Female adipose tissue-specific (aP2) - mice were generated by crossing B6.Cg-Tg(Fabp4-cre)1Rev/J mice with B6.129X1-Esr1tm1Gust (ERαfl/fl) mice, kindly provided by J.-A. Gustafsson (University of Houston, TX, USA). ERαfl/fl/aP2-Cre−/+ (atERαKO) and control littermates ERαfl/fl/aP2-Cre−/− (wt) were used for all experiments. Animals were maintained in a temperature-controlled facility with a 12 h dark/light cycle. At the age of 6 weeks, a group of atERαKO and wt-mice (n = 24), were challenged for 14 weeks with a HFD (60% kcal from fat, Brogaarden, Lynge, Denmark). The HFD groups and the CD were both fed ad-libitum. Metabolic phenotyping was performed with a metabolic cage system (TSE- Systems GmbH, Bad Homburg, Germany), based on indirect calorimetry as described before12. Body composition was assessed by nuclear magnetic resonance imaging (Echo MRI mouse, Echo Medical Systems, Houston, USA). Body temperature was measured rectally 3 to 5 days in row and mean values were calculated. Wt-mice were sacrificed during estrous phase of the cycle. atERαKO mice did not present a physiological estrous cycle, and were sacrificed independent of (non-) cycle phases; their vaginal smear was collected and analyzed to exclude a proestrous analogous stage. Organs were collected and frozen in liquid nitrogen. All animal experiments were approved by the Landesamt für Gesundheit und Soziales (LaGeSo, Berlin, Germany) and were conducted in alliance with the German Law on the Protection of Animals.
THP-1 cells (American Type Culture Collection (ATCC)) were cultured in RPMI 1640 (Life Technologies) with 1% Pen-Strep (Invitrogen) and 10% charcoal-stripped fetal bovine serum (cs-FBS; Sigma Aldrich). THP-1 cells were differentiated for 48 hours with 10 ng/ml phorbol myristate acetate (Sigma Aldrich). Prior to stimulation, cells were kept in phenol red-free medium for 48 h and starved overnight (12 h) with phenol red-free RPMI medium reducing the content of cs-FBS to 2.5%. Next, cells were stimulated for 24 h with 10 nM E2 (Sigma), 50 μM solution of stearic acid (C18:0) in 10% BSA (Sigma Aldrich), or with 10% BSA alone (control). Lipopolysaccharide (100 nM, LPS) from E. coli (Sigma) was added 2 h prior to harvesting with lysis buffer (Qiagen). For luciferase reporter assay passive lysis buffer (Promega) was used.
Bone-marrow derived macrophages (BMDM) were isolated as described before37, using 10% L929-conditioned medium for macrophage differentiation. RPMI with 10% cs-FBS and 1% Pen-Strep was used to differentiate and cultivate cells. For stimulation, phenol red-free medium was used with addition of E2, BSA and C18:0 in the same concentrations as done with THP-1 cells.
HeLa cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with addition of 10% cs-FBS and 1% Pen-Strep. Prior to stimulation with E2+BSA, or E2+C18:0 (analogously to THP-1 cells), cells were transfected as described below.
Gene expression analysis
RNA from cultured THP-1 cells, and BMDM, from adipose tissue, uteri and hypothalamus was isolated with RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. For real-time PCR analysis, RNA was reverse transcribed and relative mRNA expression was normalized to 18 S (animal tissues), β2-microglobulin (BMDM) or to β-Actin (THP-1 cells). Primer sequences will be provided on demand.
17β-estradiol measurement in plasma
Plasma samples of atERαKO and wt mice were obtained after final blood collection. The measurement of 17β-estradiol (E2) concentrations was performed by a radioimmunoassay set up as a sequential assay as previously described38. Prior to radioimmunoassay, blood plasma was extracted twice with toluene and the pooled extracts were evaporated to dryness and re-dissolved. The antiserum used was directed against E2–6-carboximethyloxim (CMO)–BSA and exhibited the following cross-reactions: estrone, 1.3%; estriol, 0.7%; all tested non-phenolic steroids <0.01%. The minimum detectable concentration was 5 pg/ml; intra- and interassay coefficient of variation (CV) were 7.1 and 17.6%, respectively.
Fatty acid profiling in plasma samples and in protein precipitates
Fatty acids were analysed in plasma samples from atERαKO mice on CD and HFD with a triplequad mass spectrometer coupled HLPC method, as described before39. Briefly, after hydrolysis, samples were diluted 1:10 in methanol containing internal standards. Samples were injected and separated by a reverse-phased column (Phenomenex Kinetex-C18 column 2.6 μm, 2.1 × 150 mm) with a solvent gradient containing aqueous formic acid (0.1%) and acetonitrile. After separation of components, fatty acid’s identity was analysed by an Agilent 6460 triplequad mass spectrometer with electrospray ionisation.
After sacrificing mice, uteri were partly fixed with 4% formalin, and paraffin-embedded. Sections were deparaffinised with xylene and rehydrated with a gradient of alcoholic solutions. Antigen heat retrieval with citric acid and incubation with goat serum was performed with slides prior to hematoxylin and eosin (H&E), monoclonal rat anti-mouse Mac3 antibody (clone M3/84, BD Pharmingen, USA), and monoclonal rat anti-mouse Ly6G antibody (clone 1A8, Biolegend, USA) staining. For evaluation of the grade of inflammation, and for Mac3 and Ly6G expression a semi-quantitative scoring system was applied, using a standardized procedure under observation of the equivalent anatomical structures of the uteri.
For FACS analysis, THP-1 macrophages were stained with PE conjugated anti-CD11b (BioLegend Inc. San Diego, USA) or PE conjugated anti-CD209 (BioLegend), and 7-AAD (Cell Viability Solution, BioLegend). FACS analysis was performed on a FACS Calibur flow cytometer (BD Biosciences, Heidelberg, Germany). As cells were only single stained, auto fluorescence controls were used to set negatives. The expression of the constitutively expressed adhesion protein CD11b and the M2-marker CD209 was quantified as mean fluorescence intensity (MFI). Relative up- or downregulation of CD11b- and CD209-expression was calculated by the following equation: MFI (CD11b (or CD209) of cells)/MFI (CD11b (or CD209) of E2+LPS treated cells). 7-AAD-staining was used to determine and exclude dead cells.
Phagocytosis assay and immunofluorescence staining
For testing of phagocytosis activity, phagocytosis Assay Kit (Cayman) was used and performed in accordance to the manufacturer’s instructions. Briefly, FITC-labelled beads were added to the supernatant and THP-1 macrophages were processed analogously as for immunostaining experiments. For immunostaining cells were fixed with formalin and blocked with goat serum. Afterwards, cells were incubated with PE conjugated anti-CD209 antibody (Biolegend), washed, stained with DAPI (1:1000, Thermo Scientific, blue) and mounted with mounting medium (Dako). Three fluorescent pictures were captured randomly from every sample (n = 6 per group) with an all-in-one fluorescence microscope (BZ-9000E, Keyence). Finally, representative pictures were selected.
Luciferase reporter assay
After plating and differentiating THP-1 cells as described above, cells were transiently transfected with hERα-pSG5 (kindly provided by P. Chambon, Institut Clinique de la Souris, Illkirch Cedex, France), or with pcDNA flag ERα wt or pcDNA flag ERα C447A (kindly provided by F. Acconcia, University Roma Tre, Rome, Italy), and with pERE-TkGL3 (kindly provided by P.J. Kushner, Metabolic Research Unit and Diabetes Center, University of California, San Francisco, USA), and pRL-CMV (Promega), a renilla luciferase vector. Transfection was performed with jetPEI®- Macrophage transfection reagent (Polyplus-transfection) according to the manufacturer’s instructions. Prior to stimulation, cells were starved for a maximum of 10 h. After harvesting, cell lysates were used to measure luciferase activity with the dual-luciferase reporter assay system (Promega).
For ERα-immunoprecipitation, HeLa cells (ATTC) were transfected with 3 μg hERα-pSG5 or pSG5 using Lipofectamine® 2000 (Thermo Scientific) and OptiMEM (Invitrogen). 24 h after transfection, cells were stimulated for 24 h with E2 and/or C18:0, in concentrations as described for THP-1 cells. Cells were lysed with RIPA (with proteinase-inhibitors, Complete Mini, Roche), sonicated (Sonopuls HD 2070, 30 s, 40–50%), and centrifuged. Supernatant was used to perform immunoprecipitation (IP) with anti-ERα-antibody (D8H8, Cell Signaling) bound to Protein A Sepharose-beads (Amersham). For western immunoblotting a distinct anti-ERα-antibody (sc-8002, Santa Cruz) was used. Washed beads-antibody-ERα complexes were analysed for fatty acids- binding as described above.
All experiments were repeated at least three times and n-numbers are indicated for each experiment. Statistical analysis was performed with GraphPad Prism Software and statistical significance was assumed at p < 0.05. Comparison of mean values was evaluated by two-way ANOVA (Bonferroni posttest), two-way ANOVA with repeated measures (Bonferroni posttest), or unpaired t-tests, as appropriate.
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