Biswadev Bishayi

Staphylococcal catalase protects intracellularly survived bacteria by destroying H2O2 produced by the murine peritoneal macrophages.

To determine the interrelationship between the hydrogen peroxide (H(2)O(2)) mediated killing and the potential role of bacterial catalase and SOD in the evasion of host defense, we examined three clinical isolates of Staphylococcus aureus and evaluated their intracellular survival mechanism within murine peritoneal macrophages. Fluorescent microscopy and bacterial colony-forming unit (cfu) count revealed that phagocytic capacity of murine peritoneal macrophages was highest after 2h of in vitro infection with S. aureus. To understand whether catalase and SOD contributing in the intracellular survival, were of bacterial origin or not, 3 amino 1,2,4 triazole (ATZ) and Diethyldithiocarbamic acid (DDC) were used to inhibit specifically macrophage derived catalase and SOD respectively. Catalase activity from the whole staphylococcal cell in presence of ATZ suggested that the released catalase were of extracellular origin. Scanning electron microscopy revealed the degraded host cell membrane integrity during prolonged infection. Purified bacterial catalase from the intracellularly survived S. aureus recovered after 5h of infection and its inhibition by ATZ in the zymography strengthened the scope of involvement of these anti-oxidants in the intracellular survival of S. aureus.

Protective effects of interleukin-6 in lipopolysaccharide (LPS)-induced experimental endotoxemia are linked to alteration in hepatic anti-oxidant enzymes and endogenous cytokines

Endogenous interleukin-6 has been considered as an important anti-inflammatory cytokine controlling both local and systemic acute inflammatory responses; the usefulness of IL-6 in endotoxemia aiming to block the production of reactive oxygen species and the release of inflammatory cytokines is under discussion The aim of the study was to evaluate the protective effects of IL-6 in experimentally induced endotoxemia in mice correlating the changes in tissue anti-oxidant enzymes and circulating cytokines. Liver injury in low-dose bacterial lipopolysaccharide (5 microg/mouse)-induced endotoxemic mice receiving IL-6 (300 ng/mouse) treatment either before or after LPS injection was detected by the estimation of serum oxaloacetate transaminase and serum glutamate pyruvate transaminase. This finding supports that liver injury during experimental endotoxemia could be lowered by IL-6. Current data also demonstrate the critical role of IL-6 in inducing SOD in liver, whereas IL-6 prior and after LPS challenge group showed reduced PMN infiltration in the liver as evident by decreased hepatic MPO content in those mice. IL-6 treatment also showed higher IL-10 production in serum than endotoxic group as IL-10 is a potent and pleiotropic anti-inflammatory cytokine that inhibits the synthesis of pro-inflammatory cytokines and also has a suppressive effect on pro-inflammatory cytokine like TNF-alpha, IFN-gamma and IL-12. It is also directly involved in the modulation of other aspects of inflammation, particularly cytokine responses and inflammatory cell infiltration.

Gentamicin in Combination with Ascorbic Acid Regulates the severity of Staphylococcus aureus Infection–Induced Septic Arthritis in Mice

To study the effects of gentamicin in combination with ascorbic acid on septic arthritis, mice were infected with Staphylococcus aureus (S. aureus) and treated with gentamicin, which was given at 5 mg/kg after 24 h of infection, followed by ascorbic acid, given at 20 mg/kg body weight after 2 h of gentamicin treatment. Mice were sacrificed at 3, 9, 15 days post‐infection (dpi). Combined treatment of infected mice with gentamicin and ascorbic acid eradicated the bacteria from the blood, spleen and synovial tissue and showed a significant gross reduction in arthritis, reduced serum levels of tumour necrosis factor alpha (TNF‐α) and interferon gamma (IFN‐γ). S. aureus‐infected mice have demonstrated the disturbed antioxidant status measured in terms of cellular antioxidants like reduced glutathione and antioxidant enzymes such as superoxide dismutase (SOD) and catalase. The same were ameliorated when the animals were co‐treated with gentamicin along with ascorbic acid.

Contribution of Catalase and Superoxide Dismutase to the Intracellular Survival of Clinical Isolates of Staphylococcus aureus in Murine Macrophages.

The present study was performed in order to carefully investigate the interaction of Staphylococcus aureus with murine macrophages and the contribution of catalase and superoxide dismutase in intracellular persistence of Staphylococcus aureus within murine macrophages during in vitro infection. We have reported that Staphylococcus aureus internalized by murine macrophages did not appear to be rapidly killed. Data indicating the contribution of a single catalase and superoxide dismutase in intracellular survival of Staphylococcus aureus were provided using established biochemical assays. The results of the present experiment suggest that the survival of Staphylococcus aureus within phagocytic cells is facilitated by its ability to resist oxidative products. Organisms in the log phase of growth clearly demonstrate a resistance to oxidative products.

Expression of CXCR1 (Interleukin-8 Receptor) in Murine Macrophages After Staphylococcus aureus Infection and its Possible Implication on Intracellular Survival Correlating with Cytokines and Bacterial Anti-Oxidant Enzymes

Interaction with the live Staphylococcus aureus promotes secretion of interleukin-8 (IL-8), although the expressions of functional CXCR1 (IL-8RA) in murine macrophages have not been identified. Expression of CXCR1 was induced in S. aureus-infected macrophages, whereas, CXCR1 was undetectable in control macrophages. CXCR1 blocking significantly reduced the phagocytosis of S. aureus and TNF-α, IL-6, IL-1β, IFN-γ, IL-12, and IL-8 production and increased release of MIP-2 and soluble TNF-R1. Increased bacterial catalase and decreased superoxide dismutase (SOD) activities by S. aureus with concomitant decrease in hydrogen peroxide (H2O2), superoxide anion, and nitric oxide (NO) release were observed in case of prior CXCR1 blocking. In the presence of cytochalasin D, S. aureus-mediated induction of IL-8 was inhibited concomitant with decreased bacterial count suggesting that internalization of S. aureus was necessary for induction of IL-8. Shedding of TNF-R1 due to CXCR1 blocking after S. aureus inoculation was critical for neutralization of TNF-α signaling and arrests the inflammation.

Possible Role of Toll‐like Receptor‐2 in the Intracellular Survival of Staphylococcus aureus in Murine Peritoneal Macrophages: Involvement of Cytokines and Anti‐Oxidant Enzymes

Effects of blocking toll‐like receptor‐2 (TLR‐2) on the survival of Staphylococcus aureus (S. aureus) and cytokine production in peritoneal macrophages of Swiss albino mice were analysed. Macrophages were infected with S. aureus in the presence and absence of anti‐TLR‐2 antibody. Tumour necrosis factor‐α (TNF‐α) interleukin‐6 (IL‐6), interferon‐gamma (IFN‐γ), interleukin‐1β (IL‐1β), interleukin‐12 (IL‐12) and interleukin‐10 (IL‐10) concentrations were measured. Expressions of TLR‐2, NF‐κB, MyD 88 were analysed by Western Blot. Expression of TLR‐2 was increased in S. aureus‐infected macrophages with respect to control and was MyD 88 independent. TLR2 blocking significantly reduced TNF‐α, IL‐6, IL‐1β and IL‐10 and increased IFN‐γ and IL‐12 production. Decreased catalase activity and increased superoxide dismutase (SOD) by S. aureus with concomitant increase in H2O2 and nitric oxide (NO) were observed in the case of prior TLR‐2 blocking. To understand whether catalase contributing in the intracellular survival, was of bacterial origin or not, 3‐amino, 1, 2, 4‐triazole (ATZ) was used to inhibit specifically macrophage‐derived catalase. Catalase enzyme activity from the whole staphylococcal cells in the presence of ATZ suggested that the released catalase were of extracellular origin. From the intracellular survival assay, it was evident that pretreatment of macrophages with ATZ reduces the bacterial burden in macrophages when infected with the recovered bacteria only from the anti‐TLR‐2 antibody‐treated macrophages after phagocytosis. Catalase protein expression from the whole staphylococcal cells recovered after phagocytosis also indicated the catalase release from S. aureus. Capturing of S. aureus via TLR‐2 induces inflammatory reactions through activation of NF‐κB‐signalling pathways which was MyD88‐independent.

Differential Induction of Inflammatory Cytokines and Reactive Oxygen Species in Murine Peritoneal Macrophages and Resident Fresh Bone Marrow Cells by Acute Staphylococcus aureus Infection: Contribution of Toll-Like Receptor 2 (TLR2)

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.

Effect of exogenous MCP-1 on TLR-2 neutralized murine macrophages and possible mechanisms of CCR-2/TLR-2 and MCP-1 signalling during Staphylococcus aureus infection.

It has been reported that Staphylococcus aureus survives within macrophages by hijacking host cell surface Toll-like receptor-2 (TLR-2). Moreover, S. aureus infection induced activation of TLR-2 has been reported to downregulate the expression of CC-chemokine receptor-2 (CCR-2), a receptor essential for binding of chemokines to propagate phagocytosis. Thus, we hypothesized that prior blocking of TLR-2 may help normal expression of CCR-2 on cell surface; thereby, administration of exogenous MCP-1 (a CCR-2 ligand) to bind to its free receptors might result in activation of downstream inflammatory signalling cascade. In order to address this, we compared the ability of S. aureus to modulate CCR-2 expression in TLR-2 free or neutralized macrophages in presence or absence of exogenous MCP-1 and associated downstream signalling. Exogenous MCP-1 by interacting CCR-2 leads to the release of nitric oxide and ROS that are important for bacterial clearance. In this experimental setup, the possible molecular pathway connecting an increase in proinflammatory cytokine levels with increased ROS/NO production, and therefore increased killing activity, possibly by involving either MyD88 dependent or RhoA GTPases dependent NF-κB activation or endogenous synthesis of MCP-1, independent of TLR-2-MyD88 pathway. Thus, induction of CCR-2/MCP-1 signalling by macrophages depending on the availability of MCP-1 during S. aureus infection may be important for regulation of septic shock by induction of reactive oxygen species and various cytokines.

Escherichia coli lipopolysaccharide administration alters antioxidant profile during hypercholesterolemia

Pathogens, especially Gram-negative bacteria or bacterial endotoxin, along with other classical factors, may be involved in inflammatory response within the aortic endothelium during the progression of cardiovascular disease. Studies have shown that bacterial endotoxin activates various inflammatory processes in the body. Our study aims to establish a correlation between endotoxemia and vascular expression of antioxidant enzymes. Swiss albino mice (4 weeks old) were fed a high fat diet for 24 weeks and then were administered Escherichia coli endotoxin intraperitonealy, for 4 weeks. Tissue antioxidant enzymes, serum levels of IL-6 and TNF alpha were measured from the mice. We report that i.p. administration of endotoxin to hyperlipidemic mice resulted in elevation of superoxide dismutase and catalase enzymes, which was paralleled by a systemic reduction of serum cholesterol and LDL expression. Myeloperoxidase levels were also found to be elevated in aortic tissue, while an increase was also observed in the serum cytokine levels.

Killing of Staphylococcus aureus in murine macrophages by chloroquine used alone and in combination with ciprofloxacin or azithromycin

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.