Supplementary information. The proton-sensing G protein-coupled receptor T-cell death-associated gene 8

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1 1 Supplementary information 2 3 The proton-sensing G protein-coupled receptor T-cell death-associated gene 8 4 (TDAG8) shows cardioprotective effects against myocardial infarction 5 Akiomi Nagasaka 1+, Chihiro Mogi 2+, Hiroki Ono 1+, Toshihide Nishi 1, Yuma Horii 1, Yuki 6 Ohba 1, Koichi Sato 2, Michio Nakaya 1, Fumikazu Okajima 2,, Hitoshi Kurose 1,*

2 7 Supplementary Figure 1. Comparison of Gpr4, Ogr1 and G2a expression levels in 8 the hearts of WT and TDAG8 mice on post-mi day 3. 9 Quantitative analysis of the expression of proton-sensing GPCR mrnas in WT mice 1 hearts (sham, n = 3; MI, n = 7) and TDAG8 mice hearts (sham, n = 3; MI, n = 5) on 11 post-mi day 3. mrna expression was normalized to GAPDH. Comparisons were 12 assessed using unpaired Student s t-tests. Error bars represent the mean ± SEM (, 13 not significant) Supplementary Figure 2. The area at risk and infarct size were comparable 16 between WT and TDAG8 mice during the initial phase after MI. 17 (a) Representative heart sections of Evans blue/ttc staining 3 h after MI. The 18 non-ischemic area is indicated in blue, the area at risk (AAR) in red and the infarct area 19 in white. (b) The infarct size and AAR were quantified as a percentage of the AAR and 2 the left ventricular area (LV) of WT (n = 5) and TDAG8 (n = 4) mice. Error bars 21 represent the mean ± SEM. Comparisons among groups were assessed using unpaired 22 Student s t-test (, not significant) Supplementary Figure 3. Comparison of infiltrated B cells in the infarcted hearts

3 25 of WT and TDAG8 mice. 26 Comparison of infiltrated B cells (B22 + ) on post-mi day 3 between WT mice (n = 4) 27 and TDAG8 mice (n = 3) was performed using flow cytometry. Representative 28 FACS profiles are shown. The comparison was assessed using unpaired Student s t-tests. 29 Error bars represent the mean ± SEM Supplementary Figure 4. No significant differences in Tnf mrna were found on 32 post-mi day Tnf mrna expression in infarcted and non-infarcted areas of WT mice (sham n = 3, 34 MI n = 7) and TDAG8 mice (sham n = 3, MI n = 5) 3 days after MI was measured 35 using real-time RT-PCR. The comparison was assessed using an unpaired Student s 36 t-test Supplementary Figure 5. Time course of Ccl2 mrna expression in cardiac 39 macrophages stimulated with TNF- and HMGB1. 4 Cardiac macrophages were isolated from WT mice on post-mi day 3 and stimulated 41 with 1-ng/ml TNF- (left) or 1- g/ml HMGB1 (right). Three heart samples were 42 combined as one sample and used for real-time RT-PCR analysis.

4 43 44 Supplementary Figure 6. Comparison of infiltrated CCR6 + T cells in the 45 infarcted hearts of WT and TDAG8 mice on post-mi day Cardiac T cells were isolated from WT and TDAG8 mice 5 days after MI; stained 47 with antibodies against CD3, TCR and CCR6; and were analysed by flow cytometry. 48 Grey histograms indicate the isotype control Supplementary Figure 7. Upregulation of IL-17a mrna in TDAG8 mice 7 51 days after MI. 52 IL-17a mrna expression in the non-infarct and infarcted areas of WT mice (n = 7) or 53 TDAG8 mice (n = 5) 7 days after MI was measured using real-time RT-PCR. 54 Comparisons were assessed with a one-way ANOVA followed by Tukey s test. Error 55 bars represent the mean ± SEM (, not significant) Supplementary Figure 8. Comparison of IL-17A-related inflammatory genes of 58 WT and TDAG8 mice. 59 Expression levels of inflammatory gene mrnas in the infarcted and non-infarcted areas 6 of WT mice (sham, n = 6; MI, n = 1) and TDAG8 mice (sham, n = 5; MI, n = 8)

5 61 on post-mi day 3. # indicates that Il8 mrna was not detected. Comparisons were 62 assessed using unpaired Student s t-tests. Error bars represent the mean ± SEM (, 63 not significant) Supplementary Figure 9. A portion of CCR6 + T cells secrete IL-17A. 66 Cardiac cells were isolated from TDAG8 mice on post-mi day 5 and stimulated 67 with PMA and ionomycin in the presence of Brefeldin A. The samples were stained 68 with antibodies against CD3, TCR, CCR6 and IL-17A. After gating visible and 69 singlet cells, CD3 + CR + CCR6 + cells were further gated, and IL-17A expression was 7 determined using flow cytometry. Mixed samples (n = 3) were used for the analysis Supplementary Figure 1. Comparison of fibrosis between WT and TDAG8 73 mice on the post-mi day (a) Expression levels of fibrosis-related gene mrnas in the infarcted and non-infarcted 75 area of WT mice (sham n = 3, MI n = 7) and TDAG8 mice (sham n = 3, MI n = 5) 76 on post-mi day 3. (b) Quantification of the fibrotic area using Picrosirius Red staining 77 in the infarcted areas on post-mi day 3 in WT (n = 3) and TDAG8 (n = 3) mice. 78 Comparisons were assessed using unpaired Student s t-tests. Error bars represent the

6 79 mean ± SEM (, not significant) Supplementary Figure 11. Quantitative analysis of the mrna expression of 82 proton-sensing GPCRs in cardiac macrophages. 83 Cardiac cells were isolated from WT mice on post-mi day 3, and cardiac macrophages 84 (CD Ly6G CD11b + CD3 ) were sorted after eliminating dead cells and doublet 85 cells. The levels of proton-sensing GPCRs (TDAG8, GPR4, OGR1 and G2A) were 86 quantified using real-time RT-PCR. Three infarcted WT mice hearts were combined for 87 this analysis

7 9 Supplemental Table 1. Sequences of primers or Assay ID used for real time 91 RT-PCR mrna Sequences of primers (Sigma) Il17a Forward: 5 -GGTCAACCTCAAAGTCTTTAACTCC-3 Reverse: 5 -GGTCTTCATTGCGGTGGAGAG-3 Probe: 5 -CCAGAAGGCCCTCAGACTACCTCAACCG-3 Ccl2 Forward: 5 -CGGCTGGAGCATCCACGT-3 Reverse: 5 -ATTGGGATCATCTTGCTGGTGAAT-3 Probe: 5 -TCAGCCAGATGCAGTTAACGCCCCAC-3 Cxcl12 Forward: 5 -GTGACGGTAAACCAGTCAGCC-3 Reverse: 5 -GCACAGTTTGGAGTGTTGAGGA-3 Probe: 5 -CGGTTCTTCGAGAGCCACATCGCCAG-3 Ccl2 Forward: 5 -CCTCTCGTACATACAGAC-3 Reverse: 5 -CGTGTGAAAGATGATAGC-3 Probe: 5 -CATCGGCCATCTGTCTTGTGAA-3 Il23 Forward: 5 -TCAAGGACAACAGCCAGTTCT-3 Reverse: 5 -AAGATGTCAGAGTCAAGCAGGT-3 Probe: 5 -AGCCAGACCTTGGCGGATCCTTTGC-3

8 92 Supplemental Table 1 (Continued) mrna Sequences of primers (Sigma) Tdag8 Forward: 5 -TGAGCTAGGGATTTACCTCTTCAG-3 Reverse: 5 -AGTCCAGTTGTCTTTATTCCAAGTG-3 Probe: 5 -CTGTCCCTGTCAGACCTGCTGTATGCG-3 Gpr4 Forward: 5 -GCACCGCTCTTCCATGATGA-3 Reverse: 5 -CGCTCCATGGGGAACTTCTC-3 Probe: 5 -TCGTGATCGCTACAACCACACCTTCTGCT-3 Ogr1 Forward: 5 -TCTGGGAGAGAAACTGTGAGTTTG-3 Reverse: 5 -TCCTCGGAGGCGGGCTAG-3 Probe: 5 -TCTATCACTTCTCCCTCCTCCTCACCAGCT-3 G2a Forward: 5 -GGTCCTGGTGGTGGTGTAC-3 Reverse: 5 -GCAGAACAGGTAGACGGCTAG-3 Probe: 5 -CCTACCAGCCAACTGCCTGACTGCCT-3 GAPDH Forward: 5 -CGTCCCGTAGACAAAATGGTGA-3 Reverse: 5 -CCACTTTGCCACTGCAAATGG-3 Probe: 5 -CCAATACGGCCAAATCCGTTCACACCGA-3 93

9 94 Supplemental Table 1 (Continued) mrna Sequences of primers (Sigma) or Assay ID (Thermo Fisher) 18S rrna Forward: 5 -GGGTCATAAGCTTGCGTTGATTAAG-3 Reverse: 5 -TCCGAGGGCCTCACTAAAC-3 Probe: 5 - TACACACCGCCCGTCGCTACTACCG-3 Cxcl15 (Il8) Forward: 5 - ACAGAAAGGAAGTGATAGCAGTCC-3 Reverse: 5 - GAGGTCCTCAGGTAGGAACCT-3 Probe: 5 - ATTGGGCCAACAGTAGCCTTCACCCATG-3 Tnf Mm443258_m1 Il1 Mm434228_m1 Il6 Mm44619_m1 Ccl2 Mm _m1 Col1a1 Mm81666_g1 Tgf 1 Mm117882_m1 Ifn Mm _m1 Tdag8 Mm _s1 18S rrna Hs33631_g1 Csf-2 Mm12962_m1

10 Gpr4 Ogr1 G2a (x1-5 ) (x1-5 ) (x1-5 ) Copy number/gapdh Copy number/gapdh Copy number/gapdh sham infarct sham infarct WT TDAG8 sham infarct sham infarct sham infarct sham infarct WT TDAG8 WT TDAG8 Supplementary Figure 1. Comparison of Gpr4, Ogr1 and G2a expression levels in the hearts of WT and TDAG8 mice on post-mi day 3. Quantitative analysis of the expression of proton-sensing GPCR mrnas in WT mice hearts (sham, n = 3; MI, n = 7) and TDAG8 mice hearts (sham, n = 3; MI, n = 5) on post-mi day 3. mrna expression was normalized to GAPDH. Comparisons were assessed using unpaired Student s t-tests. Error bars represent the mean ± SEM (, not significant).

11 a b 1 mm N.S N.S AAR /LV (%) Infarct area /AAR (%) WT 4 WT WT Supplementary Figure 2. The area at risk and infarct size were comparable between WT and TDAG8 mice during the initial phase after MI. (a) Representative heart sections of Evans blue/ttc staining 3 h after MI. The non-ischemic area is indicated in blue, the area at risk (AAR) in red and the infarct area in white. (b) The infarct size and AAR were quantified as a percentage of the AAR and the left ventricular area (LV) of WT (n = 5) and TDAG8 (n = 4) mice. Error bars represent the mean ± SEM. Comparisons among groups were assessed using unpaired Student s t-test (, not significant).

12 WT Lymphocyte singlet B cell (B22 + ) p=.14 SSC-A Lymphocyte FSC-A FSC-H singlet FSC-A Count 46. B22 (%) WT Supplementary Figure 3. Comparison of infiltrated B cells in the infarcted hearts of WT and TDAG8 mice. Comparison of infiltrated B cells (B22+) on post-mi day 3 between WT mice (n = 4) and TDAG8 mice (n = 3) was performed using flow cytometry. Representative FACS profiles are shown. The comparison was assessed using unpaired Student s t-tests. Error bars represent the mean ± SEM.

13 mrna/18s rrna (fold) WT Tnfα Sham Non-infarct Infarct Supplementary Figure 4. No significant differences in Tnfα mrna were found on post-mi day 3. Tnfα mrna expression in infarcted and non-infarcted areas of WT mice (sham n = 3, MI n = 7) and TDAG8 mice (sham n = 3, MI n = 5) 3 days after MI was measured using real-time RT-PCR. The comparison was assessed using an unpaired Student s t-test.

14 mrna/18s rrna (fold) h 2 h Ccl2 (TNF-α) 4 h 8 h 24 h h 2 h Ccl2 (HMGB1) 4 h 8 h 24 h Supplementary Figure 5. Time course of Ccl2 mrna expression in cardiac macrophages stimulated with TNF-α and HMGB1. Cardiac macrophages were isolated from WT mice on post-mi day 3 and stimulated with 1-ng/ml TNF-α (left) or 1-μg/ml HMGB1 (right). Three heart samples were combined as one sample and used for real-time RT-PCR analysis.

15 CD3ε + γδtcr + cells WT Count CCR6 Supplementary Figure 6. Comparison of infiltrated CCR6 + γδt cells in the infarcted hearts of WT and TDAG8 mice on post-mi day 5. Cardiac γδt cells were isolated from WT and TDAG8 mice 5 days after MI; stained with antibodies against CD3ε, γδtcr and CCR6; and were analysed by flow cytometry. Grey histograms indicate the isotype control.

16 Il-17a mrna/18s rrna (fold) Non-infarct WT Infarct p <.5 Non-infarct Infarct Supplementary Figure 7. Upregulation of Il-17a mrna in TDAG8 mice 7 days after MI. IL-17a mrna expression in the non-infarct and infarcted areas of WT mice (n = 7) or TDAG8 mice (n = 5) 7 days after MI was measured using real-time RT-PCR. Comparisons were assessed with a one-way ANOVA followed by Tukey s test. Error bars represent the mean ± SEM (, not significant).

17 Il6 Il8 (Cxcl15) Gm-csf (Csf2) mrna/18s rrna (fold) # # # # WT WT WT Sham Non-infarct Infarct Supplementary Figure 8. Comparison of IL-17A-related inflammatory genes of WT and TDAG8 mice. Expression levels of inflammatory gene mrnas in the infarcted and non-infarcted areas of WT mice (sham, n = 6; MI, n = 1) and TDAG8 mice (sham, n = 5; MI, n = 8) on post-mi day 3. # indicates that Il8 mrna was not detected. Comparisons were assessed using unpaired Student s t-tests. Error bars represent the mean ± SEM (, not significant).

18 CD3ε + γδtcr + CD3ε + γδtcr + CCR6 + Count CCR IL-17A Supplementary Figure 9. A portion of CCR6 + γδt cells secrete IL-17A. Cardiac cells were isolated from TDAG8 mice on post-mi day 5 and stimulated with PMA and ionomycin in the presence of Brefeldin A. The samples were stained with antibodies against CD3ε, γδtcr, CCR6 and IL-17A. After gating visible and singlet cells, CD3ε + γδtcr + CCR6 + cells were further gated, and IL-17A expression was determined using flow cytometry. Mixed samples (n = 3) were used for the a.nalysis.

19 a Col1a1 Tgfb mrna/18s rrna (fold) Sham Non-infarct Infarct WT WT b Infarct 4 WT CVF (%) μ WT Supplementary Figure 1. Comparison of fibrosis between WT and TDAG8 mice on the post-mi day 3. (a) Expression levels of fibrosis-related gene mrnas in the infarcted and non-infarcted area of WT mice (sham n = 3, MI n = 7) and TDAG8 mice (sham n = 3, MI n = 5) on post-mi day 3. (b) Quantification of the fibrotic area using Picrosirius Red staining in the infarcted areas on post-mi day 3 in WT (n = 3) and TDAG8 (n = 3) mice. Comparisons were assessed using unpaired Student s t-tests. Error bars represent the mean ± SEM (, not significant).

20 Copy number/gapdh (x1-3 ) Proton-sensing GPCRs TDAG8 GPR4 OGR1 G2A Supplementary Figure 11. Quantitative analysis of the mrna expression of protonsensing GPCRs in cardiac macrophages. Cardiac cells were isolated from WT mice on post-mi day 3, and cardiac macrophages (CD Ly6G CD11b + CD3 ) were sorted after eliminating dead cells and doublet cells. The levels of proton-sensing GPCRs (TDAG8, GPR4, OGR1 and G2A) were quantified using realtime RT-PCR. Three infarcted WT mice hearts were combined for this analysis.

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