Twelve female and six male BALB/c Specific Pathogen Free (SPF) mice aged six to eight weeks were used. The mice were obtained from the Multidisciplinary Center for Biological Research in the Science Area of the Animal Laboratory of the State University of Campinas (CEMIB/UNICAMP) and were kept under controlled conditions of light (light from 7 am to 7 pm) and temperature (23 ± 3°C), with free access to water and feed in the bioterium for mice of the Multidisciplinary Health Institute of the Federal University of Bahia (IMS/UFBA).

All experiments with mice were conducted in accordance with internationally accepted principles for the use and care of laboratory animals, as established in the Brazilian guideline for the care and use of animals in teaching or scientific research activities (DBCA) related to principles of conduct that ensure the care and ethical management of animals used for scientific or teaching purposes, and carried out after approval by the Ethics Committee on the Use of Animals (CEUA) of the University of São Paulo, under protocol 03/2013.

Females were ovariectomized bilaterally, a model in which female sex hormones are significantly decreased (Dimitrijević et al., 2013), or submitted to sham operation. The animals were anesthetized with xylazine and ketamine at doses of 5 mg/kg and 50 mg/kg respectively (de Andrade et al., 2011). The surgery was carried out according to the current method in endocrinology (Mirbaha et al., 2009). The animals received antibiotic prophylaxis with 2.5% enrofloxacin in a single dose of 2.5 mg/kg. It is worth mentioning that the half-life of this antibiotic in mice is 89 minutes (Bregante et al., 1999)

To elicit peritoneal cells, 1 ml of 3% thioglycollate medium was injected into the peritoneal cavity of male mice (n = 6), OVX females (n = 6) and sham females (n = 6) (Layoun et al., 2015). After three days, the mice were euthanized by deepening anesthesia (xylazine 30 mg/kg and ketamine 300 mg/kg). The cells were isolated from peritoneal lavage according to the protocol described in the literature (Zhang et al., 2008; Lu et al., 2013) and cultured in RPMI 1640 medium (Gibco ™) supplemented with 10% Bovine Fetal Serum (SFB) (Sigma Aldrich) and ciprofloxacin (50 µg/ml) (Sigma Aldrich). The MPM suspension (2 x 105/ml) was plated on 24-well plates.

MPMs from OVX and sham female mice were cultured with the inoculum of S. aureus (1 x 106 CFU –MOI = 1:100) or sterile saline solution for 6 hours in 5% CO2 and 95% humid air at 37 °C. Part of MPMs from OVX females were pre-treated with 17β-estradiol (10-7 M) (Sigma Aldrich) for 24 hours at 37°C before stimulation with S. aureus. To assess whether sex would influence the response of macrophages to S. aureus, we performed the same experiment with MPMs derived from male mice.

After being removed with trypsin, the MPMs were stored with RNAlater at -70°C and, later, destined for the gene expression of inflammatory markers by the RT-qPCR array methodology. The MPMs mRNA was extracted using the TRIzol® Plus RNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA) following the protocol provided by the manufacturer. The cDNA was obtained by a SuperScript III Reverse Transcriptase Kit. We used personalized plates of the target genes IL-1β, IL-6, IL-8, TNF-α, and TLR2. The SYBR® PCR Master Mix test gene expression (Applied Biosystems™) was performed on personalized plates with targets genes, endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin genes and MGDC control genes (Mouse Genomic DNA Contamination), RTC (Reverse Control Transcription) and PPC (Positive PCR Control). Classically activated macrophages in S. aureus more commonly adopt TLR ligation signaling via MyD88 and NF-κB, generating TNF-α, IL-1β, IL-6, and IL-8 (Cole et al., 2014; Tarique et al., 2015). Classically activated macrophages also express high levels of TLR2 (Cole et al., 2014), which has been greatly implicated in host defense against S. aureus (Miller and Cho, 2011; Krishna and Miller, 2012). Amplification was performed on the StepOnePlusTM Software v2.3 thermocycler, with the following parameters for all genes: 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. The data were analyzed using the comparative method (2ΔΔCt) and normalization was performed based on GAPDH expression.

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