Identification of an Endogenous Organosulfur Metabolite by Interpretation of Mass Spectrometric Data

The chemical structure of x11564, a new endogenous organosulfur metabolite, was elucidated by <i>de novo</i> interpretation of mass spectrometric data. The structure was confirmed by comparison to a synthetic standard. Metabolite x11564 is structurally related to intermediates in the methionine salvage pathway

Cytokine expression by Foxp3+ Treg in adenoidal cells.

<p>Percentage of Foxp3+ Treg in adenoidal MNC after stimulation with WCA compared with unstimulated medium control (A). Intracellular cytokine staining shows IL-10 (B) and IL-17 (C) production by Foxp3+ Treg and Foxp3− CD4+ T cells after WCA stimulation compared with unstimulated control. Only CD4+ T cell gate is shown in B and C. Result of one representative experiment of four replicates is shown.</p

Expression of CD39 and CTLA4 by Foxp3+ Treg cells.

<p>Fluorescence histograms showing CD39 (A) and CTLA4 (B) expression by Foxp3+ Treg cells in PBMC (black) and adenoidal MNC (AdMNC) (red) from one representative of 20 replicate experiments summarized in panel C. (*p<0.01 compared to PBMC).</p

Association of pneumococcal carriage and Treg cells in adenoidal MNC.

<p>Percentages of CD4+Tcells which were Foxp3+ Treg (%Treg) (A) and their expression of CD39 (B) and intracellular CTLA4 (C) in PBMC and adenoidal MNC (AdMNC) in children who were culture positive (+) and negative (−) for pneumococcus. MFI – median fluorescence intensity. (*p<0.05 compared to culture negative children.) Culture+, n = 8; culture−, n = 12.</p

Proportion of Treg in CD4+T cells and percentages of Treg expressing CD45RO and CD69 in PBMC and adenoidal MNC.

<p>Mean percentages (SD) are shown. (*p<0.05, **p<0.01 compared to PBMC).</p

Adenoidal Treg exhibit strong inhibition of CD4+ T cell responses to WCA.

<p>CD4+ T cell proliferation (A+B) and cytokine production (C) in the presence or absence of Treg after stimulation with pneumococcal WCA. A). % CD4+ T cell proliferation in adenoidal MNC and Treg-depleted MNC from pneumococcal culture+ (n = 6) and culture− children (n = 6) (*p<0.01 compared to culture− group). B). CD4+ T cell proliferation induced by WCA in Treg-depleted MNC suppressed by the repletion of purified Treg (*p<0.01 compared to Treg-depleted MNC+WCA, n = 4. C). *p<0.01, #p<0.01 compared to Treg-depleted MNC+WCA.</p

Role of T-helper-subset-associated cytokines in protection from nasopharyngeal colonization.

<p>A. Mice defective in IFN-γ, IL-4 or IL-17A receptor were immunized as described, then challenged with pneumococcal strain 0603. Mice with IFN-γ or IL-4 deficiency were significantly protected by WCV (P<0.001 vs. respective CT controls) whereas IL-17A receptor deficient mice were not protected (P>0.5 vs. CT). Dashed line represents the lower limit of detection of bacterial colonization. B. Expression of IL-17A from splenocytes of WCV-immunized mice. Cultured splenocytes from mice immunized with WCV (black columns) or CT alone (white columns) were stimulated for 72 hours with medium alone, Concanavalin A (5 µg/ml), WCA (10 µg dry weight) after which IL-17A production was measured by ELISA. Significantly more IL-17A was expressed following WCA stimulation of WCV-immunized vs. CT-immunized mice, although response to concanavalin A was similar. C. Effect of CD4+ T cell depletion upon IL-17A expression from splenocytes. Splenocytes (without or with CD4+ T cell depletion) from mice immunized with WCV were stimulated for 72 hours with medium alone or WCA after which IL-17A was measured by ELISA. IL-17A expression in splenocytes following WCA stimulation was significantly higher in the presence of CD4+ T cells compared to stimulation with medium alone or when CD4+ T cells were depleted. Repletion of CD4+ T cells restored the response. ** P<0.01 compared to cells stimulated with medium alone. D. IL-17A intracellular staining of splenocytes from WCV immunized mice. Splenocytes from WCV immunized mice were stimulated with WCA, blocked with monensin, harvested and stained for CD4+ and intracellular IL-17A as described. There is a statistically significant increase in CD4+ IL-17A positive cells following stimulation with WCA, which is not observed in the CD4- population. No increase in IL-17A positive cells could be detected in cells from unimmunized mice (data not shown). **P = 0.008 for comparison of frequency of IL-17A-positive cells in absence and presence of WCA stimulation among CD4+ cells. Data shown here are representative of three experiments, including at least 5 mice per experiment. E. Expression of IL-17A from NALT of WCV- vs. CT-immunized mice. Cultured splenocytes from mice immunized with WCV (black columns) or CT alone (white columns) were stimulated for 72 hours with medium alone or with WCA (10 µg dry weight) after which IL-17A production was measured by ELISA. Significantly more IL-17A was expressed following WCA stimulation of WCV-immunized vs. CT-immunized mice. **P<0.01 for comparison of IL-17A in WCV vs. CT-immunized mice following stimulation with WCA.</p

Correlation of IL-17A expression and density of nasopharyngeal colonization in mice.

<p>Three weeks after immunization of mice (n = 90) with CT with doses of WCA ranging from 1 to 100 µg, and one week before pneumococcal challenge, blood samples were obtained and stimulated with WCA (10 µg) for 6 days, after which supernatants were collected and assayed for IL-17A concentration. The correlation between density of colonization (cfu/nasal wash) 7 days after challenge and pre-challenge IL-17A expression was evaluated. IL-17A expression was significantly correlated with density of colonization.</p

Effect of neutrophils on WCV-induced protection against pneumococcal colonization.

<p>A. Effect of neutrophil depletion on WCV-induced protection from nasopharyngeal colonization. Each data point represents the density of nasopharyngeal colonization in cfu/nasal wash for each mouse. The horizontal bar shows the geometric mean cfu/nasal wash for each group and the dashed line shows the lower limit of detection of bacterial colonization. Mice were immunized with CT or WCV as indicated; just prior to the time of challenge, mice were randomized to receive antineutrophil antibody vs. saline. Proportion of colonized mice and density of colonization was determined 7 days post challenge. WCV-immunized mice that received saline treatment were significantly better protected than WCV-immunized mice that received antineutrophil antibody, with a lower proportion of colonized mice (P = 0.025 by Fisher's Exact) and density of colonization (P = 0.05 by Mann-Whitney U). B. Correlation between neutrophil count and density of pneumococcal colonization. Neutrophil counts following neutrophil depletion were assayed at the time of sacrifice and plotted against density of colonization. There was a strong negative association between neutrophil counts and colonization density (Spearman <i>ρ</i> = −0.75). C. Histopathology of nasopharyngeal tissue following nasopharyngeal challenge of CT- (left panel) and WCV-immunized (right panel) mice. Seven days post pneumococcal challenge, mice were euthanized, heads stored in formalin, and H&E sections of nasopharyngeal tissue prepared and examined under light microscopy at 60× magnification. The presence of a dense neutrophilic infiltrate in the submucosa at the junction of the olfactory and respiratory epithelium was noted in WCV-immunized mice following pneumococcal nasopharyngeal challenge but not in CT-immunized mice. The two slides shown are representative of a total of 15 examined specimens (8 WCV-immunized and 7 CT controls, all at day 7 post pneumococcal challenge). Lesions like those represented here were observed in 6/8 immunized mice and 0/7 controls.</p

Effect of exposure to pneumococcus on IL-17A expression from human tissues and cells.

<p>A. Expression of IL-17A from tonsillar mononuclear cells from children. Tonsillar cells (n = 8) were cultured as described and stimulated with WCA or WCA derived from an isogenic, pneumolysin-negative strain (WCAply-). Stimulation with WCA was associated with significantly increased IL-17A expression compared to exposure to medium alone (P = 0.008 by Wilcoxon signed rank test), whereas stimulation with WCAply- did not increase IL-17A production. B. Expression of IL-17A from peripheral blood of adults and umbilical cord blood. Peripheral blood samples from adults (healthy adult volunteers (n = 7), parturient women (n = 11) and umbilical cord blood (n = 11) were stimulated with WCA for 6 days after which IL-17A concentration was assayed by ELISA. IL-17A production was significantly greater in adults than cord blood (P<0.001 by Mann-Whitney U test).</p