Figure 5. HIV ssRNA40 activates NLRP3 inflammasome and inhibits the expression of its negative regulator, miR-223.

(a) HMG cells were treated with ssRNA40 for 24h and 48h; NLRP3 levels were analyzed with qPCR (top) and immunoblotting (bottom). Left, representative immunoblots for NLRP3 and β-Actin using antibody to NLRP3 and human β-Actin and right, densitometric analysis for fold change in NLRP3 expression from 3 independent donors. Results are presented as mean ± SD, n=3; *P<0.05. (b) Pharmacologic inhibition of NLRP3 by MCC950 inhibits ssRNA40 induced inflammasome activation in microglia cells. HMG cells were pre-treated with NLRP3 inflammasome inhibitor, MCC950 (10μM), or caspase-1-specific inhibitor Ac-YVAD-cmk (50μM), or vehicle control prior to their exposure to ssRNA40, ssRNA41 or vehicle control and production of IL-1β, IL-1α, and TNF-α in culture supernatants were measured at 24h post treatment by ELISA. (c) HMG cells were exposed to ssRNA40 (5μg/mL), ssRNA41 or vehicle control and monitored for expression of miR-223, and NLRP3 mRNA at 24h and 48h post treatment time points. Results are presented as mean ± SD, n=3; *P<0.05.