(CCSP006) KNOCKDOWN OF ADIPOR1 PROMOTES ADIPONECTIN'S ANTI-INFLAMMATORY ACTIONS MEDIATED THROUGH ADIPOR2 IN THP-1 MACROPHAGES

Background: Adiponectin is an adipose-tissue secreted hormone that exerts its anti-inflammatory and anti-atherogenic functions through its two main receptors, adiponectin receptor 1 (AdipoR1) and AdipoR2, which are highly expressed in monocyte-derived macrophages. In response to various stimuli, macrophages can polarize towards a more pro-inflammatory M1, or anti-inflammatory M2 phenotype. Herein, we investigated the effect of adiponectin on macrophage polarization during single AdipoR1, single AdipoR2 and double AdipoR1/R2 knockdown conditions.

METHODS AND RESULTS: siRNA-mediated knockdown of AdipoR1 and/or AdipoR2 was performed in THP-1 monocyte-derived macrophages and treated in the presence or absence of adiponectin. All knockdown conditions were compared to the non-targeting control (NTC) siRNA. Flow cytometry was performed to assess AdipoR1 and AdipoR2, M1 macrophage population (CD86 and CD80), and M2 macrophage population (CD206 and CD163). Magnetic bead-based immunoassays were performed on cell culture supernatants to assess 12 pro-inflammatory (TNF-alpha, MCP-1, IL-1beta, IL-6, IL-8, IL-12p40, IL-12p70) and anti-inflammatory (IL-1Ralpha, IL-4, IL-10, TGF-beta1 and CCL17) cytokines and chemokines.
In response to adiponectin, flow cytometry analyses for single AdipoR1, single AdipoR2, and double AdipoR1/R2 siRNA resulted in a significant increase in double AdipoR1/R2-positive macrophages. Although adiponectin treatment of single AdipoR1, single AdipoR2 and double AdipoR1/R2 siRNA significantly increased both macrophage populations, this increase was more prominent in the M2 macrophage population. Immunoassay analyses in AdipoR knockdown conditions demonstrated increased TNF-alpha, MCP-1, IL-6, and IL-12p40 levels during the presence compared to absence of adiponectin. Further, in adiponectin-treated AdipoR knockdown conditions, TNF-alpha, MCP-1, IL-6, and IL-12p40 were decreased during single AdipoR1 siRNA relative to adiponectin-treated NTC siRNA. This was consistent with decreased pro-/anti-inflammatory chemokine/cytokine ratios, including TNF-alpha/IL-10, MCP-1/IL-10, IL-12p40/IL-10, TNF-alpha/TGF-beta1, and IL-6/TGF-beta1 during adiponectin-treated single AdipoR1 siRNA compared to NTC and to single AdipoR2 siRNA.

Conclusion: Adiponectin treatment increases the amount of AdipoR1/R2 cell surface expression on macrophages despite intracellular AdipoR knockdown conditions. Furthermore, although adiponectin promoted both macrophage populations, a greater shift towards M2 macrophages was noted. Downregulation of AdipoR1 mRNA promoted adiponectin’s anti-inflammatory actions mediated through AdipoR2, by reducing levels of TNF-alpha, MCP-1, IL-6, and IL-12p40, as well as decreasing pro-/anti-inflammatory chemokine/cytokine ratios. Our current work will further unravel the underlying mechanisms of the role of the adiponectin-AdipoR pathway in regulating M1-M2 macrophage polarization.