Journal: eLife

Article Title: Nociceptor neurons control pollution-mediated neutrophilic asthma

doi: 10.7554/eLife.101988

Figure Lengend Snippet: ( 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 . 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 . ( D ) In silico analysis of GSE192987 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 . ( E–G ) Alveolar macrophages (3×10 5 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/F 0 ) 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.

Article Snippet: ELISA was used to measure artemin levels in BALF (R&D Systems, # DY1085-05) following the manufacturer’s instructions.

Techniques: Control, Multiplex Assay, Enzyme-linked Immunosorbent Assay, In Silico, Expressing, Marker, Labeling, Cell Culture, Real-time Polymerase Chain Reaction, Fluorescence

Journal: eLife

Article Title: Nociceptor neurons control pollution-mediated neutrophilic asthma

doi: 10.7554/eLife.101988

Figure Lengend Snippet: In our study, mice were exposed to PM 25 particles and ovalbumin (OVA) to model pollution-exacerbated asthma. Compared to mice exposed to OVA alone, co-exposure to PM 25 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.

Article Snippet: ELISA was used to measure artemin levels in BALF (R&D Systems, # DY1085-05) following the manufacturer’s instructions.

Techniques: Produced, Imaging

Journal: Journal of Cellular and Molecular Medicine

Article Title: KIF1B Regulates NLRP3 ‐Mediated Pyroptosis in Asthma Progression

doi: 10.1111/jcmm.70975

Figure Lengend Snippet: KIF1B inhibition reduces IL‐13‐induced inflammatory responses in BEAS‐2B cells. (A) BEAS‐2B cells were transfected with si‐KIF1B or si‐NC and treated with IL‐13 (10 ng/mL) for 24 h, followed by Western blot analysis of NLRP3, cleaved caspase‐1 and cleaved GSDMD protein expression. Conditioned medium was collected to measure cytokine levels: (B) TNF‐α, (C) IL‐1β, (D) IL‐18 and (E) IL‐10 by ELISA. β‐actin was used as the loading control for normalisation in Western blot. Data are presented as mean ± SD ( n = 3; *** p < 0.001 vs. control, p < 0.01, p < 0.001 vs. IL‐13 group).

Article Snippet: For murine samples (BALF), TNF‐α (Cat# MTA00B, R&D Systems), IL‐1β (Cat# MLB00C, R&D Systems), IL‐18 (Cat# DY7625‐05, R&D Systems) and IL‐10 (Cat# M1000B, R&D Systems) were assessed.

Techniques: Inhibition, Transfection, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: KIF1B Regulates NLRP3 ‐Mediated Pyroptosis in Asthma Progression

doi: 10.1111/jcmm.70975

Figure Lengend Snippet: KIF1B regulates IL‐13‐induced inflammation in BEAS‐2B cells via NLRP3. BEAS‐2B cells were transfected with si‐NC, si‐KIF1B + empty vector or si‐KIF1B + NLRP3‐ORF plasmid, with or without IL‐13 treatment. (A) NLRP3 gene expression was measured by qRT‐PCR following NLRP3‐ORF transfection to confirm successful overexpression. (B) Following transfection with si‐KIF1B with or without NLRP3‐ORF and IL‐13 treatment, intracellular ROS, MDA and GSH levels were measured. (C) Protein expression of NLRP3, cleaved caspase‐1 and cleaved GSDMD was assessed by Western blot. (D) Cytokine levels (TNF‐α, IL‐1β, IL‐18 and IL‐10) in conditioned medium were measured by ELISA. GAPDH gene was used as an internal reference for qPCR analysis, and β‐actin was used as the loading control for normalisation in Western blot. Data are presented as mean ± SD ( n = 3; *** p < 0.001 vs. control, p < 0.01, p < 0.001 vs. IL‐13 group, & p < 0.05, & p < 0.01 vs. IL‐13 + si‐KIF1B group).

Article Snippet: For murine samples (BALF), TNF‐α (Cat# MTA00B, R&D Systems), IL‐1β (Cat# MLB00C, R&D Systems), IL‐18 (Cat# DY7625‐05, R&D Systems) and IL‐10 (Cat# M1000B, R&D Systems) were assessed.

Techniques: Transfection, Plasmid Preparation, Gene Expression, Quantitative RT-PCR, Over Expression, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: KIF1B Regulates NLRP3 ‐Mediated Pyroptosis in Asthma Progression

doi: 10.1111/jcmm.70975

Figure Lengend Snippet: KIF1B knockdown reduces OVA‐induced inflammation and pyroptosis in mouse pulmonary tissues. C57BL/6 mice were infected with lentiviral particles carrying sh‐KIF1B or sh‐NC, and subsequently treated with OVA for 14 days to induce asthmatic responses. Lung tissues were collected at sacrifice. (A) Wright‐Giemsa staining was used to quantify total cells, macrophages, eosinophils, lymphocytes and neutrophils in BALF. (B) IgE, TNF‐α, IL‐1β, IL‐18 and IL‐10 levels in BALF were measured by ELISA. (C) Protein levels of NLRP3, cleaved caspase‐1 and cleaved GSDMD in lung homogenates were assessed by Western blot. β‐actin was used as the loading control for normalisation in Western blot. Data are presented as mean ± SD ( n = 8; *** p < 0.001 vs. control, p < 0.01, p < 0.001 vs. OVA group).

Article Snippet: For murine samples (BALF), TNF‐α (Cat# MTA00B, R&D Systems), IL‐1β (Cat# MLB00C, R&D Systems), IL‐18 (Cat# DY7625‐05, R&D Systems) and IL‐10 (Cat# M1000B, R&D Systems) were assessed.

Techniques: Knockdown, Infection, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Control