Influenza A viruses limit NLRP3-NEK7-complex formation and pyroptosis in human macrophages (2020)
Figure 1

Figure 1. Secretion of IL-1β Upon Infection with Different IAV Strains.

A, B THP-1 derived macrophages were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Viral protein expression for PB1-F2 (A) or NP (B) was detected by immunoblotting at 24 h post-infection (hpi) or indicated time points, respectively. Equal loading was confirmed by detection of beta actin.

(C) THP-1 derived macrophages were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Levels of viral M1 mRNA expression were detected by RT q‑PCR at 16 hpi. The mean ± standard deviation of three independent biological samples is shown. Statistical analysis was performed by students t-test.

(D) THP-1 derived macrophages were infected with VN and PR8 Wt and ΔF2 strains for 24 h at MOI 10. Levels of IL-1β were quantified by ELISA from the supernatants. The mean ± standard deviation of seven independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.

(E) THP-1 control and knockout cells for NLRP3 were mock treated or infected with the VN Wt and ΔF2 strains for 24 h at MOI 10 and IL-1β levels were assessed by ELISA from the supernatants. The mean ± standard deviation of five independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.

(F) Immunoblot detection for NLRP3 on control and NLRP3 knockout THP-1 cells. Equal loading was confirmed by specific blotting against beta actin. Representative blot of three independent repeats is shown.

(G) Human primary macrophages differentiated from PBMCs from two healthy donors were infected with H5N1 Wt and ΔF2 for 5 h at MOI 10. Levels of IL-1β were measured by ELISA in technical replicates. The mean of two independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.

(H) THP-1 derived macrophages were infected with a human H3N2 IAV strain (Wyo) Wt and ΔF2 for 24 h at MOI 4. Levels of IL-1β were measured by ELISA from the supernatants. The mean ± standard deviation of four independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.

(I) Detection of viral matrix protein M1 by immunoblotting in cells infected with the H3N2 Wt and ΔF2 strains.

(J) Human primary macrophages isolated and differentiated from PBMCs from three healthy donors were infected with H3N2 Wt and ΔF2 for 24 h at MOI 4. Levels of IL-1β were measured by ELISA. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.

(K-N) Primary human alveolar macrophages from BALF of five pediatric patients (patients are color coded) were infected with the human H3N2 strain (Wyo) Wt and ΔF2 for 16 h at MOI 1. (K) Levels of IL-1β were measured by ELISA from the supernatants. The mean ± standard deviation is shown. Levels of viral M1 mRNA (L), Mx1 mRNA (M) and IP10 mRNA (N) expression were detected by RT q‑PCR. The mean ± standard deviation for cells from four patients are shown. Individiual patients are color coded.