Fig 1.
IFNαβ induction by and effect of IFNαβ induction on MuHV-4 infection in vitro and in vivo.
a. RAW-264 monocyte/macrophages were infected or not with MuHV-4 (2 p.f.u. / cell) then cultured ± poly(I:C) as a positive control of IFN-I induction (50μg/ml). Cell supernatants pooled from triplicate cultures were assayed for IFNβ by ELISA 1 and 2d later. The dashed line shows the limit of assay sensitivity. A replicate experiment gave equivalent results. b. RAW264 cells were infected and cultured ± poly(I:C) to induce IFN-I as in a. Supernatants were assayed for infectious virus by plaque assay. Symbols show mean ± SEM of triplicate cultures. IFN-I induction significantly reduced virus titers from d1 onwards (p<0.03). c. Mice were given i.n. MuHV-4 (3x104 p.f.u.), with or without poly(I:C) inoculation (50μg i.p. and i.n., 6h before and at the time of virus inoculation) as a positive control of IFN-I induction. After 1d bronchial washes were assayed for IFNβ by ELISA. Each point shows 1 mouse. Bars show mean ± SEM. The dashed line shows the limit of assay sensitivity. Poly(I:C) significantly increased IFNβ production in infected mice (p<0.01). d. Mice were infected i.n. or not with MuHV-4 with or without poly(I:C) treatment as in c. 1 and 3d later Mx1 mRNA in lungs was quantitated by real time PCR and normalized by cellular nidogen-1 copy number. Numbers show the fold increase in Mx1 copy number of treated mice over the untreated controls (dashed horizontal line). Bars show mean ± SEM of 3 mice. Mx1 copies were significantly increased by poly-IC at d1 (p<0.05) but not at d3. e. Mice were infected i.n. with MHV-LUC, and treated or not with poly(I:C) as in c. Lung infection was tracked by luciferin injection and live imaging of light emission. Circles show individual mice, bars show means. Poly(I:C) significantly reduced luciferase counts at d3 but not at d2 or d4. f. Mice were infected i.n. with MuHV-4 and treated or not with poly(I:C) to induce IFN-I as in c. Lungs were titered for infectious virus by plaque assay. Circles show individual mice, bars show means. Poly(I:C) significantly reduced infection at d3 but not at other time points. g. Mice were given an anti-IFNAR blocking antibody or not (200μg i.p.) 24h before and poly(I:C) or not (50μg i.p. and i.n.) 6h before i.n. infection with MuHV-4 (3x104 p.f.u.). 3d later lungs were titered for infectious virus by plaque assay. Cont = virus only. Crosses show means, other symbols show individual mice. Poly(I:C) significantly reduced titers (p<0.01) and this effect was reversed by anti-IFNAR antibody.
Fig 2.
MuHV-4 propagation through IFNαβ-responding cells.
a. Mx1-cre mice were given MHV-4-RG i.n. (3x104 p.f.u. in 30μl under anesthesia). Viruses were recovered from lungs by plaque assay and from lymphoid tissue by intact cell explant onto BHK-21 cell monolayers. Plaques were then typed as eGFP+ (switched) or mCherry+ (unswitched). For each mouse (circles), % eGFP+ = % switched of total recovered plaques. Crosses show means. MLN = mediastinal lymph nodes. b. Mice were given MHV-RG i.n. (3x104 p.f.u. in 5μl without anesthesia) to infect just the nose, then analysed for viral fluorochrome switching as in a. SCLN = superficial cervical lymph nodes. Viruses were recovered from noses by plaque assay and from SCLN by intact cell explant (infectious centre assay). c. Mice were given i.n. or i.p. MHV-RG, with or without poly(I:C) (50μg i.n. or i.p. 6h before and at the time of infection) to maximally induce IFN-I. Viruses recovered 3d later from lungs by plaque assay (i.n.) or from spleens by infectious centre assay (i.p.) were analysed for fluorochrome expression as in a. d. Mice were infected i.n. (lung) or i.p. (spleen) and given poly(I:C) or not as in c. Virus titers were determined 3d later by plaque assay. Circles show individuals, crosses show means. e. Mx1-cre mice were depleted or not of pDCs with mAb 120G8, then infected i.p. with MuHV-RG (105 p.f.u.). 5d later spleens were titered for total recoverable virus by explant of intact splenocytes onto BHK-21 cells. Infectious centres (ICs) were also typed as mCherry+ (unswitched) or GFP+ (switched). % eGFP+ = % of total plaques that were switched. Bars show mean ± SEM, other symbols show individual mice. pDC depletion had no significant effect.
Fig 3.
Viral fluorochrome switching in infected lung and spleen cells.
a. Mx1-cre mice were given i.n. poly(I:C) (50μg) to induce IFN-I 6h before and at the time of i.n. infection with MHV-RG (3x104 p.f.u.). 5d later lung sections were stained for viral eGFP / mCherry, and the cell markers podoplanin (PDP, AEC1s), CD68 (macrophages) and CD169 (AMs). Each image is representative of 5 mice. Arrows show example positive cells. Quantitation is shown in b. The cytoplasmic rather than nuclear distribution of eGFP is typical for the relatively mild fixation of periodate-lysine-1% formaldehyde. b. Lung sections of mice infected as in a (5 per group) were analysed at d4-6, counting eGFP+ (green) and mCherry+ (red) PDP+ and CD68+ cells for 3 sections per mouse. Each section corresponds to approximately 10 fields of view as illustrated in a. Bars show mean ± SEM. At d5 CD68+ cells showed significantly more fluorochrome switching than PDP+ cells (p<0.01). c. Mx1-cre mice were infected i.p. with MHV-RG (105 pfu), and eGFP+ (green) and mCherry+ (red) MZ macrophages (CD169+) and B cells (B220+) counted 4d later. Bars show mean ± SEM counts for 3 sections each of 3 mice per group. B220+ cells were significantly more switched than unswitched, whereas CD169+ were significantly more unswitched than switched (p<0.02). d. Example images of mice infected as in c. Arrows show positive cells. e. Mx1-cre mice were infected i.p. with MHV-RG (105 pfu), as in d, but poly(I:C) (50μg i.p.) was given 6h before and at the time of infection to test the effect of increased IFN-I induction. Spleen sections were analysed at d4 for viral eGFP and mCherry expression in B220+ B cells and CD68+CD169+ MZ macrophages. Arrows show example positive cells. Quantitation is shown in f. f. Mean ± SEM counts are shown for 3 sections each of 5 mice per group, analysed as in e at d3-5 post-infection. Each field of view was 10x the area of the images shown in e, and we counted at least 10 fields of view per section (at least 200 viral fluorochrome+ cells). B220+ cells showed significantly more switched than unswitched fluorochrome expression at d3 and d4 of infection (p<10−4). CD68+ cells did not (p>0.5). g. Mice were infected as in e and infected MZ B cells identified by staining for IgM. WP B cells express lower levels of IgM and while this is detectable by flow cytometry, by confocal microscopy as used here, WP B cells are IgD+IgM- [3]. >90% of infected MZ B cells were eGFP+, confirming the results of e-f.
Fig 4.
IFNαβ responses measured by Mx1-cre activation of ROSA26-YFP.
a. Mx1-cre x ROSA26-YFP mice were infected i.n. with MuHV-4 (3x104 p.f.u.). IFN-I was induced or not with poly(I:C) (50μg i.n. and i.p., 6h before and at the time of infection). Lungs were harvested at d3 and d5 and stained for YFP, CD68 and MuHV-4 antigens. Nuclei were stained with DAPI. White arrows show example YFP+CD68+MuHV-4+ AMs; open arrows show YFP+CD68+ AMs; Grey-filled arrows show YFP-MuHV-4+ AEC1s. No AEC1s were YFP+. All YFP+ cells were AMs (n>30). Images are representative of 5 sections from each of 3 mice per group. b. Mx1-cre x ROSA26-YFP mice were infected i.p. with MuHV-4 (105 p.f.u.). IFN-I was induced or not with poly(I:C) (50μg i.p., 6h before and at the time of infection). D5 spleen sections were stained for YFP and CD169 (MZ macrophages), F4/80 (RP macrophages) or B220 (B cells). Nuclei were stained with DAPI and the cells visualized by confocal microscopy. Arrows show example YFP+ cells expressing the relevant cellular marker. For CD169 staining a dashed line shows the boundary between the B cell-dominated WP and the macrophage-dominated MZ, with CD169+ MZ macrophages lying adjacent to WP B cells. For B220 staining after virus + poly(I:C), separate channels are shown to make clear the extensive YFP expression in B cells. Images are representative of 5 sections from each of 3 mice per group. >50% of each macrophage population was YFP+, with no difference between poly-IC treated and untreated. In WP follicles, 10–50% of B220+ B cells were YFP+ after poly(I:C) treatment and 5–20% were YFP+ without poly(I:C) treatment. Quantitation for IgM+ B cells is shown in e. c. Naïve C57BL/6 and Mx1-cre x ROSA26-YFP spleen sections were stained for YFP, B220 and F4/80. YFP expression was evident in >80% of F4/80+ macrophages and <5% of B220+ B cells. Dashed lines show the MZ demarcation between F4/80+ RP macrophages and B220+ WP B cells. d. Mx1-cre x ROSA26-YFP mice were infected as in b. Spleen sections were stained for YFP and IgM to identify MZ B cells, and visualized by epifluorescence microscopy. Arrows show example YFP+IgM+ cells. e. Quantitation of YFP+IgM+ spleen cell numbers across 3 sections from each of 2 mice per group (5 fields of view per section). YFP expression in IgM+ B cells was significantly induced by both infection and poly(I:C). Bars show means, other symbols show individual mice.
Fig 5.
MuHV-4 ORF36 disruption reduces mainly splenic infection.
a. Mice were infected i.n. with wild-type (WT) or ORF36- MuHV-4 or an independently derived ORF36 mutant (ORF36-ind) (3x104 p.f.u.). IFN-I was induced or not with poly(I:C) (50μg i.p. and i.n. 6h before and at the time of infection). Lungs were titered for infectious virus by plaque assay at d5. Crosses show means, other symbols show individual mice. WT titers were not significantly affected by poly(I:C) treatment (p>0.5) whereas ORF36- titers were significantly reduced (p<0.01). b. Mice were given WT or ORF36- MuHV-4 i.p. (105 pfu). Explanting intact spleen cells onto BHK-21 cell monolayers yielded significantly fewer ORF36- than WT infectious centres (ICs) at d3 and d5 (p<0.001). Circles show individual mice, crosses show means. c. Mice were given ORF36- or WT MuHV-4 i.p. as in b. At d4, spleen sections were stained for viral antigens (green) and cellular markers (red). Arrows show example infected RP (F4/80+) and MZ macrophages (CD169+), with most ORF36- infection being in the RP. d. Quantitation of staining as in c showed significantly more WT than ORF36- infection in the WP and MZ, and significantly less in the RP (p<0.03). Bars show mean ± SEM (3 sections each of 5 mice per group).
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.
Fig 7.
Mx1-cre-dependent fluorochrome switching of MCMV.
a. Mx1-cre mice were infected i.p. with MCMV-GR, which switches fluorochrome expression when exposed to cre recombinase. IFN-I was induced or not by i.p. poly(I:C) inoculation (pIC, 50μg / mouse) 6h before and at the time of infection. 5d later virus in livers and spleens was titered by plaque assay. Circles show individuals, bars show means. Poly(I:C) had no significant effect on titers (p>0.5). b. The samples from a were assayed for viral fluorochrome switching by identifying plaques as eGFP+ or tdTomato+. c. Example image from a MCMV-GR-infected and poly(I:C)-treated spleen shows eGFP+ and tdTomato+ cells around a WP follicle. Quantitation is shown in d. d. Quantitation of eGFP+ and tdTomato+ cells on liver and spleen sections, pooled from 3 mice per group infected as in a. Bars show means, other symbols show individual sections. Poly(I:C) significantly increased infected cell fluorochrome switching in livers (p<0.01) but not in spleens (p>0.5). e. Example image of a MCMV-GR-infected, poly(I:C)-induced, liver showing only tdTomato+ cells that do not co-localize with CD68. Two other livers gave equivalent results.