Fig 2: Aging exacerbates A59 (mCoV) infection.(A) Schematic of A59 (mCoV) infection experiment with young (2–6 months) and old mice (20–24 months). (B) Probability of survival of young (n = 6) and old (n = 6) infected mice. Survival of mice was examined after infection with high dose of virus (PFU 7e3) up to 14 days. (C–E) Young (n = 8) and old mice (n = 8) were infected with low dose of virus (PFU 7e2), and weight change (%) (C), O2 saturation (D), and daily food intake (E) were recorded. (F) Plaque assay of lung from uninfected, infected young and old mice. (G–L) Flow cytometry analysis of CD4 T cell (G), CD4/CD8 T cell ratio (H), γδ T cell (I), neutrophil (J), Ly6Chi cell (K), and CD64+ MerTK+ cell (L) on day 8 post (PFU 7e2) infection. Error bars represent the mean ± S.E.M. Two-tailed unpaired t-tests were performed for statistical analysis. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig 3: Adipose eosinophil and macrophage populations are influenced by sex, diet, and RELMα.(A) t-Stochastic neighbor embedding (tSNE) analysis to identify stromal vascular fraction (SVF) leukocyte populations. (B) tSNE analyses of SVF from the different groups (male ♂, female ♀, wild-type [WT], or RELMα knockout [KO]) after 12 weeks of diet exposure (Ctr or high-fat diet [HFD]) revealed changes in eosinophil RELMα-dependent and diet-induced changes in eosinophil (red outline) and macrophage (green outline) subsets. (C, D) CD11c surface expression in CD45+MerTK+CD64+ macrophages was analyzed by tSNE, where dashed red outline shows CD11cHi cells, and quantified. (E) CD301b surface expression on SVF macrophages was examined by tSNE and quantified by mean fluorescent intensity (MFI). (F) Siglec-F surface expression on CD45+SiglecF+CD11b+ SVF eosinophils was examined by tSNE and quantified by mean fluorescent intensity (MFI). tSNE data are one representative animal per group. All other data are presented as individual points for each animal, where lines represent group means ± standard error of the mean (SEM). Statistical significance was determined by three-way analysis of variance (ANOVA) Sidak’s with multiple comparisons test (ns, no significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Data are representative of 3 experiments with 4–6 mice per group.

Fig 4: RELMα protects females from diet-induced obesity.(A) RELMα levels in serum and visceral adipose tissue from 18-week-old male (♂) and female (♀) C57BL/6 mice after exposure to control (Ctr) or high-fat diet (HFD) for 12 weeks. (B) Wild-type (WT) or RELMα knockout (KO) mice were weighed for 12–15 weeks of diet exposure. After 6- and 12-week diet exposure (C), whole body, visceral and subcutaneous fat pad weights were recorded (D). (E) CCL2, Granulocyte-macrophage colony-stimulating factor (GM-CSF), IL10, and IL5 levels in protein extracts from visceral fat pad after 12-week diet exposure. (F) Gating strategy for flow cytometric analysis of the visceral adipose stromal vascular fraction (SVF). Proportion in the SVF of CD45+ leukocytes (G), CD45+CD64+Mertk+ macrophages (H), and CD45+SiglecF+CD11b+ eosinophils (I). Males (blue), females (red), WT (open symbols), RELMα KO (filled symbols), control diet (Ctr, circles), HFD (squares); data for (B) are presented as mean ± standard error of the mean (SEM), data for (H) are representative of one animal, all other data are presented as individual points for each animal, where lines represent group means ± SEM. Statistical significance between HFD WT females and HFD RELMα KO females was determined by two- or three-way analysis of variance (ANOVA) with Sidak’s multiple comparisons tests (ND, not detected; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 are indicated for functionally relevant comparisons). Data are representative of 3 experiments with 4–6 mice per group.