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. 2022 Aug 17;11:e79794. doi: 10.7554/eLife.79794

Figure 4. Trophoblast organoids secrete high levels of antiviral IFN-λ2.

Figure 4.

(A, B) Comparison of the levels of the type I IFNs IFN-α2 and IFN-β and type III IFNs IFN-λ1 and IFN-λ2 in conditioned medium (CM) isolated from TOs (A) or DOs (B) as determined by Luminex assays. (C) Comparison of the levels of IFN-λ2 in CM isolated from TOs, matched villous tissue, or in primary human trophoblasts (PHTs). (D) IFN-λ activity shown as relative light units (RLUs) of CM isolated from TOs (blue) and DOs (orange) as assessed using an IFN-λ reporter 293T cells line. NCM (non-conditioned medium, gray) was as negative control, NCM supplemented with recombinant human IFN-λ2 (100 ng) was used as a positive control (red). (E) Human osteosarcoma U2OS cells were treated with CM isolated from TOs (blue) or with recombinant IFN-λ2 (100 ng) for 24 hr and then infected with ZIKV for 24 hr (in the presence of CM). Level of ZIKV replication was assessed by viral RNA as determined by qPCR. Data are shown as a fold change in infection from NCM-treated cells. (F) Representative confocal micrographs of DOs treated with NCM or CM isolated from TOs for 24 hr, then infected with ZIKV for 72 hr. Shown in green is viral double stranded RNA, a replication intermediate. DAPI-stained nuclei are shown in blue. Scale bar, 50 μm. (G) Automated image analysis of ZIKV infection in ZIKV-infected DOs treated with NCM or TO-derived CM, as described in (F). Imaged were captured from three different experiments. (H) Comparison of the levels of IFN-λ2 in CM isolated from matched TOs grown under conditions that promote EVT differentiation (Diff., right) or under standard culture conditions (Undiff., left). Data in all panels are mean ± standard deviation from at least three independent CM preparations. Significance was determined by a Student’s t-test, Mann-Whitney U-test or by Kruskal-Wallis U-test with multiple comparisons. *p<0.05; **p<0.01; ****p<0.001, ns, not significant. In all, each symbol represents unique CM preparations. DO, decidua organoid; TO, trophoblast organoid.