Fig 2: SAMPs activate the TLR8/MyD88/NF-κB axis in moDCs.(A) HEK293 cells stably transfected with human TLR7, TLR8, or luciferase alone (-) were stimulated with SCV2-RNA for 24 hours. NF-κB activation was evaluated as luciferase activity. Data are expressed as mean ± SEM (n = 3); *P < 0.05 versus (-) by 1-way ANOVA with Dunnett’s post hoc test. (B–E) moDCs were stimulated with SCV2-RNA as indicated (B) or pretreated with CU-CPT9a (1 μM) and then stimulated with SCV2-RNA or LPS for 1 hour (E). Nuclear extracts were blotted against NF-κB p65 and lamin B. One representative donor and densitometry of 3 donors are shown. *P < 0.05 versus untreated by 1-way ANOVA with Dunnett’s post hoc test; #P < 0.05 versus “SCV2-RNA” by paired Student’s t test. (C, left panel) moDCs were transfected with indicated siRNAs and target gene expression evaluated by qPCR. Results depict percentage of target gene expression (mean ± SEM n = 4). (C, right panel) moDCs transfected with indicated siRNAs were stimulated with SCV2-RNA for 24 hours and IL-6 production evaluated by ELISA. Data are expressed as percentage of production (n = 4); *P < 0.05 versus “ctr siRNA” by 1-way ANOVA with Dunnett’s post hoc test. (D) moDCs were transfected with indicated siRNAs and the expression of TLR8 was evaluated by qPCR (left y axis, white bars). IL-6 production upon SCV2-RNA stimulation was evaluated by ELISA (right y axis, gray bars). Data (percentage of expression/production) represent the mean ± SEM (n = 3); *P < 0.05 versus respective “ctr” by paired Student’s t test. (F) moDCs were pretreated with CU-CPT9a, then stimulated as indicated for 24 hours and IL-6 production evaluated by ELISA. Data are expressed as percentage of production for each individual stimulation (n = 3); *P < 0.05 versus respective “0” by 1-way ANOVA with Dunnett’s post hoc test.

Fig 3: PRP-1 effect on TLR receptors and adaptor proteins in human chon-drosarcoma JJ012 cell line. PRP-1 upregulated protein expression of TLR1 and TLR2 in dose-dependent manner in human JJ012 chondrosarcoma cell lysates. TLR3 expression was very weak and the data is not shown. TLR4, TLR5 proteins were expressed but PRP-1 did not have any effect. Tubulin was used as loading control. The bands were detected for TLR1, TLR2, TLR4 and TLR5 and tubulin at 120, 119, 130, 90 and 50 kDa, correspondingly. PRP-1 upregulated protein expression of TLR6 in dose-dependent manner in human JJ012 chondrosarcoma cell lysates. TLR7 was not expressed in this cell line at all. TLR8, 9 and 10 were expressed but no effect of PRP-1 was observed. Tubulin was used as housekeeping control. The bands were detected for TLR6, TLR8, TLR9, TLR10 and tubulin at 100, 86, 100, 120 and 50 kDa, correspondingly. PRP-1 upregulated TICAM2 (TRAM) adaptor protein in dose-response manner but did not have any effect on TICAM1 (TRIF) adaptor protein in human JJ012 chondrosarcoma cell line. Tubulin was used as loading control. The bands were detected for TICAM1, TICAM2 and tubulin.

Fig 4: Regulated expression of TLR7 in human macrophages. a Schematic representation of the in vitro generation of M-MØ and GM-MØ from peripheral blood monocytes. TLR7 gene expression (normalized fluorescence intensity) in M-MØ and GM-MØ, as determined in microarray experiments (GSE68061) on three independent samples. Statistically significant differences (adjp) are indicated. b TLR7 protein levels in four independent preparations of M-MØ and GM-MØ, as determined by Western blot (left panel). Relative level of TLR7, including both the full-length (140 kDa) and the proteolytically processed mature (75 kDa) forms, were determined in M-MØ and GM-MØ by densitometry. Mean ± SEM of four independent samples is shown (***p < 0.001). c Relative TLR7 gene expression in Monocytes, M-MØ and GM-MØ, as determined by RNA-sequencing (GSE188278) on three independent samples. Statistically significant differences are indicated. dTLR7 gene expression in M-MØ before and after exposure to GM-CSF (1,000 U/mL) for 24 or 48 h, and either with or without medium replacement, as determined by RT-PCR. Mean and SEM of four independent experiments are shown (**p < 0.005). e TLR7 gene expression in M-MØ generated from control individuals (2 independent donors) or an MCTO patient (two distinct samples derived from a single MCTO patient), as determined by RNA-sequencing (GSE155883). Statistically significant differences are indicated. fTLR7 gene expression in M-MØ and GM-MØ before and after LPS activation, as determined in microarray experiments (GSE99056) on three independent samples. Statistically significant differences (adjp) are indicated. g t-Distributed stochastic neighbor embedding plots (t-SNE, Perplexity:25, Color plot by k = 10) illustrating TLR7, MAFB, and FOLR2 gene expression in the human fetal-maternal interface [58] (https://www.ebi.ac.uk/gxa/sc/home). The “Expression level” bar indicates the expression level in each case (Smartseq 2 data; CPM, counts per million).

Fig 5: The acquisition of the TLR7-specific transcriptome and neutrophil-recruiting chemokine production depends on the macrophage polarization state. a Schematic representation of siRNA-mediated gene silencing before treatment of M-MØ with CL264 (1 μg/mL) for determination of the gene expression profile. b Summary of GSEA of the Clusters identified in Fig. 4 on the ranked comparison of the transcriptomes of CL264-treated (4 h) siAHR M-MØ (siAHR M-MØ + CL264 [left panel]), siMAF M-MØ (siMAF M-MØ + CL264 [middle panel]), or siMAFB M-MØ (siMAFB M-MØ + CL264 [right panel]) versus siCNT M-MØ + CL264. FDRq value is indicated in each case. c Relative expression of the indicated chemokine-encoding genes in CL264-treated (4 h) siAHR M-MØ, siMAF M-MØ, and siMAFB M-MØ (log2FC [siTF-M-MØ + CL264/siCNT-M-MØ + CL264]), as determined by RNA-Seq (*, adjp < 0.05). d Schematic representation of siRNA-mediated gene silencing before treatment of M-MØ with CL264 (1 μg/mL) for determination of the cytokine profile. e Production of the indicated chemokines in untreated M-MØ (−), siCNT M-MØ + CL264, siAHR M-MØ + CL264, siMAF M-MØ + CL264, and siMAFB M-MØ + CL264, as determined by ELISA. Data represent the production of chemokines by each cell type relative to the levels produced by siCNT M-MØ + CL264. Mean ± SEM of three independent donors are shown (*p < 0.05; **p < 0.01).