Figures and data in Nociceptor neurons control pollution-mediated neutrophilic asthma

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8 figures, 2 videos, 1 table and 2 additional files

Figures

Figure 1

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Air pollution exacerbates nociceptor neuronal activity.

(A–C) Male and female C57BL/6 mice (6–10 weeks of age) 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 hr 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 s, 30 µM at 90–120 s, 100 µM at 120–150 s) and then with KCl (40 mM at 420–435 s). 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 s prior to stimulation. Data are plots as the per dish average of AITC and KCl responsive neurons and show that AITC (10 µM) responses were 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 totaling 35 neurons (control group), 8 dishes totaling 42 neurons (OVA group), and 10 dishes totaling 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

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Air pollution reprograms the transcriptome of nociceptor neurons.

(A–F) Naïve male and female *Trpv1*^cre::*tdTomato*^fl/wt mice (6–10 weeks of age) 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

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Nociceptor neurons control pollution-exacerbated asthma.

(A–B) Male and female C57BL/6 mice (6–10 weeks of age) 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 min 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 of age) 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

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Vagal sensory neurons gatekeep alveolar macrophage (AM) motility and neutrophil numbers.

(A–D) Male and female littermate control (*Scn10a*^wt::*Dta*^fl/wt denoted as Na_V1.8^WT) and nociceptor-ablated (*Scn10a*^cre::*Dta*^fl/wt denoted as Na_V1.8^DTA) mice (6–10 weeks of age) 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 AMs (red). Scale bar: 20 µm. (B) Quantification of AM numbers per field of view (FOV). (C) Net displacement of AMs over 1 hr. (**D–F**) Representative tracks of individual AMs (each color represents a single AM) over 1 hr. While the AM numbers (**A–B**) were not impacted, their net displacement (**C–F**) was reduced in OVA-FPM-exposed Na_V1.8^DTA mice. (**G–L**) Male and female littermate control (*Scn10a*^wt::*Dta*^fl/wt denoted as Na_V1.8^WT) and nociceptor-ablated (*Scn10a*^cre::*Dta*^fl/wt denoted as Na_V1.8^DTA) mice (6–10 weeks of age) were sensitized via intraperitoneal injection with an emulsion of 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 FPM (20 µg/dose) on days 14–16, and images were acquired on day 17. Immediately prior to performing 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 min. (**J–K**) Representative tracks of individual neutrophils (each color represents a single neutrophil) over 20 min. (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 FOV (**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: 20 µm), mean ± SEM (**B, C, H**), spider plot (**D–F, J–K**), violin plot showing median (I), and stacked 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’s t-test (**I, L**). p-Values are shown in the figure.

Figure 5 with 2 supplements

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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 of age) were sensitized and challenged under the same ovalbumin (OVA)±fine particulate matter (FPM) protocol (days 0, 7, and 14–16). Bronchoalveolar lavage fluid (BALF) was collected on day 17 and assessed by multiplex array and enzyme-linked immunosorbent assay (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 (Kupari et al., 2019). 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., 2019. (D) In silico analysis of GSE192987 (Zhao et al., 2022) 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., 2022. (**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 hr post-stimulation, and *Artn* expression was assessed using quantitative PCR (qPCR). FPM exposure increased *Artn* transcript levels at both 1 and 4 hr (**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 s), capsaicin (TRPV1 agonist; 300 nM at 320–335 s), and KCl (40 mM at 720–735 s). 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’s t-test (**G, J**). p-Values are shown in the figure.

Figure 5—figure supplement 1

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In silico analysis of *Artn* expression in mouse immune cells.

(A) In silico re-analysis of *Artn* expression in mouse immune cells using the ImmGen database (Immunological Genome Project, 2020). *Artn* and *Ahr* are expressed in *Itgam*⁺ macrophages. Data are presented as per-gene z-scores of normalized gene expression, calculated by the median of ratios method. (B) In silico re-analysis of the single-cell RNA-sequencing dataset from Tavares-Ferreira et al., 2022 (Sensoryomics; dbGaP accession phs001158) shows that *Gfra3* is expressed in *Trpa1*-positive nociceptors, C-LTMRs, and silent nociceptors within the human dorsal root ganglion. Expression levels are reported as per-gene z-scores calculated with the median-of-ratios normalization method.

Figure 5—figure supplement 2

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In silico re-analysis of Ahr and Artn expression in human tissues.

(A) In silico re-analysis of data from Karlsson et al., 2021 (Human Protein Atlas, proteinatlas.org) indicates that *Ahr* and *Artn* are expressed in human lung. Expression values are reported as per-gene z-scores using the median-of-ratios normalization method. Experimental details and cell clustering are described by Karlsson et al., 2021. (B) In silico re-analysis of data from Uhlén et al., 2005 (Human Protein Atlas, proteinatlas.org) confirms *Ahr* protein expression in human lung macrophages, as shown by immunohistochemistry. These data are presented as protein-normalized expression levels. Experimental details and cell clustering are described by Uhlén et al., 2005. (C) In silico re-analysis of data from Abdulla et al., 2023 (CELLxGENE, CZI Single-Cell Biology) reveals co-expression of *Artn* and *Ahr* in lung and alveolar macrophages from patients. Expression values are provided as per-gene z-scores, calculated by the median-of-ratios normalization method. Experimental details and cell clustering are described by Abdulla et al., 2023.

Figure 6

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Schematic of nociceptor involvement in pollution-exacerbated allergic asthma.

In our study, mice were exposed to PM₂₅ particles and ovalbumin (OVA) to model pollution-exacerbated asthma. Compared to mice exposed to OVA alone, co-exposure to PM₂₅ and OVA significantly increased bronchoalveolar lavage fluid (BALF) neutrophils and lung γδ T cell levels. To counteract this heightened airway inflammation, we administered intranasal QX-314—a charged lidocaine derivative—at the peak of inflammation, effectively normalizing BALF neutrophil levels. Ablation of TRPV1⁺ nociceptor neurons produced a similar effect. Further analysis with calcium imaging revealed that neurons from the jugular-nodose complex in pollution-exposed asthmatic mice were more sensitive via their TRPA1 channels. Levels of TNFα and the growth factor artemin were also elevated in the BALF of these mice, returning to normal following nociceptor ablation. We identified alveolar macrophages as the source of artemin, which they secrete upon sensing fine particulate matter (FPM) through aryl hydrocarbon receptors. Artemin, in turn, heightened TRPA1 responsiveness to its agonist (mustard oil), thereby exacerbating airway inflammation. Our findings suggest that silencing nociceptor neurons can disrupt this pathway, offering a novel therapeutic approach to mitigate neutrophilic airway inflammation driven by pollution.

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Videos

Video 1

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posterframe for video — Intravital recording of alveolar macrophage motility.

6- to 10-week-old male and female littermate control (*Scn10a*^wt::DTA^fl/wt denoted as NaV1.8^WT) and nociceptor-ablated (*Scn10a*^cre::DTA^fl/wt denoted as Na_V1.8^DTA) mice were sensitized via intraperitoneal injection of an emulsion containing ovalbumin (OVA; 200 µg/dose) and aluminum hydroxide (1 mg/dose) on days 0 and 7. On day 10, phagocytes were labeled by intranasal injection of PKH26 (25 pmol/dose). 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. Alveolar macrophage intravital imaging was performed on day 17 and is presented as a 1 hr time-lapse video.

Video 2

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Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (Mus musculus)	C57BL/6J	The Jackson lab	Stock No: 000664; RRID:IMSR_JAX:000664	Wild-type background strain used for experiments and/or breeding.
Strain, strain background (Mus musculus)	DTAfl/fl (floxed diphtheria toxin A line; ‘DTAfl’)	The Jackson lab	Stock No: 010527; RRID:IMSR_JAX:010527	Cre-dependent DTA expression for genetic ablation when crossed to Cre driver lines.
Strain, strain background (Mus musculus)	DTAfl/fl (floxed diphtheria toxin A line; ‘DTAfl’)	The Jackson lab	Stock No: 009669; RRID:IMSR_JAX:009669	Cre-dependent DTA expression for genetic ablation when crossed to Cre driver lines.
Strain, strain background (Mus musculus)	tdTomatofl/fl (Ai14; ‘tdTomatofl’)	The Jackson lab	Stock No: 007914; RRID:IMSR_JAX:007914	Cre-dependent tdTomato reporter used to label/sort Trpv1⁺ neurons (Trpv1cre/wt::tdTomatofl/wt).
Strain, strain background (Mus musculus)	TRPV1cre/cre	The Jackson lab	Stock No: 017769; RRID:IMSR_JAX:017769	Trpv1 promoter-driven Cre; used to target Trpv1⁺ nociceptors for reporter labeling and/or DTA-mediated ablation.
Strain, strain background (Mus musculus)	NaV1.8cre/cre	The Jackson lab	Stock No: 036564; RRID:IMSR_JAX:036564	NaV1.8 (Scn10a) promoter-driven Cre; used to target NaV1.8⁺ nociceptors for DTA-mediated ablation.
Antibody	Anti-mouse CD45 [clone 30-F11] (rat monoclonal)	BioLegend	Cat# 103128; RRID:AB_493715	1:200 to 1:400
Antibody	Anti-mouse CD90.2 (Thy1.2) [clone 53–2.1] (rat monoclonal)	BioLegend	Cat# 140307; RRID:AB_10643585	1:200 to 1:400
Antibody	Anti-mouse CD11b [clone M1/70] (rat monoclonal)	BioLegend	Cat# 101243; RRID:AB_2561373	1:200 to 1:400
Antibody	Anti-mouse CD11c [clone N418] (Armenian hamster mAb)	BioLegend	Cat# 117303; RRID:AB_313772	1:200 to 1:400
Antibody	Anti-mouse Ly6C [clone HK1.4] (rat monoclonal)	BioLegend	Cat# 128004; RRID:AB_1236553	1:200 to 1:400
Antibody	Anti-mouse Ly6G [clone 1A8] (rat monoclonal)	BioLegend	Cat# 127610; RRID:AB_1134159	1:200 to 1:400
Antibody	Anti-mouse Siglec-F [clone 1RNM44N] (rat monoclonal)	Invitrogen	Cat# 12-1702-82; RRID:AB_2637129	1:200 to 1:400
Antibody	Anti-mouse TCR γ/δ [clone GL3] (Armenian hamster mAb)	BioLegend	Cat# 118128; RRID:AB_2562771	1:200 to 1:400
Antibody	Anti-mouse TCR β [clone H57-597] (Armenian hamster mAb)	BioLegend	Cat# 109201; RRID:AB_313424	1:200 to 1:400
Antibody	Anti-mouse CD19 [clone 1D3] (rat monoclonal)	BD Biosciences	Cat# 553783; RRID:AB_395047	1:200 to 1:400
Antibody	Anti-mouse NK1.1 [clone PK136] (mouse monoclonal)	Invitrogen	Cat# 25-5941-82; RRID:AB_469665	1:200 to 1:400
Antibody	Anti-mouse F4/80 [clone BM8] (rat monoclonal)	Invitrogen	Cat# 14-4801-82; RRID:AB_467558	1:200 to 1:400
Antibody	Anti-mouse FcεRIα [clone MAR-1] (rat monoclonal)	BioLegend	Cat# 134318; RRID:AB_10640122	1:200 to 1:400

Additional files

Supplementary file 1 Differentially expressed genes and pathway analysis of vagal nociceptors in pollution-exacerbated asthma. Naïve 6–10 weeks’ male and female Trpv1^cre::tdTomato^fl/wt mice were either subjected to a pollution-exacerbated asthma protocol, to the classic ovalbumin (OVA) protocol, or remained naïve. On day 17, jugular-nodose complex (JNC) neurons were harvested and dissociated, and Trpv1⁺ (tdTomato⁺) neurons were FACS-purified to remove stromal and non-peptidergic cells before being processed for RNA-sequencing. The different tabs show the DESeq2 identified for each of these conditions. Other tabs show Gene Ontology (GO) terms enriched in each condition, analyzed using the web-based tool g:Profiler.: https://cdn.elifesciences.org/articles/101988/elife-101988-supp1-v1.xlsx
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MDAR checklist: https://cdn.elifesciences.org/articles/101988/elife-101988-mdarchecklist1-v1.docx
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