15 research outputs found
Effector cytokine potential is acquired alongside CD200R expression in vivo.
<p>Dot plots show cytokine<sup>+</sup> gated CD4 cells <i>ex vivo</i> (-P/I) and after recall with pdbU + Ionomycin (+P/I, 5h); gates are based on unstimulated controls (-P/I). A. Infection with <i>S. mansoni</i> (8wk) increased CD200R<sup>+</sup> IL-4<sup>+</sup> CD4 cells in MesLN from 0.017±0.019% to 3.39±1.36% and 83±4.14% of IL-4<sup>+</sup> cells were CD200R<sup>+</sup>, while CD200R<sup>–</sup> TNFα<sup>+</sup> CD4 cells decreased from 47.5±4.66% to 30.0±0.99%. (n = 3, p = 0.01). B. Infection with <i>S. enterica</i> increased IFNγ<sup>+</sup> CD4 cells in spleen from 1.9±0.65% to 31.1±4.4% and 49.5±9.86% of IFNγ<sup>+</sup> cells were CD200R<sup>+</sup>, while CD200R<sup>–</sup> TNFα<sup>+</sup> CD4 cells decreased from 19.6±2.31% to 15.1±1.56%. (n = 5, p = 0.01). Results are representative of at least 3 (A, n = 3 mice/group) and 2 (B, n = 5 mice/group) independent experiments.</p
Progressive acquisition of CD200R correlates with effector cytokine secretion.
<p>Peripheral LN cells from BALB/c mice and 4-get mice were analysed ex vivo (Naive) or after culture for 7d in transient TCR or chronic TCR stimulation conditions, as indicated, using polarising culture conditions to promote Th2 (A) or Th1 (B) cytokine differentiation. Intracellular cytokine staining with CD200R expression is shown for TNFα, IL-2, IL-4 and IFNγ. Intracellular staining with anti-GFP was used to enhance eGFP signal in 4-get cells (control stainings for anti-GFP are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s005" target="_blank">Figure S5B & C</a>). Contour plots show percentage of cytokine<sup>+</sup> gated CD4 cells; gates were based on unstimulated controls (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s003" target="_blank">Figure S3A and S3B</a>). Data are representative of 5 independent experiments. Pairwise comparison of multifunctional cytokine loss from replicate cultures is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s003" target="_blank">Figure S3D-F</a>.</p
Chronic infection induces CD200 and CD200R expression in CD4 T cells.
<p>C57BL/6 (A–E) and 4-get (F) mice were infected with <i>S. mansoni</i>; MesLN (A–D, F) and spleen (E) were analyzed. A. Representative contour plot of CD44 with CD200, and B. CD44 with CD200R is shown for gated CD4 T cells. Upon infection (8 wk), CD44<sup>hi</sup>CD200<sup>+</sup>CD4 cells (panel A, R3) increased from 7.71±2.66% (n = 14) to 15.2±3.72% (n = 17, p<0.0001); CD44<sup>hi</sup>CD200R<sup>+</sup>CD4 cells (panel B, R3) increased from 4.36±1.69% (n = 14) to 15.3±4.67% (n = 17, p<0.0001). Data were pooled from 4 independent experiments. C. Dot plots show CD200 and CD200R co-expression in CD44<sup>lo</sup> and CD44<sup>hi</sup> CD4 cells from uninfected and infected mice. D. Graph shows absolute numbers of CD200R<sup>+</sup> (panel B, R3) and CD200R<sup>–</sup> (panel B, R2) CD44<sup>hi</sup>CD4 cells from 4 independent experiments. Upon infection CD44hiCD200R+ cells increased from (2.7±0.6)× 10<sup>5</sup> (n = 14) to (10.8±3.2)×10<sup>5</sup> (n = 17, ***p = 3.03×10<sup>–10</sup>), while CD44<sup>hi</sup> CD200R<sup>–</sup> cells increased from (7.9±3.3)×10<sup>5</sup> to (18.6±11.9)×10<sup>5</sup> (**p = 0.003). E. Contour plots show CD200R expression in proliferating (BrdU<sup>+</sup>) cells, in gated CD4 cells for spleen of uninfected and infected mice. Upon infection BrdU<sup>+</sup>CD200R<sup>+</sup> CD4 T cells increased from 4.5±0.69 (n = 4) to 46.6±17 (n = 10) p<0.0005. Specificity controls for BrdU staining are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s007" target="_blank">Figure S7A</a> & B. F. Contour plots (left) show CD200 and eGFP expression; CD200<sup>+</sup>GFP<sup>+</sup>CD4 cells increased from 0.64±0.12% to 13.7±7.24% after infection (p = 0.01, n = 4). Histogram overlays (right) show CD200 and CD200R levels in gated GFP<sup>+</sup> (green line) and GFP<sup>–</sup> (filled histograms) CD4 cells from infected mice. Control stainings for intracellular GFP staining are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s005" target="_blank">Fig S5B & C</a>. ** = p<0.0001, * = p<0.001. Results are representative of at least 4 (A–D) and 3 (E-F) independent experiments (n = 3 mice/group).</p
CD200R correlates with infection intensity in human schistosomiasis.
<p>Expression of CD200R and IL-4 (A) or IFNγ (B) in gated CD4 T cells from blood after recall with PMA + Ionomycin. Isotype control stainings are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s009" target="_blank">Figure S9</a>. Charts show mean (±s.e.) of CD200R MFI in R1-3. C. CD200R-, CD200R<sup>lo</sup> and CD200R<sup>hi</sup> αβTCR<sup>+</sup>CD4 cells were analysed for IL-4 and GATA-3 expression. One representative sample is shown in left histograms. Mean percentage (±s.e.) of cells in each population are shown in the bar graph. * = p<0.05, ** = p<0.01 (n = 8, ANOVA A–C). D. Correlation between the percentages of IL-4<sup>+</sup> and CD200R<sup>+</sup> CD4 T cells, respectively determined by intracellular and surface stain. E. Correlation between the proportions of CD200R<sup>hi</sup>CD4 T cells and infection intensity determined as urine egg counts (n = 29, D-E). Percentages of CD200R<sup>hi</sup>CD4 T cells and IL-4<sup>+</sup>CD4 T cells were arcsine square root transformed to allow the use of parametric tests. Infection intensity was Log10(x+1) transformed. To allow for potential confounding effects of sex (categorical variable) and age (continuous variable) transformed data were assessed by ANOVA. Resulting residuals were used to analyse the correlation between both CD200R<sup>hi</sup>CD4 T cells and IL-4+CD4 T cells or between CD200R<sup>hi</sup>CD4 T cells and infection intensity of which the r-correlation coefficients and p-values are indicated.</p
T cells co-express CD200 and CD200R following TCR activation.
<p>A. Naïve C57BL/6 peripheral LN cells were cultured with a titration of anti-CD3 + anti-CD28 (2 µg/ml) for 3d. Contour plots show upregulation of CD200 (upper row) and CD200R (lower row) together with CD44 (R1) in gated CD4 cells. B. DO11.10 LN and spleen cells were cultured with a titration of OVA peptide (ISQAVHAAHAEINEAGR) for 3d. CD200R:CD200 co-expression (R1) is shown in gated CD4 cells. C. Naïve LN cells were stimulated with anti-CD3 (1 µg/ml) + anti-CD28 (2 µg/ml) either transiently on Ab-coated wells for the first 2d then removed to fresh media (blue lines) or chronically with Ab-coated aAPC present throughout the culture (red lines) in Th1 (top row), Th2 (middle row) or non-polarising (IL-7, bottom row) conditions. Histograms show expression of CD200 and CD200R in gated CD4 cells at d3 (left) and d7 (right) compared to naïve levels (grey filled histograms). Data are representative of 6 independent experiments and statistical validation is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035466#pone.0035466.s001" target="_blank">Figure S1A and S3C</a>.</p
Differential abundance of host miRNAs in mouse serum during <i>S. mansoni</i> infection.
<p>miRNAs were quantified by qRT-PCR and normalized to a synthetic RNA spike-in. Each symbol represents data from one individual mouse. Fold changes are defined as the ratio of abundance in infected versus naïve serum; the signal from naïve was set as 1. (***p<0.001, ****p<0.0001).</p
Detection of parasite-derived miRNAs in mouse serum during <i>S. mansoni</i> infection.
<p>miRNAs were quantified by qRT-PCR, normalized to a synthetic RNA spike-in and fold change calculated as the ratio of abundance in infected serum compared to the background abundance level detected in naïve serum, which represents the noise in the assay likely derived from cross-hybridization with endogenous small RNAs (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).</p
Small RNA classification in serum of naïve and infected mice.
1<p>This does not include 42 reads identical to sja-miR-277 that contain 1 nucleotide mismatch to <i>S. mansoni</i> draft genome.</p
Discrimination between the <i>S. mansoni</i> infected and uninfected individuals using the combined data for 3 parasite miRNAs.
<p>Fold-changes (median with interquartile range indicated) and ROC curves for bantam, miR-277 and miR-3479-3p (Piida- panel A, Chiredzi-panel B) (**p<0.01).</p
Top miRNAs dysregulated in the liver at 7 weeks post <i>S. mansoni</i> infection as determined by microarray analysis and showing results obtained by qRT-PCR (microarray: p<0.05, Fold change ≥2, qRT-PCR: t-test).
<p>Top miRNAs dysregulated in the liver at 7 weeks post <i>S. mansoni</i> infection as determined by microarray analysis and showing results obtained by qRT-PCR (microarray: p<0.05, Fold change ≥2, qRT-PCR: t-test).</p