RNY (YRNA)-derived small RNAs regulate cell death and inflammation in monocytes/macrophages

Abstract

The recent discovery of new classes of small RNAs has opened unknown territories to explore new regulations of physiopathological events. We have recently demonstrated that RNY (or Y RNA)-derived small RNAs (referred to as s-RNYs) are an independent class of clinical biomarkers to detect coronary artery lesions and are associated with atherosclerosis burden. Here, we have studied the role of s-RNYs in human and mouse monocytes/macrophages and have shown that in lipid-laden monocytes/macrophages s-RNY expression is timely correlated to the activation of both NF-κB and caspase 3-dependent cell death pathways. Loss- or gain-of-function experiments demonstrated that s-RNYs activate caspase 3 and NF-κB signaling pathways ultimately promoting cell death and inflammatory responses. As, in atherosclerosis, Ro60-associated s-RNYs generated by apoptotic macrophages are released in the blood of patients, we have investigated the extracellular function of the s-RNY/Ro60 complex. Our data demonstrated that s-RNY/Ro60 complex induces caspase 3-dependent cell death and NF-κB-dependent inflammation, when added to the medium of cultured monocytes/macrophages. Finally, we have shown that s-RNY function is mediated by Toll-like receptor 7 (TLR7). Indeed using chloroquine, which disrupts signaling of endosome-localized TLRs 3, 7, 8 and 9 or the more specific TLR7/9 antagonist, the phosphorothioated oligonucleotide IRS954, we blocked the effect of either intracellular or extracellular s-RNYs. These results position s-RNYs as relevant novel functional molecules that impacts on macrophage physiopathology, indicating their potential role as mediators of inflammatory diseases, such as atherosclerosis.

Figure 1

Atherogenic stimuli induce s-RNY expression in monocytes/macrophages, which is timely correlated with inflammatory and cell death responses. Quantitative RT-PCR detecting the indicated s-RNYs at the indicated time in either human THP1 monocytes or BMDMs stimulated with either 100 μg/ml of oxLDL in combination with 0.25 μM of Tg (upper panels of a and b, respectively) or with 0.25 mM of PA in complex with BSA (upper panels of c and d, respectively). The data were normalized by U2 snRNA and presented as mean and S.D. (n=4). Each condition was subjected to immunoblot with the indicated antibodies (lower panels) to analyze the time correlation between the s-RNY induction and the inflammatory or cell death responses. One-way ANOVA with post-hoc Tukey test: *P<0.05; **P<0.01; ***P<0.001

Figure 2

s-RNYs promote apoptosis and inflammation in lipid-laden macrophages. Cell death data by flow cytometry from human THP1 cells (a) or mouse BMDMs (b) transfected with a cocktail of siRNAs against the terminal loop of all RNYs (siRNYs) to induce s-RNY maturation, a cocktail of 2'-OMe-RNA antisense oligonucleotides (AS) to all s-RNYs expressed, or control. Cells were stimulated with 0.25 mM of PA for 18 h (left panel), 0.25 μM Tg in combination with 100 μg/ml of oxLDL for 48 h (middle panel) or left unstimulated (right panel). Percentage of apoptotic cells was determined by staining with annexin V-FITC. Data are presented as mean and S.D. (n=3). Immunoblot analysis of IκBα, actin, total caspase 3 (casp3) and its cleaved form (c-casp3) in THP1 cells (c) or BMDMs (d) transfected with either a cocktail of AS to all s-RNYs or control. Cells were stimulated with 0.25 mM of PA for 18 h (left panel), 0.25 μM Tg in combination with 100 μg/ml of oxLDL for 48 h (middle panel) or left unstimulated (right panel). Student's t-test: *P<0.05; **P<0.01; ***P<0.001

Figure 3

Extracellular s-RNYs promote apoptosis and inflammation in lipid-laden macrophages. (a) Time and dose response of cell death by flow cytometry from human THP1 cells incubated with the immunopurified complex of s-RNY/Ro60 or RNY/Ro60. Incubation with BSA was used as control. Data are presented as mean and S.D. (n=5). One-way ANOVA with post-hoc Tukey test: *P<0.05. (b) Flow cytometry from human THP1 cells (left panel) or mouse BMDMs (right panel) incubated with 10 μg/ml of either the immunopurified complex of s-RNY/Ro60 or BSA, as control. s-RNY/Ro60 complex was previously incubated for 16 h in rotation at 4 °C with either 40 nM of 2'-OMe-RNA AS to s-RNYs or control. Data are presented as mean and S.D. (n=5). Student's t-test: *P<0.05. (c) Immunoblot analysis of IκBα, actin, total caspase 3 (casp3) and its cleaved form (c-casp3) in THP1 cells (left panel) or BMDMs (right panel) incubated s-RNY/Ro60 complex previously pretreated with either AS to s-RNYs or control

Figure 4

Intracellular s-RNYs activate TLR7 to promote apoptosis and inflammation in monocytes/macrophages. Cell death data by flow cytometry from human THP1 cells (a – left panels) or mouse BMDMs (b – left panels) transfected with a cocktail of siRNAs against the terminal loop of all RNYs (siRNYs) to induce s-RNY maturation or control (siCTL). Cells were incubated with 50 mM of chloroquine for 48 h (left panels) or with the TLR7/9 specific inhibitor IRS954 at 10 μg/ml of concentration for 48 h (middle panels). Percentage of apoptotic cells was determined by staining with annexin V-FITC. Data are presented as mean and S.D. (n=3). The same experimental conditions were analyzed by immunoblot of IκBα, actin, total caspase 3 (casp3) and its cleaved form (c-casp3) in THP1 cells (a – right panel) or BMDMs (b – right panels). Student's t-test: *P<0.05; ***P<0.001

Figure 5

Extracellular s-RNYs activate TLR7 to promote apoptosis and inflammation in monocytes/macrophages. Cell death data by flow cytometry from human THP1 cells (a – left panel) or mouse BMDMs (a – right panel) incubated with 10 μg/ml of the immunopurified complex of s-RNY/Ro60, RNY/Ro60 or BSA. Cells were incubated with 50 mM of chloroquine for 24 h or with IRS954 at 10 μg/ml of concentration for 24 h, as indicated. Percentage of apoptotic cells was determined by staining with annexin V-FITC. Data are presented as mean and S.D. (n=6 for BMDMs and 4 for THP1 cells). The same experimental conditions were analyzed by immunoblot of IκBα, actin, total caspase 3 (casp3) and its cleaved form (c-casp3) in THP1 cells (b – left panel) or BMDMs (b – right panel). Student's t-test: *P<0.05; **P<0.01

Figure 6

A model for s-RNY mode of action in lipid-laden monocytes/macrophages that exerts signaling pathway effects enhancing cell death and inflammation