Eosinophils! 40! 30! 20! 10! 0! NS!
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1 A Macrophages Lymphocytes Eosinophils Neutrophils Percentage (%) 1 ** 4 * 1 1 MMA SA B C Baseline FEV1, % predicted 15 p = 1.11 X CD4:CD8 ratio 1 Supplemental Figure 1. Cellular infiltrate in the airway lumen, FEV1 and IFN-g mrna expression in human asthma subjects. (A) Differential cell counts in BAL fluid of subjects with MMA and SA; n = 28 and 29 for MMA and SA, respectively. (B) Baseline FEV1% predicted for MM and SA subjects; n = 33 each for MMA and SA. (C) Ratio of CD4+ and CD8+ T cell percentages in the BAL fluid of MMA and SA subjects; n = 13 each for MMA and SA. For (A-C), * p 0.05, ** p 0.01, *** p 0.001, NS not significant by Mann-Whitney test.
2 A IFN-g IL-17A IL-12p40 MMA, not on CS ng/ml pg/ml MMA, on CS IL-4 IL-5 IL-13 IL pg/ml d 1 d B IFN-g IL-17A IL-12p40 ng/ml pg/ml 1 MMA, on CS SA IL-4 IL-5 IL-13 IL-9 pg/ml Supplemental Figure 2. (A) Concentration of indicated cytokines in culture supernatants of BAL cells from MMA subjects treated with or without corticosteroid therapy.; n = 6 and 8 for MMA with or without CS therapy, respectively, for IFN-g and IL-17A and n = 5 and 4 for MMA with or without CS therapy, respectively for IL-4, IL-5, IL-9, IL-12p40 and IL-13. (B) Concentration of indicated cytokines in supernatants of ex vivo cultured BAL cells from MMA patients on CS therapy and severe asthma patients. n = 5-6 and 14 for MMA + CS and SA, respectively, for IFN-g, IL-17A, IL-5 and IL-13 estimation, n = 5 and 8 for MMA + CS and SA, respectively, for IL-4, IL-9 and IL-12p40 estimation. For (A-B), * p 0.05, NS non-significant using Mann-Whitney test.
3 A Percentage (%) 1 1 NS NS Naive SA SA + Dex MA Th1 lo Th B CD4 + Cells (X 10 4 ) 6 *** Naive SA SA + Dex MA Th1 lo Th MA Th1 lo Th + Dex MA Th1 lo Th2 + Dex ** Macs Neu Eos Lymph C Ifng Il Il13 Il17a mrna Expression 8 ** Il NS 1 * NS 5 Il9 * NS ND ND ND ND Supplemental Figure 3. Cellular infiltrate in the airway lumen and cytokine mrna expression in mouse asthma models.(a) Percentages of cell types in BAL fluid of mice sensitized and challenged in SA and MA Th1 lo Th2 models ± Dex. * p 0.05, *** p 0.001, NS non-significant using Student s unpaired t-test (B) Total lung CD4 + T cell numbers in mice sensitized and challenged in SA and MA Th1 lo Th2 models ± Dex quantified by flow cytometry. **** p using One-way ANOVA with Tukey s post-hoc test. (C) Cytokine mrna expression in whole lungs of mice sensitized and challenged in SA and MA Th1 lo Th2 models ± Dex. ND not detected. * p 0.05, ** p 0.01, *** p 0.001, NS not significant using One-way ANOVA with Tukey s post-hoc test. Data shown are mean ± s.e.m and representative of 2-3 independent experiments with n = 3-4 mice per group.
4 A B Ifng Il17a Ifng Il17a NS WT SA Il17ra -/- SA 15 5 ND WT SA Ifng-/- SA Il Il13 Il Il13 mrna Expression 1 NS 5 1 NS mrna Expression 1 NS 15 5 NS 8 6 Il NS Il9 3 Il NS Il9 ND ND 1 ND ND Supplemental Figure 4. Cytokine mrna expression in WT, Il17ra-/- and Ifng -/- mice subjected to the SA model. Mice were subjected to the SA Model and analyzed 24 h after the final allergen challenge. (A) Cytokine mrna expression in whole lungs of WT and Il17ra-/- mice. (B) Cytokine mrna expression in whole lungs of WT and Ifng-/- mice. For (A-B), * p 0.05, NS not significant by Student s unpaired t-test. Data shown are mean ± s.e.m and representative of 2-3 independent experiments with n = 3-4 mice per group.
5 Table 1: Airway hyperresponsiveness-related genes (IPA output) O21Rik ICOS PIK3R1 ADAM1 IFNG PLAT ADIPOQ IgG platelet activating factor ALOX IGHG1 PRG AMBP IL PRMT arginase IL PTAFR C3 IL6 PTEN C IL9 RAC1 C3AR1 IL1 Ras C5AR1 IL13 RIPK C5AR IL18 RORC CARD11 IL2 RUNX3 CAT IL2 SLPI CCL IL27 SMAD3 Ccl IL13RA SOCS3 CCL11 IL15RA SPI1 CCL17 IL17A SPON CCL2 IL17B SPRED1 CCL28 IL17RA STAT3 CCL3L3 IL1RL1 STAT CCR3 IL1RN STAT6 CD4 IL27RA TACR1 CD86 IL2RB TBX21 CD1D IL4R Tgf beta CD300LF IL6R TICAM1 CDH13 ITK TLR CHD KIT TNF CLCA1 LAMA TNFAIP6 CMA1 LGALS3 TNFRSF CX3CR1 LTA TNFRSF9 CXCL1 LTB4R TNFRSF1A CXCR LTC4S TPSG1 DLL MAPK3 TRAF3IP F2RL1 MGAT Traj18 FLT3LG mir-1 TSLP GATA3 MYD88 U0126 GGT NFE2L VDR HAS Nos VEGFA HGF NRTN HRAS PIK3CD Table 1. List of AHR-related genes identified using Ingenuity Knowledge Base (IKB) Diseases and Functions search bar from the IPA software.
6 Supplemental Figure 5. IFN-γ-regulated AHR-related genes expressed in structural cells of the lung identified using IPKB. Using IPA s Build function, AHR-related genes shown in Table 1 were specifically selected for genes expressed in the lung, epithelial cells and smooth muscle cells. Genes from this reduced set were further tested for regulation by IFN-g in the lung using Path Explorer function.
7 A B mrna Expression Foxj1 Cdh1 Acta Smooth Muscle Pecam Endothelium Airway Epithelium Brushing Whole lung Epithelial Genes Non-Epithelial Genes Supplemental Figure 6. Characterization of mouse airway epithelial cell brushings. qrt-pcr analysis of expression of (A) Epithelial-specific genes and (B) non-epithelial genes in whole lung and brush-harvested samples. Data shown are mean ± s.e.m and representative of 3 independent experiments with n = 3-4 mice per group.
8 mrna Expression CCL NS MMA SA Supplemental Figure 7.Ccl22 mrna expression analyzed by qrt-pcr in epithelial brushings of MMA and SA subjects; n = 16 and 19 for MMA and SA, respectively. NS non-significant, Mann-Whitney test.
9 A Slpi B Slpi mrna Expression ** SA * MA Th1 lo Th 1 MA Th mrna Expression 1 NS WT SA Il17ra -/- SA Supplemental Figure 8. SLPI mrna expression in different models of asthma. (A) SLPI mrna expression in airway epithelial brushings from mice subjected to the SA, MA Th1 lo Th2 and MA Th2 asthma models. ** p 0.01, *** p using One-way ANOVA with Tukey s post-hoc test. (B) SLPI mrna expression in airway epithelial brushings of WT and Il17ra-.- mice subjected to the severe asthma model. NS not significant by Student s unpaired t-test. For (A- B), data shown are mean ± s.e.m and representative of 2-3 independent experiments with n = 3-4 mice per group.
10 A B C Il12p3 Il12p WT 80 5 * 2. Stat1-/- IL-12p (pg/ml) Untreated c-di-gmp- transfected IFN-g (pg/ml) ND ND ND ND Untreated c-di-gmp- transfected mrna Expression D mrna Expression Ifng E Slpi * mrna Expression 1 Supplemental Figure 9. c-di-gmp regulation of IFN-g production via IL-12 induction is dependent on STAT1. (A) IL-12p40 and (B) IFN-g concentrations in culture supernatants of WT and Stat1-/- BMDMs transfected with c-di- GMP. (C) Il12p35 and Il12p40 mrna expression in lymph nodes of WT and Stat1-/- sensitized in the severe asthma model. (D) Ifng mrna expression in whole lungs of WT and Stat1-/- mice sensitized and challenged in the severe asthma model. (E) Slpi mrna expression in airway epithelial brushings from WT and Stat1-/- mice sensitized and challenged in the severe asthma model. For (A-E) * p 0.05, ** p 0.01 using Student s unpaired t-test. Data shown are mean ± s.e.m and representative of 2 independent experiments with n = 3-4 mice per group.
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SUPPLEMENTARY INFORMATION
doi: 1.138/nature89 IFN- (ng ml ) 5 4 3 1 Splenocytes NS IFN- (ng ml ) 6 4 Lymph node cells NS Nfkbiz / Nfkbiz / Nfkbiz / Nfkbiz / IL- (ng ml ) 3 1 Splenocytes IL- (ng ml ) 1 8 6 4 *** ** Lymph node cells
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Supplementary Figure 1
d f a IL7 b IL GATA RORγt h HDM IL IL7 PBS Ilra R7 PBS HDM Ilra R7 HDM Foxp Foxp Ilra R7 HDM HDM Ilra R7 HDM. 9..79. CD + FOXP + T reg cell CD + FOXP T conv cell PBS Ilra R7 PBS HDM Ilra R7 HDM CD + FOXP
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Supplementary Information
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