Research ArticleIMMUNOLOGY

Local sympathetic innervations modulate the lung innate immune responses

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Science Advances  13 May 2020:
Vol. 6, no. 20, eaay1497
DOI: 10.1126/sciadv.aay1497
  • Fig. 1 Whole-tissue 3D assessment of local neural innervations in the lung tissues.

    (A) iDISCO(ace) procedure. The intact, unsectioned lung (left lobe) of the adult mouse before (top) and after (bottom) the procedure. Photo credit: Tingting Liu, Peking University. (B to I) Whole-tissue 3D assessment of local neural innervations in the mouse lung. The lungs (left lobe) of wild-type mice were processed for the whole-tissue immunolabeling of anti-synaptophysin (B and C), anti-Tuj1 (D and E), anti-TH (F and G), or anti-VChAT (H and I). (B, D, F, and H) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (C, E, G, and I) Representative optical sections (top) or 3D projection images of the 600-μm depth of the intact tissues (bottom) at ×12.6 magnification of the lightsheet imaging are shown. Arrowheads denote the clusters of neuroendocrine cells. (J to L) Whole-tissue 3D assessment of local neural innervations in the monkey lung. The lung tissues of adult macaque monkeys were processed for the whole-tissue immunolabeling of anti-PGP9.5 (J and K) or anti-TH (L). (J) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (K and L) Representative 3D projection images of the 500-μm depth of the tissues at ×12.6 magnification of the lightsheet imaging are shown. Arrowhead denotes the cluster of neuroendocrine cells.

  • Fig. 2 Genetic ablation of local sympathetic innervations promoted the LPS-elicited innate immune response in the lung.

    (A and B) Normal development of the lungs of Th-Cre; TrkAfl/fl mice. (A) Gross appearance of the lungs (left lobe) of Th-Cre; TrkA+/+ versus Th-Cre; TrkAfl/fl adult mice. Photo credit: Tingting Liu, Peking University. (B) The tissue weight of the lungs was quantified. n = 5, means ± SEM, n.s., not significant (Student’s t test). (C to H) Genetic ablation of local sympathetic innervations in the lungs of Th-Cre; TrkAfl/fl mice. The lungs (left lobe) of Th-Cre; TrkA+/+ and Th-Cre; TrkAfl/fl mice were processed for the whole-tissue immunolabeling of anti-TH (C and D), anti-VChAT (E and F), or anti-synaptophysin (G and H). (C, E, and G) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (D) TH-positive sympathetic axons were quantified. n = 5, means ± SEM, *P < 0.01 (Student’s t test). (F) VChAT-positive parasympathetic axons were quantified. n = 3, means ± SEM, n.s., not significant (Student’s t test). (H) Synaptophysin-positive total axons were quantified. n = 4, means ± SEM, *P < 0.01 (Student’s t test). (I) Spatial engagement of local sympathetic innervations with the LPS-elicited immune response in the lung. The wild-type mice were intranasally treated with LPS. The lung (left lobe) was then processed for the whole-tissue coimmunolabeling of anti-TH (green) and anti–Ly-6G (magenta). Representative 3D projection images of the 600-μm depth of the intact tissue at ×12.6 magnification of the lightsheet imaging are shown. (J to Q) Genetic ablation of local sympathetic innervations in the lung promoted the LPS-elicited immune response. Th-Cre; TrkA+/+ and Th-Cre; TrkAfl/fl mice were intranasally treated with saline control or LPS. (J and K) The lungs (left lobe) were processed for the whole-tissue anti–Ly-6G immunolabeling. (J) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (K) The density of Ly-6G+ neutrophils was quantified. n = 4, means ± SEM, *P < 0.01 (ANOVA test). (L and M) CD45+ CD11b+ Ly-6G+ neutrophils in the lungs were examined by the FACS analysis (L) and quantified (M). n = 5, means ± SEM, *P < 0.01 (ANOVA test). (N) CD45+ CD11b+ Ly-6G+ neutrophils in the bronchoalveolar lavage fluid (BALF) were quantified by the FACS analysis. n = 4, means ± SEM, *P < 0.01 (ANOVA test). (O and P) The lung tissues were assessed by H&E (hematoxylin and eosin) staining. (O) Representative images are shown. (P) The histologic scores were determined. n = 4, means ± SEM, *P < 0.01 (ANOVA test). (Q) The survival rate of the mice was followed for 6 days after the LPS treatment. n = 7, *P < 0.05 (log-rank test).

  • Fig. 3 Pharmacologic or surgical ablation of local sympathetic innervations enhanced the LPS-elicited immune response in the lung.

    (A to F) Pharmacologic ablation of local sympathetic innervations in the lung. The wild-type mice were intranasally treated with saline control or 6-OHDA. (A) Gross appearance of the lungs (left lobe) of saline-treated versus 6-OHDA–treated mice. Photo credit: Tingting Liu, Peking University. (B) The tissue weight of the lungs was quantified. n = 4, means ± SEM, n.s., not significant (Student’s t test). (C to F) The lungs (left lobe) of saline-treated and 6-OHDA–treated mice were processed for the whole-tissue immunolabeling of anti-TH (C and D) or anti-synaptophysin (E and F). (C and E) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (D) TH-positive sympathetic axons were quantified. n = 5, means ± SEM, *P < 0.01 (Student’s t test). (F) Synaptophysin-positive total axons were quantified. n = 4, means ± SEM, *P < 0.01 (Student’s t test). (G to K) Pharmacologic ablation of local sympathetic innervations in the lung promoted the LPS-elicited immune response. Saline-treated and 6-OHDA–treated mice were intranasally administered with LPS. (G and H) The lungs (left lobe) were processed for the whole-tissue anti–Ly-6G immunolabeling. (G) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (H) The density of Ly-6G+ neutrophils was quantified. n = 4, means ± SEM, *P < 0.01 (ANOVA test). (I and J) The lung tissues were assessed by H&E staining. (I) Representative images are shown. (J) The histologic scores were determined. n = 4, means ± SEM, *P < 0.01 (ANOVA test). (K) The survival rate of the mice was followed for 6 days after the LPS treatment. n = 7, *P < 0.05 (log-rank test). (L and N) Surgical ablation of local sympathetic innervations in the lung. The wild-type mice were subjected to the local sympathectomy of the lung. The lungs (left lobe) of the mice after mock surgery or sympathectomy were processed for the whole-tissue anti-TH immunolabeling. (L) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (N) TH-positive sympathetic axons were quantified. n = 4, means ± SEM, *P < 0.01 (Student’s t test). (M and O) Surgical ablation of local sympathetic innervations in the lung enhanced the LPS-elicited immune response. The mice after mock surgery or sympathectomy were intranasally administered with LPS, and the lungs (left lobe) were then processed for the whole-tissue anti–Ly-6G immunolabeling. (M) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (O) The density of Ly-6G+ neutrophils was quantified. n = 4, means ± SEM, *P < 0.01 (ANOVA test).

  • 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; TrkAfl/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; TrkAfl/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).

  • Fig. 5 Sympathetic neurotransmitter NE cell-intrinsically inhibits the innate immune responses.

    (A) Decrease in the NE content in the lungs of Th-Cre; TrkAfl/fl mice. n = 4, means ± SEM, *P < 0.01 (Student’s t test). (B) Decrease in the NE content in the lungs of 6-OHDA–treated mice. n = 3, means ± SEM, *P < 0.01 (Student’s t test). (C and D) NE cell-intrinsically suppressed the LPS- or IL-33–elicited innate immune response. Primary alveolar macrophages (C) or ILC2s (D) isolated from the lungs of wild-type mice were in vitro treated with LPS (C) or IL-33 (D) in the presence of 10 μM NE. Expression levels of cytokines and chemokines were examined by the qPCR analysis. n = 3, means ± SEM, *P < 0.01 (ANOVA test). (E and F) The expression profile of adrenergic receptors in primary alveolar macrophages (E) or ILC2s (F) was determined by the qPCR analysis. n = 3, means ± SEM. (G and H) β2-Adrenergic receptor signaling cell-intrinsically inhibited the LPS- or IL-33–elicited innate immune response. Primary alveolar macrophages (G) or ILC2s (H) were in vitro treated with LPS or IL-33 in the presence of 10 μM formoterol or clenbuterol. Expression levels of cytokines and chemokines were examined by the qPCR analysis. n = 3, means ± SEM, *P < 0.01 (ANOVA test).

  • Fig. 6 β2-Adrenergic receptor signaling suppresses the innate immune responses in the lung.

    (A and B) Normal development of local sympathetic innervations in the lungs of Adrb2−/− mice. The lungs (left lobe) of Adrb2+/+ and Adrb2−/− mice were processed for the whole-tissue anti-TH immunolabeling. (A) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (B) TH-positive sympathetic axons were quantified. n = 3, means ± SEM, *P < 0.01 (Student’s t test). (C to H) Genetic deletion of the β2-adrenergic receptor boosted the LPS-elicited immune response. Adrb2+/+ and Adrb2−/− mice were intranasally treated with saline control or LPS. (C and D) The lungs (left lobe) were processed for the whole-tissue anti–Ly-6G immunolabeling. (C) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (D) The density of Ly-6G+ neutrophils was quantified. n = 3, means ± SEM, *P < 0.01 (ANOVA test). (E) CD45+ CD11b+ Ly-6G+ neutrophils in the BALF were quantified by the FACS analysis. n = 4, means ± SEM, *P < 0.01 (ANOVA test). (F and G) The lung tissues were assessed by H&E staining. (F) Representative images are shown. (G) The histologic scores were determined. n = 4, means ± SEM, *P < 0.01 (ANOVA test). (H) The survival rate of the mice was followed for 6 days after the LPS treatment. n = 7, *P < 0.05 (log-rank test). (I to L) Genetic deletion of the β2-adrenergic receptor enhanced the IL-33–elicited immune response in the lung. Adrb2+/+ and Adrb2−/− mice were intranasally treated with saline control or IL-33. (I and J) The lungs (left lobe) were processed for the whole-tissue anti–Siglec-F immunolabeling. (I) Representative 3D projection images at ×1.26 magnification of the lightsheet imaging are shown. (J) The density of Siglec-F+ immune cells was quantified. n = 3, means ± SEM, *P < 0.01 (ANOVA test). (K and L) The lung tissues were assessed by H&E staining. (L) Representative images are shown. (K) The histologic scores were determined. n = 4, means ± SEM, *P < 0.01 (ANOVA test).

Supplementary Materials

  • Supplementary Materials

    Local sympathetic innervations modulate the lung innate immune responses

    Tingting Liu, Lu Yang, Xiangli Han, Xiaofan Ding, Jiali Li, Jing Yang

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