The signaling cascade is mainly initiated by binding of M avium

The signaling cascade is mainly initiated by binding of M. avium components AZD3965 to TLR2 followed by recruitment of the MyD88 adaptor molecule and the activation of NFκB and MAP kinases. This chain

of events ends with the induction of inflammatory cytokines [10] controlling macrophage activation and granuloma formation. We monitored the induction of cytokine expression of THP-1 macrophages by the WT and the PLX-4720 research buy mutants in order to evaluate their ability to stimulate the immune signaling. To this aim we quantified the secretion of selected cytokines: the pro-inflammatory cytokines TNF-α, IL-1β and the anti-inflammatory cytokine IL-10. Five independent experiments were normalised for WT (expression ratio 1) to determine the expression ratio for the mutants in comparison to WT. While results for TNF-α and IL-1β were not significantly different as compared to WT, IL-10 was significantly (P <0.007) up-regulated for mutant MAV_4334 (Figure  5). IL-10 can inhibit the production of inflammatory cytokines such as TNF-α in monocytes pre-activated by IFN-γ and LPS [67, 68] and therefore plays an important role in the immune response. Figure 5 Induction of IL-10 cytokine secretion

by infected macrophages. THP-1 cells (2.0×105) were infected (MOI 50) with mutants and WT. After 24 hours cytokines from supernatants were measured by ELISA. When compared to FDA-approved Drug Library WT a P value <0.01 (two-tailed, unpaired Mann–Whitney test) was considered very significant (**). Intracellular survival The ability to survive and even replicate inside the phagosomes of macrophages is an important virulence factor of mycobacteria and was therefore included in our screening options. Infection experiments with macrophages give information on the early host response to mycobacterial infections [69]. Different types of macrophages

or monocytic cells have been employed to assess mycobacterial virulence and among these the human macrophage-like cell line THP-1 has proven a suitable system for virulence testing [69, 70]. It was shown that THP-1 cells are similar to primary human monocyte-derived macrophages with respect to their ability to take up mycobacteria and limit their growth [71]. We infected THP-1 cells that had been differentiated by PMA with the WT and the mutants. Intracellular pentoxifylline mycobacteria were measured by quantitative real-time PCR and CFU by plating. Survival of mutants in THP-1 cells was not consistently different if compared to the WT (data not shown). More significant differences were obtained when using human blood monocytes for the infection experiments. The growth of mutant MAV_4334, MAV_1778 and MAV_3128 was affected the most in human monocytes (Figure  6). They were reduced significantly for the first two days (P < 0.05 to P < 0.01). Mutant MAV_4334 and MAV_1778 (Figure  6 A and C) were almost reduced to half during the first two days.

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