Defense Mechanisms of Hepatocytes Against Burkholderia pseudomallei

The Gram-negative facultative intracellular rod Burkholderia pseudomallei causes melioidosis, an infectious disease with a wide range of clinical presentations. Among the observed visceral abscesses, the liver is commonly affected. However, neither this organotropism of B. pseudomallei nor local hepatic defense mechanisms have been thoroughly investigated so far. Own previous studies using electron microscopy of the murine liver after systemic infection of mice indicated that hepatocytes might be capable of killing B. pseudomallei. Therefore, the aim of this study was to further elucidate the interaction of B. pseudomallei with these cells and to analyze the role of hepatocytes in anti-B. pseudomallei host defense. In vitro studies using the human hepatocyte cell line HepG2 revealed that B. pseudomallei can invade these cells. Subsequently, B. pseudomallei is able to escape from the vacuole, to replicate within the cytosol of HepG2 cells involving its type 3 and type 6 secretion systems, and to induce actin tail formation. Furthermore, stimulation of HepG2 cells showed that IFNγ can restrict growth of B. pseudomallei in the early and late phase of infection whereas the combination of IFNγ, IL-1β, and TNFα is required for the maximal antibacterial activity. This anti-B. pseudomallei defense of HepG2 cells did not seem to be mediated by inducible nitric oxide synthase-derived nitric oxide or NADPH oxidase-derived superoxide. In summary, this is the first study describing B. pseudomallei intracellular life cycle characteristics in hepatocytes and showing that IFNγ-mediated, but nitric oxide- and reactive oxygen species-independent, effector mechanisms are important in anti-B. pseudomallei host defense of hepatocytes

Oncolytic Virotherapy as Emerging Immunotherapeutic Modality: Potential of Parvovirus H-1

Human tumors develop multiple strategies to evade recognition and efficient suppression by the immune system. Therefore, a variety of immunotherapeutic strategies have been developed to reactivate and reorganize the human immune system. The recent development of new antibodies against immune check points may help to overcome the immune silencing induced by human tumors. Some of these antibodies have already been approved for treatment of various solid tumor entities. Interestingly, targeting antibodies may be combined with standard chemotherapy or radiation protocols. Furthermore, recent evidence indicates that intratumoral (it) or intravenous (iv) injections of replicative oncolytic viruses such as herpes simplex-, pox-, parvo- or adenoviruses may also reactivate the human immune system. By generating tumor cell lysates in situ, oncolytic viruses overcome cellular tumor resistance mechanisms and induce immunogenic tumor cell death resulting in the recognition of newly released tumor antigens.This is in particular the case of the oncolytic parvovirus H-1 (H-1PV) which is able to kill human tumor cells and stimulate an antitumor immune response through increased presentation of tumor-associated antigens, maturation of dendritic cells and release of proinflammatory cytokines. Current research and clinical studies aim to assess the potential of oncolytic virotherapy and its combination with immunotherapeutic agents or conventional treatments to further induce effective antitumoral immune responses

Original Contribution 2 PGD 2 and PGE 2 regulate gene expression of Prx 6 in primary macrophages via Nrf2

Prostaglandin 26 Free radicals 27 Peroxiredoxin 6 (Prx 6) is a bifunctional enzyme with both glutathione peroxidase and acidic Ca 2+ -28 independent phospholipase A 2 activities. We have recently shown that exposure of murine bone marrow-29 derived macrophages to LPS and IFN-γ leads to induction of COX-2 expression and secretion of PGE 2 , up-30 regulating Prx 6 mRNA levels. This study was designed to investigate various prostaglandins (PGs) for their 31 ability to induce gene expression of Prx's, in particular Prx 6, and to determine the underlying regulatory 32 mechanisms. We provide evidence that both conventional and cyclopentenone PGs enhance Prx 6 mRNA 33 expression. Treatment with either activators or inhibitors of adenylate cyclase as well as cAMP analogs 34 indicated that Prx 6 gene expression is regulated by adenylate cyclase in response to PGD 2 or PGE 2 . 35 Furthermore, our study revealed that JAK2, PI3K, PKC, and p38 MAPK contribute to the PGD 2 -or PGE 2 -36 dependent Prx 6 induction. Using stimulated macrophages from Nrf2-deficient mice or activators of Nrf2 and 37 PPARγ, we found that Nrf2, but not PPARγ, is involved in the PG-dependent increase in Prx 6 mRNA 38 expression. In summary, our data suggest multiple signaling pathways of Prx 6 regulation by PGs and 39 identified Nrf2 as a critical player mediating transcriptional induction. 40 © 2011 Elsevier Inc. All rights reserved

Distinct roles for nitric oxide in resistant C57BL/6 and susceptible BALB/c mice to control <it>Burkholderia pseudomallei </it>infection

Abstract Background Burkholderia pseudomallei is the causative agent of melioidosis, an emerging bacterial infectious disease in tropical and subtropical areas. We recently showed that NADPH oxidase but not nitric oxide (NO) contributes to resistance in innately resistant C57BL/6 mice in a B. pseudomallei respiratory infection model. However, the function of NO for resistance was shown to differ among distinct strains of mice and proved also to be stage dependent in various infection models. The present study therefore aimed to examine the role of NO in a systemic infection model of melioidosis and to test whether the function of NO differs among innately resistant C57BL/6 and susceptible BALB/c mice after B. pseudomallei infection. Results C57BL/6 iNOS-/- mice that were intravenously infected with B. pseudomallei survived several weeks, whereas most of the wild type animals succumbed during this period. The bacterial burden in liver and spleen was significantly higher in wild type animals compared to iNOS-/- mice 13 days after challenge. In contrast, BALB/c mice that were treated with amminoguanidine to inhibit NO expression in vivo showed significantly enhanced mortality rates and higher bacterial loads in liver and spleen compared to control animals. The bactericidal function of IFN-γ stimulated C57BL/6 iNOS-/- macrophages were not altered after B. pseudomallei infection, but BALB/c macrophages exhibited reduced killing activity against the pathogen when NO was inhibited. Conclusion Our present data indicate a dual role of NO among resistant and susceptible mouse strains after B. pseudomallei infection. NO mediated mechanisms are an essential component to control the infection in susceptible BALB/c mice. In contrast, NO production in B. pseudomallei infected C57BL/6 mice rather harmed the host likely due to its detrimental effects.</p

Cytokine expression in caspase-6 deficient mice 6 hours after infection with <i>B</i>. <i>pseudomallei</i>.

<p>Quantitative real-time PCR analysis of inflammatory parameters in the spleens of C57BL/6 WT (n = 8) and <i>caspase6</i>^<i>-/-</i> mice (n = 8) 6 hours after infection with 5 × 10⁴ CFU <i>B</i>. <i>pseudomallei</i> strain E8. Data were analysed using Student’s <i>t</i> test. Values are means ± standard deviations from two independent experiments.</p

Role of Inducible Nitric Oxide Synthase and NADPH Oxidase in Early Control of Burkholderia pseudomallei Infection in Mice

Infection with the soil bacterium Burkholderia pseudomallei can result in a variety of clinical outcomes, including asymptomatic infection. The initial immune defense mechanisms which might contribute to the various outcomes after environmental contact with B. pseudomallei are largely unknown. We have previously shown that relatively resistant C57BL/6 mice can restrict bacterial B. pseudomallei growth more efficiently within 1 day after infection than highly susceptible BALB/c mice. By using this model, our study aimed to investigate the role of macrophage-mediated effector mechanisms during early B. pseudomallei infection. Depletion of macrophages revealed an essential role of these cells in the early control of infection in BALB/c and C57BL/6 mice. Strikingly, the comparison of the anti-B. pseudomallei activity of bone marrow-derived macrophages (BMM) from C57BL/6 and BALB/c mice revealed an enhanced bactericidal activity of C57BL/6 BMM, particularly after gamma interferon (IFN-γ) stimulation. In vitro experiments with C57BL/6 gp91phox(−/−) BMM showed an impaired intracellular killing of B. pseudomallei compared to experiments with wild-type cells, although C57BL/6 gp91phox(−/−) cells still exhibited substantial killing activity. The anti-B. pseudomallei activity of C57BL/6 iNOS(−/−) BMM was not impaired. C57BL/6 gp91phox(−/−) mice lacking a functional NADPH oxidase were more susceptible to infection, whereas C57BL/6 mice lacking inducible nitric oxide synthase (iNOS) did not show increased susceptibility but were slightly more resistant during the early phase of infection. Thus, our data suggest that IFN-γ-mediated but iNOS-independent anti-B. pseudomallei mechanisms of macrophages might contribute to the enhanced resistance of C57BL/6 mice compared to that of BALB/c mice in the early phase of infection

Effect of IL-10 in macrophages during <i>B</i>. <i>pseudomallei</i> infection.

<p>(A) Quantitative real-time PCR analysis of IL-10 expression in macrophages from C57BL/6 WT and <i>caspase6</i>^<i>-/-</i> mice 6 h after infection with <i>B</i>. <i>pseudomallei</i> strain E8 (MOI ~ 50). The data were logarithmized to achieve normal distribution and compared using Student’s <i>t</i>-test. Values are means ± standard deviations from three independent experiments. (B) Intracellular bacterial burden of IL-10-treated and non-treated C57BL/6 macrophages after infection with <i>B</i>. <i>pseudomallei</i> strain E8 (MOI ~ 25). Data were analysed using Student’s <i>t</i>-test. Values are means ± standard deviations from triplicate determinations. The experiment was repeated three times.</p

Invasion and replication of <i>B</i>. <i>pseudomallei</i> in caspase-6 deficient macrophages.

<p>Intracellular bacterial burden of C57BL/6-WT and C57BL/6-<i>caspase6</i>^<i>-/-</i> macrophages after infection with <i>B</i>. <i>pseudomallei</i> strain E8 at an MOI of ~ 25. The CFU data were logarithmized to achieve normal distribution and compared using Student’s t-test. Values are means ± standard deviations from triplicate determinations. The experiment was repeated three times.</p