Background and Aims

TIR8/SIGIRR dampens the excessive activation mediated by ILRs and TLRs agonism and thus is a key regulator of inflammation. Aim of this study was to investigate the role of TIR8 in atherosclerosis.

Methods

8 weeks old-LDLR KO and TIR8/LDLR double KO (DKO) male mice were fed with standard diet (STD) or cholesterol-enriched diet (WTD) for 12 weeks. Plasma lipid profiling, extensive immunophenotyping and histological analysis of the atherosclerotic plaques were then performed.

Results

TIR8 deletion in STD-fed LDLR KO mice impacts circulating immune cell profile: decreased percentage of T lymphocytes (-29%, p<0.001) and increased percentage of B cells (+14%, p<0.05) were observed compared to LDLR KO mice, as well as increased mature Natural Killer cells (+13%, p<0.0001), as already described in the TIR8 KO mouse model. When fed a cholesterol rich diet for 12 weeks to induce atherosclerosis, in addition to changes observed on STD, also circulating levels of monocytes increased in DKO mice compared to LDL-R KO mice (mean 1464 vs 910 cells/ul, p<0.05). These changes in immune profile, however, did not affect atherosclerotic plaque area or stability. Similarly, no differences in plasma lipid profile were observed.

Conclusions

TIR8 deficiency in LDLR KO mice increases NKs and monocytes blood levels compared to LDL-R KO mice. Changes in these immune subsets, however, do not impact the development of atherosclerosis.

Background and Aims

Increased adiposity is associated with an increased risk of coronary artery disease (CAD). Whether adiposity has a direct causal effect on the risk of CAD, or whether the effect of adiposity on CAD is mediated indirectly through the effect of adiposity on other risk factors is unknown.

Methods

We constructed a body mass index (BMI) instrumental variable genetic score composed of 96 variants associated with BMI. We used this instrument to conduct a multivariable Mendelian randomization (MR) mediation analysis to estimate the direct and indirect effects of BMI on CAD. The study included individual level data on 445,774 participants including 23,032 cases of CAD and 28,563 cases of type II diabetes (T2D); and summary data on up to 877,643 participants including 63,746 cases of CAD and 26,676 cases of T2D.

Results

A one unit increase in BMI was associated with a 7% increased odds of CAD (OR:1.07; 95%CI:1.05-1.09, p=1.6E-10), a 25% increased odds of T2D (OR:1.25, 95%CI:1.23-1.28, p=2.2E-131); and 0.34 mmHg increase in systolic blood pressure (SBP) (95%CI:0.25-0.42, p=8.5E-15). A one unit increase in BMI was also associated with a 0.02 mmol/L increase in triglycerides (95%CI:0.02-0.03, p=4.9E-21), but not with increased apolipoprotein B levels. In MR mediation analyses, 80.0% of the effect of BMI on the risk of CAD was mediated through BMI induced increases in SBP (29.6%) and T2D risk (50.4%).

Conclusions

The vast majority of the effect of increased BMI on the risk of CAD appears to be mediated indirectly through the effect of BMI on SBP and T2D.

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

Increased lipoprotein receptor expression in the heart was associated with lipid accumulation, cardiomyocyte mitochondrial impairment and cardiac dysfunction. This project aimed at investigating whether PCSK9 regulates lipoprotein receptors expression in the heart, thus modulating heart metabolism and cardiac function.

Methods

Pcsk9 KO, Albumin CRE PCSK9LoxP/LoxP conditional KO) and double LDLr-Pcsk9 KO male mice undewent an extensive characterization for heart function followed by heart metabolomics and proteomic profiling.

Results

Pcsk9 KO hearts presented heart failure with preserved ejection fraction, reduced running resistance without muscular defects coupled to major adaptations in cardiac metabolism and mitochondrial functionality. This profile was associated with lipid accumulation in the heart. Moreover PCSK9 deficiency resulted in the accumulation of carnitine-conjugated fatty acid (C8; 0,066±0,047 Vs0,195±0,035 and C12; 0,021±0,018Vs0,11±0,069 pg/ug of prot, p<0,05) which was paralleled by a reduction of glucose 6-P, ribose-5P and erythrose-4P levels in the heart, suggesting a shift from fatty acid oxidation toward anaerobic glycolysis. Also Krebs cycle intermediates were reduced compared to WT samples. A similar phenotype was observed in LDLr Double KO confirming an effect independent of LDLr expression. The cardiac phenotype was reverted in the liver selective KO model thus excluding the involvement of circulating PCSK9 in the development of HFpEF observed in full KO mice. Translational studies showed that human subjects carrying the R46L loss of function polymorphism displayed increased left ventricular mass without alterations in ejection fraction compared to R46R BMI-matched controls.

Conclusions

These data suggest that PCSK9 deficiency impacts cardiac lipid metabolism indepedently of LDLR modulation and promotes the development of HFpEF.