Rana Adhikary

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.

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.

Clitoria ternatea flower petals: Effect on TNFR1 neutralization via downregulation of synovial matrix metalloproteases

ETHNOPHARMACOLOGICAL RELEVANCE Clitoria ternatea Linn. (C. ternatea) is a traditionally used herb in arthritis, and its anti-arthritic activity has been attributed to polyphenols (e.g. quercetins) from its flower petal. AIM OF THE STUDY The present study was designed to investigate whether C. ternatea or quercetin-3ß-D-glucoside (QG) support the antibody mediated TNFα-receptor 1 (TNFR1) neutralization to ameliorate arthritis in mice. MATERIALS AND METHODS Development of collagen-induced arthritis (CIA) in male Swiss mice (20-22g, 3-4 weeks of age) was followed by estimation of synovial polymorphonuclear cell (PMN) accumulation (in terms of myeloperoxidase activity), synovial and systemic release of cytokines, chemokines and C-reactive protein (CRP) by enzyme-linked immunosorbent assay (ELISA), biochemical estimation of synovial free radical generation and antioxidant status, as well as immunoblot assessment of synovial TNFR1, toll-like receptor 2(TLR2), cyclooxygenase-2(COX-2) and inducible nitric oxide synthase (iNOS) expression; and zymographic analysis of synovial matrix-metalloprotease-2 (MMP-2) activity. RESULTS CIA was induced from day 2 post-secondary immunizations as evidenced from arthritic scores and joint swelling in parallel to increased inflammatory and oxidative stress parameters in synovial joints. Long term supplementation with extract from Clitoria ternatea flower petals CTE (50mg/kg) and QG (2.5mg/kg) upto 24 days post booster immunization augmented anti-arthritic potential of TNFR1 neutralization with anti-TNFR1 antibody (10μg per mice) in terms of reduced MPO activity, decrease in release of pro-inflammatory cytokines, chemokines, reactive oxygen species (ROS)/ reactive nitrogen species (RNS) production in parallel to significant (p<0.05) reduction in TNFR1, TLR2, iNOS, COX-2 and MMP-2 expression. CONCLUSION CTE and QG possess potential anti-arthritic activity which targets synovial MMP-2 in arthritic joints and TNFR1 targeting followed by CTE or QG treatment might become a combinatorial approach in future therapeutic research in treatment of arthritis.