Figure 7.

Host MDR driven overproduction of IFN-β facilitates antimony resistance. (A,B) Mouse RAW264.7 wild type reporter cells were either mock transfected or with Ld-DNA [2.5 ug/ml with Lipofectamine 2000] (A) or mouse RAW264.7 reporter derived cGAS ablated [cGAS KO] and control [ISG-WT] cells were transfected with Ld-DNA [2.5 ug/ml with Lipofectamine 2000] (B). Cells were harvested and relative transcript levels of MRP-1 and P-gp were measured by qRT-PCR. Normalized levels for GAPDH are presented. (C,D) Mouse RAW264.7 cells were either treated with r-mIL-2 or r-mIFN-β or antibody (Ab) to mIFN-β. Production of IL-10 was estimated at the release level in the culture supernatant by ELISA (C) and relative transcript level of host MRP1 was measured by qRT-PCR (D). (E) Mouse RAW264.7 wild type reporter cells were either pre-treated with probenecid (200 uM) and/or verapamil (2 uM) or lovastatin (10 uM). Then these pre-treated cells were either mock transfected or transfected with Ld-DNA [2.5 ug/ml with Lipofectamine 2000]. Production of IFN-β was estimated at the release level in the culture supernatant by ELISA. (F) Mouse RAW264.7 reporter derived cGAS ablated [cGAS KO] and control [ISG-WT] cells were cells were either pre-treated with probenecid (200 uM) and/or verapamil (2 uM) or lovastatin (10 uM). Then these pre-treated cells were either left uninfected or infected with antimony sensitive (SB^S-LD) or antimony resistant (SB^R-LD) L.donovani parasites (parasite: macrophage = 10: 1) and later treated/untreated with SAG (60 ug/ml). Number of intracellular amastigotes per 100 macrophages on infection was microscopically evaluated on stained coverslip preparations from each experiment. Data represent mean ± SEM of at least three independent experiments (n = 4 for A–C and F; and n = 3 for D,E). Statistically significant results are marked as **p < 0.001.