Sensory neurons promote immune homeostasis in the lung

Abstract

Cytokines employ downstream Janus kinases (JAKs) to promote chronic inflammatory diseases. JAK1-dependent type 2 cytokines drive allergic inflammation, and patients with JAK1 gain-of-function (GoF) variants develop atopic dermatitis (AD) and asthma. To explore tissue-specific functions, we inserted a human JAK1 GoF variant (JAK1^GoF) into mice and observed the development of spontaneous AD-like skin disease but unexpected resistance to lung inflammation when JAK1^GoF expression was restricted to the stroma. We identified a previously unrecognized role for JAK1 in vagal sensory neurons in suppressing airway inflammation. Additionally, expression of Calcb/CGRPβ was dependent on JAK1 in the vagus nerve, and CGRPβ suppressed group 2 innate lymphoid cell function and allergic airway inflammation. Our findings reveal evolutionarily conserved but distinct functions of JAK1 in sensory neurons across tissues. This biology raises the possibility that therapeutic JAK inhibitors may be further optimized for tissue-specific efficacy to enhance precision medicine in the future.

Keywords: AAV; CGRP; ILC2; JAK1; afferent nerves; allergic lung inflammation; atopic disorders; neuropeptide; sensory neurons; vagus nerve.

Figure 1.. Human germline JAK1 GOF mutation is associated with AD and asthma in patients and promotes spontaneous AD-like disease in mice

(A) Human JAK1 c.1901C>A (p.A634D) GOF mutation within the pseudokinase domain. (B) Timeline of AD and asthma diagnosis, EASI assessment, and PFTs. (C) Baseline visual image of AD skin lesions from Patient A with a JAK1 GOF mutation. (D) Baseline visual image of AD skin lesions from Patient B with a JAK1 GOF mutation. (E) Baseline EASI scores from Patient A and Patient B. (F) Baseline FEV1 of Patient A and Patient B. (G) Baseline FVC of Patient A and Patient B. (H) Schematic of the targeting strategy used to introduce the germline human JAK1 GOF mutation (JAK1^GOF) into the murine Jak1 locus. (I) Representative ear skin images of WT control and JAK1^GOF mice. (J) Ear thickness measurements of WT control and JAK1^GOF mice. (K) Representative skin histology images (H&E) of WT control and JAK1^GOF mice. Scale bars indicate 100 μm. (L-O) Flow cytometry of skin lymph nodes from WT control and JAK1^GOF mice. Shown are the frequencies of (L) total CD45⁺ immune cells, (M) CD4⁺ T cells, (N) ILC2s, and (O) eosinophils. (P-Q) Representative lung histology images of WT control and JAK1^GOF mice, obtained by (P) H&E stain and (Q) PAS stain. Scale bars indicate 200 μm. (R-U) Flow cytometry of lung tissue from WT control and JAK1^GOF mice. Shown are the frequencies of (R) total CD45⁺ immune cells, (S) CD4⁺ T cells, (T) ILC2s, and (U) eosinophils. (J, L-O, R-U) Data were pooled from three independent experiments, n = 8–15 pooled mice per group. ^†p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, and not significant (n.s.) (Unpaired t-test with Welch’s correction). Data are represented as mean ± standard deviation (SD). AD – atopic dermatitis, EASI – eczema area and severity index, FEV1 – forced expiratory volume, FVC – forced vital capacity, GOF – gain-of-function, ILC2s – group 2 innate lymphoid cells, H&E – hematoxylin and eosin, JAK1 – Janus kinase 1, PAS – periodic acid-Schiff, PFT – pulmonary function test, WT – wild-type. See also Figure S1

Figure 2.. Stroma-intrinsic expression of JAK1^GOF is protective in allergic lung inflammation

(A) Schematic of the allergic lung inflammation model using the protease allergen Alternaria alternata (ALT). (B-C) Representative lung histology images of WT control and JAK1^GOF mice challenged with intranasal ALT, obtained by (B) H&E stain and (C) PAS stain. (D-E) Flow cytometry of lung tissue from WT control and JAK1^GOF mice challenged with intranasal ALT. Shown are the frequencies of (D) ILC2s and (E) eosinophils. Data were obtained from three independent experiments, n = 5 mice per group. (F) Schematic of experimental approach used to generate chimeric mice. BM derived from WT mice was transferred into WT control and experimental JAK1^GOF mice. (G) JAK1 genotype of the respective hematopoietic and stromal cell components in chimeric mice (WT→WT and WT→JAK1^GOF). (H) Schematic of experimental approach used to generate the allergic lung inflammation model in the BM chimeras. (I-J) Representative lung histology images of chimeric WT→WT and WT→JAK1^GOF mice challenged with intranasal ALT, obtained by (I) H&E stain and (J) PAS stain. (K-L) Flow cytometry of lung tissue from chimeric WT→WT and WT→JAK1^GOF mice challenged with intranasal ALT. Shown are the frequencies of (K) ILC2s and (L) eosinophils. Data were obtained from three independent experiments, n = 11–14 pooled mice per group. (M) KEGG pathway overrepresentation analysis of RNA-seq data obtained from the lung tissues of chimeric WT→JAK1^GOF vs. WT→WT mice (n = 2 mice/group). Overrepresentation analysis used Fisher’s exact test to determine the significance of enrichment. All terms shown were identified as the top four significantly suppressed pathways in terms of adjusted p-value (p-adjusted < 0.05, Benjamini-Hochberg procedure). **p < 0.01, *p < 0.05, and not significant (n.s.) (Unpaired t-test with Welch’s correction). Data are represented as mean ± SD. Scale bars indicate 200 μm. (A, F-G) Figure adapted from an image created with BioRender.com. ALT- Alternaria alternata, BM – Bone marrow, BMT – Bone marrow transplantation. See also Figure S1

Figure 3.. Chemical denervation of TRPV1⁺ sensory neurons exacerbates allergic lung inflammation

(A) Visual representation of the sensory innervation of the lung arising from the VG and DRG. (B-C) Expression of Trpv1 and Scn10a (the gene encoding Na_v1.8) within the VG, specifically the (B) JG, and (C) NG. Trpv1 expression is shown as the percentage of total neurons sequenced. Scn10a expression is shown as the percentage of Trpv1-expressing neurons. The full scRNA-seq dataset is available in Kupari et al. (D-E) Imaging of classical nociceptive markers (TRPV1 and Na_v1.8) in the VG and DRG using Rosa26^{STOPflox-tdTomato} mice crossed with the respective Trpv1^Cre and Scn10a^Cre mice. (D) TRPV1 in the VG and DRG, (E) Na_v1.8 in the VG and DRG. (F) Schematic of the allergic lung inflammation model generated following systemic denervation of vagal and spinal visceral afferents. (G) Withdrawal latency from noxious heat (hot plate test). Mice treated with s.c. vehicle control or RTX were placed on a hot plate set at 50 degrees Celsius, and withdrawal latency was measured as the time until the appearance of paw withdrawal behavior. (H-I) Representative lung histology images from mice treated with s.c. vehicle control or RTX challenged with intranasal ALT, obtained by (H) H&E stain and (I) PAS stain. (J-K) Flow cytometry of lung tissue from mice treated with s.c. vehicle control or RTX and challenged with intranasal ALT. Shown are the frequencies of (J) ILC2s and (K) eosinophils. Data were obtained from three independent experiments, n = 4–5 mice per group. (L) Schematic of the allergic lung inflammation model generated following selective denervation of vagal afferents. (M) Withdrawal latency from noxious heat (hot plate test). Mice treated with intraganglionic (i.g.) vehicle control or RTX were placed on a hot plate set at 50 degrees Celsius, and withdrawal latency was measured as the time until the appearance of paw withdrawal behavior. (N-O) Representative lung histology images of mice treated with i.g. vehicle control or RTX challenged with intranasal ALT, obtained by (N) H&E stain and (O) PAS stain. (P-Q) Flow cytometry of lung tissue from mice treated with i.g. vehicle control or RTX challenged with intranasal ALT. Shown are the frequencies of (P) ILC2s and (Q) eosinophils. Data were obtained from two independent experiments, n = 4–5 mice per group. **p < 0.01, *p < 0.05, and not significant (n.s.) (Unpaired t-test with Welch’s correction). Data are represented as mean ± SD. Scale bars indicate 200 μm. (A, F, L) Figure adapted from an image created with BioRender.com. DRG – dorsal root ganglia, i.g. – intraganglionic, JG – jugular ganglia, NG – nodose ganglia, RTX – resiniferatoxin, s.c. – subcutaneous VG – vagal ganglia See also Figures S1 and S2

Figure 4.. Disruption of sensory neuron-intrinsic Jak1 exacerbates allergic lung inflammation and alters the expression levels of neuropeptides

(A-B) Jak1 expression in Na_v1.8⁺ vagal afferents, (A) JG and (B) NG. Gene expression is shown as the percentage of Na_v1.8⁺ neurons that express Jak1. The full scRNA-seq dataset is available in Kupari et al. (C) Schematic of the allergic lung inflammation model using littermate control (Jak1^flox) and experimental Jak1^Δneuron mice. (D-E) Representative lung histology images of control and Jak1^Δneuron mice challenged with intranasal ALT, obtained by (D) H&E stain and (E) PAS stain. (F-G) Flow cytometry of lung tissue from control and Jak1^Δneuron mice challenged with intranasal ALT. Shown are the frequencies of (F) ILC2s and (G) eosinophils. Data were obtained from two independent experiments, n = 10–12 pooled mice per group. (H-I) Profiling of neuropeptide expression in Na_v1.8⁺ vagal afferents; Calca, Calcb, Tac1, Vip, and Nmu were evaluated in the (H) JG and (I) NG. Gene expression is shown as the percentage of Na_v1.8⁺ neurons that express the indicated gene. The full scRNA-seq dataset is available in Kupari et al. (J-K) Imaging of the classical neuropeptides CGRPα and CGRPβ in the VG and DRG using Rosa26^{STOPflox-tdTomato} mice crossed with Calca^Cre and Calcb^Cre/ERT mice, respectively. Shown are (J) CGRPα in the VG and DRG and (K) CGRPβ in the VG and DRG. (L) MotifMap (human)-predicted neuropeptide targets of STAT family members. Data were analyzed using the MotifMap Predicted Transcription Factor Targets dataset (see Methods). (M-R) Quantitative PCR (qPCR) of transcripts from the VG and DRG from control and Jak1^Δneuron mice challenged with intranasal ALT. Shown are (M, N) Calcb, (O, P) Calca, and (Q, R) Tac1 transcript levels. Data were obtained from two independent experiments, n = 3–4 mice per group. **p < 0.01, *p < 0.05, and not significant (n.s.) (Unpaired t-test with Welch’s correction). Data are represented as mean ± SD. Scale bars indicate 200 μm. (C) Figure adapted from an image created with BioRender.com. See also Figures S1 and S3–5

Figure 5.. CGRPβ suppresses type 2 cytokine production from lung ILC2s and allergic lung inflammation

(A) Schematic of the lung ILC2 suppression assay. (B-C) Type 2 cytokine measurement from lung ILC2s stimulated with IL-33, SP, and/or CGRPβ. Shown are data for (B) IL-5 and (C) IL-13. (D) Schematic of the induction of allergic lung inflammation in Rag1^−/− mice. (E-F) Flow cytometry of lung tissue from Rag1^−/− mice challenged with intranasal ALT as well as vehicle control or CGRPβ or CGRPβ 8–37. Shown are the frequencies of (E) ILC2s and (F) eosinophils. Data were obtained from two independent experiments, n = 4–5 mice per group. (G-H) Representative lung histology images of Rag1^−/− mice challenged with intranasal ALT as well as vehicle control or CGRPβ or CGRPβ 8–37, obtained by (G) H&E stain and (H) PAS stain. ***p < 0.001, **p < 0.01, *p < 0.05 (Unpaired t-test with Welch’s correction). Data are represented as mean ± SD. Scale bars indicate 200 μm. (A, D) Figure adapted from an image created with BioRender.com. ELISA – enzyme-linked immunosorbent assay, IL – interleukin, SP – substance P See also Figures S1 and S6

Figure 6.. Expression of human JAK1^GOF in lung sensory neurons suppresses allergic inflammation

(A) Schematic of the allergic lung inflammation model generated by delivery of AAV-Cre (control) or AAV-Cre/JAK1 ^GOF (experimental) into the airways of Rosa26^{STOPflox-tdTomato} mice. (B) Imaging of tdTomato in the DRG and VG following AAV-assisted gene delivery into lung-innervating sensory neurons. Shown are representative images of DRG and VG from mice treated with AAV-Cre/JAK1^GOF. (C) Numbers of tdTomato⁺ neurons in DRG (thoracic segments 4–8) and VG, n = 5 mice per group. Data were analyzed in mice treated with AAV-Cre/JAK1 GOF. (D-E) Representative lung histology images of mice challenged with intranasal ALT following AAV-Cre control and AAV-Cre/JAK1^GOF infection, obtained by (D) H&E stain and (E) PAS stain. (F-H) Flow cytometry of lung tissue from mice challenged with intranasal ALT following AAV-Cre control and AAV-Cre/JAK1^GOF infection. Shown are the frequencies of (F) total CD45⁺ immune cells, (G) ILC2s, and (H) eosinophils. Data were obtained from two independent experiments, n = 4–5 mice per group. (I) Schematic of the allergic lung inflammation model generated by conditional insertion of JAK1^GOF variant into sensory neurons. (J-K) Representative lung histology images of mice challenged with intranasal ALT obtained by (J) H&E stain and (K) PAS stain. (L-N) Flow cytometry of lung tissue from mice challenged with intranasal ALT. Shown are the frequencies of (L) total CD45⁺ immune cells, (M) ILC2s, and (N) eosinophils. (O) Protein levels of CGRPβ in the bronchoalveolar lavage fluid (BALF). Data were obtained from two independent experiments, n = 4–5 pooled mice per group. *p < 0.05, **p < 0.01, and not significant (n.s.) (Unpaired t-test with Welch’s correction). Data are represented as mean ± SD. Scale bars indicate 200 μm. (A, I) Figure adapted from an image created with BioRender.com. AAV – adeno-associated virus; specifically, AAV2-retro, BALF – Bronchoalveolar lavage fluid See also Figure S1, Video S1, and S2