Chitin Activates Parallel Immune Modules that Direct Distinct Inflammatory Responses via Innate Lymphoid Type 2 and T Cells

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Immunity, Volume 4 Supplemental Information Chitin Activates Parallel Immune Modules that Direct Distinct Inflammatory Responses via Innate Lymphoid Type 2 and T Cells Steven J. Van Dyken, Alexander Mohapatra, Jesse C. Nussbaum, Ari B. Molofsky, Emily E. Thornton, Steven F. Ziegler, Andrew N.J. McKenzie, Matthew F. Krummel, Hong-Erh Liang, and Richard M. Locksley Supplemental Figures 1-5 Supplemental Experimental Procedures Supplemental References

A 92.9 Yarg SSC DAPI 93.9 CD11c 2.9 CD11b 76.3 2.1 72.5 FSC Siglec F Siglec F YFP (Yarg) Neutrophils samples_9.fcs <APC-Cy7-R-A-A>, <PerCPCy5-5-B-A-A> subset <APC-R-B-A>: ly6g 1 5 1 4 Ly6G 1 3 1 2 4.2 SSC Eosinophils 73.1 1 8 6 4 2 Eosinophils 4get open YFP (Yarg).1 B CD11b 1 2 1 3 1 4 1 5 <APC-Cy7-R-A-A>: cd11b FSC GFP (4get) 1 2 1 3 1 4 1 5 <FITC-A>: 4get/yarg 9.8 1 1 SSC FSC 74. DAPI / Lin 4.5 KLRG1 Thy1.2 78.2 KLRG1 8 6 4 2 CD25 1 2 1 3 1 4 1 5 <APC-Cy7-R-A-A>: cd25 8 6 4 2 CD127 1 2 1 3 1 4 1 5 <PerCPCy5-5-B-A-A>: cd127 1 Thy1.2 - KLRG1 - Thy1.2 + KLRG + 8 6 4 2 ICOS 1 2 1 3 1 4 1 5 <APC-R-B-A>: icos Figure S1, related to Figure 1. Flow cytometric identification of lung neutrophils, eosinophils, AAMs, and ILC2. Lung cells from chitin-treated wild-type (), Il4 4get reporter (4get), and Arg1 Yarg reporter mice (Yarg) were analyzed by flow cytometry using surface markers to identify (A) neutrophils (Siglec F - CD11b + Ly6G + ), eosinophils (FSC lo SSC hi CD11c - Siglec F + ), and AAMs (YFP + CD11b + Siglec F - ), using control cells to delineate YFP and GFP reporter expression. Live cells were gated by DAPI exclusion. (B) ILC2 were identified as DAPI - Lin - (Lin=CD3ε, CD4, CD8α, CD11b, CD11c, CD19, DX5, NK1.1, F4/8, Ter119, Gr-1) KLRG1 + Thy1.2 + cells. Comparative expression of CD25, CD127, and ICOS between ILC2 and KLRG1 - Thy1.2 - cells is indicated in right panels.

A B Eotaxin-1 (pg / mg total protein) 45 3 15 PBS ** Chitin Eosinophils (x1 5 ) 7. 6. 5. 4. 3. 2. 1.. * Ccr3 -/- Figure S2, related to Figure 2. Eotaxin-1 (CCL11) expression in lung tissue and eosinophil accumulation in Ccr3 -/- mice after intranasal administration of chitin. (A) Eotaxin-1 levels in whole lung lysates from wild-type mice 6 hours after intranasal treatment with PBS or chitin. (B) Total eosinophil numbers in the lungs of wild-type () and Ccr3 -/- mice 48 hours after intranasal treatment with chitin as described in Figure 1A. Numbers calculated from cell counts and flow cytometric subset percentages as described in Figure S1. Bars represent mean±sem, n=4-6/group; *p<.5; **p<.1 (unpaired t-test), compared to PBS-treated (A) or wild-type control (B).

lung_b6 cht_3.fcs ILC2 ILC2 CD4 T cells CD8 T cells γδ T cells lung_ss cht1_1.fcs gamma delta t inkt cells 25K 25K 2K 15K 1K...1 2K 15K 1K.2. 5K 5K lung_cc SS cht_3.fcs ILC2 1 2 1 3 1 4 1 5 25K lung_cc SS_1.fcs gamma delta t 1 2 1 3 1 4 1 5 25K 2K S13 15K 1K 7.6..1 2K 15K 1K.1. 5K 5K 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 NK cells lung_ss cht2_1.fcs nk cells Basophils Eosinophils B cells lung_ss cht2_1.fcs b cells Alveolar Mφ lung_b6 cht_3.fcs alv macs 25K 1 5 1 5 2K 15K 1K 5K.2. <Qdot 655-V-A-A>: cd45.4 1 4 1 3 1 2.4 <FITC-B-C-A>: siglec f 1 4 1 3 1 2.2 lung_ss cht1_1.fcs nk cells 1 2 1 3 1 4 1 5 25K lung_ss cht1_1.fcs b cells lung_ss cht1_3.fcs alv macs 1 1 1 1 1 1 2 3 4 5 2 3 1 4 1 5 1 5 1 5 S13 2K 15K 1K 5K.2. <Qdot 655-V-A-A>: cd45.4 1 4 1 3 1 2.3 <FITC-B-C-A>: siglec f 1 4 1 3 1 2.3 1 2 1 3 1 4 1 5 hucd4 (IL-13) 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 Figure S3, related to Figure 2. IL-13 reporter (Smart13) expression among lung immune cell subsets. Lung cells from chitin-treated wild-type () or homozygous Il13 Smart/Smart (S13) reporter mice were stained for surface markers and analyzed by flow cytometry for expression of hucd4 IL-13 reporter. ILC2=Lin - Thy1.2 + CD25 + ; CD4 T cells=cd3ε + CD4 + ; CD8 T cells=cd3ε + CD8α + ; γδ T cells=cd3ε + GL3 + ; inkt cells=cd3ε + (PBS-57-loaded)CD1d tetramer + ; NK cells=nk1.1 + ; B cells=cd3 - B22 + ; basophils=dx5 + FcεRI + ; eosinophils=fsc lo SSC hi CD11c - Siglec F + ; alveolar mφ=cd11c + CD11b - Siglec F +.

ILC2 CD4 T cells CD8 T cells γδ T cells inkt cells Untreated 1 8 6 4 1 8 6 4 1 8 6 4 1 8 6 4 1 8 6 4 Red5 2 2 2 2 2 1 1 2 1 3 1 4 1 5 1 1 2 1 3 1 4 1 5 1 1 2 1 3 1 4 1 5 1 1 2 1 3 1 4 1 5 1 1 2 1 3 1 4 1 5 8 8 8 8 8 Chitin (48 hours) 6 4 6 4 6 4 6 4 6 4 2 2 2 2 2 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 NK cells B cells Basophils Eosinophils Alveolar Mφ 1 1 1 1 8 8 8 8 Untreated 6 4 6 4 6 4 6 4 2 2 2 2 1 1 2 1 3 1 4 1 5 1 1 2 1 3 1 4 1 5 1 1 2 1 3 1 4 1 5 1 1 2 1 3 1 4 1 5 8 8 8 8 Chitin (48 hours) 6 4 6 4 6 4 6 4 2 2 2 2 1 2 1 3 1 4 1 5 tdtomato (IL-5) 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 Figure S4, related to Figure 3. IL-5 reporter (Red5) expression among lung immune cell subsets. Lung cells from untreated or chitin-treated wild-type () or homozygous Il5 Red5/Red5 (Red5) reporter mice were stained for surface markers to identify indicated subsets which were analyzed for expression of Red5 IL-5 reporter (tdtomato). ILC2=Lin - Thy1.2 + CD25 + ; CD4 T cells=cd3ε + CD4 + ; CD8 T cells=cd3ε + CD8α + ; γδ T cells=cd3 + GL3 + ; inkt cells=cd3ε + (PBS-57-loaded)CD1d tetramer + ; NK cells=nk1.1 + ; B cells=cd3 - B22 + ; basophils=dx5 + FcεRI + ; eosinophils=fsc lo SSC hi CD11c - Siglec F + ; alveolar mφ=cd11c + CD11b - Siglec F +.

A B Figure S5, related to Figure 3. IL-5 reporter (Red5) expression in lung sections after intranasal administration of chitin (A) IL-5 reporter (Red5) expression (arrows) in lung slices from homozygous Il5 Red5/Red5 reporter mice imaged by 2-photon microscopy. Red=IL5+ cells; blue=collagen; green/yellow=autofluorescence. (B) Similarly imaged lung slice from wild-type B6 control mouse. Bar=1μm. Images are representative of results obtained from at least 3 independent experiments.

Supplemental Experimental Procedures Mice Additional mice used in this study included Stat6 -/- (Kaplan et al., 1996), Il4 -/- Il13 -/- (McKenzie et al., 1999), Il5 transgenic (Lee et al., 1997), Rag2 -/- -/- /γ c (Taconic), Ccr3 -/-, Rorc GFP/GFP and C57BL/6 (Jackson Laboratories). Flow cytometry / sorting Lung cells were stained using the following antibodies: phycoerythrin (PE)/cyanine (Cy)7- or Pacific Blue (PB)-anti-CD11c (N418), Brilliant Violet (BV) 65-, PB-, or allophycocyanin (APC)-anti-CD11b (M1/7), PB- or peridinin chlorophyll protein complex (PerCP)-Cy5.5-anti-Gr-1 (RB6-8C5), PB- or PerCPCy5.5-anti-CD3ε (17A2), PB-anti-CD19 (6D5), PE/Cy7-anti-CD69 (H1.2F3), PB-, BV65- or APC/Cy7-anti-CD4 (RM4-5), PB- or BV65-anti-CD8α (53-6.7), APC-anti-ICOS (C398.4A), APC- or APC/Cy7-anti-CD25 (PC61), fluorescein isothiocyanate (FITC)-anti-FcεRIα (MAR-1), PB- or APC-anti-NK1.1 (PK136), APC- or BV65-anti-Thy1.2 (53-2.1), PerCP/Cy5.5- anti-tcrγδ (GL3; BioLegend), APC-anti-Ly6G (1A8), PE-anti-Siglec-F (E5-244; BD Biosciences), efluor45-anti-f4/8 (BM8), PerCP/Cy5.5-anti-CD127 (A7R34), PE-antihuCD4 (RPA-T4), streptavidin-fitc, efluor45- or APC-anti-CD49b (DX5), PE/Cy7- anti-klrg (2F1; ebioscience), Qdot65-anti-CD45.2 (14; Life Technologies), APCanti-IL17RB (75211), PE-anti-TSLPR (polyclonal; R&D Systems), biotin-anti-t1/st2 (DJ8; MD Biosciences), and APC-CD1d tetramers (PBS-57-loaded and unloaded) were obtained from the National Institutes of Health tetramer core facility. Exclusion of DAPI (4',6-diamidine-2'-phenylindole dihydrochloride; Roche) identified live cells, which were enumerated with flow cytometric counting beads (CountBright Absolute; Life

Van Dyken et al. Technologies). Sample data were acquired with a LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Inc.). For cell sorting, ILC2 from either PBS- or chitin-treated lungs were identified as DAPI- Lin(CD3ε, CD4, CD8α, CD11b, CD11c, CD19, DX5, NK1.1, F4/8, Ter119, Gr-1)- negative, CD5 lo Thy1.2 + CD25 + and Arg1 + (Bando et al., 213) and were sorted using a MoFlo XDP (Beckman Coulter). Sorted ILC2 were cultured (37 C, 5% CO 2 ) at 25 cells/well for 48 hrs in 1 µl RPMI-164 containing 1% heat-inactivated FBS, penicillin-streptomycin-l-glutamine (1X), and 2-mercaptoethanol (Life Technologies), plus IL-7 (1 ng/ml; R&D Systems), or where indicated, ionomycin (5 ng/ml) and phorbol 12-myristate 13-acetate (4 ng/ml; PMA; Sigma). Supplemental References Bando, J.K., Nussbaum, J.C., Liang, H.E., and Locksley, R.M. (213). Type 2 innate lymphoid cells constitutively express arginase-i in the naïve and inflamed lung. J Leukoc Biol 94, 877-884. Kaplan, M.H., Schindler, U., Smiley, S.T., and Grusby, M.J. (1996). Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 4, 313-319. Lee, N.A., McGarry, M.P., Larson, K.A., Horton, M.A., Kristensen, A.B., and Lee, J.J. (1997). Expression of IL-5 in thymocytes/t cells leads to the development of a massive eosinophilia, extramedullary eosinophilopoiesis, and unique histopathologies. J Immunol 158, 1332-1344. McKenzie, G.J., Fallon, P.G., Emson, C.L., Grencis, R.K., and McKenzie, A.N. (1999). Simultaneous disruption of interleukin (IL)-4 and IL-13 defines individual roles in T helper cell type 2-mediated responses. J Exp Med 189, 1565-1572.

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