Figure 6. HIV ssRNA40 mediated NLRP3 inflammasome activation is associated with mitochondrial damage in microglial cells.

HMG cells were primed with ssRNA40, ssRNA41 or vehicle for 24h and stained with (a) MitoSOX Red (b) TMRE (black solid line, Vehicle; black dotted line, ssRNA41; black dashed line, ssRNA40), and (c) Mitotracker Green and Mitotracker Deep Red. (a) Representative histogram indicating increase in ROS generation in ssRNA40 treated HMG cells. (b) Representative histogram showing loss of mitochondrial membrane potential (Δψm) in ssRNA40 stimulated HMG cells. (c) HMG cells were treated with ssRNA40, ssRNA41 or vehicle for 24h followed by staining with Mitotracker Deep Red and Mitotracker Green. An increase in gated cells which were less brightly stained with Mitotracker Deep Red (i.e., downward shift) is indicative of loss of Δψm. Data are representative of three independent donors (n=3, *P<0.05). (d) HMG transfected with non-specific control siRNA (siControl), or NLRP3 siRNA (siNLRP3) were exposed to vehicle (LyoVec) or ssRNA40 (5µg/mL) for 24h. At 24h, cells were harvested and analyzed for NLRP3 expression by qPCR. ROS generating mitochondria were analyzed by intracellular MitoSOX red staining. Left, relative fold change in NLRP3 mRNA. Right, representative histogram showing decrease in ssRNA40 induced ROS generation in NLRP3 silenced HMG cells (top) and the respective geometric mean intensity (bottom). Data are representative of three independent donors (n=3, *P<0.05).