Fig. 6 | Nature Communications

Fig. 6

From: Leishmania RNA virus exacerbates Leishmaniasis by subverting innate immunity via TLR3-mediated NLRP3 inflammasome inhibition

Fig. 6

LRV limits NLRP3 activation via ATG5. a, b C57BL/6 BMDMs were silenced with a scramble sequence (SCR) or Atg5-specific shRNA, then infected with L.g.− or L.g.+, and the efficiency of the process (a) was confirmed by WB. β-actin: loading control. b After 24 h of infection, levels of IL-1β were determined by ELISA in cell-free supernatants. NT: Non-transduced BMDMs. c, d BMDMs from LysMCre/+/Atg5Fl/+ (Littermate control) or LysMCre/+/Atg5Fl/Fl (Knockout) mice were infected with L.g.− or L.g.+ for 24 h, and Casp1 activation was determined by FLICA assay (FACS). The percentage of Casp1 + cells (FAM-YVAD) (c) and iMFI (d) are shown. e LysMCre/+/Atg5Fl/+ and LysMCre/+/Atg5F/Fl BMDMs were infected and left untreated or treated with IFN-β (1000 U/mL) at the moment of the infection. Levels of IL-1β in cell-free SNs were quantified by ELISA, after 24 h of infection. f ELISA assay in Parkin deficient BMDMs (Prkn−/) and its respective littermate control (Prkn+/+) after 24 h of infection. g WB for inflammasome components and ATG5 in LysMCre/+/Atg5Fl/+ and LysMCre/+/Atg5Fl/Fl BMDMs after 24 h of infection with L.g.− or L.g.+. β-actin was used as a loading control. h, i BMDMs were infected with metacyclic promastigotes from either L.g.− or L.g.+, at a MOI of 1. One hour after infection, cells were washed and left in culture for 1, 48, or 96 h. The percentage of infected cells (h) and the average number of amastigotes per cell (i) was evaluated by Panotico Giemsa. The results are shown as mean ± SD. Statistical analysis was performed by unpaired Student’s t test. P < 0.05 (*) was considered statistically significant. One representative of at least two independent experiments performed with technical replicates is shown

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