Fig 2: TSLP alleviated lung inflammation in LPS-induced acute lung injury. (A) The flow chart showing the process of the experimental animal studies. (B, C) Representative sections and lung injury score evaluation of H&E staining. Scale bar = 100 μm. n = 4 per group, Mann Whitney test for statistics analysis between Veh group and LPS group, Kruskal-Wallis test for statistics analysis between LPS group, LPS+TSLP 2.5 group, LPS+TSLP 10 group. (D) Summary bar graphs with scatter plots showing the concentration of IL-6, TNF-α, and IL-1β in the BALF of groups treated with Vehicle, LPS, LPS+ 2.5 μg/kg TSLP (LPS+TSLP 2.5), and LPS+ 10 μg/kg TSLP (LPS+TSLP 10). n = 4 per group, for IL-6, unpaired Student’s t test for statistics analysis between Veh group and LPS group, Kruskal-Wallis test for statistics analysis between LPS group, LPS+TSLP 2.5 group, LPS+TSLP 10 group. For TNF-α and IL-1β, unpaired Student’s t test for statistics analysis between Veh group and LPS group, one-way ANOVA test for statistics analysis between LPS group, LPS+TSLP 2.5 group, LPS+TSLP 10 group. (E) The volcano plot showing differentially expressed genes of the lung tissues of LPS group versus Veh group. (F) The volcano plot showing differentially expressed genes of the lung tissues of LPS+TSLP group versus LPS group. (G) The bar chart of indicated enriched GO pathways in the lung tissues of LPS group versus Veh group (H) The bar chart of indicated enriched GO pathways in the lung tissues of LPS+TSLP group versus LPS group. *P < 0.05, **P < 0.01, ****P < 0.0001, ns, no significance. TSLP, Thymic Stromal Lymphopoietin, LPS, Lipopolysaccharide, Veh, Vehicle, BALF, Broncho-alveolar lavage fluid, WT, wild type, NES, normalized enrichment score. Data are represented as mean ± SEM.

Fig 3: Exogenous TSLP suppresses IFN-γ-producing iNKT cells in LPS-induced acute lung injury. (A) Representative plots and summary bar graphs with scatter plots depicting ratio and counts of iNKT cells in lung tissues of indicated groups. n = 3 (Veh), n=5 (LPS), n=4 (LPS+TSLP), unpaired Student’s t test for statistics analysis between Veh group and LPS group or LPS group and LPS+TSLP group. (B, C) Representative plots and summary bar graphs with scatter plots depicting ratios of IFN-γ-producing iNKT cells, CD8 T cells, and CD4 T cells in indicated groups. n = 3 (Veh), n=5 (LPS), n=4 (LPS+TSLP), for ratios of IFN-γ-producing iNKT cells, Mann Whitney test for statistics analysis between Veh group and LPS group, unpaired Student’s t test for statistics analysis between LPS group and LPS+TSLP group. For ratios of IFN-γ-producing CD8 T cells and CD4 T cells, unpaired Student’s t test for statistics analysis between Veh group and LPS group or LPS group and LPS+TSLP group. *P < 0.05, **P < 0.01, ns, no significance. TSLP, Thymic Stromal Lymphopoietin, LPS, Lipopolysaccharide, Veh, Vehicle. Data are represented as mean ± SEM.

Fig 4: TSLP reduces M1 macrophages polarization in vivo and in vitro. (A, B) Representative histogram and summary bar graph with scatter plots showing INOS+ or ARG1+ pulmonary macrophages in indicated groups. n = 4 per group, for ratio of INOS, Mann Whitney test for statistics analysis between Veh group and LPS group, unpaired Student’s t test for statistics analysis between LPS group and LPS+TSLP group. For ratio of ARG1, MFI of INOS and ARG1, unpaired Student’s t test for statistics analysis between Veh group and LPS group or LPS group and LPS+TSLP group. (C) Summary bar graphs with scatter plots showing the concentration of TNF-α, IL-6, and IL-1β in the supernatant of BMDM in indicated groups. n = 5 per group, unpaired Student’s t test for statistics analysis between Veh group and LPS group or LPS group and LPS+TSLP group. (D, E) Representative histogram and summary bar graph with scatter plots showing INOS and CD206 expression in BMDM in indicated groups. n = 6 per group, unpaired Student’s t test for statistics analysis between Veh group and LPS group or LPS group and LPS+TSLP group. (F) Summary bar graph with scatter plots shows Nos2, Arg1 and Chil3 mRNA foldchange in BMDM in indicated groups. n = 3 per group, unpaired Student’s t test for statistics analysis between Veh group and LPS group or LPS group and LPS+TSLP group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significance, TSLP, Thymic Stromal Lymphopoietin, LPS, Lipopolysaccharide, Veh, Vehicle, BMDM, bone marrow-derived macrophages, mac, macrophages. Data are represented as mean ± SEM.

Fig 5: Immune cell overview during LPS exposure with or without TSLP pretreatment. (A) Representative histogram and summary bar graphs with scatter plots showing neutrophil ratio and count in BALF of indicated groups. n = 4 per group, for neutrophil ratio, unpaired Student’s t test for statistics analysis between Veh group and LPS group, Kruskal-Wallis test for statistics analysis between LPS group, LPS+TSLP 2.5 group, LPS+TSLP 10 group. For neutrophil count, unpaired Student’s t test for statistics analysis between Veh group and LPS group, one-way ANOVA test for statistics analysis between LPS group, LPS+TSLP 2.5 group, LPS+TSLP 10 group. (B) Representative histogram and summary bar graphs with scatter plots showing ratio and count of macrophages in BALF of indicated groups. n = 4 per group, unpaired Student’s t test for statistics analysis between Veh group and LPS group, one-way ANOVA test for statistics analysis between LPS group, LPS+TSLP 2.5 group, LPS+TSLP 10 group. (C) Representative histogram and summary bar graphs with scatter plots showing ratio and counts of CD4 T cells and CD8 T cells in lung tissues of indicated groups. n = 4 per group, for ratio and counts of CD8 T cells and ratio of CD4 T cells, unpaired Student’s t test for statistics analysis between Veh group and LPS group, one-way ANOVA test for statistics analysis between LPS group, LPS+TSLP 2.5 group, LPS+TSLP 10 group. For counts of CD4 T cells, unpaired Student’s t test for statistics analysis between Veh group and LPS group, Kruskal-Wallis test for statistics analysis between LPS group, LPS+TSLP 2.5 group, LPS+TSLP 10 group. *P < 0.05, **P < 0.01, ns, no significance. TSLP, Thymic Stromal Lymphopoietin, LPS, Lipopolysaccharide, Veh, Vehicle, BALF, Broncho-alveolar lavage fluid. conT, conventional T cells, T#, T cell number. Data are represented as mean ± SEM.