Fig 1: 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 2: Biological activity of 9-PAHSA enantiomers R- and S-9-PAHSA. A: Insulin secretion from clonal pancreatic MIN6 β-cells: MIN6 cells were incubated with DMSO (control) or racemic, R-9-PAHSA or S-9-PAHSA (20 μM) for 1 h and stimulated with low (2.5 mM) or high (20 mM) glucose for 45 min. n = 8 wells/condition. Each isomer was tested in two independent studies. Data are the means ± SEM. ∗P < 0.05 versus high glucose DMSO by one-way ANOVA. B: GSIS from isolated human islets from donor 3. n = 50 islets/well and three wells per each condition. ∗P < 0.05 versus high glucose DMSO, #P < 0.05 versus high glucose R-9-PAHSA by one-way ANOVA. C: Calcium flux assay in mGPR40 stably transfected cells treated with the control buffer, 9-PAHSA, R-9-PAHSA, and S-9-PAHSA. n = 4/condition. Data are the means ± SEM. ∗P < 0.05 versus buffer by one-way ANOVA. D: Basal and insulin-stimulated glucose transport in 3T3L1 adipocytes treated with racemic 9-PAHSA, R-9-PAHSA, S-9-PAHSA (20 μM), or DMSO for 24 h. n = 4–6 wells/condition. ∗P < 0.05 versus DMSO. E, Effects of racemic (Rac) and R- and S-9-PAHSA on LPS-induced Tnf or Il-6 secretion from BMDCs and BMDMs. n = 4–6 wells/condition. Each isomer was tested in two independent studies. Data are the means ± SEM. ∗P < 0.05 versus LPS-induced DMSO by one-way ANOVA. BMDC, bone marrow–derived dendritic cell; BMDM, bone marrow–derived macrophage; GSIS, glucose-stimulated insulin secretion; LPS, lipopolysaccharide; PAHSA, palmitic acid hydroxy stearic acid.
Fig 3: Inhibition of inflammatory cytokine production in M2 phenotype BMDM and TM by Compound C. Enriched populations of TM (C, D) and corticosterone-induced BMDM (A, B) were pre-treated with Compound C (C. C, 0.5 μM, 30 min), then stimulated with LPS (1 ug/ml, 1 and 3 h, respectively). (A, C) The effects of AMPK inactivation on expression of Il10 and Tnfa were examined by qRT-PCR. (B, D) After pretreatment with Compound C and LPS (3 h) cell supernatants were collected and the protein levels of TNF-α and IL-10 were measured by ELISA. Results are representative of three to four independent experiments. *p < 0.05; **p < 0.01; *p <0.001 as indicated. One-way ANOVA.
Fig 4: Complex I is specifically required for IFN? signaling in human cells.(A) CD14+ monocytes from healthy human donors were differentiated into macrophages with GM-CSF. Mean fluorescence intensity (MFI) of cell surface markers PD-L1, ICAM1, CD40, and HLA-DR was determined by flow cytometry following stimulation with IFN? and/or inhibition of complex I with rotenone (10 µM) for 24 hr. Data are representative of two independent experiments, and values are normalized to donor-specific unstimulated/vehicle control. Mean ± standard deviation for six biological replicates of each condition. Differences were tested by two-way ANOVA using the Sidak–Holm method to correct for multiple hypothesis testing. (B, C) Quantification of IL-1ß and TNF production from primary human macrophages, measured by ELISA from cell supernatants following stimulation. Lines connect values for individual donors treated with vehicle (DMSO, black squares) or rotenone (empty squares). Differences were tested by repeated measures two-way ANOVA using the Sidak–Holm method to correct for multiple hypothesis testing. p-Values of 0.05, 0.01, 0.001, and 0.001 are indicated by *, **, ***, and ****, respectively.
Fig 5: 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.
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