Central role of PD-L1 in cardioprotection resulting from P2Y4 nucleotide receptor loss

Figures

• 

  Figure 1. Loss of mouse P2Y4 induces beiging of pericardial adipose tissue. (A) Representation of the biological processes related to the genes differentially expressed between PAT of P2Y4 KO and WT ischemic mice (24h post-MI) in RNA-sequencing experiments. (B) Selection of biological processes revealed as enriched for a differentially expressed number of genes (Nbr of genes) between PAT RNA of P2Y4 KO and WT ischemic mice, after a Gene Ontology enrichment analyses performed with DAVID software. (C) qPCR quantification of Ucp1, Cited1 and Prdm16 mRNAs in PAT of P2Y4 KO and WT control (Ctrl) mice or 24h after LAD ligation (myocardial infarction (MI)), normalized to Rpl32 housekeeping gene (n = 6). (D) UCP1 staining performed by immunohistochemistry on paraffin‐embedded sections of PAT of WT and P2Y4 KO ischemic mice (24h post-MI) and counterstained with hematoxylin/eosin (scale bar = 50 μm). Examples of regions positive for UCP1 staining are indicated by black arrows and images were taken at original magnification 100x. Data represent mean ± SEM. *p < 0.05; **p < 0.01.

• 

  Figure 2. Identification of PD-L1 as a marker of beige adipocytes, upregulated in P2Y4 KO PAT. (A) Quantification of lipid droplet size in adipocytes generated from WT, P2Y4 KO, Adipo KO and DKO cADSCs in the presence or absence of R+I treatment. Quantification was performed using ImageJ software (n = 4). (B) Oil red O staining of cardiac adipose-derived stem cells (cADSC) cultures after adipogenic differentiation and after treatment with two adipocyte browning agents, rosiglitazone (1 µM) plus isoproterenol (100 µM) (R+I) (200x magnification). Adipogenic differentiation leads to big lipid droplet appearance in control (Ctrl) cADSC cultures. The additional presence of browning agents (R+I) in the differentiation medium leads to small lipid droplet appearance in cADSC cultures. (C) Higher PD-L1 expression in P2Y4 KO differentiated cADSCs. Density plots, percentage of PD-L1 positive cells and mean fluorescence intensities (MFI) obtained by flow cytometry quantification of PD-L1 expression in differentiated adipocytes isolated from PAT of WT, P2Y4 KO, Adipo KO and DKO mice, with or without R+I (n = 4-6). (D) Flow cytometry analysis showing increased level of PD-L1 expression in P2Y4 KO PAT.…

• 

  Figure 3. Loss of P2Y4 induces anti-inflammatory macrophage polarization in ischemic PAT. (A) Flow cytometry data are shown as representative dotplots for F4/80 and CD206, a marker of M2 macrophages, quantified in PAT of WT, P2Y4 KO, adiponectin KO (Adipo KO) and P2Y4/adiponectin double KO (DKO) mice, without myocardial infarction (no MI), or 24h or 7 days (7D) after LAD ligation. (B) Histograms representing increase of F4/80+ macrophages after LAD ligation in PAT of WT, P2Y4 KO, Adipo KO and DKO mice (normalized to adipose tissue (AT) weight) (n = 4-5). (C) Flow cytometry quantification of M1, M2 and M2c macrophage populations in PAT using CD206 and MertK, a marker of M2c anti-inflammatory macrophages. Data are expressed as relative distribution of CD206-, CD206+, and CD206+ MertK+ macrophage populations within the PAT, before and 24h or 7D after MI (n = 4-6). Data represent mean ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001.

• 

  Figure 4. Lymphoid cluster reduced expansion and T cell polarization in ischemic PAT of P2Y4 mice. (A) Representative pictures of hematoxylin/eosin staining showing FALCs (lymphoid clusters; some are indicated with black arrows) observed in PAT of WT, P2Y4 KO, Adipo KO and DKO mice, with or without MI (200x magnification) (scale bar = 50μm). (B) Quantification of mean FALCs size detected in PAT 24 hours (24h) or 7 days (7D) after MI (a.u., arbitrary units) (n = 5). (C) mRNA level quantification of Il33 and Ccl24 expression in PAT of WT, P2Y4 KO, Adipo KO and DKO mice, without MI or 24h post-MI (relative expression normalized to Rpl32 housekeeping gene)(n = 3-6). (D) CD25-Foxp3 dot plots representing the gating used for flow cytometry quantification of regulatory lymphocyte population in PAT of mice without MI (upper dot plots) and 7 days post-MI (lower dot plots). (E) Percentage of regulatory lymphocytes in WT, P2Y4 KO, Adipo KO and DKO PAT from mice without MI, and 3 days (3D) or 7 days (7D) after MI (normalized to Treg level in control (ctrl) WT PAT) (n = 5-6). Data represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

• 

  Figure 5. T cell apoptosis is increased in ischemic PAT of P2Y4 KO mice. (A, B) Flow cytometry quantification of CD4 and CD8 T lymphocytes in PAT of WT, P2Y4 KO, Adipo KO and DKO mice, 3 or 7 days (3D or 7D) post-MI, normalized to adipose tissue (AT) weight (g)). (C, D) Quantification of naive T cells, effector memory T cells (Tem) and central memory T cells (Tcm) by quantification of CD44 and CD62L in ischemic PAT, 7 days post-MI (n = 5-9). (E) Representative flow cytometry dotplots for annexin V+ CD3+ lymphocytes in PAT WT, P2Y4 KO, Adipo KO and DKO mice, 7 days post-MI. Mice were injected intraperitoneally or not, with anti-PD-L1 blocking antibody (10 µg/g) (PD-L1), 1h before LAD ligation then at days 1, 3 and 5 post-LAD ligation. (F) Flow cytometry quantification of annexin V+ apoptotic T cells in PAT of WT, P2Y4 KO, Adipo KO and DKO mice (percentage compared to total CD3 positive cells), 7 days post-MI mice or in sham mice (no MI). Ischemic mice were injected intraperitoneally or not, with anti-PD-L1 blocking antibody (10 µg/g), 1h before LAD ligation, then at days 1, 3 and 5 post-LAD ligation (n = 5-6). Data represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

• 

  Figure 6. Anti-PD-L1 blocking antibody inhibits the reduction of T cell infiltration and fibrosis observed in ischemic P2Y4 KO hearts. (A) Representative T cell staining (CD3) in myocardial infarct sections of WT, P2Y4 KO, adiponectin KO (Adipo KO) or P2Y4/adiponectin double KO (DKO) mice, 7 days after LAD ligation using anti-CD3 (green) and Hoechst in blue (20x magnification) (scale bar = 50μm). Mice were intraperitoneally injected or not (MI), with anti-PD-L1 blocking antibody (10 µg/g), 1h before LAD ligation then at days 1, 3 and 5 post-LAD ligation. (B) CD3+ cell quantification in representative counting surface/field (0.1 mm2) of myocardial infarct sections, 7 days after LAD ligation. Data are expressed as percentages compared to T cell number in WT ischemic mice and obtained using ImageJ software by examining 30 fields per heart at 100x magnification (n = 5-6). (C) Representative Masson’s trichrome staining of cardiac fibrosis (in blue) in WT, P2Y4 KO, Adipo KO or DKO mice, 7 days after LAD ligation, injected or not with anti-PDL1 blocking antibody (10 µg/g)) (scale bar = 2.5 mm). (D) Fibrosis area represents collagen staining in blue quantified by color image analyser ImageJ in…