B lymphocytes trigger monocyte mobilization and impair heart function after acute myocardial infarction

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1 Supplementary Figures to 3 B lymphocytes trigger monocyte moilization and impair heart function after acute myocardial infarction Yasmine Zouggari, Hafid Ait-Oufella, Philippe Bonnin, Taassome Simon, Andrew P Sage, Coralie Guérin, José Vilar, Giuseppina Caligiuri, Dimitrios Tsiantoulas, Ludivine Laurans, Edouard Dumeau, Salma Kotti, Patrick Bruneval, Israel F. Charo, Christoph J. Binder, Nicolas Danchin, Alain Tedgui, Thomas F. Tedder, Jean-Séastien Silvestre, Ziad Mallat Nature Medicine: doi:.38/nm.38

2 a cells/mg tissue 8 6 Neutrophils (CD + Ly6G + 7/ hi ) MI sham Cells/mg tissue 3 7/ hi and 7/ lo Monocytes 7/ hi MI 7/ hi MI sham 7/ lo MI 7/ lo MI sham c Cells/mg tissue MΦ/Dendritic cells (CDc + ) MI sham d Cells/mg tissue. 3 7 NK cells (CD + Ly6G - 7/ - NK. + ) MI sham e Cells/mg tissue CD3 + T lymphocytes MI sham f Cells/mg tissue B neutro 7/ hi 7/ lo Nature Medicine: doi:.38/nm.38

3 Supplementary Fig.. Myocardial infarction induces time-dependent recruitment of specific inflammatory cell susets in cardiac tissue. Cell suspensions from sham-operated or infarcted hearts of C7BL/6J mice were stained and analyzed y flow cytometry at different time points after MI. (a) Neutrophils were identified as CD + Ly6G + 7/ hi (gated on CD + cells). () Monocytes were identified as CD hi Ly6G 7/ hi or 7/ lo (gated on CD + cells). (c) Dendritic cells or macrophages were stained with the anti-cdc antiody. (d) NK cells as CD + Ly6G 7/ NK. + cells. (e) CD3-positive T lymphocytes. Data are representative of - mice per group. Mean values ± SEM are shown. P <.; P <.; P <. vs sham-operated mice. Nature Medicine: doi:.38/nm.38

4 a MI D Heart B+ IgM+ B Cells/mg of tissue Sham D IgM IgM B. 3 MI sham 3 7 B MI Sham D3 MI Sham D3 D D c MI Heart D3 IgD.7% 99.3%.87% 3 3 CD9 3 IgM Supplementary Fig.. The injured myocardium triggers B lymphocytes recruitment. (a) Representative dot plots of B+ IgM+ B cells (gated on CD+ cells) in sham-operated (sham) and infarcted (MI) hearts on day after surgery. Numers of infiltrating B cells were determined per milligram of tissue at specified days following MI (n= to mice per time point). Mean values ± SEM are represented. P <.. () Representative sections of B immunostaining on days 3 and after surgery in healthy hearts and in the order infarct area. Bars, µm. (c) Representative dot plots of CD9+IgD+IgM+ B cells (gated on CD+ cells) in hearts on day 3 after MI. Nature Medicine: doi:.38/nm.38

5 a c 3 D % 3 IgM CD9.% B 3 CD9 αcd D3 3 CD3 IgM 3 CD3 3 3 B + IgM + B Cells/mg tissue αcd 3 Heart D3 α-cd B 3 CD FO B cells 37.% MZ B cells 3.% 3 CD FO B cells.% MZ B cells.% B + IgM + B Cells/Spleen [x ] Cells/mg of spleen [x ] Cells/mg of spleen [x 3 ] B + IgM + B Cells/mg tissue Heart D Spleen D FO B cells Spleen D3 α-cd α-cd MZ B cells Spleen D3 α-cd α-cd Supplementary Fig. 3. CD ma therapy reduces B cells in the cardiac tissue and in the spleen. Representative examples and quantitative analysis of B hi IgM + B cells stainings in the injured myocardium (a) and spleens () of anti-cd antiody treated mice compared to controls (n= to per group). (c) Quantitative analysis of Follicular (CD3 + CD int ) and marginal zone (CD3 - CD hi ) B cells (n=7 per group). Mean values ± SEM are represented. Bars, µm. P <.; P <.; P <.. Nature Medicine: doi:.38/nm.38

6 α-cd Tunel (cells/field) D α-cd capillaries/myocyte a D.. c α-cd Arterioles/mm D α-cd Supplementary Fig.. CD ma therapy impairs myocardial cell death and improves cardiac healing. Representative photomicrographs and quantitative analysis of myocardial cell apoptosis (a), capillary density () and arterioles numers (c) in the order zone of the infarct on day (n=8- per group). Mean values ± SEM are represented. Bars, panels a and c, µm; Bars, panel, µm. P <. vs. Nature Medicine: doi:.38/nm.38

7 a IgM (mg/ml) D IgM D3 D3 αcd D D αcd IgG (mg/ml) D D3 IgG D3 αcd D D αcd c IgGc (mg/ml) D D3 IgGc D3 αcd D D αcd Supplementary Fig.. Circulating IgM and IgG levels after acute MI. ELISA for concentrations of total IgM (a), IgG (), and IgGc (c) in plasma at aseline (day ), day 3 as well as day following coronary artery ligation in mice either treated with CD A or (n=-8 per group). P <. vs.. Nature Medicine: doi:.38/nm.38

8 mrna level (A.U.) mrna level (A.U.) a ILβ TNFα IL8 IL TGFβ αcd αcd αcd αcd αcd ILβ TNFα IL8 IL TGFβ αcd αcd αcd αcd αcd Supplementary Fig. 6. CD ma treatment reduces systemic pro-inflammatory cytokine production. Representative histograms of mrna levels of the pro-inflammatory cytokines IL-β, TNF-α, IL-8 and the anti-inflammatory cytokines IL- and TGF-β, in spleens (a) and in lymph nodes () of B cells depleted mice on day after MI (n=8- per group). Mean values ± SEM are represented. P <.; P <. vs. Nature Medicine: doi:.38/nm.38

9 D3 Ly6-G 3.3% Cells/spleen [x ] Monocytes 7/ hi 3 3 CD 3 7/ (Ly6-C) α-cd Ly6-G 3 3.8% 3 CD α-cd D / (Ly6-C) Cells/spleen [x ] Monocytes 7/ lo..... α-cd Supplementary Fig. 7. CD ma-induced B cell depletion enhances 7/ hi monocyte accumulation in spleen. Representative examples of 7/ hi and 7/ lo monocyte stainings in spleen of B cell-depleted mice compared to controls on day 3 after MI (n=8- per group). Mean values ± SEM are represented. P <. vs. Nature Medicine: doi:.38/nm.38

10 a Heart D3 Monocytes 7/ hi cells/mg of tissue p=.7 αcd Monocytes 7/ lo cells/mg of tissue 6 3 p=.8 αcd c Heart D3 Neutrophils cells/mg of tissue Heart D Neutrophils αcd CD3 + T lymphocytes cells/mg of tissue CD3 + T cells αcd NK cells cells/mg of tissue 8 6 NK cells p=.6373 p=.698 p=.39 αcd Neutrophils cells/mg of tissue 7 Neutrophils CD3 + T lymphocytes cells/mg of tissue CD3 + T cells NK cells cells/mg of tissue 3 αcd αcd NK cells p=.373 p=.387 p=.736 αcd Supplementary Fig. 8. Effect of CD ma-induced B cell depletion on accumulation of leukocyte susets in the ischemic heart. Quantitative analysis of 7/ hi and 7/ lo monocytes (a), neutrophils, CD3 + T cells, NK cells on days 3 () and (c) after MI in cardiac tissue of B cell-depleted mice compared to controls (n=8- per group). Mean values ± SEM are represented. Nature Medicine: doi:.38/nm.38

11 Ccl mrna (A.U.) Ccl7 mrna (A.U.) a Plasma Ccl Ccl7 Ccl Ccl (pg/ml) 7 Ccl7 (pg/ml) 3 Ccl (pg/ml) Ischemic Heart Ccl Ccl7 Ccl Ccl mrna (A. U.) Ccl (pg/mg of protein) c Ccl (pg/ml) 3 7 Plasma Ccl Ccl7 (pg/mg of protein) αcd Ccl (pg/mg of protein) Supplementary Fig. 9. Ccl, Ccl7 and Ccl production after acute MI. (a) Ccl, Ccl7 and Ccl protein levels in plasma of C7BL/6J mice after acute MI. () mrna and protein levels of Ccl, Ccl7 and Ccl within the injured myocardium at different time point after MI (n= mice). (c) Ccl protein levels in the plasma of B cell-depleted mice compared to untreated group. Mean values ± SEM are represented. P <.; P <.; P <. vs Day Nature Medicine: doi:.38/nm.38

12 a Spleen D Ccl7 WT Ccl7 Ccl7 -/ CD9 3 CD9 Spleen D Sham MI c Spleen D CD9 3 CD9 7.%.% IgM IgM Ccl7 + CD9 + IgM + B cells (cells/mg of spleen) % 3 Ccl7 MI Sham 7.6% % Ccl7 + cells among CD9 + B cells 3 3 Sham MI WT MyD88 -/- TRIF -/- d mrna levels (A.U.) ND WT ND Ccl7 -/- WT Activation WT Ccl7 -/- WT Activation Ccl7 Gapdh Nature Medicine: doi:.38/nm.38

13 Supplementary Fig.. B cells upregulate Ccl7 production in response to myocardial infarction in part through Myd88/Trif pathway. (a) Representative examples of Ccl7 protein production in B cells evaluated y flow cytometry compared to Ccl7 -/- mice. () Flow cytometry and quantification of the percentage of Ccl7 + CD9 + cells in the spleen on day after MI compared to sham-operated mice. (c) Quantification of the numer of Ccl7 + CD9 + cells in the spleen of Myd88 -/- Trif -/- mice compared to controls on day after MI (n= per group). (d) Representative and quantitative analysis of Ccl7 mrna levels in B cells evaluated y RT-PCR on day after MI compared to Ccl7 -/- and sham-operated mice (n=6 per group). Mean values ± SEM are represented. P <.; P <.; P <.. Nature Medicine: doi:.38/nm.38

14 Cell/ml of Blood [x ] SF (%) a Monocytes 7/ hi 6 8 Blood D Monocytes 7/ lo Blood D WT Ccl7 -/- WT Ccl7 -/- 3 3 D WT Ccl7 -/- Cell/femur [x ] BM D WT Ccl7 -/- 8 6 BM D WT Ccl7 -/- Supplementary Fig.. Ccl7 deficiency reduces 7/ hi monocyte moilization and improves cardiac function following acute MI. (a) Representative histograms of 7/ hi monocyte counts in lood and one marrow of wild-type and Ccl7 / mice on day after MI. () Echocardiography analysis of wild-type and Ccl7 / mice showing the results of the assessment of the shortening fraction (SF) (n=-8 per group). Mean values ± SEM are represented. P <.; P <. vs wild-type mice. Nature Medicine: doi:.38/nm.38

15 a 3% Before purification 98.% After purification B cells/spleen [x ] WT Spleen D3 αcd # IgM αcd +WT B cells ## αcd +Ccl7 -/- B cells CD9 Cells/ml [x ] Blood D3 WT αcd αcd αcd +WT +Ccl7 -/- B cells B cells Monocytes 7/ lo Percentage of CD + 7/ lo Monocytes IgM IgG d e f IgM (mg/ml) αcd +WT B cells D αcd +Ccl7 -/- B cells c IgG (mg/ml). D αcd +WT B cells αcd +Ccl7 -/- B cells IgGc (mg/ ml) 3 3 WT αcd Heart D3 # IgGc. D αcd +WT B cells αcd +WT B cells $$ αcd +Ccl7 -/- B cells αcd +Ccl7 -/- B cells Nature Medicine: doi:.38/nm.38

16 Supplementary Fig.. B lymphocytes reduce 7/ lo monocyte moilization and recruitment into the injured myocardium without affecting circulating IgM and IgG levels in Rag -/- mice after acute MI. (a) B cells purity after isolation. Representative examples of CD9 + IgM + B cells efore and after purification, using the B cell isolation kit (Miltenyi Biotec) according to manufacturer s instructions. () Quantification of B cell counts in spleens of Rag -/- mice injected with either wild-type splenocytes, B cell-depleted splenocytes, or B cell-depleted splenocytes resupplemented with wild-type or Ccl7 -/- B cells. (c) Quantification of 7/ lo monocytes in lood (left) and infarcted myocardium (right) of the groups of mice, 3 days after MI. ELISA for concentrations of total IgM (d), IgG (e), and IgGc (f) in plasma at day following coronary artery ligation in Rag -/- mice either re-supplemented with WT or Ccl7 -/- B cells. Data are representative of to mice per group in three independent experiments. Mean values ± SEM are shown. P <. and P <. vs WT splenocytes ; # P <.; ## P <. vs α-cd splenocytes; $$ P <. vs α-cd splenocytes with WT B cells. Nature Medicine: doi:.38/nm.38

17 a Blood D3 % Total Cells 3 BAFFR+/- BAFFR-/- T T MZ B 7/ lo monocytes Cells/ml WT Baff-R -/- c Infarct size d Blood D3 Infarct Size (%) Baff-R +/- Baff-R -/- 7/ lo monocytes Cells/ml αbaff e Infarct Size (%) Infarct size αbaff Supplementary Fig. 3. Complementary results on Baff-r / mice and anti-baff antiody treated mice. (a) cytes from Baff-r +/ and Baff-r / littermates were stained and analyzed y flow cytometry for B cell susets gated on B + IgM + cells, defined as T cells (CD3 -/lo CD -/lo ), T (CD3 + CD hi ), MZ (CD3 -/lo CD hi ) and B (CD3 + CD + ). Data is expressed as % total live cells. 7/ lo monocytes lood levels of Baff-r / mice () and anti-baff-treated mice (d) compared to controls. Quantitative analysis of the infarct size of Baff-r / mice compared to Baff-r +/ mice (c) and anti-baff treated mice (e) compared to controls. Mean values ± SEM are shown. Nature Medicine: doi:.38/nm.38

18 Supplementary Tale : Baseline Characteristics and hospital management of patients according to Ccl7 plasma levels Ccl7 not detectale Ccl7 detectale p (n = 79) (n = 6) Demographic and risk factors Male Sex 63 (7%) 39 (67%).3 Age, yr 66 ± 67 ±. Hypertension 8 (6%) 37 (67%). Hypercholesterolemia (%) 8 (%).69 Diaetes mellitus 8 (3%) 67 (33%).7 Family history of CAD (%) (%).3 Previous or current smokers 8 (6%) (9%). Prior myocardial infarction 3 (7%) 37 (8%).6 Prior PCI or CABG (8%) 9 (%). Prior stroke or TIA 6 (8%) (%).9 Prior Heart Failure 3 (%) 3 (6%).6 Chronic Renal Failure 3 (%) (%).6 Clinical Presentation Body mass index (kg/m²) 7 ± 7 ±. Systolic lood pressure at admission 39 ± 8 ± 3.8 Diastolic lood pressure at admission 8 ± 7 8 ± 8. Heart rate at admission 79 ± 8 ±. STEMI, No (%) (3%) 7 (%).8 Killip entree class = or more (7%) 3 (6%).68 GRACE Score 63 ± ± 37.7 Left ventricular ejection fraction 3 ± ±. Baseline iological exams Ccl7 (pg/ml) 3.9 [ ] <. Baff (pg/ml) [8-7] 66.7 [86.7-3].3 CRP (mg/l). [ ].7 [ ]. In-hospital Management PCI, No (%) 7 (69%) 8 (6%).7 Thromolysis, No (%) 9 (6%) 33 (6%).9 Coronary artery ypass surgery, No (%) 3 (%) (%).8 Statins 6 (78%) 9 (77%).7 Beta-lockers 69 (7%) 9 (7%).8 Calcium Channel lockers 9 (%) (%). ACE inhiitors or ARB 3 (3%) (%).78 Nitrated Derivatives 8 (%) (6%).6 Aspirin 79 (9%) 9 (93%).33 Clopidogrel 7 (89%) 8 (88%).7 Heparin 69 (8%) 7 (83%).68 Low Molecular Weight Heparin 9 (6%) 3 (6%).6 Diuretics 6 (33%) 7 (3%).6 Glycoprotein II/IIIa inhiitor 38 (%) 66 (3%). Digitalis glycosides 8 (%) (%). p is given y Wilcoxon rank-sum or Kruskal-Wallis (continuous variales) and exact Pearson Χ² or Fisher test (categorical variales) mean ± sd, Median, Q, Q3 Nature Medicine: doi:.38/nm.38

19 ACE : angiotensin-converting enzyme inhiitors or ARB : angiotensin receptor lockers, PCI : percutanoeus coronary angioplasty, CABG: coronary artery ypass surgery Nature Medicine: doi:.38/nm.38

20 Supplementary Tale. Baseline Characteristics and hospital management of patients according to Baff tertiles Baff (pg/ml) 88 ] [ 76 p (n =39) (n =39) (n =3) Demographic and risk factors Male Sex (76%) 8 (68%) 9 (6%) <. Age, yr 6 ± 3 6 ± 3 69 ± <. Hypertension (63%) 8 (6%) 7 (68%). Hypercholesterolemia 6 (%) 6 (%) 69 (3%).8 Diaetes mellitus (33%) 9 (9%) (3%).63 Family history of CAD 87 (7%) 7 (%) 77 (%).33 Previous or current smokers 66 (%) 8 (8%) 69 (3%). Prior myocardial infarction (%) 3 (7%) 7 (%). Prior PCI or CABG 9 (%) (6%) 69 (%).7 Prior stroke or TIA 9 (6%) (8%) 3 (%). Prior Heart Failure 9 (3%) (3%) 7 (8%) <. Chronic Renal Failure (%) 6 (%) (7%). Clinical Presentation Body mass index (kg/m²) 7 ± 7 ± 7 ±.8 Systolic lood pressure at admission ± 7 39 ± 8 38 ± 3 <. Diastolic lood pressure at admission 8 ± 7 8 ± 6 78 ± 8 <. Heart rate at admission 77 ± 8 79 ± 3 8 ± <. STEMI, No (%) 7 (%) 63 (%) 7 (3%).8 Killip entree class = or more 7 (8%) 7 (3%) (39%) <. GRACE Score 7 ± 36 9 ± 36 7 ± 36 <. Left ventricular ejection fraction 6 ± 3 ± 9 ± 3 <. Baseline iological exams Ccl7 (pg/ml) 8. ± 9 8 ±.9 ±.6 Baff (pg/ml) 7.7 ± 3 ± ± 7 <. CRP (mg/l) 8.97 ± 9. ±. ± 7 <. In-hospital Management PCI, No (%) 36 (7%) 9 (66%) 99 (6%) <. Thromolysis, No (%) 8 (%) 8 (%) 7 (8%).6 Coronary artery ypass surgery, No (%) (3%) (%) 9 (3%).3 Statins (78%) (79%) 9 (78%).9 Beta-lockers 38 (7%) 39 (7%) 8 (68%).8 Calcium Channel lockers 6 (9%) 7 (%) 68 (%).66 ACE inhiitors or ARB 63 (%) 7 (%) 83 (7%).3 Nitrated Derivatives 6 (%) 67 (%) 7 (%).9 Aspirin 8 (89%) 97 (93%) 89 (9%). Clopidogrel 9 (9%) 89 (9%) 69 (8%) <. Heparin 68 (8%) 63 (8%) (79%).3 Low Molecular Weight Heparin 9 (66%) 7 (6%) 8 (6%). Diuretics 7 (%) 9 (9%) (7%) <. Glycoprotein II/IIIa inhiitor 37 (3%) 37 (3%) 97 (3%) <. Digitalis glycosides 3 (%) (%) (%) <. p is given y Wilcoxon rank-sum or Kruskal-Wallis (continuous variales) and exact Pearson Χ² or Fisher test (categorical variales) Tertiles are constructed among the entire population sample, mean ± sd Nature Medicine: doi:.38/nm.38

21 ACE : angiotensin-converting enzyme inhiitors or ARB : angiotensin receptor lockers, PCI : percutanoeus coronary angioplasty, CABG: coronary artery ypass surgery Nature Medicine: doi:.38/nm.38