Fig 2: Inhibition of autophagy inhibits interleukin (IL)-6 and IL-10 release by macrophages in vitro. Mouse RAW 264.7 macrophages were treated with different concentrations of 3-methyladenine (3-MA) in combination with (a, c) 100 ng mL-1 lipopolysaccharide (LPS) or (b, d) 1 µg mL-1 R-848 for (a, b) 6 h or (c, d) 16 h and IL-6 release was measured by ELISA. (e, f) RAW 264.7 cells were transfected with nontargeting (scrambled) or small interfering RNA (siRNA) targeting Becn1 and treated with different concentrations of (e) LPS or (f) R-848 for 16 h and IL-6 release was measured by ELISA. (g) Western blot confirmation of Beclin-1 protein knockdown in RAW 264.7 cells. The image is representative of three independent experiments. The full Western blot is shown in Supplementary figure 7. (h, i) RAW 264.7 macrophages were treated with different concentrations of 3-MA in combination with (h) 100 ng mL-1 LPS or (i) 1 µg mL-1 R-848 for 16 h and IL-10 release was measured by ELISA. (j, k) RAW 264.7 cells were transfected with nontargeting (scrambled) or Becn1 siRNA and treated with different concentrations of (e) LPS or (f) R-848 and IL-10 release was measured by ELISA. (l) RAW 264/7 cells were treated with LPS (100 ng mL-1) with or without 3-MA (10 mM) for 4 h and Il10 messenger RNA (mRNA) expression was measured by quantitative PCR (qPCR). (m) RAW 264.7 cells were transfected with scrambled or Becn1 siRNA and treated with LPS or R-848 for 4 h. Expression of Il10 mRNA was measured by qPCR. N = 3 independent experiments. **P < 0.01, ***P < 0.005, ****P < 0.001. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig 3: Corticosterone induces M2 macrophage polarization in BMDM. (A–D) BMDM were derived by culture in RPMI 1640 medium supplemented with GM-CSF (50 ng/ml), before the addition of corticosterone (50 ng/ml) for 7 days. (A, B) The macrophage markers F4/80 and CD206 were identified by flow cytometry. Representative plots are shown (A); alongside average percentage of macrophages expressing CD206 (B). (C) The mRNA expression levels of Cd206, Il10, and Tnfa were quantified by qRT-PCR and normalized against Actin. (D, E) BMDM cultured with or without corticosterone (D), and primary TM and PM (E) were seeded on XF96- Seahorse culture plates (104 per well). The ECAR and OCR were tested in XF-96 assay medium (see Methods) and normalized against protein concentration. After establishing a baseline, glucose (Glu, 10 mM), oligomycin (OM, 0.25 µM), 2-deoxyglucose (2-DG, 100 mM), or oligomycin (OM, 0.25 µM), FCCP (1 µM) and rotenone/antimycin A (ROT/AA, 1 µM) were sequentially added. Continuous ECAR and OCR values (pmoles/min/µg protein) are shown. Each repetition involved 4–5 mice per group and three replicates were performed (mean ± SD, Welch’s t-test). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Corticosterone treatment protects testis from UPEC-induced orchitis. (A) WT C57BL/6J mice were injected with saline or uropathogenic Escherichia coli into the vas deferens. Mice were then injected daily with corticosterone (0.1mg/day) ± Compound C (C.C, 0.4 mg/kg). Seven days post-infection mice were sacrificed and macrophage populations were measured in cells from testes by flow cytometry. Data are representative of two independent experiments. (B) Mean percentages of F4/80hiCD11blo resident TM, F4/80loCD11bhi monocyte-derived macrophages (upper panel) and the F4/80hi CD206+ M2 TM (lower panel) in testes of the different treatment groups are presented. (C) The number of resident F4/80hi macrophages and CD11bhi monocyte-derived macrophages in testes of the different treatment groups is presented. (D) Percentage of CD206-expressing TM among F4/80hiCD11blo resident macrophages in the different groups is shown; (E) TM were isolated and the relative expression of Tnfa, Il10, and Acadm were detected by RT-PCR. Mean ± SD; n = 5 for each group). The unpaired one-way ANOVA was employed for statistical analysis. *** p < 0.001, **p < 0.01, * p < 0.01. (F) Day 7 testicular tissue sections from each treatment group were stained with hematoxylin and eosin (magnification = 20× objective).

Fig 5: ELISA estimations in mice plasma. Insulin (A), IL10 (B), IL6 (C), TNF-a (D) were estimated in Type 1 DM and Type 2 DM mice plasma using ELISA. All values are expressed in mean + SEM. N=6. a,b,c p < 0.05 compared to control, STZ and PDX respectively. STZ – Streptozotocin, PDX – 10(S),17(S)-DiHDoHE, T1DM – Type 1 diabetes mellitus, T2DM – Type 2 diabetes mellitus.