Fig 2: PBMC gene expression of TLR7 was higher in post-pubertal females. In healthy volunteers, PBMC gene expression was assessed by Nanostring and normalized gene transcript counts are shown (n = 50). p-values represent significance of the coefficient for the variables shown after correcting for the other variable (sex or puberty). (A) There was an overall difference in TLR7 expression (p = 0.016). After post hoc testing with Bonferroni correction, post-pubertal females had a higher TLR7 expression than post-pubertal males (p = 0.024) and pre-pubertal females (p = 0.03) (B) PBMC TLR9 gene expression was lower in post-pubertal volunteers. (C) There were no pubertal (p = 0.274) differences in the PBMC IFN score. (D) After stimulation with IFNa, the IFN score (post stim IFN score) was significantly higher in post pubertal volunteers (p = 0.001, n = 33). *p < 0.05; **p < 0.005.

Fig 3: Supernatants from co-cultures of M-CSF MΦ and HCMV-infected RPE cells inhibit viral spread.Cell-free supernatants from uninfected or HCMV-infected RPE (MOI 12) or M-CSF-MΦ (MOI 3) mono-cultures and RPE/M-CSF-MΦ 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-α 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-α2b, IFN-γ, or TNF-α 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 4: IL-36 enhances the production of IFN-a downstream of toll-like receptor 9. (a) PBMCs were stimulated with CpG for 6 hours, in the presence or absence of IL-36 pretreatment (6 hours). The expression of IFN signature genes was measured by real-time PCR. Data represent the mean ± SEM of results obtained in three independent donors, each stimulated in triplicate. (b) After PBMC stimulation, IFN-a production was measured by ELISA. Data represent the mean ± SEM of results obtained in two independent donors, each stimulated in triplicate. (c) After PBMC stimulation, the percentage of IFNa+ pDCs was determined by flow cytometry. A representative set of plots is shown (left), together with the data obtained in three independent healthy donors (right). *P < 0.05; **P < 0.01 (Friedman test, with Dunn posttest). CpG, CpG-containing DNA; PBMC, peripheral blood mononuclear cell; pDC, plasmacytoid dendritic cell; SEM, standard error of the mean.

Fig 5: IL-36 upregulates PLSCR1. (a) After treatment of PBMCs with IL-36, PLSCR1 expression was measured by real-time PCR. (b) After IL-36 treatment of pDCs, PLSCR1 MFI was measured by flow cytometry, in gated PSLCR1+ pDCs. A representative histogram is shown on the left. (c) After pretreatment with SB203580 (MAPKi), PBMCs were stimulated with IL-36. PLSCR1 expression was then determined by real-time PCR. (d) Proposed pathogenic model. IL-36 produced by mDC upregulates PLSCR1 in pDCs, potentiating TLR-9 dependent IFN-a release. IFN-a induces further PLSCR1 transcription, thus propagating an inflammatory feed-forward loop. All data are shown as mean ± SEM of results obtained in at least three donors, each stimulated in triplicate. *P < 0.05 (Wilcoxon signed rank test [a, b] and Friedman test with Dunn posttest [c]) mDC, myeloid dendritic cell; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cell; pDC, plasmacytoid dendritic cell; TLR, toll-like receptor; SEM, standard error of the mean.