Air pollution exacerbates nociceptor neurons’ activity.

(A-C) Male and female C57BL6 mice aged 6-10 weeks were sensitized via intraperitoneal injection with an emulsion containing ovalbumin (OVA, 200 µg/dose) and aluminum hydroxide (1 mg/dose) on days 0 and 7. They were then challenged intranasally with OVA (50 µg/dose) with or without fine particulate matter (FPM, 20 µg/dose) on days 14-16. Bronchoalveolar lavage fluid (BALF) was collected, and JNC neurons were cultured on day 17. (B-C) JNC neurons were harvested and cultured for 24 hours. Following culturing, the cells were loaded with the calcium indicator Fura-2AM and sequentially stimulated with AITC (10 µM at 60-90 seconds, 30 µM at 90-120 seconds, 100 µM at 120-150 seconds) and KCl (40 mM at 420-435 seconds). Calcium flux was recorded throughout these stimulations (B), and the amplitude of AITC responses was calculated as the ratio of peak F340/F380 after stimulation to the baseline F340/F380 30 seconds before stimulation (C). Data are presented as means ± 95% CI (B), means ± SEM (C)

(B-C) Representative data from 2 independent experiments. AITC-responsive neurons were included in amplitude analysis. n=35 neurons from control mice, n=42 neurons from OVA-treated mice, and n=76 neurons from OVA-FPM-treated mice were analyzed.

Statistical significance is indicated by *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Air pollution reprogram nociceptor neurons’ transcriptome.

(A-F) Naïve male and female TRPV1cre::td-Tomatocre/wt mice, aged 6-10 weeks, were subjected to either a pollution-exacerbated asthma protocol, the classic ovalbumin (OVA) protocol, or were kept naïve. On day 17, at the peak of inflammation, JNC neurons were harvested, dissociated, TRPV1+ neurons FACS-purified (td-tomato+) from stromal cells and non-peptidergic neurons, and isolated for RNA sequencing.

Differentially expressed genes were calculated and displayed as volcano plots and heatmaps for the comparisons: OVA-FPM vs. naïve (A-B), OVA-FPM vs. OVA (C-D), OVA alone vs. naïve (E-F). Significant neuronal reprogramming was observed and characterized by the overexpression in the OVA-FPM vs. naïve groups (Lifr, Oprm3), OVA-FPM vs. OVA alone groups (Oprm1, Nefh, P2ry1, Prkcb, Gabra1, Kcnv1), and OVA alone vs. naïve groups (Npy1r, Kcna1).

Data are presented as volcano plot (p < 0.05; A, C, E) or heatmap (B, D, F) showing normalized gene expression (log2 (0.01 + transcripts per million reads (TPM)) − mean).

The sequencing experiment was not repeated. n consisted of 2-3 mice.

Nociceptor neurons control pollution-exacerbated asthma.

(A-B) ale and female C57BL mice aged -10 weeks were sensitized via intraperitoneal injection with an emulsion containing ovalbumin (200 µg/dose in 100 μl) and aluminum hydroxide (1 mg/dose in 100 μl) on days 0 and 7. They were then challenged intranasally with OVA (50 µg/dose in 50 μl) with or without fine particulate matter (FP, 20 µg/dose in 50 μl) on days 14-1 and received intranasal injections of QX-314 (5 nmol/dose in 50 μl) on day 1, 30 minutes after the last challenge. Bronchoalveolar lavage fluid (BALF) was collected on day 17 and analyzed immune influx immunophenotyped by flow cytometry. Compared to naïve mice or OVA-exposed mice, the one co-challenged with OVA-FP showed increased BALF infiltration of neutrophils (A) and eosinophils (B). The mice treated with QX-314 were protected from these increases (A-B).

(C-G) ale and female littermate control (TRPV1wt::DTAfl/wt) and nociceptor ablated (TRPV1cre::DTAfl/wt) mice aged -10 weeks were sensitized via intraperitoneal injection with an emulsion containing ovalbumin (OVA, 200 µg/dose) and aluminum hydroxide (1 mg/dose) on days 0 and 7. They were then challenged intranasally with OVA (50 µg/dose) with or without fine particulate matter (FP, 20 µg/dose) on days 14-1. Bronchoalveolar lavage fluid (BALF) was collected on day 17, and immune influx was immunophenotyped by flow cytometry.

Compared to naïve mice or OVA-exposed mice, the one co-challenged with OVA-FP showed increased BALF infiltration of neutrophils (C) and γδ T-cells (E). The nociceptor ablated (TRPV1cre::DTAfl/wt) mice were protected from these increases (C, E) but showed similar levels of BALF eosinophils (D).

(F-G) BALF cytokines were measured using a cytokine multiplex array and ELISA, and increased levels of IL-5, CP1 (CCL2), TNFα, and artemin were found. TNFα (F) and artemin (G) rise were absent in nociceptor-ablated mice.

(A-G) Data are presented as means ± SEM.

(A-B) Pooled data from 2 independent experiments. n=6-12 mice per group. (C-E) Pooled data from 2 independent experiments. n=9-18 mice per group. (F) Representative data from 2 independent experiments. n=3-6 mice per group.

(G) Representative data from 2 independent experiments. n=2-8 mice per group. (J) Pooled data from 2 independent experiments. n=5-12 mice per group.

Statistical significance is indicated by *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Artemin sensitizes TRPA1 activity in vagal sensory neurons.

(A) In-silico analysis of the dataset GSE124312 generated a heatmap showing the transcript expression levels of the pan neural-crest lineage transcription factor (Prdm12), voltage-gated sodium channels (Scn9a, Scn10a), jugular subset markers (Wfdc2, Mrgprd, Osmr, Sstr2, Nefh, Trpm8), peptidergic neuron markers (Trpa1, Trpv1, Calca, Tac1, Gfra3), and the pan placodal lineage marker (Phox2b). Experimental details and cell clustering are defined in Kupari et al.41.

(B) In-silico analysis of GSE192987 presenting the co-expression of Gfra3 with TRPA1 or other inflammatory markers. The data are presented as a heatmap displaying the row z-scores (A) or cells that has TPTT>1 in UMAPs.

(C-E) Alveolar macrophages from naïve male and female C57BL6 mice (3 × 10^5 cells/well) were cultured overnight and stimulated with DMSO or FPM (100 µg/ml). RNA was extracted at 1 and 4h post-stimulation, and Artn expression was analyzed using qPCR (C). Data showed that FPM increased artemin transcript expression 1 and 4h post-stimulation (D-E).

(F-H) JNC neurons were harvested, pooled, and cultured overnight with either vehicle or artemin (100 ng/mL). The cells were then sequentially stimulated with AITC (TRPA1 agonist; 300 µM at 240-270 seconds), capsaicin (TRPV1 agonist; 300 nM at 320-335 seconds), and KCl (40 mM at 720-735 seconds). The percentage of AITC-responsive neurons among KCl-responsive cells was normalized to the vehicle-treated dish from each batch of experiments (F). Artemin-exposed neurons showed increased responsiveness to AITC, while the responses to capsaicin and KCl were not impacted (G-H).

Data are presented as heatmap (A), TSNe plot (B), means ± SEM (D, E, H) or means ± 95% CI of the maximum Fura-2AM (F/F0) fluorescence recorded every 15 seconds (G).

(D) Data from 1 experiment with n=2 technical repeats. (E) Pooled data from 2 independent experiments with n=8 technical repeats. (G) Representative data from 2 independent experiments with n=107 vehicle-treated and n=122 artemin-treated neurons were analyzed. (H) Pooled data from 2 independent experiments with n=4 fields of view per group.

Statistical significance is indicated by *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

In silico analysis of Artn expression in mouse immune cells

In silico analysis of Artn expression in mouse using the Immgen database70.

Data are presented as per-gene z-scores of normalized gene expression using the median of ratios method.

Schematic of nociceptor involvement in air pollution-exacerbated allergic asthma.

In our study, we modeled pollution-exacerbated asthma in mice by exposing them to PM2.5 particles and ovalbumin. This exposure significantly increased bronchoalveolar lavage fluid (BALF) neutrophils and γδ T-cells compared to exposure to ovalbumin alone. To address the heightened airway inflammation, we applied intranasal QX-314, a charged derivative of lidocaine, during peak inflammation, which effectively normalized the inflammation. Similarly, ablating TRPV1-expressing nociceptor neurons also normalized lung neutrophil levels.

Further analysis using calcium microscopy on neurons from the jugular-nodose complex showed increased sensitivity of TRPA1 channels in neurons from pollution-exacerbated asthmatic mice. Elevated TNFα and the growth factor artemin levels were found in the BALF of pollution-exposed mice, which normalized following nociceptor neuron ablation.

We pinpointed alveolar macrophages as the source of artemin, which, expressing aryl hydrocarbon receptors, responded to fine particulate matter (FPM) by releasing artemin. This release increased TRPA1 responsiveness to its agonist, mustard oil, exacerbating airway inflammation. Our findings indicate that silencing nociceptor neurons can interrupt this mechanistic pathway and offer a novel approach to mitigating neutrophilic airway inflammation in pollution-driven asthma.