IL-4/IL-13 polarization of macrophages enhances Ebola virus glycoprotein-dependent infection
A) TIM-4 surface expression. Matured bone marrow derived macrophages and pmacs from C57BL/6 Ifnar-/- mice were lifted from tissue culture plates, stained with directly conjugated F4/80 and CD11b mAb. Positively gated cells were analyzed for TIM-4 (grey histogram) or isotype control (white histogram) by flow cytometry. B) TIM-4 expression is required for robust rVSV/EBOV GP infection. C57BL/6 Ifnar-/- or Ifnar/Timd-4 -/- pmacs were infected with the indicated infectious units of rVSV/EBOV GP. Twenty-four hours following infection, cells were quantified for GFP expression by flow cytometry. C) Infection of BMDMs is not affected by the absence of TIM-4 expression. Bone marrow cells were isolated from C57BL/6 Ifnar-/- or Ifnar/Timd-4 -/- mice. Adherent cells were matured into macrophages by incubating for 6 days in the presence of 50ng/mL MCSF. Matured macrophages were infected with the indicated infectious units of rVSV/EBOV GP and number of infected cells were quantified by GFP expression at 24 hour following infection by flow cytometry. D) Pmacs were harvested from C57BL/6 Ifnar-/- or Ifnar/Timd-4 -/- mice and incubated with VLPs expressing EBOV GP and EBOV VP40 fused to beta lactamase. Virus/cell membrane fusion was subsequently quantified by loading cells with CCF2-AM, a β-lactam-containing fluorescent substrate, and analyzed on flow cytometry to determine the relative amount of cleaved and un-cleaved substrate. Data are shown as mean ± S.D. Statistics were calculated using Student’s t-test, *p<0.05.