Supplemental Information. The Microbial Metabolite Butyrate Stimulates. Bone Formation via T Regulatory Cell-Mediated. Regulation of WNT10B Expression

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1 Immunity, Volume 9 Supplemental Information The Microbial Metabolite yrate Stimulates Bone Formation via T Regulatory Cell-Mediated Regulation of WNT1B Expression Abdul Malik Tyagi, Mingcan Yu, Trevor M. Darby, Chiara Vaccaro, Jau-Yi Li, Joshua A. Owe, Emory Hsu, Jonathan Adams, M. Neale Weitzmann, Rheinallt M. Jones, and Roberto Pacifici

CD+ cells/ cells A B C. 1. CD Ab 1... Figure S1 (Related to Figure 1).

cells. Following red cells lysis, single cell suspeio were prepared from BM, and stained with antibodies to the indicated antige and live/dead cell dye. A.

The figure shows one representative flow cytometric plot per group. The numbers in each plot represent the frequency of cells in the corresponding quadrant. C.

2 CD+ cells/ cells A B C. 1. CD Ab 1... Figure S1 (Related to Figure 1). Gating strategy used to identify bone marrow (BM) Treg cells by flow cytometry and effects of, butyrate (but) or anti-cd Ab on the relative frequency of BM CD+ and cells. Following red cells lysis, single cell suspeio were prepared from BM, and stained with antibodies to the indicated antige and live/dead cell dye. A. Gated regio are numbered from R1 to R. The figure shows one representative gating of flow cytometric plot. B. The figure shows one representative flow cytometric plot per group. The numbers in each plot represent the frequency of cells in the corresponding quadrant. C. Average CD+/ cells ratio. n = 1 samples per group. Data were expressed as Mean + SEM. Data were normally distributed according to the Shapiro-Wilk normality test. Data and analyzed by two-way AN and post hoc tests applying the Bonferroni correction for multiple compariso. = p<.1, = p<.1 and = p<.1 compared to the indicated group. = not significant.

3 Femur Ct.Th (mm) N.Ob/BS (1/mm) Ob.S/BS (%) Femur Tt.Ar (mm ) Femur Ct.Ar (mm ) Femur Ct.Ar/Tt.Ar(%) Femur Tb.N (1/mm) Femur Tb.Sp (mm) Femur Tb.Th (mm) Spine Tb.N (1/mm) Spine Tb.Sp (mm) Spine Tb.Th (mm) A B C 7 CD Ab CD Ab CD Ab D E F 1 CD Ab CD Ab CD Ab G.. CD Ab H..1 CD Ab I CD Ab J K L CD Ab CD Ab 8 CD Ab

4 Figure S (Related to Figure ). Treatment with anti-cd Ab prevents the bone anabolic activity of and butyrate (). A-C. Prospective measurements of vertebral trabecular number (Tb.N), trabecular separation (Tb.Sp) and trabecular thickness (Tb.Th) by in vivo CT scanning. D-J. Cross-sectional measurements of femoral Tb.N, Tb.Sp, Tb.Th, total cross-sectional area (Tt.Ar), cortical bone area (Ct.Ar), cortical area fraction (Ct.Ar/Tt.Ar), cortical thickness (Ct.Th) by in vitro CT scanning. K, L. The number of osteoblasts per mm bone surface (N.Ob/BS), and the percentage of bone surface covered by osteoblasts (Ob.S/BS) as measured by bone histomorphometry. n=11-17 mice per group. Data were expressed as Mean + SEM. All data were normally distributed according to the Shapiro-Wilk normality test. For Panel A-C data were analyzed by AN for repeated measures. = p<., = p<.1, and = p<.1 compared to baseline, = p<., = p<.1, = p<.1 and = p<.1 compared to Irrelevant (Irr) Ab vehicle. All other data were analyzed by twoway AN and post hoc tests applying the Bonferroni correction for multiple compariso. = p<., = p<.1, and = p<.1 compared to the indicated group. = not significant.

5 mrna levels egfp- T cells Wnt1b mrna egfp-cd+ T cells Wnt1b mrna egfp+cd+ T cells Wnt1b mrna Caspase- Activity (pmol pna/ug protein) Osteoblast differentiation Genes Wnt target genes BM IL-1 mrna (Relative Expression) egfp+cd+ cells IL-1 mrna (Relative Expressiom) BrdU Incorporation (OD nm) BM RANKL mrna (Relative Expression) egfp+cd+ cells RANKL mrna (Relative Expression) BM OPG mrna (Relative Expression) A B C D G CD Ab CD Ab CD Ab H 8 E CD Ab CD Ab CD Ab RUNX OSX Col-I OCN BSP J K L I F CD Ab CD Ab CD Ab CD Ab CD Ab Ahr Cyrb1 NKD Tagln TGFb Thbs1 Wisp1 M. CD Ab CD Ab CD Ab IL-1β TNFα IL- IL-17 IL- IFNγ

6 Figure S (Related to Figure ). Effects of and butyrate () on BM and Treg cells production of cytokines, stromal cell (SC) proliferation, apoptosis differentiation and Wnt dependent gene expression, and T cell expression of Wnt1b mrna. A. BM RANKL mrna expression. B. RANKL mrna levels in sorted BM T cells from Foxp.eGFP reporter mice. C. BM OPG mrna expression. D. BM IL-1 mrna expression. E. IL-1 mrna levels in sorted BM T cells from Foxp.eGFP reporter mice. F. SC proliferation as measured by BrdU incorporation. G. SC apoptosis as measured by Caspase- activity. H. mrna levels of factors representative of the differentiation of SCs into osteoblasts, I. Tracripts of genes that are specifically increased by Wnt signaling in SCs. The analyzed genes were: aryl-hydrocarbon receptor (Ahr), axin, cysteine rich protein 1 (Cyr1), naked cuticle homolog (Nkd), tragelin (tagln), traforming growth factor (TGF ), thrombospondin 1 (Thbs1), Twist gene homolog 1 (Twist1) and Wnt1 inducible signaling pathway protein 1 (Wisp1). J-M. Wnt1b mrna levels in sorted BM T cells from Foxp.eGFP reporter mice and cytokine production by unfractionated BM cells. Panel J: egfp- T cells. Panel K: egfp-cd+ T cells. Panel L: Treg cells (egfp+cd+ cells). Panel M: cytokine production by unfractionated BM cells. For each cytokine, there were no significant differences among groups. n = -1 mice per group. Data were expressed as Mean + SEM. All data were normally distributed according to the Shapiro-Wilk normality test. All data and analyzed by two-way AN and post hoc tests applying the Bonferroni correction for multiple compariso. = p<., = p<.1, and = p<.1 compared to the indicated group. = not significant.

7 N.Ob/BS (1/mm) Ob.S/BS (%) Femur Ct.Ar (mm ) Femur Ct.Ar/Tt.Ar(%) Femur Ct.Th (mm) Femur Tb.N (1/mm) Femur Tb.Th (mm) Femur Tb.Sp (mm) Femur Tt.Ar (mm ) Spine Tb.Th (mm) Spine Tb.Sp (mm) Spine Tb.Sp (mm) Spine Tb.Sp (mm) Spine Tb.N (1/mm) Spine Tb.N (1/mm) Spine Tb.Th (mm) SpineTb.Th (mm) A..... DEREG+ DEREG+ DEREG+ Spine Tb.N (1/mm)..... DEREG+DT+ DEREG+DT+ DEREG+DT WTLM+DT+ WTLM+DT+ WTLM+DT DEREG+ DEREG+ DEREG DEREG+DT+ DEREG+DT+ DEREG+DT+ B WTLM+DT+ WTLM+DT+ WTLM+DT DEREG+ DEREG+ DEREG DEREG+DT+ DEREG+DT+ DEREG+DT WTLM+DT+ WTLM+DT+ WTLM+DT+ 1 DEREG DEREG+DT WTLM+DT..1 DEREG DEREG+DT WTLM+DT 17 DEREG DEREG+DT WTLM+DT DEREG DEREG+DT WTLM+DT C.1.. DEREG DEREG+DT WTLM+DT 1 8 DEREG DEREG+DT WTLM+DT 1 1 DEREG DEREG+DT WTLM+DT DEREG DEREG + DT WTLM + DT DEREG DEREG + DT WTLM+DT

8 Figure S (Related to Figure ). Treatment with Diphtheria Toxin (DT) prevents the bone anabolic activity of and butyrate () in DEREG mice. A. Prospective measurements of vertebral trabecular number (Tb.N), trabecular separation (Tb.Sp) and trabecular thickness (Tb.Th) by in vivo CT scanning. n = 8-1 mice per group. B. Cross-sectional measurements of femoral Tb.N, Tb.Sp, Tb.Th, total cross-sectional area (Tt.Ar), cortical bone area (Ct.Ar), cortical area fraction (Ct.Ar/Tt.Ar), cortical thickness (Ct.Th) by in vitro CT scanning. n = 8-1 mice per group. C. The number of osteoblasts per mm bone surface (N.Ob/BS), and the percentage of bone surface covered by osteoblasts (Ob.S/BS) as measured by bone histomorphometry. n=-11 mice per group. All data were expressed as Mean + SEM. All data were normally distributed according to the Shapiro-Wilk normality test. Panel A: Data were analyzed by AN for repeated measures. = p<., = p<.1, = p<.1 and = p<.1 compared to baseline, = p<., = p<.1, = p<.1 and = p<.1 compared to Irr. Ab vehicle. Panels B,C. Data were analyzed by two-way AN and post hoc tests applying the Bonferroni correction for multiple compariso. =p<., =p<.1, and = p<.1 compared to the indicated group. = not significant.

9 Ct.Ar (mm ) Ct.Ar/Tt.Ar (%) Ct.Th (mm) Tb.N (1/mm) Tb.Th (mm) Tb.Sp (mm) Tt.Ar (mm ) Spine Tb.Sp (mm) Spine Tb.Sp (mm) Spine Tb.Th (mm) Spine Tb.Th (mm) Spine Tb.Th (mm) Spine Tb.N (1/mm) Spine Tb.N (1/mm) Spine Tb.N (1/mm) Spine Tb.Sp (mm) BM Foxp+ T cells (% TCRb+CD+ cells) Splenic Foxp+ T cells (%TCRb+CD+ cells) PP Foxp+ T cells (%TCRb+CD+ cells) BM Foxp+ T cells (Cells/Samplex1 ) Spleen Foxp+ T cells (Cells/Samplex1 ) A B.. TCRb-/- TCRb-/- TCRb-/ Wnt1b CDCD8 Wnt1b CDCD8 Wnt1b CDCD8. Wnt1b CDDCD8 Wnt1b CDDCD8 Wnt1b CDDCD TCRb-/- TCRb-/- TCRb-/- Wnt1b CDDCD8 Wnt1b CDDCD8 Wnt1b CDDCD TCRb-/- TCRb-/- TCRb-/ Wnt1b CDCD8 Wnt1b CDCD8 Wnt1b CDCD Wnt1b CDDCD8 Wnt1b CDDCD8 Wnt1b CDDCD8 Wnt1b CDCD Wnt1b CDCD8 Wnt1b CDCD C TCRβ-/- mice Wnt1b CDCD8 Wnt1b CDDCD TCRβ-/- mice Wnt1b CDCD8 Wnt1b CDDCD8 TCRβ-/- mice Wnt1b CDCD8 Wnt1b CDDCD TCRβ-/- mice Wnt1b CDCD8 Wnt1b CDDCD TCRβ-/- mice Wnt1b CDCD8 Wnt1b CDDCD TCRβ-/- mice Wnt1b CDCD8 Wnt1b CDDCD TCRβ-/- mice Wnt1b CDCD8 Wnt1b CDDCD8

10 Figure S (Related to Figure ). Effects of and butyrate () on the number of Treg cells, and indices of spinal and vertebral trabecular structure measured by in vivo and in vitro CT scanning in TCR -/- mice recotituted with WT CD+ and T cells or WT CD+ and Wnt1b-/- T cells. A. Relative and absolute number of bone marrow (BM), splenic, and Peyer s Patches Treg cells in T cell deficient TCR -/- mice adoptively traferred with WT CD+ T cells and WT T cells. Since the enumeration of the absolute number of PP Treg cells is inaccurate, PP Treg cells are shown as % only. n = 9-1 mice per group. B. Prospective measurements of vertebral trabecular number (Tb.N), trabecular separation (Tb.Sp) and trabecular thickness (Tb.Th) by in vivo CT scanning in TCR -/- mice recotituted with WT CD+ and T cells or WT CD+ and Wnt1b-/- T cells. n = 1-1 mice per group. C. Cross-sectional measurements of femoral Tb.N, Tb.Sp, Tb.Th, total cross-sectional area (Tt.Ar), cortical bone area (Ct.Ar), cortical area fraction (Ct.Ar/Tt.Ar), cortical thickness (Ct.Th) by in vitro CT scanning in TCR -/- mice recotituted with WT CD+ and T cells or WT CD+ and Wnt1b-/- T cells. n = 9-1 mice per group. Data were expressed as Mean + SEM. All data were normally distributed according to the Shapiro-Wilk normality test. Panel A: data were analyzed by one-way AN and post hoc tests applying the Bonferroni correction for multiple compariso. =p<.1, =p<.1, and =p<.1 compared to the indicated group. = not significant. Panel B: Data were analyzed by AN for repeated measures and post hoc tests applying the Bonferroni correction for multiple compariso. = p<., = p<.1, and =p<.1 compared to baseline, = p<. and = p<.1 compared to vehicle. Panel C: Data were analyzed by two-way analysis-of-variance and post hoc tests applying the Bonferroni correction for multiple compariso. = p<., = p<.1, and = p<.1 compared to the indicated group. = not significant

11 +MHC-II+CD+Cells (Number/Sample) +MHC-II+CD+CD8+Cells (Number/Sample) cells (% +MHC-II+CD+) CD8+ cells (% +MHC-II+CD+) A CD Ab B 8 CD Ab C D 1 1 CD Ab 1 1 CD Ab Figure S (Related to Figure 7). Effects of and butyrate (but) on the expression of CD8 and CD8 by BM mature dendritic cells (DCs). A. Relative frequency of BM mature DCs. B. Relative frequency of BM CD8+ mature DCs. C. Absolute frequency of BM mature DCs. D. Absolute frequency of BM CD8+ mature DCs. n = 1 mice per group. Data were expressed as Mean + SEM. All data were normally distributed according to the Shapiro-Wilk normality test. Data were analyzed by twoway AN and post hoc tests applying the Bonferroni correction for multiple compariso. = p<.1, = p<.1 and = p<.1 compared to vehicle or the indicated group. = not significant.

12 SMAD (Fold Enrichment) Wnt 1b mrna T cells Wnt1b mrna NFAT1 (Fold Enrichment) NFAT (Fold Enrichment) SMAD (Fold Enrichment) SMAD (Fold Enrichment) Wnt1b mrna Wnt1b mrna (Relative Expression) NFAT1 (Fold Enrichment) NFAT (Fold Enrichment) SMAD (Fold Enrichment) A B C D 1 itreg CD8 Treg E 1 1 itreg CD8 Treg F 1 itreg CD8 Treg itreg CD8 Treg DCs/ T cells Tregs Tregs (1 x 1 ) G 1 H 1 I J CTLA-Ig CTLA-Ig K CTLA-Ig CTLA-Ig L CTLA-Ig CTLA-Ig µm 1 µm µm CTLA-Ig CTLA-Ig DCs/ T cells CTLA-Ig Anti CD Ab- Anti CD Ab+

13 Figure S7 (Related to Figure 7). Effects of Treg cells and CTLA-Ig on NFATs and SMADs binding to the Wnt1b promoter and Wnt1b mrna expression. OT-I T cells were cocultured with peptide pulsed + DCs. A-D. Addition of induced Treg cells increases the binding of NFAT1/ and SMAD to the Wnt1b promoter, as assessed by ChIP assays. E. Addition of induced Treg cells increases the expression of Wnt1b by T cells. In panels A-E T cells were either utimulated, activated with peptide pulsed DCs and cultured with and without Treg cells. F. Treg cells increase the expression of Wnt1b by T cells in a dose dependent manner. T cells were activated with peptide pulsed DCs and cultured with the indicated numbers of Treg cells. G-J CTLA--Ig increases the binding of NFAT1/ and SMAD to the Wnt1b promoter, as assessed by ChIP assays. K. CTLA--Ig increases the expression of Wnt1b by T cells. In panels G-K T cells were utimulated, activated with peptide pulsed DCs and cultured with and without CTLA--Ig. L. Addition of butyrate () to cultures of resting and anti-cd Ab stimulated T cells does not increase Wnt1b mrna expression. Data were expressed as mean + SEM. Panels A-D, G-J and L. n = samples per group. Panels E and K n = samples per group. Panels F n = samples per group. Data were analyzed by Kruskal-Wallis and Dunn s multiple compariso non-parametric test as they were not normally distributed as assessed by Shapiro-Wilk normality test. = p<.1, = p<.1 and = p<.1 compared to ol or the indicated group. = p<.1 and = p<.1 compared to the indicated group.

14 Table S1 (Related to STAR methods). Primer sequences for real-time PCRs, ChIP assays, plasmid cotruction and mutation of binding sites in murine samples. Primer Name Mouse Wnt1b Mouse BSP Mouse Col1a1 Mouse Ocn Mouse Osx Mouse Runx Mouse TNF Mouse IL- Mouse IL- Mouse IL-1 Mouse IFNγ Mouse IL-17A Mouse TGFβ1 18s ribosomal RNA Mouse IL-1 Mouse RANKL Mouse OPG Forward primer sequence ('-') GGGACCTCGGGTGACA ATAA GCACTCCAACTGCCCAA GA CCCTACTCAGCCGTCTG TGC GCCTTCATGTCCAAGCA GGA GTGTTAGTAACCTGGCC GGG CTGTGGTTACCGTCATG GCC AACTCCAGGCGGTGCCT AT GGTCTCAACCCCCAGCT AGT TAGTCCTTCCTACCCCA ATTTC CCTGGCTCTTGCTTGCC TT ACAGCAAGGCGAAAAAG GAT TGACGCCCACCTACAAC ATC CCACCTGCAAGACCATC GAC ATTCGAACGTCTGCCCT ATCA GCTCTTACTGACTGGCA TGAG CCTGATGAAAGGAGGG AGCA Reverse primer sequence ('-') CCTCTGTCCTTTTCCAAC CG TTTTGGAGCCCTGCTTTC TG GGGTTCGGGCTGATGTA CC GCGCCGGAGTCTGTTCA CTA CATTGGACTTCCCCCTTC TTG GGAGCTCGGCGGAGTAG TTC TGCCACAAGCAGGAATG AGA GCCGATGATCTCTCTCAAA GTGAT TTGGTCCTTAGCCACTCC TTCC GGTCTTGTGTGATGTTGC TCA TGGTGGACCACTCGGAT GAG CATCATGCAGTTCCGTCA GC CTGGCGAGCCTTAGTTT GGAC GTCACCCGTGGTCACCA TG CGCAGCTCTAGGAGCAT GTG TGGAATTCAGAATTGCCC GA

15 Mouse Wnt1b promoter (ChIP) Mouse Wnt1b - bp (Clone) Mouse Wnt1b -1 bp (Clone) Mouse Wnt1b -7 bp (Clone) Mouse Wnt1b +1 bp (Clone) NFAT mut SMAD mut CACCTTGAAGGGCCTGA TGT GGGAGGGAGTGATCCA GATA CGACGCGTAGCAAGACT TCAGAGAGGTTAG TTACGCGTACTCAGAAA GAGCACCTCCCG CGACGCGTCGATGAAA GACCCTGTCATTG taacgatgaaagaccctg TCATTGG gcgtccgtcctcagcgtg TCAA TTTTGGGAAAGTGGGAT GTTTT CAGAACCACCCGTGAGT TAG TTTAAGCTTGGGAAACTG TCGGGTTCAG agctttgcctccgagagtg GGT tatggaccttagggttact GACG

Conv. R GF Col. GF. Sham Ovx. E. coli. Veh. E. coli. E. coli. Veh. E. coli

Conv. R GF Col. GF. Sham Ovx. E. coli. Veh. E. coli. E. coli. Veh. E. coli B A Uterus weight (mg) 5 5 Body Weight (g) - - Uterus Weight (mg) - - Supplemental Figure. Effects of prolide and treatment with probiotics on uterus weight and body weight. Data are expressed as ean +