Impact of MyD88 reactivation on the expression of factors that govern MO/DC function in infection and inflammation.

<p>(A, B) WT, MyD^OFF, CD11c-MyD^ON and LysM-MyD^ON mice were infected orally with <i>C</i>. <i>rodentium</i>. On day 4 p.i. leukocytes were isolated from the cLP and sorted for DC (lin⁻CD64⁻MHC-II⁺CD26⁺CD11c⁺) and MO (lin⁻CD64⁺F4/80⁺MHC-II⁺). As lineage marker, antibodies against CD3, CD19, B220 and NK1.1 were included (A) Heat map displaying expression of genes involved in MNP function in infection and inflammation analyzed by real-time PCR from sorted DC/MO fractions. Data presented as mean log2 value of relative mRNA expression (see color scale). (B) Bar graphs show mean expression of selected genes in sorted DC/MO fractions relative to the expression of 5 housekeeping genes (<i>Actb</i>, <i>B2m</i>, <i>Gapdh</i>, <i>Gusb</i>, <i>Hsp90ab1</i>). Data were pooled from three individual experiments with n = 4–6 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p<0.05, **p<0.01, ns–not significant.</p

IEC intrinsic MyD88 signaling promotes barrier function of the epithelium.

<p>(A, B) Gene expression in IEC isolated before (control) or on day 4 p.i. (infected) with <i>C</i>. <i>rodentium</i> from the colon of WT, MyD^OFF and IEC-MyD^ON mice. Data shown as mean relative expression to <i>Actb</i>. (C) Principal component analysis of the intestinal microbiota in individual mice before (control) or on day 4 p.i. (infected) with <i>C</i>. <i>rodentium</i>. (D) Intestinal permeability in WT, MyD^OFF and IEC-MyD^ON mice before (control) or on day 8 p.i. (infected) with <i>C</i>. <i>rodentium</i>. FITC-dextran serum levels were determined 4 h after oral administration of this compound. Data were pooled from two (C), three (D) or four (A, B) independent experiments with n = 3–5 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test (A, B, D); *p<0.05, **p<0.01, ***p<0.001.</p

MyD88 signaling in T cells and ILC3 is not sufficient to induce host immune responses.

<p>(A) WT, MyD^OFF and Rorc-MyD^ON mice were infected orally with <i>C</i>. <i>rodentium</i> and monitored for survival. (B) Representative flow cytometry plots showing the frequency of colonic IL-22⁺ cells within live ILC3 on day 4 p.i. Graphs represent frequency (%) of IL-22⁺ cells amongst live ILC3. (C) Colonic T cell response on day 8 p.i. Representative flow cytometry plots of live CD3⁺CD4⁺ T isolated from the cLP and stained for IL-17A and IFN-γ. Graphs represent frequency (%) of colonic IL-17A⁺ (IFN-γ^+/) and IFN-γ⁺ (IL-17^+/−) cells amongst live CD3⁺CD4⁺ T cells. (D) Representative H&E staining of colon sections of WT, MyD^OFF and Rorc-MyD^ON mice on day 8 p.i. with <i>C</i>. <i>rodentium</i>. Scale bar represents 100 μm. Black arrow head indicates distinct necrotic epithelial injury and gangrenous mucosal damage in the colon of Rorc-MyD^ON mice. Bar graph shows inflammation score based on infiltration and epithelial hyperplasia. (E) Bacterial load in the feces and the liver on day 8 p.i. Data are representative for two independent experiments with n = 6 mice per group (A) or were pooled from two (B-D) or three (E) independent experiments with n = 2–5 mice per group. Horizontal bar represents mean. Error bar represents +SEM. Dashed line indicates the limit of detection. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p<0.05, **p<0.01, ***p<0.001, ns–not significant.</p

MyD88 signaling in CD11c⁺ cells is sufficient to induce an inflammatory response in the colon of mice infected with <i>C</i>. <i>rodentium</i>.

<p>(A, B) Representative H&E staining of colon sections of WT, MyD^OFF, CD11c-MyD^ON and LysM-MyD^ON mice on day 8–10 p.i. with <i>C</i>. <i>rodentium</i>. Black asterisks indicate inflammatory infiltration into the cLP; black arrow heads indicate crypt elongation (A) or gangrenous-like necrotic structures (B). Scale bar represents 100 μm. Bar graph shows inflammation score based on infiltration and epithelial hyperplasia. (C) Leukocytes were isolated from the cLP of mice before (control) or on day 8 p.i. (infected) with <i>C</i>. <i>rodentium</i> and the T cell response was analyzed by flow cytometry. Representative flow cytometry plots of live CD3⁺CD4⁺ T cells stained for IL-17A and IFN-γ (upper panel). Graphs represent frequency (%) of IL-17A⁺ (IFN-γ^+/−), IFN-γ⁺ (IL-17^+/−) and IL-22⁺ cells amongst live CD3⁺CD4⁺ T cells (lower panel). (D) Expression of <i>Reg3g</i> in colonic epithelial cells isolated from mice before (control) or on day 8 p.i. (infected) with <i>C</i>. <i>rodentium</i>. Gene expression is shown relative to <i>Actb</i>. (E) Bacterial load in the feces and the liver on day 8–10 p.i. Data were pooled from two (A) or four (C, D) independent experiments with n = 3 mice (A) or n = 1–4 mice (C, D) per group. Horizontal bar represents mean. Error bar represents +SEM. Dashed line indicates the limit of detection. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p<0.05, **p<0.01, ***p<0.001, ns–not significant.</p

(A) mice were treated with DSS in the drinking water for 6 d, followed by water for 10 d

This protocol was repeated for a total of three cycles. After the last cycle, cells isolated from the colon were restimulated in vitro with PMA/ionomycin for 5 h and subjected to intracellular staining for GFP, IL-17, and Foxp3. Histograms (from left to right) report percent GFPTCR-β cell subsets in total T cells (see ), total numbers of RORγt Tαβ cells present in the organ, and the ratio of IL-17–producing to Foxp3 cells within RORγt Tαβ cells (see ). Right panels show immunofluorescence histology of a colon from a healthy or a treated mouse. Bar, 100 μm. (B) mice were infected intranasally with 100 PFUs of influenza A virus for 7 d. Cells were then isolated from the lung and processed as in A. Right panels show immunofluorescence histology of a lung from healthy or an infected mouse. Bar, 50 μm. (C) Cells were isolated from the mesenteric LNs of a 4-mo-old × mouse and processed as in A. Right panels show immunofluorescence histology of a mesenteric LN from a normal or a tumor-bearing mouse. Bar, 100 μm. Data shown are representative of at least three independent experiments. Three to four mice were analyzed per group. *, P < 0.05 as compared with control (mock-treated or WT mice).<p><b>Copyright information:</b></p><p>Taken from "In vivo equilibrium of proinflammatory IL-17 and regulatory IL-10 Foxp3 RORγt T cells"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1381-1393.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413035.</p><p></p

(A and B) Flow cytometry analysis of cells isolated from the organs of 8–12-wk-old mice

Plots are gated on CD3 cells (A) or GFP CD3 cells (B). Numbers indicate mean percent cells in quadrants ± SD obtained with at least three mice. LPLs, lamina propria lymphocytes isolated from small intestine; mLN, mesenteric LNs; BM, bone marrow. (C) Immunofluorescence histology of RORγt cells in the small intestine of mice. Most RORγt cells in villi are T cells, whereas RORγt cells in cryptopatches located between crypts are CD3 LTi cells. Bar, 50 μm. (D) Expression of CD4 and CD8α by spleen GFPTCR-β and lung or GFPTCR-δ cells.<p><b>Copyright information:</b></p><p>Taken from "In vivo equilibrium of proinflammatory IL-17 and regulatory IL-10 Foxp3 RORγt T cells"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1381-1393.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413035.</p><p></p

(A) Cells isolated from the spleen and mesenteric LNs of mice were sorted into eight distinct populations based on their expression of GFP, CD3, TCR-β, TCR-δ, CD4, and CD25 (Fig

S1), and gene expression was assessed using real-time PCR. Ct values were normalized to the mean Ct of five housekeeping genes. Data are the mean of two or three independent experiments. (B) Foxp3 RORγt T cells express IL-10. Cells isolated from LNs of mice were restimulated in vitro with PMA/ionomycin for 5 h and subjected to intracellular staining for GFP, IL-17, Foxp3, and IL-10 or an isotype control. Numbers indicate percent cells in quadrants. Results are representative of at least three individual experiments.<p><b>Copyright information:</b></p><p>Taken from "In vivo equilibrium of proinflammatory IL-17 and regulatory IL-10 Foxp3 RORγt T cells"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1381-1393.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413035.</p><p></p

MACS-sorted naive (CD62L) CD4 T cells from the spleens of mice were stimulated in duplicates with anti-CD3 and anti-CD28 in the presence of blocking anti–IFN-γ and anti–IL-4 antibodies and the indicated cytokines or RA

After different periods of time, cells were restimulated with PMA/ionomycin for 5 h and analyzed by flow cytometry for the expression of GFP, Foxp3, IL-17, and IL-10. All plots are gated on TCR-β cells, except plots for IL-10 that are gated on GFPTCR-β cells. Numbers indicate percent cells in quadrants. Data are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "In vivo equilibrium of proinflammatory IL-17 and regulatory IL-10 Foxp3 RORγt T cells"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1381-1393.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413035.</p><p></p

pUL11Fc modulates T cell proliferation in a dose dependent manner.

<p>PBMCs were either left unstimulated or stimulated with anti-CD3 (OKT3) together with the indicated concentrations of UL11Fc derived from HCMV strain (A) TB40 or (B) Merlin or with the Fc control protein. Single representative experiments from four (A) or two (B) replicates are shown; error bars show standard deviation of technical replicates.</p

Treatment with pUL11Fc affects phosphorylation of the regulatory tyrosines of Lck.

<p>PBMC were incubated with anti-CD3 (OKT3) in the presence or absence of UL11Fc and Fc at the indicated concentrations for three days. Cells were then labelled extracellularly with anti-CD3 and anti-CD4 and intracellularly with anti-Lck pY505 or anti-Lck pY394. The experiment was repeated three times using cells from different donors. Mean fluorescence intensities are shown for Y505 (i) and Y394 (ii) phosphorylation of live CD3+CD4+ cells, fold increases in geometric mean fluorescent intensity normalised to anti-CD3 treated cells are indicated. A) depicts one representative experiment (values for UL11 treated cells). B) shows fold increases in phosphorylation over all three experiments for UL11Fc and Fc treated cells. Error bars indicate standard deviation. Statistical significance of differences between groups was determined by ANOVA. i)*p = 0.0014 for the difference in Y505 phosphorylation between pUL11Fc and Fc treated cells. ii)*p = 0.0043 for the difference in Y394 phosphorylation between pUL11Fc and Fc treated cells.</p