FTO-ACKR3 Axis Regulates Polycyclic Aromatic Hydrocarbons-Induced Asthma Attacks via AhR-Mediated m⁶A RNA Methylation

Abstract

Airborne polycyclic aromatic hydrocarbons (PAHs) are increasingly linked to asthma onset and exacerbation, yet their molecular mechanisms remain unclear. Building on our previous finding that PM2.5 alters m6A RNA methylation, we hypothesized that PAHs drive asthma progression through epitranscriptomic regulation. Here, we show that PAH exposure aggravated lung injury, airway remodeling, and fibrosis while reducing global m6A levelsan effect reversed by AhR inhibition. Mechanistically, PAH-activated AhR suppressed transcription of the m6A demethylase FTO, leading to widespread hypomethylation. Reduced m6A deposition on ACKR3 mRNA enhanced its stability through IGF2BP1/2/3 binding, thereby increasing ACKR3 expression. Elevated ACKR3 promoted autophagy and inflammatory signaling in bronchial epithelial cells. Consistently, FTO-deficient mice displayed exacerbated airway inflammation, fibrosis, and ACKR3-driven autophagy. Moreover, PAHs activated the TNFα/NF-κB pathway, further amplifying autophagy and inflammation. Together, these findings define a previously unrecognized AhR–FTO–ACKR3 epitranscriptomic axis through which PAHs potentiate NF-κB signaling, autophagy, and airway injury, providing new mechanistic insight and potential therapeutic targets for pollution-driven asthma.

Introduction

Environmental pollutants, particularly fine particulate matter (PM2.5), pose a severe global public health risk. Over 99% of the world’s population is exposed to PM2.5 levels exceeding WHO guidelines. PM2.5 carries toxic polycyclic aromatic hydrocarbons (PAHs), which are mutagenic and carcinogenic, − and are linked to pulmonary diseases including asthma, fibrosis, and COPD. Asthma alone affects over 300 million people worldwide, with PM2.5 and PAHs contributing to its development and exacerbation.

The airway epithelium serves as the first line of defense against pollutants. , PAHs disrupt tight junction proteins in epithelial cells, promoting inflammation. Nevertheless, there is still a lack of comprehensive understanding regarding the intricate mechanisms that cause asthma and worsen its symptoms due to PAHs exposure.

Epigenetic modifications, especially RNA methylation (m⁶A), play a critical role in lung development and disease. PAHs-induced DNA methylation changes have been well documented, but its effect on m⁶A RNA methylation is largely unknown. − m⁶A is regulated by writers (e.g., METTL3), erasers (e.g., FTO), and readers, influencing mRNA stability and translation. − Although m⁶A dysregulation is implicated in lung cancer and fibrosis, − its role in PAH-induced asthma is unclear. Recent evidence suggests that the aryl hydrocarbon receptor (AhR), activated by PAHs, may mediate m⁶A alterations, contributing to inflammatory responses. ,

We hypothesized that AHR activation by PM2.5-bound PAHs disrupts m⁶A methylation, accelerating asthma progression. Our previous work showed that PM2.5 alters m⁶A levels via METTL3 and FTO, affecting gene expression and promoting lung injury. Here, we treated bronchial epithelial cells with PAHs and observed suppressed FTO expression and increased m⁶A methylation, leading to pulmonary inflammation and fibrosis in mice. We identified ACKR3 (CXCR7) as a key target regulated by m⁶A-dependent mRNA stability and translation, triggering autophagy and inflammation. Furthermore, FTO deficiency exacerbated lung autophagy and fibrosis. PAHs induced autophagy and promoted FTO degradation by suppressing p62 transcription. These findings reveal a novel mechanism through which PAHs impair FTO-mediated m⁶A modification, activating autophagy and inflammation, thereby worsening asthma. This study underscores the significance of epitranscriptomic regulation in environmental asthma pathogenesis.

Materials and Methods

Reagents

The antibodies used in this study were Anti-FTO (ab92821), Anti-NF-κB (ab32536), Anti-ACKR3 (ab72100), Anti-GAPDH (ab8245), Anti-α-SMA (ab314895), Antifibronectin (ab2413), Pro-caspase1 (ab179515), Anti-p62 (ab109012), NLRP3 (ab263899), Anti-LC3B (ab192890), Anticollagen-I (ab260043), Anti-IL-1β (ab283818), METTL3 (ab195352), and Anti-MUC5AC (ab3649), all obtained from Abcam. Cell Signaling Technology provided antibodies for METTL14 (48699), Anti-ATG5 (12994), Anti-ATG7 (8558), Anti-IGF2BP1 (8482), Anti-IGF2BP2 (14672), and Anti-IGF2BP3 (57145). We would like to thank Abcam for kindly providing us with the secondary antibodies (Abcam, ab150113 and ab150). The Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17–700) was purchased from Sigma-Aldrich, located in Missouri. The Pierce BCA Protein Assay Reagent A was sourced from Beyotime, a leading company based in Shanghai, China. The ChamQ Universal SYBR qPCR Master Mix was acquired from Vazyme, a top-tier company based in Nanjing, China. We used the ECL kit to measure m⁶A RNA methylation (Beyotime, P0018FS). Hydroxychloroquine (HCQ, HY-17589A), TNF-α (HY-P70426A), and Actinomycin D (HY-17559) were obtained from MCE (Shanghai, China). The Hoechst 33258 stain solution (1 mg/mL, C0021) and 4′,6-diamidino-2-phenylindole (DAPI) solution (1 mg/mL) were acquired from Solarbio (Beijing, China). Compounds CS1 (5 mg/kg, Bisantrene, NSC-337766, CL 216,942, Cat. S9946) and CS2 (5 mg/kg, Brequinar, Cayman, Cat. 24445) were also employed.

Plasmids

Genechem Co., Ltd. (Shanghai, China) provided lentiviral vectors expressing short hairpin RNA targeting negative control (shNC), FTO (shFTO), and ACKR3 (shACKR3). Cloning two separate shRNAs targeting FTO and ACKR3 produced these vectors. The plasmid pGL4 NF-κB-Luc was procured from Promega. Adapted from, CRISPR was used to create FTO-knockout cells. Two guide RNA sequences (Table S1) were bought from Genechem Co., Ltd. (Shanghai, China) for the Cas9 lentiviral vector.

Murine Model of Asthma and Treatments

PM2.5 samples were collected on 47 mm quartz membranes using a Tianhong TH-150C sampler (Wuhan, China), as reported. The organic extractable matter (OEM) was isolated from ambient PM2.5 via Soxhlet extraction with dichloromethane, a method known for high efficiency and solubility. Filter pieces (5 per tube, 3 tubes) were immersed in 40 mL dichloromethane and sonicated (50 min, 100 W, 20 °C). The extracts were pooled, filtered through 0.45 μm cellulose acetate filters (Sartorius), and evaporated under nitrogen to a final volume of 5 mL. From this, 1 mL was used for chemical characterization. To prevent c-PAH loss during evaporation, 100 μL of 1,2-propanediol was added as a keeper. The residue was redissolved in 4 mL DMSO to yield the OEM stock, which was stored at −80 °C for toxicological assays.

In total, 212.5 mg of PM2.5 from 15 filter pieces yielded OEM at 42.5 mg PM-equivalent/mL (PM-eq/mL). This concentration was used to calculate exposure levels (μg PM-eq/cm²) for BEAS-2B cells. PAH quantification was performed by GC–MS (Agilent 7890B/5977A) with a 60 m DB-5MS column (J&W Scientific, Folsom), using helium as the carrier gas (1 mL/min), after dichloromethane extraction of pristine filters.

Six- to eight-week-old C57BL/6J female mice (Beijing HFK Bioscience) and FTO knockout females (Cyagen, Guangzhou, China) were acclimated for 7 days before exposure. Mice were housed in individually ventilated cages (IVC) under controlled conditions, with ad libitum access to water and chow. Animals were randomly assigned to PBS control (Ctrl), PAHs, asthma (HDM), or asthma + PAHs (HDM+PAHs, HP) groups.

A total of 36 C57BL/6J or FTO-KO females were used, with six mice per subgroup (pulmonary function, histopathology, and other analyses). Chronic asthma was induced with 3 μg HDM extract (Greer Laboratories) in 20 μL PBS administered intranasally on days 0 and 10, followed by 10 μg HDM every 2 days from day 14. Intervention groups additionally received intranasal instillations of PAHs (10 μL, 10 μg/μL) 5 days per week for 6 weeks, while controls received equal volumes of DMSO. Except during exposure, mice were maintained in filtered-air cages. All animals were euthanized with 1% pentobarbital sodium the day after the final exposure, and lung tissues were harvested. Animal protocols were approved by the West China Hospital of Sichuan University (Approval No. 20240528109).

Pulmonary Function Test, BALF Collection, Cell Counting

The FinePointe Pulmonary Function Test instrument (DSI) was used to evaluate pulmonary function in mice (FTO^+/+ and FTO^–/–, with or without PAHS treatment) 24 h after exposure, the protocol adapted from. Mice were terminally anaesthetized and had their right lungs lavaged four times with 1 mL PBS. The collected cell suspension was centrifuged at 1000g for ten min to pellet cells then resuspended with PBS and hemocytometer counted.

ELISA

The concentration of interleukin-4/5/13 in BALFs was measured by ELISA kits (MEIMIAN) following manufacturer procedures.

Immunofluorescence Staining

An established protocol was followed for immunofluorescence staining. The tissues were subjected to a brief fixation, permeabilization, and blocking process with 10% serum, followed by an incubation period with primary antibodies, including anti-LC3B, anti-FTO, and anti-ATG5. Secondary antibodies matching primary antibodies were then added. The analysis of nuclei stained with DAPI was conducted using confocal microscopy (Olympus, Japan).

Histological Analysis

Lungs were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 5 μm. Sections were stained with H&E, PAS, Masson’s trichrome, or MUC5AC antibody. Slides were examined microscopically; PAS-positive cells were quantified as a percentage of airway epithelial cells, and collagen deposition as stained area using ImageJ.

Cell Culture and Transfection

BEAS-2B cells were cultured in DMEM with 10% FBS and antibiotics at 37 °C in 5% CO₂. Gene knockdown or overexpression was achieved using lentiviral shRNAs/overexpression vectors (shNC, shFTO, shACKR3, FTO-OE, METTL3/14-OE) or siRNAs (IGF2BP1/2/3, ATG5, ATG7, METTL3/14) with siNC as control. Transfections were performed using Entranster reagent. Sequences are listed in Table S1.

Cell Viability

Cell viability under PAH exposure was assessed using the CCK-8 kit (Servicebio, China) according to the manufacturer’s instructions. BEAS-2B cells were seeded in 96-well plates and treated with PAHs (0, 5, 10, 20, 40, 80 mg/L) for 72 h, with six replicates per concentration. Concentrations were selected based on our previous work. Triton X-100 (0.05%) served as a positive control. After treatment, 10 μL of CCK-8 solution was added to each well and incubated for 3 h at 37 °C. Absorbance was measured at 450 nm using a microplate reader (Molecular Devices).

Western Blot Analysis

WB was adapted from. Membranes were blocked with 5% nonfat milk in TBST and incubated overnight at 4 °C with primary antibodies against LC3B (1:1000, ab192890), Collagen I (1:5000, ab260043), α-SMA (1:1000, ab314895), NLRP3 (1:1000, ab263899), Pro-caspase-1 (1:1000, ab179515), NF-κB (1:2000, ab32536), P62/SQSTM1 (1:1000, ab109012), METTL3 (1:1000, ab195352), GAPDH (1:20000, ab8245), FTO (1:1000, ab92821), ACKR3 (1:2000, ab72100), IL-1β (1:1000, ab283818), METTL14 (1:1000, 48699), ATG5 (1:1000, 12994), ATG7 (1:1000, 8558), IGF2BP1 (1:1000, 8482), IGF2BP2 (1:1000, 14672), and IGF2BP3 (1:1000, 57145). After washing, membranes were incubated for 1 h with HRP-conjugated secondary antibodies (Cell Signaling). Protein bands were visualized using the Chemiluminescence Touch Imaging System (e-BLOT, Shanghai, China).

m⁶A Dot Blot Assay, MeRIP-qPCR

m6A dot blot and MeRIP-qPCR assays were performed as previously described. Total RNA was immunoprecipitated with an anti-m6A antibody and analyzed by MeRIP-qPCR. Primer sequences for full-length mRNAs are listed in Table S2.

qPCR was conducted using a CFX system (Bio-Rad, CA) with ChamQ Universal SYBR Master Mix (Vazyme, Nanjing, China). Each sample was tested in triplicate. Ct values for FTO, ALKBH5, MUC5AC, COL1A1, α-SMA, ACKR3, ATG5, ATG7, p62, LAMP1, YTHDF1–3, YTHDC1–2, METTL3/14, WTAP, NFKB1, TNFAIP3, TNFRSF1A, NFKBA, RELA, RELB, IL1β, IL6, IL4, IL5, IL13, and TGF-α were normalized to ACTIN and GAPDH.

Dual-Luciferase Reporter Assay

To analyze XRE sites in the FTO promoter, a DNA fragment was amplified by PCR (primers in Table S2), cloned into the pGL4.17­[luc2/Neo] vector, and mutagenized by site-directed PCR. For luciferase assays, the constructs were cotransfected with pRL-SV40 as an internal control. In parallel, 293T cells were transfected with pGL3 NF-κB-Luc and pRL-TK using GenJet Plus reagent. After 48 h, firefly and Renilla luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Promega).

RNA Stability and RIP

ACKR3 mRNA stability was assessed using actinomycin D (5 mg/mL), with cells collected at 0, 3, and 6 h after treatment. RNA-binding protein immunoprecipitation (RIP) was performed with the Magna RIP Kit (Millipore) according to the manufacturer’s protocol. RNase T1 was used to lyse and digest PAH-exposed and control cells. Antibodies against IGF2BP1/2/3 were mixed at a 1:1:1 volume ratio, with IgG as control, and applied to the RIP lysate supernatant. Input and coimmunoprecipitated RNAs were extracted with TRIzol (Invitrogen) and analyzed by RT-qPCR.

Chromatin Immunoprecipitation (ChIP) Assays

ChIP was performed following Bell et al. Cells were treated with glycine and micrococcal nuclease, then incubated with FTO antibody, H3 [D2B12] (positive control), or IgG (negative control). Antibody–protein G bead complexes were immunoprecipitated, eluted, and reverse cross-linked. Enrichment was assessed by qPCR.

Statistics and Reproducibility

Statistical analyses were done with R or Prism 8 (GraphPad). The means of three independent experiments were reported. SDs and SEs of means are shown by error bars. To compare the two groups, a t test was used. Tukey’s post hoc test and ANOVA were used for multiple group comparisons. Statistical significance was indicated by a p-value less than 0.05.

Results

PAHs Facilitated Inflammation and PF in Murine Asthma Models

We established a chronic HDM-induced asthma model in C57BL/6 female mice (Figure A) to assess PAH effects. PAHs markedly intensified airway pathology. H&E staining revealed pronounced inflammatory infiltration, with the HDM+PAHs (HP) group showing the highest histopathology scores (Figure B–C). Periodic Acid-Schiff (PAS) and MUC5AC staining confirmed increased goblet cell hyperplasia and mucin secretion, further amplified by PAHs (Figure B,E–F). Fibrotic remodeling was similarly aggravated. Masson’s trichrome staining showed greater collagen deposition in HDM and PAH groups, maximized in HP mice (Figure B,D). Collagen I and α-SMA expression was also strongly upregulated in HP lungs (Figure G–I). BALF analysis revealed elevated total cells, neutrophils, and eosinophils in asthmatic and PAH-exposed mice, with synergistic increases in the HP group (Figure J–L). Collectively, PAHs synergize with HDM to amplify airway inflammation, mucus hypersecretion, and fibrotic remodeling, driving more severe asthma phenotypes.

1.

PAHs exposure aggravated inflammation and fibrosis in lung of mouse chronic asthma model. (A) Schematic diagram of mouse chronic asthma model. Mice were sensitized with intraperitoneal injection of 3 μg HDM and then challenged by repeated intranasal challenges once a day with 10 μg of HDM (with a two-day interval) and PAHS over a 6-week period as described in the Methods. Lungs were harvested at day 58. (B) H&E, Masson’s trichrome, PAS staining and immunohistochemistry (IHC) staining of MUC5AC was performed. Original magnification, × 400. (C) Inflammation score and (D) Collagen deposition area (%) of proximal airways in each group (n = 9). (E) The quantification histograms of PAS staining in proximal airways (n = 9). (F) The quantification histograms of IHC MUC5AC staining in proximal airways (n = 9). (G) Representative immunoblots of collagen-I and α-SMA proteins in lung lysates. (H–I) Changes in protein levels of α-SMA (H) and collagen-I (I). The numbers of total inflammatory cells (J), neutrophil (K) and Eosinophil (L) cells in BALF were counted by Hemocytometer, n = 6. Data is shown as mean ± SEM of 6 individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (versus control), ^# p < 0.05, ^### p < 0.001, ^#### p < 0.0001 (versus asthma). P values were determined by one-way ANOVA followed by the Bonferroni’s test.

FTO Mediates PAHs-Induced Airway Remodeling in Bronchial Epithelial Cell

FTO demethylation has been reported to suppress asthmaprogression. In line with this, we observed markedly reduced FTO expression in HDM-challenged mice compared with controls, and this effect was further exacerbated by PAH exposure (Figure A,B,A1,B1). In BEAS-2B cells, PAHs decreased viability at concentrations above 10 mg/L (81% at 20 mg/L; Figure S1A), leading us to select 10 mg/L for mechanistic studies. At this dose, PAHs significantly reduced both FTO protein and mRNA levels (Figure D,E,D1). Restoration of FTO through lentiviral overexpression effectively reversed this suppression (Figures C; S1B–D). Functionally, PAHs activated the NLRP3 inflammasome and promoted fibrotic remodeling, as evidenced by elevated NLRP3, pro-caspase-1, IL-1β, fibronectin, collagen I, and intracellular MUC5AC (Figures F–K; S1E,F). Overexpression of FTO attenuated these increases. Likewise, PAHs enhanced transcript levels of collagen I, α-SMA, and MUC5AC, whereas FTO overexpression significantly reduced their induction (Figure L). Conversely, FTO knockdown further amplified PAH-induced upregulation of these genes (Figure S1G–I). Collectively, these findings position FTO as a critical negative regulator of PAH-driven airway inflammation and remodeling.

2.

FTO down-regulated by PAHs exposure and participated in PAHs-induced airway remodeling in vitro and in vivo. In the mouse asthma model induced by HDM and PAHs, FTO protein contents were measured by Western blotting (A and A1), and immunofluorescence (B and B1). (C) BEAS-2B cells were treated with PAHs (10 mg/L) for 24 h. Whole-cell protein lysates were prepared for Western blot analysis using anti-FTO (D and D1, n = 3), or qPCR (E, n = 6). (F–K) After transfection with control vector or FTO expression vector for 72 h, BEAS-2B cells were treated with PAHs (10 mg/L) for 24 or 48 h. (F–K) Representative blot images (F) and quantification (G-K) of NLRP3, Pro-caspase 1, IL-1β, fibronectin and collagen-I in BEAS-2B cells. (L) mRNA changes of collagen-I, α-SMA and MUC5AC in BEAS-2B cells. Data are mean ± SEM of 3 individual experiments, *p < 0.05, **p < 0.01, ***p < 0.001. P values were determined by Student’s t test or one-way ANOVA followed by the Dunnett’s test.

FTO Deficiency Exacerbates PAHs-Induced Lung Injury

To examine FTO’s role in airway inflammation and fibrosis, female FTO knockout (KO, FTO^–/–) and wild-type (WT, FTO^+/+) mice were exposed to PAHs for 6 weeks (Figure A). Dynamic spirometry revealed restrictive ventilatory impairments in both genotypes post-PAHs, with KO mice showing greater declines. After 6 weeks, KO-PAHs mice exhibited significant reductions in all measured lung function markers compared to KO controls (p < 0.05), while WT-PAHs mice showed decreases only in inspiratory capacity (IC), chord compliance, and total lung capacity (TLC) (p < 0.05, Figure S2A–F). Notably, KO-PAHs mice had 19.9, 29.3, and 15.5% lower FVC, FRC, and VC than WT-PAHs mice (Figure S2B,C,F).

Histopathology confirmed that PAHs aggravated lung injury in both WT and KO mice, but the effect was more pronounced in FTO^–/^– animals. H&E and Masson’s trichrome staining showed enhanced inflammation and fibrosis in KO-PAHs compared with WT-PAHs (Figure A,B,E,F), with a 2.1-fold increase in Ashcroft scores (Figure C,G). PAS and MUC5AC immunostaining further demonstrated intensified goblet cell hyperplasia and mucin secretion in KO-PAHs lungs (Figure C,D,G,H).

3.

FTO deficiency aggravated inflammation and fibrosis in lung of mice induced by PAHs. FTO^–/– mice and FTO^wt/wt 34 mice were sensitized and challenged with HDM as described in the “Methods”. Lungs of mice were harvested at day 58. Representative pictures of lung tissue sections stained with H&E (A), Masson staining (B), PAS staining (C) and immunohistochemistry staining of MUC5AC (D). Scale bars = 20 μm. Inflammation score (E) and collagen deposition area (%) of proximal airways (F) in each group (n = 5). (G) The quantification histograms of PAS staining in proximal airways (n = 5). The quantification histograms of IHC MUC5AC staining in proximal airways (H, n = 5). Data mean ± SEM of n individual experiments. Representative protein bands of collagen-I, fibronectin and α-SMA in lungs of WT and FTO^–/– mice, GAPDH was used for normalization (I). The expressions of collagen-I, fibronectin and α-SMA in lungs of WT and FTO ^–/– mice after PAHS exposure (J–L), respectively. Data are expressed as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 between indicated conditions (One-way ANOVA followed by the Bonferroni’s test).

At the molecular level, fibronectin, collagen I, and α-SMA were elevated in both WT- and KO-PAHs groups, but KO mice exhibited the strongest induction (1.4-, 1.3-, and 1.6-fold higher than WT-PAHs, respectively; Figure I–L). Consistent with these findings, PAH exposure downregulated FTO expression in WT mice, with further loss observed in KO animals (Figure S3A–C). In asthmatic models, PAHs also synergistically increased IL-4, IL-5, and IL-13 in BALF compared with either stimulus alone (Figure S3D–F). Together, these in vivo and in vitro data demonstrate that FTO protects against PAH-induced airway inflammation and fibrotic remodeling, and its deficiency sensitizes the lung to more severe injury.

PAHs Inhibits Global m⁶A RNA Methylation via AHR-Mediated Downregulation of FTO

We next examined whether PAHs modulate m6A machinery in asthma models. After 6 weeks of exposure, FTO and ALKBH5 were markedly reduced in murine lungs, while METTL3 and METTL14 were upregulated compared with controls (Figure A,B). Consistently, PAHs decreased FTO mRNA but increased METTL3 transcripts, whereas METTL14 and ALKBH5 were only modestly affected (Figure C).

4.

FTO m⁶A modification is downregulated by PAHs in vivo and in vitro. (A, B) Representative protein bands and quantitation of demethylase FTO and ALKBH5, methylase METTL3 and METTL14 were detected in the lungs of mice following an 8-week period of PAHs exposure. (C) The mRNA changes of m6A-associated proteins. (D) Dot blot shows the m⁶A level in FTO-OE, ALKBH5-OE and METTL3/14-OE or shFTO and shMETTL3 cells. (E, F) Representative images (E) and quantitative results of m⁶A immunofluorescence staining (F); (G) ChIP-qPCR assay showed that AhR was significantly bound to FTO, and the binding decreased after PAHs exposure. (H) Dot blot shows m⁶A enrichment (IP/Input) in BEAS-2B cells with knockdown FTO and METTL3. **p < 0.01, ***p < 0.001, **** or ^#### p < 0.0001.

In BEAS-2B cells, PAHs reduced m6A enrichment as shown by dot blot and MeRIP-qPCR. Forced expression of FTO or ALKBH5 attenuated this effect, whereas METTL3/14 overexpression further increased global m6A (Figures D, S4A–D). Thus, FTO downregulation emerges as a central driver of PAH-induced m6A hypomethylation.

Mechanistically, AHR signaling mediated this regulation. Pharmacological (CH) or genetic inhibition of AHR rescued PAH-induced hypomethylation (Figure S4E,F). Knockdown of AHR also restored FTO mRNA and protein expression, whereas CH treatment reversed PAH-induced FTO suppression (Figure S4G,H). ChIP-qPCR demonstrated AHR binding to the FTO promoter, which decreased after PAH treatment (Figure G). Eight xenobiotic response elements (XREs) were identified within the FTO promoter (Figure S4L); luciferase assays confirmed that PAHs specifically reduced reporter activity at the wild-type XRE4 site but not its mutant (Figure S4M). These findings establish AHR as a direct transcriptional regulator of FTO.

Functionally, FTO knockdown increased global m6A levels in total and purified RNA, whereas METTL3 silencing mitigated this effect (Figures H, S4I–K), indicating that PAH-induced m6A dysregulation requires coordinated FTO–METTL3 activity. Collectively, these results suggest that PAHs impair airway homeostasis by activating AHR, which represses FTO transcription, disrupts m6A balance, and promotes epithelial injury.

MeRIP-Seq and Transcriptome Analyses Identified ACKR3 as a Key Target for PAHs-Induced FTO Downregulation

To uncover m6A-modified transcripts affected by PAHs, BEAS-2B cells were exposed to PAHs (10 μg/mL, 24 h) and subjected to MeRIP-seq and RNA-seq A total of 1868 m6A peaks corresponding to 1125 genes were detected, with 1003 peaks enriched and 865 reduced relative to controls (Figure A). Peaks were predominantly located near stop codons within 3′UTRs, where PAH exposure enhanced m6A deposition and altered distribution patterns (Figure B). Motif analysis confirmed conserved RRACH sites (Figure C). Notably, ACKR3 exhibited a downregulated m6A peak (Figure A,D).

5.

Identification of the potential target of PAHs-induced FTO downregulation via transcriptome-wide m6A-seq and RNA-seq analyses. (A) The differential m⁶A peaks (p < 0.05). (B) The proportion of m⁶A peak distribution in the indicated regions across the entire set of mRNA transcripts in control and PAHs treated cells (C) Top consensus m⁶A motif identified by HOMER with m⁶A peaks in BEAS-2B cells with or without PAHs treatment. (D) Distribution of m6A peaks in “Exon” 2 of the ACKR3 transcript in control and PAHS treated cells. (E) Venn diagram showing the overlap between genes with altered expression or m⁶A enrichment in four data sets: m⁶A seq (control/PAHs), RNA seq (PAHs/control), RNA seq [human asthma (GSE144770)], and RNA seq [Mouse asthma (GSE195996)]. ACKR3 is found in all groups. (F) Immunoblot and qPCR analysis of ACKR3 in control and PAHs treated cells. (G) m⁶A IP qPCR analysis of m6A enrichment across the ACKR3 transcript in control and PAHs treated cells (n = 3). (H) qPCR analysis of ACKR3 mRNA stability following treatment with actinomycin (ActD, 2 μmol/L) in control and PAHs treated cells (n = 3). (I) m⁶A IP qPCR analysis of m⁶A enrichment across the ACKR3 transcript in PAHs treated cells with or without FTO deletion (n = 3). (J) qPCR analysis of ACKR3 mRNA level in cells with or without FTO knockdown (n = 3). (K, L) qPCR analysis of ACKR3 mRNA stability following treatment with ActD in PAHS treated cells with or without FTO knockdown (n = 3). (M-N) Immunoblot analysis of ACKR3 and FTO (M), and mRNA level of ACKR3 (N) in FTO-KO cells transfected with wild-type (WT) or demethylase-inactive mutant FTO (Mutant 1, H231A/D233A; Mutant 2, R316Q/R322Q). (O, P) Immunoblot analysis of ACKR3 and FTO (O), and qPCR analysis (P) of ACKR3 mRNA in PAHs treated BEAS-2B cells plus treated with vehicle, CS1 (200 nmol/L), and CS2 (200 nmol/L). All data were performed on n ≥ 3 biologically independent samples. Error bars are shown as mean ± SD. p-value by two-tailed unpaired t test is indicated. *p < 0.05, **p < 0.01, ****p < 0.0001, ns, no significance.

To prioritize functional targets, we integrated m6A- and RNA-seq data sets with human and murine asthma transcriptomes (GSE144770, GSE195996). Five genes showed consistent alterations across data sets: ACKR3, METTL3, and IL24 (upregulated) and RAET1G and FTO (downregulated) (Figure E). ACKR3 was selected for further study given its known upregulation in allergic airway inflammation. ,

PAH exposure reduced global m6A levels and FTO expression, implicating FTO in ACKR3 regulation. Indeed, PAHs increased ACKR3 mRNA and protein expression (Figure F) while decreasing m6A enrichment across its transcript, particularly in exon 2 (Figures G, S5A). This was accompanied by reduced mRNA stability (Figure H). FTO deletion elevated ACKR3 transcript abundance, m6A enrichment, and stability (Figure I–L), suggesting that increased m6A deposition stabilizes ACKR3 mRNA. Functional assays confirmed this dependency on FTO activity. Wild-type FTO, but not catalytically inactive mutants, suppressed ACKR3 mRNA and protein in FTO-deficient cells (Figure M, N). Conversely, pharmacological inhibition of FTO with CS1 or CS2 elevated ACKR3 expression without altering FTO protein levels (Figure O,P).

Together, these data demonstrate that PAHs downregulate FTO, leading to reduced demethylation and increased stabilization of ACKR3 mRNA, thereby identifying ACKR3 as a key effector of PAH-induced airway remodeling.

Identification of ACKR3 as the Functional Target of FTO

Given ACKR3′s role in airway inflammation, we examined whether it is directly regulated by FTO. shRNA-mediated knockdown of FTO significantly increased ACKR3 mRNA, whereas FTO overexpression suppressed its transcript and protein levels (Figure A–C), indicating that FTO negatively regulates ACKR3 expression.

6.

ACKR3 is critical downstream target gene of FTO in PAHs induced asthma. The transcripts changes of FTO, and ACKR3 in BEAS-2B stable cells with or without knockdown FTO (A) or ACKR3 (B), and forced overexpression of FTO (C), respectively. The alteration of ACKR3 (D) and FTO (E) in PAHs-HDM-treated lungs in mice asthma model. (F–H) Immunoblot bands (F) and quantification (G) and mRNA levels (H) of ACKR3 in WT and FTO KO mice treated with PAHs, HDM or both. (I–L) Immunoblot (I, J), qPCR (K) and histology (L) analysis to confirm ACKR3 knockdown on the inflammatory response in the lungs with or without FTO deletion. All data were performed on n ≥ 3 biologically independent samples. Error bars are shown as mean ± SD or mean ± SE. p-values by two tailed unpaired t tests. ^###, ***p < 0.0001 and ^####, ****p < 0.00001. *: Control comparisons; #: FTO KO comparisons.

To assess this interaction in vivo, we analyzed lungs from mice exposed to HDM, PAHs, or both. Across all treatments, ACKR3 was upregulated while FTO was downregulated (Figure D–E). Notably, FTO deletion further enhanced ACKR3 expression in HDM/PAH-treated mice (Figure F–H), suggesting an inverse regulatory relationship during asthma pathogenesis.

Functional relevance was confirmed by silencing ACKR3 in FTO-deficient mice. ACKR3 knockdown partially rescued the heightened lung inflammation caused by FTO loss (Figures I–L, S5B) and reduced expression of inflammatory cytokines, including IL1β, IL4, IL5, IL6, IL13, and TGFα (Figure S5C).

Collectively, these findings identify ACKR3 as a pivotal downstream effector of FTO, linking PAH-induced FTO downregulation to airway inflammation and asthma progression.

FTO Regulates ACKR3 mRNA Stability Through m⁶A and IGF2BPs

To dissect the mechanism underlying FTO-mediated regulation of ACKR3, we first manipulated the m6A methyltransferases METTL3 and METTL14. In FTO-deficient cells, additional knockdown of METTL3/14 further enhanced ACKR3 transcript abundance, protein expression, and mRNA stability (Figures A–B, S5D–G). Conversely, METTL3 overexpression suppressed ACKR3 expression even in PAH-treated cells lacking both FTO and METTL3 (Figure C–D), indicating that FTO acts in opposition to METTL3/14 in regulating ACKR3.

7.

FTO regulates ACKR3 through m⁶A. (A, B) schematic diagram and qPCR analysis of ACKR3 mRNA stability in BEAS-2B cells with or without FTO knockdown and/or METTL3/14 knockdown (n = 3). (C, D) Immunoblot (C) and qPCR (D) analysis of ACKR3 and METTL3 in cells with FTO and METTL3 knockdown transfected with METTL3. (E) qPCR analysis of mRNA levels of critical m⁶A regulators in BEAS-2B stable cells of shNC or shFTO. (F) Immunoblot analysis of ACKR3, IGF2BP1, IGF2BP2, IGF2BP3, and FTO in cells with or without FTO knockdown in combination with transfection with siRNA targeting negative control (siNC) or all three IGF2BP1–3 (siALL). (G) qPCR analysis of ACKR3 mRNA level in cells as in E (n = 6). (H) qPCR analysis of ACKR3 mRNA stability in cells as in E (n = 3). (I) RIP-qPCR showing the binding of IGF2BPs to the ACKR3 transcript in control and PAHS treated cells (n = 3). All data were performed on n ≥ 3 biologically independent samples. Error bars are shown as mean ± SD or mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance. A Two-way ANOVA with Tukey’s or Dunn’s post-tests were used to verify statistical differences.

We next examined whether m6A “reader” proteins contribute to this process. While FTO depletion did not broadly affect other m6A regulators (Figure E), it significantly stabilized ACKR3 mRNA. Given that IGF2BP1–3 are known to protect m6A-modified transcripts, we tested their involvement. Silencing IGF2BP1, 2, or 3 reduced ACKR3 protein levels (Figure F), and combined knockdown of IGF2BPs abolished the stabilizing effect of FTO loss on ACKR3 mRNA (Figure G–H). RIP-qPCR further confirmed direct binding of IGF2BPs to ACKR3 mRNA, which was attenuated following PAH exposure (Figure I).

Together, these results demonstrate that FTO regulates ACKR3 mRNA stability through an m6A-dependent pathway involving IGF2BP proteins.

FTO Regulates Autophagy in BEAS-2B Cells via ACKR3

To explore how FTO modulates PAH-induced airway epithelial inflammation, we performed RNA sequencing in BEAS-2B cells. Pathway analysis revealed significant enrichment of ACKR3-associated signaling networks, including autophagy and cytokine–cytokine receptor interactions, consistent with previous reports. , Although FTO is recognized as a key regulator of autophagy, , its precise role in PAH-driven bronchial epithelial cells (BECs) remained unclear.

PAH exposure increased both protein and mRNA levels of autophagy-related markers, including LC3B, collagen I, fibronectin, α-SMA, p62/SQSTM1, and ATG5 in BEAS-2B cells (Figure S6A–D), and elevated ATG5 and LC3B in asthmatic mice (Figure S6F). FTO overexpression attenuated these effects, reducing LC3B, ATG5, ATG7, and p62 expression (Figures A,B, S6E). Conversely, asthma-challenged FTO^–/– mice displayed elevated autophagy marker levels relative to WT animals (Figure C).

8.

FTO regulated autophagy in bronchial epithelial cells via ACKR3. (A, B) After being treated with PAHs for 24h, representative Western blots (A) and protein changes (B) of autophagy-related markers, including collagen-I, fibronectin, α-SMA, LC3B, p62 and ATG5 in control and PAHS treated BEAS-2B cells (n = 3). (C) FTO^–/– and FTO^wt/wt mice were sensitized and challenged with HDM and PAHS as described in the Methods. Lungs of mice were harvested at day 58. Representative immunofluorescence pictures of lung tissue sections stained with ATG5 and LC3B. Scale bars = 50 μm. (D) After transfection with control vector or overexpression vector of FTO (FTO-OE) and ACKR3 (ACKR3-OE) for 72h, BEAS-2B cells were treated with PAHs (10 mg/L) for 48h. Representative blot images of ACKR3, ATG5, Collagen-1, LC3B and p62 in BEAS-2B cells. Data means SEM of n individual experiments (Student’s t test), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To confirm the functional role of autophagy, BEAS-2B cells were treated with hydroxychloroquine (HCQ) or ATG5 siRNA. Both interventions suppressed PAH-induced autophagy, with HCQ blocking LC3B–II accumulation and attenuating ATG5, p62, and MUC5AC expression, while ATG5 knockdown reduced LC3B–II levels (Figure S7A–E). These findings implicate autophagy as a mediator of PAH-induced airway inflammation.

Finally, we assessed whether ACKR3 mediates FTO-dependent autophagy regulation. FTO overexpression suppressed both ACKR3 expression and autophagy, whereas enforced ACKR3 expression restored autophagy despite high FTO levels (Figure D). Together, these results demonstrate that PAH-induced autophagy in BECs is regulated through the FTO–ACKR3 axis.

PAHs Upregulates ACKR3 via Activation of the TNF/NF-κB Pathway

KEGG pathway analysis revealed that PAH exposure significantly enriched cytokine–cytokine receptor signaling, potentially explaining ACKR3 overexpression in asthma (Figure A). PAHs also enhanced NF-κB and TNF pathway activity, along with lysosomal function. qPCR confirmed that PAH-treated cells exhibited elevated expression of TNF-related genes (TNFRSF1A, TNFAIP3, NFKB2, RELA, RELB, NFKBIA) and autophagy markers (ATG5, p62) relative to controls (Figure B). Given that NF-κB activation drives p62 expression, we hypothesized that PAHs promote ACKR3 expression through TNFα–NF-κB signaling.

9.

PAHs upregulates ACKR3 expression through TNFα/NF-κB signaling. (A) KEGG pathway analysis of upregulated genes shared in PAHS treated cells as compared with control cells. (B) qPCR analysis of TNFRSF1A, TNFAIP3, NFKB2, RELA, RELB in control and four PAHS treated cells (n = 6). (C) Luciferase reporter analysis of NF-κB activity in control and PAHs treated cells (n = 3). (D) Luciferase reporter analysis of NF-κB activity in cells treated with or without TNFα (10 and 50 ng/mL) for 20 h (n = 3). (E-F) Immunoblot analysis and quantification of ACKR3, ATG5 and FTO in PAHs treated cells plus with or without TNFα for 20 h. (G) Immunoblot analysis of FTO, RELA and ACKR3 in PAHS treated cells plus with or without TNF (50 ng/mL) following transfection with or without siRNA targeting RELA. All data were performed on n ≥ 3 biologically independent samples. Error bars are shown as mean ± SD. p-values of all data by two-tailed unpaired t test are indicated.

Consistent with this, PAH exposure increased NF-κB activity compared to controls (Figure C,D). TNFα treatment suppressed NF-κB activation and correspondingly reduced ACKR3 and ATG5 protein levels, while restoring FTO expression (Figure E,F). Moreover, inhibition of RELA (p65), a core NF-κB subunit, abrogated TNFα-mediated regulation of ACKR3 via FTO (Figure G). In conclusion, these findings establish that PAHs induce ACKR3 upregulation through activation of the TNF/NF-κB pathway, which concurrently represses FTO.

Discussion

A growing body of evidence indicates that even low concentrations of airborne pollutants such as PM2.5 can exert deleterious effects on human health, contributing significantly to respiratory diseases including asthma. , PAHs, key toxic constituents of PM2.5, are known agonists of the AhR and have been implicated in the initiation and exacerbation of asthma. However, the molecular mechanisms through which PAHs promote asthma pathogenesis remain incompletely elucidated. Our study provides novel evidence that PAHs downregulate the RNA demethylase FTO via AhR activation, leading to increased m⁶A methylation on ACKR3 mRNA, enhanced ACKR3 expression, and subsequent activation of autophagy-inflammation pathways culminating in asthma exacerbation and pulmonary fibrosis.

An important finding of our work is the demonstration that PAH-induced suppression of FTO is AhR-dependent. Both pharmacological inhibition and genetic knockdown of AhR restored FTO expression and global m⁶A levels, underscoring a direct transcriptional regulatory link. This is consistent with prior studies indicating tissue-specific reduction of FTO in asthmatic patients and heightened inflammatory responses in Fto-KO mice. Interestingly, contrary to reports that low-level PM2.5 can elevate FTO expression, our data indicate that PAHsas distinct componentssuppress it, highlighting the complex and composition-dependent effects of air pollution mixtures. A central contribution of this study is the identification of ACKR3 as a key downstream target of FTO-mediated m⁶A erasure. We found that PAH exposure stabilizes ACKR3 mRNA in an m⁶A-dependent manner, and this effect is reversed upon FTO overexpression. Although our m⁶A-IP and qPCR data support the role of FTO in regulating ACKR3 methylation, direct evidencesuch as RNA immunoprecipitation demonstrating FTO binding to ACKR3 mRNAis currently lacking. Future studies using CLIP-seq or similar approaches are needed to confirm direct binding and elucidate the precise epitranscriptomic mechanism.

ACKR3 has previously been implicated in modulating allergic airway inflammation. Our results align with and extend these findings by positioning ACKR3 within the PAH–FTO–autophagy axis. Knockdown of ACKR3 attenuated pulmonary inflammation and fibrosis even in the context of FTO deletion, supporting its functional importance in this pathway. Furthermore, we provide evidence that m⁶A-mediated stabilization of ACKR3 is facilitated by IGF2BP family readers, suggesting a complex regulatory circuit involving writers, erasers, and readers. Another layer of regulation involves autophagy, which we found to be upregulated following PAH exposureconsistent with prior studies involving PM2.5. Importantly, we demonstrate that FTO deficiency exacerbates autophagic activity and that ACKR3 overexpression drives autophagy-related gene expression (Atg5, Atg7, p62), supporting the proposed FTO→ACKR3→autophagy cascade. Nevertheless, the exact mechanism by which ACKR3 activation stimulates autophagy remains unclear and warrants further investigation.

The clinical and translational implications of our findings are notable. FTO and ACKR3 represent promising candidate biomarkers for assessing environmental asthma risk or progression. Therapeutically, targeting the AhR–FTO–ACKR3 axis may offer novel avenues for intervention. Potential strategies could include: (i) Development of FTO activators or mimetics to restore m⁶A homeostasis; (ii) Use of ACKR3 inhibitors currently under exploration in cancer contexts; (iii) Repurposing of NF-κB or NLRP3 inflammasome inhibitors, given their downstream roles in airway inflammation and remodeling.

In a summer, our study delineates an epitranscriptomic pathway through which PAHs promote asthma pathogenesis via AhR-mediated suppression of FTO, culminating in m⁶A hypermethylation and stabilization of ACKR3 mRNA, activation of autophagy, and enhanced NF-κB–NLRP3 signaling. While these findings provide mechanistic insight, several limitations remain, including the need for direct binding assays between FTO and ACKR3 mRNA, in vivo validation of IGF2BP involvement, and clarification of ACKR3′s role in autophagic induction. Future studies should also explore the therapeutic efficacy of FTO enhancers or ACKR3 antagonists in preclinical models of PAH-induced asthma.

Environmental and Health Implications

PM2.5 is a major global health threat due to its complex composition and ultrafine size, enabling deep lung penetration and contributing to impaired respiratory function, increased infections, chronic inflammation, fibrosis, and even lung cancer. Among its constituents, toxic PAHs are especially concerning because of their mutagenic and carcinogenic properties and their strong links to asthma, pulmonary fibrosis, and COPD.

Here, we uncover a previously unrecognized epigenetic mechanism by which PAHs inactivate the RNA demethylase FTO, leading to ACKR3 upregulation via an m6A–IGF2BP–dependent pathway. This axis drives autophagy-mediated airway remodeling and exacerbates asthma, as confirmed in both cellular and animal models.

These findings position PAHs as potent epigenetic hazards and identify the FTO–ACKR3 signaling cascade as a druggable pathway. Pharmacological modulation of FTO activity, blockade of ACKR3, or interference with downstream autophagy may provide novel therapeutic strategies to mitigate pollution-induced asthma. Beyond mechanistic insight, this work highlights the urgent need to integrate epigenetic targets into the development of precision interventions for populations chronically exposed to air pollution.

Conclusion

Our findings demonstrate that chronic PAH exposure suppresses FTO expression and reduces global m6A enrichment in airway epithelial cells, thereby aggravating inflammation and pulmonary fibrosis. Mechanistically, PAHs promote FTO degradation through p62-dependent selective autophagy, while simultaneously enhancing p62 expression via activation of the TNFα/NF-κB pathway. We further identify ACKR3 as a key functional target of FTO, with its mRNA stability governed by m6A modification through FTO–IGF2BP interactions. Collectively, these results define an FTO–m6A–ACKR3 epitranscriptomic axis as a central driver of PAH-induced asthma. Targeting this pathway may provide innovative strategies to prevent or treat pollution-related asthma and ultimately reduce disease burden in exposed populations.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/envhealth.5c00261.

• siRNA, shRNA and gRNA used in the study (Table S1); primers used in the study (Table S2); FTO participated in PAHs‑induced airway remodeling in vitro (Figure S1); FTO deficiency aggravated the pulmonary function decreases in mice after PAHs exposure for 6 weeks (Figure S2); FTO deficiency aggravated inflammation and fibrosis in lung of mice induced by PAHs (Figure S3); PAHs inhibits global m6A RNA methylation through AhR-mediated downregulation of FTO (Figure S4); ACKR3 is the functional target of FTO (Figure S5); FTO regulated autophagy in bronchial epithelial cells via ACKR3 (Figure S6); inhibition of autophagy by HCQ and ATG5 siRNA reversed PAHs induced LC3B, ATG5, MUC5AC and p62 expression in bronchial epithelial cells in vitro (Figure S7) (PDF)

⊥.

S.L., J.C., and J.X. contributed equally to this work. S.L., S.Q. and X.S. conceived and supervised the study. Z.H., X.S. and S.Q. contributed to project initiation and cosupervision. S.L. played a central role in study design, experimental planning, and manuscript preparation. J.C., J.X. and X.Y. conducted animal experiments, laboratory assays, and data analysis, and contributed to manuscript drafting and revision. All authors reviewed and approved the final manuscript.

The research received financial support by a key research project grant from the Chengdu Technology Bureau (2024-YF05–00875-SN), and a grant from the Health Commission of Chengdu (2021021), the National Key Research and Development Program of China (2023YFC3403200), the National Natural Science Foundation of China (No. 82300113) and the Sichuan Province Science and Technology Support Program (25GJHZ0376).

Animal experiments were performed by the principles of laboratory animal care (NIH publication no. 85–23, revised 1985) and approved by the Sciences Animal Care and Use Committee of West China Hospital, Sichuan University (Approval No. 20240528109).

The authors declare no competing financial interest.