107 Immunomodulatory Effects of Manumycin-type Antibiotics on Human Macrophages

BACKGROUND: Polyketide-derived antibiotics including macrolides are known to exert potent anti-inflammatory and immunomodulatory effects beyond their purely antibacterial action. The mechanisms of their biological activities are still being investigated but the effect on signalling pathways of transcription factors which regulate a number of pro-inflammatory and/or pro-fibrotic genes might be preferentially involved. The aim of our study was to assess the effect of manumycin and structurally related compounds asukamycin and collabomycin on a release of proinflammatory cytokines IL-1beta and IL-18 from THP-1 monocyte/macrophage cell line. Furthermore, the level of mRNA expression of multiple genes associated with immune regulation has been studied. METHODS: The THP-1 cells were cultured in RPMI1640 with 5% fetal calf serum and then stimulated with TNF alpha (20 ng/mL) under serum free conditions in the presence or absence of manumycin and asukamycin (both at 0.3 μg/mL). The concentrations of cytokines in culture supernatants were measured by ELISA (IL-18, MBL) or Luminex (IL-1 beta, R&D). Quantitative RT-PCR (SABiosciences) was used for the evaluation of 84 different gene expressions in TNF alpha and manumycin stimulated cultures. RESULTS: IL-1 beta was not detectable in culture supernatants of unstimulated THP-1 cells but appeared in response to TNF alpha (4.96 + 0.59 pg/mL). Both manumycin (0.34 + 0.48 pg/mL) and asukamycin (1.06 + 0.81) inhibited IL-1 beta release induced by TNF alpha. IL-18 was found to be constitutively produced (14.68 + 7.83 pg/mL) and the release was doubled by TNF alpha (30.98 + 2.21 pg/mL) and inhibited to basal values by both manumycin (18.04 + 10.21 pg/mL) and asukamycin (12.96 + 2.32 pg/mL). Manumycin inhibited mRNA expression of several genes associated with proinflammatory responses including IL-1 beta, IL-6, and TLR8. Among the genes upregulated in response to manumycin, HMOX1, gene for heme oxigenase 1, showed the highest mRNA induction. CONCLUSIONS: We assume from our study that manumycin and asukamycin represent potent inhibitors of IL-1 beta and IL-18 release from human macrophages. Some of the potentially proinflammatory genes are regulated on the level of transcription.