Immunological parameters of macrophages infected by methicillin resistant / sensitive Staphylococcus aureus Parâmetros imunológicos de macrófagos frente à infecção por Staphylococcus aureus meticilina sensível / resistente

First submission on 28/06/12; last submission on 29/12/12; accepted for publication on 05/01/13; published on 20/04/13 1. Master’s degree in Tropical Medicine; attending doctorate in Tropical Medicine at Universidade Federal de Pernambuco (UFPE). 2. Biomedical scientist; attending master in Public Health at UFPE. 3. Master’s degree in Tropical Medicine, attending doctorate in Entomology at University of California Riverside. 4. Post-doctorate in Immunology at Universidad de Salamanca-Spain; associate professor at UFPE. ABStrACt

The organism invasion by pathogens may cause deregulation of microbicide response in immune system cells (23) .This mechanism is triggered by several microorganisms, since it enables the development of the infection process and it also optimizes hostage survival (11) .
In order to spread infection, Staphylococcus aureus has developed several mechanisms to resist to the immunological system, which involve countless virulence factors that are coordinately expressed during the different stages of infection.This wide range of factors, either occurring on the surface or secreted in the external medium, may reduce phagocytic microbicide response, induce cellular death and/or inhibit antibiotic action (8,23) .Despite its scientific relevance, there are hardly any studies that evaluate the activation and survival of macrophages after infection by multi-resistant bacterial strains.
Since the 1960's, infections by methicillin resistant S. aureus (MRSA) have become a major public health problem worldwide, inasmuch as it is linked with higher mortality rates (5) .It is believed that MRSA strains have distinctive virulence mechanisms, which may be more exacerbated in comparison with MSSA strains (8) .
Several clinical studies based on morbidity and mortality rates show that MRSA is more virulent than MSSA.Nevertheless, laboratory studies that assess the presence and magnitude of pathogenic mechanisms and virulence factors of MSSA and MRSA strains have yielded conflicting results (12,20) .Thus, the present investigation had the objective to evaluate immunological parameters of alveolar macrophages of rats after in vitro cellular infection with MSSA and MRSA strains.

MAtEriAL AnD MEthoDS
The study protocol was approved by the Ethics Committee in Animal Experiments from Universidade Federal de Pernambuco -Centre of Biological Sciences (Protocol 239/09 -24/11/2009).

Animals
We applied 20 male, albino, Wistar rats (90-120 days) from the vivarium of UFPE-Nutrition Department.The animals were kept in propylene cages with galvanized wire lid, containing 3 animals per cage at most, with free access to water and food.The room temperature was controlled at 22 ± 1ºC, and the animals were exposed to 12:12h photoperiodic light-dark cycles.

Staphylococcus aureus strains
We used methicillin resistant Staphylococcus aureus (The American Type Culture Collection [ATCC] 33591) and methicillin sensitive S. aureus (ATCC 29213) due to its distinctive resistance profile and its relevance concerning public health.The bacteria were maintained in Tryptic Soy Broth (TSB) with 20% glycerol at -20ºC.24 hours prior to each experiment, strains were seeded in blood agar dishes (agar with 5% lamb blood) and incubated at 37ºC.Some colonies were transferred to tubes containing sterile PBS in order to achieve approximately 0.15 nm turbidity by spectrophotometer at 570 nm.According to Lu and McEwan (12) , this absorbance corresponds to 10 6 UFC/ml of PBS.

Bronchoalveolar lavage (BAL)
The animals were anesthetized with chloralose-urethane (0.5 and 12.5%, respectively) at the proportion of 8 ml/kg administered intraperitoneally.The BAL was collected with a saline injection (0.9%) through a plastic cannula inserted in the trachea.Several aliquots of 3 ml were injected and collected in 50 ml conical polypropylene tubes (Falcon, Sigma).We obtained approximately 30 ml of BAL per animal.

Alveolar macrophage culture
The BAL was centrifuged at 1,500 RPM for 15 minutes.The precipitate was resuspended in RPMI 1640 (Gibco-Invitrogen Corporation ® ) with 3% fetal bovine serum (Gibco-Invitrogen Corporation ® ) and antibiotics (penicillin 100 U/ml and streptomycin 100 μg/ml).After dilution in Trypan blue (1:10), the BAL cells were counted with the aid of a Newbauer camera.The concentration 10 6 cells/ml of RPMI was standardized.Subsequently, the cells were transferred to 35 mm diameter culture dishes (6 wells, Falcon ® ).After an hour at 37ºC and 5% CO 2 , the supernatant with non-adherent cells was discarded and 1 ml of RPMI medium was added.

Systems
Two systems were applied for the assessment of phagocytic index: • MSSA, MA and 100 µl of bacterial inoculum of sensitive strain (ATCC 29213); • MSSA, MA and 100 µl of bacterial inoculum of resistant strain (ATCC 33591).
Three systems were established for the adhesion index (AI) and superoxide anion kinetics: negative control (NC), containing exclusively alveolar macrophages, MSSA and MRSA, as described previously.The positive control (PC) was included for the assessment of nitric oxide kinetics and for the viability of alveolar macrophages, which contained MA and 10 µl of LPS (Escherichia coli serotype; 055:B5, Sigma ® ).The plates were incubated at 37ºC in humid atmosphere and 5% of CO 2 .

Adhesion index
After incubation for an hour, 10 µl of culture supernatant was collected and added to Trypan blue at 1:10 dilution.The counting was performed with Newbauer camera.The AI was calculated through the formula described by Fuente et al. (2) : IA = 100-non-adherent cells/ml divided by the initial number of cells/ml × 100.The results were expressed in percentage of non-adherent cells.

Phagocytic index (FI)
In this research, two systems (MSSA and MRSA) were established in test tubes.The contents were homogenized and uniformly distributed onto slides for optic microscopy.The slides were incubated at 37ºC for an hour.Subsequently, they were washed for the removal of non-adherent cells, stained and assessed through optic microscopy by a skilled observer, who was unaware of the systems surveyed herein.The results were expressed in percentage of phagocytic cells after total counting of 100 cells.

Kinetics of anion superoxide production (O 2-)
O 2-was detected by its ability to reduce iron-cytochrome C chemically (30 mg/ml in Hank's Balanced Salt Solution [HBSS], 2.4 × 10-3 M, firon-cytochrome C of horse mitochondrion, type VI, SIGMA Chemical Company, St. Louis, MO).After adding phorbol miristate acetate/ PMA (Sigma ® ) to Hank's solution (HBSS, Gibco-Invitrogen Corporation ® ), 3 systems of discontinuous analysis (NC, MSSA and MRSA) were prepared at 2 μg/ml with assessment between the first and the second hour of incubation.The reading was performed

Kinetics of nitric oxide production (NO)
The NO production was determined through the concentration of supernatant nitric in culture cells (10) .100 µl of supernatant cells was collected every two hours totaling 24 hours of incubation.50 µl of Griess reagent (1.5% of sulfanilamide in 5% of H 3 PO 4 , 0.1% in H 2 O of naphthyl ethylenediamine dihydrochloride) was added to the cell culture supernants for nitric quantification.After 15 minutes at rest and at room temperature, the reading was performed by Elisa (BIORAD, Model 680) with a 550 nm filter.The nitric concentration was calculated through mean values of NaNO 2 standard curve and the collected data were expressed in µM of nitrite/ml.

Viability of alveolar macrophages
The cellular viability was assessed by mitochondrial reduction of 3-[4.5-dimethylthiazol-2-il]-2.5-diphenyltetrazolium bromide (MTT) to formazan, as described by Mosmann (15) .After a 24-hour incubation period, the cell cultures were washed with PBS (1×) at room temperature.Subsequently, they were incubated with 550 µl of PBS and 55 µl of MTT solution for two hours under light protection.After this period, 200 µl of PBS and 200 µl of dimethylsulfoxide (DMSO) were added and, afterwards, the cellular monolayer was scraped.The quantification of formazan solution was performed with ELISA reader (BIORAD, Model 680) with a 570 nm filter.The results were expressed in percentage of viable cells, applying NC results as reference.

Statistical analysis
The analysis of variance (ANOVA) was applied for the comparison of multiple means.When ANOVA results revealed significant difference, Tukey test was performed in order to identify inconsistency in results.In the analysis of only two systems, which were used in the assessment of superoxide kinetics and FI, test t Student was employed for parametric data and Mann-Whitney test for non-parametric data.The results were expressed by mean ± standard error.The significance was defined by p < 0.05.The statistical program employed in the analysis was Sigma Stat ® .
Furthermore, there was no difference as to phagocytic index when comparing MSSA and MRSA strains.Similar results were reported by Salgado et al. (21) , who compared phagocytic susceptibility to MSSA and MRSA.Mekontso-Dessap et al. (14) also observed no difference neither in the phagocytic function of Wistar rat neutrophils nor in the survival rate of bacteria inside the cell when compared with methicillin sensitive and resistant strains.

Kinetics of NO production
The kinetics of NO revealed an increase in the production of NO when compared with other incubation periods (p < 0.001).Similar results were detected in PC, MSSA and MRSA, with p < 0.001.The maximum liberation peak of this free radical occurred after a 22h incubation period in PC (CP-22h: 7.55 ± 0.295 µM/ml), 4h in the MSSA system (MSSA-4h: 11.778 ± 0.413 µM/ml) and 8h in the MRSA system (MRSA-8h: 11.663 ± 0.16 µM/ml).
When comparing NC and PC, there was a higher production of nitrogen reactive species in PC after an 8h incubation period, remaining steady until the end of the analysis (p < 0.001).In the MSSA and MRSA systems, there were differences in the production of NO in the following periods: 4h, 6h, 8h and 10h.Nevertheless, after 12h the nitrite concentration within the systems was similar to NC.During the kinetic period, the mean nitrite production was lower in MRSA in contrast with MSSA, p < 0.05 (Figure 4).

DiSCuSSion
In the present investigation, the coincubation of alveolar macrophages with Staphylococcus aureus strains did not induce any change in the adhesion index of these cells.This finding may be justified by the capacity of Staphylococcus aureus to evade immune system recognition by means of polysaccharide capsules or biofilm The phagocytosis of microorganisms activate oxidase dependant on adenine dinucleotide phosphatese (NADPH), which induces the production of high levels of superoxide, a process commonly denominated respiratory burst.Studies on the release of this anion describe that there is increasing production of O 2-during the first 6h after antigenic stimulus with phorbol myristate acetate (17) .Herein, the generation of O 2-in supernatant of alveolar macrophage culture increased in the first to the second hour of the incubation period in the control systems as well as in MSSA and MRSA strain systems.According to Mandell (13) , regardless of the resistance antimicrobial profile, S. aureus seems to neutralize oxygen intermediates formed after its internalization through the release of catalase and staphyloxanthin enzymes.
As to NO production, there was an increase after stimulation with LPS in all analyzed periods.Ferreira-Silva et al. (24) yielded similar results evaluating the production of NO in Wistar rat macrophages after contact with LPS, which corroborates our investigation.According to Hwang et al. (9) , LPS is regarded as an efficient macrophage stimulator, insofar as it induces the production of NO, hence being a positive control for the activation of these cells.
Pumerantz et al. (16) claimed that No produced by alveolar macrophages play a major microbicide role against S. aureus.According to Sasaki et al. (22) , the expression of induced nitric oxyde synthase is initiated 3h after S. aureus infection.During the kinetics, a maximum peak of NO release was observed after 4h in the MSSA system and 8h in the MRSA system.These results evinced that the resistant strain activates a more delayed microbicide response of macrophages in comparison with sensitive strains.Thus, this may enable the multiplication of resistant bacteria inside the phagocytes, inasmuch as they do not present antimicrobial mechanisms until the macrophagic activation occurs.
After No production peaks, a reduction in the concentration of nitrite was detected in the supernatant of MSSA and MRSA systems, and after a 12 hour incubation period, it was similar to basal levels of NC.According to Richardson et al. (18) , S. aureus may evade multiple components of the innate immune response, including the microbicide action of NO.These researchers proved that S. aureus is able to adapt metabolically to nitrosative stress, inasmuch as it expresses inducible dehydrogenase (NO-L-lactato).Its production allows S. aureus to maintain homeostasis during nitrosative redox stress.
The kinetics of NO production in MSSA and MRSA strains showed that there was oscillation during 4h, 6h, 8h and 10h of incubation.The mean concentration of nitrite during this period was lower in MRSA in comparison with MSSA.This result implies that macrophages may have been activated with lower intensity during the infection by MRSA or that this bacterium may have distinctive mechanisms of evasion to microbicide response.Vaudaux e Waldvogel (25) analyzed PI cytosis of polimorphonuclear leucocytes (PMN) through an in vitro assay.After 30 and 60 minutes of incubation, MRSA strains showed a better survival to phagocytic action of PMN in comparison with MSSA strains.
According to Cutler (1) , methicillin resistant staphyloccocus present thicker cellular walls and different lipidic content in contrast with those sensitive to this antibiotic.Accordingly, it is possible to claim that the morphology of resistant strains enables a better protection against destruction by lysosomal enzymes or by the oxidative metabolism of activated phagocytes.In 2011, Ferreira et al. (6) affirmed that MRSA is a major pathogen in nosocomial infections and it is associated with high rates of mortality opposed to infections by MSSA.
As to the viability rate of Wistar rat alveolar macrophages, there was a reduction after a 24h incubation period with MSSA and MRSA in comparison with PC.This finding may explain the low levels of nitrite in culture supernatant.According to Deleo et al. (3) , there are two possible interactions between bacterium-phagocyte.The bacterial phagocytosis triggers the production of oxygen and nitrogen reactive species as well as lysosome degranulation.This process occurs along with apoptosis induction, hence combating the infection.Alternatively, some bacterial pathogens may cause lysis of phagocytes or induce apoptosis, enabling the bacteria to remain in the medium or the development of the disease.
In order to escape cytoplasmatic confinement, S. aureus induces phagocyte death by triggering pyroptosis or by releasing leukocidins (11) .The main characteristic of pyroptosis is the rupture of the plasmatic membrane with the release of pro-inflammatory intracellular content via caspase-1 (cytoplasmatic protease) (7) .The action mechanism of leukodicins causes leukocyte destruction due to the formation of pores in the cellular membrane.Kubica et al. (11) confirmed that after phagocytosis by macrophages, S. aureus, regardless of its antimicrobial resistance profile, survives intracellularly in vacuoles for 3-4 days prior to escaping into the cytoplasm and ultimate cellular lysis.Besides remaining several days inside macrophages, the bacterium was able to escape intracellular confinement and to proliferate in the hostage organism.
Different bacterial strains may express a higher or lower number of virulence factors such as adhesins and toxins or differ in their ability to produce biofilm and resist to phagocytosis.According to Rossney et al. (19) , the expression regulation of virulence factors plays a pivotal role in the pathogenicity of S. aureus.According to Gordon e Lowy (7) , MRSA strains may contain genetic factors that maximize virulence, which allow these bacteria to cause particular clinical syndromes.Further studies with molecular biology techniques could elucidate the differences in macrophage immune response to methicillin resistant and sensitive strains.

ConCLuSionS
There are some evidences that methicillin resistance constitutes a factor that influences the ability to evade microbicide response in alveolar macrophages from adult Wistar rats.Although the adhesion capacity, PI, O 2-production and viability of infected phagocytes were not altered, there were oscillations in the kinetics of ON production, which may contribute significantly to its pathogenicity and bacterial survival.
linked with enzyme (ELISA-BIORAD, Model 680) with a 55 nm filter.The quantification of O 2-was expressed in nM/ml.

figurE 1 -
figurE 1 -AI of alveolar macrophages from adult Wistar rats for the following systems: NC, MSSA and MRSA.Values expressed as mean ± standard error (n = 20) AI: adhesion index; NC: negative control; MSSA: methicillin sensitive S. aureus; MRSA: methicillin resistant S. aureus.