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.

Miltefosine increases macrophage cholesterol release and inhibits NLRP3-inflammasome assembly and IL-1ß release.

Miltefosine is an FDA approved oral drug for treating cutaneous and visceral leishmaniasis. Leishmania is a flagellated protozoa, which infects and differentiates in macrophages. Here, we studied the effects of Miltefosine on macrophage's lipid homeostasis, autophagy, and NLRP3 inflammasome assembly/activity. Miltefosine treatment conferred multiple effects on macrophage lipid homeostasis leading to increased cholesterol release from cells, increased lipid-raft disruption, decreased phosphatidylserine (PS) flip from the cell-surface, and redistribution of phosphatidylinositol 4,5-bisphosphate (PIP2) from the plasma membrane to actin rich regions in the cells. Enhanced basal autophagy, lipophagy and mitophagy was observed in cells treated with Miltefosine vs. control. Miltefosine treated cells showed marked increased in phosphorylation of kinases involved in autophagy induction such as; Adenosine monophosphate-activated protein kinase (AMPK) and Unc-51 like autophagy activating kinase (ULK1). The Toll like receptor (TLR) signaling pathway was blunted by Miltefosine treatment, resulting in decreased TLR4 recruitment to cell-surface and ~75% reduction in LPS induced pro-IL-1ß mRNA levels. Miltefosine reduced endotoxin-mediated mitochondrial reactive oxygen species and protected the mitochondrial membrane potential. Miltefosine treatment induced mitophagy and dampened NLRP3 inflammasome assembly. Collectively, our data shows that Miltefosine induced ABCA1 mediated cholesterol release, induced AMPK phosphorylation and mitophagy, while dampening NLRP3 inflammasome assembly and IL-1ß release.