SUPPLEMENTARY MATERIAL

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1 SUPPLEMENTARY MATERIAL IL-1 signaling modulates activation of STAT transcription factors to antagonize retinoic acid signaling and control the T H 17 cell it reg cell balance Rajatava Basu 1,5, Sarah K. Whitley 1,5, Suniti Bhaumik 1, Carlene L. Zindl 1, Trenton R. Schoeb 2, Etty N. Benveniste 3, Warren S. Pear 4, Robin D. Hatton 1 and Casey T. Weaver 1 Department of 1 Pathology, 2 Genetics and 3 Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA 4 Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA USA 5 These authors contributed equally to this work. Correspondence: cweaver@uab.edu

2 Supplementary Figure 1. Effects of IL-1β on expression of RA and IL-1 receptor components. (a,b) FACS-sorted naïve CD4 T cells (CD4 + CD25 - CD62L high CD44 low ) from Il17f thy1.1 mice were activated with plate-bound αcd3 and soluble αcd28 under Th17 polarizing conditions in presence or absence of IL-1β under indicated concentrations of at-ra (1nM 100nM). Cells were collected at 60 h for analysis by RT-PCR for expression levels of Rara,b,g (above) and Rxra,b,g (below) transcripts (a) and for expression levels of Il1r1, Ilrap and Il1r2 transcripts (b). Values are normalized to Th0 controls. Data are means +/- SEM (*p<0.05, **p<0.01).

3 Supplementary Figure 2. IL-17-expressing cells and IL-1 signaling are required for host protection during infection with Citrobacter rodentium. (a) Histopathology of distal colonic tissues from untreated Il17f Thy-1.1 mice, Il17f Thy-1.1 mice treated with depleting anti-thy1.1 mab or Il1r1 / mice collected eight days post inoculation with 2 x 109 cfu C. rodentium. H&E-stained sections (scale bars: 100µm). (b) Histopathological scoring of distal colons from groups in a was performed at d8 PI as described in Materials and Methods (Epi indicates epithelial; Infl indicates inflammation; Total indicates aggregate histopathology score). (c) ELISA quantitation of IL-22 in supernatants from homogenates of colonic tissue collected from Il17f Thy-1.1 and Il1r1 / mice at the indicated times after inoculation with C. rodentium and cultured ex vivo for 24 hours. (d) Schematic representation of experiment in Main Fig. 2e,f. Data are representative of one of two similar experiments (a,b) or two independent experiments with 6 mice per group (b,c) (means and s.e.m.). *p<0.05, **P<0.01 (two-tailed unpaired T-test).

4 Supplementary Figure 3. IL-1 receptor deficiency alters the balance of Foxp3 and IL-17 expressing T cells during enteropathogenic bacterial infection. (a) WT B6 mice were inoculated with C. rodentium (2 x 10 9 cfu) and assessed for frequencies of CD4 + Foxp3 + lymphocytes in MLN and LP at indicated time-points post-infection by flow cytometry (CD4 T cell gate). (b) Pooled data from a showing frequencies of CD4 + Foxp3 + lymphocytes in MLN and colonic LP at indicated timepoints post-infection. (c) Il1r1 +/+ Il17f Thy-1.1.Foxp3 gfp and Il1r1 / Il17f Thy-1.1.Foxp3 gfp mice were inoculated with 2 x 10 9 cfu C. rodentium and analyzed without restimulation ex vivo for frequencies of IL-17F (Thy1.1 + ) and Foxp3 (GFP + ) CD4 + T cells within the activated pool of T cells isolated from colonic lamina propria (LP) at indicated time-points post-infection. Numbers are percentages of cells in the each quadrant. (d) Pooled data from c

5 showing frequencies of IL-17F (Thy1.1 + ) and Foxp3 (GFP + ) isolated from colonic LP T cells at indicated time points post-infection. Data are representative of one of two similar experiments (a,c) or two independent experiments with 6 or more mice per group as depicted by individual data points corresponding to one mouse (b,d) (means and s.e.m.). *p<0.05, **p<0.01 (two-tailed unpaired T-test). (e) Schematic representation of experiment in Main Fig. 3a,b.

6 Supplementary Figure 4. In vivo blockade of RA-mediated signaling reduces intestinal bacterial load in absence of IL-1 signaling. (a) Serial whole-body imaging of Il1r1+/+ Il17f Thy-1.1.Foxp3gfp and Il1r1 / Il17f Thy Foxp3gfp mice inoculated with 2 x 109 cfu C. rodentium and gavaged with vehicle alone or with retinoic acid inhibitor (LE135) on days 3-7 post infection and imaged at the indicated days post infection. (b) Colonization kinetics data from a. (c) Body

7 weight kinetics of Il1r1 +/+ Il17f Thy Foxp3 gfp and Il1r1 / Il17f Thy Foxp3 gfp mice inoculated with C. rodentium and gavaged with vehicle alone or with retinoic acid inhibitor (LE135) on indicated days post infection. Data are representative of one of two similar experiments (a) or two independent experiments with 6 mice per group (b, c) (means and s.e.m.). *p<0.05 (Il1r1 +/+ vs Il1r1 / ); #p<0.05 (Il1r1 / + Vehicle vs Il1r1 / + LE135); **p<0.01 (Il1r1 +/+ vs Il1r1 / ) (two-tailed unpaired T-test).

8 Supplementary Figure 5. IL-1, but not IL-23, overrides RA-mediated repression of Th17 development. Serial whole-body imaging of Il1r1+/+ Il17f Thy-1.1.Foxp3gfp and Il1r1 / Il17f Thy Foxp3gfp mice inoculated with 2 x 109 cfu C. rodentium and gavaged with vehicle alone or with retinoic acid inhibitor (LE135) on days 3-7 post infection and imaged at the indicated days post infection. (b) Colonization kinetics data from a. (c) Body weight kinetics of Il1r1+/+ Il17f Thy Foxp3gfp and Il1r1 / Il17f Thy Foxp3gfp mice inoculated with C. rodentium and gavaged with vehicle alone or with retinoic acid inhibitor (LE135) on indicated days post infection. Data are representative of one of two similar experiments (a) or two independent experiments with 6 mice per group (b, c) (means and s.e.m.). *p<0.05 (Il1r1+/+ vs Il1r1 / ); #p<0.05 (Il1r1 / + Vehicle vs Il1r1 / + LE135); **p<0.01 (Il1r1+/+ vs Il1r1 / ) (two-tailed unpaired T-test).

9 Supplementary Figure 6. IL-1 increases IL-21-mediated STAT3 tyrosine phosphorylation in Th17 cells. (a) Naïve CD4 + T cells were cultured under Th17 polarizing conditions for 4 days, then restimulated with IL-21 alone, IL-1β alone, or both for the indicated time periods. Cell lysates were harvested, immunoblotted with antibody directed against phospho-tyrosine(705)-stat3 (py-stat3; upper panel) or total STAT3 (lower panel). (b) Pooled data representing integrated band density values (IDVs) of py-stat3 normalized to total STAT3 from a. Data are representative of one of two independent experiments (a) or are pooled from two independent experiments representing integrated band density values (IDVs) of py-stat3 normalized to total STAT3 from a (means and s.e.m. in b). *p<0.05 (two-tailed unpaired T-test).

10 Supplementary Figure 7. Comparative effects of NF-κB and MAP kinase pathway inhibitors on IL-1 induced augmentation of tyrosine and serine phosphorylation of STAT3. (a) Naïve CD4 + T cells were cultured under Th17 polarizing conditions for 5 days, then restimulated with IL-23 alone, IL-1β alone, or both for indicated time periods. Cell lysates were harvested, immunoblotted with antibody directed against phospho-serine(727)-stat3 (ps-stat3), with anti-stat3 serving as a loading control. (b,c) Naïve CD4 + T cells polarized as in a were isolated and pre-treated with nothing or the indicated signaling inhibitors for 1 hour, then either left unstimulated or treated with IL-23 +/- IL-1β before cell lysates were harvested and immunoblotted for ps727-stat3 (b), py705-stat3 (c), or a STAT3 loading control (b,c). Phospho-STAT integrated band density values (IDVs) were normalized to total STAT3 and expressed as fold change over unstimulated cells. Data are representative of one of two independent experiments (a) or are pooled from two independent experiment (b,c) (means and s.e.m.). *p<0.05, **p<0.01 (two-tailed unpaired T test).

11 Supplementary Figure 8. IL-1β and IL-6 are required for FoxP3 + it reg developmental plasticity. (a) FACS-sorted naïve CD4 + T cells (CD4 + CD25 CD62L high CD44 low ) from WT B6 mice were activated with plate-bound αcd3 and soluble αcd28 under it reg cell polarizing conditions in presence or absence of IL-23 or IL-1β and analyzed for intracellular Foxp3 and IL-17A. (b) Pooled data from a. (c) FACS-sorted naïve CD4 + T cells from Il17f Thy-1.1.Foxp3 gfp mice were activated with plate-bound αcd3 and soluble αcd28 under it reg polarizing conditions. On day 4 of primary culture the Thy1.1 - GFP + fraction of cells were sorted by flow cytometry and further cultured under Th17 conditions with or without IL-1β (20ng/ml), in the presence or absence of at-ra (1nM). On day 4 of secondary culture, frequencies of IL-17F (Thy1.1) versus Foxp3-GFP + cells were determined after surface staining of Thy1.1 (IL-17F) and flow cytometry. Numbers are percentages of cells in the each quadrant. (d) Pooled data from secondary cultures from c. Data are: representative of one of two similar independent experiments a; pooled from two independent experiments with 6 samples per group b; one of two similar independent experiments c; or pooled from two independent experiments with 6 samples per group where individual data points depict each sample d (means and s.e.m. in b,d). **p<0.01 (two-tailed unpaired T-test).

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