Abstract
Background
Atherosclerosis is accompanied by an imbalance between resolving and pro-inflammatory lipid mediators. Targeting lipid signaling pathways might offer a new anti-inflammatory therapy for improving the clinical outcome in cardiovascular disease patients. We considered lysophosphatidylinositol (LPI) and its receptor G protein-coupled receptor (GPR)55 as a potential modulator of atherosclerosis. Its role in regulating atherosclerosis and B cell function is unknown.
Hypothesis
We assessed the hypothesis that GPR55 signaling causally affects atherosclerosis and whether it has a specific role in regulating B cell function in this disease.
Methods
Atherosclerotic plaques were compared between apolipoprotein E deficient (ApoE−/−) and ApoE−/−Gpr55−/− mice after 4 to 16 weeks Western Diet (WD; 0.15% cholesterol; n=12–15 per group). To specifically test the role of B cell GPR55 in atherosclerosis, we generated mixed chimeras by lethally irradiating low density lipoprotein receptor deficient (Ldlr−/−) mice and reconstituting with a mixture of μMT and wildtype (control) or μMT and Gpr55−/− bone marrow cells. Circulating B cells were sorted and bulk RNA sequencing analysis was performed. We performed lipid and immunostainings of murine aortic root plaques, qPCR and ELISA of tissue lysates, as well as multiplex analysis of plasma immunoglobulins. Leukocyte plasma and tissue counts were determined by flow cytometry.
Results
GPR55 expression in mouse and human atherosclerotic plaques was detected by immunostaining. Furthermore, we confirmed murine Gpr55 mRNA expression on sorted circulating B220+B cells via qPCR, which was higher compared to CD3+ T cells, while CD11+ myeloid cells as well as NK cells had only low Gpr55 mRNA levels. ApoE−/−Gpr55−/− mice had significantly larger plaques after 4&16 weeks WD compared to ApoE−/− controls, with more pronounced body weight increases and higher cholesterol levels at the 16 weeks WD time point. In addition, global Gpr55 deficiency resulted in enhanced aortic pro-inflammatory cytokine mRNA expression (IL-1β, IL-6, TNFα) and a massively upregulated IgG1 plasma levels and increased percentages of splenic germinal center and plasma cells. B-cell RNA-seq analysis showed 460 differential expressed regulated genes in the ApoE−/−Gpr55−/− compared to ApoE−/−. The main pathways affected were calcium ion transport, immunoglobulin production, negative regulation of phosphorylation, and cellular component morphogenesis, suggesting a dsysregulation of B cell function. B cell specific Gpr55 deficiency blunted the metabolic effects on body weight and cholesterol, but still translated in larger atherosclerotic plaques and elevated plasma IgG levels compared to the respective controls.
Conclusion
Both global and B cell-restricted Gpr55 deficiency promotes atherosclerosis and is associated with a more pro-inflammatory phenotype. Our findings suggest a novel role for GPR55 in regulating B cell development and function.
Funding Acknowledgement
Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)
Increased estrogen to androgen ratio enhances immunoglobulin levels and impairs B cell function in male mice
