Early recruitment of highly activated monocytes and neutrophils after IL-10R signaling blockade.

<p>Cells from naïve ears or from the ears of 1 week infected mice from control or anti-IL-10R treated animals were collected and analyzed by flow cytometry. Representative flow analysis (left) of CD11b⁺, Ly6C⁺, Ly6G⁺ (A), MHCII⁺ and iNOS⁺ (B) expression and bar graph (right) of frequency and number of cells recovered per ear at 1 week after infection. Numbers shown in Ly6C⁺, Ly6G⁺ and iNOS⁺ plots represent the percentage of expression in the CD11b⁺ gated population. MHCII⁺ expression is depicted as the percentage of expression in Ly6C⁺ cells. Values represent the mean ± SEM of 5 mice per group. The data shown are from one experiment and are representative of at least three experiments (*, <i>p</i><0.05).</p

IL-10R signaling blockade increases IFN-γ and IL-17A production.

<p>Cells from naïve or 5 week infected draining lymph nodes or from ears were analyzed for IFN-γ, IL-17A and IL-4 production. Antigen-specific cytokine release by draining lymph node cells from 5 week infected mice was determined by ELISA after restimulation with <i>L. major</i> freeze-thawed antigen (FTAg). Values represent mean ± SEM of 5 mice per group (ND: not detectable) (A). Cells from the ears of 5 week infected mice were stimulated with PMA and ionomycin for 4 h and analyzed for intracellular cytokine production. Contour plots and bar graphs show the frequency and number of IFN-γ (B) and IL-17A (C) in gated CD4 T cells at 1 (top) and 5 (bottom) weeks after infection. Frequency of double positive IL-17A/IL-10 (D) or IL-17A/IFN-γ (E) producer CD4 T cells at 1 or 5 weeks after infection, respectively. Quadrant values are the percentages from total gated population. Numbers between parentheses indicate the mean fluorescence intensity (MFI) for the IL-17 within the CD4 T cells. The data shown are from one experiment and are representative of at least three experiments (*, <i>p</i><0.05).</p

IL-17 neutralization reverses the pathology induced after blockade of IL-10R signaling.

<p>C57BL/6, or <i>Il-10^−/−</i> mice were infected intradermally with <i>L. major</i> metacyclic promastigotes. Either isotype, anti-IL-10R or anti-IL-17 or both mAbs were administrated at day-1 and twice weekly during 4 weeks. Lesion development was assessed by measuring ear thickness (A, F). Values represent mean induration in mm (mean ± SEM) of 5 mice per group. Numbers of parasites in ear lesions were quantified using limiting dilution assays at 6 and 5 wk after infection (B, F). Mean ± SEM parasite numbers are shown as individual parasite counts per ear. Bar graph showing number of Ly6G⁺ cells per ear (C). Level of cytokines was measured in supernatants from <i>in vitro</i> stimulated draining lymph node cells with <i>L. major</i> FTAg (D, E) (ND: not detectable). (G) H&E staining of histological sections of paraffin-embedded ears at 6 wks of infection showing epithelial hyperplasia (head arrow), leukocyte infiltration in epidermis (black arrows) and localized neutrophils infiltration in deep dermis marked as * in the anti-IL-10R treated sections (Bars, 100 µm; EP: epidermis, D: dermis). The data shown are from one experiment and are representative of at least three experiments (*, <i>p</i><0.05).</p

CD4 and CD8 T cells are primary source of IL-10 during early <i>L. major</i> infection.

<p>IL-10-eGFP reporter mice (Vert-X mice) were infected intradermally in the ear <i>L. major</i> metacyclic promastigotes. Some mice were treated with anti-IL-10R mAb as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003243#s4" target="_blank">material and methods</a>. One week after infection, cells from the ear were prepared and stained for CD11b, CD19, TCRγδ, NK1.1, CD8 and CD4 T cells. Additional intracellular staining was performed for Foxp3 and IFN-γ. Frequency of IL-10-eGFP⁺ cells on different populations (A). Gated Foxp3⁺ or Foxp3⁻ CD4⁺ T cells depicting IL-10-eGFP expression (B) and IFN-γ production (C) in infected mice are shown. Number represents percentage of IL-10-eGFP on each gated population. Histogram showing the frequency (right) and number (left) of Foxp3⁺ or Foxp3⁻ CD4T cells expressing IL-10-eGFP (D). Values are mean ± SEM of 5 mice per group and representative of two experiments (*, <i>p</i><0.05).</p

IFN-γ inhibits neutrophil recruitment and IL-17 production after IL-10R blockade.

<p>Mice were infected intradermally with <i>L. major</i> metacyclic promastigotes. Isotype, anti-IL-10R or anti-IFN-γ monoclonal antibodies were administrated at day −1 and twice weekly for 1 week. Cells from the ears were collected, stained and analyzed by flow cytometry. Bar graph showing the frequency of Ly6C⁺, MHCII⁺ and iNOS⁺ expression (A). Numbers on Ly6C⁺ and iNOS⁺ represents percentage of expression on CD11b⁺ Ly6G⁻ gated population. MHCII expression is depicted as the percentage of expression in Ly6C⁺ cells. Ear thickness was measured after 1 week of intradermal inoculation of 2×10⁶<i>L. major</i> metacyclic promastigotes (B) Values represent mean induration in mm (mean ± SEM) of 5 mice per group. Numbers of parasites in ears were quantified using limiting dilution assays (B). Bar graph showing frequency of Ly6G⁺ cells in CD11b⁺ (C). Antigen-specific cytokine release by draining lymph node cells after restimulation with <i>L. major</i> FTAg measured by ELISA (D). Value is the mean ± SEM of 5 mice per group. The data shown are from one experiment and are representative of at least two experiments (*, <i>p</i><0.05). PBMCs from patients with leishmaniasis were cultured with soluble <i>leishmania</i> antigen in the presence or absence of IL-10 or anti-IFN-γ for 4 days. Antigen-specific IL-17 production was analyzed by ELISA (E, F).</p

IL-10 protects mice from developing severe pathology following infection with <i>L. major.</i>

<p>Mice were infected intradermally with <i>L. major</i> metacyclic promastigotes. Isotype or anti-IL-10R monoclonal antibodies were administrated at day −1 and twice weekly for 4 weeks. Lesion development was assessed (mean ± SEM) in C57BL6 (WT) or <i>Il10</i>^−/− mice by measuring ear thickness after intradermal inoculation of 2×10⁶ (A, C) <i>L. major</i> metacyclic promastigotes. Values represent mean induration in mm (mean ± SEM) of 5 or more mice per group. Numbers of parasites in ear lesions were quantified using limiting dilution assays at 5 wk (B) and 20 wk (D) after infection. Mean ± SEM parasite loads are shown as individual parasite counts per ear. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003243#s2" target="_blank">Results</a> are pooled from four independent experiments (B) or representative of two independent experiments (D). Representative photograph of C57BL/6 mice ears, 3 wks after inoculation of <i>L. major</i> metacyclic promastigotes (E). H&E staining of histological sections of paraffin-embedded ear at 5 wks of infection showing epithelial hyperplasia (head arrow), leukocyte infiltration in epidermis (black arrows) and localized neutrophils infiltration in deep dermis marked as * (Bars, 100 µm; EP: epidermis, D: dermis). The lower panels are at a higher magnification (F). *, <i>p</i><0.05 compared with isotype control mice.</p

Increased IL-17 production after IL-10R blockade is IL-1R1 dependent.

<p>C57BL/6 and <i>Il1r1^−/−</i> mice were infected intradermally with <i>L. major</i> metacyclic promastigotes. Isotype or anti-IL-10R mAb were administrated at day-1 and twice at week during 4 weeks after infection. Real-time PCR analysis was performed to quantify mRNA expression of cytokines in lesions after 1 week of infection from C57BL/6 control and treated mice. Data are expressed as the fold change relative to naïve ear (A). Lesion development was assessed by measuring ear thickness (B). Values represent mean induration in mm (mean ± SEM) of 5 or more mice per group. Numbers of parasites in ear lesions were quantified using limiting dilution assays at 4 wk after infection (C). Mean ± SEM parasite numbers are shown as individual parasite counts per ear. Number of Ly6G⁺ cells per ear (D). Antigen-specific cytokine release by draining lymph node cells from 4 weeks infected mice was determined by ELISA after restimulation with <i>L. major</i> FTAg (E). Values are mean ± SEM of 5 mice per group (ND: not detectable). The data shown are from one experiment (*, <i>p</i><0.05, difference statistically).</p