Azithromycin suppresses interleukin-12p40 expression in lipopolysaccharide and interferon-γ stimulated macrophages

Abstract

Azithromycin (AZM), a 15-member macrolide antibiotic, possesses anti-inflammatory activity. Macrophages are important in innate and acquired immunity, and produce pro-inflammatory cytokines such as interleukin (IL)-12, which are composed of subunit p40 and p35. The key function of IL-12 is the induction and maintenance of T-helper-1 responses, which is associated with the pathogenesis of chronic inflammatory diseases. We investigated the effect of azithromycin on IL-12p40 production in macrophages after lipopolysaccharide (LPS)/interferon (IFN)-γ stimulation. RAW264.7 macrophage cell line was pre-treated with vehicle or AZM, followed by the stimulation with LPS/IFN-γ. We measured IL-12 production by RT-PCR and ELISA. IL-12 transcriptional regulation was assessed by electrophoretic mobility shift assay and reporter assay. Phosphorylation of activator protein (AP)-1 and interferon consensus sequence binding protein (ICSBP) was assessed by immunoprecipitation using phosphotyrosine antibody, and immunoblotting using specific antibodies against JunB and ICSBP. AZM reduced the induction of IL-12p40 by LPS/IFN-γ in a dose dependent manner. AZM inhibited the binding of AP-1, nuclear factor of activated T cells (NFAT), and ICSBP, to the DNA binding site in the IL-12p40 promoter. AZM also reduced LPS/IFN-γ-induced IL-12p40 promoter activity. Phosphorylation of JunB and ICSBP was inhibited by azithromycin-treatment in stimulated cells. In conclusion, AZM reduced IL-12p40 transcriptional activity by inhibiting the binding of AP-1, NFAT, and ICSBP to the promoter site. This may represent an important mechanism for regulating the anti-inflammatory effects of AZM in macrophages.

Azithromycin inhibits macrophage interleukin-1β production through inhibition of activator protein-1 in lipopolysaccharide-induced murine pulmonary neutrophilia

“…Our results obtained both in vitro and in vivo are in accordance with previous in vitro findings showing macrolide-mediated inhibition of AP-1 activation and DNA binding in a murine macrophage cell line and human monocytes, following LPS + interferon-γ and LPS stimulation, respectively [18,41]. In addition, in line with our data, azithromycin did not influence ERK1/2 activation in the LPS-stimulated human monocytic cell line, THP-1, either [42].…”

Azithromycin use in patients with cystic fibrosis

“…It also inhibits the neutrophil release of a large number of cytokines and chemokines, including IL-1β, IL-6, IL-10, tumour necrosis factor (TNF)-α, chemokine ligand (CCL) 1, CCL3, CCL5, CCL20 and CCL22, and granulocyte colony-stimulating factor (G-CSF), increases phagocytosis, attenuates the secretion of IL-12 by macrophages (thus reducing Th-1 responses and subsequent chronic inflammatory disease) and modulates the differentiation and maturation of dendritic cells [45][46][47][48].…”

Anti-Inflammatory Macrolides to Manage Chronic Neutrophilic Inflammation

“…29 Azithromycin reduces IL-12p40 transcriptional activity by inhibiting the binding of AP-1 to the promoter site. 30 The p40 subunit is a 216 Chapter 6…”