Melanie R. Power Coombs

TLR2 Mediates Recognition of Live Staphylococcus epidermidis and Clearance of Bacteremia

Background Staphylococcus epidermidis (SE) is a nosocomial pathogen that causes catheter-associated bacteremia in the immunocompromised, including those at the extremes of age, motivating study of host clearance mechanisms. SE-derived soluble components engage TLR2; but additional signaling pathways have also been implicated, and TLR2 can play complex, at times detrimental, roles in host defense against other Staphylococcal spp. The role of TLR2 in responses of primary blood leukocytes to live SE and in clearance of SE bacteremia, the most common clinical manifestation of SE infection, is unknown. Methodology/Principal Findings We studied TLR2-mediated recognition of live clinical SE strain 1457 employing TLR2-transfected cells, neutralizing anti-TLR antibodies and TLR2-deficient mice. TLR2 mediated SE-induced cytokine production in human embryonic kidney cells, human whole blood and murine primary macrophages, in part via recognition of a soluble TLR2 agonist. After i.v. challenge with SE, early (1 h) cytokine/chemokine production and subsequent clearance of bacteremia (24–48 h) were markedly impaired in TLR2-deficient mice. Conclusions/Significance TLR2 mediates recognition of live SE and clearance of SE bacteremia in vivo.

Piperine, an alkaloid from black pepper, inhibits growth of human colon cancer cells via G1 arrest and apoptosis triggered by endoplasmic reticulum stress.

Piperine, a piperidine alkaloid present in black pepper, inhibits the growth of cancer cells, although the mechanism of action is not well understood. In this study, we show that piperine (75–150 µM) inhibited the growth of several colon cancer cell lines but had little effect on the growth of normal fibroblasts and epithelial cells. Piperine inhibited HT‐29 colon carcinoma cell proliferation by causing G1 phase cell cycle arrest that was associated with decreased expression of cyclins D1 and D3 and their activating partner cyclin‐dependent kinases 4 and 6, as well as reduced phosphorylation of the retinoblastoma protein and up‐regulation of p21/WAF1 and p27/KIP1 expression. In addition, piperine caused hydroxyl radical production and apoptosis that was partially dependent on the production of reactive oxygen species. Piperine‐treated HT‐29 cells showed loss of mitochondrial membrane integrity and cleavage of poly (ADP‐ribose) polymerase‐1, as well as caspase activation and reduced apoptosis in the presence of the pan‐caspase inhibitor zVAD‐FMK. Increased expression of the endoplasmic reticulum stress‐associated proteins inositol‐requiring 1α protein, C/EBP homologous protein, and binding immunoglobulin protein, and activation of c‐Jun N‐terminal kinase and p38 mitogen‐activated protein kinase, as well as decreased phosphorylation of Akt and reduced survivin expression were also observed in piperine‐treated HT‐29 cells. Furthermore, piperine inhibited colony formation by HT‐29 cells, as well as the growth of HT‐29 spheroids. Cell cycle arrest and endoplasmic reticulum stress‐associated apoptosis following piperine treatment of HT‐29 cells provides the first evidence that piperine may be useful in the treatment of colon cancer.

Exposure of breast cancer cells to a subcytotoxic dose of apigenin causes growth inhibition, oxidative stress, and hypophosphorylation of Akt

Epidemiological studies show that fruit- and vegetable-rich diets are associated with a reduced risk of developing certain forms of cancer, including breast cancer. In this study we demonstrate that a subcytotoxic concentration of apigenin, which is a flavone found at high concentrations in parsley, onions, grapefruit, oranges, and chamomile tea, inhibited DNA synthesis in a panel of human breast cancer cell lines (MDA-MB-231, MBA-MB-468, MCF-7, SK-BR-3). Decreased proliferation of MDA-MB-468 cells in the presence of apigenin was associated with G2/M phase cell cycle arrest and the production of reactive oxygen species. Apigenin-treated MDA-MB-468 cells also showed reduced phosphorylation of Akt (protein kinase B), which is an essential effector serine/threonine kinase in the phosphatidylinositide 3-kinase pathway that promotes tumor growth and progression. However, exposure to the antioxidant reduced glutathione failed to reverse apigenin-mediated inhibition of Akt phosphorylation and cell proliferation, indicating that these effects were not due to oxidative stress. Taken together, these findings suggest that low-dose apigenin has the potential to slow or prevent breast cancer progression.

Neonatal Host Defense against Staphylococcal Infections

Preterm infants are especially susceptible to late-onset sepsis that is often due to Gram-positive bacterial infections resulting in substantial morbidity and mortality. Herein, we will describe neonatal innate immunity to Staphylococcus spp. comparing differences between preterm and full-term newborns with adults. Newborn innate immunity is distinct demonstrating diminished skin integrity, impaired Th1-polarizing responses, low complement levels, and diminished expression of plasma antimicrobial proteins and peptides, especially in preterm newborns. Characterization of distinct aspects of the neonatal immune response is defining novel approaches to enhance host defense to prevent and/or treat staphylococcal infection in this vulnerable population.

A neonatal model of intravenous Staphylococcus epidermidis infection in mice <24 h old enables characterization of early innate immune responses.

Staphylococcus epidermidis (SE) causes late onset sepsis and significant morbidity in catheterized preterm newborns. Animal models of SE infection are useful in characterizing disease mechanisms and are an important approach to developing improved diagnostics and therapeutics. Current murine models of neonatal bacterial infection employ intraperitoneal or subcutaneous routes at several days of age, and may, therefore, not accurately reflect distinct features of innate immune responses to bacteremia. In this study we developed, validated, and characterized a murine model of intravenous (IV) infection in neonatal mice <24 hours (h) old to describe the early innate immune response to SE. C57BL/6 mice <24 h old were injected IV with 106, 107, 108 colony-forming units (CFU) of SE 1457, a clinical isolate from a central catheter infection. A prospective injection scoring system was developed and validated, with only high quality injections analyzed. Newborn mice were euthanized between 2 and 48 h post-injection and spleen, liver, and blood collected to assess bacterial viability, gene expression, and cytokine production. High quality IV injections demonstrated inoculum-dependent infection of spleen, liver and blood. Within 2 h of injection, SE induced selective transcription of TLR2 and MyD88 in the liver, and increased systemic production of plasma IL-6 and TNF-α. Despite clearance of bacteremia and solid organ infection within 48 h, inoculum-dependent impairment in weight gain was noted. We conclude that a model of IV SE infection in neonatal mice <24 h old is feasible, demonstrating inoculum-dependent infection of solid organs and a pattern of bacteremia, rapid and selective innate immune activation, and impairment of weight gain typical of infected human neonates. This novel model can now be used to characterize immune ontogeny, evaluate infection biomarkers, and assess preventative and therapeutic modalities.

Adenosine modulates Toll-like receptor function: basic mechanisms and translational opportunities.

Adenosine is an endogenous purine metabolite whose concentration in human blood plasma rises from nanomolar to micromolar concentrations during the inflammatory process. Leukocytes express seven-transmembrane adenosine receptors whose engagement modulates Toll-like receptor-mediated cytokine responses, in part via modulation of intracellular cyclic adenosine monophosphate. Adenosine analogs are used clinically to treat arrhythmias and apnea of prematurity. Herein, we consider the potential of adenosine analogs as innate immune response modifiers to prevent and/or treat infection.

Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4(+) T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca(2+) release to inhibit IκB phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4(+)CD25(+) regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

Inhibitory effect of iron withdrawal by chelation on the growth of human and murine mammary carcinoma and fibrosarcoma cells

Since iron uptake is essential for cell growth, rapidly dividing cancer cells are sensitive to iron depletion. To explore the effect of iron withdrawal on cancer cell growth, mouse and human mammary carcinoma cells (4T1 and MDA-MB-468, respectively) and mouse and human fibrosarcoma cells (L929 and HT1080, respectively) were cultured in the absence or presence of DIBI, a novel iron-chelating polymer containing hydroxypyridinone iron-ligand functionality. Cell growth was measured by a colorimetric assay for cell metabolic activity. DIBI-treated 4T1, MDA-MB-468, L929 and HT1080 cells, as well as their normal counterparts, showed a dose- and time-dependent reduction in growth that was selective for human cancer cells and mouse fibrosarcoma cells. The inhibitory effect of DIBI on fibrosarcoma and mammary carcinoma cell growth was reversed by addition of exogenous iron in the form of iron (III) citrate, confirming the iron selectivity of DIBI and that its inhibitory activity was iron-related. Fibrosarcoma and mammary carcinoma cell growth inhibition by DIBI was associated with S-phase cell cycle arrest and low to moderate levels of cell death by apoptosis. Consistent with apoptosis induction following DIBI-mediated iron withdrawal, fibrosarcoma and mammary carcinoma cells exhibited mitochondrial membrane permeabilization. A comparison of DIBI to other iron chelators showed that DIBI was superior to deferiprone and similar to or better than deferoxamine for inhibition of fibrosarcoma and mammary carcinoma cell growth. These findings suggest that iron withdrawal from the tumor microenvironment with a selective and potent iron chelator such as DIBI may prevent or inhibit tumor progression.

Structure–function relationships in histidine-rich antimicrobial peptides from Atlantic cod

Gad-1 and Gad-2 are antimicrobial peptide (AMP) sequences encoded by paralogous genes. They are rich in histidine, which suggests that their activity might be pH-dependent. We examined their structure-function relationships with a view to learning how to improve AMP therapeutic ratios. Activity assays with Gram-negative bacteria and cancer cell lines demonstrate that Gad-2 is substantially more active at slightly acidic pH than it is at neutral pH. By contrast, the activity of Gad-1 at lower pH is similar to its activity at pH7. Circular dichroism spectra indicate that the greater functional plasticity of Gad-2 correlates with a greater structural plasticity; Gad-2s percent helicity varies dramatically with altered pH and lipid environment. Interestingly, Gad-2s highest levels of helicity do not correspond to the conditions where it is most active. High resolution solution NMR structures were determined in SDS micelles at pH5, conditions that induce an intermediate level of helicity in the peptides. Gad-1 is more helical than Gad-2, with both peptides exhibiting the greatest helical tendencies in their central region and lowest helicity in their N-termini. The high resolution structures suggest that maximum activity relies on the appropriate balance between an N-terminal region with mixed hydrophobic/hydrophilic structure features and an amphipathic central and C-terminal region. Taken together with previous studies, our results suggest that to improve the therapeutic ratio of AMPs, consideration should be given to including sequential histidine-pairs, keeping the overall charge of the peptide modest, and retaining a degree of structural plasticity and imperfect amphipathicity.

Apigenin inhibits the inducible expression of programmed death ligand 1 by human and mouse mammary carcinoma cells

Programmed death ligand 1 (PD-L1) is expressed by many cancer cell types, as well as by activated T cells and antigen-presenting cells. Constitutive and inducible PD-L1 expression contributes to immune evasion by breast cancer (BC) cells. We show here that the dietary phytochemical apigenin inhibited interferon (IFN)-γ-induced PD-L1 upregulation by triple-negative MDA-MB-468 BC cells, HER2(+) SK-BR-3 BC cells, and 4T1 mouse mammary carcinoma cells, as well as human mammary epithelial cells, but did not affect constitutive PD-L1 expression by triple-negative MDA-MB-231 BC cells. IFN-β-induced expression of PD-L1 by MDA-MB-468 cells was also inhibited by apigenin. In addition, luteolin, the major metabolite of apigenin, inhibited IFN-γ-induced PD-L1 expression by MDA-MB-468 cells. Apigenin-mediated inhibition of IFN-γ-induced PD-L1 expression by MDA-MB-468 and 4T1 cells was associated with reduced phosphorylation of STAT1, which was early and transient at Tyr701 and sustained at Ser727. Apigenin-mediated inhibition of IFN-γ-induced PD-L1 expression by MDA-MB-468 cells also increased proliferation and interleukin-2 synthesis by PD-1-expressing Jurkat T cells that were co-cultured with MDA-MB-468 cells. Apigenin therefore has the potential to increase the vulnerability of BC cells to T cell-mediated anti-tumor immune responses.