Arnab Majhi

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.

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.

In Vitro Anti-inflammatory and Immunomodulatory Effects of Ciprofloxacin or Azithromycin in Staphylococcus aureus-Stimulated Murine Macrophages are Beneficial in the Presence of Cytochalasin D

ABSTRACTWe 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.

Increased resistance of immobilized-stressed mice to infection: correlation with behavioral alterations.

Immobilization is an easy and convenient method to induce both psychological and physical stress resulting in restricted motility and aggression and is believed to be the most severe type of stress in rodent models. Although it has been generally accepted that chronic stress often results in immunosuppression while acute stress has been shown to enhance immune responses, the effects of IS on the host resistance to Escherichia coli (E. coli) infection and associated behavioral changes are still not clear. In a series of experiments aimed at determining the level of hypothalamic COX-2, HSP-90, HSP-70, SOD-1 and plasma level of corticosterone, cytokine, antibody titer and their association with behavioral activities, mice were infected with viable E. coli during acute and chronic IS by taping their paws. In this study we show that acute and chronic IS enhances the resistance of mice to E. coli infection via inhibiting the production of pro-inflammatory cytokines, free radicals, and by improving the exploratory behavior. Altogether, our findings support the notion that cytokines released during immune activation and under the influence of corticosterone can modulate the open field behavior both in terms of locomotor activity as well as exploration. One of the features observed with chronic stressor was a lower ability to resist bacterial infection, although in case of acute stress, a better clearance of bacterial infection was observed in vivo with improvement of exploratory behavior and cognitive functions.

Levofloxacin-Ceftriaxone Combination Attenuates Lung Inflammation in a Mouse Model of Bacteremic Pneumonia Caused by Multidrug-Resistant Streptococcus pneumoniae via Inhibition of Cytolytic Activities of Pneumolysin and Autolysin

ABSTRACT In this study, our objective was to determine whether a synergistic antimicrobial combination in vitro would be beneficial in the downregulation of pneumococcal virulence genes and whether the associated inflammation of the lung tissue induced by multidrug-resistant Streptococcus pneumoniae infection in vivo needs to be elucidated in order to consider this mode of therapy in case of severe pneumococcal infection. We investigated in vivo changes in the expression of these virulence determinants using an efficacious combination determined in previous studies. BALB/c mice were infected with 106 CFU of bacteria. Intravenous levofloxacin at 150 mg/kg and/or ceftriaxone at 50 mg/kg were initiated 18 h postinfection; the animals were sacrificed 0 to 24 h after the initiation of treatment. The levels of cytokines, chemokines, and C-reactive protein (CRP) in the serum and lungs, along with the levels of myeloperoxidase and nitric oxide the inflammatory cell count in bronchoalveolar lavage fluid (BALF), changes in pneumolysin and autolysin gene expression and COX-2 and inducible nitric oxide synthase (iNOS) protein expression in the lungs were estimated. Combination therapy downregulated inflammation and promoted bacterial clearance. Pneumolysin and autolysin expression was downregulated, with a concomitant decrease in the expression of COX-2 and iNOS in lung tissue. Thus, the combination of levofloxacin and ceftriaxone can be considered for therapeutic use even in cases of pneumonia caused by drug-resistant isolates.

In vitro susceptibility of a penicillin-resistant and tolerable isolate of Streptococcus pneumoniae to combination therapy

INTRODUCTION Preference for combination therapy to treat infection due to multidrug-resistant S. pneumoniae (MDRSP) has not been well elucidated in previous studies. METHODOLOGY In the present study, 19 antibiotics in combinations were tested against an MDRSP isolate. In vitro susceptibility studies including minimum inhibitory concentration (MIC), minimal bactericidal concentrations (MBC) and disk agar diffusion (DAD), tolerance to resistant antibiotics, checkerboard assay, time-kill curve, hemolytic assay, and autolysis assay were performed on the test strain to study its in vitro susceptibility to combination therapy. RESULTS From the checkerboard assay and time-kill curve, it was observed that a combination of levofloxacin (MIC, 16 µg/mL) and ceftriaxone (MIC, 2 µg/mL), at sub-MIC concentration was synergistic and most effective against the MDRSP isolate (penicillin MIC, > 64 µg/mL). Hemolytic activities also increased significantly with combination therapy compared to monotherapy (p < 0.05). Moreover, the hemolytic activity of levofloxacin in combination with ceftriaxone was better than ciprofloxacin plus ceftriaxone or cefepime. The autolysis rate was also found to increase rapidly within one hour of exposure to levofloxacin plus ceftriaxone, and this was found to be significantly different from the other combinations at the fifth and sixth hour post incubation (p < 0.05). CONCLUSIONS This data suggests that this combination is bactericidal in vitro, and requires further studies in in vivo models for treatment against MDRSP infections.

Systemic Staphylococcus aureus infection in restraint stressed mice modulates impaired immune response resulting in improved behavioral activities

Stress leads to immune malfunction and increased susceptibility to infection resulting in impaired cognitive behavior and depression. Working with an animal model of Staphylococcus aureus infection and restraint stress we demonstrated impaired immune response and altered behavior against the S. aureus infection after exposure to acute or chronic restraint stress. This enhanced the resistance of mice to S. aureus infection via inhibiting the production of pro-inflammatory cytokines, free radicals, and upregulating corticosterone and anti-inflammatory cytokine production, resulting in altered exploratory behavior, compared to non-stressed infected group (P<0.05), thereby helping the animals to recover from depressive-like symptoms due to stress.