P2Y6 receptor potentiates pro-inflammatory responses in macrophages and exhibits differential roles in atherosclerotic lesion development

Abstract

Background: P2Y(6), a purinergic receptor for UDP, is enriched in atherosclerotic lesions and is implicated in pro-inflammatory responses of key vascular cell types and macrophages. Evidence for its involvement in atherogenesis, however, has been lacking. Here we use cell-based studies and three murine models of atherogenesis to evaluate the impact of P2Y(6) deficiency on atherosclerosis.

Methodology/principal findings: Cell-based studies in 1321N1 astrocytoma cells, which lack functional P2Y(6) receptors, showed that exogenous expression of P2Y(6) induces a robust, receptor- and agonist-dependent secretion of inflammatory mediators IL-8, IL-6, MCP-1 and GRO1. P2Y(6)-mediated inflammatory responses were also observed, albeit to a lesser extent, in macrophages endogenously expressing P2Y(6) and in acute peritonitis models of inflammation. To evaluate the role of P2Y(6) in atherosclerotic lesion development, we used P2Y(6)-deficient mice in three mouse models of atherosclerosis. A 43% reduction in aortic arch plaque was observed in high fat-fed LDLR knockout mice lacking P2Y(6) receptors in bone marrow-derived cells. In contrast, no effect on lesion development was observed in fat-fed whole body P2Y(6)xLDLR double knockout mice. Interestingly, in a model of enhanced vascular inflammation using angiotensin II, P2Y(6) deficiency enhanced formation of aneurysms and exhibited a trend towards increased atherosclerosis in the aorta of LDLR knockout mice.

Conclusions: P2Y(6) receptor augments pro-inflammatory responses in macrophages and exhibits a pro-atherogenic role in hematopoietic cells. However, the overall impact of whole body P2Y(6) deficiency on atherosclerosis appears to be modest and could reflect additional roles of P2Y(6) in vascular disease pathophysiologies, such as aneurysm formation.

Figure 1. P2Y₆ expression mediates release of calcium and pro-inflammatory cytokines in 1321N1 cells.

Evaluation of calcium release and cytokine secretion to cell culture media in 1321N1 astrocytoma cells stably expressing human P2Y₆ receptor. A) Isolated 1321N1 clones expressing varying levels of P2Y₆ mRNA and mock transfected cells (Parental) were incubated with increasing concentrations of UDP and 3P-UDP. Calcium mobilization was measured to generate concentration response curves with examples shown for 3G10 clone and parental cell line. For each clone, achieved maximal calcium response is expressed as percentage relative to 3G10 clone (set as 100%) together with calculated EC₅₀ values (ND  =  not determined) and relative P2Y₆ mRNA levels (relative to parental cells, which had very low but detectable P2Y₆ mRNA). B) 1321N1 clones were incubated for 20 hr with increasing concentrations of 3P-UDP and cytokines in cell culture medium quantified using multiplex ELISA as described in Materials and Methods. Each cytokine was normalized to cellular protein content. The data are presented as mean +/− SD of replicate wells.

Figure 2. P2Y₆ receptor agonism modulates inflammatory cytokine release from macrophages.

Activation of P2Y₆ with 3P-UDP potentiates secretion of pro-inflammatory cytokines in macrophages. A) Differentiated THP-1 macrophages were incubated for 16 hr with increasing concentrations of 3P-UDP and cytokines in cell medium analyzed using multiplex ELISA. Cytokines which were affected by 3P-UDP treatment are shown (left graph). On the right, THP-1 monocytes were differentiated in presence of control non-targeting (NT) or P2Y₆ siRNA (siRNA) and treated with 33 µM 3P-UDP for 16 hr to determine cytokine levels in medium. The level of P2Y₆ mRNA knockdown was 78%. The data are presented as mean +/−SD of replicate wells from a representative experiment. Significance P2Y₆ siRNA vs non-targeting siRNA: *p<0.01. B) Adhered mouse peritoneal macrophages were incubated in serum-free medium for 16 hr with combinations of 100 µM 3P-UDP either alone or in the presence of additional inflammatory stimulus (0.1 ng/mL TNF-α or 10 ng/mL LPS) and analyzed for cytokines levels in cultured medium using ELISA. C) Human primary monocyte-derived macrophages were incubated with 100 µM 3P-UDP either alone or in the presence of 10 pg/mL TNF-α and analyzed for cytokine levels as described in Materials and Methods. Relative expression of P2Y₆ mRNA in each cell line was compared using quantitative PCR Threshold Cycle (Ct) values. P2Y₆ Ct values were normalized to RPL30 (set at Ct  =  20) and plotted to illustrate relative expression compared to abundant RPL30 housekeeping gene (Ct  =  20) or genes expressed at the very low levels (Ct> 35).

Figure 3. P2Y₆ knockout mice exhibit reduced responsiveness to inflammatory challenge.

Generation and evaluation of P2Y₆ KO mice. A) Targeting strategy used to replace the 3^rd exon of the P2Y₆ gene containing the entire open reading frame with a pKOS-17 vector containing β-galactosidase (LacZ) reporter and neomycin resistance (Neo) cassette. The second and third exon of the P2Y₆ gene are depicted as grey boxes with indicated translational start and stop codons. Location of Southern Blot probes is shown in green and blue, and PCR genotyping primers are shown in red. B) MPMs from P2Y₆ knockout mice (KO) and wild type littermates (WT) were incubated with increasing concentrations of 3P-UDP to generate calcium concentration response curves. Calculated EC₅₀ value for WT MPMs is shown. C) P2Y₆ KO males and WT male littermates (n =  4–5 per group) were subjected to intra-peritoneal challenge with vehicle, MSU or combination of MSU and 3P-UDP (1mg/mouse each) as described in Materials and Methods. Blood was collected 1 hr after injection and cytokines in plasma measured by multiplex ELISA. Cytokines that displayed differential responses in KO mice are shown. Baseline (unchallenged mice) and vehicle treated mice are also depicted for MIP-1α. Significance KO vs WT: **p<0.01 *p<0.05. Not significant, n.s.

Figure 4. Atherosclerosis development with P2Y₆-deficient bone marrow reconstitution.

A) Quantification of en face lesion area with oil red O staining in the whole aorta from the ascending aorta to the iliac bifurcation and B) aortic arch. C) Representative images of aortic arch atherosclerosis. Group sizes: n = 9 P2Y₆ WT transplanted mice; n = 14 P2Y₆ KO transplanted mice. Significance vs. control: *p<0.05. Not significant, n.s.

Figure 5. Atherosclerosis development with Western Diet Feeding.

En face plaque quantification of excised mouse aortas using oil red O. A) Lesion area measured in the whole aorta from the ascending aorta to the iliac bifurcation. B) Regional atherosclerosis in the aortic arch. The percentage of positive stained area with respect to total analyzed aortic area. Group sizes: n = 12 P2Y₆ WT mice; n = 13 P2Y₆ KO mice.

Figure 6. Accelerated Atherosclerosis development with Angiotensin II infusion.

Atherosclerosis development measured in the aorta with oil red O. A) Quantification of en face lesion area in the thoracic aorta (ascending aorta to the diaphragm). Regional atherosclerosis in B) aortic arch and C) descending thoracic aorta segments. Group sizes: n = 9 P2Y₆ WT mice; n = 14 P2Y₆ KO mice. The fold change in P2Y₆ lesion area is given relative to P2Y₆ wild type levels. Not significant, n.s.

Figure 7. Abdominal Aortic Aneurysms.

A) Incidence of aneurysm development in the abdominal aorta. B) Representative images of abdominal aortic aneurysms for P2Y₆ KO mice and P2Y₆ wild-type mice. Aneurysm-positive and -negative aortas are given for P2Y₆ KO wild type mice. Aneurysms are demarcated by the white boxes. C) Trichrome-stained histological sections of aneurysms taken from the supra-renal section of the abdominal aorta. Sections were cut from the center of the aneurysm mass. The single asterisk denotes the highly inflamed and remodeled adventitial mass. The double asterisk denotes a region of apparent hemorrhage. Neo-intimal plaque is shown by the arrows. The image at the far right shows an abdominal aorta cross-section taken from P2Y₆ wild type mice. The magnification scale for each image is shown.