Genome-Wide Analysis Reveals Mucociliary Remodeling of the Nasal Airway Epithelium Induced by Urban PM(2.5).

Primary nasal AEC ALI cultures - five-donor dose-response set

Primary nasal AEC ALI cultures - 12-donor moderate OE set

Primary nasal AEC ALI cultures - chronic stimulation set

Differentially expressed gene burden across the PM2.5 extract dose series

Paired mock-versus-treated RNA-seq across the full acute dose series in five donor cultures. Water-soluble extract produced no DEGs at either 3 or 30 ug/cm2. Organic extract produced a dose-dependent DEG burden: 11 DEGs at the low dose (0.045 ug/cm2), 124 at the moderate dose (0.45 ug/cm2) and 1,296 at the high dose (4.5 ug/cm2) - roughly a 10-fold rise in DEG count per 10-fold rise in concentration. The NIST 2786 reference material at 30 ug/cm2 gave 111 DEGs, comparable in size to the moderate OE dose. DEG counts are study-level totals rather than per-gene measurements, so they are recorded in these descriptions and no QuantityValue is invented for them.

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Evidence

CYP1A1 and AHR battery enzyme expression - moderate OE dose

In the 12-donor moderate OE dose characterization (424 DEGs: 281 up, 143 down), CYP1A1 was the most highly induced gene at LFC 6.21. Other AHR-inducible xenobiotic metabolizing enzymes were also significantly induced (CYP1B1, NQO1, ALDH3A1), consistent with the PAH content of the organic extract acting through AHR-mediated transcriptional activation. The paper reports no effect sizes for CYP1B1, NQO1 or ALDH3A1 in the cached text, so only CYP1A1 carries a value.

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Evidence

IL1A and IL1B expression - moderate OE dose

IL1A (LFC 0.91) and IL1B (LFC 1.22) were among the most highly upregulated genes at the moderate OE dose. In the eight-metagroup OE response network built with FGNet, CYP1A1, IL1A and IL1B were shared across metagroups and had especially high betweenness centrality, marking the IL-1 axis as a hub of the response rather than an incidental signal. Both were further upregulated at the high OE dose.

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Evidence

SPDEF and FOXA3 expression - moderate OE dose

Coexpression analysis of the OE-upregulated mucus secretory genes put SPDEF - the canonical transcription factor driving mucus production and metaplasia - at the centre of a module containing MUC5AC, BPIFA1, BPIFB1 and the ER stress factor XBP1. FOXA3, a further mucus metaplasia transcription factor, was upregulated in a different cluster. The cached text reports no LFC for SPDEF or FOXA3 at the moderate dose (the chronic-stimulation SPDEF LFC is recorded on GE:montgomery-spdef-chronic-qpcr), so these output records carry no value.

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Evidence

SCGB1A1 expression - high OE dose

The activation of mucus secretory genes at the high OE dose was matched by repression of SCGB1A1, the club secretory cell marker gene (LFC -0.92, Padj = 1.13e-3), which the paper reads as mucus metaplasia of club secretory cells of the kind reported for IL-13.

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Evidence

SPDEF expression - chronic OE stimulation, with IL-13 benchmark

Five days of repeat high-OE stimulation raised SPDEF expression (LFC 0.84, P = 3.19e-2), alongside MUC5AC upregulation - the signature of mucus metaplasia. Chronic IL-13, used as a benchmark for metaplastic expansion of mucus secretory cells, produced a larger SPDEF rise (LFC 2.91, P = 2.54e-2). The matching increase in SPDEF protein (1.5-fold) is recorded in response_comparisons as COMP:montgomery-spdef-protein-chronic.

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Evidence

SCGB1A1 expression - chronic OE stimulation, with IL-13 benchmark

Chronic OE stimulation decreased SCGB1A1 expression (LFC -1.51, P = 2.94e-2), as did the IL-13 benchmark (LFC -1.74, P = 4.09e-2) - loss of club cell identity accompanying the mucus metaplasia phenotype.

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Evidence

Gel-forming mucin transcripts and MUC5AC/MUC5B ratio - acute OE dose series

MUC5AC was strongly induced at the moderate OE dose (LFC 0.90) together with the normally gut-restricted gel-forming mucin MUC2 (LFC 1.34). MUC5B trended down only at the high dose (LFC -0.79, Padj = 1.0e-1). The resulting MUC5AC/MUC5B ratio, which governs the viscoelastic properties of airway mucus, rose significantly at the moderate dose (P = 3.19e-2) and further at the high dose (P = 2.68e-2), both by two-tailed paired t test against mock-treated cultures. A high-dose-specific induction of mucin O-glycosylation enzymes (GALNT3, 5, 6, 7, 12 and GCNT3) accompanied the ratio shift. Mucin mRNA values are log2 fold changes; the ratio itself is reported only as a significant increase, with no ratio value, so no QuantityValue is recorded for it.

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Evidence

MUC5AC+ and MUC5B+ cells and mucin transcripts - chronic OE stimulation

After five days of repeat high-OE stimulation, MUC5AC mRNA rose (LFC 1.72, P = 1.27e-2; IL-13 benchmark LFC 3.71, P = 8.86e-3) while MUC5B trended down (LFC -0.72, P = 9.74e-2; IL-13 LFC -5.24, P = 2.55e-2). Immunofluorescence for the two gel-forming mucins showed more MUC5AC+ cells in all four donor pairs (FC 2.88, P = 3.19e-2) and fewer MUC5B+ cells in all four (FC 0.74, P = 8.84e-2). AB-PAS staining of the same cultures is recorded separately as GCM:montgomery-abpas-chronic. The cell-count fold changes and their p-values are in response_comparisons; the transcript values here are log2 fold changes.

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Evidence

AB-PAS mucin-positive cells - chronic OE stimulation

Alcian blue-periodic acid-Schiff staining of culture sections found more mucin-positive cells after chronic OE stimulation in three of the four donor pairs examined (average fold change 2.01, P = 6.52e-2). The paper attributes the extra staining primarily to MUC5AC. The fold change and p-value are in response_comparisons; the paper reports no absolute mucin-positive cell percentage.

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Evidence

ATP-induced MUC5AC and MUC5B secretion - chronic OE stimulation

In an ATP-induced mucus secretion assay read by colorimetric ELISA in cultures from five donors, chronic OE raised total secreted MUC5AC 2.2-fold (P = 1.54e-2, comparable with the 3.87-fold rise under the IL-13 benchmark, P = 3.85e-2) and lowered MUC5B secretion to 0.81-fold (P = 3.59e-3; IL-13 0.70-fold, P = 5.20e-2). All four fold changes are recorded in response_comparisons with their p-values; the paper reports no absolute mucin concentrations.

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Evidence

Ciliated cell expression programs - high acute OE dose

Downregulated DEGs specific to the high OE dose were dominated by cilia development and function terms, and were highly enriched for ciliated cell marker genes (Padj = 3.57e-118). Mean expression of the ciliary movement (P = 3.05e-3) and ciliary assembly (P = 4.20e-4) GO modules fell only at the high dose, by two-tailed paired t test. The canonical ciliated cell differentiation transcription factors MCIDAS, MYB, TP73, RFX2, RFX3 and FOXJ1 were consistently decreased. The cached text gives no per-gene LFC for these transcription factors at this dose, so no value is recorded on the output record.

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Evidence

FOXJ1 expression and nuclear localization - chronic OE stimulation

Chronic OE stimulation lowered FOXJ1 transcript levels (LFC -0.36, P = 1.92e-2; IL-13 benchmark LFC -1.15, P = 6.34e-3) and, by confocal immunofluorescence in three donors, produced a 75% average decrease in FOXJ1+ nuclei versus mock (P = 1.20e-2) - more pronounced than the nonsignificant 40% decrease under IL-13 (P = 1.02e-1). Ciliary structure and abundance were relatively consistent across conditions, although acetylated alpha-tubulin staining appeared weaker in OE-stimulated cultures, so the paper reads this as loss of nuclear FOXJ1 rather than loss of ciliated cells. The percent decreases are recorded in response_comparisons.

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Evidence

MUC5AC+ cells, chronic OE vs mock

Increase seen in all four donor pairs examined. Figure 5B quantifies the number of MUC5AC+ cells by immunofluorescence, so the compared measurement is a cell count (of MUC5AC+ mucus secretory cells), not a protein expression level.

Statistical test: paired, one-tailed t test

MUC5B+ cells, chronic OE vs mock

Decrease seen in all four donor pairs examined; P above the 0.05 threshold. Figure 5B quantifies the number of MUC5B+ cells by immunofluorescence, so the compared measurement is a cell count (of MUC5B+ mucus secretory cells), not a protein expression level.

Statistical test: paired, one-tailed t test

AB-PAS mucin-positive cells, chronic OE vs mock

Average fold change across four donor pairs; three of the four increased. P above the 0.05 threshold.

Statistical test: paired, one-tailed t test

ATP-induced MUC5AC secretion, chronic OE vs control

Statistical test: paired, one-tailed t test

ATP-induced MUC5B secretion, chronic OE vs control

Statistical test: paired, one-tailed t test

ATP-induced MUC5AC secretion, chronic IL-13 vs control (benchmark)

IL-13 benchmark for metaplastic expansion of mucus secretory cells, run on the same donors.

Statistical test: paired, one-tailed t test

SPDEF protein, chronic OE vs control

Western blot band intensity normalized to actin, five independent donors.

Statistical test: paired, one-tailed t test

FOXJ1+ nuclei, chronic OE vs mock

Average percent decrease in FOXJ1+ nuclei by confocal immunofluorescence in cultures from three donors.

Statistical test: paired, one-tailed t test

FOXJ1+ nuclei, chronic IL-13 vs mock (benchmark)

Nonsignificant average decrease under the IL-13 benchmark, less pronounced than under chronic OE.

Statistical test: paired, one-tailed t test

AhR pathway activation leads to IL-1 inflammatory program activation

The paper places AHR signaling activation by PM2.5 PAHs as the early, central event underlying the wider epithelial response, with IL1A and IL1B as the hub genes of the resulting inflammatory network.

IL-1 inflammatory program activation leads to goblet cell hyperplasia

The paper's central mechanistic claim: the IL-1-driven inflammatory network initiates the mucus secretory program, whose transcription factors SPDEF and FOXA3 drive mucus metaplasia, confirmed cellularly by the chronic exposure model.

PM2.5 collection, fractionation and chemical characterization

PM2.5 collected on 47-mm polytetrafluoroethylene filters at three California monitoring sites in 2011, pooled by region and fractionated into water-soluble (WE) and organic (OE) extracts. WE species quantified by inductively coupled plasma mass spectrometry and ion chromatography; OE PAH species by gas chromatography-mass spectrometry. Blank filters were extracted to produce the mock extract controls used for stimulations.

Equipment: 47-mm polytetrafluoroethylene filters; Inductively coupled plasma mass spectrometer; Ion chromatograph; Gas chromatograph-mass spectrometer

Nasal AEC air-liquid interface culture and apical extract stimulation

Primary nasal AECs expanded and differentiated at air-liquid interface. On mucociliary differentiation (Day 28-32 of ALI culture) cultures were washed with PBS and apically treated with mock extract or PM2.5 extract in a final volume of 20 ul culture media; the wash and stimulation were repeated at 24 hours and RNA was collected 24 hours later. For chronic experiments the wash and stimulation were repeated every 24 hours for five stimulations, with a high OE dose or 10 ng/ml IL-13.

Equipment: Transwell inserts; ALI culture media; RNA lysis buffer (Zymo Research) with 40 mM DTT

Whole transcriptome RNA-seq of stimulated ALI cultures

KAPA mRNA HyperPrep (Roche) whole transcriptome libraries from 250 ng RNA per sample with 12 cycles of amplification, sequenced on the Illumina NovaSeq 6000. Paired differential expression analysis between mock-stimulated and extract-stimulated cultures from the same donor; DEG enrichment tested with Enrichr against GO/KEGG/Reactome and against scRNA-seq-derived cell type marker lists, with P values corrected for multiple testing. Networks built with FGNet and GeneTerm Linker.

Equipment: KAPA mRNA HyperPrep kit (Roche); Illumina NovaSeq 6000

Quantitative PCR of chronically stimulated cultures

Quantitative PCR of chronic OE- and IL-13-stimulated cultures against paired mock/media controls. The paper places the detailed qPCR conditions in its online data supplement, which is not part of the cached article text.

Immunofluorescence labeling for MUC5AC and MUC5B

Immunofluorescence labeling of culture histological sections for the gel-forming mucins MUC5AC and MUC5B with DAPI nuclear counterstain, followed by quantification of MUC5AC+ and MUC5B+ cells. Representative of labeling in cultures from four donors.

Equipment: Fluorescence microscope; Anti-MUC5AC antibody; Anti-MUC5B antibody; DAPI

Immunofluorescence labeling for FOXJ1 and acetylated alpha-tubulin

Confocal fluorescence microscopy maximum projections of intact epithelial cultures labeled for E-cadherin, acetylated alpha-tubulin (cilia) and FOXJ1, with DAPI nuclear counterstain, followed by quantification of FOXJ1+ nuclei. Representative of labeling in cultures from three donors.

Equipment: Confocal fluorescence microscope; Anti-FOXJ1 antibody; Anti-acetylated alpha-tubulin antibody; Anti-E-cadherin antibody; DAPI

Alcian blue-periodic acid-Schiff staining of culture sections

Alcian blue-periodic acid-Schiff staining of culture histological sections for mucopolysaccharides/mucins, with quantification of AB-PAS+ cells. Four donor pairs examined.

Equipment: AB-PAS staining reagents; Light microscope

Western blot for SPDEF protein

Western blot analysis of total protein for SPDEF, quantified by band intensity normalized to actin as a loading control. Representative of data from five independent donors.

Equipment: SDS-PAGE apparatus; Anti-SPDEF antibody; Densitometry imaging system

ATP-induced mucin secretion assay with colorimetric ELISA

ATP-induced mucus secretion assay followed by colorimetric ELISA absorbance readings at 490 nm for MUC5AC and MUC5B in the secretions of cultures from five donors.

Equipment: Plate reader (490 nm); MUC5AC ELISA; MUC5B ELISA; ATP stimulation reagent