eng3, Vivian Medina4, Jennifer R. Elhawary3, Sam S. Oh3, Jose Rodriguez-Santana4, Eszter K. Vladar5,6, Esteban G. Burchard3,7, and Max A. Seibold1,5,8 1Center for Genes, Environment, and Health, and 8Department of Pediatrics, National Jewish Health, Denver, Colorado; 2RTI International, Research Triangle Park, North Carolina; 3Department of Medicine and 7Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California; 4Centro de Neumologıa´ Pediatrica, ´ San Juan, Puerto Rico; and 5Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine and 6Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, Colorado ORCID IDs: 0000-0002-6748-2329 (M.T.M.); 0000-0001-9821-6667 (S.-H.C.); 0000-0002-1935-4672 (J.L.E.); 0000-0001-8774-7454 (K.C.G.); 0000-0003-3326-1680 (J.R.E.); 0000-0002-2815-6037 (S.S.O.); 0000-0002-4160-8894 (E.K.V.); 0000-0001-7475-2035 (E.G.B.); 0000-0002-8685-4263 (M.A.S.).

Abstract mucus secretory expression program (.100 genes), which included transcriptional drivers of mucus metaplasia (SPDEF , FOXA3 fi Air pollution particulate matter 2.5 mm (PM2.5) exposure is and ). Exposure to a higher OE dose modi ed the associated with poor respiratory outcomes. Mechanisms underlying expression of 1,240 genes and further exacerbated expression PM2.5-induced lung pathobiology are poorly understood but likely responses observed at the moderate dose, including the mucus involve cellular and molecular changes to the airway epithelium. We secretory program. Moreover, the higher OE dose significantly extracted and chemically characterized the organic and water-soluble increased the MUC5AC/MUC5B gel-forming mucin expression ratio components of air pollution PM2.5 samples, then determined the and strongly downregulated ciliated cell expression programs, whole transcriptome response of human nasal mucociliary airway including key ciliating cell transcription factors (e.g., FOXJ1 and epithelial cultures to a dose series of PM2.5 extracts. We found that MCIDAS). Chronic OE stimulation induced mucus metaplasia–like 1 PM2.5 organic extract (OE), but not water-soluble extract, elicited a remodeling characterized by increases in MUC5AC secretory cells potent, dose-dependent transcriptomic response from the and MUC5AC mucus secretions. This epithelial remodeling may fi mucociliary epithelium. Exposure to a moderate OE dose modi ed underlie poor respiratory outcomes associated with high PM2.5 the expression of 424 genes, including activation of aryl hydrocarbon exposure. receptor signaling and an IL-1 inflammatory program. We generated an OE-response gene network defined by eight functional Keywords: air pollution particulate matter 2.5; whole enrichment groups, which exhibited high connectivity through transcriptome RNA-seq; human nasal airway epithelium; mucus CYP1A1, IL1A, and IL1B. This OE exposure also robustly activated a metaplasia; airway remodeling

(Received in original form December 20, 2019; accepted in ﬁnal form April 9, 2020 ) Supported by U.S. National Institutes of Health (NIH) grants R01MD010443, R01HL128439, R01HL135156, and P01HL132821 and Department of Defense grant W81WH-16-2-0018 (M.A.S.). Also supported by the Sandler Family Foundation; the American Asthma Foundation; the Robert Wood Johnson Foundation Amos Medical Faculty Development Program; Harry Wm. and Diana V. Hind Distinguished Professor in Pharmaceutical Sciences II; NIH grants R01HL117004, R01HL128439, R01HL135156, 1X01HL134589, R01HL141992, and R01HL141845; NIH and Environmental Health Sciences grants R01ES015794 and R21ES24844; the National Institute on Minority Health and Health Disparities grants P60MD006902, RL5GM118984, R01MD010443, and R56MD013312; the Tobacco-Related Disease Research Program under Award Numbers 24RT-0025 and 27IR-0030; and the National Human Genome Research Institute U01HG009080 (E.G.B.). Author Contributions: Conception and experimental design: M.T.M., S.P.S., S.-H.C., J.L.E., C.L.R., S.S.O., and M.A.S. Acquisition of data: M.T.M., S.-H.C., J.L.E., C.L.R., M.C., C.E., V.M., J.R.E., S.S.O., J.R.-S., E.K.V., E.G.B., and M.A.S. Analysis of data: M.T.M., S.P.S., S.-H.C., N.D.J., B.S., J.R.E., and S.S.O. Interpretation of work: M.T.M., S.P.S., S.-H.C., J.L.E., K.C.G., N.D.J., S.S.O., E.K.V., E.G.B., and M.A.S. Manuscript preparation for intellectual content: M.T.M., S.P.S., S.-H.C., J.L.E., K.C.G., N.D.J., S.S.O., J.R.-S., E.G.B., and M.A.S. Correspondence and requests for reprints should be addressed to Max A. Seibold, Ph.D., National Jewish Health, 1400 Jackson Street, Denver, CO 80206. E-mail: [email protected]. This article has a related editorial This article has a data supplement, which is accessible from this issue’s table of contents at www.atsjournals.org. Am J Respir Cell Mol Biol Vol 63, Iss 2, pp 172–184, August 2020 Copyright © 2020 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2019-0454OC on April 10, 2020 Internet address: www.atsjournals.org

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PM2.5 was associated with 4.2 million the GALA II (Genes-Environments and Clinical Relevance deaths and 103.1 million disability-adjusted Admixture in Latino Americans II) life years globally in 2015 (5). These childhood asthma study. To comprehensively This transcriptomic study details the respirable particles are usually generated as determine epithelial PM2.5 responses, we molecular mechanisms that are combustion by-products and serve as a performed gene and network analysis of dysregulated in the airway epithelium vehicle for transport of potentially noxious whole transcriptome RNA sequencing during exposure to increasing heavy metals, hydrocarbons, and reactive (RNA-seq) data from these cultures. We doses of air pollution particulate ions deep into the respiratory tract to the then harnessed powerful single-cell RNA- , matter 2.5 mm (PM2.5) organic sites of gas exchange. seq (scRNA-seq) data sets to infer how fi constituents, and connects deleterious On inhalation, PM2.5 rst encounters PM2.5 affects epithelial cell type reprogramming of gene expression the mucociliary barrier formed by highly composition and function and confirmed programs to disease-relevant tissue specialized secretory and ciliated cells of the many of these effects in a model of chronic remodeling processes at the cellular airway epithelium. However, most studies PM2.5 exposure. These analyses reveal and protein level. We highlight investigating molecular/cellular responses linked networks of PM2.5-induced fl potential mechanisms by which PM2.5 of the airway epithelium to PM2.5 have used in ammation coinciding with and likely exposure may initiate disease-related transformed lung epithelial cells that lack driving pathologic mucociliary remodeling airway pathobiology, thereby the cellular diversity and functional of the airway epithelium. Some of the providing targetable pathways for capability of the in vivo airway epithelium results of this study have been previously future study and clinical intervention (6, 7). In addition, cellular responses to reported in the form of an abstract (12). in air pollution–mediated/exacerbated PM2.5 have typically been determined by airway disease. Furthermore, our using PM standard reference material from fi Methods PM2.5 response pro le can be the National Institute of Standards and leveraged in future nasal gene Technology (NIST) in place of real-world – PM Collection and Characterization expression studies by aiding in air pollution derived PM2.5. Unfortunately, 2.5 identification of subjects with prior these NIST standard reference materials PM2.5 samples collected on 47-mm fi polytetrafluoroethylene filters from PM2.5 exposure and contributing to were collected in bulk from air ltration the identification of a PM molecular systems in Europe and baghouses in three cities in California in 2011 2.5 fi endotype of airway disease. Washington D.C. over 40 years ago; (Bakers eld/Fresno, Sacramento, and Yuba therefore, the composition and particle size City) were provided by the California of these samples are poorly representative Air Resources Board. Material collected on filters was pooled by region and In 2014, the World Health Organization of ambient air pollution PM2.5 generated cited air pollution as the world’s most and inhaled in modern U.S. cities (8). Only fractionated into WE and OE. Chemical fi fi prominent single environmental health risk a few studies have evaluated the effects of species were identi ed and quanti ed – using inductively coupled plasma mass (1). Exposure to indoor and outdoor air pollution derived PM2.5 extracts in pollution is a major causal factor for primary mucociliary airway epithelial spectrometry and ion chromatography mortality due to cardiovascular and cultures (9, 10). In general, these studies for WE and gas chromatography-mass fi respiratory conditions, including stroke, determined responses of a limited number spectrometry for OE. Blank lters were also ischemic heart disease, lung cancer, chronic of specific candidate genes, pathways, or extracted to produce mock extract controls respiratory diseases, and acute respiratory mechanisms, generating only an isolated for stimulations. infections. Furthermore, chronic exposure piece of the full epithelial molecular to air pollution components has been response to PM2.5. These studies have Human Subject Information linked to impaired lung function reported effects on metabolic and Nasal AECs used for culture came from development in children, and even short- inflammatory pathways, mucus production, subjects recruited as part of the GALA II term exposure has been shown to be and perturbation of defense mechanisms, childhood asthma study, which was associated with poor health outcomes in reinforcing the significant influence of approved by local institutional review subjects with childhood asthma, including PM2.5 on epithelial functions (11). boards (University of California San increased hospitalization and mortality However, no study has evaluated genome- Francisco, institutional review board (2–4). wide responses to contemporary urban air number 10-00889, reference number Air pollution is a heterogeneous pollution PM2.5 constituents in primary 153543, National Jewish Health HS-2627). mixture, primarily composed of gaseous mucociliary epithelial cultures generated All subjects and their parents provided pollutants (e.g., carbon monoxide, nitrogen from a large number of donors. written informed assent and consent, oxides, ozone, and sulfur dioxide) and Here, we obtained air pollution PM2.5 respectively (13, 14). suspended solid or liquid droplet particulate from California urban areas in 2011, and – matter (PM) derived from both fractionated the PM2.5 into chemically Mucociliary Air Liquid Interface anthropogenic and geogenic sources. Fine characterized, water-soluble (WE) and Culture and Stimulation air pollution particle matter with an organic-soluble (OE) extracts. We used Nasal AECs from five healthy subjects were , – aerodynamic diameter 2.5 mm (PM2.5) these PM2.5 extracts in dose response selected randomly for California extract has proven particularly detrimental to studies of mucociliary airway epithelial cell dose–response experiments, whereas NIST human health, as exposure to ambient (AEC) cultures generated from donors in 2786 stimulations were conducted on four

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donors. Full characterization of moderate Gene Network Analysis the effects of our unique PM2.5 extracts, we OE dose–response was conducted with six FGNet and GeneTerm Linker databases also stimulated cultures from these donors healthy donors and six donors with asthma were used to construct gene networks on the with an unseparated fine PM standard (n = 12). Expansion and differentiation at basis of GO- and KEGG-enriched terms for reference material, NIST 2786 (,4 mm air–liquid interface (ALI) in vitro was groups of genes significantly upregulated by diameter; stimulation concentration 30 conducted according to established moderate OE stimulation (17–19). mg/cm2 culture area) (8). Experiments were protocols (15). On mucociliary conducted by performing two consecutive differentiation (Day 28–32 of ALI culture), stimulations at the indicated concentrations cultures were washed with PBS and then Results 24 hours apart, with culture harvest 24 apically treated with mock extract or hours after the second stimulation. We did fi fi Characterization of California Urban fi , Bakers eld/Fresno PM2.5 extracts in a nal not observe signi cant toxicity ( 1% by volume of 20 ml culture media. PBS wash PM2.5 Reveals Diverse Organic and lactate dehydrogenase assay) with either and stimulation were repeated at 24 hours, Water-Soluble Chemical Components PM extract or NIST 2786 at the tested fi 2.5 followed by on-membrane lysis using RNA PM2.5 collected on lters from three concentrations (data not shown). To lysis buffer (Zymo Research) containing California air pollution monitoring sites evaluate whether these PM types mediated 40 mM DTT for total RNA collection (Bakersfield/Fresno, Sacramento, and significant transcriptional changes in our after an additional 24 hours of culture Yuba City) in 2011 was extracted and differentiated mucociliary model of the incubation. fractionated into WE and OE as described airway epithelium, we generated whole For chronic stimulation experiments, (see Figure E1 in the data supplement). transcriptome RNA-seq data using total Chemical characterization of WE revealed cultures were washed with PBS and then 2 1 RNA isolated from all stimulated as well as apically treated with media control or high nitrate (NO3 ), ammonium (NH4 ), mock-stimulated cultures. We performed 22 OE dose from the Yuba City PM2.5 sample and sulfate (SO4 ) (listed in order of paired differential gene expression analyses (because of limited Bakersfield/Fresno abundance; Figure 1A). In contrast to the between the mock-stimulated cultures and fi 32 sample) in a nal volume of 20 ml media. other two sites, phosphate (PO4 ) was cultures treated with each of the PM2.5 WE, Alternatively, cultures were treated with not present at measurable levels in the OE, and NIST dosages to identify all genes 10 ng/ml IL-13 in media on apical (20 ml) Yuba City sample. Among the inorganic with significantly modified expression. No and basolateral sides (500 ml). PBS washes elements, metals that are characteristic genes exhibited significantly modified and stimulations were repeated every of traffic-related pollution, including expression with either WE stimulation 24 hours for a total of five stimulations copper (Cu), zinc (Zn), barium (Ba), concentration tested. In contrast, OE before cultures were analyzed. Details for and strontium (Sr), were measured at a stimulations resulted in DEGs with a dose- quantitative PCR, immunofluorescence greater mass fraction in the Sacramento site dependent increase in the number of DEGs labeling, Western blot, and mucin secretion sample than in the other site samples detected (Figure 2B and Table E2). assays can be found in the data supplement. (Figure 1B). Chemical characterization of Specifically, each 10-fold increase in OE OE revealed an array of carcinogenic and concentration resulted in an z10-fold immunotoxic polycyclic aromatic increase in the number of DEGs detected, Whole Transcriptome RNA-seq hydrocarbon (PAH) species, including from 11 (low dose) to 124 (moderate dose) KAPA mRNA HyperPrep (Roche) whole benzo[a]pyrene, indeno[1,2,3-c,d]pyrene, to 1,296 (high dose). The genes detected transcriptome libraries were constructed and dibenz[a,h]anthracene (Figure 1C). across these OE exposures appeared to be with 250 ng RNA per sample and 12 cycles The Bakersfield/Fresno OE consistently highly conserved, as 9 of the 11 low dose of library amplification. Libraries were contained the highest level for individual as DEGs were also DEGs for the moderate sequenced using the Illumina NovaSeq 6000 well as combined PAH species measured dose, and 71% of the moderate dose DEGs system. Details for RNA-seq data processing per microgram of extracted material were also DEGs for the high dose and analysis can be found in the data (Figures 1C and 1D). (Figure 2C). We also detected 111 DEGs supplement. induced by NIST 2786 stimulation, a PM2.5 Modifies Airway Epithelial Gene response comparable in size to the moderate Gene Enrichment Analysis Expression through Organic OE dose stimulation (Table E2). Moreover, Enrichment of differentially expressed genes Components 35% of the moderate OE DEGs were also (DEGs) was performed with Enrichr To model the acute transcriptional effects DEGs for NIST, and the log2 fold changes (16), separately for upregulated and of PM2.5 components on the human (LFCs) of all DEGs resulting from these downregulated genes, using the most airway epithelium, we leveraged mature two stimulants were highly correlated current gene ontology (GO) databases (GO mucociliary ALI cultures derived from (Pearson correlation coefficient, 0.75; 2 Biological Processes 2018, GO Molecular primary human nasal AECs (Figure 2A). P =1.683 10 34; Figures 2C and 2D). Function 2018, GO Cellular Component To determine biologically active, yet Together, these results indicate that PM2.5 2018, KEGG 2016, and Reactome 2016). nontoxic concentrations of WE and OE, we OE exposure can modify airway epithelial We also tested for DEG enrichment within performed stimulations of five independent gene expression in a dose-dependent manner cell type marker lists derived from scRNA- donor cultures using a range of extract and that WE does not induce significant seq data as described in the online concentrations (WE: 3 and 30 mg/cm2 transcriptomic changes in our model, even at supplement. Enrichment P values were culture area; OE: 0.045, 0.45, and greater than six times higher concentration corrected for multiple testing. 4.5 mg/cm2 culture area). To benchmark than the highest OE dose tested.

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A B 1,000 100

10 100

0.1

1 ng/µg Water Extract (log scale) ng/µg Water Extract (log scale) 0.01

0.1 0.001 - +

3 2- 3- K V

Ti 4

Cr 4 4

Fe Zn

Sn Sb Ba Pb

Na Ca Cu Cd

Mg Mn NO NH SO PO Ionic Species Elemental Species

C D 0.04 0.15 Bakersfield/Fresno Sacramento Yuba City 0.03 0.10

0.02

0.05 0.01 ng/µg Organic Extract ng PAH/µg Organic Extract

0.00 0.00

BkF

BaP BeP

BbjF

CHR

DaiP

DahA BghiP Fresno Yuba City Bakersfield/ Sacramento

7,12-DMB(a)A PAH Analyte Collection Site

Figure 1. Chemical analysis of urban air pollution particulate matter ,2.5 mm (PM2.5) extracts. (A) Relative abundance of ionic chemical species per microgram of water-soluble extract (WE) detected by ion chromatography. (B) Relative abundance of inorganic elemental chemical species per microgram of WE detected by inductively coupled plasma mass spectrometry. (C) Relative abundance of polycyclic aromatic hydrocarbon (PAH) species per microgram of organic extract (OE) detected by gas chromatography–mass spectrometry. (D) Combined concentration of all assayed PAH species for each pool. BaP = benzo[a]pyrene; BbjF = benzo[bj]ﬂuoranthene; BeP = benzo[e]pyrene; BghiP = benzo[g,h,i]perylene; BkF = benzo[k]ﬂuoranthene; CHR = chrysene; DahA = dibenz[a,j]anthracene; DaiP = dibenz[ai]pyrene; IP = indeno[1,2,3-c,d]pyrene.

Moderate Levels of PM2.5 Organic effects of OE exposure on the airway identified 424 DEGs consisting of 281 Extract Trigger Transcriptional epithelium. We performed this moderate upregulated and 143 downregulated genes Upregulation of Inflammatory and OE dose stimulation on mature airway (Table E2). Principal component analysis of Mucus Secretory Cell Gene epithelial cultures from an additional 7 these DEGs verified consistency in this Expression donors (for 12 donors total), followed by response across donors (Figure 3A). On the basis of our dose–response whole transcriptome RNA-seq, increasing Examining this gene set we found that the experiment, we selected our moderate OE our power to characterize the genome- most highly OE-induced gene was the dose (0.45 mg/cm2) for further stimulation wide response. Paired differential gene xenobiotic metabolizing cytochrome P450 experiments to elucidate the biological expression analysis of these donors enzyme, CYP1A1 (LFC, 6.21; Figure 3B).

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A C Stimulants High OE Cali PM2.5 OE Cali PM2.5 WE NIST 2786 NIST 2786 51 16 Isolate/expand basal nasal AECs 1,157 39 5

Harvest 49 31 Differentiation Expansion Stimulation (28-32 days) Mod. OE (48h) (2x24h)

BD 1,500 2 1,296

1 1,000

0 500 Shared DEG Mod. OE DEG 124 Mod. OE Log2 Fold Change -1 NIST DEG Differentially Expressed Genes 11 0 Low Mod. High -1 012 OE Dose NIST 2786 Log2 Fold Change

Figure 2. OE drives the airway epithelial response to PM2.5 stimulation. (A) Schematic of experimental design starting from isolation of nasal airway epithelial cells (AECs) to mucociliary air–liquid interface culture differentiation and acute (48 h) stimulation with PM2.5 extracts (Cali PM2.5) or National Institute of Standards and Technology (NIST) 2786. (B) Number of differentially expressed genes (DEGs) after stimulation with three separate PM2.5 OE doses. (C) Venn diagram showing shared and unique DEGs across moderate (Mod.) OE dose, high OE dose, and NIST 2786. (D) Comparison of effect sizes between the union of DEGs from moderate OE and NIST 2786 stimulations.

CYP1A1 is known to be induced by PAHs, significant enrichment terms, thereby was strongly induced by OE stimulation, as a confirmed component of our OE extract, constructing an OE functional gene response was another gel-forming mucin, MUC2 through aryl hydrocarbon receptor (AHR)- network (Figure 3C and Table E3). This OE (mucin 2), which is normally expressed in the mediated transcriptional activation (20). response network was composed of eight gut and lowly expressed in the airway (LFC, Consistent with OE activation of the AHR significant clusters (metagroups). These 0.90 and 1.34, respectively; Table E2) (22). receptor, we observed significant induction metagroups included several defined by Together, these results suggest that the of other AHR-inducible xenobiotic functional terms relating to an inflammatory number and/or function of mucus secretory metabolizing enzymes, including CYP1B1 response, including cytokine-mediated cells within the epithelium is increasing with and genes encoding the AHR battery signaling, IFN-g signaling, antigen processing OE exposure. To more comprehensively enzymes NQO1 (NAD(P)H quinone and presentation, response to liposaccharide, investigate whether OE exposure DEGs are dehydrogenase 1) and ALDH3A1 (aldehyde and chemotaxis. The CYP1A1, IL1A,and enriched in genes expressed by mucus dehydrogenase 3 family member A1) (21). IL1B genes were shared by multiple secretory or other epithelial cell types, we Interestingly, we found that IL1A and metagroups and exhibited especially high tested all moderate OE DEGs for enrichment IL1B (IL-1a and -1b)inflammatory betweenness centrality, indicating the likely among a catalog of epithelial cell type marker cytokines were both among the most highly critical nature of these genes in the genes generated from scRNA-seq of the upregulated genes with OE stimulation (LFC, transcriptional response to OE stimulation. human airway epithelium (Figure 3D and 0.91 and 1.22, respectively; Figure 3B), We also found significant enrichments Table E4). Although several cell type indicating that OE stimulation triggered for Golgi subcompartment and endoplasmic enrichments were observed, the most epithelial inflammation. To interrogate this reticulum (ER) protein processing terms significant enrichment was for mucus and other biological processes underlying (Figure 3C and Table E3), both of which secretory cell marker genes among OE- P 3 274 OE-upregulated genes, we performed gene set contained genes involved in the production of upregulated DEGs ( adj =1.19 10 ). enrichment analyses using FGNet (17). Genes mucin proteins. Furthermore, we found that To confirm a functional relationship were filtered and organized based on the one of the two primary gel-forming mucin among mucus secretory genes that were relationship between, and similarity of, genes in the airway, MUC5AC (mucin 5AC), upregulated by moderate OE, we tested

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A B D 15 Mock Up DEGs Mod. OE Down DEGs 75 Gene Log2FC Padj Function 10 CYP1A1 6.21 2.55 x 10–87 CYP1B1 1.59 4.14 x 10–9 AHR response 50 5 NQO1 0.17 4.88 x 10–2 battery –2 ALDH3A1 0.64 4.42 x 10 25 0 –3 PC2 IL1A 0.91 9.26 x 10 inflammatory IL1B 1.22 7.59 x 10–4 mediators 0 −5 SPDEF 0.66 4.92 x 10–17 –6 FOXA3 0.80 7.81 x 10 airway mucus -log10 Enrichment P Value −10 MUC5AC 0.90 2.48 x 10–4 program 25 MUC2 1.34 6.81 x 10–6 basal basal

−20 −10 0102025 Mucus Ciliated

PC1 secretory KRT8 high Proliferating intermediate Differentiating Cell Type C Hydrolase activity Protein amino acid dephosphorylation ER lumen SAA1 ER protein processing PTPRH DAPP1 PREP PDP1 RNP2 XBP1 TSPAN1 PKN1 Endosome PRSS22 HYOU1 PTPRJ TGM2 Lysosomal membrane DNAJB9 HLA-DPA1 CYGB TMEM63A SCARB1 LAPTM5 SEC24D TYMP CD74 RDH10 NQO1 FUCA1 FTH1 TIMP1 PLIN3 TPRA1 PTGS2 ADRB2 DUSP4 OSMR MAP3K8 Response to cytokines HSD17B2 PLAT LUM FADS3 ADAM8 Response to organic ANGPTL4 IRAK3 cyclic substance TLR2 SOCS3 ALDH3A1 SAT1 LCN2 SEC61A1 AOX1 HSP90B1 IL1B IL1A LIF CXCL5 DHRS9 CYP1A1 DNAJC10 GGT1 TNFSF15 LRRC59 IFNGR1 CXCL6 LYN WNT7A CYP1B1 IDO1 IFNGR2 HSPA5 MMP1 HLA-DRA Chemokine signaling KYNU CCL20 Chemotaxis NOX1 PLAU CYBA FAR2 KMO PDIA4 TNFRSF10A ITGB7 FDFT1 PTAFR IFI6 SLC7A11PTPRN2 HMGCS1 PTGES ACLY HLA-DRB1 IFI27 RAC2 HLA-B LDLR QSOX1 SRD5A2 FBLN5 CD63 CLDN7 ME1 HLA-C ADM2 ITGA5 TUBA1A BIRC3 HLA-A PFKP B4GALNT2 GDF15 EHD1 Antigen processing/presentation SQLE TUBB2A Xenobiotic metabolism MDK IFN-γ signaling CACNG6 CLDN10 ICA1 Electron carrier activity NEK6 ER to Golgi transport NFKB2 MT2A Oxygen binding S100P Cytokine activity Node color - metagroup Response to lipopolysaccharide Black node - > 2 metagroups MAPK signaling pathway Node size - betweenness

Figure 3. PM2.5 OE stimulates an inﬂammatory network that induces a mucus secretory cell program. (A) Principal component (PC) analysis on 424 DEGs from moderate OE and mock comparison. (B) Table of select DEGs induced by moderate OE stimulation. (C) Functional gene network of moderate OE- upregulated DEGs. Node colors refer to metagroup membership, and black nodes represent genes shared between two or more metagroups. Node size represents betweenness across entire network. (D) Enrichment of moderate OE DEGs for cell type–speciﬁc marker genes. AHR = aryl hydrocarbon receptor; ER = endoplasmic reticulum.

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fi P 3 2100 P 3 24 whether any of these genes were coexpressed signi cant ( adj =8.27 10 ;Figure4C ( = 4.20 10 ) GO terms exhibited in the data set (Figure E2). Interestingly, this and Table E4), as was observed with the significant downregulation in expression analysis resulted in a mucus secretory moderate OE dose, the high dose exhibited only with high OE dose (Figure 4E). coexpression module containing MUC5AC even higher average levels of upregulation for Supporting this loss in ciliated cell gene as well as two of the BPI-fold containing master mucus secretory TFs (SPDEF, expression, we found canonical TFs genes (BPIFA1 and BPIFB1), which encode FOXA3,andXBP1) (Figure 4B and Table controlling ciliated cell differentiation antimicrobial defense proteins produced by E6). Moreover, this activation of mucus (MCIDAS, MYB, TP73, RFX2, RFX3, and secretory epithelial cells (23). This cluster secretory genes with high OE dose FOXJ1) were consistently decreased with also contained XBP1 (X-box binding stimulation was matched by repression high OE dose (Figure 4B and Table E2) protein 1), a cellular stress response of SCGB1A1 (secretoglobin family 1A (33–38). Together, these data show that 2 P 3 23 transcription factor (TF) that initiates ER member 1) (LFC, 0.92; adj = 1.13 10 ; high OE dose mediates the same mucus programs to accommodate accumulation of Figure 4B and Table E2), the marker gene of secretory changes observed with moderate secretory proteins (24). Most interestingly club secretory cells. This is consistent with OE dose but also downregulates ciliary and perhaps most central to this cluster was mucus metaplasia of club secretory cells, as expression programs. SPDEF (SAM pointed domain containing has been reported for IL-13–induced mucus ETS transcription factor), a canonical TF metaplasia (27). In line with this mucus Chronic OE Stimulation Results in reported to drive mucus production and metaplastic signal, MUC5AC expression was Mucus Secretory Remodeling of the metaplasia in response to the type 2 increased with high OE dose, similar to its Airway Epithelium cytokine, IL-13 (25). Although found in a induction with moderate OE dose (Figure 4D To investigate whether transcriptional different cluster, we also observed and Table E2). However, we observed a trend reprogramming induced by high OE dose upregulation of FOXA3 (forkhead box A3), toward decreased expression of the other culminates in demonstrative changes to an additional mucus metaplasia TF major gel-forming mucin, MUC5B (mucin protein expression, cellular composition, functionally related to SPDEF (26). 5B),onlywiththehighOEdose(LFC, and in the mucus secretions of the airway 2 P 3 21 Together, these results suggest that 0.79; adj =1.0 10 ). The viscoelastic epithelium, we chronically stimulated moderate OE exposure triggers AHR properties of airway mucus are largely mature airway epithelial cultures with a high signaling and IL-1–mediated inflammation, determined by the ratio of levels of the dose of OE (5 days of repeat exposure). As a which in turn initiates a mucus secretory “healthy” homeostatic mucin MUC5B and benchmark for metaplastic expansion of program in the airway epithelium. the inflammation-induced MUC5AC mucus secretory cells, we also performed (28–32). It is notable that a significant chronic stimulations of additional cultures High Levels of PM2.5 Organic Extract increase in the MUC5AC/MUC5B ratio with from the same donors with the cytokine IL- 2 Cause Dysregulation of Mucociliary moderate OE dose (P =3.193 10 2)was 13. We found that both chronic OE and IL- Transcriptional Programs increased further with the high OE dose 13 stimulation resulted in upregulation of 2 2 On the basis of the number of DEGs induced, (P =2.683 10 2; Figure 4D), suggesting an MUC5AC (OE LFC, 1.72; P = 1.27 3 10 2; 2 our OE dose–response analysis suggested that important relationship between OE IL-13 LFC, 3.71; P = 8.86 3 10 3) paired the highest OE dose (4.5 mg/cm2) induced a stimulation and alteration of mucus with an increase in SPDEF expression 2 more extensive epithelial gene expression properties. Accompanying this change in the (OE LFC, 0.84; P = 3.19 3 10 2; IL-13 2 response than the moderate dose. Using gel-forming mucin ratio was a high OE LFC, 2.91; P = 2.54 3 10 2), characteristic Enrichr (16), we performed gene set dose–specific induction of genes involved in of mucus metaplasia (Figure E3A). enrichment analysis of upregulated and the O-linked glycosylation of mucins, Moreover, these stimuli resulted in downregulated DEGs across moderate and including five members of the GalNac-T decreased expression of SCGB1A1 (OE 2 high OE dose to identify shared terms as well family of glycosyltransferase enzymes LFC, 21.51; P = 2.94 3 10 2; IL-13 LFC, 2 asthoseuniquetothehighdose(TableE5). (GALNT3, 5, 6, 7,and12)and 21.74; P = 4.09 3 10 2) and a trend toward Most of the significant enrichments for the N-acetylglucosaminyltransferase enzyme decreased MUC5B expression (OE LFC, 2 moderate OE dose DEGs (from n =12data GCNT3 (Table E2), which likely contribute to 20.72; P = 9.74 3 10 2; IL-13 LFC, 25.24; 2 set) were also enrichments for the high OE modifications in the viscoelastic properties of P = 2.55 3 10 2), as well as downregulation dose DEGs (from n = 5 data set). In addition, mucus induced by the OE-stimulated airway of FOXJ1 (forkhead box J1) (OE LFC, 2 we found that of the 169 upregulated DEGs epithelium (31). 20.36; P = 1.92 3 10 2; IL-13 LFC, 21.15; 2 that were shared between doses, 164 were Enrichment analysis of downregulated P = 6.34 3 10 3). These results support that further upregulated with the high dose, genes yielded multiple terms specific to the OE induces a mucus metaplasia process suggesting that a stronger stimulus high OE dose, the majority of which were similar to IL-13 (Figure E3A). exacerbates dysregulated expression of these related to the development and function of To confirm these results histologically, genes (Figure 4A and Table E6). In cilia (Table E5). Furthermore, in contrast to we examined cultures with alcian particular, we noticed that genes central to moderate OE DEGs, we found highly blue–periodic acid-Schiff staining to the moderate OE dose–response (IL1A, IL1B, significant enrichment of high OE identify mucin-positive cells and found and CYP1A1) were further upregulated with dose–downregulated DEGs within ciliated higher numbers of mucin-positive cells in 2118 the high OE compared with moderate OE cell marker genes (Padj = 3.57 3 10 ; chronic OE-stimulated versus mock- dose (Figure 4B and Table E6). In addition, Figure 4C and Table E4). Mean expression stimulated cultures for three of the four although the enrichment of high dose DEGs of genes within the ciliary movement donor pairs examined (average fold change 2 2 within mucus secretory cell markers was (P = 3.05 3 10 3) and ciliary assembly [FC] = 2.01; P = 6.52 3 10 2; Figure 5A).

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A B 1:1 OE Dose 5 genes 164 genes Low Mod. High 0.8 CYP1A1 CYP1B1 0.6 IL1A 0 10 IL1B SPDEF 0.4 FOXA3

Density MUC5AC MUC5B 0.2 -2 3 SCGB1A1

Log2 Fold Change MCIDAS 0.0 MYB 024 6 8 TP73 RFX2 Ratio of Log2 Fold Change (High/Mod.) -1 0 RFX3 FOXJ1

C D E 125 Up DEGs 11.00 100 Down DEGs MUC5AC MUC5B 5AC/5B 3 75 *** * 50 2 10.75 * 25 * 0 1 ** 10.50 25

50 0 Log2 Fold Change -log10 Enrichment P Value 75 10.25

Mean Expression of Enriched Genes *** 100 -1 Cil. Movement Cil. Assembly 125 Low Low Low Low Mod Mod Mod Mod High High High High basal basal Mock Mucus Ciliated OE Dose OE Dose secretory KRT8 high Proliferating intermediate Differentiating Cell Type

Figure 4. High OE dose induces transcriptional remodeling of the airway epithelium. (A) Histogram of the ratio of log2 fold change (High/Mod.) for shared upregulated DEGs between moderate and high OE dose stimulation. (B) Heat map of the log2 fold change in expression between mock and OE dose- stimulated donor culture pairs (columns) for key response genes across OE dose. (C) Strength of enrichment for high OE dose DEGs within groups of cell type–specific marker genes. (D) First and second panels: log2 fold change of MUC5AC (mucin 5AC) and MUC5B (mucin 5B) across OE dose (*Padj , 0.05, and ***Padj , 0.001); third panel: ratio of log2 fold changes (MUC5AC/MUC5B). Statistically significant differences in ratio compared with mock-treated cultures (*P , 0.05) were determined by two-tailed, paired t test. (E) Mean expression of cilia (Cil.) assembly and cilia movement genes across mock and OE dose. Statistically significant differences in module gene expression between mock and OE dose (**P , 0.01 and ***P , 0.001) were determined by two-tailed, paired t test. For all plots, mock and moderate OE, n = 12 independent donors; low and high OE, n = 5 independent donors.

Using immunoﬂuorescent labeling for increased alcian blue–periodic acid- Consistent with these changes in mucin MUC5AC and MUC5B, we found an increase Schiff staining with chronic OE expression being mediated by the action 1 in MUC5AC cells with chronic OE stimulation. In contrast, cultures for all of SPDEF, we found that chronic OE stimulation for all four donors (FC = 2.88; four donors exhibited a decrease in stimulation resulted in a 1.5-fold increase 2 1 2 P =3.193 10 2;Figure5B),suggestingthat MUC5B cells with chronic OE exposure in SPDEF protein levels (P =1.073 10 2; 2 this mucin was the primary source of (FC, 0.74; P =8.843 10 2;Figure5B). Figure 5C).

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A B p=0.065 50 µm MUC5ACMUC5BDAPI 30 * 40 Mock High OE IL-13 Mock Mock

30 20 50 µm

p=0.088 Cells/Field Cells/Field 20 + + 10 Mucin

AB-PAS 10

0 IL-13 High OE 0 IL-13 High OE High OE Response MUC5AC MUC5B

C D E ** ** p=0.052 60 1.5 * 0.15 53 kD SPDEF 45 kD

Actin 35 kD 40 1.0 * 0.10

IL-13 * Mock

High OE 20 0.5 0.05 MUC5B Absorbance (A490) MUC5AC Absorbance (A490) Normalized SPDEF Band Intensity 0 0.0 0.00 High OE IL-13 High OE IL-13 High OE IL-13 Stimulant Response Stimulant Response Stimulant Response Figure 5. Chronic OE stimulation increases the number of MUC5AC1 cells and MUC5AC secretion. (A) Left panel: alcian blue–periodic acid-Schiff (AB- PAS) staining of culture histological sections for mucopolysaccharides/mucins. Right panel: quantification of AB-PAS1 cells. Scale bar, 50 mm. (B) Left panel: immunofluorescence labeling of culture histological sections for gel-forming mucins MUC5AC (red) and MUC5B (green). Nuclei were stained with DAPI (blue). Right panel: quantification of MUC5AC1 and MUC5B1 cells. Scale bar, 50 mm. Images for A and B are representative of labeling performed in cultures from four donors. (C) Left panel: Western blot analysis of total protein for SPDEF (SAM pointed domain containing ETS transcription factor) expression. Images are representative of data from five independent donors. Right panel: quantification of SPDEF Western blot band intensity normalized to actin expression as a loading control. (D and E) Colorimetric ELISA absorbance readings at 490 nm (A490) for MUC5AC (D) and MUC5B (E) detection in ATP-induced secretions of cultures from five donors. Statistically significant differences (*P , 0.05 and **P , 0.01) were determined by paired, one-tailed t test (on the basis of direction suggested by transcriptomic analyses). Independent donors are represented by data point shape, and connecting lines indicate paired mock- and OE- or IL-13–treated cultures.

To examine the effect of these mucus data demonstrate that chronic OE projections of labeled cultures and revealed secretory cell changes on mucin secretion, stimulation induces simultaneous that ciliary structure and abundance were 1 we used an ATP-induced mucus secretion expansion and reduction of MUC5AC relatively consistent across all conditions 1 assay (39), followed by examination of and MUC5B mucus secretory cells, (Figure 6). We did note that the acetylated mucin content in these secretions with respectively, resulting in corresponding a-tubulin staining (cilia) appeared weaker ELISA assays. Chronic OE induced a 2.2- changes in secretion of these mucins. in OE-stimulated cultures than in either fold increase in total secreted MUC5AC Together, these effects are consistent mock or IL-13–stimulated cultures. 2 (P = 1.54 3 10 2), comparable with with the observed increase in However, OE-stimulated cultures the robust induction in MUC5AC SPDEF protein levels with chronic OE displayed a 75% average decrease in 1 secretion stimulated by IL-13 (FC, 3.87; stimulation. FOXJ1 nuclei compared with mock- 2 2 P = 3.85 3 10 2; Figure 5D). Moreover, we We next investigated the consequences treated cultures (P =1.203 10 2;Figures observed a significant decrease in the of chronic OE stimulation on ciliated cells 6 and E3B). Interestingly, this effect was secretion of MUC5B with chronic OE (FC, by immunofluorescence labeling of ciliated more pronounced than the nonsignificant 2 1 0.81; P = 3.59 3 10 3), again similar to the cells for the transcription factor FOXJ1 and 40% average decrease in FOXJ1 nuclei effect mediated by IL-13 stimulation (FC, for cilia with acetylated a-tubulin. Confocal observed for IL-13–treated cultures 2 2 0.70; P = 5.20 3 10 2; Figure 5E). These microscopy was used to produce maximum (P =1.023 10 1). The lack of nuclear

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DAPI E-cadherin Acetylated D-Tub. FOXJ1 Merge Mock High OE IL-13 Mock 2,000 25 µm p=0.102 * 1,500

Nuclei/Field 1,000 + High OE

FOXJ1 500

0 High OE IL-13 IL-13 Stimulant Response

Figure 6. Chronic OE impairs FOXJ1 (forkhead box J1) expression in ciliated cells. Left panel: confocal fluorescence microscopy maximum projections of intact epithelial cultures labeled for E-cadherin (white), acetylated a-tubulin (red), and FOXJ1 (green). Nuclei were stained with DAPI (blue). Images are representative of labeling performed in cultures from three donors. Scale bar, 25 mm. Right panel: quantification of FOXJ11 nuclei. Statistically significant differences (*P , 0.05) were determined by paired, one-tailed t test (on the basis of direction suggested by transcriptomic analyses). Independent donors are represented by data point shape and connecting lines indicate paired mock-treated and OE- or IL-13–treated cultures.

FOXJ1 in ciliated cells from chronic Our study represents one of the first to networks, leading to changes in the cellular OE-stimulated cultures is consistent examine molecular responses of the nasal composition of the airway epithelium. The with the decrease in ciliated cell gene epithelium to the organic and water-soluble high network interconnectedness of expression observed in our transcriptome components of PM2.5, independently. CYP1A1 suggests that activation of AHR fi analysis. Although it is dif cult to determine how signaling by PM2.5 PAHs is an early and closely the concentrations we tested relate central event underlying the larger in vivo to exposures, we attempted to use epithelial response to PM2.5 observed. In concentrations that were not toxic and did Discussion addition, our network analysis also not have any significant effect on epithelial identified IL1A and IL1B as hub genes in integrity, yet that influenced the molecular the PM response. These potent cytokines Decades of epidemiologic research have 2.5 function of the epithelium. The limited were part of, and the likely drivers of, an confirmed the detrimental influence of quantities of the sample prevented us from intense inflammatory expression profile we PM2.5 exposure on the development and identifying a concentration of PM2.5 WE found to be activated by PM OE. Prior exacerbation of lung diseases. Yet, the 2.5 which altered molecular function of the b cellular and molecular mechanisms, as well studies have established IL-1 , and to a epithelium. Despite this, considering heavy a as the chemical components through which lesser extent IL-1 , as major drivers in metals present in PM2.5 WE are known to chronic inflammatory lung diseases, PM2.5 drives these poor outcomes, are incompletely understood. As the first and trigger epithelial responses, we anticipate including asthma and chronic obstructive primary interface through which the higher WE concentrations would mediate pulmonary disease (COPD) (42). For an epithelial response of sorts. In contrast, example, IL-1b secretion has been found to respiratory tract interacts with PM2.5 and other inhaled matter, the nasal airway all tested concentrations of PM2.5 OE be increased in the sputum of patients with epithelium acts as a sentinel, capable of altered epithelial gene expression in a dose- COPD and asthma at baseline and further directing responses by other structural and dependent manner, providing dosage increased during acute exacerbations – IL1B immune cells of the lower airways and lung. guidelines for future primary epithelial (43 46). In addition, expression of Building on prior studies investigating exposure studies. Our results in primary in sputum and serum is positively candidate mechanisms involved in airway airway epithelial cells are in line with and correlated with systemic inflammation epithelial responses to PM2.5,we extend previous studies identifying OEs and and negatively correlated with pulmonary agnostically and comprehensively defined PAHs as the most harmful components of function (forced expiratory volume in 1 airway epithelial responses to the organic PM2.5 for transformed lung epithelial cells second percent predicted) in patients with components of PM2.5 using whole (40, 41). COPD (46, 47). In asthma, IL-1a and IL-1b transcriptome analysis and further Rather than just a simple perturbation are reported to independently regulate examined whether chronic OE exposure of xenobiotic metabolism pathways, we find immune subtypes of disease characterized modulates cellular composition and that the organic components of PM2.5 by neutrophilic and eosinophilic function of the epithelium. modulate the expression of many genes and inflammation, respectively (48). Together,

Montgomery, Sajuthi, Cho, et al.:PM2.5-induced Remodeling of the Nasal Airway Epithelium 181

ORIGINAL RESEARCH these data make clear that the IL-1 epithelial genes whose expression is IL-13, whereas it appears that IL-1 fl in ammatory axis is involved in affected also scales with the PM2.5 OE cytokines drive PM2.5-mediated epithelial respiratory inflammation and disease, and dose. Importantly, we found the highest dysfunction. Moreover, differences in the our work suggests that extensive exposure OE dose tested was sufficient to strongly secretory proteins upregulated and to air pollution PM2.5 may underlie downregulate ciliated cell gene expression, downregulated by PM2.5 versus IL-13 activation of these mechanisms in many suggesting OE-mediated dysfunction of (e.g., BPIFA1, BPIFA2,andMMP2)and patients. ciliated cells. Supporting this, we observed many other genes suggest the nature of Perhaps most interestingly, our a dramatic decrease in the expression the metaplastic mucus secretory cells and study of primary mucociliary epithelial of transcriptional regulators of ciliated other cell states differs between these cultures allowed us to observe dramatic cell specification and ciliogenesis stimuli as well. Importantly, these MCIDAS FOXJ1 MYB PM2.5 effects on mucus secretory cells. (e.g., , ,and ). differences suggest a highly plastic airway Importantly, we found the inflammation- Moreover, our chronic OE exposure model epithelium capable of remodeling to related mucin, MUC5AC, was strongly provides evidence for the epithelial multiple cell compositions and containing upregulated by PM2.5 OE. Moreover, the remodeling suggested by our whole different cell states, likely underlying the MUC5AC/MUC5B gel-forming mucin transcriptome analysis. Epithelial complexity in mechanisms driving ratio, which is known to be critical for the remodeling included an increase in common airway disease. In the future, our 1 fi physical properties of airway mucus, was MUC5AC cells and a decrease in PM2.5 response pro lecouldbeusedasa 1 strongly upregulated in OE-stimulated MUC5B cells, matched by a biomarker in nasal gene expression cultures. In addition, the activation corresponding change in secretion of these studies to identify subjects with excessive of an array of enzymes involved in mucins. These changes also occur with IL-13 PM2.5 exposure and possibly a PM2.5 O-glycosylation of mucins further supported stimulation, revealing similarities in the airway endotype. Identification of these the likely highly modified nature of mucus epithelial response to these two stimuli. subjects in expression studies would allow produced by the PM2.5-stimulated airway. Although we did not observe a decrease in direct in vivo comparison of endotype fi We also found that PM2.5 induced the ciliated cell frequency, we did nd that OE mechanisms as well as precise XBP1 transcription factor and target ER induces a decrease in nuclear FOXJ1 determination of the clinical chaperone genes, which are activated by labeling among ciliated cells. Loss of consequences of PM2.5 exposure. cellular stress caused by increased mucin nuclear FOXJ1 may underlie the There are several limitations to the production. Enrichment analysis of downregulation in ciliated cell gene present study and multiple areas of upregulated DEGs within mucus secretory expression we observed in our whole investigation that should be pursued based cell marker genes revealed strong evidence transcriptome analysis. Although these on our findings. Although our results that this increase in the expression of ciliated cells appeared structurally normal, suggest that PM2.5 OE activates cellular mucin production and secretion pathways continued OE exposure may eventually remodeling of the epithelium, these resulted from an increase in the proportion impede the maintenance of ciliated cells changes will need to be replicated and of mucus secretory cells within the andresultinlossofthiscriticalcelltypeor investigated in depth by methods such as epithelium. The strong activation of both block ciliated cell regeneration in response lineage tracing and scRNA-seq. SPDEF and FOXA3 expression, coupled to injury. On the basis of these results, we Moreover, longer-term chronic PM2.5 with a loss in SCGB1A1 expression, hypothesize that long-term exposure to stimulation experiments will need to be suggests that the generation of new mucus PM2.5 could lead to decreased mucociliary conducted to model effects on the secretory cells may occur through mucus clearance and increased mucostasis/mucus epithelium occurring in subjects with in vivo metaplasia of club secretory cells, not plugging . In fact, the mechanisms years of PM2.5 exposure. Finally, although dissimilar to the mucus metaplasia process we outline here could partially explain our study involved more human primary in vitro stimulated by the type 2 cytokine IL-13 the strong inverse correlation between epithelial donors than any PM2.5 (27). We note recent work by Chen and PM2.5 and forced expiratory volume in study to date, in the future, larger studies fi colleagues nding that either IL-1a or 1 second (2), as well as the increased will be needed to determine if PM2.5 IL-1b stimulation can induce mucus cell susceptibility to respiratory infections response mechanisms are enhanced or metaplasia in mice and that SPDEF is a among subjects with high exposures to different among subjects with respect to critical downstream effector in this process PM2.5 (50). variables such as race/ethnicity, age, sex, (49). We speculate that the IL-1 cytokine- The PM2.5-induced changes to the and disease status. driven OE inflammatory response network airway epithelium we observed approach In summary, we found that the triggers SPDEF, FOXA3, and XBP1 the level of an airway endotype organic components of PM2.5 are production, which form a regulatory axis (pathobiological mechanism), not sufficient to drive broad-scale that drives mucus metaplasia of the airway dissimilar to that exhibited by individuals transcriptomic, cellular, and mucin epithelium. with type 2–high asthma (51, 52). These secretory changes to the human airway Our dose–response analysis strongly similarities include the induction of epithelium. These changes appear to 1 supports worldwide public health efforts to SPDEF and accompanying MUC5AC emanate from PM2.5 induction of AHR mitigate ambient air pollution levels. In mucus metaplasia, loss of SCGB1A1 signaling and IL-1–driven inflammation fact, not only do we find the magnitude of expression, as well as downregulation in of the epithelium, resulting in mucus gene expression alteration increases with ciliated cell gene expression. However, secretory changes and ciliated cell higher PM2.5 OE doses, but the number of type 2 epithelial disease is driven through dysfunction. These effects on the

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ORIGINAL RESEARCH epithelium appear to occur through Author disclosures are available with the text with tissue processing and staining. The alteration of core transcription factors in of this article at www.atsjournals.org. authors also thank Alicia Adams and the ﬁ California Air Resources Board for the speci cation of these cell types. These supplying air pollution ﬁlters that served results provide potential mechanisms Acknowledgment: The authors thank the asthesourceofPM2.5 samples used in underlying PM2.5 exposure effects on Genomics and Microarray Core Facility at this study. The authors thank the respiratory health and provide targetable the University of Colorado Anschutz patients, families, recruiters, health care pathways for further study and clinical Medical Campus for sequencing services, providers, and community clinics for – the NJH Regenerative Medicine and their participation. In particular, the intervention in air pollution mediated Genome Editing Program for access to authors thank Sandra Salazar for her or -exacerbated lung and airway scRNA-seq data sets, and Dave Heinz support as the GALAII/SAGE II study diseases. n (NJH Pathology Laboratory) for assistance coordinator.

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184 American Journal of Respiratory Cell and Molecular Biology Volume 63 Number 2 | August 2020