Athero-immunometabolism Flashcards

Question

how have lactate carbons been shown to be used for fatty acid biosynthesis?

Answer 1

Carbon tracing analysis - Fed activated T cells with carbon-labelled lactate - Followed lactate carbons into biosynthesis of palmitate: these carbons are integrated into newly synthesised palmitate - When mAb to SLC5A12 is added, this prevents lactate uptake, which reduces palmitate biosynthesis - Direct evidence that lactate-derived carbons are used for biosynthesis of fatty acids

Answer 2

Palmitate is one of the most abundant lipids synthesised in cells

Answer 3

Unbiased lipidomics to look at lipids generated via lactate-feeding of CD4 T cells - When T cells are fed lactate, they abundantly form lipid droplets compared to control - Lipid droplets have been rediscovered as immune hubs – they are key for biosynthesis of cytokines in immune cells - T cells from slc5a12 K/O mouse have reduced formation of lipid droplets – proves that lactate uptake and lactate carbons is needed for biosynthesis for lipid

Answer 4

Atherosclerosis involves build up of immune cells - CD4 T cells are key in atherosclerosis, not just macrophages - Atheroma is a hypoxic niche with lactate accumulation – relevant to T cell function

Answer 5

Adenovirus-8 that encodes PCSK9 to model atherosclerosis - Usually AS model uses background of APOE or LDL receptor K/O mice on western/high fat diet - develop atheroma - To study impact of gene on AS, need to back cross K/O mice into APOE/LDLR – long and expensive process

Answer 6

Now cheaper and quicker method: don’t need background – take SLC5A12 K/O (CRISPR-Cas9) and inject with AAV8 encoding PCSK9 - This adenovirus has tropism for hepatocytes – the hepatocytes pick up the virus and produce PCSK9 - PCSK9 degrades LCL receptor in hepatocytes - generates conditional K/O of LDL receptor in the liver - Put mouse on high fat diet – over 12 weeks they develop AS

Answer 7

AS plaques develop in aortic arches of WT mice – pink staining for AS plaques – high deposition of immune cells - In SLC K/O mice, there are fewer AS plaques, and less dense area of plaques (reduced pink staining = smaller atheroma) – area is smaller Within the heart, plaques within the sinuses are not different between the WT and K/O mice - No immune cell deposition Suggests that plaques and effects are mediated by immune cells deficient in SLC5A12

Answer 8

PCR analysis: IL-17 and IL-21 are reduced in SLC5A12 K/O, while foxp3 is upregulated

Answer 9

Looked at 2 cohorts of AS patients – compare healthy vessels vs AS vessels In both cohorts, SLC5A12 is upregulated in disease, whereas common MCTs such as MCT4, MCT1 are not upregulated - SLC5A12 expression is in the tissue, not in the blood

Answer 10

Therapy against SLC5A12 will therefore unlikely have systemic effects – will have effects in the diseased tissue and avoids systemic cytokine release like TNF, IL-1 – means reduced side effects

Answer 11

Looked at cross sections of coronary arteries from explanted hearts of heart transplant AS patients -- 3 patients’ cross-sections of same artery at same position - Patients’ cross-sections scored as mild, moderate and severe – can see progression of disease - Mild patient near normal arteries – vessel is mostly normal, and mild where vessel wall has slightly thickened - In overall moderate disease, can see shoulder of plaque - In severe disease – 2 coronary arteries are almost completely occluded Across the 9 cross-sections in 3 artery, can see whole history of AS progression Usually we can only study the carotid artery at end-stage disease – loss of vessel wall and complete occlusion – can’t see progression

Answer 12

Immunostained for CD3 - CD3+ cells present as immune aggregates in core lumen of plaque and in adventitia of severe lesion Stained for SLC5A12 - Can see some T cells in both plaque and adventitia are positive for SLC5A12 In severe lesions, immune cells accumulate into clusters in adventitia - Forms ectopic lymphoid structure – TLS formed in human plaque

Answer 13

Nanostring platform to perform spatial transcriptomics - Can get gene expression of cells in an anatomical fashion – tissue structure is mainatained and doesn’t need to be resuspended in solution - Can know the gene expression of different cells at different sites in tissue - Looks at gene expression in pool of hundreds of cells

Answer 14

Stained tissue for CD45 – markers immune cells, and then stained for CD4 - Regions of interest were either CD45+CD4-poor or CD45+CD4-rich – discriminates signals of CD4 from other immune cells Circles are ectopic lymphoid structures - 2 regions of interest where there is clustering of immune cells in that region - One area is CD4 rich, the other is CD4 poor - Can distinguish the gene expression in that area

Answer 15

Unsupervised clustering of genes found in geoMx - Applied whole human transcriptome (18000 transcripts) - On left = gene expression of CD4 T cells in adventitia of severe lesions - Right = gene expression of CD4 T cells in adventitia of mild lesions - The gene expression profiles are opposites in severe and mild lesions, indicating T cells contribute to the disease progression - Middle = CD4 T cells in plaque of severe lesions – mixed phenotype between severe and mild extremes – maybe more similar to CD4 in adventitia of severe lesions

Answer 16

Looked at genes of interest e.g. SLC5A12 in regions of interest of plaque and adventitia - muscle of tissue was negative control – negative CD45 and CD4 expression – low expression of SLC5A12 in muscle I- n mild, moderate and severe, SLC5A12 is upregulated in adventitia of more early-stage disease than in late-stage plaques - SLC5A12 may be gatekeeper of disease

Answer 17

Genes that are downstream of SLC5A12 e.g. FASN, STAT3 - In an unbiased way, they show similar trends to SLC5A12 expression - Whereas IL-21, which is enriched in severe disease, is highly expressed in moderate and severe disease - HSD3B2 is upregulated in CD4 cells in the adventitia severe disease

Answer 18

Pathway analysis of gene enrichment of volcano plot - Many CD4-specific pathways are differentially upregulated in adventitia of mild lesions and not in severe - Suggests that CD4 T cells change phenotype to stem-like phenotype – still not understood - Identified 26 relevant pathways significantly regulated, including translational elongation and complement cascade

Answer 19

Complement can be an IC mechanism in immune cells, controlling inflammasome and cytokine production - Suggests that complement proteins are upregulated in CD4 T cells in mild lesions to protect from progression of disease

Answer 20

HSD3B2 is upregulated in severe disease - HSD underpins metabolism of lipids – key in AS disease of lipids

Answer 21

Volcano plot comparing CD45 cells in adventitia vs plaque (not including severity factor) - Number of genes differentially regulated - Specifically PLA2G2A is differentially regulated in adventitia - PLA2G2A follows same pattern of SLC5A12 - But the regulation spans across 3 logs – huge regulation - PLA2G2A encodes a phospholipase – not sure how its involved

Answer 22

In AS - There is big involvement of adaptive immunity: Th1, Th17, Tregs - These are involved in formation of ectopic lymphoid structures - In AS, it is debated whether TLS are good or bad for disease – not clear - Lactate build up in anatomical area can contribute to disease - Can we reduce lactate sensing to resolve disease? Using K/O mice, mAbs, acetylation, lactylation, lipidomics to understand how lactate is used in this system