Steroids 102 (2015) 17–26

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Estradiol protects female rats against sepsis induced by Enterococcus faecalis improving leukocyte bactericidal activity Rafael Simone Saia a,⇑, Fabíola Morales Garcia b, Evelin Capellari Cárnio b a b

Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil Department of General and Specialized Nursing, College of Nursing of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil

a r t i c l e

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Article history: Received 9 March 2015 Received in revised form 25 April 2015 Accepted 30 June 2015 Available online 2 July 2015 Keywords: Nitric oxide Cell migration Macrophage Neutrophil Cytokines Phagocytosis Tumor necrosis factor

a b s t r a c t Enterococcus faecalis is a Gram-positive bacteria described as an important causative agent of sepsis. The contact between host leukocytes and bacteria activates the innate immunity, participating as the first defense mechanism against infection. Pro-inflammatory cytokines [including tumor necrosis factor (TNF)-a and interleukin-1b] and nitric oxide (NO) are essential to recruitment of leukocytes into the infectious focus as well as their activation for phagocytosis. Beyond the bacteria species, gender has been considered another factor to predict outcome in septic patients. Studies suggest that females exhibit a protective advantage during sepsis models, being gonadal hormones possible modulators of functions of immune cells. Nevertheless, the role of estradiol during Gram-positive infection remains a literature gap. Our aims were to investigate whether estradiol protects rats against bacterial dissemination during E. faecalis-induced sepsis. We determined whether estradiol modulates the local and systemic inflammatory response, as well as the cell migration into the infectious focus and the bactericidal capacity of leukocytes. Our findings demonstrated that estradiol pre-treated rats showed a dose-dependent reduction in bacterial counts in peritoneal lavage fluid (PLF) and in liver. Moreover, TNF-a and nitrate levels were increased in plasma, while only TNF-a was increased in the PLF in estradiol-treated rats. The prevention of bacterial dissemination may be related to the enhanced neutrophil and macrophage migration into the peritoneal cavity. Furthermore, estradiol improved the phagocytic and bactericidal ability of these both inflammatory cells. Taken together, the present study clearly demonstrates an important protective role of estradiol against sepsis induced by E. faecalis in female rats. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Sepsis and its most deleterious complication, septic shock, represent the systemic inflammatory response to infection, which may microbiologically be classified as confirmed, probable or possible [1,2]. Since the 1990’s, an inversion on the profile of causative microorganisms of infections (such as bloodstream, intra-abdominal and sepsis) has been reported, raising the incidence of Grampositive instead Gram-negative bacteria in isolates around the world [3–5]. In this scenario, Enterococcus faecalis has been considered as a healthcare-associated emerging pathogen due its multiple antimicrobial resistance (especially those of restrictive hospital use, including vancomycin), extended length of hospital stay and increased risk of mortality [6,7]. ⇑ Corresponding author at: Department of Physiology, School of Medicine of Ribeirão Preto/USP, Avenida dos Bandeirantes, 3900, 14049-900 Ribeirão Preto, São Paulo, Brazil. E-mail address: rssaia@rfi.fmrp.usp.br (R.S. Saia). http://dx.doi.org/10.1016/j.steroids.2015.06.016 0039-128X/Ó 2015 Elsevier Inc. All rights reserved.

Innate immunity participates as the first defense mechanism for host protection against microbial infections. Structural constituents of pathogens activate several intracellular signaling pathways responsible for triggering the innate immune response, the synthesis of inflammatory mediators and also phagocytosis [8]. The production of pro-inflammatory cytokines, mainly tumor necrosis factor (TNF)-a, interleukin (IL)-1b and interferon (IFN)-c, in the infectious focus is essential to signaling, recruitment and activation of leukocytes preventing the systemic dissemination of microorganisms [9]. Therefore, whether the host’s immune cells fail to restrict the pathogens to a localized compartment, the systemic inflammatory response initiates a massive production of cytokines, nitric oxide (NO) and cytotoxic factors [10]. This pathophysiological condition, commonly present in the initial phase of experimental and clinical sepsis, has been characterized by the ‘‘dysregulation of the inflammatory response’’ and the disturbance of the homeostatic state [10,11]. Beyond the bacteria species, gender has been considered another provocative factor to predict outcome in septic patients.

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Whereas epidemiologic studies report a higher incidence of sepsis and also mortality rate in males [3,12], the clinical findings are still inconsistent. Given this divergence it is impossible to determine whether females could be protected or not from developing this syndrome [13]. Meanwhile, experimental studies suggest that females exhibit a protective advantage during different sepsis models, showing increased survival rate [14,15] as well as attenuated hypothermia [16]. Despite that the gender-based differences involve multiple factors, one possible explanation for these previous findings has been associated with the ability of gonadal hormones to modulate diverse functions of immune cells [17]. This intercommunication between the immune and endocrine systems has been extensively reported and has been reinforced by the expression of estradiol receptors (ERs) in leukocytes [18]. There are two receptors identified, ER-a and ER-b, which exhibit similar binding activities by the main physiological estrogen agonist, the 17b-estradiol [19]. During inflammatory conditions, the estradiol administration has demonstrated protection of gastrointestinal membrane integrity [20] and gut perfusion [21], changes on thermoregulatory control [16,22], reduced synthesis of inflammatory mediators [22,23] as well as changed leukocyte functionality and chemotaxis [24]. With this information put together, we hypothesized that estradiol could exert a protective role against sepsis induced by the emerging nosocomial pathogen E. faecalis. Furthermore, estradiol could modulate the local and systemic inflammatory response, as well as the cell migration into the infectious focus and the bactericidal capacity of leukocytes.

2. Materials and methods 2.1. Animals Experiments were performed on adult female Wistar rats weighing 200–250 g and being five- to seven-weeks old. The animals were housed in a ventilated and controlled temperature chamber (25 ± 2 °C) and exposed to a daily 12:12 h light–dark cycle (lights on at 0700 AM). They were housed in polypropylene cages, grouped in 4–5 rats per cage and given food and water ad libitum. Animal handling followed the recommendations of the Guide for the Care and Use of Laboratory Animals [25,26]. All procedures were conducted in accordance with the Ethics Committee of the University of São Paulo (CEUA) – Campus Ribeirão Preto, University of São Paulo, São Paulo, Brazil (protocol number 10.1.179.53.3).

2.2. E. faecalis inoculum The vancomycin-resistant E. faecalis strain was obtained from the American Type Culture Collection (ATCC) number 51299. Bacterial stocks were kept at 80 °C in a brain heart infusion (BHI; Himedia Laboratories, Mumbai, India) broth, supplemented with 40% (v/v) glycerol. Before each experiment, the frozen aliquots were cultured overnight in BHI broth at 37 °C to recover viability. Subsequently, fresh grown bacteria was spread onto BHI agar plates for 18–20 h at 37 °C. The optical density of bacterial suspensions was adjusted spectrophotometrically at 625 nm (approximately, 2–3  109 CFU/mL) and were prepared in sterile saline for intraperitoneal (i.p.) administration to the animals. The number of CFU of the inoculum was quantified through serial dilutions, plating on BHI agar dishes and counting colonies after a 24 h incubation period.

2.3. Bilateral ovariectomy Rats were generally anesthetized with 2,2,2-tribromoethanol (250 mg/kg, i.p.; Aldrich, Milwaukee, WI, USA). The ovaries were accessed by a small incision made between the last rib and the upper pelvic ridge. A small portion of the uterus was knotted with sterile cotton suture and the ovaries were removed. After surgery, all animals were treated with antibiotics (100,000 UI of benzylpenicillins and 50 mg of streptomycin, i.m.) and analgesic flunixin (2.5 mg/kg, s.c.; Banamine, Schering, São Paulo, SP, Brazil). They were returned to collective cages and allowed a recovery period of 10–12 days before the experiment took place. 2.4. Experimental design To evaluate the effect of estradiol in changes of microbiological parameters, inflammatory response and cell migration to the peritoneal cavity, ovariectomized rats were treated for three consecutive days with estradiol cypionate (Pfizer, Paulínia, SP, Brazil) intraperitoneally at two different doses, 50 or 100 lg/kg. These doses were chosen based on previous studies [22,27]. The control groups (vehicle) received an i.p. injection of corn oil, which was the estradiol diluent. On the third day, 2 h after the estradiol or vehicle administration, the rats received an intraperitoneal E. faecalis inoculation (0.5–1  1010 CFU/animal) (time ‘‘zero’’ hour). At 6 and 24 h, rats were deeply anesthetized (ketamine and xylazine at 55 and 10 mg/kg, respectively, i.p.) for sample collection. As detailed in the subsequent sections, the blood was obtained by cardiac puncture, the peritoneum was washed for peritoneal lavage fluid (PLF) collection and the liver was aseptically removed and homogenized. The samples were separated in aliquots and were kept on ice. For the cytokines and nitrate dosage, the blood and the PLF were immediately centrifuged for 20 min at 2000g at 4 °C to separate the plasma and inflammatory cells. All samples were rapidly distributed in multiple aliquots and stored at 70 °C. 2.5. Microbiological analysis At the given time points (6 and 24 h after i.p. E. faecalis inoculation), rats were deeply anesthetized (ketamine and xylazine at 55 and 10 mg/kg, respectively, i.p.) for samples collection under aseptically conditions. The blood was collected by cardiac puncture (5 mL) in chilled 15 mL tubes (containing 1500 IU/tube of sodium heparin), gently homogenized and kept on ice. Fifty microliters of total blood were plated in duplicates on BHI agar dishes and incubated at 37 °C under aerobic condition [28]. The PLF was obtained by washing the cavity with sterile heparinized saline, after skin disinfection and exposure of the abdominal muscle without injury. The saline was injected inside the peritoneal cavity (10 mL containing 500 IU of sodium heparin), gently homogenized (during 30 s) avoiding blood vessels rupture, aspirated out, transferred to sterile plastic tubes and kept on ice. The entire liver was excised, washed in saline to remove the blood excess, homogenized in 20 mL of sterile saline and kept on ice. Serial dilutions of PLFs and livers were also plated in duplicates on BHI agar dishes and incubated at 37 °C under aerobic condition. The number of viable bacteria in all samples were analyzed after incubating the plates for 20–22 h and the results were expressed as the number of CFU per mL or per organ. 2.6. Cytokines levels measurements The plasma and PLF IL-10, IL-1b and TNF-a concentrations were determined using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The IL-10, IL-1b and TNF-a kit detection limits were 10, 5 and 5 pg/mL, respectively.

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2.7. Nitrate determination Nitrate was determined by using the purge system from Sievers Instruments Nitric Oxide Analyzer (NOA model 280i, Boulder, CO, USA). Plasma and PLF samples were deproteinized using cold absolute ethanol and were injected in a reaction vessel containing vanadium trichloride (VCl3), which converts nitrate to NO. The NO produced was detected as ozone induced by chemiluminescence. Peak NO values of samples were measured using a standard curve established with sodium nitrate solutions of various concentrations (5, 10, 25, 50 and 100 lM). 2.8. Cell migration to the peritoneal cavity The cell influx to the peritoneal cavity was evaluated at 6 and 24 h after E. faecalis inoculation. The rats were deeply anesthetized (ketamine and xylazine at 55 and 10 mg/kg, respectively, i.p.) and the PLF was collected by injecting 10 mL of heparinized PBS (50 IU/mL). Total counts were made in a hematocytometer and differential cell counts were made on centrifuge slides (Cytospin 3, Shandon Southern Products, Atsmoore, UK) stained by the May– Grünwald–Giemsa method. At least 200 cells were evaluated per slide and the number of neutrophils and macrophages expressed per lL of PLF. 2.9. In vitro experiments 2.9.1. Neutrophil culture Naive rats received an i.p. injection containing 10 mL of oyster glycogen (1% w/v; Sigma–Aldrich) to recruit neutrophils into the peritoneal cavity. Four hours later, the rats were euthanized by decapitation and the cells were collected through washing the peritoneum with RPMI 1640 medium. Cells were resuspended with RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 lg/mL streptomycin (Sigma) (complete medium), counted and then cultured at 1  106 cells/mL in microtubes. Neutrophils were allowed to equilibrate at the appropriate temperature and pH (at 37 °C on a humid heater with 5% of CO2) for 1–2 h prior the start of phagocytosis or killing assays. The collected cells were composed by 80–90% of neutrophils and showed viability, evaluated by the trypan blue exclusion test, higher than 95%. 2.9.2. Peritoneal macrophage culture Naive rats were injected i.p. with 10 mL of thioglycolate medium (3% w/v; Sigma–Aldrich, USA) to recruit macrophages into the peritoneal cavity. Four days later, the rats were euthanized by decapitation and the cells were collected through washing the peritoneum with cold RPMI 1640 medium. Macrophages were resuspended with complete medium, counted and then cultured at densities 1  106 cells/mL in 24-well plastic plates (for killing assay) or 1  105 cells/200 lL in 8-well chambers glass slides (Lab Tek, Nalgene NUNC, Rochester, NY, USA) (for phagocytosis assay). After incubation at 37 °C on a humid heater with 5% of CO2 for 2 h, the non-adherent cells were removed washing each well once with PBS and twice with RPMI 1640 medium. The remaining cultured cells were at least 97% macrophages and a trypan blue exclusion test showed viability higher than 92%. 2.9.3. Phagocytosis assay The neutrophils and macrophages phagocytic activity were evaluated using Candida albicans, according to the methods described previously [29] and modified by us [30]. C. albicans was grown in a BHI broth for 48 h, washed three times, heat-killed in sterile saline (100 °C for 30 min) and stored at 20 °C. The yeast particles were opsonized incubating the cells with 50% fresh rat

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serum (v/v) for 30 min at 37 °C, washed twice and resuspended in PBS. During this period, macrophages and neutrophils were cultured as outlined above and treated with dimethylsulfoxide (DMSO; vehicle) or different 17b-estradiol concentrations (Sigma; 10 12, 10 10 or 10 8 M) for 2 h. After that, the cells were incubated with opsonized C. albicans at 37 °C with mild shaking, for 1 h (1 cell: 10 yeast particles) in neutrophil culture and for 2 h (1 cell: 20 yeast particles) in macrophage culture. Then, the cells were washed three times with PBS to remove the extracellular and non-phagocytized yeast cells, deposited onto microscope slides in a cytocentrifuge, fixed with a 4% paraformaldehyde for 15 min and stained by the May–Grünwald–Giemsa method. Under oil immersion microscopy, 100 cells were analyzed and the number of leukocytes that ingested and did not ingest particles was counted. The phagocytic index (the measure of particle uptake) was calculated multiplying the percentage of cells containing at least one yeast cell by the mean number of particles per positive cell. The image of representative slides was obtained by a system connecting a PC to the optic microscope (Zeiss KS 300, Thornwood, NY, USA) under oil immersion (100). 2.9.4. Killing assays The neutrophil and macrophage killing activity was evaluated by two different assays. In the first set of experiments, we used a methodology described previously by us [30]. The neutrophil and macrophage (1  106 cells) cultures were treated with DMSO or different 17b-estradiol concentrations (Sigma; 10 12, 10 10 or 10 8 M) for 2 h, in a RPMI 1640 medium supplemented exclusively with a 10% heat-inactivated fetal bovine serum. After this period, the cells were incubated with E. faecalis at a pre-optimized ratio of 1 leukocyte per 5 bacteria for 2 h at 37 °C in a humid heater. In the second protocol, the macrophage culture (1  106 cells) was treated with DMSO, different 17b-estradiol concentrations or E. faecalis heat-killed (ratio 1 cell per 100 bacteria) for 2 h, in complete RPMI 1640 medium. Subsequently, a suspension containing live C. albicans was added (1  103 yeast cells per well) and incubated for 14 h at 37 °C in a humid heater. In the both methods, after the incubation period, the cells were washed five times with 1 mL PBS and lysed with distillated water for 10 min, followed by vortex agitation to complete the release of live intracellular bacteria. Samples were serially diluted, 50 lL aliquots were spread on BHI agar plates and incubated at 37 °C for 20–22 h. Results were expressed as the number of viable colonies of E. faecalis or C. albicans per respective volume used for the cell lysis. 2.10. Statistical analysis All values are expressed as means ± S.E.M. Data showed in figures are representative of at least two or three independent experiments. All samples were measured in the same session to reduce experimental inter-assay variation. Data was compared using oneway analyses of variance (ANOVA) and significant differences were obtained using the Tukey multiple variances post hoc test (GraphPad Software version 3.0, San Diego, CA, USA). For all tests, statistical significance was considered when p < 0.05. 3. Results Our experimental model of infection induced by E. faecalis has reproduced the typical early clinical signs of human sepsis, including lethargy, piloerection and fever. 3.1. Estradiol protects female rats against sepsis induced by E. faecalis Our model of sepsis induced by E. faecalis exhibited positive blood culture in a limited number of samples (50% at 6 h and

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36.4% at 24 h; Fig. 1A), in comparison to other Gram-positive infections [30]. Meanwhile, a persistent bacterial counting in the PLF and liver cultures were found throughout the experimental period of 24 h. The hormone treatment with estradiol did not modify bacteremia, regardless of the tested dose or the time after sample collection (Fig. 1A). Although no statistical difference was observed, the estradiol administration at 100 lg/kg produced negative cultures in all samples 24 h after inoculation. Additionally, the estradiol treatment reduced the CFU counts in the PLF, in a dose-dependent manner, at both time-points investigated (Fig. 1B). Only the higher estradiol dose differed in comparison to the vehicle group at 6 (3.04 ± 0.85  106 vs. 18.37 ± 0.48  107 CFU/mL; p < 0.05; F2,13 = 6.88) and also at 24 h (0.24 ± 0.07  106 vs. 1.03 ± 0.14  106 CFU/mL; p < 0.05; F2,20 = 10.10) after inoculation. The results of liver cultures showed a similar pattern to that found in the PLF, i.e. estradiol reduced the bacteria counting when compared with the control animals (p < 0.05; Fig. 1C) in both time-points. 3.2. Estradiol modulates the systemic and local inflammatory response during sepsis induced by E. faecalis The estradiol pre-treatment per se had no effect on cytokine and nitrate production neither in plasma nor in PLF (Figs. 2 and 3). The pro-inflammatory cytokines, TNF-a and IL-1b, presented a transient profile of synthesis in plasma and also in PLF, reaching the maximum values at 6 h after sepsis induction. Interestingly, a significant increase of plasma TNF-a levels at both time-points investigated were detected in ovariectomized female rats treated with estradiol prior E. faecalis inoculation (p < 0.05; Fig. 2A). In

relation to PLF, the TNF-a concentration was also enhanced in estradiol-treated rats only at the initial phase of sepsis (409.95 ± 58.14 pg/mL), when compared with the vehicle group (274.56 ± 9.53; p < 0.05; F5,54 = 89.06; Fig. 3A). Furthermore, the plasma and PLF IL-1b levels showed no alterations amongst groups, with the exception of a discrete reduction in PLF in rats whose estradiol was administered at 50 lg/kg (Figs. 2B and 3B). Different from the pro-inflammatory mediators, the anti-inflammatory cytokine IL-10 showed a distinct profile of synthesis in plasma. The IL-10 levels increased just slightly in the initial phase of sepsis, without statistical significance when compared with control animals (Fig. 2C). Moreover, the estradiol treatment did not change the IL-10 production, regardless of the time of inoculation. In PLF, sepsis increased IL-10 synthesis only at 6 h (p < 0.05), which decreased progressively up to 24 h after inoculation. As was found correspondingly in plasma, the estradiol did not alter the IL-10 concentration in the PLF (Fig. 3C). The NO formation was evaluated by the nitrate concentration, which is a stable product from NO oxidation. The nitrate production showed a particular profile, reaching the maximum values in the late phase of infection. Furthermore, the hormone treatment with the higher dose of estradiol intensified the nitrate levels only in the plasma, but at both the time-points evaluated (Figs. 2D and 3D). 3.3. Estradiol stimulates neutrophil and macrophage migration into the peritoneal infectious focus The estradiol pre-treatment did not change leukocyte migration into the peritoneal cavity in non-septic rats (Fig. 4A and B). On the

Fig. 1. Estradiol protects against Enterococcus faecalis-induced sepsis in female rats. Ovariectomized rats were treated intraperitoneally with estradiol or corn oil (vehicle) for three consecutive days. On the third day, 2 h after the hormone treatment, the rats received an intraperitoneal E. faecalis inoculum administration (time ‘‘zero’’ hour). At the indicated time-points, blood (A), peritoneal lavage fluid (PLF) (B) and liver (C) were collected for microbiological analysis. Values are expressed as the median of the bacterial counts per mL. n = 5–12 rats per group. #p < 0.05 vs. vehicle + E. faecalis group.

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Fig. 2. Estradiol modulates TNF-a and nitrate synthesis in plasma during Enterococcus faecalis-induced sepsis. Ovariectomized rats were treated intraperitoneally with estradiol or corn oil (vehicle) for three consecutive days. On the third day, 2 h after the hormone treatment, the rats received an intraperitoneal E. faecalis inoculum administration (time ‘‘zero’’ hour). At 6 and 24 h after injection, blood was collected and the plasma concentrations of TNF-a (A), IL-1b (B) and IL-10 (C) were determined using ELISA kits, while plasma nitrate concentration (D) was quantified by chemiluminescence. Values are expressed as means ± S.E.M. n = 5–8 rats per group. *p < 0.05 vs. vehicle group and #p < 0.05 vs. vehicle + E. faecalis group.

Fig. 3. Estradiol modulates TNF-a synthesis peritoneal lavage fluid (PLF) during Enterococcus faecalis-induced sepsis. Ovariectomized rats were treated intraperitoneally with estradiol or corn oil (vehicle) for three consecutive days. On the third day, 2 h after the hormone treatment, the rats received an intraperitoneal E. faecalis inoculum administration (time ‘‘zero’’ hour). At 6 and 24 h after injection, PLF was collected and the concentrations of TNF-a (A), IL-1b (B) and IL-10 (C) were determined using ELISA kits, while plasma nitrate concentration (D) was quantified by chemiluminescence. Values are expressed as means ± S.E.M. n = 5–8 rats per group. *p < 0.05 vs. vehicle group and #p < 0.05 vs. vehicle + E. faecalis group.

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other hand, sepsis induced by E. faecalis produced a rapid and massive neutrophil migration to the peritoneal cavity at hour 6 after the inoculation (17146.57 ± 569.93 vs. 383.29 ± 52.54 cells/lL; p < 0.001; F5,21 = 79.32), maintaining a high number of cells until the end of the experimental period (9772.41 ± 1033.2 vs. 492.32 ± 92.15 cells/lL; p < 0.001; F5,41 = 45.56). As expected, the macrophage influx into the peritoneal infectious focus increased slowly and differed in relation to the vehicle group in the late phase of sepsis at 24 h (7259.32 ± 508.97 vs. 1715.24 ± 26.02 cells/lL; p < 0.001; F5,43 = 20.33). Having established that the estradiol treatment controls the bacterial dissemination and also modulates the inflammatory response, we investigated whether these findings may be correlated with increased cell influx to the peritoneal cavity and an ability to restrict the infection inside this compartment. Our findings confirmed this hypothesis since rats treated with a high estradiol dose showed increased neutrophil and macrophage migration to the infectious focus at the late phase of sepsis (Fig. 4A and B) (p < 0.05).

3.4. Estradiol maximizes neutrophil and macrophage bactericidal ability Our results demonstrated that systemic and local bacterial loads were reduced by treatment with estradiol at 100 lg/kg and one possible mechanism involved was the induction of cell migration into the peritoneal cavity. Apart from the role of estradiol in chemotaxis [24,31], few studies attempted to explain whether this hormone could also modulate de bactericidal ability of neutrophils and macrophages. The incubation of neutrophils and macrophages with different 17b-estradiol concentrations, including 10 10 and 10 8 M, enhanced the phagocytic activity of heat-killed C. albicans particles (Fig. 5A and B). Representative images of neutrophil and macrophage phagocytosis confirm the increased ability of these cells promoted by estradiol treatment (Fig. 5C and D). Moreover, not only was the number of phagocytized yeast cells increased by estradiol pre-treatment, but also the ability of these cells to kill the engulfed bacteria. Since neutrophils and macrophages were incubated with estradiol at concentrations 10 10 and 10 8 M, they became more efficient in destroying E. faecalis, thereby reflecting a reduced number of viable intracellular bacteria (Fig. 6A and B). In another assay protocol, we evaluated the long-lasting effect of estradiol incubation in macrophage killing. The estradiol treatment increased the macrophage’s capacity to kill live C. albicans, not only in the presence of heat-killed E. faecalis but also in the absence of bacteria (Fig. 6C).

4. Discussion The present study demonstrates that estradiol pre-treatment plays a protective role controlling the E. faecalis dissemination from the primary infectious focus in female rats. Moreover, we showed for the first time that estradiol also promotes macrophage and neutrophil migration into the peritoneal cavity and also improves the bactericidal activity of these inflammatory cell types. Epidemiological studies have highlighted a significant increase on the incidence of nosocomial E. faecalis infections around the world, being associated with high risk of mortality and prolonged length of hospital stay [4,6,7]. The increment in enterococcal isolation in bloodstream infections, the intrinsic resistance to antimicrobials and the high ability to acquire resistance [4,7], reinforce the importance of understanding the pathogenesis of sepsis induced by this bacteria and new therapeutic strategies. Clinical reports have recognized that gender may influence the host susceptibility to infections [17]. The fact that females exhibit a more robust inflammatory response against pathogens evidences its protective role as well as why sepsis occurs later in life in comparison to their male counterparts [3,13]. Interestingly, pre-puberty male patients are more predisposed to sepsis and acute respiratory distress syndrome [32], indicating that gender-based differences may be a combination of gene encoded regulatory factors and also reproductive hormones during their development. Additionally, similar findings have been made in experimental models of infection. When it is induced by Coxiella burnetii [33], Mycobacterium avium [34] or cecal ligation and puncture [35], the estradiol treatment reduced bacteremia, bacterial counts in tissue cultures (liver and spleen) and consequently, improved the animal’s survival. Using a sepsis model induced by E. faecalis, the ovariectomized rats treated with estradiol reduced the number of bacteria in the peritoneal cavity and also in the liver. Our results are in accordance with previous studies and suggest that estradiol may protect the host, avoiding bacterial spreading. Surprisingly, our septic rats presented only a discrete bacteremia in comparison to other Gram-positive bacteria [30,36], independently of the treatment adopted. This fact could more likely be related to the characteristics of enterococcal infections since this pathogen has efficient strategies for immune system evasion and also has an exceptional ability of biofilm formation. As expected, it colonizes tissues preferentially leading to common clinical life-threatening complications, such as endocarditis and catheter contamination [37]. In the sepsis context, it is noteworthy that the production of inflammatory mediators exerts a dual effect. Although the

Fig. 4. Estradiol enhances neutrophil and macrophage migration into the peritoneal cavity during Enterococcus faecalis-induced sepsis. Ovariectomized rats were treated intraperitoneally with estradiol or corn oil (vehicle) for three consecutive days. On the third day, 2 h after the hormone treatment, the rats received an intraperitoneal E. faecalis inoculum administration (time ‘‘zero’’ hour). At 6 and 24 h after injection, PLF was collected and total cell counts were made. The differential cell counts of neutrophils (A) and macrophages (B) were made on cytocentrifuge slides stained by the May–Grünwald–Giemsa method. Values are expressed as means ± S.E.M. n = 5–8 rats per group. * p < 0.05 vs. vehicle group and #p < 0.05 vs. vehicle + E. faecalis group.

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Fig. 5. Estradiol improves neutrophil and macrophage phagocytic ability. The neutrophil and macrophage cultures were treated with vehicle (DMSO) or one of the estradiol concentrations (10 12, 10 10 or 10 8 M) for 2 h before the tests. Then, cells were incubated with opsonized heat-killed Candida albicans in a neutrophil culture (A) and in a macrophage culture (B). Leukocytes were deposited onto microscope slides, fixed and stained. The phagocytic index (the measure of particle uptake) was calculated multiplying the percentage of cells containing at least one yeast cell by the mean number of particles per positive cell. Representative images of neutrophil (C) and macrophage (D) phagocytosis were obtained by optical microscope (100). Values are expressed as means ± S.E.M. n = 5–8 samples per group. #p < 0.05 vs. respective vehicletreated group.

Fig. 6. Estradiol improves neutrophil and macrophage bactericidal capacity. The neutrophil and macrophage cultures were treated with vehicle (DMSO) or one of the estradiol concentrations (10 12, 10 10 or 10 8 M) for 2 h before the tests. Then, neutrophil (A) and macrophage cultures (B) were incubated with live E. faecalis for 2 h. Subsequently, the cells were washed to remove the extracellular bacteria and lysed to release of intracellular bacteria. Surviving bacteria were enumerated by a quantitative culture and expressed as the number of viable S. aureus per mL. The long-lasting macrophage bactericidal ability was evaluated incubating the culture with live Candida albicans for 14 h (C). After this period, the extracellular yeast particles were removed and macrophage lysed. The remaining yeasts were quantified and expressed as the number of viable C. albicans per mL. Values are expressed as means ± S.E.M. n = 5–8 samples per group. #p < 0.05 vs. respective vehicle-treated group.

overproduction of cytokines and NO may lead to the systemic inflammatory response syndrome, they consist an important goal of innate immune response, triggering the host immune defense

against bacterial infections and maintaining the organism integrity [10,38]. Among them, TNF-a and IL-1b are essential for leukocytes activation and have been considered biomarkers predicting fatal

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outcome of severe Staphylococcus aureus bacteremia [38,39]. Our results showed that estradiol treatment elevated plasma TNF-a levels in the early and late phases of sepsis, which may be associated with reduced bacterial counts in the PLF and in the liver. On the other hand, rats pre-treated with estradiol presented higher PLF TNF-a concentrations only at 6 h after E. faecalis challenge. The stimulatory effect of estradiol on TNF-a synthesis was demonstrated by in vivo and in vitro studies [40,41]. The hormone exposure sensitizes mononuclear cells by inducing the protein expression and cell surface availability of Toll-like receptors (TLR)-2 [42]. The TLR-2 is the main receptor responsible for detection of Gram-positive constituents and the first step to initiating the inflammatory cascade. Furthermore, NO exists in another essential bactericidal molecule, whose synthesis is stimulated by pro-inflammatory cytokines (TNF-a, IL-1b, interferon-c) as well as microbial products (LPS or lipotheicoic acid). The NO-related antimicrobial activity has been demonstrated in a broad spectrum of pathogenic microorganisms [43]. In experimental models, the abrogation of inducible NO synthase (iNOS) activity produces an increase in the bacteria burden, suggesting that NO formation is directly correlated with the host ability to contain the infection and reduce mortality [36,43]. Our data shows that E. faecalis induces a progressive NO production in systemic and also in the focus of infection, possibly by the expression of iNOS [44]. However, the estradiol treatment increases the NO formation only in plasma, a fact that could contribute to protect rats against sepsis, as previously observed in M. avium [34]. Meanwhile, IL-10 represents an anti-inflammatory cytokine, prevents mortality during sepsis [45] and its magnitude response has also been positively correlated with the severity of the inflammatory insult [46]. In the present study, the maximal values did not reach those found in endotoxemia [44,45] or S. aureus infection [30], suggesting that this cytokine could not represent a marker of the infectious insult in our model. In addition, the IL-10 levels were not affected by estradiol treatment excluding a possible modulation of an anti-inflammatory pathway. In opposition to other models of bacterial infection [47], the inflammatory response due to estradiol treatment creates a pro-inflammatory profile, essential for host protection and the appropriate activation of immune cells to destroy E. faecalis. The rapid recruitment of neutrophils into the peritoneal cavity is a crucial mark in bacterial clearance during sepsis. However, the stages involved in blood-derived neutrophils migration are influenced by the severity of the infectious challenge, as well as the local production of chemokines [48]. In lethal peritonitis induced by CLP [49] or S. aureus inoculation [36], the failure of neutrophil migration has been accompanied by increased bacterial counts in blood and PLF, high amounts of circulating cytokines and consequently an elevated mortality rate. Similar to other studies [30,36,49,50], our non-lethal model of sepsis presented an intense neutrophil influx after E. faecalis inoculation, possibly promoted by the local production of TNF-a and IL-1b, two potent chemoattractant mediators. Although a decrease in bacterial counting in the PLF was found after estradiol treatment, it was correlated with the maintenance of a significant migration of polymorphonuclear leukocytes only in the late phase of sepsis. During this phase, estradiol could create an environment that propitiates the neutrophil influx, induced by elevated TNF-a levels compartmentalized in the peritoneum at hour 6 after inoculation. Previously, it has been demonstrated that estradiol promotes leukocyte-endothelial interaction in the presence of inflammatory mediators, particularly TNF-a and IFN-c, inducing cell surface expression of various adhesion molecules [31]. However, other studies diverged from our findings albeit the involvement of estradiol has not been investigated in an infectious context. With the hormone treatment leukocytes become less responsive to in vitro

chemotaxis induced by fMLP [51] and also to rolling and endothelial adhesion decreases the in vivo migration induced by carrageenan [52]. On the other hand, in the initial phase of sepsis the lack of migration in those estradiol-treated rats may be explained by the high production of TNF-a and NO (two inhibitory factors on neutrophil chemotaxis) [36,49]. This hypothesis is supported by studies showing that intravenous administration of cytokines, such as TNF-a, IL-8 and IL-10, reproduces the inhibition on neutrophil adhesion, rolling and transmigration from the intravascular compartment [48,53,54]. Although TNF-a could contribute to failure of neutrophil migration in the early phase of infection, the reduced bacterial counting in the PLF in estradioltreated rats suggest that this cytokine exerts an essential influence on activation of inflammatory cells, consequently enhancing their ability in the phagocytosis process [55,56]. Peritoneal resident macrophages are regarded as sentinels of the innate immune response, important for sensing pathogens and producing pro-inflammatory cytokines and chemokines [54]. Moreover, the inflammatory context created in the peritoneal cavity by these cells will assist in the recruitment of neutrophils and the clearance of microorganisms from the infectious focus [57,58]. In the late phase of inflammation, the pattern of leukocyte recruitment switches from neutrophils to the monocyte/macrophage lineage, which are stimulated by chemotactic factors, mainly TNF-a, IL-1b, IFN-c and monocyte chemotactic protein (MCP)-1 [59]. In the present study, the macrophage migration into the peritoneal cavity increased progressively up to the 24 h after inoculation. Similar to neutrophil migration, the treatment with estradiol increased the macrophage influx only at the late phase of sepsis. Interestingly, our study is the first to demonstrate the monocyte chemotactic activity of estradiol in an experimental model of infection. The administration of contraceptive doses of estradiol in naive mice alters the monocyte/macrophage distribution, increasing their number in the peripheral blood but reducing it in the peritoneal exudate [47]. However, in vitro studies showed an inhibitory effect of estradiol in monocyte chemotaxis induced by MCP-1 [60] or even adhesion in TNF-a-stimulated endothelial cells [61]. In our case, the divergence among our results and the previous in vitro studies could be explained by an indirect effect of estradiol, through changes in the inflammatory environment in the presence of bacterial antigens. Since estradiol increased the TNF-a production in the PLF, it promotes the monocyte transmigration to the focus of infection, furthermore stimulates the cytotoxic/tumor cell killing macrophage phenotype and also enhances their capability to phagocytosis [47,62]. Our hypothesis highlighting the importance of the inflammatory response in cell migration may be clarified by comparing it with the Listeria monocytogenes infection. In this model, estradiol exerts an immune suppressive role decreasing synthesis of pro-inflammatory mediators (such as TNF-a and IL-12) [47], which results in reduced peritoneal monocyte influx and increased mice mortality [63]. E. faecalis accumulates a repertoire of virulence factors capable of evading the host innate immune defenses from initial phagocyte recruitment to the process of opsonization and intracellular effectors of bacterial killing [37]. Nowadays the infectious diseases therapies have been focused only on the combat of pathogens, exploring in minor extension strategies for improvement of cellular microbicidal activity. Therefore, whether estradiol restricts bacterial dissemination inducing leukocyte capability remains poorly investigated. Our results confirmed that estradiol not only favored phagocytosis, but also enhanced E. faecalis killing by macrophages and neutrophils. Previous studies demonstrated a potentiating role of estradiol on phagocytic ingestion by human neutrophils [56], murine peritoneal macrophages and peritoneal exudate cells [47]. Moreover, the same response was found by in vivo experiments in estradiol-treated mice suggesting that resident

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macrophages, such as Küpffer cells, could also be stimulated to the phagocytic process and become more able to destroy bacteria in liver tissue. Furthermore, the enhancement of killing activity of neutrophils and macrophages by estradiol has also been demonstrated, suggesting a direct effect of this hormone on the regulation of microbicidal capacity by inflammatory cells [47,55,56]. In summary, the data presented here demonstrates a protective role for the gonadal hormone estradiol in a Gram-positive sepsis model induced by E. faecalis. This hormone modulates the systemic and local inflammatory response, which possibly influences the neutrophil and macrophage migration into the site of infection. Moreover, the most interesting mechanism evidenced from our results is that estradiol improved the microbicidal capacity of two important phagocytes, neutrophils and macrophages. These results suggest that estradiol might be used as a promising adjuvant therapeutic agent, for its modulatory ability of the leukocytes’ functions in favor of host integrity. Disclosure statement The authors have nothing to disclose. Conflict of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Acknowledgements We would like to thank Marcelo Eduardo Batalhão for the technical assistance. The authors gratefully acknowledge the English grammar review by Renata Brancaleoni Mitchell and Ian Mitchell. This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). References [1] T. Calandra, J. Cohen, The international sepsis forum consensus conference on definitions of infection in the intensive care unit, Crit. Care Med. 33 (2005) 1538–1548. [2] M.M. Levy, M.P. Fink, J.C. Marshall, E. Abraham, D. Angus, D. Cook, et al., 2001 SCCM/ESICM/ACCP/ATS/SIS international sepsis definitions conference, Intensive Care Med. 29 (2003) 530–538. [3] G.S. Martin, D.M. Mannino, S. Eaton, M. Moss, The epidemiology of sepsis in the United States from 1979 through 2000, N. Engl. J. Med. 348 (2003) 1546–1554. [4] A.R. Marra, L.F. Camargo, A.C. Pignatari, T. Sukiennik, P.R. Behar, E.A. Medeiros, et al., Nosocomial bloodstream infections in Brazilian hospitals: analysis of 2,563 cases from a prospective nationwide surveillance study, J. Clin. Microbiol. 49 (2011) 1866–1871. [5] J.L. Vincent, Y. Sakr, C.L. Sprung, V.M. Ranieri, K. Reinhart, H. Gerlach, et al., Sepsis in European intensive care units: results of the SOAP study, Crit. Care Med. 34 (2006) 344–353. [6] M.E. de Kraker, V. Jarlier, J.C. Monen, O.E. Heuer, N. van de Sande, H. Grundmann, The changing epidemiology of bacteraemias in Europe: trends from the European Antimicrobial Resistance Surveillance System, Clin. Microbiol. Infect. 19 (2013) 860–868. [7] S.J. McBride, A. Upton, S.A. Roberts, Clinical characteristics and outcomes of patients with vancomycin-susceptible Enterococcus faecalis and Enterococcus faecium bacteraemia – a five-year retrospective review, Eur. J. Clin. Microbiol. Infect. Dis. 29 (2010) 107–114. [8] K. Miyake, Innate recognition of lipopolysaccharide by Toll-like receptor 4MD-2, Trends Microbiol. 12 (2004) 186–192. [9] M. Verdrengh, A. Tarkowski, Role of neutrophils in experimental septicemia and septic arthritis induced by Staphylococcus aureus, Infect. Immun. 65 (1997) 2517–2521. [10] L. Ulloa, K.J. Tracey, The ‘‘cytokine profile’’: a code for sepsis, Trends Mol. Med. 11 (2005) 56–63. [11] F.A. Bozza, J.I. Salluh, A.M. Japiassu, M. Soares, E.F. Assis, R.N. Gomes, et al., Cytokine profiles as markers of disease severity in sepsis: a multiplex analysis, Crit. Care 11 (2007) R49. [12] J. Schroder, V. Kahlke, K.H. Staubach, P. Zabel, F. Stuber, Gender differences in human sepsis, Arch. Surg. 133 (1998) 1200–1205.

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