Figures
Abstract
Background
Dietary selenium is of fundamental importance to maintain optimal immune function and enhance immunity during infection. To this end, we examined the effect of selenium on macrophage bactericidal activities against Staphylococcus aureus.
Methods
Assays were performed in golden Syrian hamsters and peritoneal macrophages cultured with S. aureus and different concentrations of selenium.
Results
Infected and selenium-supplemented animals have significantly decreased levels of serum nitric oxide (NO) production when compared with infected but non-selenium-supplemented animals at day 7 post-infection (p < 0.05). A low dose of 5 ng/mL selenium induced a significant decrease in macrophage NO production, but significant increase in hydrogen peroxide (H2O2) levels (respectively, p = 0.009, p < 0.001). The NO production and H2O2 levels were significantly increased with increasing concentrations of selenium; the optimal macrophage activity levels were reached at 20 ng/mL. The concentration of 5 ng/mL of selenium induced a significant decrease in the bacterial arginase activity but a significant increase in the macrophage arginase activity. The dose of 20 ng/mL selenium induced a significant decrease of bacterial growth (p < 0.0001) and a significant increase in macrophage phagocytic activity, NO production/arginase balance and S. aureus killing (for all comparisons, p < 0.001).
Conclusions
Selenium acts in a dose-dependent manner on macrophage activation, phagocytosis and bacterial killing suggesting that inadequate doses may cause a loss of macrophage bactericidal activities and that selenium supplementation could enhance the in vivo control of immune response to S. aureus.
Citation: Aribi M, Meziane W, Habi S, Boulatika Y, Marchandin H, Aymeric J-L (2015) Macrophage Bactericidal Activities against Staphylococcus aureus Are Enhanced In Vivo by Selenium Supplementation in a Dose-Dependent Manner. PLoS ONE 10(9): e0135515. https://doi.org/10.1371/journal.pone.0135515
Editor: Laurel L. Lenz, University of Colorado School of Medicine, UNITED STATES
Received: February 14, 2015; Accepted: July 22, 2015; Published: September 4, 2015
Copyright: © 2015 Aribi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
Introduction
S. aureus is one of the most frequently isolated pathogens that often cause severe nosocomial infections, such as bloodstream infections, skin and soft tissue infections, and pneumonia. It is also responsible for a variety of suppurative infections and toxin-mediated diseases [1]. The nasal carriage has been associated with several infections including bacteremia, postoperative and diabetic foot ulcer infections, subjects colonized with S. aureus being at greater risk of subsequent infection than uncolonized individuals [2].
Macrophages are the primary professional scavenger cells. They can engulf microorganisms, proteins and other smaller cells using several mechanisms, such as Fc receptor- and complement-mediated phagocytosis, pinocytosis and endocytosis [3]. Macrophages are known to produce various molecules such as nitric oxide (NO) and reactive oxygen species (ROS) in response to phagocytosis and ligands of pattern recognition receptors (PRRs) [4]. The production of ROS and reactive nitrogen species (RNS) radicals are under the control of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and inducible nitric oxide synthase (iNOS), respectively [5].
Activated macrophages produce NO by inducible NO synthase (iNOS), encoded by the NOS2 gene [6], by two successive monooxygenations of L-arginine (L-Arg) [7]. Hydrogen peroxide (H2O2) is one of the most active oxygen species, which is produced in the mitochondria by MnSOD (manganese-containing superoxide dismutase, SOD2) as an end product of plasma membrane associated-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase during the respiratory burst in activated macrophage [8]. Both NO and H2O2 play an important role as cell-signaling molecules and are effector agents for the microbicidal and cytotoxic response of macrophages after stimulation [9]. S. aureus cells can protect themselves against microbicidal agents generated by phagocytes by the expression of numerous virulence factors that allow them to colonize host tissues, to proliferate, and to escape the killing effect of the immune system [10].
Of note, S. aureus can survive intracellularly within phagocytes including neutrophils [11] and macrophages [12], and consequently evade host defenses and antibiotic treatment. Among S. aureus resistance mechanisms to phagocytosis, its ability to cleave the heavy chains of opsonic antibodies, using V8 protease (staphylococcal serine protease A, SspA) has been described [13]. This and other proteolytic enzymes may also be able to degrade the host antimicrobial peptide agents and tissue components [14]. The pathogenicity of S. aureus can also be linked to its ability to produce arginase. S. aureus arginase production modulates the immune system by consumption of the host arginine, resulting in reduced substrate for iNOS, thereby generating reduced amount of NO [15].
In the context of translational medicine, new therapeutic strategies against infectious pathogens using trace elements like selenium have been suggested to counteract pathogen immune evasion. Hence, selenium was used to prevent bacterial colonization on biomaterial surfaces [16] and S. aureus biofilm formation [17]. Supplementation with this micronutrient is usually required for optimum immune response through several enzymes, known as selenoproteins, involved in both innate and adaptive immunity [18]. Here, we tested the role of selenium supplementation on macrophage activities during S. aureus infection.
Materials and Methods
Ethics statement
The study was carried out with Good Laboratory Practices (GLP), and was reviewed and approved by the Institutional Ethics Committee of Tlemcen University. The necessary measures have been planned to prevent and minimize potential animal suffering if pain and distress are observed. In order to assess health and welfare, animals were regularly monitored and ongoing reviews of several criteria of such discomforts were used, including poor grooming, anorexia, decrease in water consumption, dehydration, decreased urination or stool output, vomiting or diarrhea, weight loss or loss of body condition, disorder in body temperature, disorder in pulse or respiratory rate, etc.
Staphylococcus aureus strain
The S. aureus isolate was obtained from the Neonatology Department of Tlemcen University Medical Centre, Algeria. The isolate was a methicillin-susceptible S. aureus (MSSA) as determined by both phenotypic, i.e., disk diffusion method using oxacillin (5 mg) and cefoxitin (30 mg) disks (Bio-Rad) performed and interpreted according to recommendations of the Antibiogram Committee of the French Society of Microbiology, and genotypic methods performed as described previously [19] and showing no amplification of the mecA gene (S1 Fig).
In vivo assays: animals, selenium supplementation and infection methods
A total of eighty (80) male golden Syrian hamsters (Mesocricetus auratus), six-week of age, weighing 60–80 g, were enrolled for a cross-sectional case/control study over six weeks. Hamsters were randomly divided into four equal groups; group 1, SA+/Se+ (n = 20, infected by the MSSA isolate and supplemented with selenium); group 2, SA+/Se- (n = 20, infected by the MSSA isolate and not supplemented with selenium); group 3, SA-/Se+ (n = 20, not infected but supplemented with selenium); group 4, SA-/Se- (n = 20, not infected and not supplemented with selenium, control group). Each group was divided into four subgroups of five hamsters to assess the concentration of circulating nitric oxide in regular time intervals and the levels of immunoglobulins and specific antibodies to evaluate the in vivo effect of selenium supplementation on feedback loop regulating immune response. Groups 1 and 2 were infected at the second week by intraperitoneal injection of S. aureus in a dose of 1 X 107 CFU per mL in 1 mL sterile physiological saline with incomplete Freund's adjuvant. The control group was injected with 1 mL of 0.9% sterile physiological saline. Animals were monitored for clinical signs of S. aureus infection, such as hunched posture and reduced activity [20]. No mortality was recorded. In addition to clinical symptoms, infection was checked by microbiological culture procedure on Chapman medium [21]. The animals did not show signs of pain or distress during the experiment, except for the infection signs seen quite early. Intraperitoneal fluid samples were collected from the third day post-infection to isolate S. aureus and quantify bacterial load in infected groups (SA+/Se+ and SA+/Se-). Selenium supplementation was performed by oral administration of sodium selenite [22], at 0.6 mg/kg/day [23–25]. The amount of selenium in the basal diet was 0.1 mg/kg/day feed as minimal nutrient needs for animals [26]. Supplementation began 2 weeks before infection and continued until 1 day before sacrifice. On days 7, 14, 21 and 28 post-infection, the animals were weighed, and blood samples and intraperitoneal macrophages were collected. For the animal sacrifices, hamsters were euthanized with an overdose of intraperitoneal injection of sodium pentobarbital (150 mg/kg).
Study design
Our experiments were carried out on serum samples, cell lysates and peritoneal macrophages (Fig 1). The evaluation of selenium effects on the serum levels of immunoglobulins, S. aureus-specific antibodies and NO was carried out in hamsters, supplemented (Se+) or not (Se-) with selenium and infected (SA+) or not (SA-) by S. aureus. Bacterial and macrophage lysates were used to measure the effect of selenium on the bacterial and the macrophage arginase activity, respectively. The effect of selenium on the levels of macrophage NO production, H2O2, bacterial growth, phagocytosis and bacterial killing were performed on a mixture of macrophages (2 × 106 cells/mL) from normal hamsters, cultured with S. aureus cells (2 × 107 CFU/mL) and diverse concentrations of selenium (0, 5, 10, 20, 30 and 40 ng/mL). The ex vivo macrophage activation was performed using the NO production and H2O2 assays. Fetal bovine serum (FBS) used in ex-vivo assays was quantified for each selenium level added to the medium. Each experiment was repeated at least four times.
Sample preparation
For the preparation of peritoneal macrophages, hamster macrophages were aseptically harvested from the peritoneal cavity at various time-points by lavage with at least 10 mL sterile ice-cold phosphate-buffered saline (PBS) twice, injected intraperitoneally with gentle abdominal massage, as described elsewhere [27,28]. Macrophages were enriched after the incubation of total peritoneal cells harvested for 2 hours at 37°C to allow macrophages to adhere to tissue culture plastic [28–30]. Macrophages were seeded at 2 × 106 cells/mL in cell culture media. Cells were counted using a hemocytometer under microscopy [31]. Macrophage cells were cultured as reported [32] in Dulbecco's modified Eagle's medium-high glucose (DMEM-HG, Sigma, Germany) with L-glutamine and supplemented with 10% (vol/vol) FBS, without antibiotics.
For the bacterial lysis assay, S. aureus was initially cultured using Brain-Heart Infusion Broth (BHIB) enrichment medium. Nine test tubes each containing 10 mL of BHIB medium were prepared, and 5, 10, 20, 30 and 40 ng/mL of selenium standard solution were respectively added to five of them. Then, each of the 9 tubes were inoculated with a bacterial suspension, and incubated at 37°C for 24 hours. In the next step, the bacterial cells were lysed. To this end, cells were firstly centrifuged at 20,000 x g for 20 min. After removal of the supernatants, the pellets were washed at least 3 times, to remove any remaining culture medium until the medium became colorless. The wash was performed by adding to the cell pellet, 1 mL of PBS buffer, and then continued by centrifugation at 1000 x g for 10 min. The pellet was suspended in 1 mL of lysis solution, 1% Triton X-100, 10 mM Tris-HCl pH 7. After a rigorous mixing followed by centrifugation at 20,000 x g for 20 min, the bacterial lysate was collected from the supernatant and protein content was measured. For the macrophage lysis assay, peritoneal macrophages were washed three times with 1.0 mL of PBS and then lysed by exposure to ice-cold 1% Triton X-100 buffer as reported [33,34]. The protein content of the supernatants was then measured.
Experimental procedures
Protein assay.
Protein concentration was measured at 540 nm by the colorimetric Biuret method using Thermo Scientific kit (Thermo Fisher Scientific Inc., Middletown, USA).
Immunoglobulins and specific antibodies assays.
Total serum IgM and IgG antibody levels were measured quantitatively by agarose gel single radial immunodiffusion (SRID) assay using anti-golden Syrian Hamster IgG (goat, H + L) and IgM (rabbit, μ-chain specific) (Rockland Immunochemicals Inc., Gilbertsville, PA, USA), and respective isotype controls from serum of normal hamsters (BioLegend, San Diego, CA, USA). Specific antibodies were detected spectrophotometrically by ELISA reader at 490 nm using cell lysate, S. aureus antigen and plates treated with peroxidase-conjugated anti-IgG or anti-IgM as described [35].
Nitric oxide assay.
NO levels were assayed by measuring the accumulation of stable oxidative metabolites (NOx, nitrite and nitrate) with the sensitive colorimetric Griess reaction, using trichloracetic acid (TCA), Vanadium (III) chloride and Griess reagent, as previously described [36]. For macrophage NO production, peritoneal macrophages were incubated overnight. One hundred microliters of supernatant was treated with 100μL of Griess reagent (1% sulphanilamide, 0.1% naphtylethylenediamine dihydrochloride, and 5% orthophosphoric acid), and the mixture was incubated at room temperature for 5min. The absorbance was measured at 540nm in a microplate reader. The amount of nitrite in the sample was determined using sodium nitrite for the standard curve [37].
Arginase activity assays.
The tests were carried out using a spectrophotometric assay based on the determination of the production of urea after the addition of L-arginine [38,39]. Bacterial and macrophage lysates were used to measure the effect of selenium on the bacterial and macrophage arginase activities, respectively: the bacterial arginase assay was carried out on a culture without macrophages; the macrophage arginase was performed on removed adherent macrophage cells from the tissue culture plastic after culture in the presence of S. aureus. Briefly, 25 μL of bacterial or macrophage cell lysate were added to 200 μL aliquot of arginine buffer (10 mM L-arginine, pH 6.4), and the mixture was incubated at 37°C for 60 min. The reaction was stopped by adding 750 μL of acetic acid [40], and the amount of urea generated by arginase [41] was analyzed at 600 nm using a commercial kit (UREA/BUN—COLOR, BioSystems, S.A. Costa Brava 30, Barcelona, Spain). The arginase activity was expressed as nmol urea/mg proteins/1hr.
Hydrogen peroxide assay.
H2O2 measurement was carried out spectrophotometrically using commercial kit according to the manufacturer instructions (Sigma-Aldrich, St. Louis, Missouri, USA).
Bacterial growth and CFUs count assay.
The effect of diverse concentrations of selenium (0, 5, 10, 20, 30 and 40 ng/mL) added to the media on bacterial growth was assayed according to the arginase activity. After culture for one hour with macrophage cells, a volume of 0.2 mL of diluted bacterial suspension was plated on the surface of Chapman medium. Following incubation for 24 hours at 37°C, a counting of bacterial colony forming units (CFUs) was performed using ImageJ software (NIH, USA), as reported [42].
Phagocytosis and bacterial killing assays.
Tests of phagocytosis and bacterial killing were performed using a bactericidal assay as described in detail elsewhere [43], with some modifications. Assays were made at 0 and 60 minutes on six-well plates containing either a mixture of macrophages and S. aureus cells with the different concentrations of selenium or bacterial cells alone. The number of viable bacteria was determined microbiologically based on the counting of CFUs on Chapman medium as reported above. The results were expressed as a percentage of phagocytosis and bacterial cells killing, calculated as follows:
with bacterial growth = C60/C0 where C0 and C60 correspond to control sample at 0 minute and 60 minutes respectively. The percentage of viable bacteria =
. M0 corresponds to mixture assay sample at 0 minute and M60 corresponds to mixture assay sample at 60 minutes. EC, mixture assay sample supernatant after centrifugation.
Statistical analysis
All data are expressed as the means ± standard error. Non-parametric Mann-Whitney U test was used to compare the differences between two groups. Comparison of more than two independent groups was carried out using Kruskal-Wallis test. Statistical analyses were performed using SPSS 16.0 (Statistical Package for the Social Sciences, SPSS Inc., Chicago, IL, USA) software. A p-value less than 0.05 was considered statistically significant.
Results
In vivo effect of selenium
All the clinical and behavioral characteristics of the four animal groups were similar at the beginning of the experiment. Additionally, all the last group of hamsters resumed normal and same habit at the end of the experiment, i.e. at day 28 post-infection. For both groups of infected animals, we observed that the number of bacteria decreases progressively and significantly from the third day post-infection (p < 0.001). Although circulating levels of S. aureus-specific antibodies were significantly higher in selenium-supplemented animals compared with those not supplemented (S2 Fig, S1 File), the bacterial eradication appeared later in the first week after infection in selenium-supplemented group (p < 0.01). At day 7 post-infection, the circulating levels of NO were significantly decreased in infected animals compared to non-infected animals, supplemented or not with selenium (respectively, p < 0.01, p < 0.05). They remain decreased until the end of the experiment in infected animals compared to non-infected animals, but the difference did not reach statistical significance (p > 0.05). Additionally, infected and selenium-supplemented animals have significantly decreased levels of serum NO when compared with infected but not selenium-supplemented animals at day 7 post-infection (p < 0.05). In contrast, no significant difference was revealed between the four groups of animals from day 14 post-infection (Fig 2).
SA+/Se+: animals infected by S. aureus and with oral supplementation by 0.6 mg/kg/day selenium, SA+/Se-: infected animals without selenium supplementation, SA-/Se+: non-infected selenium-supplemented animals, controls: not infected and not supplemented group (SA-/Se-). NO: nitric oxide production [NOx, nitrite (NO2-) and nitrate (NO3-)]. d7, d14, d21, d28: numbers of days post-infection. The asterisks indicate a significant difference between infected and control animals as follows: •,*p < 0.05, **p < 0.01. The black dots indicate the difference between SA+/Se+ and SA+/Se-.
Effect of selenium on peritoneal macrophage activation
The addition of a low selenium concentration (5 ng/mL) induced a significant decrease in NO production, but significant increase in H2O2 levels (Fig 3). Additionally, the NO production and H2O2 levels were significantly increased with increasing concentrations of selenium, but then significantly decreased again at 40 ng/mL; the optimal macrophage activity levels were reached between 20 ng/mL and 30 ng/mL (for both variables, p < 0.0001 by Kruskal-Wallis test).
The ex vivo macrophage activation was performed using the NO production and H2O2 assays. NO: nitric oxide production [NOx, nitrite (NO2-) and nitrate (NO3-)], H2O2: hydrogen peroxide. P-values are given with significance degree indicated by asterisks. ***p < 0.0001 by Kruskal-Wallis test.
Effect of selenium on bacterial and peritoneal macrophage arginase activity
The concentration of 5 ng/mL of selenium induced a significant decrease in the activity of the bacterial arginase. This activity varied in the Gaussian form according to selenium concentration and reached a maximum at 20 ng/mL (Fig 4). In contrast, the concentration of 5 ng/mL selenium induced a significant increase in the macrophage arginase activity. This activity also changed as a Gaussian distribution, but reached the optimum at 10 ng/mL. The Kruskal-Wallis test gave p-value < 0.0001 for the two comparisons.
Arginase activity assays were carried out using a spectrophotometric method based on the determination of the production of urea after the addition of L-arginine. P-values are given with significance degree indicated by asterisks: ***p < 0.0001 by Kruskal-Wallis test.
Effect of selenium on peritoneal macrophage NO production/arginase and arginase/NO production ratios
The NO production/arginase ratio decreased significantly with a low selenium concentration corresponding to 5 ng/mL, and then increased significantly from 10 ng/mL to reach the optimal level at 20 ng/mL (Fig 5). Additionally, NO production/arginase ratio changed as a Gaussian distribution. In contrast, the arginase/NO production ratio reached the optimal level with a concentration of 5 ng/mL selenium, and then decreased significantly from 10 ng/mL to reach a lowest level with a dose of 20 ng/mL selenium. For both comparisons, p-value = 0.0001 were obtained by Kruskal-Wallis test.
P-values are given with significance degree indicated by asterisks. **p < 0.001 by Kruskal-Wallis test.
Effect of selenium on S. aureus growth according to the overall arginase activity
The bacterial growth rate was simultaneously increased with increased of overall arginase activity at selenium concentrations between 0 and 10 ng/mL. From 10 to 20 ng/mL selenium, both the rate of bacterial growth and overall arginase activity decreased; nevertheless, the decrease in the arginase activity does not reach the significance level (respectively, p < 0.0001; p = 0.120 by Mann-Whitney U test). Between the doses of 20–40 ng/mL selenium, arginase activity significantly decreased (p = 0.022), but the S. aureus growth rate significantly increased (p = 0.000). For all comparisons using the Kruskal-Wallis test, p-values were < 0.0001 and < 0.001 for bacterial growth and arginase activity, respectively (Fig 6).
Bacterial culture was performed on Chapman medium and counting of CFUs was determined using ImageJ software (NIH, USA). Overall arginase activity was determined as the amount of arginase activity of both peritoneal macrophage and bacteria cultured in the presence of diverse concentrations of selenium (0, 5, 10, 20, 30 and 40 ng/mL). CFU: colony forming unit. P-values are given with significance degree indicated by asterisks. **p < 0.001, ***p < 0.0001 by Kruskal-Wallis test.
Effect of selenium on peritoneal macrophage phagocytosis and bacterial killing
Selenium progressively decreased the phagocytic activity in the range of 5 ng/mL to 10 ng/mL selenium; this activity increased significantly to reach an optimal level with a dose of 20 ng/mL selenium (Fig 7). Inversely, selenium gradually increased the activity of bacterial lysis from a dose of 5 ng/mL. Similarly to the phagocytic activity, bacterial lysis reached an optimal level with a dose of 20 ng/mL selenium. The activity of S. aureus killing by peritoneal macrophage changed as a Gaussian distribution. P-value was < 0.0001 for the two comparisons using the Kruskal-Wallis test.
Macrophages were isolated from non-infected and non-selenium-supplemented hamsters. Assays were made at 0 and 1hr on six-well plates containing either a mixture of macrophages and S. aureus cells with the differente concentrations of selenium or bacterial cells alone. The results were expressed as a percentage of phagocytosis and bacterial cells killing. P-values are given with significance degree indicated by asterisks. ***p < 0.0001 by Kruskal-Wallis test.
Discussion
Selenium is the main component of selenoproteins and functions as a redox centre [44]. It is transported in blood with selenoprotein P (SePP) and can be stored as selenomethionine (SeMet) in different tissues and organs, such as liver, kidney, pancreas, heart, brain and spleen. Sodium selenite, a common dietary form of selenium, is an essential micronutrient as well as a toxic trace element in animal and human nutrition.
The strong involvement of selenoproteins in various metabolic pathways may explain the importance of selenium status in several vital functions like immune defense. It has been suggested that it modifies the interactions between macrophages and lymphocytes or acts as an antioxidant on the cells involved in immunological reactions [45]. It also acts as a modulator of inflammatory and immune responses by helping to control peroxide concentration at sites of inflammation, and by decreasing leukotriene synthesis and stimulating cellular immunity [46]. Additionally, selenium: i) plays an important role in the regulation of the expression of pro-inflammatory genes and cytokines and of their receptors on immune cells [47], ii) can suppress the expression of cyclooxygenase (COX)-2, prostaglandin E2 (PGE2), interferon (IFN)-regulatory factor 3 (IRF3) and tumour necrosis factor (TNF)-α, and modulate both myeloid differentiating factor 88 (MyD88)- and TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent signaling pathways of toll-like receptors (TLRs) leading to decreased inflammatory gene expression [48], iii) can reduce markers of inflammation in patients with systemic inflammatory response syndrome (SIRS) and sepsis [49], and iv) can mediates inhibition of the activation of the transcription factor NF-kB, which regulates genes that encode inflammatory cytokines [50]. In this context, our study aimed at investigating the role of selenium supplementation on macrophage activity during S. aureus infection and significant effects of selenium were demonstrated on macrophage activation, phagocytosis and bacterial killing.
In vivo effect of selenium on serum nitric oxide
The activation of the immune system by bacterial products induces the release of reactive oxygen or nitrogen species. Among these, NO is useful in the initial phase of the innate immune response by acting as a proinflammatory molecule and can act as a potent microbicidal agent, which prevents the development of a number of microorganisms, including viruses, bacteria, fungi, and parasites. The antimicrobial properties of NO can be the result of several actions on DNA, proteins and lipids. Besides these antimicrobial properties, it can also induce apoptosis and cell cycle arrest [51].
On the other hand, NO is also critically involved in the regulation of a number of diverse biological processes, including innate immunity and immunological host defense against invading pathogens and wound healing [52]. It is produced from L-arginine by the enzyme NOS from different cells. Various inflammatory cytokines or TLR agonists may induce the expression of iNOS. The production of NO particularly through expression of the isoform iNOS induced by inflammatory cytokines or TLR agonists occurs in a variety of microbial infections as an effective antimicrobial agent. Several studies have shown that serum NO levels were significantly higher in many animal species during inflammation due to microbial infection, and that these levels decrease under anti-inflammatory treatment [53,54]. Consequently, NO can be used as a clinical marker to evaluate the efficacy of anti-inflammatory drugs. Contrarily to these previous observations, the serum NO levels were significantly decreased in early S. aureus infection in our study. Moreover, selenium supplementation induces further decreasing of NO levels, which may reflect the role of selenium as a modulator of inflammatory responses. Thereby, our results corroborate those of previous researches showing the inhibitory effect of selenium on the levels of NO production [48,55].
The inhibition of bacterial growth was shown less important during the first week of infection in selenium-supplemented animals compared with those not supplemented. Although S. aureus-specific antibodies levels were higher in supplemented animals compared with those of not supplemented, those of NO were decreased. Altogether, these results suggested that the process of in vivo eradication of S. aureus would be much more effective in the presence of mediators of non-adaptive immunity, like NO.
Effect of selenium on peritoneal macrophage activation
Macrophages are considered as key components of the innate immune system, but also as orchestrator of adaptive immunity [56]. They can directly kill microorganisms and promote their clearance by phagocytosis. Their activation and microbicidal function are characterized by the release of many effectors, like NO and H2O2 [57,58].
H2O2 generated from activated macrophage is produced by respiratory/oxidative burst based on the increased activation of membrane-associated NADPH oxidase complexes [59], and may be induced by pathogens opsonized with complement factor C3b/iC3b, IgG-coated bacteria to Fc receptors, C3a- and C5a-mediated chemotaxis, etc. [60,61]. It can enhance the bacterial killing power, but can also have adverse effects on the cells and the surrounding healthy tissue. H2O2 also functions as a signaling agent, particularly in higher organisms [62]. It is freely diffusible within and between cell membranes, but can be decomposed biologically by catalase, produced by some bacteria, such as S. aureus, to protect against the host respiratory burst [63], and consequently to evade hosts' immune defense to a certain extent.
It has been recently demonstrated that optimal selenium concentration is critical for macrophage activation and resolution of inflammation [64]. We observed that selenium affects macrophage activation in a dose-dependent manner, a low dose impairing NO production but increasing H2O2 levels. These results are in accordance with previous studies showing that during infection, antioxidant nutrients commonly included in the diet such as selenium are absolutely necessary to regulate the reactions that release free radicals [65]. Indeed, selenium affects NF-κB activation, which regulates genes that encode inflammatory cytokines [66], may be able to downregulate the LPS-induced expression of iNOS and other proinflammatory genes in macrophages, and its deficiency increases the expression of iNOS [67,68].
Effect of selenium on bacterial arginase activity
Among many virulence factors, arginases secreted by S. aureus can compete with iNOS for their common substrate, the amino acid L-arginine (L-Arg) [69]. Consequently, the NO production in activated macrophages will be reduced, since its translation is strongly dependent on the availability of L-Arg in the macrophage. The decreased levels of NO then influence negatively on bactericidal activity and phagocytosis, and impact the expression of an NO-inducible L-lactate dehydrogenase (Sa-LDH-1) by S. aureus [70]. This ultimately results in improved resistance of S. aureus to oxidative stress, increased growth, bacterial survival and persistence.
We showed a dose-dependent effect of selenium on the activity of both bacterial and macrophage arginase. A low concentration of selenium of 5 ng/mL induced a strong alteration of the bacterial arginase activity, thereby counteracting the effects described above for this virulence factor.
Effect of selenium on peritoneal macrophage arginase activity and NO production/arginase balance
The lower concentration of selenium tested (5 ng/mL) also induced a significant decrease in the concentration of NO production, a significant increase in the macrophage arginase activity, and, consequently, a significant decrease in NO production/macrophage arginase activity ratio. In contrast, high concentrations of selenium could allow optimum expression of NO and a sharp decrease in the arginase activity. It is therefore important to remember that arginases and various NOS compete for the same substrate L-Arg and that their enzymatic activities can be inhibited reciprocally [71–73]. The use the NO production/arginase balance and not the results of NO production or arginase activity alone to evaluate the bactericidal efficacy against the bacterial escape is therefore essential.
Nevertheless, the concentration of NO produced by macrophages in vitro may not fully reflect the concentration of NO in vivo. The differences between the in vivo and in vitro assays could partly be due to dose effects, selenium metabolism and pharmacology.
Effect of selenium on S. aureus growth according to the overall arginase activity
Several new therapeutic approaches using various selenium compounds and nanoparticles have been developed to inhibit S. aureus growth and pathogenesis. Among them, selenium nanoparticles synthesized from sodium selenite were shown to inhibit S. aureus growth in vitro [74,75]. Selenium concentration that may be delivered by such nanoparticules at the infection site has to be considered with caution because we showed that dose-dependent effects of selenium on S. aureus growth, 20 ng/mL selenium causing both significant growth inhibition of S. aureus and decrease of arginase activity while doses higher than 20 ng/mL could, on the contrary, increase bacterial colony number despite their ability to decrease the overall arginase activity.
Effect of selenium on peritoneal macrophage phagocytosis and bacterial killing
We showed that selenium supplementation can enhance phagocytosis and bactericidal capacity in a dose-dependent manner reaching a maximum level for a 20 ng/mL selenium dose. However, mechanisms impacted by selenium could not be totally deciphered because of the complexity and diversity of involved factors. Indeed, several well-defined surface components present in most clinical isolates of S. aureus also prevent opsonization and phagocytosis, such as polysaccharide capsule [76]. Additionally, most strains produce chemotaxis inhibitory protein [77] that can delay leukocyte migration toward the site of infection [78] and secrete various C3 complement inhibitors [79]. Finally, the ability of S. aureus to form three-dimensional biofilms during the infectious cycle [80] can also prevents phagocytosis [81]. Currently, the impact of selenium on these different factors remain largely unknown despite selenium has been shown to influence several cells or functions involved in innate immunity like lymphokine activated killer cells and macrophages, the tumor cytotoxicity by mouse macrophages, and the phagocytic function of neutrophils and macrophages [82–85].
Conclusions and Future Prospects
Selenium supplementation can enhance the in vivo control of immune response to S. aureus. To the best of our knowledge, we reported here for the first time that during the initial phase of infection, the in vivo bacterial eradication processes would strongly be based on the mediators of the non-adaptive immunity, such as NO. In fact, during this phase, the selenium administration induced a decrease in circulating levels of NO, and simultaneously a low inhibition of bacterial growth. In the ex vivo experiments, we observed that selenium acts in a dose-dependent manner on macrophage activation, phagocytosis and bacterial killing, suggesting that inadequate doses may cause a loss of macrophage bactericidal activities. These results warrant further investigations and among others, it would be of particular interest to: i) complete arginase activity study by qPCR-based expression or western immunoblotting assays, ii) conduct a kinetic study in hamsters using different doses of sodium selenite, iii) check whether it would be possible to replicate our results using other cellular models of macrophage from different origins, such as bone marrow and spleen, and iv) monitor bacterial load in peritoneal fluid samples and follow disease progression and outcome. In fine, clinical evaluation will still be required to determine the in vivo impact and optimal conditions of selenium administration in patients with S. aureus infection.
Supporting Information
S1 Fig. mecA gene amplification for the Staphylococcus aureus isolate.
Lanes 1 to 4, positive control (amplification product of 528 pb), GeneRuler 100-bp DNA Ladder (Fermentas), negative control, methicillin-suceptible S. aureus (MSSA) isolate. Positive and negative controls are clinical isolates of S. aureus, respectively mecA+ and mecA- (Laboratoire de Bactériologie, Centre Hospitalier Régional Universitaire de Montpellier).
https://doi.org/10.1371/journal.pone.0135515.s001
(TIF)
S2 Fig. Effect of selenium supplementation on circulating levels of immunoglobulins and specific antibodies.
SA+/Se+: infected and selenium-supplemented animals, SA+/Se-: infected animals without selenium supplementation, Controls: not infected and not supplemented group (SA-/Se-), Sp: specific immunoglobulin M (SpIgM) or G (SpIgG). *p < 0.05.
https://doi.org/10.1371/journal.pone.0135515.s002
(TIF)
Acknowledgments
The authors are grateful to Dr. MC Smahi and Dr. SA Rebiahi for providing S. aureus clinical isolate.
Author Contributions
Conceived and designed the experiments: MA JLA HM. Performed the experiments: WM SH YB. Analyzed the data: MA JLA HM. Contributed reagents/materials/analysis tools: MA HM. Wrote the paper: MA. Critical reading of the manuscript: HM JLA.
References
- 1. Rasigade JP, Thomas D, Perpoint T, Peyramond D, Chidiac C, Etienne J, et al. T-cell response to superantigen restimulation during menstrual toxic shock syndrome. FEMS Immunol Med Microbiol 2011; 62: 368–371. pmid:21492259
- 2. Weidenmaier C, Goerke C, Wolz C. Staphylococcus aureus determinants for nasal colonization. Trends Microbiol 2012; 20: 243–250. pmid:22494802
- 3. Woods J, Lu Q, Ceddia MA, Lowder T. Special feature for the Olympics: effects of exercise on the immune system: exercise-induced modulation of macrophage function. Immunol Cell Biol 2000; 78: 545–553. pmid:11050538
- 4. Horta MF, Mendes BP, Roma EH, Noronha FS, Macêdo JP, Oliveira LS, et al. Reactive oxygen species and nitric oxide in cutaneous leishmaniasis. J Parasitol Res 2012; 2012: 203818. pmid:22570765
- 5. Karimian P, Kavoosi G, Amirghofran Z. Anti-oxidative and anti-inflammatory effects of Tagetes minuta essential oil in activated macrophages. Asian Pac J Trop Biomed 2014; 4: 219–227. pmid:25182441
- 6. Myers JT, Tsang AW, Swanson JA. Localized reactive oxygen and nitrogen intermediates inhibit escape of Listeria monocytogenes from vacuoles in activated macrophages. J Immunol 2003; 17: 5447–5453.
- 7. Stuehr DJ, Kwon NS, Nathan CF, Griffith OW, Feldman PL, Wiseman J. N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine. J Biol Chem 1991; 266: 6259–6263. pmid:1706713
- 8. Forman HJ, Torres M. Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. Am J Respir Crit Care Med 2002; 166: 4–8.
- 9. Vatansever F, de Melo WC, Avci P, Vecchio D, Sadasivam M, Gupta A, et al. Antimicrobial strategies centered around reactive oxygen species—bactericidal antibiotics, photodynamic therapy, and beyond. FEMS Microbiol Rev 2013; 37: 955–989. pmid:23802986
- 10. Fraunholz M, Sinha B. Intracellular Staphylococcus aureus: live-in and let die. Front Cell Infect Microbiol 2012; 2: 43. pmid:22919634
- 11. Thwaites GE, Gant V. Are bloodstream leukocytes Trojan Horses for the metastasis of Staphylococcus aureus. Nat Rev Microbiol 2011; 9: 215–222. pmid:21297670
- 12. Kubica M, Guzik K, Koziel J, Zarebski M, Richter W, Gajkowska B, et al. A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages. PLoS One 2008; 3: e1409. pmid:18183290
- 13. Nair SP, Williams RJ, Henderson B. Advances in our understanding of the bone and joint pathology caused by Staphylococcus aureus infection. Rheumatology (Oxford) 2000; 39: 821–834.
- 14. Aslam R, Marban C, Corazzol C, Jehl F, Delalande F, Van Dorsselaer A, et al. Cateslytin, a chromogranin A derived peptide is active against Staphylococcus aureus and resistant to degradation by its proteases. PLoS One 2013; 8: e68993. pmid:23894389
- 15. Lahiri A, Das P, Chakravortty D. New tricks new ways: exploitation of a multifunctional enzyme arginase by pathogens. Virulence 2010; 1: 563–565. pmid:21178497
- 16. Mathews SM, Spallholz JE, Grimson MJ, Dubielzig RR, Gray T, Reid TW. Prevention of bacterial colonization of contact lenses with covalently attached selenium and effects on the rabbit cornea. Cornea 2006; 25: 806–814. pmid:17068458
- 17. Tran PL, Lowry N, Campbell T, Reid TW, Webster DR, Tobin E, et al. An organoselenium compound inhibits Staphylococcus aureus biofilms on hemodialysis catheters in vivo. Antimicrob Agents Chemother 2012; 56: 972–978. pmid:22123688
- 18.
Gladyshev VN. Selenoproteins and selenoproteomes. In: Hatfield DL, Berry MJ, Gladyshev VN, editors. Selenium: Its molecular biology and role in human health. 2nd ed. New York: Springer; 2006. pp. 99–114.
- 19. Predari SC, Ligozzi M, Fontana R. Genotypic identification of methicillin-resistant coagulase-negative staphylococci by polymerase chain reaction. Antimicrob Agents Chemother 1991; 35: 2568–2573. pmid:1810190
- 20. Krut O, Utermöhlen O, Schlossherr X, Krönke M. Strain-specific association of cytotoxic activity and virulence of clinical Staphylococcus aureus isolates. Infect Immun 2003; 71: 2716–2723. pmid:12704146
- 21. de Andrade D, Angerami EL, Padovani CR. A bacteriological study of hospital beds before and after disinfection with phenolic disinfectant. Rev Panam Salud Public 2000; 7: 179–184.
- 22. Poulsen HD, Danielsen V, Nielsen TK, Wolstrup C. Excessive dietary selenium to primiparous sows and their offspring. I. Influence on reproduction and growth. Acta Vet Scand 1989; 30: 371–378. pmid:2640772
- 23. Chen K, Fang J, Peng X, Cui H, Chen J, Wang F, et al. Effect of selenium supplementation on aflatoxin B1-induced histopathological lesions and apoptosis in bursa of Fabricius in broilers. Food Chem Toxicol 2014; 74: 91–97. pmid:25261862
- 24. Krstić B, Jokić Z, Pavlović Z, Zivković D. Options for the production of selenized chicken meat. Biol Trace Elem Res 2012; 146: 68–72. pmid:21986861
- 25. Akil M, Bicer M, Menevse E, Baltaci AK, Mogulkoc R. Selenium supplementation prevents lipid peroxidation caused by arduous exercise in rat brain tissue. Bratisl Lek Listy 2011; 112: 314–317. pmid:21692404
- 26. Zeng H, Cao JJ, Combs GF Jr. Selenium in bone health: roles in antioxidant protection and cell proliferation. Nutrients 2013; 5: 97–110. pmid:23306191
- 27. Johnston MJ, Wang A, Catarino ME, Ball L, Phan VC, MacDonald JA, et al. Extracts of the rat tapeworm, Hymenolepis diminuta, suppress macrophage activation in vitro and alleviate chemically induced colitis in mice. Infect Immun 2010; 78: 1364–1375. pmid:20028812
- 28. Dai JH, Iwatani Y, Ishida T, Terunuma H, Kasai H, Iwakula Y, et al. Glycyrrhizin enhances interleukin-12 production in peritoneal macrophages. Immunology 2001; 103: 235–243. pmid:11412311
- 29. Shi C, Sakuma M, Mooroka T, Liscoe A, Gao H, Croce KJ, et al. Down-regulation of the forkhead transcription factor Foxp1 is required for monocyte differentiation and macrophage function. Blood 2008; 112: 4699–4711. pmid:18799727
- 30. Nomura F, Akashi S, Sakao Y, Sato S, Kawai T, Matsumoto M, et al. Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression. J Immunol 2000; 164: 3476–3479. pmid:10725699
- 31. Urao N, Razvi M, Oshikawa J, McKinney RD, Chavda R, Bahou WF, et al. IQGAP1 is involved in post-ischemic neovascularization by regulating angiogenesis and macrophage infiltration. PLoS One 2010; 5: e13440. pmid:20976168
- 32. Trevino-Villarreal JH, Vera-Cabrera L, Valero-Guillén PL, Salinas-Carmona MC. Nocardia brasiliensis cell wall lipids modulate macrophage and dendritic responses that favor development of experimental actinomycetoma in BALB/c mice. Infect Immun 2012; 80: 3587–3601. pmid:22851755
- 33. Dace DS, Khan AA, Kelly J, Apte RS. Interleukin-10 promotes pathological angiogenesis by regulating macrophage response to hypoxia during development. PLoS One 2008; 3: e3381. pmid:18852882
- 34. Hassall DG, Graham A. Changes in free cholesterol content, measured by filipin fluorescence and flow cytometry, correlate with changes in cholesterol biosynthesis in THP-1 macrophages. Cytometry 1995; 21: 352–362. pmid:8608733
- 35. Teixeira FM, Fernandes BF, Rezende AB, Machado RR, Alves CC, Perobelli SM, et al. Staphylococcus aureus infection after splenectomy and splenic autotransplantation in BALB/c mice. Clin Exp Immunol 2008; 154: 255–263. pmid:18782329
- 36. Guevara I, Iwanejko J, Dembińska-Kieć A, Pankiewicz J, Wanat A, Anna P, et al. Determination of nitrite/nitrate in human biological material by the simple Griess reaction. Clin Chim Acta 1998; 274: 177–188. pmid:9694586
- 37. Sánchez-Miranda E, Lemus-Bautista J, Pérez S, Pérez-Ramos J. Effect of kramecyne on the inflammatory response in lipopolysaccharide-stimulated peritoneal macrophages. Evid Based Complement Alternat Med 2013; 2013: 762020. pmid:23573152
- 38. Klasen S, Hammermann R, Fuhrmann M, Lindemann D, Beck KF, Pfeilschifter J, et al. Glucocorticoids inhibit lipopolysaccharide-induced up-regulation of arginase in rat alveolar macrophages. Br J Pharmacol 2001; 132: 1349–1357. pmid:11250887
- 39. Corraliza IM, Soler G, Eichmann K, Modolell M. Arginase induction by suppressors of nitric oxide synthesis (IL-4, IL-10 and PGE2) in murine bone-marrow-derived macrophages. Biochem Biophys Res Commun 1995; 206: 667–673. pmid:7530004
- 40. Zhang X, Zhang J, Zhang R, Guo Y, Wu C, Mao X, et al. Structural, enzymatic and biochemical studies on Helicobacter pylori arginase. Int J Biochem Cell Biol 2013; 45: 995–1002. pmid:23454280
- 41. Li Z, Zhao ZJ, Zhu XQ, Ren QS, Nie FF, Gao JM, et al. Differences in iNOS and arginase expression and activity in the macrophages of rats are responsible for the resistance against T. gondii infection. PLoS One 2012; 7: e35834. pmid:22558235
- 42. Geissmann Q. OpenCFU, a new free and open-source software to count cell colonies and other circular objects. PLoS One 2013; 8: e54072. pmid:23457446
- 43. Mehrzad J, Duchateau L, Burvenich C. Phagocytic and bactericidal activity of blood and milk-resident neutrophils against Staphylococcus aureus in primiparous and multiparous cows during early lactation. Vet Microbiol 2009; 134: 106–112. pmid:18954946
- 44. Rayman MP. The importance of selenium to human health. Lancet 2000; 356: 233–241. pmid:10963212
- 45. Giadinis N, Koptopoulos G, Roubles N, Siarkou V, Papasteriades A. Selenium and vitamin E effect on antibody production of sheep vaccinated against enzootic abortion (Chlamydia psittaci). Comp Immunol Microbiol Infect Dis 2000; 23: 129–137. pmid:10670702
- 46. Ducros V, Favier A. Selenium metabolism. EMC Endocrinol 2004; 1: 19–28.
- 47. Vunta H, Davis F, Palempalli UD, Bhat D, Arner RJ, Thompson JT, et al. The anti-inflammatory effects of selenium are mediated through 15-deoxy-Delta12,14-prostaglandin J2 in macrophages. J Biol Chem 2007; 282: 17964–17973. pmid:17439952
- 48. Youn HS, Lim HJ, Choi YJ, Lee JY, Lee MY, Ryu JH. Selenium suppresses the activation of transcription factor NF-kappa B and IRF3 induced by TLR3 or TLR4 agonists. Int Immunopharmacol 2008; 8: 495–501. pmid:18279804
- 49. Valenta J, Brodska H, Drabek T, Hendl J, Kazda A. High-dose selenium substitution in sepsis: a prospective randomized clinical trial. Intensive Care Med 2011; 37: 808–815. pmid:21347869
- 50. Maehira F, Miyagi I, Eguchi Y. Selenium regulates transcription factor NF-kappaB activation during the acute phase reaction. Clin Chim Acta 2003; 334: 163–171. pmid:12867288
- 51. Meraz IM, Savage DJ, Segura-Ibarra V, Li J, Rhudy J, Gu J, et al. Adjuvant cationic liposomes presenting MPL and IL-12 induce cell death, suppress tumor growth, and alter the cellular phenotype of tumors in a murine model of breast cancer. Mol Pharm 2014; 11: 3484–3491. pmid:25179345
- 52. Fox S, Wilkinson TS, Wheatley PS, Xiao B, Morris RE, Sutherland A, et al. NO-loaded Zn(2+)-exchanged zeolite materials: a potential bifunctional anti-bacterial strategy. Acta Biomater 2010; 6: 1515–1521. pmid:19861185
- 53. Shih YT, Wu DC, Liu CM, Yang YC, Chen IJ, Lo YC. San-Huang-Xie-Xin-Tang inhibits Helicobacter pylori-induced inflammation in human gastric epithelial AGS cells. J Ethno pharmacol 2007; 112: 537–544.
- 54. Fritzsche C, Schleicher U, Bogdan C. Endothelial nitric oxide synthase limits the inflammatory response in mouse cutaneous leishmaniasis. Immunobiology 2010; 215: 826–832. pmid:20576313
- 55. Yun CH, Yang JS, Kang SS, Yang Y, Cho JH, Son CG, et al. NF-kappaB signaling pathway, not IFN-beta/STAT1, is responsible for the selenium suppression of LPS-induced nitric oxide production. Int Immunopharmacol 2007; 7: 1192–1198. pmid:17630198
- 56. Silva MT. When two is better than one: macrophages and neutrophils work in concert in innate immunity as complementary and cooperative partners of a myeloid phagocyte system. J Leukoc Biol 2010; 87: 93–106. pmid:20052802
- 57. Xia Y, Zweier JL. Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci U S A 1997; 94: 6954–6958. pmid:9192673
- 58. Ding AH, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol 1988; 141: 2407–2412. pmid:3139757
- 59. More P, Pai K. In vitro NADH-oxidase, NADPH-oxidase and myeloperoxidase activity of macrophages after Tinospora cordifolia (guduchi) treatment. Immunopharmacol Immunotoxicol 2012; 34: 368–372. pmid:22295977
- 60. Wrann CD, Winter SW, Barkhausen T, Hildebrand F, Krettek C, Riedemann NC. Distinct involvement of p38-, ERK1/2 and PKC signaling pathways in C5a-mediated priming of oxidative burst in phagocytic cells. Cell Immunol 2007; 245: 63–69. pmid:17507002
- 61. Rotllant J, Parra D, Peters R, Boshra H, Sunyer JO. Generation, purification and functional characterization of three C3a anaphylatoxins in rainbow trout: role in leukocyte chemotaxis and respiratory burst. Dev Comp Immunol 2004; 28: 815–828. pmid:15043949
- 62. Stone JR, Yang S. Hydrogen peroxide: a signaling messenger. Antioxid Redox Signal 2006; 8: 243–270. pmid:16677071
- 63. Kraus D, Peschel A. Staphylococcus aureus evasion of innate antimicrobial defense. Future Microbiol 2008; 3: 437–451. pmid:18651815
- 64. Nelson SM, Lei X, Prabhu KS. Selenium levels affect the IL-4-induced expression of alternative activation markers in murine macrophages. J Nutr 2011; 141: 1754–1761. pmid:21775527
- 65. Puertollano MA, Puertollano E, de Cienfuegos GÁ, de Pablo MA. Dietary antioxidants: immunity and host defense. Curr Top Med Chem 2011; 11: 1752–1766. pmid:21506934
- 66. Maehira F, Miyagi I, Eguchi Y. Selenium regulates transcription factor NF-kappaB activation during the acute phase reaction. Clin Chim Acta 2003; 334: 163–171. pmid:12867288
- 67. Vunta H, Belda BJ, Arner RJ, Channa Reddy C, Vanden Heuvel JP, Sandeep Prabhu K. Selenium attenuates pro-inflammatory gene expression in macrophages. Mol Nutr Food Res 2008; 52: 1316–1323. pmid:18481333
- 68. Prabhu KS, Zamamiri-Davis F, Stewart JB, Thompson JT, Sordillo LM, Reddy CC. Selenium deficiency increases the expression of inducible nitric oxide synthase in RAW 264.7 macrophages: role of nuclear factor-kappaB in up-regulation. Biochem J 2002; 366: 203–209. pmid:12006087
- 69. Bronte V, Zanovello P. Regulation of immune responses by L-arginine metabolism. Nat Rev Immunol 2005; 5: 641–654. pmid:16056256
- 70. Richardson AR, Libby SJ, Fang FC. A nitric oxide-inducible lactate dehydrogenase enables Staphylococcus aureus to resist innate immunity. Science 2008; 319: 1672–1676. pmid:18356528
- 71. Rutschman R, Lang R, Hesse M, Ihle JN, Wynn TA, Murray PJ. Cutting edge: Stat6-dependent substrate depletion regulates nitric oxide production. J Immunol 2001; 166: 2173–2177. pmid:11160269
- 72. Colleluori DM, Ash DE. Classical and slow-binding inhibitors of human type II arginase. Biochemistry 2001; 40: 9356–9362. pmid:11478904
- 73. Szabó C, Southan GJ, Wood E, Thiemermann C, Vane JR. Inhibition by spermine of the induction of nitric oxide synthase in J774.2 macrophages: requirement of a serum factor. Br J Pharmacol 1994; 112: 355–356. pmid:7521253
- 74. Chudobova D, Cihalova K, Dostalova S, Ruttkay-Nedecky B, Rodrigo MA, Tmejova K, et al. Comparison of the effects of silver phosphate and selenium nanoparticles on Staphylococcus aureus growth reveals potential for selenium particles to prevent infection. FEMS Microbiol Lett 2014; 351: 195–201. pmid:24313683
- 75. Tran PA, Webster TJ. Selenium nanoparticles inhibit Staphylococcus aureus growth. Int J Nanomedicine 2011; 6:1553–1558. pmid:21845045
- 76. Foster TJ. Immune evasion by staphylococci. Nat Rev Microbiol 2005; 3: 948–958. pmid:16322743
- 77. Wright AJ, Higginbottom A, Philippe D, Upadhyay A, Bagby S, Read RC, et al. Characterisation of receptor binding by the chemotaxis inhibitory protein of Staphylococcus aureus and the effects of the host immune response. Mol Immunol 2007; 44: 2507–2517. pmid:17258808
- 78. de Haas CJ, Veldkamp KE, Peschel A, Weerkamp F, Van Wamel WJ, Heezius EC, et al. Chemotaxis inhibitory protein of Staphylococcus aureus, a bacterial antiinflammatory agent. J Exp Med 2004; 199: 687–695. pmid:14993252
- 79. Amdahl H, Jongerius I, Meri T, Pasanen T, Hyvärinen S, Haapasalo K, et al. Staphylococcal Ecb protein and host complement regulator factor H enhance functions of each other in bacterial immune evasion. J Immunol 2013; 191: 1775–1784. pmid:23863906
- 80. Vanhommerig E, Moons P, Pirici D, Lammens C, Hernalsteens JP, De Greve H, et al. Comparison of biofilm formation between major clonal lineages of methicillin resistant Staphylococcus aureus. PLoS One 2014; 9: e104561. pmid:25105505
- 81. Thurlow LR, Hanke ML, Fritz T, Angle A, Aldrich A, Williams SH, et al. Staphylococcus aureus biofilms prevent macrophage phagocytosis and attenuate inflammation in vivo. J Immunol 2011; 186: 6585–6596. pmid:21525381
- 82. Erickson KL, Medina EA, Hubbard NE. Micronutrients and innate immunity. J Infect Dis 2000; 182: S5–S10. pmid:10944478
- 83. Kiremidjian-Schumacher L, Roy M. Selenium and immune function. Z Ernahrungswiss 1998; 37 Suppl 1: 50–56. pmid:9558729
- 84. Urban T, Jarstrand C. Selenium effects on human neutrophilic granulocyte function in vitro. Immunopharmacology 1986; 12: 167–172. pmid:3771194
- 85. Safir N, Wendel A, Saile R, Chabraoui L. The effect of selenium on immune functions of J774.1 cells. Clin Chem Lab Med 2003; 41: 1005–1011. pmid:12964805