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Cells often adopt different phenotypes, dictated by tissue-specific or local signals such as cell-cell and cell-matrix contacts or molecular micro-environment. This holds in extremis for macrophages with their high phenotypic plasticity. Their broad range of functions, some even opposing, reflects their heterogeneity, and a multitude of subsets has been described in different tissues and diseases. Such micro-environmental imprint cannot be adequately studied by single-cell applications as cells are detached from their context, while histology-based assessment lacks the phenotypic depth due to limitations in marker combination. Here, we present a novel, integrative approach in which 15-color multispectral imaging allows comprehensive cell classification based on multi-marker expression patterns, followed by downstream analysis pipelines to link their phenotypes to contextual, micro-environmental cues such as their cellular (“community”) and metabolic (“local lipidome”) niches in complex tissue. The power of this approach is illustrated for myeloid subsets and associated lipid signatures in murine atherosclerotic plaque.

Genome-wide association studies (GWASs) provide an unprecedented opportunity to examine, on a large scale, the association of common genetic variants with complex diseases like type 2 diabetes (T2D) and cardiovascular disease (CVD), thus allowing the identification of new potential disease loci. Using this approach, numerous studies have associated SNPs on chromosome 9p21.3 situated near the cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) locus with the risk for coronary artery disease (CAD) and T2D. However, identifying the function of the nearby gene products (CDKN2A/B and ANRIL) in the pathophysiology of these conditions requires functional genomic studies. We review the current knowledge, from studies using human and mouse models, describing the function of CDKN2A/B gene products, which may mechanistically link the 9p21.3 risk locus with CVD and diabetes.

Background/Aims The molecular mechanisms leading to Non-Alcoholic Steatohepatitis (NASH) are not fully understood. In mice, NASH can be inhibited by fenofibrate, a synthetic agonist for the nuclear receptor peroxisome proliferator activated receptor alpha, which regulates hepatic triglyceride metabolism. This study aimed to elucidate the relation between steatosis and inflammation in NASH in a human-like hyperlipidemic mouse model. Methods Liver phenotype and gene expression were assessed in APOE2 knock-in mice that were fed a western-type high fat diet with or without co-administration of fenofibrate. Results In response to a western diet, APOE2 knock-in mice developed NASH characterized by steatosis and inflammation. Strikingly, macrophage accumulation in the liver preceded the steatosis during progression of the disease. This phenotype was in line with gene expression patterns, which showed regulation of two major groups of genes, i.e. inflammatory and lipid genes. Fenofibrate treatment decreased hepatic macrophage accumulation and abolished steatosis. Moreover, a marked reduction in the expression of inflammatory genes occurred immediately after fenofibrate treatment. Conclusions These data indicate that inflammation might play an instrumental role during the development of NASH in this mouse model. Inhibition of NASH by fenofibrate may be due, at least in part, to its inhibitory effect on pro-inflammatory genes.

Introduction Des analyses d'association de genes montrent que le locus CDKN2A/B qui code notamment la proteine p16INK4a, pourrait etre associe au developpement des maladies coronariennes, de l'atherosclerose et du diabete de type 2. Ces pathologies sont associees a l'expansion et a l'inflammation de differents depots de tissu adipeux (TA), notamment le tissu adipeux perivasculaire (PVAT). p16INK4a est un regulateur du cycle cellulaire cependant sa fonction dans l'adipogenese reste encore inconnue. Materiels et methodes Dans cette etude, nous avons etudie le role de p16INK4a dans l'adipogenese in vitro en utilisant des preadipocytes 3T3L1 transfectes par un siRNA-CDKN2A ou des fibroblastes embryonnaires (MEF) isoles des souris p16+/+ et p16−/−, et in vivo, chez l'homme, en etudiant l'expression de p16INK4a dans le PVAT, et chez la souris, en etudiant le developpement des depots de TA induit par un traitement Rosiglitazone. Le role de p16INK4a dans le developpement du PVAT a partir de la moelle osseuse a ete etudie chez des souris chimeres p16−/−LDLRKO et p16+/+LDLRKO soumises a un regime western. Resultats La diminution de l'expression de p16INK4a dans les pre-adipocytes 3T3L1 augmente l'adipogenese, mesuree par une augmentation de l'expression de PPARgamma, adiponectine, perilipine et CEBPalpha et une accumulation de lipides, sans affecter l'expansion clonale. Des resultats similaires ont ete obtenus dans les MEF p16−/−. Chez l'homme, p16INK4a est fortement exprime dans le PVAT cardiaque compare aux autres depots de TA. Chez la souris, bien que la deficience de p16INK4a n'influence pas le developpement des differents depots de TA, le traitement des souris p16−/− par la Rosiglitazone augmente specifiquement le developpement du PVAT, associe a une augmentation de l'expression de marqueurs de cellules precurseurs des adipocytes de la moelle. La deficience de p16INK4a dans la moelle osseuse de souris chimeres p16−/− LDLRKO augmente le developpement du PVAT induit par un regime western. Conclusion L'ensemble de ces donnees demontre un nouveau role de p16INK4a dans l'adipogenese et le developpement du PVAT.

Rationnel Recently, it has been shown that the genomic locus CDKN2A, which partially codes for p16 ink4a , is associated with the development of type 2 diabetes (T2D) in several populations. P16 ink4a is a tumour suppressor that controls the cell cycle and cellular senescence. Macrophages are known to play an instrumental role during the development of T2D. They can present different polarization states : classical (M1 : pro-inflammatory) or alternative (M2 : anti-inflammatory). We therefore aim to investigate the role of p16 ink4a during differentiation and polarization of human monocyte-derived macrophages. Materiels et Methodes p16 ink4a mRNA and protein levels were measured in classically differentiated and in alternatively differentiated (by IL4) human monocyte-derived macrophages. To explore the role of p16 ink4a , its levels were lowered by RNA interference, after which macrophage phenotype was analyzed. In addition, p16 ink4a expression levels were compared between ATMs and monocyte-derived macrophages isolated from obese patients. Resultats p16 ink4a was induced during classical macrophage differentiation. Interestingly, alternative differentiation inhibited p16 ink4a expression. Silencing p16 ink4a resulted in an increase of a number of M2 marker genes (MR, AMAC1, TGFbeta, IL1Ra, IL10, MMP2, ItgB). Surprisingly, despite this M2 phenotype, these cells had increased responses to LPS, shown by amplified expression of pro-inflammatory markers as Cox2, Mcp1, and TNF, possibly as the result from an increase in Tlr4 expression. Additionally, these cells secreted higher levels of TNF protein. Discussion This phenotype closely resembles the one of adipose tissue macrophages (ATMs). Confirming the inverse correlation of p16 ink4a expression levels with an ATM phenotype, we found that in ATMs from obese patients, p16 ink4a levels were lower than in monocyte-derived macrophages of the same subjects. Conclusion Suppression of p16 ink4a in human macrophages leads to a phenotype resembling that of ATMs. P16 ink4a may thus play a role in ATM development and function.