The NLRP3 inflammasome is involved with the pathogenesis of Mayaro virus
Fig 2
The NLRP3 inflammasome is activated in macrophages infected with MAYV.
(A-D) WT bone marrow-derived macrophages (BMDMs) were infected with Mayaro virus (MAYV) at an MOI of 5 (MOCK was used as a control), or treated with RPMI medium (NI) or ultrapure LPS (500 ng mL-1) as negatives and positive controls, respectively. After 3 and 6 hours of infection, cells were lysed and the RNA was extracted for qPCR analysis of Casp1 (A), Nlrp3 (B), Aim2 (C) and Asc (D). (E) PAM(3)CSK(4)-primed BMDMs derived from WT, Nlrp3–/–, Asc–/–, Casp1/11–/–and Aim2–/–mice were infected with MAYV at a MOI of 5. After 24 hours of infection, cell-free supernatants were harvested and IL-1β was quantified by ELISA. (F) Primed WT and Nlrp3–/–BMDMs were infected with either fresh MAYV, heat inactivated (HI) or UV-inactivated (UVI) virus. After 24 hours, the levels of IL-1β were measured by ELISA. (G) Western Blotting was performed in WT BMDMs after 24 hours of infection. Supernatants (SN) and cellular extracts (CE) were harvested from MAYV-infected (or MOCK) BMDMs, and levels of cleaved caspase-1 (p20) and IL-1β (p17) were detected in the SN. As loading controls, levels of pro-caspase-1 and β-actin were assessed in the CE. (H-J) WT and Nlrp3–/–BMDMs were infected with MAYV at a MOI of 5. After 24 hours of infection the cells were stained for active-caspase-1 with FAM-YVAD and analyzed as shown by the representative histograms (G). The percentage (H) and integrated mean of fluorescence (iMFI) (I) of activated cells is shown. Data shown are means ± SD of triplicate samples (A-E, G-I) and are representative of the data obtained from two (A-D, F) or three (E, G-H) independent experiments. Statistical analysis was performed by student’s t test. *, P < 0.05.