Abstract
Background: Chronic neutrophilic lung inflammation in lung transplantation may be driven by IL-17 producing T lymphocytes. Azithromycin (AZM) has proven to (partially) restore pulmonary function and decrease IL-17 in patients samples (lavage fluid and biopsies). However, the direct effect of azithromycin on the production of IL-17 by T helper lymphocytes has never been investigated.
Materials and methods: Total CD4+T cells were magnetically sorted from Balb/c mice splenocytes using a CD4 negative isolation kit. FACS analysis was performed after magnetic isolation to check the purity of the CD4+ T cell population. Cells (2x105 cells/well) were then polyclonally activated with plate-bound anti-CD3 and soluble anti-CD28 in 96-wells flat-bottom plates and exposed to diverse azithromycin concentrations (0-10-20-40-80 µg/ml). Cultures were incubated at 37°C in a 5% CO2 atmosphere in appropriate medium (RPMI 1640 supplemented with 2mM L-glutamine, 1 mM nonessential amino acids, 5% FCS and 1 mM sodium pyruvate). IL-17A production was measured in culture supernatants after 72h using ELISA (detection threshold 20pg/ml).
Results: FACS analysis demonstrated a CD4+ T cell purity of 95%. The level of IL-17 in control condition (anti-CD3/CD28 alone) reached 218±22 pg/ml. When exposed to increasing concentrations of azithromycin, IL-17 decreased in a dose-dependent fashion as follows: 187±27 pg/ml at 10µg/ml AZM, 133±24 pg/ml at 20µg/ml AZM, 73±33 pg/ml at 40 µg/ml AZM and 23±5 pg/ml at 80 µg/ml AZM (p=0.0079).
Discussion: We observed that azithromycin negatively regulates IL-17 production by activated CD4+ T cells, confirming for the first time a direct inhibitory effect of azithromycin on Th17 cells.
- Copyright ©ERS 2015