Fig. 3: CRH^PVN neurons influence neutrophil infiltration and responses during ALI.

a Experimental scheme for chemogenetic activation of CRH^PVN neurons in CRH-IRES-Cre mice subject to ALI. Flow cytometry analysis of CD45⁺ leukocytes and Ly6G⁺ neutrophils in BALF of PBS-treated, LPS-treated control, and LPS-treated hM3Dq mice receiving CNO after 8 h (b) and 48 h (c) treatment (n = 8 mice per group). d Representative immunohistochemical staining images of Ly6G (upper images) and MPO (lower images) in lung tissues of PBS-treated, LPS-treated control, and LPS-treated hM3Dq mice at 48 h post-LPS challenge. Scale bar: 50 μm. e Quantification of Ly6G⁺ and MPO⁺ cells in ×400 fields (n = 4 mice per group). f Volcano plot of RNA sequencing of lung tissues showing DEGs in control mice and hM3Dq mice subjected to ALI. g Heatmap of neutrophil-associated lung DEGs between control mice and hM3Dq mice treated with LPS. h Experimental scheme for chemogenetic inhibition of CRH^PVN neurons in CRH-IRES-Cre mice subjected to ALI. Numbers of CD45⁺ leukocytes (i) and Ly6G⁺ neutrophils (j) in BALF of PBS-treated mice, LPS-treated control mice, and LPS-treated hM3Dq mice analyzed by flow cytometry (n = 6 mice per group). Immunohistochemical staining (k) and quantification (l) (in a 400× field) of Ly6G⁺ and MPO⁺ cells in the lung of PBS-treated, LPS-treated control, and LPS-treated hM3Dq mice (n = 5 mice per group). Scale bar: 50 μm. Statistical differences were determined by 1-way ANOVA with Tukey’s post-hoc multiple comparisons test. Each dot represents an individual mouse. Unless specified otherwise, the data are presented as mean ± SEM. a, h were created in BioRender. Liu, T. (2025) https://BioRender.com/p48gmjd. All experiments were repeated three times, yielding similar results. Source data are provided as a Source data file. ALI acute lung injury, BALF bronchoalveolar lavage fluid, CRH corticotropin-releasing hormone, MPO myeloperoxidase, PVN paraventricular nucleus, DEGs differentially expressed genes.