Figure 2. IFN-γ primes human ECs for MAC-induced NLRP3 inflammasome activation.

(A) ECs were primed with IFN-γ (50ng/mL) for 24 hours and upregulation of IL-1β, NLRP3, NLRC4/IPAF, Caspase-1, Gasdermin D and HLA-DR α-chain transcript levels were assessed by qRT-PCR (n=3, Student’s t-test, SEM). (B) ECs were stimulated with IFN-γ for the duration indicated and pro-IL-1β, NLRP3, pro-caspase-1, and gasdermin D expression were assessed by immunoblot. (C) Unprimed and IFN-γ-primed ECs were transfected with control, STAT1, MyD88, or TRIF siRNA and protein expression of NLRP3, IPAF, pro-caspase-1 and pro-IL-1β were assessed by immunoblot. (D) Unprimed and IFN-γ-primed ECs were incubated with complement-fixing anti-human endoglin IgG_2a mouse monoclonal antibody (mAb) (20μg/mL) and human complement prior to flow cytometry analysis of IgG binding, HLA-DR, C4d and C5b-9 (n=3, Student’s t-test, SEM). (E) Unprimed and IFN-γ-primed ECs were incubated with anti-human endoglin IgG_2a mAb and varying concentrations of human complement. Cell lysates and culture supernatants were assessed for cleaved caspase-1 and cleaved IL-1β, respectively, by immunoblot. (F) ELISA measurement of IL-1β secretion in culture supernatants by unprimed and IFN-γ-primed ECs after incubation with anti-human endoglin mAb and varying levels of human complement (n=3, one-way ANOVA and Tukey’s multiple comparisons test, SEM). *P<0.05, **P<0.01, ***P<0.001, n.s., non-significant.