Genetic variant in 3' untranslated region of the mouse pycard gene regulates inflammasome activity.

Quantitative trait locus mapping for interleukin-1ß release after inflammasome priming and activation was performed on bone-marrow-derived macrophages (BMDM) from an AKRxDBA/2 mouse strain intercross. The strongest associated locus mapped very close to the Pycard gene on chromosome 7, which codes for the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). The DBA/2 and AKR Pycard genes only differ at a single-nucleotide polymorphism (SNP) in their 3' untranslated region (UTR). DBA/2 vs. AKR BMDM had increased levels of Pycard mRNA expression and ASC protein, and increased inflammasome speck formation, which was associated with increased Pycard mRNA stability without an increased transcription rate. CRISPR/Cas9 gene editing was performed on DBA/2 embryonic stem cells to change the Pycard 3'UTR SNP from the DBA/2 to the AKR allele. This single base change significantly reduced Pycard expression and inflammasome activity after cells were differentiated into macrophages due to reduced Pycard mRNA stability.

Quantitative trait locus mapping identifies the Gpnmb gene as a modifier of mouse macrophage lysosome function.

We have previously shown that the DBA/2J versus AKR/J mouse strain is associated with decreased autophagy-mediated lysosomal hydrolysis of cholesterol esters. Our objective was to determine differences in lysosome function in AKR/J and DBA/2J macrophages, and identify the responsible genes. Using a novel dual-labeled indicator of lysosome function, DBA/2J versus AKR/J bone marrow derived macrophages had significantly decreased lysosome function. We performed quantitative trait loci mapping of lysosome function in bone marrow macrophages from an AKR/J × DBA/2J strain intercross. Four distinct lysosome function loci were identified, which we named macrophage lysosome function modifier (Mlfm) Mlfm1 through Mlfm4. The strongest locus Mlfm1 harbors the Gpnmb gene, which has been shown to recruit autophagy protein light chain 3 to autophagosomes for lysosome fusion. The parental DBA/2J strain has a nonsense variant in Gpnmb. siRNA knockdown of Gpnmb in AKR/J macrophages decreased lysosome function, and Gpnmb deletion through CRISP/Cas9 editing in RAW 264.7 mouse macrophages also demonstrated a similar result. Furthermore, a DBA/2 substrain, called DBA/2J-Gpnmb+/SjJ, contains the wildtype Gpnmb gene, and macrophages from this Gpnmb-preserved DBA/2 substrain exhibited recovered lysosome function. In conclusion, we identified Gpnmb as a causal modifier gene of lysosome function in this strain pair.

Fine Mapping of the Mouse Ath28 Locus Yields Three Atherosclerosis Modifying Sub-Regions.

A mouse strain intercross between Apoe-/- AKR/J and DBA/2J mice identified three replicated atherosclerosis quantitative trait loci (QTLs). Our objective was to fine map mouse atherosclerosis modifier genes within a genomic region known to affect lesion development in apoE-deficient (Apoe-/-) mice. We dissected the Ath28 QTL on the distal end of chromosome 2 by breeding a panel of congenic strains and measuring aortic root lesion area in 16-week-old male and female mice fed regular laboratory diets. The parental congenic strain contained ~9.65 Mb of AKR/J DNA from chromosome 2 on the DBA/2J genetic background, which had lesions 55% and 47% smaller than female and male DBA/2J mice, respectively (p < 0.001). Seven additional congenic lines identified three separate regions associated with the lesion area, named Ath28.1, Ath28.2, and Ath28.3, where the AKR/J alleles were atherosclerosis-protective for two regions and atherosclerosis-promoting for the other region. These results were replicated in both sexes, and in combined analysis after adjusting for sex. The congenic lines did not greatly impact total and HDL cholesterol levels or body weight. Bioinformatic analyses identified all coding and non-coding genes in the Ath28.1 sub-region, as well as strain sequence differences that may be impactful. Even within a <10 Mb region of the mouse genome, evidence supports the presence of at least three atherosclerosis modifier genes that differ between the AKR/J and DBA/2J mouse strains, supporting the polygenic nature of atherosclerosis susceptibility.

Confirmation of Ath26 locus on chromosome 17 and identification of Cyp4f13 as an atherosclerosis modifying gene.

BACKGROUND AND AIMS: We previously demonstrated that Apoe-/- mice on DBA/2 vs. AKR genetic background have >10-fold larger atherosclerotic lesions. Prior quantitative trait locus mapping via strain intercrossing identified a region on chromosome 17, Ath26, as the strongest atherosclerosis-modifying locus. We aimed to confirm Ath26, identify candidate genes, and validate the candidate gene effects on atherosclerosis. METHODS: We bred chromosome 17 interval congenic mice to confirm that Ath26 locus contains atherosclerosis modifying gene(s). Bone marrow derived macrophage transcriptomics was performed to identify candidate genes at this locus whose expression was correlated with lesions in a strain intercross. The Cyp4f13 candidate gene was tested via a gene knockout approach and in vivo and ex vivo phenotype analyses. RESULTS: A congenic mouse strain containing the DBA/2 interval on chromosome 17 on the AKR Apoe-/- background demonstrated that this interval conferred increased lesion area. Transcriptomic analysis of bone marrow macrophages identified that expression of the Cyp4f13 gene, mapping to this locus, was highly associated with lesion area in an F2 cohort. AKR vs. DBA/2 macrophages had less Cyp4f13 mRNA expression, and their livers had lower leukotriene B4 (LTB4) 20-hydroxylase enzymatic activity. A Cyp4f13 knockout allele was bred onto the DBA/2 Apoe-/- background and this conferred less enzymatic activity, decreased macrophage migration in response to LTB4, and smaller aortic root atherosclerotic lesions. CONCLUSIONS: Allelic differences in the Cyp4f13 gene may in part be responsible for the Ath26 QTL conferring larger lesions in DBA/2 vs. AKR Apoe-/- mice.

Quantitative Trait Locus Mapping of Macrophage Cholesterol Metabolism and CRISPR/Cas9 Editing Implicate an ACAT1 Truncation as a Causal Modifier Variant.

OBJECTIVE: Cholesterol metabolism is a dynamic process involving intracellular trafficking, cholesterol esterification, and cholesterol ester hydrolysis. Our objective was to identify genes that regulate macrophage cholesterol metabolism. APPROACHES AND RESULTS: We performed quantitative trait loci mapping of free and esterified cholesterol levels and the ratio of esterified to free cholesterol in acetylated low-density lipoprotein-loaded bone marrow-derived macrophages from an AKR×DBA/2 strain intercross. Ten distinct cholesterol modifier loci were identified, and bioinformatics was used to prioritize candidate genes. The strongest locus was located on distal chromosome 1, which we named Mcmm1 (macrophage cholesterol metabolism modifier 1). This locus harbors the Soat1 (sterol O-acyltransferase 1) gene, encoding Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), which esterifies free cholesterol. The parental AKR strain has an exon 2 deletion in Soat1, which leads to a 33 amino acid N-terminal truncation in ACAT1. CRISPR/Cas9 editing of DBA/2 embryonic stem cells was performed to replicate the AKR strain Soat1 exon 2 deletion, while leaving the remainder of the genome unaltered. DBA/2 stem cells and stem cells heterozygous and homozygous for the Soat1 exon 2 deletion were differentiated into macrophages and loaded with acetylated low-density lipoprotein. DBA/2 stem cell-derived macrophages accumulated less free cholesterol and more esterified cholesterol relative to cells heterozygous and homozygous for the Soat1 exon 2 deletion. CONCLUSIONS: A Soat1 deletion present in AKR mice, and resultant N-terminal ACAT1 truncation, was confirmed to be a significant modifier of macrophage cholesterol metabolism. Other Mcmm loci candidate genes were prioritized via bioinformatics.

Physiological difference in autophagic flux in macrophages from 2 mouse strains regulates cholesterol ester metabolism.

OBJECTIVE: DBA/2 apoE(-/-) mice have ≈10-fold larger lesions than AKR apoE(-/-) mice. The objective of this study was to determine whether macrophages from these 2 strains had altered cholesterol metabolism that might play a role in their divergent atherosclerosis susceptibility. APPROACH AND RESULTS: AKR and DBA/2 macrophages incubated with acetylated low-density lipoprotein resulted in higher cholesterol ester (CE) and lower free cholesterol accumulation in the DBA/2 cells. However, these strains had equivalent acetylated low-density lipoprotein uptake and cholesterol esterification activity. Cholesterol efflux from unloaded cells to apolipoprotein A-I or high-density lipoprotein was similar in the 2 strains. However, on acetylated low-density lipoprotein loading, cholesterol efflux was impaired in the DBA/2 cells, but this impairment was corrected by loading in the presence of an inhibitor of cholesterol esterification. Thus, the cholesterol efflux capabilities are similar in these strains, but there seemed to be a defect in lipid droplet-stored CE mobilization in DBA/2 cells. Lalistat 1, a specific inhibitor of lysosomal acid lipase, completely blocked the hydrolysis of lipid droplet-stored CE, implying that lipid droplet autophagy is responsible for CE turnover in these cells. CE turnover was 2-fold slower in DBA/2 versus AKR cells. Autophagic flux, estimated by a fluorescent light chain 3-II reporter and the increase in p62 levels after chloroquine treatment, was higher in AKR versus DBA/2 macrophages, which had an apparent decrease in autophagosome fusion with lysosomes. When autophagy was activated by amino acid starvation, CE levels decreased in DBA/2 cells. CONCLUSIONS: Physiological regulation of autophagy in macrophages controls CE accumulation and may modify atherosclerosis susceptibility.

Red blood cells play a role in reverse cholesterol transport.

OBJECTIVE: Reverse cholesterol transport (RCT) involves the removal of cholesterol from peripheral tissue for excretion in the feces. Here, we determined whether red blood cells (RBCs) can contribute to RCT. METHODS AND RESULTS: We performed a series of studies in apolipoprotein AI-deficient mice where the high-density lipoprotein-mediated pathway of RCT is greatly diminished. RBCs carried a higher fraction of whole blood cholesterol than plasma in apolipoprotein AI-deficient mice, and as least as much of the labeled cholesterol derived from injected foam cells appeared in RBCs compared with plasma. To determine whether RBCs mediate RCT to the fecal compartment, we measured RCT in anemic and control apolipoprotein AI-deficient mice and found that anemia decreased RCT to the feces by over 35% after correcting for fecal mass. Transfusion of [(3)H]cholesterol-labeled RBCs led to robust delivery of the labeled cholesterol to the feces in apolipoprotein AI-deficient hosts. In wild-type mice, the majority of the blood cholesterol mass, as well as [(3)H]cholesterol derived from the injected foam cells, was found in plasma, and anemia did not significantly alter RCT to the feces after correction for fecal mass. CONCLUSIONS: The RBC cholesterol pool is dynamic and facilitates RCT of peripheral cholesterol to the feces, particularly in the low high-density lipoprotein state.