DCs injected at the time of infection with <i>L. mexicana</i> lead to a more robust Th1 response.

<p>C57BL/6 mice were infected as before in the ear with <i>L. mexicana</i>, however, one group also received DCs that were derived in vitro. At two weeks post-infection ears and dLNs were processed. (<b>A</b>) Levels of IFN-γ (ng/mL) from the supernatants of single cell suspensions from the dLN of each group that were stimulated for 72 hours with <i>L. mexicana</i> freeze-thaw antigen. (<b>B</b>) Percentage and absolute number of iNOS-producing cells in the ear of <i>L. mexicana</i> infected mice or <i>L. mexicana</i> infected mice that received DCs. Cells are previously gated on live, singlets that are CD11b^hi CD11c⁺. (<b>C</b>) The cellularity of the dLN in <i>L. mexicana</i> infected mice or <i>L. mexicana</i> infected mice that received DCs. The results expressed are the mean percentage (± SD for FACS plots) or the mean number of cells (± SE for bar graphs) of 2–4 mice per group. The results are representative of two experiments. * significantly higher (p<0.05) compared to <i>L. mexicana</i> infected mice.</p

Fewer FITC+ endogenous mo-DCs migrate to the dLN during <i>L. mexicana</i> infection compared to <i>L. major</i>.

<p>C57BL/6 mice were infected as above for two weeks. FITC isomer was applied to the ears of naïve, <i>L. major</i> or <i>L. mexicana</i> infected mice on day 14 and dLNs were harvested and processed forty-eight hours later. FITC⁺ MHCII^hi CD11c⁺ cells were enumerated in the dLNs. These cells are previously gated on live, singlets that are CD11b^hi. The results expressed are the mean number of cells (± SE) of 3 mice per group. The results are representative of two experiments. * significantly different (p<0.05) between indicated groups.</p

Fewer transferred monocytes migrate to the dLN during <i>L. mexicana</i> infection compared to <i>L. major</i>.

<p>Monocytes enriched from CD45.1 C57BL/6 mice were injected into the ear of CD45.2 C57BL/6 mice that were infected for two weeks with either <i>L. major</i> or <i>L. mexicana</i>. Eighteen hours following injection of the monocytes, ears and dLNs were harvested and processed. (<b>A</b>) Absolute number of transferred monocytes (CD11b⁺ CD45.1⁺) in the ear. Cells are previously gated on total, live cells that are singlets. (<b>B</b>) Mean fluorescence intensity (MFI) of Ly6C on transferred cells recovered from the ears of infected mice. Percentage (<b>C</b>) or absolute number (<b>D</b>) of CD45.1⁺ cells in the dLN. These cells are previously gated on live, singlets that are CD11b^hi cells. The results expressed are the mean percentage (± SD for FACS plots) or the mean number of cells (± SE for bar graphs) of 3 mice per group. The results are representative of two experiments. * significantly different (p<0.05) compared to <i>L. major</i> infected mice.</p

Mo-DCs produce significantly less iNOS during infection with <i>L. mexicana</i> compared to <i>L. major</i>.

<p>C57BL/6 mice were infected and ears were processed as above. In addition to staining for surface markers, intracellular staining for iNOS was performed. Percentage (<b>A</b>) or absolute number (<b>B</b>) of iNOS-producing cells in the ear of naïve or infected mice on day 14. Cells are previously gated on live, singlets that are CD11b^hi CD11c⁺. The results expressed are the mean percentage (± SD for FACS plots) or the mean number of cells (± SE for the bar graph) of 3 mice per group. The results are representative of two experiments. * significantly lower (p<0.05) compared to <i>L. major</i> infected mice.</p

Production of IL-10 during <i>L. mexicana</i> infection contributes to less recruitment of monocytes.

<p>Ears from naïve, <i>L. mexicana</i> infected or <i>L. mexicana</i> infected and α-IL-10R treated C57BL/6 mice were processed as above. (<b>A</b>) Histograms of monocytes on day 7 and 14 or mo-DCs on day 14 in the ear of naïve (grey shaded histogram), <i>L. mexicana</i> infected mice (black line, top) or <i>L. mexicana</i> infected and α-IL-10R treated mice (black line, bottom). Absolute numbers of monocytes and mo-DCs are also shown. Monocytes are previously gated on live, singlets that are CD11b^hi CD11c⁻. Mo-DCs are previously gated on live, singlets that are CD11b^hi CD11c⁺. (<b>B</b>) Levels of IFN-γ (ng/mL) from the supernatants of single cell suspensions from the dLN of each group that were stimulated for 72 hours with <i>L. mexicana</i> freeze-thaw antigen. (<b>C</b>) Percentage and absolute number of iNOS-producing cells in the ear of naïve, <i>L. mexicana</i> infected mice or <i>L. mexicana</i> infected and α-IL-10R treated mice on day 14. Cells are previously gated on live, singlets that are CD11b^hi CD11c⁺. The results expressed are the mean percentage (± SD for FACS plots) or the mean number of cells (± SE for bar graphs) of 3–5 mice per group. The results are representative of two experiments. * significantly higher (p<0.05) compared to <i>L. mexicana</i> infected mice in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001858#pntd-0001858-g006" target="_blank">Fig. 6A and 6C</a> (FACS plots) or p<0.05 between indicated groups in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001858#pntd-0001858-g006" target="_blank">Fig. 6A, 6B and 6C</a> (bar graphs).</p

Fewer CD11b^hi Ly6C⁺ cells in the ear following <i>L. mexicana</i> infection compared to <i>L. major</i>.

<p>Ears from naïve, as well as <i>L. major</i> or <i>L. mexicana</i> infected C57BL/6 mice were processed to single cell suspensions. (<b>A</b>) Percentage of CD11b^hi cells present in the ear of naïve or infected mice on day 3 and 14. Cells are previously gated on total, single live cells. Histograms of monocytes (<b>B</b>) or mo-DCs (<b>C</b>) in the ear of naïve (grey shaded histogram) or infected mice (black line) on day 3 and 14 and absolute number of monocytes (<b>B</b>) or mo-DCs (<b>C</b>) from naïve, day 3 and day 14 infected mice. Monocytes are pre-gated on CD11b^hi CD11c⁻ cells and mo-DCs are previously gated on CD11b^hi CD11c⁺ cells. The results expressed are the mean percentage (± SD for FACS plots) or the mean number of cells (± SE for bar graphs) of 3 mice per group. The results are representative of two experiments. * significantly lower (p<0.05) compared to <i>L. major</i> infected mice in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001858#pntd-0001858-g001" target="_blank">Fig. 1A</a> or p<0.05 between indicated groups in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001858#pntd-0001858-g001" target="_blank">Fig. 1B</a> and C.</p

<i>L. mexicana</i> infection does not lead to a defect in differentiation of monocytes to mo-DCs.

<p>C57BL/6 mice were infected in the ear with <i>L. major</i> or <i>L. mexicana</i> and ears were processed as above. Percentage of monocytes and mo-DCs on day 3 and 14 were used to determine a ratio of differentiation from monocytes to mo-DCs in <i>L. major</i> or <i>L. mexicana</i> infected mice. The results expressed are the mean ratio (± SE) of 3 mice per group. The results are representative of two experiments.</p

IL-22 does not modulate the skin microbiome at the steady state.

<p>Swabs were collected from <i>Il22</i>^<i>+/+</i>, <i>Il22</i>^<i>+/-</i>, and <i>Il22</i>^<i>-/-</i> cohoused littermates and bacterial DNA was isolated and sequenced. <b>(A)</b> Within sample diversity was calculated using four commonly utilized alpha metrics: Chao I, Shannon Index, Observed Species, and Faith’s Phylogenetic Diversity. <b>(B)</b> The microbiome composition was calculated at multiple phylogenetic levels. The outer ring represents the relative contributions of the 12 most prevalent genera. The inner ring represents the corresponding class for each genus. The remaining genera were compiled into the “Other” category depicted in gray. Data are representative of 2 independent experiments, with 4–5 mice per group.</p

IL-22 limits pathology during leishmania infection.

<p><b>(A)</b> C57BL/6 (wild-type) and <i>Il22</i>^<i>-/-</i> mice were intradermally infected with 2 x 10⁶<i>L</i>. <i>major</i> promastigote metacyclics and euthanized at various time-points after infection. The lesions were assessed by measuring ear thickness for 6 weeks. <b>(B)</b> Pictures were taken at 5 weeks post-infection. <b>(C)</b> Lesion pathology was determined based on a pathology score. <b>(D)</b> Number of parasites in the lesions was quantified using a limiting assay at 2, 5, and 12 weeks post-infection. <b>(E)</b> Wild-type and <i>Il22</i>^<i>-/-</i> mice were intradermally infected with 2 x 10⁶<i>L</i>. <i>braziliensis</i> promastigote metacyclics and lesions were assessed by measuring ear thickness and given a pathology score for 12 weeks and <b>(F)</b> parasite numbers were quantified using a limiting dilution assay in the lesions at 12 weeks post-infection. Data are representative of at least 2 independent experiments, with 3–5 mice per group. Error bars indicate mean ± SEM, *p < 0.05.</p

The requirement for IL-22 is parasite dose dependent.

<p>Lesion sizes and pathology scores were compiled from several experiments at 5 weeks post-infection from wild-type and <i>Il22</i>^<i>-/-</i> mice that were intradermally infected with <b>(A)</b> 2 x 10³, <b>(B)</b> 2 x 10⁶, or <b>(C)</b> 2 x 10⁷<i>L</i>. <i>major</i> metacyclics. RNA was isolated from the lesions of wild-type mice infected with <i>L</i>. <i>major</i> to assess <b>(D)</b><i>Il22</i> and <b>(E)</b><i>Il22BP</i> expression. Data are represented as relative expression to housekeeping gene <i>rps11</i> and are representative of at least 2 independent experiments, with 3–5 mice per group. Error bars indicate mean ± SEM, *p < 0.05,*p < 0.01, ***p < 0.001.</p