FAM45A-Mediated Persistent Memory-Like Fibroblast Reprogramming Drives PM2.5-Exacerbated Allergic Asthma

Abstract

Chronic coexposure to fine particulate matter (PM2.5) and house dust mite (HDM) allergen worsens allergic asthma outcomes, yet the stromal cell mechanisms linking combined environmental and allergic risk factors remain incompletely defined. Airway fibroblasts are traditionally viewed as structural matrix-producing cells, with limited published data describing their capacity to acquire antigen-presenting (APC) features after dual pollutant-allergen stimulation. Bronchoalveolar lavage fluid (BALF) fibroblasts represent a less invasive alternative to endobronchial biopsy tissue for primary stromal cell research, reducing procedural risks and sampling limitations in human asthma studies. BALF fibroblasts were isolated from 8 HDM-sensitized asthmatic participants and 8 HDM-sensitized nonasthmatic control subjects. An in vitro costimulation model was established to mimic concurrent PM2.5 and HDM exposure, with matched single-stimulus, vehicle, and TLR4 inhibitor control groups. We applied paired RNA-seq and ATAC-seq multiomics, antigen-specific CD4⁺ T cell coculture assays, CRISPR-Cas9 genetic editing, lentiviral complement, and humanized NSG mouse intervention experiments to characterize fibroblast functional shifts linked to air pollution. BALF fibroblasts from HDM-sensitized asthmatics displayed elevated HLA-DR, CD80, and CD86 surface markers alongside enhanced IL-33 secretion, and supported robust antigen-specific CD4⁺ T cell proliferation. Fibroblasts cultured with PM2.5 and HDM together acquired persistent memory-like functional alterations not observed under single-agent treatment. Integrated multiomic profiling identified FAM45A as the top transcript selectively upregulated only under dual exposure. In vitro assays demonstrated FAM45A interacts with V-ATPase complexes to slow lysosomal allergen degradation and amplifies NF-κB/AP-1 inflammatory signaling. CRISPR knockout and lentiviral rescue experiments support that FAM45A is required for the dual-stimulation-induced fibroblast phenotype. Neutralizing anti-FAM45A antibody reversed APC-like fibroblast activity in vitro and alleviated PM2.5/HDM-triggered airway inflammation in humanized mice. In a power-calculated expanded clinical cohort, fibroblast FAM45 protein levels positively correlated with residential PM2.5 exposure duration, serum HDM IgE, and GINA asthma severity. Concurrent PM2.5 and HDM exposure drives persistent memory-like functional and chromatin remodeling of human airway fibroblasts. FAM45A acts as a key molecular mediator of pollution-amplified allergic inflammation. BALF fibroblast FAM45A levels may serve as a correlative tissue marker reflecting combined environmental and allergic disease burden. Targeted FAM45A inhibition may offer a more tissue-restricted intervention relative to broad corticosteroid therapy, though comprehensive preclinical safety assessment across systemic immune compartments is needed to clarify off-target risks.

1. Introduction

Airway fibroblasts are long categorized as static structural cells that synthesize extracellular matrix; prior asthma immunology research has prioritized dendritic cells and macrophages as primary drivers of HDM-mediated allergic responses. , Few published studies have evaluated whether resident bronchial fibroblasts gain immune-modulating properties after simultaneous PM2.5 and HDM exposure, a common real-world environmental pairing linked to elevated asthma hospitalization risk. − Endobronchial biopsy provides gold-standard primary fibroblast samples but carries high procedural invasiveness, low cell yield, and strict institutional ethical constraints that limit large-scale human experimental work. − BALF-derived fibroblasts offer a practical alternative harvested during routine bronchoscopy, retaining native cellular responsiveness to both PM2.5 and HDM without biopsy-related limitations.

Existing in vitro investigations separately assessed fibroblast activation induced by isolated PM2.5 or HDM stimulation. − However, we found limited evidence of integrated chromatin-transcriptome analyses specifically addressing gene programs uniquely triggered by combined pollutant-allergen coexposure. Most single-stressor transcriptomic screens of airway stromal cells cannot resolve synergistic cellular phenotypes that only emerge when PM2.5 and HDM are administered together, creating an unaddressed research gap in environmental asthma pathogenesis.

We first formulated a broad mechanistic hypothesis to guide this work: Co-exposure to ambient PM2.5 and HDM allergen induces persistent functional and chromatin remodeling of human bronchial fibroblasts, which augments antigen-presenting capacity and amplifies allergic airway inflammation. To identify molecular mediators of this synergistic cellular response, we performed paired RNA-seq and ATAC-seq multiomic profiling across unstimulated, single-stimulus, and dual PM2.5 + HDM fibroblast groups. FAM45A emerged as the highest-ranked candidate gene showing coordinated chromatin opening and transcriptional upregulation exclusively under combined pollutant-allergen challenge. We therefore designed a tiered experimental framework to characterize FAM45A’s functional role in pollution-driven fibroblast reprogramming.

The sequential research aims of this study were: (1) compare APC-like fibroblast phenotypes between HDM-sensitized asthmatic and nonasthmatic participants, and test linear correlations between fibroblast FAM45A expression and clinical markers of PM2.5 burden, HDM sensitization, and GINA-defined asthma severity; (2) establish an in vitro dual-exposure model with TLR4 inhibitor controls to isolate synergistic functional and chromatin shifts independent of endotoxin confounding effects; (3) use CRISPR knockout and lentiviral complementation to test whether FAM45A is required for the PM2.5 + HDM-specific fibroblast phenotype; (4) dissect two linked FAM45A-regulated pathways governing intracellular allergen processing and pro-inflammatory transcription; (5) evaluate in vitro and humanized mouse in vivo effects of FAM45A neutralization on pollution-amplified allergic airway inflammation.

2. Materials and Methods

2.1. Human and Animal Ethical Approval

All human research protocols were approved by the Institutional Ethics Committee of Shenzhen University (Approval No. 202300103). All enrolled participants provided written informed consent prior to BALF collection, serological testing, and pulmonary function assessment. All human biospecimen handling followed the Declaration of Helsinki and international human tissue research standards. Minimally invasive BAL sampling reduced procedural risk relative to endobronchial biopsy and simplified institutional ethical oversight. All animal experiments received approval from the Shenzhen University Animal Ethics Committee (Approval No. 202300011) and fully complied with ARRIVE 2.0 reporting guidelines. Mice were randomly allocated to treatment groups before pollutant/allergen challenge; all histological, flow cytometric, and airway function readouts were assessed by investigators blinded to group assignment. A full ARRIVE compliance checklist is provided within Supporting Information.

2.2. Human Clinical Cohort Design

Primary discovery cohort (n = 16): 8 HDM-sensitized moderate-severe asthmatic adults and 8 HDM-sensitized nonasthmatic control participants with no chronic respiratory or autoimmune disease. All subjects underwent standardized bronchoscopy for BALF fibroblast isolation. HDM sensitization was validated via skin prick testing and ImmunoCAP (IgE ≥0.7 kUA/L for Dermatophagoides pteronyssinus/farinae).

Expanded correlation cohort (n = 32 HDM-sensitized asthmatics): Sample size was calculated via a priori power analysis (Pearson r = 0.76, α = 0.05, statistical power = 0.95; minimum n = 27). Thirty-two participants were enrolled to reduce sampling bias. Stratification criteria: GINA grade (mild n = 8, moderate n = 12, severe n = 12); 6 month continuous residential PM2.5 monitoring (high exposure >75 μg/m³, low exposure <35 μg/m³); serum HDM-IgE tier (low: 0.7–3.4 kUA/L; moderate: 3.5–17.4 kUA/L; high ≥17.5 kUA/L). Full participant clinical metadata are listed in Table S1 within Supporting Information.

2.3. BALF-Derived Bronchial Fibroblast Isolation and Culture

BALF samples were processed immediately postcollection via density gradient centrifugation and selective adherent culture to purify bronchial fibroblasts. Briefly, BALF was centrifuged at 300g for 10 min at 4 °C. Cell pellets were resuspended in PBS and layered over Ficoll–Paque Plus for mononuclear separation (400g, 20 min, room temperature). Cells were seeded in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C, 5% CO₂. Nonadherent immune cells were removed via medium exchange at 48 h. Adherent fibroblasts were expanded through serial passaging and exclusively cultured at passages 3–6 to retain native primary phenotypes. Fibroblast purity was validated by flow cytometry (vimentin⁺CD45^–EpCAM^–α-SMA^–, >98% purity). qRT-PCR confirmed stable expression of canonical airway fibroblast transcripts (COL1A1, FSP1, TGFB1, IL33). Parallel stimulation assays verified functional equivalence between BALF fibroblasts and biopsy-derived fibroblasts (Figure S1). Commercially sourced human lung fibroblasts (Lonza CC-2512) were used for cross-validation of all memory-like reprogramming assays.

For multiomic sequencing, fibroblasts sourced from identical donor batches were evenly split and assigned to each of the five experimental groups (unstimulated control, PM2.5-only, HDM-only, PM2.5 + HDM costimulation, patient asthmatic fibroblasts). Three independent biological replicates were generated per group at passage 4 to eliminate interdonor and interbatch confounding variation.

2.4. PM2.5, HDM Stimulation and TLR4 Inhibitor Treatment Protocols

Urban PM2.5 particles were collected in Shenzhen urban air; full physicochemical characterization including aerodynamic diameter (2.1 ± 0.4 μm), ICP–MS elemental profiling, and TGA thermal analysis are listed in Table S2. Recombinant D. pteronyssinus HDM extract (Greer c163) was applied at 10 μg/mL, a physiological airway concentration validated by prior airway cell models. All stimulation doses were prevalidated via LDH release assay to exclude cytotoxicity artifacts. Three parallel stimulation arms were established for all in vitro experiments: PM2.5 alone (50 μg/mL), HDM alone (10 μg/mL), PM2.5 + HDM costimulation; vehicle-only medium served as baseline negative control.

TLR inhibitor specifications: TAK-242 (Merck Millipore, catalog 614316), working concentration 1 μM, 2 h preincubation prior to pollutant/allergen exposure to eliminate LPS-mediated confounding inflammation.

Memory priming workflow: Fibroblasts received 6 days of assigned stimulation, followed by 5 days of stimulant-free washing and culture. A matched unprimed fibroblast group received identical 100 ng/mL LPS restimulation to isolate memory-specific inflammatory responses. The sustained APC phenotype was tracked for ≥14 days post stimulus withdrawal (Figure S2).

2.5. CRISPR-Cas9 Knockout and Lentiviral Rescue

FAM45A sgRNA sequence: 5′-GCTGCTGATGAAGATGCTGG-3′; nontargeting scrambled sgRNA as negative control. Recombinant Cas9 delivered via Lipofectamine 3000. FAM45A transcript/protein depletion efficiency >90% confirmed by qRT-PCR and flow cytometry (Figure S3). For rescue assays, full-length human FAM45A lentivirus (GeneCopoeia EX-Z1456-Lv105) or empty backbone transduced into KO fibroblasts; APC functional recovery assessed 72 h post-transduction.

2.6. Flow Cytometry, Immunocytochemistry & Complete Antibody Catalog

All primary antibodies (clone, vendor, catalog):

Surface markers: HLA-DR (L243, BioLegend 307618), CD80 (2D10, BioLegend 305208), CD86 (IT2.2, BioLegend 305408), vimentin (O91D3, CST 5741S), CD45 (HI30, BioLegend 304014), EpCAM (9C4, BioLegend 324208), α-SMA (1A4, Thermo MA1-06116).

Intracellular targets: FAM45A (Novus NBP1-87061), IL-33 (396118, R&D MAB3625), Der p1 (15A11, Greer 10-P10).

Secondary antibodies: Alexa Fluor 488/594/647 goat anti-IgG (Thermo Fisher).

Intracellular staining utilized Foxp3 Fix/Perm buffer. Flow cytometry run on BD LSRFortessa; analysis via FlowJo v10.8. Immunocytochemistry used Vectastain ABC kit; Zeiss LSM880 confocal imaging with ImageJ signal quantification.

2.7. Antigen Presentation & CD4⁺ T Cell Coculture

Fibroblasts pulsed with 10 μg/mL OVA323-339 or HDM extract for 4 h. HDM-sensitized human CD4⁺ T cells isolated via EasySep kit, expanded 7 days with IL-2 (purity >95%). Fibroblast:T cell coculture ratio fixed at 1:10 for 72 h. CFSE dilution measured proliferation; sandwich ELISA quantified supernatant IFN-γ and IL-4 (Figure S9). All cocultures included unstimulated and single-stimulus fibroblast controls.

2.8. RNA-seq & ATAC-seq, Data Deposition & Metadata

RNA-seq libraries: TruSeq Stranded mRNA; ATAC-seq: Active Motif kit, NovaSeq 6000 PE150 sequencing. Bioinformatics: STAR alignment, DESeq2 (FC > 2, adj.P < 0.01); Bowtie2, MACS2 (FC > 1.5, adj.P < 0.05). Synergy analysis identified transcripts/peaks with exclusive dual-exposure upregulation. All raw sequencing fastq files deposited to NCBI (PRJNA1291257). Full QC metrics in Figure S4; qRT primers in Table S3.

2.9. Phagosomal Function, Co-IP & PLA Assays

pHrodo-OVA/Der p1 tracked phagosomal pH; DQ fluorogenic substrates measured proteolysis. Cathepsin S/L activity via commercial fluorometric kits. Co-IP + LC–MS/MS identified V-ATPase interactors ATP6 V1A/ATP6 V0D2; Duolink PLA validated in situ binding (Figure S5). Parallel moDC vs fibroblast phagosomal kinetics in Figure S6. Transient V-ATPase inhibition is a known feature of myeloid APCs.

2.10. NF-κB/AP-1 Dual-Luciferase Reporter Assays

Fibroblasts cotransfected with firefly reporter, Renilla normalization vector, FAM45A overexpression/empty plasmid. Luminescence read at 24 h post-transfection; parallel KO fibroblast testing under single/dual stimulation (Figure S7). NF-κB/AP-1 are established allergic inflammatory transcription factors.

2.11. FAM45A Neutralization Antibody Characterization

Anti-FAM45A polyclonal antibody titrated 0–10 μM; maximal target suppression at 10 μM. SPR and CETSA validated antibody specificity (Figure S8). Washout assay: 24 h antibody treatment followed by 72 h inhibitor-free culture to assess phenotype recovery.

2.12. Humanized NSG Mouse Model & Standardized Histopathology Scoring

Six–eight week female NSG mice (Jackson 005557) reconstituted with 1 × 10⁷ HDM-sensitized human PBMC i.p. Four weeks later, thrice-weekly intranasal PM2.5 + HDM challenge for 14 days, followed by intratracheal fibroblast injection (1 × 10⁶ in Matrigel). Five experimental groups (n = 6 per mouse):

• (1)Nonasthmatic fibroblast + vehicle.
• (2)Asthmatic fibroblast + vehicle.
• (3)Asthmatic fibroblast + daily 100 μg anti-FAM45A i.p. (14 days).
• (4)FAM45A-KO asthmatic fibroblast + vehicle.
• (5)PM2.5 + HDM challenge only (no fibroblast) + vehicle.

End point BAL collected for inflammatory cell, CD4⁺ T cell, HDM-IgE, IL-33 quantification via flow/ELISA. Lung tissue fixed 4% PFA, paraffin embedded, 5 μm sections stained H&E and Masson’s trichrome.

Complete histological scoring protocol: For each lung lobe, 5 nonoverlapping 20× random fields captured per section. Two blinded board-certified pathologists scored peribronchial infiltrate on a 0–4 ordinal scale (0 = no infiltration, 4 = full transmural leukocyte accumulation). Subepithelial collagen thickness measured at 8 equidistant positions per bronchial lumen, averaged per airway for morphometric comparison.

2.13. Statistical Analysis

All in vitro data presented as mean ± SD, minimum three independent biological replicates with technical triplicates. Two-way ANOVA tested PM2.5 × HDM synergistic interactions. Two-group comparisons used unpaired two-tailed t-test; multigroup analysis via one-way ANOVA + Tukey posthoc correction. Pearson correlation assessed linear clinical associations. P < 0.05 defined statistical significance. Software: GraphPad Prism 9, R v4.5.2.

3. Results

3.1. BALF Fibroblast FAM45A Expression Correlates with Asthma Severity, PM2.5 Exposure and HDM-IgE

We isolated BALF fibroblasts from 8 HDM-sensitized asthmatic patients and 8 HDM-sensitized nonasthmatic control participants (Table S1). Portable monitors quantified 72 h continuous residential PM2.5 exposure for all enrolled subjects. Flow cytometric analysis demonstrated elevated HLA-DR surface expression in fibroblasts from asthmatic donors (Figure A). Asthma-derived fibroblasts also displayed significantly higher CD80 and CD86 costimulatory marker abundance (Figure B). IL-33 secretion was elevated across all stimulation conditions, with maximal levels detected after PM2.5 + HDM coexposure (Figure C). Composite APC phenotype scores (weighted normalized MFI for HLA-DR, CD80, CD86) were significantly higher in patient-derived fibroblast cultures under all treatment regimens. Fibroblasts isolated from asthmatic participants supported robust OVA and HDM-specific CD4⁺ T cell proliferation and mixed Th1/Th2 cytokine release, whereas control fibroblasts lacked functional APC activity (Figure S9). Consistent APC phenotypic differences were replicated across independent primary fibroblast isolates, aligning with prior reports of stromal immune priming capacity.

1.

FAM45A expression in BALF-derived bronchial fibroblasts increases with asthma severity and correlates with PM2.5 exposure and HDM-specific IgE. (A–C) Primary bronchial fibroblasts isolated from BALF of HDM-sensitized asthma patients and HDM-sensitized nonasthmatic controls were analyzed for APC-like phenotypic and functional properties. (A) Flow cytometry (left) and qRT-PCR (right) revealed significantly increased surface expression and transcript levels of HLA-DR, CD80, and CD86 in asthma-associated fibroblasts compared with controls, consistent with an antigen-presenting cell–like phenotype. (B) Within the asthma cohort, fibroblasts from patients with high PM2.5 exposure and high HDM-specific IgE exhibited significantly higher composite APC-like scores than those from patients with low PM2.5 exposure and low HDM-specific IgE, indicating synergistic upregulation. (C) Asthma-associated fibroblasts secreted elevated levels of IL-33 under basal, PM2.5, HDM, and combined PM2.5 + HDM stimulation, with maximal secretion observed under costimulation conditions. (D–F) Primary BALF-derived bronchial fibroblasts from an expanded clinical cohort of HDM-sensitized asthma patients (n = 32) were analyzed for FAM45A expression by flow cytometry and immunocytochemistry (ICC). (D) FAM45A expression was significantly increased in fibroblasts from patients with moderate and severe asthma compared with those from mild asthma patients. (E) FAM45A expression (flow cytometry) showed strong positive correlations with asthma severity (GINA grade), residential PM2.5 exposure, and serum HDM-specific IgE levels; linear regression lines are shown for visualization. (F) Severe asthma patients with high PM2.5 exposure and high HDM-specific IgE exhibited the highest FAM45A expression, whereas mild asthma patients with low PM2.5 exposure and low HDM-specific IgE showed levels approaching those of nonasthmatic controls. Data are presented as mean ± SD with individual data points overlaid. Statistical significance is indicated by symbols (**P < 0.01; ****P < 0.0001).

We recruited a power-calculated expanded cohort of 32 HDM-sensitized asthmatic participants. Flow cytometry and immunocytochemistry quantified fibroblast FAM45A protein abundance, which was significantly higher in patients with moderate-severe asthma relative to mild cases (P < 0.001). FAM45A expression exhibited strong positive linear correlations with three independent clinical metrics: GINA asthma severity grade (Pearson r = 0.78, P < 0.001, Figure D), 72 h average residential PM2.5 concentration (r = 0.72, P < 0.001, Figure E), and serum HDM-specific IgE titer (r = 0.69, P < 0.001, Figure F). Patients with severe asthma, sustained high PM2.5 exposure, and elevated HDM IgE displayed maximal FAM45A levels, while mild asthmatics with low environmental allergen burden expressed FAM45A at levels comparable to nonasthmatic controls. This correlation pattern supports FAM45A as a tissue marker integrating particulate pollution and allergic disease risk.

3.2. Dual PM2.5 and HDM Exposure Induces Persistent Memory-like Fibroblast Functional Shifts

We tested whether combined pollutant-allergen stimulation drives long-lasting fibroblast reprogramming distinct from single-agent exposure. After 6 days of priming followed by 5 days of stimulant-free rest, LPS restimulation induced significantly higher IL-6, IL-33, and TNF-α secretion in fibroblasts previously coexposed to PM2.5 and HDM (Figure A). Pretreatment with selective TLR4 inhibitor TAK-242 failed to ablate this synergistic inflammatory response, ruling endotoxin contamination as a confounding driver of the observed phenotype. Seahorse glycolysis stress testing revealed a synergistic shift toward glycolytic metabolism exclusive to dual PM2.5 + HDM costimulation (Figure B). ATAC-seq chromatin profiling identified thousands of uniquely open genomic loci in coprimed fibroblasts, with peak motif analysis showing significant enrichment of NF-κB and AP-1 binding sequences (Figure C,D). The APC-like phenotype (elevated MHC-II, CD86, enhanced T cell priming capacity) persisted for a minimum of 14 days after complete withdrawal of PM2.5 and HDM stimuli (Figure S10). This persistent functional shift aligns with prior descriptions of trained-like memory responses in lung stromal populations.

2.

PM2.5 and HDM coexposure induces memory-like inflammatory, metabolic, and epigenetic reprogramming in HDM-sensitized bronchial fibroblast precursors. (A) HDM-sensitized bronchial fibroblast precursors were exposed to PM2.5 alone, HDM alone, or PM2.5 + HDM for 6 days, followed by a 5 day rest period in stimulant-free medium and subsequent restimulation with LPS in the absence of additional PM2.5 or HDM. Cytokine production after LPS restimulation was used to assess memory-like inflammatory reprogramming. Cells previously exposed to PM2.5 + HDM produced significantly higher levels of IL-33, IL-6, and TNF-α than those exposed to PM2.5 or HDM alone, indicating a synergistic enhancement of trained inflammatory responsiveness. (B–D) PM2.5 and HDM copriming induces coordinated metabolic and epigenetic remodeling in bronchial fibroblasts. Fibroblasts were primed with PM2.5 alone, HDM alone, or PM2.5 + HDM prior to analysis. (B) Seahorse XF Glycolysis Stress Test demonstrated a marked increase in extracellular acidification rate (ECAR) in coprimed fibroblasts, consistent with a synergistic shift toward glycolysis relative to single-stimulation conditions. (C) Co-primed fibroblasts exhibited a significant increase in global chromatin accessibility, reflecting widespread epigenetic remodeling. (D) ATAC-seq motif enrichment analysis revealed that accessible chromatin regions in coprimed fibroblasts were enriched for binding motifs of pro-inflammatory transcription factors, including NF-κB, AP-1, and STAT6, compared with single-stimulation controls. Data are presented as mean ± SD with individual data points overlaid. Statistical significance is indicated above bars with connecting lines (***P < 0.001, ****P < 0.0001, as appropriate).

3.3. FAM45A Is Required for PM2.5/HDM-Induced Fibroblast APC Reprogramming

Integrated RNA-seq and ATAC-seq multiomic screening across five fibroblast experimental groups identified FAM45A as the top-ranked transcript with >12-fold selective upregulation only under dual PM2.5 + HDM coexposure (Figure A–C). No meaningful FAM45A induction was detected after isolated PM2.5 or HDM treatment. CRISPR-Cas9 FAM45A knockout completely eliminated PM2.5/HDM-triggered upregulation of HLA-DR, CD80, CD86, and IL-33, and abolished fibroblast capacity to drive antigen-specific CD4⁺ T cell activation (Figure D–H). Lentiviral reintroduction of FAM45A into knockout fibroblasts partially rescued the APC phenotype exclusively under dual pollutant-allergen costimulation. Combined loss- and gain-of-function genetic data support FAM45A as a required mediator of the coexposure-specific fibroblast pathogenic program.

3.

Integrated multiomics and validation analyses identify FAM45A as a synergistically induced target in asthma-associated and PM2.5 + HDM coprimed bronchial fibroblasts BALF-derived bronchial fibroblasts from HDM-sensitized normal donors, asthma-associated samples, and cells primed with PM2.5 alone, HDM alone, or PM2.5 + HDM were analyzed to identify genes associated with synergistic reprogramming. (A) Integrated ATAC-seq and RNA-seq analysis identified FAM45A as a top candidate gene, showing concomitant increases in promoter chromatin accessibility and transcript expression in both asthma-associated and PM2.5 + HDM coprimed fibroblasts. In the coprimed group, RNA-seq expression exceeded a 12-fold increase relative to single-stimulation controls. (B) qRT-PCR validation confirmed markedly elevated FAM45A mRNA levels in asthma-associated and PM2.5 + HDM coprimed fibroblasts compared with HDM-sensitized normal and single-stimulation control groups. (C) Western blot analysis similarly demonstrated increased FAM45A protein abundance in asthma-associated and PM2.5 + HDM coprimed fibroblasts, whereas PM2.5-only and HDM-only priming did not significantly increase FAM45A expression. (D–H) FAM45A is a required and sufficient causal mediator of the APC-like phenotype in respiratory fibroblasts. Quantification of the antigen-presenting cell (APC)-like phenotype in HDM-sensitized BALF-derived bronchial fibroblasts. Fibroblasts were subjected to CRISPR-Cas9-mediated knockout (KO) of FAM45A or lentiviral-mediated rescue of FAM45A expression following PM2.5 and HDM costimulation. (D) IL-33 secretion measured by ELISA; (E–G) surface expression of MHC-II, CD80, and CD86 measured by mean fluorescence intensity (MFI) via flow cytometry; and (H) the capacity of coprimed fibroblasts to induce OVA/HDM-specific CD4⁺ T-cell proliferation (%). Data are presented as mean ± SD with individual data points overlaid. Statistical significance is indicated above bars with connecting lines (***P < 0.001, ****P < 0.0001, as appropriate).

3.4. FAM45A Interacts with V-ATPase to Suppress Phagosomal Acidification and Slow Allergen Degradation

We next characterized FAM45A’s regulatory function at the phagosomal compartment. FAM45A overexpression raised average phagosomal pH and reduced lysosomal cathepsin S/L enzymatic activity, slowing proteolytic breakdown of internal OVA and HDM allergen (Figure A–F). Co-immunoprecipitation and Duol PLA in situ binding assays confirmed direct physical interaction between FAM45A and V-ATPase complex subunits ATP6 V1A and ATP6 V0D2 (Figure S11). Myeloid dendritic cells utilize transient V-ATPase inhibition to balance antigen processing and presentation (Niu et al., 2021), whereas airway fibroblasts exhibit sustained FAM45A-dependent proton pump suppression leading to prolonged intracellular allergen retention (Figure S12). In vitro anti-FAM45A antibody treatment restored physiological phagosomal acidification and normalized allergen proteolysis in both patient-derived and coprimed fibroblasts (Figure E–Figure F).

4.

FAM45A-mediated inhibition of phagosomal acidification in respiratory fibroblasts. (A,B): Quantification of phagosomal pH in HDM-sensitized BALF-derived bronchial fibroblasts overexpressing FAM45A (FAM45A-OE) or empty vector control. pH-sensitive fluorescent conjugates of (A) OVA and (B) HDM (Der p1) were used to measure phagosomal acidification via flow cytometry. Higher pH values in FAM45A-OE cells indicate impaired acidification and reduced V-ATPase-mediated proton pumping. (C,D). FAM45A prevents lysosomal antigen degradation via inhibition of phagosomal protease activity. Evaluation of antigen processing kinetics and enzymatic degradation in HDM-sensitized BALF-derived bronchial fibroblasts with high FAM45A expression (FAM45A-high) vs control. (C) Antigen Retention: Quantification of internalized OVA and HDM (Der p1) antigen retention within phagosomes 4 h postuptake, expressed as a percentage of initial load. FAM45A-high fibroblasts show significantly prolonged antigen retention. (D) Protease Activity: Intraphagosomal enzymatic activity of Cathepsin S and Cathepsin L measured using specific fluorogenic substrates. Activity is normalized to the mean of the control group. (E,F). Pharmacological or genetic inhibition of FAM45A restores phagosomal function in PM2.5 + HDM coprimed fibroblasts. (E) Phagosomal pH in control fibroblasts, PM2.5 + HDM coprimed fibroblasts, and coprimed fibroblasts treated with anti-FAM45A Ab or FAM45A KO fibroblasts. Anti-FAM45A treatment and genetic deletion normalize acidification to control levels. (F) Intraphagosomal Cathepsin S activity under the same conditions. Anti-FAM45A treatment or FAM45A deletion restores protease activity to near-control values. Bars represent mean ± SD; dots indicate individual cells or samples. Statistical significance was determined by unpaired two-tailed t-test or one-way ANOVA with post hoc comparisons. ***P < 0.001; ****P < 0.0001.

3.5. FAM45A Amplifies NF-κB/AP-1 Inflammatory Signaling Only under Dual Stimulation

FAM45A silencing ablated PM2.5 + HDM-dependent IL-33 and costimulatory molecule upregulation, with no functional impact under single-agent culture conditions. FAM45A overexpression selectively amplified NF-κB and AP-1 luciferase reporter activity after dual pollutant-allergen coexposure (Figure A–D). NF-κB and AP-1 are well-documented master transcription factors governing allergic inflammatory cascades. Confocal immunofluorescence localized FAM45A primarily to intracellular phagosomal compartments (Figure E), implying an uncharacterized lysosome-to-nucleus signal relay linking V-ATPase dysfunction to downstream inflammatory transcription. FAM45A knockout fibroblasts lost the capacity to induce robust antigen-specific CD4⁺ T cell proliferation under combined PM2.5 and HDM challenge (Figure F.G).

5.

FAM45A is both necessary and sufficient for the acquisition of an APC-like functional phenotype in bronchial fibroblasts under synergistic stimulation. (A,B) FAM45A modulates the expression of pro-inflammatory cytokines and costimulatory molecules. (A) Secretion of IL-33 (ELISA) in BALF-derived asthmatic fibroblasts transfected with control siRNA (siControl) or FAM45A-targeted siRNA (siFAM45A) following stimulation with PM2.5, HDM, or costimulation. FAM45A silencing specifically abrogates the synergistic induction of IL-33. (B) Surface expression (Mean Fluorescence Intensity, MFI) of costimulatory molecules CD80/CD86 in nonasthmatic control fibroblasts following empty vector or FAM45A-overexpression (FAM45A-OE). Overexpression induces an APC-like profile only upon PM2.5 + HDM costimulation. (C–D) FAM45A is essential for synergistic NF-κB and AP-1 signaling. Luciferase reporter activity of NF-κB (C) and AP-1 (D) in HDM-sensitized BALF-derived bronchial fibroblasts. PM2.5 + HDM costimulation significantly increases transcriptional activity in WT cells, whereas FAM45A knockout (KO) abrogates this synergistic effect with negligible impact on single-stimulus activation. (E) FAM45A primarily localizes to the phagosomal compartment. Subcellular localization of FAM45A determined via organelle fractionation and cross-ELISA. FAM45A protein levels are significantly enriched in the phagosomal fraction compared to whole-cell lysates (WCL) and cytosolic fractions. (F,G) FAM45A drives fibroblast-mediated CD4⁺ T-cell activation. (F) Proliferation of OVA/HDM-specific CD4⁺ T cells cocultured with fibroblasts under PM2.5 + HDM costimulation. FAM45A deficiency in asthmatic fibroblasts (siFAM45A) reduces T-cell proliferation, while ectopic FAM45A expression in control fibroblasts (FAM45A-OE) confers activating capacity. (G) IFN-\gamma concentration in coculture supernatants (ELISA) parallels the proliferation patterns observed in (F). Data Statistics: Data are presented as mean ± SD (n = 4 per group). Individual data points are indicated by dots. Statistical significance was determined by Student’s t-test or one-way ANOVA + Tukey post hoc test: **P < 0.01; ***P < 0.001; ****P < 0.0001.

3.6. FAM45A Neutralization Alleviates Pollution-Amplified Airway Inflammation in Humanized Mice

In vitro anti-FAM45A antibody treatment reversed the full APC-like pathogenic phenotype of PM2.5 + HDM coprimed fibroblasts, restoring baseline phagosomal function and suppressing IL-33, MHC-II, and CD86 expression; this phenotypic suppression persisted after antibody washout (Figure S14). In humanized NSG mice reconstituted with HDM-sensitized human PBMC, intratracheal delivery of asthmatic patient fibroblasts induced severe peribronchial inflammatory infiltrates, elevated BAL CD4⁺ T cell counts, increased HDM-specific IgE, and higher BAL IL-33 concentrations alongside augmented subepithelial collagen remodeling (Figure A–K). Systemic 14 day anti-FAM45A antibody administration reduced BAL CD4⁺ T cell infiltration by 72%, lowered BAL HDM-IgE and IL-3 levels by approximately 65–70%, and attenuated histological inflammation and collagen deposition. Mice receiving FAM45A-knockout asthmatic fibroblasts displayed airway pathology comparable to nonasthmatic fibroblast control groups.

6.

Asthma-associated BALF-derived bronchial fibroblasts drive PM2.5-exacerbated allergic airway inflammation in humanized NSG mice and are suppressed by anti-FAM45A therapy or FAM45A knockout. (A–F) Asthma-associated BALF-derived bronchial fibroblasts amplify PM2.5-exacerbated allergic airway inflammation in humanized NSG mice reconstituted with PBMCs from HDM-sensitized donors. Mice were challenged with PM2.5 + HDM and intratracheally injected with either nonasthmatic fibroblasts or HDM-sensitized asthma-associated fibroblasts. (A) Total inflammatory cell count in BALF. (B) CD4⁺ T-cell infiltration in BALF. (C) HDM-specific IgE levels in BALF. (D) IL-33 levels in BALF. (E) Semiquantitative peribronchial inflammatory score from H&E-stained lung sections. (F) Representative H&E-stained lung images (×400). These data show significantly increased airway inflammation in mice receiving asthma-associated fibroblasts relative to nonasthmatic fibroblasts. (G–K) Systemic anti-FAM45A Ab treatment or injection of FAM45A KO asthma-associated fibroblasts attenuates fibroblast-driven allergic airway inflammation. Mice receiving asthma-associated fibroblasts exhibited elevated BALF CD4⁺ T-cell infiltration, HDM-specific IgE, IL-33, and peribronchial inflammation/remodeling compared with controls. Systemic anti-FAM45A Ab administration (100 μg/mouse daily for 2 weeks) significantly reduced these pathogenic responses, and mice injected with FAM45A KO fibroblasts showed near-control levels. (G) BALF CD4⁺ T-cell infiltration. (H) BALF HDM-specific IgE. (I) BALF IL-33. (J) Peribronchial inflammation score. (K) Peribronchial remodeling score. Bars represent mean ± SD; individual dots represent individual mice. One-way ANOVA with Tukey post hoc test was performed. **P < 0.01; ****P < 0.0001.

4. Discussion

This study addresses key technical and ethical barriers limiting airway stromal cell research by validating BALF-derived fibroblasts as a practical alternative to invasive endobronchial biopsy tissue. , Prior asthma immunology literature centers on dendritic cell and macrophage APC activity, , while few publications characterize fibroblast immune functions following combined PM2.5 and HDM exposure. Most existing in vitro models only test single pollutant or allergen stimulation and cannot resolve synergistic stromal transcriptional programs triggered by co-occurring environmental risk factors. , The present work identifies a FAM45A-dependent persistent memory-like fibroblast reprogramming pathway specific to dual PM2.5 + HDM exposure, with integrated clinical, in vitro, multiomic and preclinical data supporting its role in pollution-worsened allergic asthma.

4.1. BALF Fibroblasts as a Clinically Practical Stromal Research Platform

Endobronchial biopsy remains limited by low cell yield and strict ethical oversight requirements. , BALF fibroblasts collected during routine bronchoscopy achieve >98% purity and recapitulate transcriptional, metabolic, and functional profiles of biopsy-derived airway fibroblasts (Figure S1). This surrogate system enables high-throughput CRISPR and multiomic experimentation that would be logistically unfeasible with scarce biopsy tissue. All clinical participants enrolled in this study were uniformly confirmed HDM-sensitized to reflect the dominant global allergic asthma endotype prone to PM2.5 exacerbation. Fibroblasts isolated from asthmatic donors consistently displayed robust APC activity absent in nonasthmatic control subjects, a phenotypic distinction rarely compared across stratified clinical cohorts in prior stromal research. ,

4.2. Synergistic PM2.5/HDM Fibroblast Reprogramming Relies on FAM45A Induction

Individual PM2.5 or HDM exposure only induced mild, transient fibroblast activation in our culture system. Concurrent dual challenge triggered stable functional and chromatin remodeling that persisted for at least 14 days after stimulus removal (Figure S10). This sustained cellular shift aligns with published descriptions of trained memory phenotypes in other lung stromal populations. FAM45A transcriptional upregulation occurred exclusively under combined pollutant-allergen treatment, marking this gene as a unique molecular integrator of overlapping environmental and allergic stress signals. TLR4 inhibitor control experiments eliminate endotoxin contamination as a confounding driver of the synergistic phenotype, strengthening the specificity of the PM2.5/HDM combinatorial signaling cascade identified here. CRISPR knockout and lentiviral rescue experiments genetically confirm FAM45A is required to establish the coexposure APC-like fibroblast state, whereas single-agent stimulation does not induce FAM45A-dependent pathogenic remodeling.

4.3. Dual FAM45A Effector Axes Coordinate Allergen Retention and Inflammatory Signaling

Mechanistic characterization uncovered two interconnected FAM45A-regulated pathways shaping fibroblast APC function. First, FAM45A physically binds V-ATPase proton pump subunits to suppress phagosomal acidification and slow lysosomal allergen proteolysis (Figure ). Myeloid dendritic cells employ transient V-ATPase inhibition to balance controlled antigen epitope generation. Airway fibroblasts exhibit prolonged FAM45A-mediated proton pump suppression that preserves intact HDM antigen for extended local T cell activation, a cell-type-specific functional divergence that appears understudied in existing airway biology literature. Second, FAM45A amplifies NF-κB/AP-1-dependent transcription to drive secretion of the pro-allergic alarmin IL-33 and surface costimulatory ligands. Confocal imaging localizes FAM45A to phagosomal compartments, highlighting an uncharacterized lysosome-to-nucleus signal transduction axis that requires follow-up adaptor protein screening to fully resolve.

4.4. Clinical Translational Value of BALF Fibroblast FAM45A

Standard clinical asthma assessment tools cannot quantify tissue-level molecular interactions between chronic particulate pollution burden and HDM sensitization status. Our power-calculated expanded patient cohort demonstrates consistent linear correlations between BALF fibroblast FAM45A levels and three core clinical readouts: GINA severity grading, long-term residential PM2.5 exposure, and circulating HDM-specific IgE titers (Figure D–F). Quantification of FAM45A from routine BAL samples provides a tissue-resident molecular marker that integrates environmental and allergic disease risk, though prospective longitudinal patient cohorts will be required to test whether baseline FAM45A levels predict long-term exacerbation risk or therapeutic responsiveness.

4.5. Translational Outlook for FAM45A-Targeted Intervention

In vitro neutralizing antibody treatment reversed the full spectrum of pathogenic fibroblast phenotypes triggered by PM2.5 + HDM coexposure. Humanized mouse preclinical data show FAM45A blockade reduces airway inflammatory infiltrate and collagen remodeling driven by asthmatic patient fibroblasts. This preferential suppression of the coexposure-specific pathogenic program suggests FAM45A may provide a more tissue-restricted intervention relative to broadly immunosuppressive corticosteroid therapies, though comprehensive evaluation of systemic and pulmonary host defense pathways is necessary to fully characterize potential off-target risks associated with long-term inhibition.

5. Study Limitations

• (1)Although BALF fibroblasts functionally match biopsy-derived stromal cells, future independent replication using endobronchial biopsy isolates will strengthen translational generalizability of our findings.
• (2)The complete lysosome-to-nucleus signaling cascade linking V-ATPase suppression to NF-κB/AP-1 activation remains incompletely defined and requires targeted adaptor protein screening in follow-up work.
• (3)All clinical correlative analyses rely on cross-sectional patient data; prospective longitudinal cohorts are needed to validate whether baseline BALF FAM45A predicts long-term asthma progression and therapeutic response.
• (4)Extended preclinical safety profiling across multiple organ systems and infectious challenge models is required to assess pulmonary host defense risks associated with sustained V-ATPase modulation via FAM45A inhibition.
• (5)Additional testing across pollen and mold-sensitized patient cohorts will clarify whether FAM45A-mediated fibroblast reprogramming represents a universal pollution-amplified asthma mechanism or HDM-specific pathway.

6. Conclusion

Concurrent PM2.5 particulate pollution and HDM allergen exposure drives persistent memory-like functional and chromatin remodeling of human airway fibroblasts toward an antigen-presenting cell phenotype. FAM45A acts as a key molecular mediator of pollution-amplified allergic airway inflammation via two coordinated effector programs: direct physical interaction with V-ATPase complexes to suppress phagosomal acidification and prolong intact HDM allergen retention, and amplification of NF-κB/AP-1 transcriptional activity to boost pro-inflammatory IL-33 and costimulatory molecule expression. CRISPR genetic manipulation and humanized mouse antibody intervention support that FAM45A activity contributes to pollutant-exacerbated allergic airway pathology. Fibroblast FAM45A quantified from routine BAL specimens may serve as a correlative tissue marker integrating environmental particulate and allergic asthma burden. Targeted FAM45A neutralization offers a potential tissue-restricted intervention approach for PM2.5-worsened HDM allergic asthma, though comprehensive long-term preclinical safety assessment and prospective human cohort validation are needed to confirm translational utility. This study also establishes BALF-derived fibroblasts as a minimally invasive, scalable experimental platform overcoming longstanding technical and ethical barriers limiting stromal cell research in allergic airway disease.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/EHP.6c00353.

• Supplementary Figure S1-Figure S13, Supplementary Tables 1–3 (clinical metadata, PM2.5 physicochemical characterization, qRT-PCR primer sequences), complete multiomics quality control metrics, ARRIVE 2.0 compliance checklist, and extended in vitro/in vivo experimental protocols (PDF)

• Table S1. Demographic and Clinical Characteristics of Human Study Participants (PDF)

• Table S2: Elemental and Compositional Characteristics of Shenzhen Urban PM2.5 Particles Used in the Study (PDF)

• Table S3. Human qPCR Primer Sequences (SYBR Green, Exon-spanning) (PDF)

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B.L. and Y.F. equally contributed to this work.

The authors declare no competing financial interest.