mRNA analysis of IL-12a and IL-33.

(A,B) mRNA analysis of IL-12a and IL-33 at week two in different treatment groups. Data are from one experiment with a total of five to six mice in each group (A,B). Error bars show SEM. Two-tailed unpaired Student’s t-test with Welch’s correction. ns, not significant. (TIF)</p

Number of total ILCs and Ki67⁺ ILCs.

(A) Number of ILCs in WT and Batf3-/- mice at week two in different treatment groups. (B) Number of Ki67+ ILCs at week two in WT mice in different treatment groups. Data are from one experiment with a total of five to six mice in each group (A,B). Error bars show SEM. Two-tailed unpaired Student’s t-test with Welch’s correction. ns, not significant, *p (TIF)</p

Analysis of IL-33R, IL-1R and IL-12R on IL-17A⁺ and IL-17A^- ILCs.

Flow cytometry analysis of IL-33R, IL-1R and IL-12R on IL-17A+ and IL-17A- ILCs in the S. epi colonized and L. major infected mice at week two. Total ILCs were used as isotype control. Data are from two experiments with a total of three to four mice in each group. Error bars show SEM. Two-tailed unpaired Student’s t-test with Welch’s correction. ns, not significant ***p (TIF)</p

Early IFN-γ and IL-13 induction in ILCs after <i>L. major</i> infection.

(A,B) Percent and number of IFN-γ+ ILCs from ILCs in None (uninfected) and L. major infected mice at week one. (C,D) Percent and number of IL-13+ ILCs from ILCs in None (uninfected) and L. major infected mice at week one. Cells were stimulated with Pma/Ino for 4 hours. Data are from two experiments with a total of five to eight mice in each group (B,D). Numbers within the flow plot show percent of cells within the gated box. Error bars shows SEM. ns, not significant. Two-tailed unpaired Student’s t-test with Welch’s correction. (TIF)</p

IFN-γ and IL-5 in <i>Rag1^-/-</i> mice.

(A) Representative flow cytometry plot of IFN-γ and IL-5 staining from ILCs in Rag1-/- mice treated with S. epi. or L. major alone or co-treated with S. epi and L. major. Numbers represent percent of cells within the gated box. (B) Number of IFN-γ and IL-5 producing ILCs in Rag1-/- mice treated with S. epi. or L. major alone or co-treated with S. epi and L. major. Cells were stimulated with Pma/Ino for 4 hours. Number within the flow plot shows percent of cells within the gated box. Error bars shows SEM. Data are from one or two experiments with a total of three to six mice in each group. (TIF)</p

IL-17A⁺ ILCs at the peak of inflammation.

(A) Experimental model for S. epi colonization and L. major infection. (B) Skin thickness measurement during the course of infection. (C) Percent and number of IL-17A+ ILCs in different treatment groups at week 5. Cells were stimulated with Pma/Ino for 4 hours. Data are from one experiment with a total of four to eight mice in each group (B,C,D). Number within the flow plot show percent of IL-17A+ cells with SEM. Error bars shows SD (B) and SEM (D). Two-tailed unpaired Student’s t-test with Welch’s correction. ns, not significant, *p (TIF)</p

<i>S</i>. <i>epidermidis</i> dependent IL-17A⁺ILCs require IL-23.

(A) qRT-PCR analysis of IL-1α, IL-1β and IL-23a in WT and Batf3-/- mice in different treatment groups at week two. (B) IL-23R expression on IL-17A+ and IL-17A-ILCs in S. epi colonized and L. major infected mice at week two. (C) Schematic representation of anti-IL-23 and anti-Ly6G treatment protocol in S. epi colonized and L. major infected WT mice. (D) IL-17A production from ILCs in control (IgG) and anti-IL-23 treated in S. epi colonized and L. major infected at week two. (E) Ear thickness measurement and pathology score in IgG and anti-IL-23 treated L. major and in S. epi colonized and L. major infected mice at week one and two. (F,G) Number of neutrophils and monocytes in different treatment groups at week two. (H) Ear thickness measurement and pathology score in IgG and anti-Ly6G treated L. major and in S. epi colonized and L. major infected mice at week one and two. Number within the flow plot show percent of IL-17A+ cells within the gated box. Data are from one experiment with a total of three to five mice in each group (A,B,D,E,F,G,H) Error bars show SEM (A,D,F) or SD (E,H). Two-tailed unpaired Student’s t-test with Welch’s correction or one-way ANOVA with Tukey’s multiple comparison analysis (A,F,G). ns, not significant; *p<0.05, **p<0.01, ***p<0.001.</p

Early IFN-γ induction in T cells after <i>L</i>. <i>major</i> infection.

(A,B) Percent and number of IFN-γ+ γδlow T cells in None (uninfected) and L. major infected mice at week one. (C,D) Percent and number of IFN-γ+ αβ T cells in None (uninfected) and L. major infected mice at week one. Cells were stimulated with Pma/Ino for 4 hours. Data are from two experiments with a total of five to six mice in each group (B,D). Numbers within the flow plot show percent of cells in within the gated box. Error bars shows SEM. ns, not significant. Two-tailed unpaired Student’s t-test with Welch’s correction. (TIF)</p

. <i>S</i>. <i>epidermidis and S</i>. <i>xylosus</i> colonization before <i>L</i>. <i>major</i> infection increase the inflammatory responses and IL-17A⁺ILCs.

(A) Schematic representation of S. epidermidis (S. epi) and L. major treatment protocol in C57BL/6 mice. (B) Recovered total colony forming units (CFUs) in the ear of different treatment groups at week two. (C) Recovered pink (S. epi-2W) colony forming units (CFUs) in the ear of different treatment groups at week two. (D) Ear thickness measurement and pathology score in mice associated with S. epi or unassociated prior to L. major infection at week one and two. (E) Percent of IL-17Α+ILCs in the skin of different treatment groups at week two. (F) Number of IL-17Α+ILCs in the skin of different treatment groups at week two. (G) Ki67 staining on IL-17A+ ILCs in S. epi colonized and L. major infected mice at week two. Total ILCs were used as isotype. (H) Percent of RORγt+ILCs in the skin of different treatment groups at week two. (I) IL-17A production from RORγt+ILCs and RORγt-ILCs in S. epi colonized and L. major infected mice at week two. (J) Number of RORγt+ILCs in the skin of different treatment groups at week two. (K) Number of RORγt+IL-17A+ILCs in the skin of different treatment groups at week two. (L) Ear thickness measurement and pathology score in mice associated with S. xylosus (S. xylo) or unassociated prior to L. major infection at week one and two. (M, N) Percent and number of IL-17Α+ILCs in the skin of different treatment groups at week two. The number within the flow plots show percent of IL-17A+ cells (E,M) or RORγt+ cells (H) with SEM or IL-17A+ cells with in the gated box (I). Data are from three experiments with a total of 10 to 16 mice in each group (E,F,M,N) except control groups in N which is from one experiment with three mice or from one experiment representative of two with five mice in each group (B,C,D,L) or from one experiment with three to five mice in each group (G,H,J,K). Error bars shows SEM (B,H,I.J,N) or SD (D,L). Two-tailed unpaired Student’s t-test with Welch’s correction or one-way ANOVA with Tukey’s multiple comparison analysis (B,F,N). ns, not significant; *p<0.05, **p<0.01, ***p<0.001.</p

Immunopathology in WT and <i>Rag1^-/-</i> mice.

(A) Experimental model for S. epi colonization and L. major infection in WT and Rag1-/- mice. (B,C) Skin thickness and pathology score during the course of infection. Data are from one experiment with a total of three to five mice in each group. Error bars show SEM. Two-tailed unpaired Student’s t-test with Welch’s correction. ns, not significant, *p (TIF)</p