Fig 1: IL-5 signal regulates eosinophils and intra-adipose axonal outgrowth. (A and B) The WT mice housed at the TN condition were subjected to cold challenge for 4 d. The mice received the intraperitoneal injection of anti–IL-5 antibody or control IgG antibody twice, 3 d prior to cold challenge and 1 d at cold treatment. The abundance of eosinophils was analyzed by flow cytometry post–antibody treatment (A). Cells were gated on CD45+Ter119- cells. The percent of eosinophils (CD11b+Siglec-F+) in CD45+Ter119- cells was quantified (B). n = 3 mice. (C–F) The iWAT from the antibody-treated mice were processed for the whole-mount immunostaining and 3D volume fluorescence imaging at 12.6× magnification on the lightsheet microscope. The 3D-projection images of TH (C) and STMN2 (E) stained with the respective antibodies were shown from a depth of 500 µm. The nerve fiber length was quantified (D and F). n = 2 mice for STMN2 anti-IL5 cold, n = 3 for the rest. (G) Expression levels of the beiging-related genes in iWAT were determined by the qPCR analysis. n = 3 for TN and n = 14 for the cold challenge. (H and I) Appearance of multilocular beige cells in iWAT was examined by HE staining, and the percentage of beige cells was quantified. n = 4 lobules for TN and n = 5 for cold. Data are presented as mean ± SEM. P values were calculated by two-tailed unpaired t test (B) and two-way ANOVA and Tukey's post hoc test (D, F, G, I). ns P > 0.05, *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001. Ab, antibody; APC, allophycocyanin.
Fig 2: Immunohistochemical staining of cytokines deposited in the dorsal skin tissue of experimental animals. (A) Expression of IL-4. (B) Expression of IL-5. (C) Expression of IFN-?. (D) Expression of IL-17. Scale bar indicates 100 µm (200X). The small black square indicates the location of antibody staining, which is subsequently magnified in the corners of the image. (i- Control, ii- Compound 1 alone, iii- Compound 2 alone, iv- DNCB, v- DNCB+1, vi- DNCB+2).
Fig 3: IL-33 maintains eosinophils via IL-5 production by ILC2s and regulates intra-adipose sympathetic axonal outgrowth. (A–C) The iWAT from “Red5” IL-5-tdTomato reporter mice were analyzed by flow cytometry. IL-5 reporter was mainly detected in ILC2s (A and B), which were predominantly ST2+ identified within lin-CD90.2+ population (C). n = 5 mice for each group. (D) ILC2s isolated from adipose tissues and expanded in culture were treated with IL-33 (10 ng/mL) for 6 h. Cell culture media were collected for the IL-5 ELISA analysis. n = 3 wells for each group. (E) The iWAT from mice housed at TN or cold condition for one day were analyzed by flow cytometry. The Lin-CD90.2+ST2+ ILC2s showed increased proportion in CD45+ population. n = 5 mice for each group. (F–L) The WT and Il33-/- mice housed at thermoneutral conditions were subjected to cold challenge. (F and G) The iWAT from the indicated genotypes at 4 d post–cold stimulation were processed for the whole-mount immunostaining and volume fluorescence imaging. The 3D-projection images of Siglec-F (F) were shown with a depth of 500 µm. The eosinophil (eos) number was counted and presented (G). n = 6 for WT group and n = 5 for Il33-/- group. (H) The iWAT from the indicated genotypes post–cold stimulation were homogenized, and the levels of NGF protein were determined by ELISA. n = 7 for each group. (I–L) The iWAT from the indicated genotypes post–cold stimulation were processed for the whole-mount immunostaining and volume fluorescence imaging. The 3D-projection images of TH and STMN2 stained with the respective antibodies are shown (I and K). The nerve fiber length is quantified and shown for TH and STMN2 (J and L). n = 3 for TN and n = 4 to 5 for cold. Data are presented as mean ± SEM. P values were calculated by two-tailed unpaired t test (B, C, H), two-tailed unpaired Welch’s t test (D, E, G), and two-way ANOVA and Tukey's post hoc test (J, L). ns P > 0.05, *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001. APC, allophycocyanin.
Fig 4: CD146 deficiency decreased Cryptococcus neoformans infection. (A) CFU analysis of Cryptococcus neoformans in the lungs. WT, wild type, n = 6; CD146 KO: CD146 Knock out, n = 6; PM2.5 group: mice were pretreated with PM2.5 (10 mg/kg) 24 h before Cryptococcus neoformans infection. Control group: mice were pretreated with PBS 24 h before Cryptococcus neoformans infection. All mice were sacrificed 4 h post fungal infection. (B) Representative images of lung sections stained with PAS; arrow indicates the fungi. (C) ELISA analysis of TNF-a in BALF; n = 6. (D) ELISA analysis of IL-1ß in BALF; n = 6. (E) ELISA analysis of IL-10 in BALF; n = 6. (F) ELISA analysis of IL-4, IL-5, and IL-13 in the lungs; n = 6. Student’s t-test was performed after the normal distribution analysis. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 5: PM2.5 promoted Cryptococcus neoformans pulmonary infection. (A) Flow chart of the Cryptococcus neoformans infection model. (B) CFU analysis of Cryptococcus neoformans in the lungs. Naïve: mice treated without PM25 and Cryptococcus neoformans, n = 7. Control: mice treated with PBS and Cryptococcus neoformans, n = 7; PM2.5: mice treated with 10 mg/kg PM2.5 and Cryptococcus neoformans; n = 7. (C) Representative images of lung sections stained with PAS, arrow indicated the fungi. (D) ELISA analysis of TNF-a in bronchoalveolar lavage fluid (BALF); n = 7. (E) ELISA analysis of IL-1ß in BALF; n = 7. (F) ELISA analysis of IL-10 in BALF; n = 7. (G) ELISA analysis of IL-4, IL-5, and IL-13 in the lungs; n = 7. (H) Western blot analysis of CD146 in the lungs. Student’s t-test was performed after the normal distribution analysis. *P < 0.05; **P < 0.01; ***P < 0.001.
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