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

Abstract

Human primary monocytes comprise a heterogeneous population that can be classified into three subsets based on CD14 and CD16 expression: classical (CD14^high/CD16^-), intermediate (CD14^high/CD16⁺), and non-classical (CD14^low/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.

Figure 1. High microRNA-146a (miR-146a) level in non-classical monocytes does not limit the LPS response.

a Gating of enriched monocytes to generate the three monocyte subsets based on CD14 and CD16 expression. b Basal miR-146a expression in the three monocyte subsets. c TNF-α mRNA up-regulation in the three monocyte subsets, as an acute response to 2 h LPS stimulation. TNF-α mRNA level expressed as a fold change with respect to basal level before LPS stimulation. The data represent the means ± SD; n ≥ 3; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. CL: classical, ITM: intermediate, NC: non-classical

Figure 2. Non-classical monocytes are the least proliferative.

a Expression of Ki67 in total peripheral blood mononuclear cells. Ki67⁺ cells were gated using actively proliferating lymphocytes as a guide. b–c Percentage of Ki67⁺ cells in the three monocyte subsets. d Relative expression levels of Ki67 in the three monocyte subsets. e-f Relative telomere length in the three monocyte subsets. Each line represents one donor; n ≥ 3. *p < 0.05; ****p < 0.0001. CL: classical, ITM: intermediate, NC: non-classical, MFI: median fluorescence intensity

Figure 3. Non-classical monocytes exhibit several features of senescence.

a Relative total cellular ROS levels as measured using H₂DCFDA ROS indicator. b Relative mitochondrial ROS levels as measured using MitoSOX mitochondrial superoxide indicator. c–d Relative mitochondrial membrane potential (MMP) as measured using DIOC₆ and JC-1. e Relative expression levels of p-ERK. All the parameters were measured using flow cytometry. Each line represents one donor; n ≥ 3. *p < 0.05; **p < 0.01; ****p < 0.0001. CL: classical, ITM: intermediate, NC: non-classical, MFI: median fluorescence intensity, MMP: mitochondrial membrane potential, ROS: reactive oxygen species

Figure 4. Non-classical and intermediate monocytes secrete high levels of pro-inflammatory cytokines basally.

TNF-α, CCL3, CCL4, CCL5, IL-6, IL-8 and IL-1β secretion was analyzed by Luminex assay after overnight culture. The data represent the means ± SD; n = 3. *p < 0.05; **p < 0.01; ****p < 0.0001. CL: classical, ITM: intermediate, NC: non-classical

Figure 5. Plasma levels of cytokines correlate with non-classical monocyte count in the blood.

IL-8, CCL4 and CCL3 levels in the plasma was analyzed by Luminex assay, and correlated with the absolute number of non-classical monocytes present in a µL of whole blood. Each dot represents one donor; n = 20. NC: non-classical

Figure 6. Non-classical and intermediate monocytes express high levels of NF-κB (p65) and membrane-bound IL-1α.

a Western blot analysis of total p65 and GAPDH protein levels in the three monocyte subsets. b Quantification of Western blot data shown in (a): p65 protein level was normalized to GAPDH (loading control) and expressed as a fold change with respect to CL subset. The data represent the means ± SD; n = 3. c Relative levels of phosphorylated-p65 (p-p65), measured by flow cytometry. Each line represents one donor; n = 7. d IL-1α secretion by the three monocyte subsets, measured by Luminex assay. The data represent the means ± SD; n = 3. e–f Relative expression levels of membrane-bound IL-1α (e) and cytoplasmic IL-1α (f), analyzed by flow cytometry. Each line represents one donor; n = 9. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. CL: classical, ITM: intermediate, NC: non-classical, MFI: median fluorescence intensity

Figure 7. Classical monocytes can be induced to exhibit senescence-associated secretory phenotype following exposure to rh IL-1α.

Fold change increase, with respect to no treatment, in TNF-α, IL-6, and IL-8 secretion in the three monocyte subsets. The data represent the means ± SD; n = 3. CL: classical, ITM: intermediate, NC: non-classical, rh: recombinant human

Figure 8. The elderly has higher non-classical monocyte count and plasma level of SASP cytokines.

a Number of monocytes from each subset expressed as a percentage of the total number of monocytes. b Absolute number of monocytes from each subset present in a µL of whole blood. c TNF-α and IL-8 levels in the plasma, analyzed by Luminex assay. Each dot represents one donor; line represents median; (a–b) n = 30 each for both young and elderly individuals; (c) n = 20 for young, n = 30 for elderly; ***p < 0.001. CL: classical, ITM: intermediate, NC: non-classical