p16ink4a mRNA and protein levels were measured in classically differentiated and in alternatively differentiated (by IL4) human monocyte-derived macrophages. To explore the role of p16ink4a, its levels were lowered by RNA interference, after which macrophage phenotype was analyzed. In addition, p16ink4a expression levels were compared between ATMs and monocyte-derived macrophages isolated from obese patients.
p16ink4a was induced during classical macrophage differentiation. Interestingly, alternative differentiation inhibited p16ink4a expression. Silencing p16ink4a 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.
This phenotype closely resembles the one of adipose tissue macrophages (ATMs). Confirming the inverse correlation of p16ink4a expression levels with an ATM phenotype, we found that in ATMs from obese patients, p16ink4a levels were lower than in monocyte-derived macrophages of the same subjects.
Suppression of p16ink4a in human macrophages leads to a phenotype resembling that of ATMs. P16ink4a may thus play a role in ATM development and function.