PIC880209 Supplemental Material - Supplemental material for Fluid flow analysis of a turbulent offset jet impinging on a wavy wall surface

Supplemental material, PIC880209 Supplemental Material for Fluid flow analysis of a turbulent offset jet impinging on a wavy wall surface by Tej Pratap Singh, Amitesh Kumar and Ashok Kumar Satapathy in Proceedings of the Institution of Mechanical Engineers, Part C: Journal of Mechanical Engineering Science</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

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

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

. <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

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

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

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

IL-17A⁺ILCs are sufficient to drive <i>S</i>. <i>epidermidis</i> mediated inflammation.

(A) Schematic representation of S. epi and L. major treatment protocol in Rag1-/- mice. (B) Ear thickness measurement and pathology score in Rag1-/- mice colonized with S. epi or uncolonized prior to L. major infection at week one and two. (C,D) Percent and number of IL-17A- producing ILCs at week two in Rag1-/- mice colonized with S. epi or uncolonized prior to L. major infection. (E) Schematic representation of ILC depletion or IL-17 neutralization treatment protocol in Rag1-/- mice. (F) Representative flow cytometry plot of ILC depletion as identified as CD45+CD90+ cells in Rag1-/- mice. (G) Number of CD90+ cells in control and anti-CD90.2 treated mice. (H) Ear thickness measurement and pathology score in Rag1-/- mice with and without (Control; IgG) ILC depletion in different groups. (I) Ear thickness measurement and pathology score in Rag1-/- mice with and without (Control; IgG) IL-17A neutralization by anti-IL-17A antibody in different groups. Number within the flow plot show percent of IL-17A+ cells with SEM (C). Data are from two experiments with a total of six to seven mice in each group. Statistical comparison within the flow plot is with L. major group. Error bars shows SEM (D,G) or SD (B,H,I). Two-tailed unpaired Student’s t-test with Welch’s correction. *p<0.05, **p<0.01, ***p<0.001.</p

<i>S</i>. <i>epidermidis</i> dependent IL-17A⁺ILCs and inflammatory responses require CD103⁺ DCs.

(A) Schematic representation of S. epi and L. major treatment protocol in WT and Batf3-/- mice. (B) Ear thickness measurement and pathology score in WT and Batf3-/- mice colonized with S. epi or uncolonized prior to L. major infection at week one and two. (C,D) Percent and number of CD103+ DCs at week two in WT and Batf3-/- mice colonized with S. epi or uncolonized prior to L. major infection at week two. (E) L. major parasite load in WT and Batf3-/- mice colonized with S. epi or uncolonized prior to L. major infection at week two. (F,G) Percent and number of IFN-γ+αβ T cells in WT and Batf3-/- mice colonized with S. epi or uncolonized prior to L. major infection at week two. (H,I) Percent and number of IL-17A+ILCs in WT and Batf3-/- mice colonized with S. epi or uncolonized prior to L. major infection at week two. (J,K) Percent and number of RORγt+IL-17A+ILCs in the skin of S. epi colonized and L. major infected WT and Batf3-/- mice at week two. Number within the flow plot show percent of CD103+CD11c+ (C), IFN-γ+ (F) and IL-17A+ (H,J) cells with SEM. Data are from two experiments with a total of six to eight mice in each group (C,D,F,G,H,I) or from one experiment representative of two with five mice in each group (B,E). Statistical comparison with in the flow plot in C is with L. major group of Batf3-/- mice. Error bars shows SEM (D,E,G,I,J,K) or SD (B). Two-tailed unpaired Student’s t-test with Welch’s correction or one-way ANOVA with Tukey’s multiple comparison analysis (D,I). ns, not significant; *p<0.05, **p<0.01, ***p<0.001.</p