Tyrosine phosphorylation patterns of major signaling modules of T helper differentiation upon enrichment of membrane cholesterol.

<p>(<b>A</b>) SDS-PAGE silver stain of pooled, negatively-sorted CD4⁺ splenic T-cell lysates from F1 mice (n = 4/group) untreated (<i>left lane</i>) or squalene treated (single dose of 180 µg, <i>right lane</i>) 3 days post-injection shows no detectable quantitative alterations in the protein bands between the two groups of mice. (<b>B</b>) Tyrosine phosphorylation patterns of the same samples in panel <i>A</i> were blotted with anti-phosphorylated tyrosine Ab-HRP conjugate. Of note, the amount of 55–100 kDa phosphorylated protein bands is increased in mice treated with squalene. (<b>C</b>) Immunoprecipitation of pooled splenic CD4⁺ T-cell lysates from F1 mice treated or not with squalene (180 µg/mouse, n = 4/group) 3 days post-injection was carried out for IL-12Rβ2, IL-2Rα, IL-4Rα, CD28, or CD3 receptors using specific Abs, and probed with specific anti-phospho Abs for STAT4, STAT5, STAT6, PI3K, and ZAP-70 kinases. Only the phosphorylated STAT-4, STAT-5, and ZAP-70 in squalene treated mice were significantly enhanced. Shown is one of two representative experiments.</p

Squalene induced accumulation of membrane cholesterol in resting CD4 T cells favors Th1 polarization under antigen-specific or non antigen specific stimulation.

<p>(A) Isolated adherent splenocytes (APCs, 5×10⁵) from individual F1 mice treated or not with 180 µg of squalene, were pulsed (+) or not pulsed (-) with HA_110–120 synthetic peptide (40 µg/mL/10⁶ cells) <i>in vitro</i> 7 days after squalene injection, and co-cultured with negatively-sorted CD4 splenic T-cells (10⁶ cells) from the same groups of mice, treated or not with squalene (Sq) (n = 4/group). Various cell co-culture combinations are shown on the X-axis, where (+) indicates presence and (–) indicates absence from the culture. Cell-culture supernatants were collected 24–48 h later, and secretion of IL-2, IL-4, and IFN-γ (Y-axis) was measured in pg/ml by Luminex. Bars represent average ± SD. Differences among groups were highly significant (<i>p</i><0.001) for cytokines with the following exceptions denoted as §: For IL-4, co-culutre in lane 6 differed significantly from lane 5 (<i>p</i> = 0.047) but not from lane 3 (<i>p</i> = 0.097). No significant difference was observed between co-culture in lane 3 and 5 for any of the three cytokines. (<b>B</b>) Intracellular cytokine staining for IFN-γ (<i>left panels</i>) and IL-4 (<i>right panels</i>) in splenic cells and CD4-gated splenic cells from individual untreated (<i>top panels</i>) and squalene treated F1 mice (<i>bottom panels</i>) (n = 3/group) were stimulated for 48 h with anti-CD3/CD28 Abs (2.5 µg each/10⁶ cells). Shown are the overlapped FACS histograms of gated CD4⁺ T cells synthesizing IL-4 or IFN-γ (red cell events), and total splenic cells (dark cell events) from squalene treated or untreated F1 mice, 7 days after squalene treatment. R1 gate indicates the low-proliferating cell population whereas the R2 gate indicates the high proliferating cell population in each experiment. Dead cells are shown in the un-gated cell population below the R1 gate. Shown is one of two representative experiments.</p

Co-localization of cytokine receptors with lipid rafts of resting CD4 T-cells before and after squalene treatment.

<p>Resting CD4 T-cells from individual F1 mice (n = 5/group) before and 7 days after squalene treatment (180 µg/mouse) were analyzed for interleukin receptor expression and distribution at the single-cell level by CLSM. Cells were stained with IL-4Rα-, IL-12Rβ2-, or IL-2Rα-PE conjugates, and co-stained for GM1 ganglioside by CTB- FITC conjugate and for nuclei with DAPI. First column indicates single-channel color for DAPI staining (blue), second column indicates GM1 staining (green), third column indicates interleukin receptor (IL-Rs) staining (red), and last column indicates merged channels at X63 magnification. <i>Top-two rows</i>, indicate cells from untreated (<i>upper row</i>) and squalene treated mice (<i>lower row</i>) stained for IL-4Rα. <i>Middle-two rows</i>, indicate cells from untreated (<i>upper row</i>) and squalene treated mice (<i>lower row</i>) stained for IL-12Rβ2. <i>Bottom-two rows</i>, indicate cells from untreated (<i>upper row</i>) and squalene treated mice (<i>lower row</i>) stained for IL-2Rα. Arrows indicate presence of IL-Receptor co-expression with the GM1 resident of lipid rafts. Enlargements of the merged channels are depicted to the <i>right</i> along with two different angles of the membrane for each IL-Receptor at X220 magnification. Shown are representative images in one of three experiments.</p

Alteration in cytokine receptors mRNA expression after squalene enrichment of membrane cholesterol in resting lymphocytes.

<p>(<b>A</b>) Quantitative real-time RT-PCR of IL-4Rα, IL-12Rβ2, and IL-2Rα mRNA extracted from peripheral blood lymphocytes of individual F1 mice (n = 5/group) analyzed before squalene treatment (dark bars) and 7 days after squalene injection (180 µg/mouse) (light bars). Y axis indicates the mean fold increase in mRNA expression level relative to the endogenous 18S rRNA expression level (control ± SD). Shown are two combined separate experiments (*<i>p</i> values<0.05). (<b>B</b>) Aliquots samples in panel A were stained with CD4-FITC conjugate, co-stained either with IL-4Rα-PE or IL-12Rβ2-PE or IL-2Rα-PE conjugates, and analyzed by FACS at the single-cell level for the surface IL-Rs expression level based on MFI measurements. Shown are the IL-Rs MFI values ± SD measured in individual mice before and after squalene treatment. Of note, no significant changes occurred in the IL-Rs expression on cell surface after squalene treatment (*<i>p</i> values>0.05).</p

Squalene administration leads to accumulation of membrane cholesterol in resting CD4 T-cells.

<p>(<b>A</b>) F1 hybrid mice (n = 5/group) were injected i.p. or not (purple line) with a single dose (black line) or 4 doses of squalene (red line) within a week interval (180 µg/dose/mouse). Seven days after the last injection, negatively-sorted splenic CD4 cells from individual mice were co-stained with CD3-PE, CD4-FITC and Filipin III. Shown is the amount of cholesterol in plasma cell membrane of gated CD3⁺CD4⁺ splenic cells as measured by MFI of Filipin III in FACS at single-cell level in one representative mouse from each group (<i>left panel</i>). <i>Right panel</i>, F1 hybrid mice (n = 7/group) were injected i.p. (black line) or not (red line) with a single dose of squalene (180 µg/mouse) and 7 days later negatively-sorted splenic CD4 cells from individual mice were co-stained with CD3-PE, CD4-FITC, and Filipin III. Shown is the percentage of gated CD4⁺ T-cells ± standard deviation (SD) and MFI values of Filipin III ± SD before and after squalene injection as collected among 700 cell events in gated population of CD3⁺4⁺ T-cells for one of three representative experiments. (<b>B</b>) Cholesterol accumulation in the spleen was identified by Oil Red O (ORO) staining of frozen spleen sections, counter-stained with hematoxylin from untreated or squalene treated mice (180 µg/mouse) given one or four doses, and analyzed 7 days post-injection (n = 3/group). <i>Left panel,</i> spleen section from untreated mouse. <i>Middle panels,</i> spleen section from squalene treated mice. <i>Right panel,</i> positive control for ORO lipid droplet staining in adipocytes. Shown is one representative ORO stained section in each group. Dark arrows indicate ORO stain. (<b>C</b>) Quantitative real-time RT-PCR of HMG-CoA reductase mRNA and Squalene epoxidase mRNA extracted from negatively-sorted CD4 splenocytes isolated from individual F1 hybrid mice (n = 5/group) that were treated (light bars) or not treated (dark bars) with a single dose of squalene (180 µg/mouse) and analyzed 7 days post-injection. Y axis indicates the mean fold increase in mRNA expression level relative to the endogenous 18S rRNA expression level (control ± standard deviation (SD). Shown are two combined separate experiments (*<i>p</i> value<0.05). (<b>D</b>) FACS measurements of CD3⁺4⁺<i>Foxp3</i>⁺ T-reg cells from negatively-sorted CD4⁺ splenic cells of the same F1 mouse groups analyzed in panel A that were co-stained with CD3-PE and Filipin III. Shown is the percentage of gated CD4⁺Foxp3⁺ T-reg cells ± SD and MFI values of Filipin III ± SD collected among 500 cell events in the gated population of GFP⁺-<i>Foxp3</i>/GFP⁺ cells from one mouse in each group from two representative experiments.</p