The absence of IL-4Rα and Relmα affects the phenotype of mononuclear phagocytes recovered from the skin.

<p>Total number of viable DEC recovered from pinnae of WT and <b>A.</b> IL-4RαKO, and <b>B</b>. RelmαKO mice. Absolute numbers of <b>C & F.</b> F4/80^-MHC-II^high DCs (R4), <b>D & G.</b> F4/80⁺MHC-II^high tissue resident macrophages (R4A), and <b>E & H.</b> F4/80⁺MHC-II^int macrophages (R3) in 1x and 4x infected WT, IL-4RαKO and RelmαKO mice. <b>I.</b> Absolute numbers of CD3⁺CD4⁺ T cells in the DEC of 1x and 4x infected WT and IL-4RαKO mice. Symbols are values for individual mice; horizontal bars are means ±SEM (n = 4–5 mice per group). n.s. denotes ‘not significant’ p>0.05; * = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001 as determined by ANOVA and Tukey’s post-test analysis, or Student’s t test.</p

Increased CD4⁺cellularity of sdLN from 4x infected mice in the absence of IL-4Rα and RELMα but not a reversal of CD4⁺ cell hypo-responsiveness.

<p>Number of <b>A</b> & <b>B.</b> total cells, and <b>C</b> & <b>D</b>. CD4⁺ T cells in the sdLN of naïve, 1x and 4x infected WT, IL-4RαKO, and RelmαKO mice. <b>E & F</b> Number of CD4⁺ BrdU⁺ cells, and <b>G & H.</b> percentage of CD4⁺ T cells that are BrdU⁺ in the sdLN of naïve, 1x and 4x infected WT mice compared to IL-4RαKO and RelmαKO mice. Symbols are values for individual mice; horizontal bars are means ±SEM (n = 4–5 mice per group). n.s. denotes ‘not significant’ p>0.05; * = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001 as determined by ANOVA and Tukey’s post-test analysis, or Student’s t test.</p

Genes related to alternative activation cell phenotype are up-regulated in dermal exudate cells (DEC) populations after repeated infection.

<p><b>A.</b> Representative flow cytometry dot plots for DEC showing the distribution of cells based upon their expression of F4/80 and MHC-II. Cells were gated into R4, R4A and R3 populations recovered from 1x (left) and 4x (right) infected mice. Values in bold are the percent positive cells found within each gate expressed as a proportion of the total DEC recovered. Cells were sorted by fluorescent activated cell sorting (FACS), and RNA from the sorted R4, R4A and R3 cell populations was applied to microarray analysis. Heat maps showing the clustering of genes within each sorted DEC population for three biological replicates recovered after <b>B.</b> a single (1x) infection, and <b>C.</b> repeated (4x) infections. <b>D.</b> Identity of the top 15 up-regulated genes in each population after 4x compared to 1x infection. Number in brackets represents the fold up-regulation of that gene in the sorted cell population from 4x infected mice. Grey shaded genes represent those that are associated with alternative activation.</p

Expression of Fas/FasL and viability of CD4⁺ T cells in the sdLN is enhanced in the absence of IL-4Rα.

<p>Percentage of CD4⁺ T cells that are <b>A.</b> Fas⁺ and <b>B.</b> FasL⁺ in 1x and 4x infected WT compared to IL-4RαKO mice. <b>C-E</b> Percentage of viable (AnnV^-PI^-), apoptotic (AnnV⁺PI^-) and dead (AnnV⁺PI⁺) CD4⁺ T cells in the sdLN of 1x and 4x infected mice as determined by an AnnexinV assay. n = 3–4 mice per group; n.s. denotes ‘not significant’ p>0.05; * = p<0.05; ** = p<0.01; **** = p<0.0001 as determined by ANOVA and Tukey’s post-test analysis.</p

IL-10 producing dermal CD4⁺ cells do not express markers of conventional regulatory T cells.

<p>(<b>A</b>) Representative flow cytometry dot plots for thymic (Nrp1⁺Helios⁺) T_reg cells, peripheral (FoxP3⁺Helios^-) T_reg cells, and Tr1 (CD223⁺CD49b⁺) cells. (<b>B</b>) Mean proportion +SEM and (<b>C</b>) absolute numbers of dermal CD4⁺ thymic and peripheral T_reg cells, as well as Tr1, and other CD3⁺CD4⁺ (Helios^-FoxP3^-, not co-expressing CD223 plus CD49b) dermal cell populations recovered from 1x (grey), or 4x (black) infected mice on day 4 post-final exposure. (<b>D</b>) Mean proportions +SEM and (<b>E</b>) absolute numbers of dermal T cells defined in <b>A</b> gated on IL-10^GFP+ CD4⁺ T cells from 1x or 4x infected mice. ANOVA and Sidak’s multiple comparisons test were performed to compare the means of selected groups (* = p<0.05; **** = p<0.0001; ns = p>0.05).</p

CD4⁺ T cells are the predominant source of IL-10 in DEC.

<p>(<b>A</b>) Representative flow cytometry dot plots, and (<b>B</b>) percentages ± SEM (n = 4–6 pinnae), of IL-10^GFP+ DEC from 1x and 4x infected mice; gate shows proportions IL-10^GFP+ cells. Representative flow cytometry dot plots of IL-10^GFP+ DEC from infected (<b>C</b>) 1x and (<b>D</b>) 4x IL-10^+/GFP mice on day 4 after final exposure to infectious cercariae. Density plots show initial F4/80 <i>vs</i>. MHC-II gating strategy (left-hand plots) to yield R1 (double negative F4/80^-MHC-II^-), R2 (F4/80⁺MHC-II^-), R3 (F4/80⁺MHC-II^mid), R4 (F4/80^-MHC-II^high), and R4A (F4/80⁺MHC-II^high) cell populations. R1 was then gated according to CD4 <i>vs</i>. CD3 expression (right-hand plot). Proportions of IL-10^GFP+ DEC from infected (<b>E</b>) 1x and (<b>F</b>) 4x mice. (<b>G</b>) Proportion of IL-10^GFP+ CD3⁺CD4⁺ T cells of total IL-10^GFP+ cells in DEC from naïve, 1x or 4x infected mice on day 4 post-final infection. Symbols are values for cells obtained from independent tissue samples; horizontal bars are the means ± SEM; n = 4–6 pinnae. ANOVA and multiple comparisons tests (Sidak’s and Tukey’s) were performed to compare the means of selected groups (** = p<0.01; *** = p<0.001; **** = p<0.0001; ns = p>0.05).</p

Dermal CD4⁺ T cells from skin exposed to <i>S</i>. <i>mansoni</i> cercariae respond <i>in vitro</i> to parasite and skin commensal antigens.

<p>(<b>A</b>) Representative flow cytometry density plots of CFDA-SE labelled CD3⁺CD4⁺ dermal T cells from 1x or 4x infected WT mice recovered on day 1 or 4 after infection; DEC were obtained from skin biopsies and stimulated <i>in vitro</i> for 96h in the presence, or absence of parasite antigen (SSAP). Gate is set for CFDA-SE^dim cells previously gated on CD3⁺CD4⁺cells. (<b>B</b>) Percent CFDA-SE^dim CD3⁺CD4⁺ cells (mean +SEM; n = 4–5). (<b>C</b>) Mean percentage +SEM of CFDA-SE^dim CD3⁺CD4⁺ T cells from DEC cultures in the presence or absence of SSAP, or antigen from skin commensals (SC), from 1x infected mice on day 1 or 4 after infection. Production of (<b>D</b> & <b>E</b>) IL-10, (<b>F</b> & <b>G</b>) IL-4 and (<b>H</b> & <b>I</b>) IFN-γ in culture supernatants of skin biopsies from 1x or 4x infected mice obtained on day 1 and 4 post-final infection, cultured in the presence, or absence of SSAP or SC antigen; bars are means + SEM, n = 4–5. ANOVA and Sidak’s multiple comparisons test analysis show statistically significant differences between selected groups (* = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001; ns = p>0.05).</p

IL-10^GFP+CD4⁺ T cells from the skin of mice infected with <i>S</i>. <i>mansoni</i> suppress proliferation of CD4⁺ T cells in the skin draining lymph nodes.

<p>(<b>A</b>) Representative flow cytometry dot plots of CFDA-SE^dim CD3⁺CD4⁺ cells from the sdLN of 1x infected mice after co-culture for 96h with dermal CD4⁺ T cells from 4x infected WT or IL-10^-/- mice in the presence of parasite antigen SSAP. (<b>B</b>) Data is given as sdLN CD3⁺CD4⁺ T cell proliferation expressed as the fold-change observed in each of seven corresponding mice co-cultured in the presence of dermal CD4⁺ T cells from either WT or IL-10^-/- mice relative to sdLN CD3⁺CD4⁺ T cells cultured without added dermal CD4⁺ T cells. Symbols are individual mice; horizontal bars are means ± SEM; n = 7. (<b>C</b>) Representative flow plots and (<b>D</b>) fold change ± SEM of CFDA-SE^dim sdLN CD3⁺CD4⁺ T cells from 1x infected mice (n = 8) co-cultured with GFP⁺, or GFP^Neg dermal CD4⁺ T cells from 4x infected mice in the presence of SSAP. Differences between the means of selected groups were compared by ANOVA and Tukey’s multiple comparisons test analysis (* = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001; ns = p>0.05).</p

IL-10 produced in the skin following repeated exposures to <i>S</i>. <i>mansoni</i> cercariae limits CD4⁺ T cell proliferation and mediates recruitment of granulocytes.

<p>(<b>A</b>) IL-10 released from overnight <i>in vitro</i> cultures of skin (pinnae) biopsies from naïve, 1x or 4x infected mice measured by ELISA. Data are means +SEM (6–8 pinnae per group). (<b>B</b>) Pinnae thickness (x10^-1 μm) (n = 8 pinnae) and (<b>C</b>) DEC numbers (n = 6–13 pinnae) for naïve, 1x and 4x infected WT, or IL-10^-/- mice, on day 4 post-final exposure. Symbols are values for individual tissue samples; horizontal bars are the means ±SEM. (<b>D</b>) IL-12p40 detected in overnight cultures of skin biopsies from naive, infected 1x, or 4x WT and IL-10^-/- mice. Values are means +SEM (n = 8 pinnae). (<b>E</b>) CD11b^highGr1⁺ neutrophils as a proportion of DEC recovered from the skin infection site of 1x, or 4x infected WT and IL-10^-/- mice. Symbols are values for independent tissue samples; horizontal bars are the means ±SEM; n = 6–8 pinnae. (<b>F</b>) Mean percentage ±SEM of SiglecF⁺F4/80⁺MHC-II^- eosinophils in DEC recovered from infected 1x, or 4x WT and IL-10^-/- mice. Symbols are values for cells obtained from independent tissue samples; horizontal bars are the means ±SEM; n = 4–5 pinnae. (<b>G</b>) Representative flow cytometry dot plots and (<b>H</b>) mean percentages ± SEM of dermal CD3⁺CD4⁺ T cells (gated on CD11b^-F4/80^-MHC-II^- DEC) from 1x, or 4x infected WT and IL-10^-/- mice on day 4 post-final exposure. (<b>I</b>) Percentage of proliferating BrdU⁺ dermal CD3⁺CD4⁺ T cells obtained from 1x, or 4x infected WT and IL-10^-/- mice. Symbols are values for cells obtained from independent tissue samples; horizontal bars are the means ± SEM; n = 6–8 pinnae. ANOVA and multiple comparisons tests (Bonferroni’s, Sidak’s and Tukey’s) were performed to compare the means of selected groups (* = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001; ns = p>0.05). (<b>J</b>) Merged confocal Z-stack images (9) from cryosections of pinnae incubated with mAbs specific for CD4 (green), MHC-II (red) and CD11b (yellow), and counterstained with DAPI nuclear stain (blue), from (<b>i</b>, <b>ii & iii</b>) 4x WT or (<b>iv, v & vi</b>) 4x IL-10^-/- skin. Scale bar = 100μm (i, ii, iv & v); = 10μm (iii & vi).</p

Lack of CD4⁺ T cells ablates IL-10 production induced in skin exposed to <i>S</i>. <i>mansoni</i> cercariae.

<p>(<b>A</b>) Proportions of CD11b^highGr1⁺ neutrophils and (<b>B</b>) SiglecF⁺F4/80⁺MHC-II^- eosinophils in total DEC recovered from 1x, or 4x infected WT and Rag2^-/- mice on day 4 post infection. Symbols are values for cells obtained from independent tissue samples; horizontal bars are the means ± SEM; n = 4–6 pinnae per group. Production of (<b>C</b>) IL-10 and (<b>D</b>) IL-4 in overnight culture supernatants of skin biopsies from 1x or 4x infected WT and Rag2^-/- mice obtained on day 4 post-final infection; bars are means + SEM, n = 4–6 pinnae per group, as measured by ELISA. Significant differences means were compared between of selected groups by ANOVA and post-hoc tests (Sidak’s and Tukey’s) (* = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001; ns = p>0.05).</p