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.

Altered expression of CXCR1 (IL-8R) in macrophages utilizing cell surface TNFR1 and IL-1 receptor during Staphylococcus aureus infection.

Currently, very few studies are available on the expression of CXCR1 in mouse macrophages having both intact TNFR1 and IL-1R or their deficiency in relation to acute S. aureus infection. Peritoneal macrophages from mice neutralized singly for TNFR1or IL-1R, or for both TNFR1 and IL-1R were infected with S. aureus in vitro and their ability to secrete cytokines and reactive oxygen species (ROS) were determined. It was observed that the release of TNF-α and IL-1ß in response to S. aureus infection was decreased in macrophages when both TNFR1 and IL-1R were neutralized. The amount of H2O2, superoxide anion, nitric oxide release and bacterial CFU were significantly decreased in TNFR1 plus IL-1R blocked macrophages when compared with macrophages having intact receptors at 60 min of S. aureus infection. There was decrement of CXCL8 (IL-8) release and expression of CXCR1 in macrophages during dual receptor (TNFR1 plus IL-1R) blocking prior to stimulation with S. aureus. Expression of CXCR1 on murine peritoneal macrophages was evaluated by immunoblots from lysate at 60 min after S. aureus infection. It was observed that at 60 min after S. aureus infection in murine peritoneal macrophages, the expression of CXCR1 was increased significantly (p < 0.05) in comparison to the control groups. CXCR1 expression was decreased significantly (p < 0.05) in macrophages pre-incubated separately with anti-TNFR1 antibody (10 µg/ml) or IL-1R antagonist protein (240 ng/ml) at 60 min after S. aureus infection. However, blocking of both TNFR1 as well as IL-1R in macrophages downregulated the CXCR1expression in comparison to the groups either pre-incubated with anti-TNFR1 antibody or IRAP alone.

Expression of CXCR1 (IL-8 receptor A) in splenic, peritoneal macrophages and resident bone marrow cells after acute live or heat killed Staphylococcus aureus stimulation in mice.

Literature reveals that interaction with live Staphylococcus aureus (S. aureus) or heat killed S. aureus (HKSA) promotes secretion of CXCL-8 or interleukin-8 (IL-8) from leukocytes, however, the expressions of CXCR1 in murine splenic (SPM), peritoneal macrophages (PM) and resident fresh bone marrow cells (FBMC) have not been identified. Currently, very few studies are available on the functional characterization of CXCR1 in mouse macrophage subtypes and its modulation in relation to acute S. aureus infection. SPM, PM and FBMCs were infected with viable S. aureus or stimulated with HKSA in presence and absence of anti-CXCR1 antibody in this study. We reported here that CXCR1 was not constitutively expressed by macrophage subtypes and the receptor was induced only after S. aureus stimulation. The CXCR1 band was found specific as we compared with human polymorphonuclear neutrophils (PMNs) as a positive control (data not shown). Although, we did not show that secreted IL-8 from S. aureus-infected macrophages promotes migration of PMNs. Blocking of cell surface CXCR1 decreases the macrophage's ability to clear staphylococcal infection, attenuates proinflammatory cytokine production and the increased catalase and decreased superoxide dismutase (SOD) enzymes of the bacteria might indicate their role in scavenging macrophage derived hydrogen peroxide (H2O2). The decreased levels of cytokines due to CXCR1 blockade before S. aureus infection appear to regulate the killing of bacteria by destroying H2O2 and nitric oxide (NO). Moreover, functional importance of macrophage subpopulation heterogeneity might be important in designing new effective approaches to limit S. aureus infection induced inflammation and cytotoxicity.

Neutralization of MMP-2 and TNFR1 Regulates the Severity of S. aureus-Induced Septic Arthritis by Differential Alteration of Local and Systemic Proinflammatory Cytokines in Mice.

Despite advancement in the field of antibiotics septic arthritis remains a serious concern till date. Staphylococcus aureus is the most common bacterium that causes septic arthritis. Severity of this disease is directly correlated with chronic inflammation induced by proinflammatory cytokines like TNF-α, interleukin (IL)-1ß, IL-6, and induction of matrix metalloproteinases (MMPs) including MMP-2. The objective of our study was to evaluate the role of MMP-2 and tumor necrosis factor receptor 1 (TNFR1) in the pathogenesis of S. aureus infection-induced septic arthritis. Mice were infected with live S. aureus (5 × 106 cells/ml) followed by administration of MMP-2 inhibitor and TNFR1 antibody. Arthritis index showed highest reduction in severity of arthritis in mice treated with both MMP-2 inhibitor and TNFR1 antibody after infection. Combined neutralization of MMP-2 and TNFR1 led to marked diminution in bacterial count in the combined group. Lowest levels of pro inflammatory cytokines like TNF-α, IL-1ß, IL-6, and IFN-γ were observed in both serum and synovial tissues indicating maximum protection in S. aureus arthritis during combination treatment. Increment in the level of IL-10 in the combination group could be positively correlated with the recovery of arthritis. Similarly, expressions of COX-2 and iNOS, markers of acute inflammation were also significantly reduced in the combination group due to resolution of inflammation. Levels of O2.- and NO also showed a significant fall in case of the group treated with MMP-2 inhibitor and TNFR1 antibody both. Neutralization of both MMP-2 and TNFR1 caused rapid decline in recruitment of neutrophil and macrophages in the synovial tissues as evident from reduced MPO and MCP-1 levels, respectively, compared to other groups. Overall, it can be suggested that administration of MMP-2 inhibitor and TNFR1 antibody in combination is protective against the severity of inflammation and cartilage destruction associated with S. aureus infection-induced septic arthritis by altering the levels of cytokines.

Beneficial Effects of Exogenous Melatonin in Acute Staphylococcus aureus and Escherichia coli Infection-Induced Inflammation and Associated Behavioral Response in Mice After Exposure to Short Photoperiod.

The administration of melatonin during acute bacterial infection was evaluated in this study. Mice pre-exposed to normal photoperiodic (NP), short photoperiodic (SP), and long photoperiodic (LP) day lengths were infected separately with live Staphylococcus aureus (5 × 106 cells/ml) or Escherichia coli (2.5 × 107 colony-forming units/ml) and treated with melatonin (10 mg/kg body weight). Behavioral studies were performed before bacterial infection and after melatonin administration. In mice pre-exposed to SP, exogenous melatonin administration resulted in better clearance of bacteria from blood and behavioral improvement. Reduced glutathione content and superoxide dismutase activities were increased, with concomitant decrease in lipid peroxidation content and catalase activities in the liver, brain, and spleen after exogenous melatonin administration. The overproduction of tumor necrosis factor-α, interferon-γ, and interleukin-6 during acute bacterial infection in mice exposed to different photoperiods was probably regulated by the administration of exogenous melatonin, by reducing neutrophil recruitment to spleen, expression of inducible nitric oxide synthase and cyclooxygenase-2 in hypothalamus, and C-reactive protein in the serum, and was also associated with improved behavioral response. Photoperiodic variations in inflammatory and oxidative stress markers might be correlated to serum melatonin and corticosterone levels. This study suggests that the administration of melatonin during SP exposure is protective in infection-induced inflammation than NP and LP exposure.

Neutralization of MMP-2 protects Staphylococcus aureus infection induced septic arthritis in mice and regulates the levels of cytokines.

Matrix metalloproteinases (MMPs) are crucial players in Staphylococcus aureus mediated synovial tissue destruction in the pathogenesis of septic arthritis. Bacterial insult increases proteolytic matrix fragments by activated chondrocytes and synovial fibroblasts leading to induction of matrix metalloproteinases. Tissue destruction via MMPs induced by bacterial products, necrotic tissues and proinflammatory cytokines have been reported. Cytokines like TNF-α, IL-1ß released from host cells in response to S. aureus infection promote cartilage degradation by stimulating the production of MMPs. Antibiotic treatment can eradicate invading bacteria but elevated levels of cytokines and cytokines induced MMPs activation lead to progressive and devastating bone and cartilage destruction even after bacterial clearance. Like other MMPs, MMP-2 also contributes to extracellular matrix degradation in different types of arthritis. Release of certain pro inflammatory cytokines can also be regulated by MMP-2 activation leading to further tissue destruction. The role of MMP-2 in the pathogenesis of S. aureus infection induced septic arthritis and its influence on cytokines regulation needs further investigation. Whether neutralization of MMP-2 provides protection against Staphylococcus aureus infection induced septic arthritis in mice is an obvious question. Here we reported that neutralization of MMP-2 during S. aureus infection induced septic arthritis might be beneficial for preventing infection induced extracellular matrix destruction thereby decreasing bacterial burden in synovial tissues and regulating inflammatory cytokines in arthritic mice.

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.

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.

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-1ß, 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.

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.

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 10(6) 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.

Combination therapy with ampicillin and azithromycin in an experimental pneumococcal pneumonia is bactericidal and effective in down regulating inflammation in mice.

OBJECTIVES: Emergence of multidrug resistance among Streptococcus pneumoniae (SP), has limited the available options used to treat infections caused by this organism. The objective of this study was to compare the role of monotherapy and combination therapy with ampicillin (AMP) and azithromycin (AZM) in eradicating bacterial burden and down regulating lung inflammation in a murine experimental pneumococcal infection model. METHODS: Balb/C mice were infected with 106 CFU of SP. Treatments with intravenous ampicillin (200 mg/kg) and azithromycin (50 mg/kg) either alone or in combination was initiated 18 h post infection, animals were sacrificed from 0 - 6 h after initiation of treatment. AMP and AZM were quantified in serum by microbiological assay. Levels of TNF-α, IFN-γ IL-6, and IL-10 in serum and in lungs, along with myeloperoxidase, inflammatory cell count in broncho alveolar lavage fluid, COX-2 and histopathological changes in lungs were estimated. RESULTS: Combination therapy down regulated lung inflammation and accelerated bacterial clearance. This approach also significantly decreased TNF-α, IFN-γ, IL-6 and increased IL-10 level in serum and lungs along with decreased myeloperoxidase, pulmonary vascular permeability, inflammatory cell numbers and COX-2 levels in lungs. CONCLUSIONS: Combinatorial therapy resulted in comparable bactericidal activity against the multi-drug resistant isolate and may represent an alternative dosing strategy, which may help to alleviate problems with pneumococcal pneumonia.