In vitro stimulation of monocytes with supernatants from KSHV-infected cells. HV-isolated monocytes were plated (1 × 106 cells per well) and incubated overnight with complete media containing 50% of supernatant from either uninfected or KSHV-infected HEK293T cells, in the presence or absence of the NLRP3 inflammasome inhibitor MCC950 (5 μM). After stimulation, culture supernatants were harvested for quantification of IL-1β and IL-18 by enzyme-linked immunosorbent assay (ELISA), and monocytes were stained for detection of inflammasome complex formation by imaging flow cytometry. (A) Numbers of monocytes showing FLICA+ ASC-speck formation per mL were quantified inside the monocyte gate. Lines represent mean values with standard error of the mean (SEM). Data were analyzed using the Welch t test to compare the uninfected with the KSHV-infected group within each monocyte subset. ∗∗P < .01; ∗∗∗P < .001. (B) The levels of (B) IL-1β and (C) IL-18 produced by stimulated monocytes were quantified by ELISA and compared across the different groups. Lines represent mean values with SEM. Data were analyzed using the Kruskal-Wallis test. ∗∗P < .01; ∗∗∗∗P < .0001. Data are presented as a pool of 2 different batches of HEK293T culture–derived supernatants.
