Fig 2: Nociceptor neurons are necessary for IgE production in allergic airway inflammation.HDM challenge increased the frequency of (A) total and (B) IgE+ B cells as well as (C) total and (D) IgE+ germinal center (GC) B cells in lung draining lymph nodes (dLNs). Nociceptor ablation significantly reduced the frequency of (B) IgE+ total B cells and (D) IgE+ GC B cells. HDM challenge increased the numbers of (E) total and (F) IgE+ antibody-secreting cells (ASCs) in the lungs of littermate control mice. Nociceptor ablation prevented this. HDM challenge increased IgG1 (G and I) and IgE (H and J) levels in bronchoalveolar lavage fluid (BALF) (G and H) and serum (I and J). These effects were absent in HDM-exposed TRPV1Cre/wt DTAfl/wt mice. Graphs show range, median, and “+” as mean. P values determined using 1-way ANOVA and Tukey’s multiple comparisons test. * denotes comparison with vehicle-exposed, nociceptor-intact mice and + comparison with HDM-exposed, nociceptor-intact mice. P < 0.05 is indicated by * or +; P < 0.01 is indicated by ** or ++; P < 0.001 is indicated by ***; P < 0.0001 is indicated by ****. Representative experiment is shown; n = 4–7/group. Experiments were replicated at least 3 independent times.

Fig 3: Substance P increases formation of antibody-secreting cells and their release of IgE.Splenic B cells were isolated from naive C57BL6 mice and cultured with or without LPS (10 µg/mL) and IL-4 (20 ng/mL) and exposed to vehicle, substance P (1 µM), and CGRP (300 nM). (A) After 4 days, the cells were immunophenotyped by flow cytometry, and immunoglobulin release was assessed by ELISA. LPS and IL-4 triggered the formation of (B) total, (C and E) IgG1+ and (D and F) IgE+ CD95+ (germinal center; C and D) and CD138+ (antibody-secreting cells; E and F) B cells. Additionally, LPS+IL-4 enhanced B cell release of (G) IgG1 and (H) IgE. These effects were augmented when B cells were coexposed to the neuropeptide substance P (1 µM). CGRP (300 nM) exposure had minimal impact on these parameters. Graphs show median and range. P values determined using 1-way ANOVA and Tukey’s multiple comparisons test. P < 0.05 is indicated by *; P < 0.01 is indicated by **. Representative experiment is shown; n = 3/group. Experiments were replicated at least 2 independent times.

Fig 4: Nociceptor neurons affect allergic skin inflammation.(A) Timeline for induction of allergic skin inflammation using calcipotriol (vitamin D analog). Calcipotriol or vehicle (EtOH) was applied once daily on mouse dorsal skin for 8 days to littermate control or genetically ablated (TRPV1cre/wt DTAfl/wt) mice. Groups of WT mice were additionally treated once daily with QX-314 (10 mg/mL, topical) from days 3 to 8. Animals were sacrificed on day 17. (B) Skin histology of day 5 control and calcipotriol-treated mice showed skin thickening, ECM deposits, loss of dendritic epidermal CD3+ T cells, recruitment of CD3+ T cells to inflamed tissue, and loss of PGP9.5 hair follicle innervation after sensitization. Scale bar represents 100 µm. Calcipotriol treatment increased numbers of (C) CD45+ cells, (D) dendritic cells, (E) CD3+ T cells, (F) CD4+ T cells, (G) regulatory T cells, and (H) CD8+ memory T cells in skin tissue. Nociceptor ablation (TRPV1Cre/wt DTAfl/wt) reversed calcipotriol-mediated skin inflammation and significantly decreased (J) serum IgE levels. (I) Serum IgG1 levels were not affected by nociceptor ablation. Graphs show range, median, and “+” as mean. P values determined using 1-way ANOVA and Tukey’s multiple comparisons test. * denotes comparison with vehicle-exposed, nociceptor-intact mice and + comparison with calcipotriol-exposed, nociceptor-intact mice. P < 0.05 is indicated by +; P < 0.01 is indicated by ** or ++; P < 0.001 is indicated by +++; P < 0.0001 is indicated by **** or ++++. n = 3–5/group. Experiments were replicated at least 2 independent times.

Fig 5: Hpb numbers are increased in microbiota-depleted animals despite evidence of a strong type 2 immune response.a SPF and antibiotic-treated mice (enrofloxacin for 2 weeks then amoxicillin and clavulanic acid for the rest of the experiment) were infected for 14 to 42 days with 200 Hpb larvae. a Number of adult worms in the intestine were assessed at each time points. Data are pooled from four independent experiments and analysed using two-way ANOVA. The number of mice per time point are: D14 = 16, D28 = 15, D35 = 16 and D38-42 = 8 for each group. b–d SPF and antibiotic-treated mice (enrofloxacin for 2 weeks then amoxicillin and clavulanic acid for the rest of the experiment) were infected for 0 to 21 days with 200 Hpb larvae. Concentration of IL-4 (b), IL-13 (c) and IFNγ (d) were measured by ELISA. Data were pooled from four independent experiments and analysed using two-way ANOVA. The number of mice per time point are: (1) for SPF mice D0 = 19, D9-10 = 17, D14 = 19 and D21 = 13; (2) for antibiotic-treated mice D0 = 22, D9-10 = 18, D14 = 20 and D21 = 10. e–g SPF and antibiotic-treated mice (enrofloxacin for 2 weeks then amoxicillin and clavulanic acid for the rest of the experiment) were infected for 0 to 42 days with 200 Hpb larvae. Concentration of total circulating IgE (e), IgG1 (f) and HES specific IgG1 (g) were measured using ELISA. Data are pooled from two independent experiments and analysed using two-way ANOVA. e, f The number of mice per time point are: (1) For SPF mice D0 = 8, D9 = 10, D14 = 10, D21 = 10, D30 = 9 and D42 = 10; (2) for antibiotic-treated mice D0 = 9, D9 = 10, D14 = 10, D21 = 7, D30 = 10 and D42 = 10. g The number of mice per time point are: (1) For SPF mice D0 = 5, D9 = 5, D14 = 5, D21 = 5, D30 = 5 and D42 = 5; (2) for antibiotic-treated mice D0 = 5, D9 = 5, D14 = 5, D21 = 4, D30 = 5 and D42 = 5. *display statically significant differences between groups and # display statistically significant differences compared to the naïve group (0 days post-infection).