The pro-inflammatory phenotype of the human non-classical monocyte subset is attributed to senescence

Siew-Min Ong,Crystal Tze-Ying Tan,Wei-Hseun Yeap,Truong-Minh Dang,Eva Hadadi,Anis Larbi,Siew-Cheng Wong,Tze-Pin Ng

doi:10.1038/s41419-018-0327-1

Abstract

Human primary monocytes comprise a heterogeneous population that can be classified into three subsets based on CD14 and CD16 expression: classical (CD14high/CD16−), intermediate (CD14high/CD16+), and non-classical (CD14low/CD16+). The non-classical monocytes are the most pro-inflammatory in response to TLR stimulation in vitro, yet they express a remarkably high basal level of miR-146a, a microRNA known to negatively regulate the TLR pathway. This concurrence of a pro-inflammatory status and a high miR-146a level has been associated with cellular senescence in other cell types. Hence, we assessed the three monocyte subsets for evidence of senescence, including proliferative status, telomere length, cellular ROS levels, and mitochondrial membrane potential. Indeed, the non-classical subset exhibited the clearest hallmarks of senescence, followed by the intermediate and then the classical subset. In addition, the non-classical subset secreted pro-inflammatory cytokines basally in vitro. The highly pro-inflammatory nature of the non-classical monocytes could be a manifestation of the senescence-associated secretory phenotype (SASP), likely induced by a high basal NF-κB activity and IL-1α production. Finally, we observed an accumulation of the non-classical monocytes, in conjunction with higher levels of plasma TNF-α and IL-8, in the elderly. These factors may contribute to inflamm-aging and age-related inflammatory conditions, such as atherosclerosis and osteoarthritis. With our new understanding that the non-classical monocyte subset is a senescent population, we can now re-examine the role of this subset in disease conditions where this subset expands.

Highlights

Human primary monocytes comprise a heterogeneous population, of which 10-20% can be distinguished by their expression of surface antigen CD161
The high level of miR-146a expression in the non-classical monocytes does not inhibit their response to LPS We first enriched for monocytes from peripheral blood mononuclear cells (PBMCs) by depleting the lymphocytes using anti-CD15, anti-CD56, anti-CD3, and anti-CD19coated microbeads
The enriched monocytes were sorted by fluorescence-activated cell sorting (FACS) into three subsets: classical (“CL”; CD14high/CD16−), intermediate (“ITM”; CD14high/CD16+), and non-classical (“NC”; CD14low/CD16+) (Fig. 1a)

Summary

Introduction

Human primary monocytes comprise a heterogeneous population, of which 10-20% can be distinguished by their expression of surface antigen CD161. These CD16+ cells are known as the “inflammatory” subset due to their potent pro-inflammatory activity[2]. Our previous microRNA (miR) profiling study on monocyte subsets found the basal level of miR-146a to be significantly higher in CD16+ cells compared to CD16−cells[8]. The higher miR-146a level in the non-classical CD16+ monocytes is not consistent with the pro-inflammatory nature of these cells. These data suggest, that up-regulation of miR-146a may have other functions other than being a negative regulator

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Results

Conclusion