Polar Lipids of Burkholderia pseudomallei Induce Different Host Immune Responses

Melioidosis is a disease in tropical and subtropical regions of the world that is caused by Burkholderia pseudomallei. In endemic regions the disease occurs primarily in humans and goats. In the present study, we used the goat as a model to dissect the polar lipids of B. pseudomallei to identify lipid molecules that could be used for adjuvants/vaccines or as diagnostic tools. We showed that the lipidome of B. pseudomallei and its fractions contain several polar lipids with the capacity to elicit different immune responses in goats, namely rhamnolipids and ornithine lipids which induced IFN-γ, whereas phospholipids and an undefined polar lipid induced strong IL-10 secretion in CD4(+) T cells. Autologous T cells co-cultured with caprine dendritic cells (cDCs) and polar lipids of B. pseudomallei proliferated and up-regulated the expression of CD25 (IL-2 receptor) molecules. Furthermore, we demonstrated that polar lipids were able to up-regulate CD1w2 antigen expression in cDCs derived from peripheral blood monocytes. Interestingly, the same polar lipids had only little effect on the expression of MHC class II DR antigens in the same caprine dendritic cells. Finally, antibody blocking of the CD1w2 molecules on cDCs resulted in decreased expression for IFN-γ by CD4(+) T cells. Altogether, these results showed that polar lipids of B. pseudomallei are recognized by the caprine immune system and that their recognition is primarily mediated by the CD1 antigen cluster

Mass spectrometry analysis by MALDI-TOF-TOF of the key molecular ions of lipid spot #17.

<p>Arrows highlight the differences between two major ions representing the loss of rhamnose. Molecular ions representing fragmentation ions are labeled.</p

Characterization of caprine peripheral blood monocyte-derived dendritic cells.

<p>Panel A shows a photograph of a classic morphology of a caprine dendritic cell. The same cultures were examined for cell surface expression of myeloid dendritic cell markers: CD14, MHC class II DR, and CD1w2 antigens. In panel B are representative histograms for cell surface expression of CD1w2 antigen (dark line) or isotype matched control (dotted line) in caprine dendritic cells from goat #19 (top and framed in red) and goat #29 (bottom and framed in blue) when cultured in media alone. Panels C and D correspond to cell surface expression of CD1w2 antigen in caprine dendritic cells when cultured for 16 hours in culture media alone or culture media with ConA, first extraction of total cell lipids of <i>B. pseudomallei</i> (F1), second lipid extract (F2), or lipid fraction of the second lipid extract (F4 to F7). Panel C represents the mean Fluorescence Channel (MFC) for CD1w2 expression in each culture. Panel D represents the percentage of positive cells for CD1w2 when compared to media alone.</p

Surface expression of MHC class II DR antigens on caprine dendritic cells and effect of polar lipid fractions on surface expression for these molecules.

<p>Panel A shows representative histograms of surface expression of MHC class II DR molecules on cDCs cultured (dark line) or isotype matched control (dotted line) with culture media alone. Panels B and C correspond to cell surface expression of MHC class II DR molecule on cDCs when cultured for 16hr in culture media alone or culture media containing ConA or each of the polar lipid fractions (F1 to F7) extracted from <i>B. pseudomallei</i> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080368#pone-0080368-g001" target="_blank">Figure 1</a>. Panel C represents the Mean Fluorescence Channel (MFC) for MHC class II DR antigen expression in each cell culture. Panel D represents the % of positive cells for MHC class II DR antigen over the percentage of positive cells expressing MHC class II DR antigen on cDCs cultured in media alone.</p

CD1w2 blocking effects on IFN-γ expression by CD4+ T cells when stimulated with polar lipids.

<p>Panel A show the histogram representing the MFC for CD1w2-PE antibody bound to cDC cultures incubated with fluorescence -CD1w2 antibody-PE (thick line, MFC = 9540) or with purified CD1w2 antibody plus CD1w2 –PE antibody [thin-dotted line, MFC 5070]. Panels B and C show representative histograms (B) and dot plots (C) of CD4 T cells expressing IFN-γ recovered from cultures after seven days incubation where the cDCs primed with each of the polar lipids when incubated with CD1w2 antibody (right column of dot plots) or without (left column of dot plots). Numbers inside the quadrants indicated the percentage (upper number) and the MFC (lower number) for IFN-γ expression in the CD4+ (right quadrants) and CD4- negative cells (left quadrants).</p

Composite sketch of polar lipids of <i>B. pseudomallei</i>.

<p>Lipids are shown as found within the second extract in the highly polar solvent system B. Colors of various lipid spots represent different staining and detection methods: Maroon Red: Spots detected as dark spot under UV light; Strawberry Red: Spots detected as a bright spot under UV light; Steel gray: Spots detected by spraying with CuSO₄ in H₃PO₄; Blue: Spots detected by spraying with Dittmer-Lester; Plum purple: Spots detected by spraying with ninhydrin as molecules containing primary amino groups; Red: Spots detected by spraying with ninhydrin as molecules containing secondary amino groups; Orange: Spots detected by spraying with ninhydrin as molecules containing tertiary amino groups/proline; Magenta: Spots detected by spraying with α-naphthol.</p

Mass spectrometry of the four major fractions of polar lipids.

<p>Lipids derived from the second lipid extract of <i>B. pseudomallei</i>. Each mass spectrometric trace has the lipid composition of its fraction indicated by a 2D-TLC plate in the upper right corner. Lipid spot numbers correspond to those used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080368#pone-0080368-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080368#pone-0080368-g002" target="_blank">2</a>. Major ions of each spot are highlighted in colored boxed. Molecular ions are in bold and enlarged in panels they first appeared. Panel A represents lipid fraction V (F4); panel B represents lipid fraction VI (F5); panel C represents lipid fraction VII (F6); and panel D represents lipid fraction VIII (F7).</p

IL-10 expression in CD4⁺ T cells.

<p>Panel A shows the representative flow cytometry plots demonstrating positive staining for CD4 and IL-10 staining in cells recovered from co cultures of autologous T cells and cDCs with media alone, fraction F4 and F7 extracted from <i>B. pseudomallei</i>. Panel B represents the mean fluorescence channel (MFC) for CD4/IL-10 positive cells in each cell culture. Panel C represents the percentage CD4/IL-10 positive cells over the percentage of positive cells expressing CD4/IL-10 cultured in media alone.</p

Mass spectrometry analysis by MALDI-TOF-TOF of the four major molecular ions of lipid spot #16.

<p>Arrows highlight the difference between two major ions representing the loss of ornithine.</p

Composite sketch of total lipids of <i>B. pseudomallei</i>.

<p>Lipids shown were found within the first extract in five different solvent systems from very polar to highly non-polar (B, A, E, C, D). Colors of various lipid spots represent different staining and detection methods: Maroon Red: Spots detected as dark spot under UV light; Strawberry Red: Spots detected as a bright spot under UV light; Steel gray: Spots detected by spraying with CuSO₄ in H₃PO₄; Blue: Spots detected by spraying with Dittmer-Lester reagent; Plum purple: Spots detected by spraying with ninhydrin as molecules containing primary amino groups; Red: Spots detected by spraying with ninhydrin as molecules containing secondary amino groups; Orange: Spots detected by spraying with ninhydrin as molecules containing tertiary amino groups/proline; Magenta: Spots detected by spraying with α-naphthol.</p