Background and Aims

Activation of T lymphocytes combines functional to metabolic rewiring of cell machinery, including cholesterol homeostasis. Here we evaluated the role of LDLR on T cell biology

Methods

Immunophenotypic characterization of CD8T cells from WT and LDLR KO mice was performed in vitro (anti-CD3/CD28) and in vivo (vaccination, homeostatic proliferation) coupled to proteomic and confocal analysis on isolated CD8T cells. In parallel, T cells from FH were tested.

Results

LDLR deficiency dampened CD8+ proliferation (-35%, p<0.01) paralleled by a reduction in INFγ production (-39.6%, p<0.01). In vivo antigen-specific activation by ovalbumin vaccination, but not homeostatic proliferation, resulted in a decreased proliferation and cytokines production (↓IFNγ p<0.001,↓IL13 p<0.01,↓perforin p<0.05) in CD8+ of KO mice. Incubation with LDL significantly increased the proliferation in WT but not KO CD8T cells (+11%,p<0.01), a phenotype that was compensated by a reprogramming of de-novo synthesis in KO CD8T cells. Proteomic analysis revealed an impairment in glycolysis and OXPHOS associated to downregulation of pathways downstream to mTORC1 activation, possibly link to its reduced lysosomal localization observed in KO CD8T cells.

CD8+ T cells from FH subjects proliferated less (-36%, p>0.05) compared to sex- and age-matched controls and presented a decreased granzyme production (-60.3%, p<0.01) when CD8 memory response was tested in vitro with virus-derived peptides in seasonal influenza vaccinated matched controls and FH subjects.

Conclusions

LDLR plays a critical role in regulating the immunometabolic responses in CD8+ Tcells, and thus might represent a checkpoint linking cellular cholesterol metabolism to adaptive immune response.

Background and Aims

Chronic inflammation is a hallmark of atherosclerosis and results from an imbalance between pro-inflammatory and pro-resolving signaling. The human GPR32 receptor serves in tandem with the ALX/FPR2 receptor to transduce biological actions of pro-resolving mediators that stimulate resolution of inflammation. However, since no murine homologs of the human GPR32 exist and comprehensive in vivo studies are lacking, we generated a novel transgenic mouse model expressing human GPR32 while lacking Fpr2 on a hyperlipidemic background to investigate the role of GPR32 in vivo.

Methods

Human atherosclerotic lesions from carotid endarterectomies and normal controls were used for mRNA microarray analysis and immunofluoresence staining. A novel transgenic mouse model expressing human GPR32 on a Fpr2×apolipoprotein E double KO background (hGPR32_myc×Fpr2^-/-×ApoE^-/-) was generated. Mice were either subjected to HFD induced atherosclerosis or zymosan peritonitis, and organs collected for cellular and biochemical analysis, e.g. RT-qPCR, immunohistochemistry and fow cytometry.

Results

GPR32 mRNA was reduced in human atherosclerotic lesions and correlated with immune cell markers Arg1, iNOS and FoxP3. Atherosclerotic lesions and aortic inflammation were reduced in hGPR32_mycTg×Fpr2^-/-×ApoE^-/- transgenic mice as compared to Fpr2^-/-×ApoE^-/- non-transgenic littermates. In a zymosan induced peritonitis model, the hGPR32_mycTg×Fpr2^-/-×ApoE^-/- transgenic mice had ~55% less exudate neutrophils at 4h and enhanced pro-resolving macrophage responses at 24h. Treatment with the GPR32 agonist aspirin-triggered resolvin D1 (AT-RvD1) reduced neutrophil numbers in hGPR32_mycTg×Fpr2^-/-×ApoE^-/- transgenic mice but not in the Fpr2^-/-×ApoE^-/- non-transgenic littermates.

Conclusions

Altogether these results provide the first evidence that GPR32 regulates resolution of inflammation and is atheroprotective in vivo.

Background and Aims

Macrophages are key regulators of inflammatory responses in atherosclerosis. Since epigenetic processes are important in controlling macrophage function, interfering with epigenetic pathways might be a novel approach to combat atherosclerosis. Histone H3K27 trimethylation is a repressive histone mark catalyzed by the Polycomb Repressive Complex 2 (PRC2) with EZH2 as the catalytic subunit. We previously showed that myeloid deletion of Kdm6b, an enzyme that in contrast to PRC2 removes repressive H3K27me3 marks, results in advanced atherosclerosis. Because of its opposing function, we here studied macrophage EZH2 and JARID2, both part of the PRC2 complex, in macrophage activation and atherosclerosis.

Methods

Myeloid-specific Ezh2 (Ezh2^del) and Jarid2 (Jarid2^del) deficient mice strains were generated (LysM-Cre+ x Ezh2^fl/fl or Jarid2^fl/fl) and bone marrow from Ezh2^del or Ezh2^wt mice was transplanted to Ldlr^-/- mice which were fed a high fat diet for 9 weeks to study atherosclerosis.

Results

Atherosclerotic lesion size was significantly decreased in Ezh2^del transplanted mice compared to control. The percentage of macrophages in the lesions was similar. However neutrophil numbers were lower in Ezh2^del transplanted mice. Correspondingly, the migratory capacity of neutrophils was decreased. Moreover, peritoneal Ezh2^del foam cells showed a reduction in the inflammatory response with reduced production of IL-6, IL-12 and NO. Currently, we are investigating the effects of JARID2 as important co-factor for the PRC2 complex on macrophage inflammatory responses by use of the Jarid2^del mouse strain and siRNA’s against JARID2 in human macrophages.

Conclusions

Myeloid Ezh2 deficiency impairs neutrophil migration and reduces macrophage foam cell inflammatory responses, both contributing to reduced atherosclerosis.

Background and Aims

DOT1L is the only histone methyltransferase for H3K79 and has recently emerged as a central player in the immune system.

Methods

Here we investigate the role of DOT1L in macrophages by application of a selective DOT1L inhibitor on either mouse or human macrophages and using myeloid-specific Dot1l deleted mice. Furthermore, we investigated myeloid Dot1l in vivo in a mouse model for atherosclerosis including scRNAseq on atherosclerotic plaques.

Results

Using RNA-seq and in vitro assays, we found that Dot1l represses macrophage activation and impacts cellular lipid metabolism. ChIP-seq for H3K79me revealed that DOT1L regulates H3K79 methylation of RXRα in macrophages, leading to reduced RXRα mRNA and protein expression, both upon Dot1l deletion as well as DOT1L inhibition. Given the established role for RXRα in controlling macrophage activation and lipid homeostasis, the phenotype we observe can thus be at least partly explained by impaired RXRα signaling. Moreover, we could induce a similar suppressed lipid phenotype using an RXRα antagonist in wild-type macrophages.

In vivo, we confirmed that myeloid Dot1l deletion increases the activation of plaque macrophages. Moreover, although plaque area was not affected, Dot1l deficiency led to enhanced necrosis, indicating potential plaque destabilization. Apart from these direct effects, we found that myeloid Dot1l also affects other immune cell subsets illustrated by reduced production of IgG and IgM antibodies targeting atherosclerosis-related antigens.

Conclusions

Our data show that myeloid DOT1L is a critical regulator of macrophage inflammatory responses and lipid homeostasis and that it impacts in vivo immune responses and atherosclerosis development.

Background and Aims

Aim: In humans, very high plasma HDL-cholesterol concentrations are associated with increased all cause- and atherosclerotic cardiovascular disease (ASCVD)-associated death. The HDL receptor-deficient mouse (Scarb1^-/-), a robust model of this phenotype, is characterized by high free cholesterol (FC) bioavailability due to too many HDL particles that are FC-rich. Clinically, plasma LDL and HDL are quantified according to their total cholesterol content, the sum of FC and esterified cholesterol, which likely contribute to ASCVD pathophysiology differently. A Western diet induces ASCVD in Scarb1^-/- mice, despite an attendant increase in HDL, the so-called “good cholesterol.” We tested the hypothesis that high HDL-FC bioavailability contributes to ASCVD in Scarb1^-/- mice by increasing FC flux into macrophage cells and erythrocytes.

Methods

Methods: Influx of HDL-FC and efflux of macrophage FC were determined between WT and SCARB1^-/- HDL and J774 macrophage cells. HDL of both genotypes were radiolabelled with [³H]FC, injected into autologous mice, and the rates of plasma clearance and erythrocyte uptake were determined.

Results

Results: Compared to WT mice, in Scarb1^-/- mice, autologous HDL-FC cleared faster and more HDL-FC transferred to macrophages. The FC/phospholipid ratios of HDL, LDL, erythrocytes, as well as some tissues, were higher. The high FC/phospholipid ratios of ovaries, erythrocytes, heart, and macrophages in response to the higher HDL-FC bioavailability in Scarb1^-/- mice is associated respectively with female infertility, impaired cell maturation, cardiac dysfunction, and ASCVD.

Conclusions

Conclusion: These findings provide a rationale for human studies to determine the utility of HDL-FC bioavailability as a risk factor for ASCVD and other pathologies.