Fig. 4 | Nature Communications

Fig. 4

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

Fig. 4

LRV triggers TLR3 to limit inflammasome activation by L.g. a, b FLICA analysis showing Casp1 activation (FAM-YVAD) of C57BL/6, Nlrp3−/− and Tlr3−/− BMDMs infected with either L.g.− or L.g.+ for 24 h, at a MOI of 10. A representative histogram (a) and the integrated MFI (iMFI) (b) are shown. c Western blotting for cleaved Casp1 (p20) in the SN, and pro-caspase-1 and in the cellular extracts (CE). d, e ELISA assay for IL-1β in the cell-free supernatants of LPS-primed BMDMs after 24 h of infection with MOI 10 stationary-phase (SP) (d) or MOI 5 metacyclic (Metac., e) parasites. f C57BL/6 (WT) and Tlr3−/− BMDMs were infected with L.g.− or L.g.+ at a MOI of 10, and after 20 h of infection, were infected with L. pneumophila (flaA Legionella). Four hours later, supernatants were collected and levels of IL-1β were assessed by ELISA. g, h C57BL/6 BMDMs were infected with L.g.− or L.g.+ at a MOI of 10, and treated with Poly:IC (5 μg/mL) (g) or IFN-β (1000 U/mL) (h) at the moment of infection. Twenty-four hours later, supernatants were collected and ELISA for IL-1β was performed. il C57BL/6, Nlrp3−/−, Tlr3−/−, and Trif−/− BMDMs were infected with metacyclic promastigotes from either L.g.− or L.g.+, at a MOI of 1. After 1 h of infection, cells were washed and left in culture for 1 or 48 h. Killing of the parasites was evaluated by Panotico Giemsa staining. i Percentage of infected BMDMs 1 h after infection; j average number of amastigotes per cell 1 h after infection; k percentage of infected BMDMs 48 h after infection; l average number of amastigotes per cell 48 h after infection. The results are shown as mean ± SD. Statistical analysis was performed by unpaired Student’s t test, and P < 0.05 (*) was considered statistically significant. One representative of at least two independent experiments performed in technical triplicates is shown

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