Effect of Dexamethasone, Lipopolysaccharide or Interferon-Gamma on the Recovery of Viable Mycobacterium Avium Subspecies Paratuberculosis from In Vitro-Infected Primary Bovine Macrophages

Abstract

ABSTRACT: Study background: The study was designed to evaluate if the addition of Dexamethasone, IFN-g, or LPS into culture media of primary bovine Monocyte-Derived Macrophages (MDMs), could support in vitro infection with Mycobacterium avium subspecies paratuberculosis. Methods: Primary bovine Monocyte-Derived Macrophages (MDMs) were infected in vitro with a reference strain of Map at 5:1 MOI for 2h. Map-infected MDMs were stimulated with IFN-g, LPS, Dexamethasone or medium alone for 24h. At 0, 6, 72 and 120h of culture, it was evaluated the presence of Map by bacterial culture and amplification of the IS900 fragment by real-time PCR. The function of Map-infected MDM was evaluated by measurement of TNF-a, IL-6, IL-8, IL-10, IL-12, IP-10, and CCL3 in culture supernatants by Luminex. Data were analyzed by Kruskal-Wallis test. Results: The IS900 segment was amplified in samples of Map-infected MDM from all stimuli. The growth of Map in bacterial culture was observed at each time-point evaluated without statistically significant differences between groups. Map-infected-MDMs stimulated with Dexamethasone significantly reduced cytokine production compared with control, excepting for IP-10 production from 6 to 120h (P<0.01). Overall cytokine production at 72h was significantly higher in Map-infected MDM treated with LPS (P<0.01) excepting for IP-10 and CCL3 production at 120h. IL-8 and IL-12 production at 72h and IP-10 production at 120h were significantly higher in Map-infected MDM treated with IFN-g (P<0.01). Conclusion: Primary bovine MDM obtained from peripheral blood mononuclear cells could be used for growth of Map in vitro. The addition of LPS or IFN-gamma reduced the capability of MDM for sustaining the growth of Map until 120h post-infection, although Dexamethasone sustained the recovery of viable Map until 120h in culture.