NMED-A65251A Supplementary Figures. Sup. Fig. 1. ILC3 cells are the main source of in obese mice a. We gated on T cells (upper panels) or T cells (lower panels), and examined production. b. CD45 + - IL-13 + cells were assessed by FACS (upper panels). CD45 + - IL- 17A + cells were further analyzed for IL-13 production (lower panels). c. HFD induce -, but not IL-22- or IFN- -producing CD45 + - innate lymphoid cells. Sup. Fig. 2. Mice from both Jackson Labs and Taconic develop HFD-induced AHR. To compare the effect of SFB in the development of ILC3 cells, mice from Taconic or Jackson lab fed a HFD for 12wks and AHR was measured. a. Graph represents the changes in lung resistance ( ). p<.1, and p<.5 ( chow vs HFD in mice from Jackson) and # p<.5 ( chow vs HFD in mice from Taconic) (Two-way ANOVA). b. ILC3 cells in mice from Jackson (upper panel) or Taconic (lower panel) were assessed. Total number of ILC3 cells was calculated (right panel). Sup. Fig. 3 IL-23 induces AHR in Rag2 -/- mice. a. FACS dot plots of Treg, M1/M2 macrophages in the lung (left panels) and adipose tissue (right panels) from chow or HFD fed mice. b. IL-1 production from M1 (CD11c +, upper panels) or M2 (CD26 +, lower panels) macrophages was analyzed by FACS. c.to induce AHR,.1 g of ril-6 or ril-23 were administered to Rag2 -/- mice intranasally for 3 consecutive days. Graph represents the changes in lung resistance ( ). p<.5 (ril-23), and not significant (IL-6) (Two-way ANOVA). d. Cells in BAL fluid were analyzed after measurement of AHR. p<.1, ril-6 or ril-23 treated mice were compared to saline treated mice (Student s t-test). e. ILC3 cells (CD45 + Lin - + cells) from ril-6, ril-23 or ril-1 treated mice were analyzed by flow cytomerty after PMA/Ionomycin stimulation. Sup. Fig. 4. IL-1 -induced ILCs express and ROR t, but not IL-17F. Nature Medicine: doi:1.138/nm.3423 1
NMED-A65251A a., IL-17F, GM-CSF, ROR t and T-bet expression was analyzed in lung cells from Rag2 - /- mice. ril-1 treatment induces -producing ILCs, and most of + cells express ROR t. b. ril-1 induced CD45 + lineage - CCR6 + cells (ILC3 cells) from Rag2 -/- mice. Sup. Fig. 5 ril-1 restores AHR in Nlrp3 -/-, IL-6 -/-, and TCR -/- mice but not in Rorc -/- mice. Changes in lung resistance ( ) in Nlrp3 -/- mice (a), Il6 -/- mice, TCR -/- mice (d), and Rorc -/- mice (g), after ril-1 treatment. p<.5 (Nlrp3 -/- mice), ril-1 treated mice were compared to saline treated mice (Two way- ANOVA). Other comparisons not significant. b, e,h. Cells in BAL fluids from a, d, g. p<.1 (Nlrp3 -/- mice), ril-1 treated group was compared to saline treated group. (Student s t-test). Other comparisons not significant. ril-1 induced IL-17 + ILC3 cells in Nlrp3 -/- mice (c), Il6 -/- mice (f), TCR -/- mice (f), and Rorc -/- mice (i). Sup. Fig. 6. is required for the development of AHR. a. r induced minimal or no AHR. Graph represents the changes in lung resistance ( ) in treated WT mice. NS (Two way- ANOVA). b. Changes in lung resistance ( ) of r and + ril-1 treated Il17A -/- mice. p<.5, vs + ril-1 treated Il17A -/- mice (Two way- ANOVA). c. Inflammatory cells in BAL fluid of (b). p<.5, p<.1, and p<.1 (Student s t-test). d. Changes in lung resistance ( ) of anti-il-17 mab injected Rag1 -/- mice before ril-1 treatment (Two way- ANOVA). e. Inflammatory cells in BAL fluid of (d). p<.5, and p<.1 (Student s t-test). f. producing ILC3 cells assessed by intracellular staining. Sup. Fig. 7. Human ILC cells in the BAL fluid from patients with lung disease. a. Severe asthma (left), mild asthma (middle) or control (right panels). First, we gated on CD45 + cells (1 st rows), on lymphocytes (2 nd rows), then on Lin - cells (3 rd rows), before analyzing for IL-7R (CD127) expression (bottom rows). The numbers in the histograms in the 4 th rows represent the percent of Lin - cells that were CD127 +, and these numbers were then used to calculate the percent of BAL fluid lymphocytes that were Lin - IL-7R +, as shown in Supplemental Table 1, 9 th column. Nature Medicine: doi:1.138/nm.3423 2
NMED-A65251A b. The top row of each pair of rows represents unstimulated cells, and the bottom row of each pair represents cells activated with PMA and ionomycin. The numbers in the upper left hand quadrants represent the percent of the Lin - CD127 + cells that were +. These numbers (in the PMA and ionomycin stimulated populations) were then used to calculate the percent of BAL fluid lymphocytes that were Lin - IL-17 +, as shown Supplemental Table 1, right most column. Nature Medicine: doi:1.138/nm.3423 3
Sup. Fig. 1 a TCR b Chow HFD Chow HFD 26.4. 39.5 1 5 1 4 1 3 1 2.11 1 2 1 3 1 4 1 5.14 TCR IL-13 TCR 1.88 Chow 2.77 HFD 1 5.32 1 5 1 4 HFD 1 3 99.1.57 1 2 1.33 1.33 1 2 1 3 1 4 1 5 99.1.57 TCR IL-13 c 25K 2K 15K 1K 4.19 5K 1 3 1 4 1 5 1 5 33.5 1.9 1 5 34.2.7 1 4 1 4 1 3 1 3 IL-22 65.3.7 1 3 1 4 1 5 IFN- 65.7.42 1 3 1 4 1 5 Nature Medicine: doi:1.138/nm.3423
arl(% of baseline) Sup. Fig. 2 4 JAX Chow JAX HFD TAC Chow 3 TAC HFD # 2 # 1 2 4 6 8 b chow HFD JAX TAC 1 5 1 4 1 3 1 2 1 3 1 4 1 5.28.58.21.52 ILC3 cells(% of lymphocyte) 1.5 1..5. NS chow HFD chow HFD JAX TAC Nature Medicine: doi:1.138/nm.3423
1 2 1 3 1 4 1 5 Sup. Fig.3 a Treg Foxp3 Macrophage CD26 1 5 1 4 1.93 1 3 1 2 4 1 2 1 3 1 1 5 1 5 2.86 1 5 2.86 1 4 1 3 1 2 Lung C how H FD CD4 41.6 2.3 M2 M2 AM M1 11.5 11.5 CD11c 1 2 1 3 1 4 1 5 2.3 29.3 AM M1 16.4 16.4 Adipose tissue C how H FD 11.4 3.31 56.8 34.9 M2 M2 M1 M1 17.7 39.1 b M1 IL-1β 1 M2 5.27 1.96 IL-1β Chow HFD 1 5 4.2 2.9 1 4 1 3 1 2 1 2 1 3 1 4 1 5 CD11c 1 4 1 3 1 2 1 2 1 3 1 4 1 5 CD26 c (%of baseline) 3 25 2 15 ril-23 ril-6 1 1 2 3 4 n.s d Cells in BALF (X1 4 ) 1. 7.5 5. 2.5. n.s ril-23 ril-6 n.s n.s n.s n.s Macs Neu Eos Lymph e 1 5 1 4 1 3 1 2 1 5 ril-6 ril-23 1 2 1 3 1 4 1 5.99 1.18 2.3 ril-1α 1 4 1 3 1.29 6.88 1 3 1 4 1 5 Nature Medicine: doi:1.138/nm.3423
Sup. Fig. 4 a 25K ril-1β b ril-1β 2K 15K 1K 12.7 14.8 5K 1 2 1 3 1 4 1 5 FCS 1 5 3.31 2.82 46.4.44 1 4 1 3 1 2 92.9.92 1 2 1 3 1 4 1 5 IL-17F 1 5 6.45 1.65 53.5.99 32.6 4.2 1 5 cells- + 3.71 CCR6 1.65 7.92 1 4 1 4 1 3 1 3 1 2 85.4 GM-CSF 6.47 1 2 1 3 1 4 1 5 62.5.7 T-bet RoRγT 94.8 1 2 1 3 1 4 1 5 Nature Medicine: doi:1.138/nm.3423
Sup. Fig. 5 a b c (% of baseline) 3 2 1 Nlrp3 ril-1β 4 ril-1β Nlrp3 1 Nlrp3 ril-1β 5 Nlrp3 3 1 4 2 4 Cells in BALF(X1 4 ) 2 1 Macs Neu Eos Lymph Nlrp3 1 3 1 2.35 1 2 1 3 1 4 1 5 3.2 d e WT ril-1β f (% of baseline) 4 3 2 1 1 2 3 4 WT ril-1β 4 WT Rorc ril-1β Rorc 3 2 WT ril-1β δtcr ril-1β Il6 ril-1β WT δtcr Il6 NS 1 1 2 3 4 6 5 4 3 2 1 3 2 1 Macs Neu Eos Lymph g h WT ril-1β i (% of baseline) Cells in BALF(X1 4 ) Cells in BALF(X1 4 ) TCR ril-1β Il6 ril-1β WT δtcr Il6 WT Rorc ril-1β Rorc Macs Neu Eos Lymp 1 5 1 4 1 3 1 2 RoRγT Rorc 1 5 1 4 1 3 1 2 WT 1 2 1 3 1 4 1 5.88.88 4.35.17 1 2 1 3 1 4 1 5 δtcr 4.46 ril-1β.51 Il6.6 3.63 Nature Medicine: doi:1.138/nm.3423
Sup. Fig.6 a (% of baseline) (% of baseline) d 3 25 2 15 WT r WT 1 2 4 3 25 2 15 NS Rag1 ril-1β Rag1 anti- Ab+rIL-1β Rag1 1 1 2 3 4 n.s (% of baseline) e Cells in BALF(X1 4 ) b 3 25 2 15 1 1 2 3 4 4 3 2 1 Il17 Il17 ril-1β Il17 ril-1β+r Macs Neu Eos Lymp c 75 Cells in BALF(X1 4 ) f 5 25 Rag1 ril-1β Rag1 anti- Ab+rIL-1β Rag1 1 5 1 4 1 3 Macs Neu Eos Lymph Rag1 RoRγT 1 3 1 4 1 5 Il17 Il17 ril-1β Il17 ril-1β+r ril-1β anti- Ab+rIL-1β.62 16.1 2.81 Nature Medicine: doi:1.138/nm.3423
Sup. Fig. 7 a Severe Asthma Mild Asthma Control 25K 2K 15K 1K 5K CD45 25K P1 P2 P3 4.42 13.2 1.32 12 13 14 15 P4 P5 P6 P7 P8 P9 25K 25K 66.4 62.2 38.1 7.51 21.3 15.9 2K 2K 15K 1K 5K CD45 25K 12 13 14 15 15K 1K 5K CD45 25K 12 13 14 15 P1 26.3 2K 2K 2K 15K 15K 15K 1K 1K 1K 5K FSC 37.4 5K 1K 15K 2K 25K 29.2 17.4 17.1 14.4 5K FSC 18.7 5K 1K 15K 2K 25K 37.2 5K 27.9 14.5 5K 1K 15K 2K 25K FSC 21.4 25K 25K 25K 2K 2K 2K 15K 1K 5K 1 8 6 4 19.6 17.9 19.5 13 14 15 28.1 46.3 17.7 8.86 21.2 7.1 34.4 15K 1K 5K 7.53 12 13 14 15 1 8 6 4 13.8 11.5 25.9 1K 2.22 5.66 15K 6 24.7 22 5K 1 8 4 12 13 14 15 4.9 25.2 2 2 2 13 14 15 CD127 12 13 14 15 CD127 12 13 14 15 CD127 b (-) Severe Asthma Mild Asthma Control P1 P2 P3 P5 P7 P8 P9 8.63 4.32.38 3.12.78 1.46.58 4.75 (-) (-) P1 P+I 87.1 1 5 22.7 3.3 1 4 99.6 6.39 5.79 95.3.78 17.4 1.47 1 7 P+I 1 5 1 4 97.4.58 3.29 99.9.14 4 P+I 1 5 1 4 95.3 12.9 1 3.28 1 3 72.7 IL-13 1.52 12 13 14 15 84.8 2.99 81.2 93 1 3 93.8 IL-13 2.87 13 14 15 95 1 1 3 87.1 IL-13 13 14 15 96.7 Nature Medicine: doi:1.138/nm.3423
Supplemental Table 1.Ten Consecutive Subjects with Pulmonary Disease Tested for ILC3-like Cells Patient Asthma severity 1 Sex Age (years) Race Co-illness FEV1 (L/sec) (%) BMI Lin - IL-7R + (% of lymphocyte) Lin - IL-17 + (% of lymphocyte) P1 Severe F 42 Asian Depression 1.52(62%) 25.6 4.7 2.5 P2 Severe M 21 African None 4.5(9%) 28.8 7.1 2.29 P3 Severe F 5 American None 2.33(77%) 35.2 3.2 2.91 P4 Severe F 39 Hispanic P5 Severe M 52 White Obesity with gastric bypass Allergic rhinitis,ge reflux 1.5(48%) 23.2 3.1 n/a 1.63(49%) 25.72 1.6.62 P6 Mild M 29 Hispanic none 4.68(118%) 28.4 1.4.8 P7 Mild M 26 White Sinusitis, Exsmoker 4.7(13%) 23.19 2.1 n/a P7 Mild F 64 White Chronic rhinitis 3.26 (119%) 23.1.4.8 P8 No asthma F 42 White Ulcerative colitis 3.42(12%) 27.1.9.46 P1 No asthma F 57 White Celiac d, diabetes 2.5(92%) 3.4 1..16 1 Based on NHLBI Guidelines In last 2 columns, LIN - IL-7R + or LIN - IL-17 + populations werecalculated as a percentage of total lymphocytes(the lymphocyte gate was considered as 1%). The gating strategies are provided in Supplemental Figures 7. GE= gastro-esophageal. Nature Medicine: doi:1.138/nm.3423