Type I Interferons Direct Gammaherpesvirus Host Colonization
Fig 6
Effect of IFNAR blockade on MuHV-4 infection of MZ macrophages and WP B cells.
a. C57BL/6 mice were given an anti-IFNAR blocking antibody i.p. or not (control) then infected i.n. or i.p. with wild-type MuHV-4. Virus in lungs (i.n.) or spleens (i.p.) was titered by plaque assay after 4d. Circles show individuals, bars show means. The IFNAR blockade significantly increased virus titers in both sites, but the difference was significantly greater in spleens (p<0.01). b. Spleens of mice infected i.p. as in a were stained for MuHV-4 antigens. Nuclei were stained with DAPI. Dashed lines show approximate MZ / WP boundaries, based on CD169 staining of adjacent sections. IFNAR blockade increased MZ infection >100 fold and WP infection <5-fold. In the MZ >90% of viral antigen+ cells had typical myeloid cell rather than B cell morphology. c. Co-staining of a representative anti-IFNAR-treated, i.p.-infected mouse after 4d shows extensive viral antigen expression in CD169+ splenic MZ macrophages (arrows). Quantitation is shown in d. d. Quantitation of MZ and WP viral antigen staining for spleen sections from 5 mice per group, treated or not with anti-IFNAR antibody and infected i.p. as in a. Bars show group means, other symbols show mean counts for 3 sections per mouse. IFNAR blockade significantly increased viral antigen expression in all populations (p<0.05), most markedly in CD169+ MZ macrophages (p<0.01). e. Spleen sections of mice infected as in b, showing additional co-localization of viral antigen with CD206+ and F4/80+ macrophages after IFNAR blockade (arrows). Again IFNAR blockade significantly increased MuHV-4 antigen expression in these populations relative to no antibody controls (p<0.01). f. Mice were given anti-IFNAR antibody or not (control) and 2d later infected i.p. with MHV-GFP. Spleens were titered for infectious virus by plaque assay after 4d and for total reactivatable virus by infectious centre (IC) assay after 4 and 7d. Circles show individuals, horizontal bars show means. g. Spleens of mice infected as in f were analysed at d7 for viral eGFP and antigen expression in CD169+ macrophages and B220+ B cells. Bars show mean ± SEM of 5 mice. IFNAR blockade significantly increased viral eGFP expression in all populations (p<0.05), most markedly in WP B cells (p<10−4). h. Example images of mice infected as in f, stained for viral eGFP (green) and cell markers (red). Nuclei were stained with DAPI (blue). The dashed lines show approximate WP / MZ boundaries, based on CD169 staining. Arrows show example eGFP+ cells. Quantitation is shown in g.