Adenosine, acting through four types of receptors (A1, A2a, A2b, A3), is anti-inflammatory and cardioprotective. Since Toll-Like Receptor 4 (TLR4), a receptor involved in innate immunity, has recently been shown to mediate adverse left ventricular remodeling after myocardial infarction (MI), we sought to determine whether adenosine acts on the TLR4 pathway.
Primary human macrophages obtained after in vitro differentiation of blood monocytes isolated from healthy volunteers and patients with acute MI were treated with adenosine (10μm), adenosine analogs, and/or lipopolysaccharide (LPS, 100ng/mL). Transgenic mice bearing a cardiac-specific and externally-regulatable overexpression of A1 or A2a receptors were used to determine the receptor involved. Flow cytometry, immunoblotting, quantitative PCR and ELISA were used to measure expression levels of TLR4, Tumor Necrosis Factor-⍺ (TNF-⍺), TNF receptor-associated factor -6 (TRAF-6), and β-arrestins.
Consistently with others, we observed that the TLR4 pathway is activated following MI. In LPS-treated macrophages, adenosine decreased cell surface expression of TLR4, resulting in a decrease of TNF-⍺ production. This anti-inflammatory effect was also observed when macrophages were challenged with endogenous TLR4 ligands such as hyaluronic acid or heparan sulfate. Inhibition of TNF-⍺ production by adenosine was stronger in macrophages isolated from MI patients compared with healthy volunteers. The effect was replicated by agonists of A2a receptors and blocked by antagonists of A2a receptors. Over-expression of the A1 and A2a receptors in mice both resulted in a decrease of TLR4 and TRAF-6 expression in the heart. Co-immunoprecipitation studies indicated that adenosine enhances the binding of β-arrestins to TRAF-6, thus interrupting TLR4 signaling.
Adenosine down-regulates inflammation through interference with the TLR4 pathway. This mechanism involves enhanced binding of β-arrestins to TRAF-6 in the presence of adenosine. This result may have important implications as it explains some anti-inflammatory and cardioprotective properties of adenosine.
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