Molecular Mechanism of Anti-inflammatory Conversion by B2-AR Agonist Salmeterol

Objectives: Salmeterol, a long-acting β2-adrenergic receptor (β2-AR) agonist, is anti-inflammatory in vitro and in vivo where lipopolysaccharide (LPS) from E. coli or Porphyromonas gingivalis was used to initiate chronic inflammation. It has now been determined that Salmeterol can convert the pro-inflammatory response into an anti-inflammatory response by inhibiting the production of TNF-α and enhancing the production of IL-10 by innate immune cells. In the present study, we assessed the molecular action of Salmeterol on innate immune cells in vitro.
Methods: Immune cells were pretreated with Salmeterol and followed by exposure to LPS. The pro-inflammatory responses were measured by the production of inflammatory mediators and immunoblot analysis was performed to measure signaling molecules protein and phosphorylation levels. Silencing of signaling molecules was accomplished using siRNA (b-arrestin-2) or by using a pharmacologic inhibitor specific for intracellular signaling molecules. Binding affinity of TAK1 (transforming growth factor-beta activated kinase 1) and TAB1 (TAK1-binding protein 1) with β-arrestin2 were examined by co-immunoprecipitation assay. The statistical significance between groups was determined by one-way and two-way ANOVA followed by Bonferroni’s multiple comparison post-test using GraphPad Prism 5.0 software.
Results: Salmeterol inhibited LPS-induced release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nitric oxide, and enhanced the production of the anti-inflammatory cytokine IL-10. We found that Salmeterol increases the expression of β-arrestin2 and enhanced expression of β-arrestin2 led to the inhibition of NF-kB activation and pro-inflammatory responses, as well as enhancing the anti-inflammatory IL-10 response. In addition, silencing of β-arrestin2 abrogates both the pro and the anti-inflammatory effects of Salmeterol in LPS-stimulated immune cells.
Conclusions: Taken together, our findings suggest that both the anti-inflammatory properties of Salmeterol are β-arrestin2 dependent and also offers novel drug targets in the convergent β2-AR/β-arrestin2 and inflammatory pathways to regulate innate immune cell activation in chronic inflammation.