Volume 27, Issue 13, Pages 3799-3807.e3 (June 2019) Panicle-Shaped Sympathetic Architecture in the Spleen Parenchyma Modulates Antibacterial Innate Immunity  Xiaofan Ding, Huanhuan Wang, Xinmin Qian, Xiangli Han, Lu Yang, Ying Cao, Qi Wang, Jing Yang  Cell Reports  Volume 27, Issue 13, Pages 3799-3807.e3 (June 2019) DOI: 10.1016/j.celrep.2019.05.082 Copyright © 2019 The Author(s) Terms and Conditions

Cell Reports 2019 27, 3799-3807.e3DOI: (10.1016/j.celrep.2019.05.082) Copyright © 2019 The Author(s) Terms and Conditions

Figure 1 Panicle-Shaped Sympathetic Architecture in the Spleen Parenchyma Revealed by the ImmuView Procedure (A) The intact, unsectioned spleen of an adult mouse before (top) and after (bottom) the ImmuView procedure. (B and C) Panicle-shaped neural architecture in the spleen parenchyma. The spleens were processed for the whole-tissue immunolabeling of synaptophysin (B) or PGP9.5 (C). Representative 3D images at 1.26× magnification on the light sheet microscope (left) and representative optical sections of the light sheet scanning (right) are shown. (D) Neural innervations in the spleen are predominantly sympathetic. The spleen was processed for the whole-tissue co-immunolabeling of tyrosine hydroxylase (TH, green) and synaptophysin (red). Representative 3D images of 300-μm depth tissue of the intact spleen at 12.6× magnification on the light sheet microscope are shown. (E and F) Panicle-shaped sympathetic architecture in the spleen parenchyma. The spleens were processed for the whole-tissue immunolabeling of TH (E) or vesicular acetylcholine transporter (VChAT, F). Representative 3D images at 1.26× magnification on the light sheet microscope (left) and representative optical sections of the light sheet scanning (right) are shown. (G and H) 3D distribution of the sympathetic architecture in the spleen. The spleen was processed for the whole-tissue co-immunolabeling of TH (green) and CD169 (magenta). (G) Representative 3D images at 1.26× magnification on the light sheet microscope are shown. (H) Representative 3D images of 300-μm depth tissue of the intact spleen at 12.6× magnification on the light sheet microscope are shown. (I) Spatial engagement of the sympathetic innervations with the vasculatures in the spleen. The spleen was processed for the whole-tissue co-immunolabeling of TH (green) and PECAM1 (red). Representative 3D images of 300-μm depth tissue of the intact spleen at 12.6× magnification on the light sheet microscope are shown. See also Figure S1 and Videos S1 and S2. Cell Reports 2019 27, 3799-3807.e3DOI: (10.1016/j.celrep.2019.05.082) Copyright © 2019 The Author(s) Terms and Conditions

Figure 2 TrkA-Dependent Establishment of the Sympathetic Architecture in the Spleen (A–H) 3D distribution of the sympathetic and parasympathetic innervations in the lymph nodes, Peyer’s patch, and thymus. The intact, unsectioned subcutaneous lymph node (A, E), mesenteric lymph node (B, F), Peyer’s patch (C, G) and thymus (D, H) of adult mice were processed for the whole-tissue immunolabeling of TH (A–D) or VChAT (E–H). Representative 3D images at 2.5× magnification on the light sheet microscope (left) and representative optical sections of the light sheet scanning (right) are shown. (I) Sympathetic innervations in the spleen originate from the celiac ganglia. The spleens were locally labeled with the Alexa 594-conjugated cholera toxin subunit B (CTB), and the celiac ganglia were processed for conventional immunohistochemistry. (J and K) Normal development of the spleens of Th-Cre; TrkAfl/fl mice. (J) Gross appearance of the spleens of Th-Cre; TrkA+/+ versus Th-Cre; TrkAfl/fl mice. (K) Tissue weight of the spleens of Th-Cre; TrkA+/+ versus Th-Cre; TrkAfl/fl mice; n = 5, means ± SEMs, n.s., not significant (Student’s t test). (L–O) Ablation of the sympathetic architecture in the spleens of Th-Cre; TrkAfl/fl mice. The spleens of Th-Cre; TrkA+/+ and Th-Cre; TrkAfl/fl mice were processed for the whole-tissue immunolabeling of TH (L and M) or synaptophysin (N and O). (L and N) Representative 3D images at 1.26× magnification on the light sheet microscope are shown. (M and O) Sympathetic axons (M) or total axons (O) in the spleens of Th-Cre; TrkA+/+ versus Th-Cre; TrkAfl/fl mice were quantified; n = 5, means ± SEMs, ∗p < 0.01 (Student’s t test). See also Figure S2 and Video S3. Cell Reports 2019 27, 3799-3807.e3DOI: (10.1016/j.celrep.2019.05.082) Copyright © 2019 The Author(s) Terms and Conditions

Figure 3 Sympathetic Architecture Negatively Modulates the Spleen Antibacterial Innate Immunity (A and B) Genetic ablation of the sympathetic innervations did not affect the overall development of immune cells in the spleen. (A) The spleens of Th-Cre; TrkA+/+ and Th-Cre; TrkAfl/fl mice were processed for whole-tissue CD169 immunolabeling. Representative 3D images at 1.26× magnification on the light sheet microscope are shown. (B) Major populations of immune cells in the spleens of Th-Cre; TrkA+/+ versus Th-Cre; TrkAfl/fl mice were examined by FACS analysis; n = 5, means ± SEMs, n.s., not significant (ANOVA test). (C–E) Genetic ablation of the sympathetic innervations enhanced the LPS-elicited innate immunity in the spleen. Th-Cre; TrkA+/+ versus Th-Cre; TrkAfl/fl mice were administered with LPS. (C) Expression levels of cytokines and chemokines in the spleens were determined by the qPCR analysis; n = 8, means ± SEMs, ∗p < 0.01 (ANOVA test). (D and E) Recruitment of CD11b+ Ly-6G+ neutrophils was examined using FACS analysis (D) and quantified (E); n = 8, means ± SEMs, ∗p < 0.01 (ANOVA test). (F–H) Enhancement of the antibacterial innate immunity in Th-Cre; TrkAfl/fl mice. Th-Cre; TrkA+/+ versus Th-Cre; TrkAfl/fl mice were inoculated with Gram-negative bacteria (E. coli, 1.5 × 107 colony-forming unit [CFU] per mouse). (F and G) Bacterial clearance was analyzed by the colony-formation assay (F) and quantified (G; log10 scale); n = 5, means ± SEMs, ∗p < 0.01 (Student’s t test). (H) The survival rate of the mice was monitored after the infection; n = 24, ∗p < 0.01 (log-rank test). Cell Reports 2019 27, 3799-3807.e3DOI: (10.1016/j.celrep.2019.05.082) Copyright © 2019 The Author(s) Terms and Conditions

Figure 4 Cell-Intrinsic Suppression of Antibacterial Innate Immunity by Norepinephrine (A) Loss of the norepinephrine content in the spleens of Th-Cre; TrkAfl/fl mice; n = 5, means ± SEMs, ∗p < 0.01 (Student’s t test). (B and C) Norepinephrine suppressed the LPS-elicited innate immunity in the spleen. The mice were treated with LPS in the absence or presence of exogenously administered norepinephrine. (B) Expression levels of cytokines and chemokines in the spleens were determined by the qPCR analysis; n = 8, means ± SEMs, ∗p < 0.01 (ANOVA test). (C) Recruitment of CD11b+ Ly-6G+ neutrophils was examined using FACS analysis; n = 4, means ± SEMs, ∗p < 0.01 (ANOVA test). (D) Norepinephrine inhibited the antibacterial innate immunity. The mice were inoculated with Gram-negative bacteria (E. coli, 5 × 106 CFU/mouse) in the absence or presence of exogenously administered norepinephrine. Bacterial clearance was analyzed by the colony-formation assay (log10 scale); n = 5, means ± SEMs, ∗p < 0.01 (Student’s t test). (E) Cell-intrinsic suppression of the LPS-elicited immune response by norepinephrine via the β2-adrenergic receptor. The cultured Adrb2+/+ and Adrb2−/− bone marrow-derived macrophages (BMDMs) were treated with LPS in the absence or presence of norepinephrine. Expression levels of cytokines and chemokines were examined using qPCR analysis; n = 3, means ± SEMs, ∗p < 0.01 (ANOVA test). (F) Cell-intrinsic PKA signaling inhibited the LPS-elicited immune response. The cultured BMDMs were treated with LPS in the absence or presence of forskolin. Expression levels of cytokines and chemokines were examined using qPCR analysis; n = 3, means ± SEMs, ∗p < 0.01 (ANOVA test). See also Figure S3. Cell Reports 2019 27, 3799-3807.e3DOI: (10.1016/j.celrep.2019.05.082) Copyright © 2019 The Author(s) Terms and Conditions