Fig 2: Supernatants from co-cultures of M-CSF MF and HCMV-infected RPE cells inhibit viral spread.Cell-free supernatants from uninfected or HCMV-infected RPE (MOI 12) or M-CSF-MF (MOI 3) mono-cultures and RPE/M-CSF-MF co-cultures were harvested 2 dpi and transferred onto fresh RPE cells infected with HCMV-GFP at MOI 0.1. (a) 8 dpi virus plaque formation was analyzed by fluorescence microscopy (scale bar = 1 mm) and (b) the size of individual plaques was determined. Cell-free supernatants prepared as described above were analyzed for (c) secreted IFN-a and (d) IFN-I activity using an ELISA method or an Mx2-Luc reporter cell line, respectively. (e) RPE cells were infected with HCMV-GFP at MOI 0.1 and incubated with recombinant (rec.) IFN-a2b, IFN-?, or TNF-a for 8 d. Then the plaque size was analyzed. (f) Cell-free supernatants prepared as described above were analyzed for different secreted proteins using a bead-based cytokine array. Data visualize log (2) values of the measured cytokine concentrations in [pg/ml]. Mean size of (b) 67–371 plaques using 6 different donors or (e) 49–100 plaques from 3 independent experiments. Mean ±SEM of (c/d) 6 different donors from 3 independent experiments or data of (f) 3 different donors from 2 independent experiments. SN = supernatant, uninf. = uninfected, *: p = 0.05, **: p = 0.01, ***: p = 0.001 (one-tailed Mann-Whitney test).

Fig 3: Co-culture of monocyte-derived cells with HCMV-infected RPE cells reduces viral spread.RPE cells were infected with HCMV-GFP at MOI 0.1, washed, and co-cultured with moDC, GM-CSF MF, or M-CSF MF at a 1:4 ratio. (a) 10 dpi HCMV plaque formation was analyzed by fluorescence microscopy (scale bar = 100 µm). (b) 6, 8, and 10 dpi the size of individual HCMV plaques was determined by using ImageJ software. (c) Secreted IFN-a was monitored by ELISA analysis of cell-free supernatants. HUVEC were infected with HCMV-GFP at MOI 1, co-cultured with M-CSF MF, and 10 dpi (d) HUVEC were analyzed for percentages of GFP expressing cells by flow cytometry and (e) supernatants were analyzed for the amount of infectious virus particles. RPE cells were infected with RV-TB40-BACKL7-SE-EGFP (RV-HCMV), co-cultured with M-CSF MF, and 13 dpi (f) the size of individual HCMV plaques was determined and (g) supernatants were analyzed for the amount of infectious virus particles. Data in (d/e/g) show values as percent of control infections in HUVEC or RPE mono-cultures. Mean ±SEM of (b/c) 4–7 ((b) 26–102 plaques analyzed), (d/e) 4, and (f/g) 6 ((f) 212–368 plaques analyzed) different donors from 2–3 independent experiments. DL = detection limit, dpi = days post infection, *: p = 0.05, ***: p = 0.001 (one-tailed Mann-Whitney test).

Fig 4: Co-culture of myeloid cells with HCMV-infected RPE cells induces enhanced IFN-a responses by pDC, but not by monocyte-derived cells.pDC, moDC, GM-CSF MF (GM-MF), or M-CSF MF (M-MF) were directly infected with HCMV-GFP at MOI 3. Furthermore, RPE cells were infected with HCMV at MOI 12, washed to remove cell-free virus, and co-cultured with myeloid cell subsets at a 1:4 ratio for 24 h and (a) percentages of intracellular IFN-a+ myeloid cells or (b) the IFN-a content in cell-free supernatants was determined by flow cytometry or an ELISA method, respectively. (c) RPE cells were infected with HCMV-GFP (MOI 12), washed, and co-cultured with M-CSF MF either from day (d) 0–4 post infection or from d 3–4 post infection and (d) percentages of intracellular IFN-a+ M-CSF MF, (e) HCMV-GFP+ M-CSF MF, (f) HCMV-GFP+ RPE cells, or (g) infectious HCMV particles in the supernatant of such cultures were determined. Mean ± SEM of (a) 4–8, (b) 3–4, or (d-g) 4 different donors from 2–4 independent experiments. inf. = infected, ns = not significant, *: p = 0.05, **: p = 0.01 (one-tailed Mann-Whitney test).

Fig 5: Co-cultures of macrophages and infected RPE cells incubated for four days show IFN-I independent inhibition of HCMV propagation.(a) Different concentrations of B18R were added to rec. IFN-a2b [100 U/ml] and after 1 h incubation the IFN-I activity was determined using an Mx2-Luc reporter cell line. (b) Co-cultures of M-CSF-MF and HCMV-infected RPE cells were set up, treated with 100 ng/ml B18R, and 0, 24, and 48 h later cultures were stimulated with 100 U/ml rec. IFN-a2b for 4 h and then Mx induction was analyzed by qPCR. (c-e) RPE cells were infected with HCMV-GFP at MOI 12, washed, and co-cultured with M-CSF MF from d 0–4 or d 3–4 post infection with (black symbols) or without (grey symbols) the daily addition of 100 ng/ml B18R. (c) Percentages of intracellular IFN-a+ M-CSF-MF, (d) HCMV-GFP+ RPE cells, or (e) infectious HCMV particles in the supernatants of such cultures were determined. Mean ±SEM of (a) 3 independent experiments and (b) 3 or (c/d/e) 4 different donors from 2 independent experiments. inf. = infected, *: p = 0.05 (one-tailed Mann-Whitney test, (b)), ns = not significant (p = 0.29) (one-tailed Wilcoxon signed rank test (c/d/e)).