Differential intracellular fate of Burkholderia pseudomallei 844 and Burkholderia thailandensis UE5 in human monocyte-derived dendritic cells and macrophages

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia pseudomallei </it>(<it>Bp</it>) is a category B biothreat organism that causes a potentially fatal disease in humans and animals, namely melioidosis. <it>Burkholderia thailandensis </it>(<it>Bt</it>) is another naturally occurring species that is very closely related to <it>Bp</it>. However, despite this closely related genotype, <it>Bt </it>is considered avirulent as it does not cause the disease. In the present study, we compared the growth kinetics of <it>B. pseudomallei </it>strain 844 (<it>Bp</it>-844) in human monocyte-derived dendritic cells (MoDCs) and macrophages (Mφs), as well as its ability to stimulate host cell responses with those of <it>B. thailandensis </it>strain UE5 (<it>Bt</it>-UE5).</p> <p>Results</p> <p>Primary human MoDCs and Mφs were infected with <it>Bp</it>-844 and its intracellular growth kinetics and ability to induce host cell responses were evaluated. The results were compared with those obtained using the <it>Bt</it>-UE5. In human MoDCs, both bacteria were similar in respect to their ability to survive and replicate intracellularly, induce upregulation of costimulatory molecules and cytokines and bias T helper cell differentiation toward a Th1 phenotype. By contrast, the two bacteria exhibited different growth kinetics in human Mφs, where the intracellular growth of <it>Bt</it>-UE5, but not <it>Bp</it>-844, was significantly suppressed. Moreover, the ability of Mφs to kill <it>Bp</it>-844 was markedly enhanced following stimulation with IFN-γ.</p> <p>Conclusion</p> <p>The data presented showed that while both strains were similar in their ability to survive and replicate in human MoDCs, only <it>Bp</it>-844 could readily replicate in human Mφs. Both bacteria induced similar host cellular responses, particularly with regard to their ability to bias T cell differentiation toward a Th1 phenotype.</p

In Vitro Anti-Inflammatory Activity of Morus alba L. Stem Extract in LPS-Stimulated RAW 264.7 Cells

Morus alba L., also known as white mulberry or Mhon, has long been used in traditional medicines. This study was aimed to investigate anti-inflammatory activities of mulberry stem ethanolic extract (MSE) in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophage cell line. The MSE was first prepared and then investigated for cell viability using the MTT assay. The anti-inflammatory activities were investigated through the inhibition of inducible nitric oxide synthase (iNOS), cyclooxygenase- (COX-) 2 mRNA expression, and iNOS protein expression using reverse transcription-polymerase chain reaction (RT-PCR) assay and immunoblotting analysis, respectively. The inhibition of nitric oxide production of the MSE was also investigated using the Griess reaction assay. The MSE concentration ranging from 10 to 40 µg/ml yielded cell viability higher than 80%. The MSE at concentrations of 20 and 40 µg/ml demonstrated anti-inflammatory activity through the inhibition of nitric oxide production via suppression of both the iNOS mRNA and protein. It was also found to inhibit the expression of COX-2 mRNA in LPS-induced RAW 264.7 cells. This study is the first to report the anti-inflammatory potential of the extract prepared from the stem of mulberry

Activation of human monocyte-derived dendritic cells by Burkholderia pseudomallei does not require binding to the C-type lectin DC-SIGN

Dendritic cells (DCs) are essential in regulating adaptive immunity. DC-SIGN (DC-specific ICAM-grabbing nonintegrin) is a C-type lectin receptor that is expressed mainly by DCs. Accumulating evidence supports that certain pathogens target DC-SIGN to escape host immunity. To investigate a possible role of DC-SIGN in Burkholderia pseudomallei infection, we initially screened its DC-SIGN binding activity by an ELISA method utilizing a DC-SIGN-Fc chimeric protein and found that all of the B. pseudomallei strains tested failed to bind DC-SIGN. However, one strain, the LPS mutant SRM117, which lacks the type II O-polysaccharide expression, actually bound DC-SIGN, in contrast to its wild-type counterpart 1026b (P<0.001). We also found that, although the LPS mutant could readily activate monocyte-derived human DCs, it induced lower levels of IL-12p70 and IL-10 production than its wild-type counterpart (P<0.01). By contrast, the wild-type and the LPS mutants were indistinguishable from one another in terms of T(H)1/T(H)2 differentiation. Altogether, these data suggest that, unlike other certain host pathogen interactions, activation of DCs by B. pseudomallei is not dependent on DC-SIGN. We also found evidence that the LPS mutant that binds DC-SIGN has a suppressive effect on DC cytokine production

Lysosome repositioning as an autophagy escape mechanism by Mycobacterium tuberculosis Beijing strain

International audienceAbstract Induction of host cell autophagy by starvation was shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of Mycobacterium tuberculosis reference strain H37Rv. Our previous study showed that strains belonging to M. tuberculosis Beijing genotype resisted starvation-induced autophagic elimination but the factors involved remained unclear. Here, we conducted RNA-Seq of macrophages infected with the autophagy-resistant Beijing strain (BJN) compared to macrophages infected with H37Rv upon autophagy induction by starvation. Results identified several genes uniquely upregulated in BJN-infected macrophages but not in H37Rv-infected cells, including those encoding Kxd1 and Plekhm2, which function in lysosome positioning towards the cell periphery. Unlike H37Rv, BJN suppressed enhanced lysosome positioning towards the perinuclear region and lysosomal delivery to its phagosome upon autophagy induction by starvation, while depletion of Kxd1 and Plekhm2 reverted such effects, resulting in restriction of BJN intracellular survival upon autophagy induction by starvation. Taken together, these data indicated that Kxd1 and Plekhm2 are important for the BJN strain to suppress lysosome positioning towards the perinuclear region and lysosomal delivery into its phagosome during autophagy induction by starvation to evade starvation-induced autophagic restriction