Figure 6.
Figure 6. MKs exposed to dengue release type I IFNs to induce IFITM3 and limit viral infection. (A-E) CD34+-derived human MKs were infected with DENV2 (MOI 1.0; culture day 12; t = 18 hours) or mock control. (A-C) Type I IFN (IFNα and IFNβ) protein expression was measured according to immunoblot in MK cell lysates and quantified according to densitometry. Immunoblots show n = 3 independent biological replicates. (D-E) Supernatants from MKs infected with DENV2 were also measured for IFNα and IFNβ by using an enzyme-linked immunosorbent assay (n = 10-11). (F) MKs were infected with DENV2 in the presence of blocking antibodies specifically against IFNα (1 μg/mL), IFNβ (1 μg/mL), or both or immunoglobulin G (IgG) controls. IFITM3 mRNA expression in MKs was measured by using quantitative RT-PCR (qRT-PCR) (n = 4-5). (G) MKs were preconditioned with DENV2 (MOI 0.1) or mock virus control (mock) on culture day 10 (t = 18 hours). After 18 hours, supernatants were harvested from DENV2 or mock preconditioned MKs. Freshly cultured MKs were then treated with harvested supernatants from DENV2 preconditioned MKs (+DENV2 sup) or left alone (NT), and infected with DENV (DENV2 virus, MOI 1.0) or mock control (Mock). After 18 hours, dengue infection in MKs was determined by using immunocytochemistry with an antibody against DENV2 (orange, top row). In parallel, IFITM3 protein expression was examined (magenta, top row). Nuclei were stained by using TO-PRO-3 (blue) and shown in the bottom panels depicting the transmission channel. The white arrows point to IFITM3-positive cell bodies, and the yellow arrows highlight cells that stained positive for DENV2. Scale bars = 25 µm. (H) Images were quantified by using CellProfiler as described in “Methods.” The bar graph shows the percentage of DENV2-infected MKs. (I-J) Human HSCs were pretreated with harvested supernatants from mock (Mock sup) or dengue (DENV2 sup) infected MKs, under conditions described in (E). (I) DENV2 mRNA was measured by using qRT-PCR, and (J) the number of dengue-infected HSCs were quantified by microscopy. (K) HSCs were infected with DENV2 in the presence of blocking antibodies specifically against IFNα or IFNβ or both or IgG controls. IFITM3 mRNA expression in MKs was measured by using qRT-PCR (n = 7).

MKs exposed to dengue release type I IFNs to induce IFITM3 and limit viral infection. (A-E) CD34+-derived human MKs were infected with DENV2 (MOI 1.0; culture day 12; t = 18 hours) or mock control. (A-C) Type I IFN (IFNα and IFNβ) protein expression was measured according to immunoblot in MK cell lysates and quantified according to densitometry. Immunoblots show n = 3 independent biological replicates. (D-E) Supernatants from MKs infected with DENV2 were also measured for IFNα and IFNβ by using an enzyme-linked immunosorbent assay (n = 10-11). (F) MKs were infected with DENV2 in the presence of blocking antibodies specifically against IFNα (1 μg/mL), IFNβ (1 μg/mL), or both or immunoglobulin G (IgG) controls. IFITM3 mRNA expression in MKs was measured by using quantitative RT-PCR (qRT-PCR) (n = 4-5). (G) MKs were preconditioned with DENV2 (MOI 0.1) or mock virus control (mock) on culture day 10 (t = 18 hours). After 18 hours, supernatants were harvested from DENV2 or mock preconditioned MKs. Freshly cultured MKs were then treated with harvested supernatants from DENV2 preconditioned MKs (+DENV2 sup) or left alone (NT), and infected with DENV (DENV2 virus, MOI 1.0) or mock control (Mock). After 18 hours, dengue infection in MKs was determined by using immunocytochemistry with an antibody against DENV2 (orange, top row). In parallel, IFITM3 protein expression was examined (magenta, top row). Nuclei were stained by using TO-PRO-3 (blue) and shown in the bottom panels depicting the transmission channel. The white arrows point to IFITM3-positive cell bodies, and the yellow arrows highlight cells that stained positive for DENV2. Scale bars = 25 µm. (H) Images were quantified by using CellProfiler as described in “Methods.” The bar graph shows the percentage of DENV2-infected MKs. (I-J) Human HSCs were pretreated with harvested supernatants from mock (Mock sup) or dengue (DENV2 sup) infected MKs, under conditions described in (E). (I) DENV2 mRNA was measured by using qRT-PCR, and (J) the number of dengue-infected HSCs were quantified by microscopy. (K) HSCs were infected with DENV2 in the presence of blocking antibodies specifically against IFNα or IFNβ or both or IgG controls. IFITM3 mRNA expression in MKs was measured by using qRT-PCR (n = 7).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal