BackgroundMacrophages serve as intracellular reservoirs of S. aureus. Recent in vitro studies have confirmed high level resistance by S. aureus to macrophage mediated killing and the intracellular persistence of Staphylococci may play an important role in the pathogenesis. Since this localization protects them from both cell-mediated and humoral immune responses, therefore, a successful anti-staphylococcal therapy should include the elimination of intracellular bacteria, further protecting the host cells from staphylococci-induced cell death. So, only antibiotic therapy may not be helpful, successful therapy needs combination of drugs not only for elimination of pathogen but also for rescuing the host cell for S. aureus induced cell death.MethodsIn keeping with this idea an in vitro study has been done to examine the effect of Riboflavin along with antibiotics on phagocytosis, hydorgen peroxide, superoxide production, antioxidant enzyme levels, and cytokine levels in mouse macrophages for amelioration of the Staphylococcus aureus burden. The immune boosting effects of Riboflavin have been validated through perturbations of redox homeostasis and pro-inflammatory cytokines measurements.ResultsIt was observed that the supplementation of Vitamin B-2 (Riboflavin) not only enhances macrophage function as previously reported but also decreases pro-inflammatory responses in Staphylococcus aureus infected macrophages. The observed influence of Riboflavin on enhanced antimicrobial effects such as enhanced phagocytosis of macrophages exposed to S. aureus, hydrogen peroxide or superoxide production when combined with either ciprofloxacin (CIP) or Azithromycin (AZM) and decrease in pro-inflammatory responses of IFN-γ, IL-6, IL-1β. Riboflavin treatment also decreased NO and TNF-α level possibly by inhibiting the NF-κβ pathway. The increased antioxidant enzymes like glutathione reductase, SOD and GSH level helped in maintaining a stable redox state in the cell.ConclusionRiboflavin plus antibiotic pretreatment not only enhances macrophage functions but also decreases proinflammatory responses in Staphylococcus aureus infected macrophages indicating better bacterial clearance and regulated inflammation which may be considered as a novel and important therapeutic intervention.

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Among the known Toll-like receptors (TLRs), Toll-like receptor 2 (TLR2) is a key sensor for detecting Staphylococcus aureus invasion. But the function of TLR2 during S. aureus infection in different cell populations is unclear. Two different cell subtypes were chosen to study the interaction of S. aureus with TLR2 because macrophages are extremely different from one compartment to another and their capacity to respond to live bacteria or bacterial products differs from one site to another. The contribution of TLR2 to the host innate response against acute live S. aureus infection and heat-killed S. aureus (HKSA) using anti-TLR2 antibody in murine peritoneal macrophages and resident fresh bone marrow cells has been investigated here. TLR2 blocking before infection induces the release of interleukin (IL)-10 by macrophages thereby inhibiting excessive production of oxidants by activating antioxidant enzymes. TLR2-blocked peritoneal macrophages showed impaired release of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and IL-6 in response to both live and heat-killed S. aureus infection except bone marrow cells. TLR2-mediated free radical production and killing of S. aureus were modulated by TLR2 blocking in peritoneal macrophages and resident bone marrow cells. This study supported that S. aureus persists in resident bone marrow cells in a state of quiescence.

We hypothesized that if internalization of Staphylococcus aureus could be blocked by using cytochalasin D (an inhibitor of phagocytosis and phagolysosome fusion), then the intracellular entry and survival of the pathogen in host's phagocytic cells recruited to the inflammatory site can be restricted. At the same time, if we use antimicrobial agents (e.g., ciprofloxacin and azithromycin) having potent intracellular and extracellular microbicidal activity against the bacterium that have not entered into the phagosome and remains adhered to the phagocytic cell membrane, then they can be eradicated from the site of infection without compromising the host cell. To validate this, role of ciprofloxacin (CIP) and azithromycin (AZM) in eliminating S. aureus by suppressing the phagocytic activity of macrophages with cytochalasin D before infection was investigated. CIP and AZM were used either alone or in combination with cytochalasin D. Supernatant and lysate obtained from the culture of macrophages were used for quantification of reactive oxygen species, lysozymes, antioxidant enzymes, and cytokines produced. Azithromycin was better than ciprofloxacin in combination with cytochalasin D for eradicating S. aureus and regulating cytokine release. Further studies are required for ensuring proper delivery of this combination at the site of infection.

This study aimed to determine any alteration in the killing of Staphylococcus aureus in murine peritoneal macrophages when chloroquine (CQ) is used alone compared with when it is used in combination with ciprofloxacin (CIP) or azithromycin (AZM). The study also aimed to find out the implication of reactive oxygen species (ROS) production and cytokine release in the intracellular killing of S. aureus in macrophages. We present here data obtained with a model of S. aureus-infected mouse peritoneal macrophages in which the intracellular growth of the bacteria and the influence of antibiotics was monitored for 30, 60, and 90 minutes in the presence or absence of CQ along with the production of ROS and alteration in levels of antioxidant enzymes and cytokines. It was observed that S. aureus-triggered cytokine response was regulated when macrophages were co-cultured with CQ and AZM as compared with CQ stimulation only. It can be suggested that action of AZM in mediating bacterial killing is enhanced by the presence of CQ, indicating enhanced uptake of AZM during early infection that may be essential for bacteria killing by AZM. Reduction of oxidative stress burden on the S. aureus-infected macrophages may pave the way for better killing of internalized S. aureus by CQ plus ciprofloxacin (CIP) or CQ plus AZM. Based on these observations, one may speculate that in an inflammatory milieu, CQ loaded with AZM elicits a stronger proinflammatory response by increasing the intracellular uptake of AZM or CIP, thus enabling the immune system to mount a more robust and prolonged response against intracellular pathogens.