NOCICEPTOR NEURONS CONTROL POLLUTION-MEDIATED NEUTROPHILIC ASTHMA

Abstract

The immune and sensory nervous systems, having evolved in parallel, communicate through shared receptors and transmitters to maintain homeostasis and respond to both external and internal disruptions. Although neural responses often confer protective benefits, they can also exacerbate inflammation during allergic reactions such as asthma. In our study, we modeled pollution-exacerbated asthma by exposing mice to ambient PM_2.5 particles alongside ovalbumin. Compared to exposure to ovalbumin alone, this co-exposure significantly increased the numbers of neutrophils and γδ T cells in bronchoalveolar lavage fluid. We found that silencing nociceptor neurons at the peak of inflammation using intranasal QX-314 or ablating TRPV1-expressing neurons reduced lung neutrophil accumulation. Live in vivo intravital imaging confirmed that neuronal ablation reduced neutrophil numbers and increased their net displacement capacity. In neurons isolated from mice with pollution-exacerbated asthma, the chemical-sensing TRPA1 channel exhibited heightened sensitivity to its cognate ligand. Elevated levels of artemin were detected in the bronchoalveolar lavage fluid of pollution-exposed mice but returned to baseline in mice with ablated nociceptor neurons. Alveolar macrophages expressing the pollution-sensing aryl hydrocarbon receptor were identified as a putative source of artemin following exposure to PM_2.5. This molecule enhanced TRPA1 responsiveness and, in turn, drove nociceptor-mediated neutrophil recruitment, revealing a novel mechanism by which lung-innervating neurons respond to air pollution in the context of allergy. Overall, our findings suggest that targeting artemin-driven pathways could provide a therapeutic strategy for controlling neutrophilic airway inflammation in asthma, a clinical condition typically refractory to treatment.

Figure 1.. Air pollution exacerbates nociceptor neuronal activity.

(A–C) Male and female C57BL/6 mice (6–10 weeks old) were sensitized via intraperitoneal injection with an emulsion of ovalbumin (OVA; 200 μg/dose) and aluminum hydroxide (1 mg/dose) on days 0 and 7. On days 14–16, mice were challenged intranasally with OVA (50 μg/dose), either alone or in combination with fine particulate matter (FPM; 20 μg/dose). Bronchoalveolar lavage fluid was collected, and jugular-nodose complex neurons were cultured on day 17 for 24 hours before being loaded with the calcium indicator Fura-2AM. Cells were sequentially stimulated with the TRPA1 agonist AITC (successively to 10 μM at 60–90 seconds, 30 μM at 90–120 seconds, 100 μM at 120–150 seconds) and then with KCl (40 mM at 420–435 seconds). Calcium flux was continuously monitored throughout the experiment. The amplitude of AITC responses was measured by calculating the ratio of peak F340/F380 fluorescence after stimulation to the baseline F340/F380 fluorescence measured 30 seconds prior to stimulation. Data are plots as the per dish average of AITC and KCl responsive neurons and show that AITC (10 μM) responses was higher in JNC neurons from OVA-FPM exposed mice when compared to vehicle or OVA alone (C). Data in are presented as means ± SEM (B-C). N are as follows: B: n = 35 neurons (control group), 19 neurons (OVA group), and 38 neurons (OVA + FPM group), C: n = 5 dishes totalling 35 neurons (control group), 8 dishes totalling 42 neurons (OVA group), and 10 dishes totalling 76 neurons (OVA + FPM group). P-values were determined by nested one-way ANOVA with post-hoc Bonferroni’s. P-values are shown in the figure.

Figure 2.. Air pollution reprograms the transcriptome of nociceptor neurons.

(A–F) Naïve male and female TRPV1^cre::tdTomato^fl/wt mice (6–10 weeks old) underwent either a pollution-exacerbated asthma protocol, the classic OVA protocol, or remained naïve. On day 17 (peak inflammation), jugular-nodose-complex (JNC) neurons were harvested and dissociated, and TRPV1⁺ neurons (tdTomato⁺) were sorted via FACS to remove stromal cells and non-peptidergic neurons. RNA was then isolated for sequencing. Volcano plots (A, C, E) and heatmaps (B, D, F) show differentially expressed genes (DEGs) for three comparisons: OVA + FPM vs. naïve (A-B), OVA + FPM vs. OVA alone (C-D), and OVA alone vs. naïve (E-F). Notable genes with increased expression include Lifr and Oprm3 in OVA + FPM vs. naïve, Oprm1, Nefh, P2ry1, Prkcb, Gabra1, and Kcnv1 in OVA + FPM vs. OVA, and Npy1r and Kcna1 in OVA alone vs. naïve. Data are presented either as volcano plots (A, C, E), showing the log2 fold change of TPM between groups along with the corresponding −log10 p-values from DESeq2 analysis, or as heatmaps (B, D, F), showing the z-scores of rlog-transformed normalized counts. The experimental groups were naïve (n=2; A–B, E–F), OVA (n=3; C–F), and OVA-FPM (n=3; A–D). P-values were determined by DESeq2 (A, C, E) and are indicated in the figure.

Figure 3.. Nociceptor neurons control pollution-exacerbated asthma.

(A–B) Male and female C57BL/6 mice (6–10 weeks old) were sensitized intraperitoneally with ovalbumin (OVA; 200 μg/dose in 200 μL) and aluminum hydroxide (1 mg/dose in 200 μL) on days 0 and 7. On days 14–16, mice were challenged intranasally with OVA (50 μg/dose in 50 μL) alone or with fine particulate matter (FPM; 20 μg/dose in 50 μL). On day 16, 30 minutes after the final challenge, mice received intranasal QX-314 (5 nmol/dose in 50 μL). Bronchoalveolar lavage fluid (BALF) was collected on day 17 and analyzed by flow cytometry. Compared with naïve or OVA-exposed mice, those co-challenged with OVA + FPM showed increased BALF neutrophils (A). QX-314 treatment normalized these levels, while BALF eosinophil levels remained comparable (B). (C–E) Male and female littermate control (TRPV1^WT) and nociceptor-ablated (TRPV1^DTA) mice (6–10 weeks old) were sensitized and challenged under the same OVA ± FPM protocol (days 0, 7, and 14–16). BALF or lungs were collected on day 17 and assessed by flow cytometry. Compared with naïve or OVA-exposed mice, OVA + FPM co-challenged mice exhibited higher BALF neutrophils (C) and lung γδ T cells (E). Nociceptor ablation protected against these increases (C, E), while BALF eosinophil levels remained comparable (D). Data are shown as mean ± SEM (A–E). Experiments were replicated twice, and animals pooled (A-E). N are as follows: A-B: control (n=6), OVA (n=7), OVA-FPM (n=12), OVA-FPM+QX-314 (n=10), C-D: TRPV1^WT + control (n=9), TRPV1^WT + OVA (n=13), TRPV1^WT + OVA-FPM (n=18), TRPV1^DTA + OVA-FPM (n=19), E: TRPV1^WT + control (n=3), TRPV1^WT + OVA (n=3), TRPV1^WT + OVA-FPM (n=4), TRPV1^DTA + OVA-FPM (n=5). P-values were determined by a one-way ANOVA with post-hoc Tukey’s (A-E). P-values are shown in the figure.

Figure 4.. Vagal sensory neurons gatekeep alveolar macrophage motility and neutrophil numbers.

(A–D) Male and female littermate control (Na_V1.8^wt::DTA^fl/wt denoted as Na_V1.8^WT) and nociceptor-ablated (Nav1.8^cre::DTA^fl/wt denoted as NaV1.8^DTA) mice (6–10 weeks old) were sensitized via intraperitoneal injection with an emulsion of ovalbumin (OVA; 200 μg/dose) and aluminum hydroxide (1 mg/dose) on days 0 and 7. Phagocytes were labeled by intranasal injection of PKH26 Red Fluorescent Cell Linker Kit (25 pmol/dose) on day 10. Mice were then challenged intranasally with OVA (50 μg/dose) alone or in combination with fine particulate matter (FPM; 20 μg/dose) on days 14–16, and images were acquired on day 17. (A) Representative maximum-intensity projection of alveolar macrophages (AMs, red). Scale bar: 20 μm. (B) Quantification of AM numbers per field of view (FOV). (C) Net displacement of AMs over 1 hour. (D-F) Representative tracks of individual AMs (each color represents a single AM) over 1 hour. While the AM numbers (A-B) were not impacted, their net displacement (C-F) was reduced in OVA-FPM-exposed NaV1.8^DTA mice. (G-L) Male and female littermate control (Na_V1.8^wt::DTA^fl/wt denoted as Na_V1.8^WT) and nociceptor-ablated (Na_V1.8^cre::DTA^fl/wt denoted as Na_V1.8^DTA) mice (6–10 weeks old) were sensitized via intraperitoneal injection with an emulsion of ovalbumin (OVA; 200 μg/dose) and aluminum hydroxide (1 mg/dose) on days 0 and 7. Mice were then challenged intranasally with OVA (50 μg/dose) alone or in combination with fine particulate matter (FPM; 20 μg/dose) on days 14–16, and images were acquired on day 17. Immediately prior to perform the intravital imaging, we administered an intravenous Ly6G antibody to label neutrophils. (G) Representative maximum-intensity projection of neutrophils (black). Scale bar: 20 μm. (H) Quantification of neutrophil numbers per FOV. (I) The total displacement of neutrophils over 20 minutes. (J-K) Representative tracks of individual neutrophils (each color represents a single neutrophil) over 20 minutes. (L) Frequency of neutrophil behaviors per FOV (adherent, crawling, patrolling, or tethering). Data show that OVA-FPM-exposed littermate control mice present an increase in neutrophil numbers per field of view (G-H), an effect absent in Na_V1.8^DTA. Interestingly, OVA-FPM-exposed Na_V1.8^DTA mice show an increase in neutrophil net displacement (I-K), an effect irrespective to one of the specific behaviors tested (L). Data are presented as representative image (A, G; scale bar: 20μm), mean ± SEM (B, C, H), spider plot (D-F, J-K), violin plot showing median (I), and staked bar graph showing mean ± SEM (L). N are as follows: B: Na_V1.8^WT + control (n=6), Na_V1.8^WT + OVA-FPM (n=6), Na_V1.8^DTA + OVA-FPM (n=6), C: Na_V1.8^WT + control (n=42), Na_V1.8^WT + OVA-FPM (n=42), Na_V1.8^DTA + OVA-FPM (n=42), H: Na_V1.8^WT + control (n=6), Na_V1.8^WT + OVA-FPM (n=6), Na_V1.8^DTA + OVA-FPM (n=6), I: Na_V1.8^WT + OVA-FPM (n=385), Na_V1.8^DTA + OVA-FPM (n=469), J: Na_V1.8^WT + OVA-FPM (n=10), Na_V1.8^DTA + OVA-FPM (n=11). P-values were determined by a one-way ANOVA with post-hoc Tukey’s (B, C, H) or unpaired Student T-test (I, L). P-values are shown in the figure.

Figure 5.. Artemin sensitizes TRPA1 activity in vagal sensory neurons.

(A-B) Male and female littermate control (TRPV1^WT) and nociceptor-ablated (TRPV1^DTA) mice (6–10 weeks old) were sensitized and challenged under the same OVA ± FPM protocol (days 0, 7, and 14–16). BALF was collected on day 17 and assessed by multiplex array and ELISA. Compared with naïve or OVA-alone groups, OVA + FPM co-challenged mice exhibited levels of TNFα, and artemin. Notably, ablating nociceptors prevented these increases. (C) In-silico analysis of the GSE124312 dataset. The heatmap displays transcript expression levels for 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). Gfra3 expression is enriched in the peptidergic neuron cluster labeled JG4. Experimental details and cell clustering are described by Kupari et al. (D) In-silico analysis of GSE192987⁵² showing co-expression of Gfra3 with Trpa1 and other inflammatory markers. Data are visualized as row z-scores in a heatmap or via UMAPs (TPTT > 1). Experimental details and cell clustering are described by Zhao et al. (E-G) Alveolar macrophages (3 × 10⁵ cells/well) from naïve male and female C57BL/6 mice were cultured overnight and then stimulated with vehicle (DMSO) or FPM (100 μg/mL). RNA was extracted 1- and 4-hours post-stimulation and Artn expression was assessed using qPCR. FPM exposure increased Artn transcript levels at both 1 and 4 hours (F, G). (H-J) Naïve mice jugular-nodose-complex neurons were harvested, pooled, and cultured overnight with either vehicle or artemin (100 ng/mL). Cells were 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 all KCl-responsive cells) was normalized to vehicle-treated controls for each batch of experiments. Artemin-treated neurons showed increased responsiveness to AITC, while responses to capsaicin and KCl were unchanged (I-J). Data are presented as means ± SEM (A-B, F-G, J), heatmap displaying the z-score of DESeq2 normalized counts (C), tSNE plots (D), schematics (E, H), means ± 95% CI of maximum Fura-2AM (F/F₀) fluorescence (I). N are as follows: A: TRPV1^WT + control (n=2), TRPV1^WT + OVA (n=3) TRPV1^WT + OVA-FPM (n=3), TRPV1^DTA + OVA-FPM (n=8), B: TRPV1^WT + OVA (n=6) TRPV1^WT + OVA-FPM (n=8), TRPV1^DTA + OVA-FPM (n=14), F: n=2/time point, G: n=8/group, I: vehicle (n=107 neurons), Artemin (n=122 neurons); J: n=4/group. P-values were determined by a one-way ANOVA with post-hoc Tukey’s (A, B) or unpaired Student T-test (G, J). P-values are shown in the figure.