Abstract 15261: Adipor2 Sirna-Mediated Knockdown Predominantly Polarizes Towards a M1 Phenotype in THP-1 Macrophages

Introduction: Monocyte-derived macrophages (Mϕs) are the major innate immune cells in plaques, where they assume a more pro-inflammatory (M1) or anti-inflammatory (M2) Mϕ phenotype. Notably, M1 Mϕs are more abundant in unstable plaques, while M2 in stable plaques. Adiponectin receptor 1 (AdipoR1) and AdipoR2 are highly expressed in the monocyte/Mϕ lineage and largely mediate immune responses. Herein, we investigated the specific contribution of AdipoR1 and AdipoR2 on polarization towards a M1 or M2 Mϕ phenotype. Methods: siRNA-mediated knockdown of AdipoR1 and/or AdipoR2 was performed in THP-1 monocyte-derived Mϕs, and collected to perform, 1) qRT-PCR analyses on AdipoR1 and AdipoR2, and 2) flow cytometry analyses on M1 Mϕ markers: CD86, CD80, MHC-II, and M2 Mϕ markers: CD206, CD163. Results: For qRT-PCR analyses, siRNA-mediated knockdown of single AdipoR1 and AdipoR2 significantly decreased the mRNA levels of their respective receptor (p<0.01, p<0.0001), while the opposing receptor was unaffected. Double knockdown resulted in a significant decrease in both AdipoR1 and AdipoR2 mRNA levels (p<0.001, p<0.0001). For flow cytometry analyses, single AdipoR1 and double AdipoR1/R2 siRNA led to a significant decrease in ΔMFI (mean fluorescence intensity) of MHC-II (p<0.01), while a significant increase was observed in single AdipoR2 siRNA (p<0.01). Intracellular siRNA-mediated knockdown of AdipoR1 and/or AdipoR2 did not result in a significant loss of their cell surface expression, as confirmed via dot plots analyses. Lastly, single AdipoR1 and double AdipoR1/R2 siRNA exhibited decreased % MHC-II/CD86-positive cells (p<0.01, p<0.001), while single AdipoR2 siRNA increased % MHC-II/CD86-positive cells (p<0.05). Conclusions: Despite intracellular siRNA-mediated knockdown of AdipoR1 and/or AdipoR2, their cell surface expression remains intact. Furthermore, single AdipoR2 siRNA was associated with an increase in M1 Mϕ population (MHC-II/CD86), which is in line with our previous evidence showing decreased AdipoR2 signalling in unstable plaques. Thus, downregulation of AdipoR2 may play a role in polarization predominantly towards a M1 phenotype in THP-1 Mϕs and hence could be mechanistically relevant in the development of unstable plaques.