Fig. 4. Local sympathetic innervations negatively modulate the IL-33–elicited type 2 innate immunity in the lung.

(A) Spatial engagement of local sympathetic innervations with the IL-33–elicited immune response in the lung. The wild-type mice were intranasally treated with IL-33. The lung (left lobe) was then processed for the whole-tissue coimmunolabeling of anti-TH (green) and anti–Siglec-F (magenta). Representative 3D projection images of the 600-μm depth of the intact tissue at ×12.6 magnification of the lightsheet imaging are shown. (B to F) Genetic ablation of local sympathetic innervations in the lung boosted the IL-33–elicited type 2 innate immunity. Th-Cre; TrkA^+/+ and Th-Cre; TrkA^fl/fl mice were intranasally treated with saline control or IL-33. The lungs were harvested at 24 hours after the second instillation. (B and C) The lungs (left lobe) were processed for the whole-tissue anti–Siglec-F immunolabeling. (B) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (C) The density of Siglec-F⁺ immune cells was quantified. n = 4, means ± SEM, *P < 0.01 (ANOVA test). (D) CD45⁺ CD11c⁻ Siglec-F⁺ eosinophils were quantified by the FACS analysis. n = 5, means ± SEM, *P < 0.01 (ANOVA test). (E and F) The lung tissues were assessed by H&E staining. (E) Representative images are shown. (F) The histologic scores were determined. n = 4, means ± SEM, *P < 0.01 (ANOVA test). (G) Local sympathetic innervations negatively regulate the IL-33–elicited early immune response. The lungs of Th-Cre; TrkA^+/+ and Th-Cre; TrkA^fl/fl mice were harvested at 2 hours after the first instillation of IL-33. Expression levels of cytokines were determined by the qPCR analysis. n = 5, means ± SEM, *P < 0.01 (ANOVA test). (H to J) Pharmacologic ablation of local sympathetic innervations in the lung enhanced the IL-33–elicited immune response. Saline-treated and 6-OHDA–treated mice were intranasally administered with IL-33. The lungs were harvested at 24 hours after the second instillation. (H and I) The lungs (left lobe) were processed for the whole-tissue anti–Siglec-F immunolabeling. (H) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (I) The density of Siglec-F⁺ immune cells was quantified. n = 4, means ± SEM, *P < 0.01 (ANOVA test). (J) The lung tissues were assessed by H&E staining, and representative images are shown. (K and L) Surgical ablation of local sympathetic innervations in the lung promoted the IL-33–elicited immune response. The wild-type mice after mock surgery or local sympathectomy were intranasally administered with IL-33. The lungs (left lobe) were harvested at 24 hours after the second instillation and processed for the whole-tissue anti–Siglec-F immunolabeling. (K) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (L) The density of Siglec-F⁺ immune cells was quantified. n = 3, means ± SEM, *P < 0.01 (ANOVA test).