Figure 3.
Evidence for enhanced reactivation of TLR7 from the inactive X chromosome in pDCs of SSc women. (A and B) Representative RNA FISH analysis of pDCs from SSc women and HDs. Z-projection of 3D RNA FISH confocal microscopy planes of cell nuclei hybridized with fluorescent probes for transcripts arising from (A) TLR7 (red) and (B) TLR8 (green). Nuclei were counterstained with DAPI (gray). The arrowheads indicate TLR7 or TLR8 transcript foci occurring on a single X chromosome (monoallelic cells; left panels) or on both X chromosomes (biallelic cells; right panels). Scale bar, 1 µm. (C and D) Quantification of nuclei with one or two RNA FISH signals (mono + biallelic) in pDCs obtained from age-matched control HD (n = 16) and SSc women (n = 21) from four independent experiments (P#01 to P#04); see Table S1. (E) Frequency of TLR7 biallelic nuclei with reference to the total number of positive nuclei (mono + biallelic) in control HD and SSc women from P#01 to P#04 as described in C and D. Statistical differences were analyzed using a paired Student’s t test and indicated as **P < 0.01. (F) Quantification of allelic expression for TLR7 and TLR8 primary transcripts in pooled pDC nuclei from HDs and SSc patients from experiments P#01 to P#04 (C and D). Statistical differences were analyzed using a Fisher’s exact test and indicated as *P < 0.05. (G)TLR7 mRNA relative expression from pooled single-cell sorted pDCs from HDs (n = 500 cells, mean cell number/donor = 100, n = 5) and SSc patients (n = 732, mean cell number/donor = 134, n = 5). Statistical analysis was performed using a Mann–Whitney test and indicated as ***P < 0.001. (H) pDCs from HDs (n = 3) were single-cell sorted before and after overnight incubation with IFN-β (1 ng/ml) and TLR7 expression level was quantified by RT-qPCR as described (Abbas et al., 2022). Statistical differences were analyzed using a paired Student’s t test and is indicated as *P < 0.05, ***P < 0.001. (I–K) pDCs were purified from frozen PBMCs and cultured for 2 days with IL-3 (5 ng/ml) and then incubated with IFN-β (1 ng/ml) for 2 h and processed for RNA FISH analysis with TLR7 (red) and XIST (cyan) specific probes. Representative images showing TLR7 RNA signals colocalized (expressed from the Xi) or far from the XIST cloud (expressed from the Xa), in cells without (left panel) or with IFN-β stimuli (right panel). (J and K) (J) Quantification of TLR7 primary transcripts expression (mono + biallelic cells) and (K) robust XIST RNA cloud formation (Types I–II). (L and M) Sequential RNA-DNA FISH of TLR7 expression from the active (Xa) and/or XIST-coated inactive (Xi) chromosomes (RNA FISH) and localization within the X territory (DNA FISH) in female human pDCs. The left panels show single confocal sections of RNA FISH for TLR7 primary transcripts (red) and XIST RNA (cyan) and right panels show DNA FISH for the TLR7/8 locus (red) and the X chromosomes (green) in the same nuclei. Top row: example of a nucleus with TLR7 expressed from both the Xa (arrowhead) and XIST-coated Xi (white arrow) (biallelic). Bottom row: example of a nucleus with a TLR7 RNA signal from the Xa (arrowhead) but not from the Xi (yellow arrow) (monoallelic). DAPI is shown in gray. Scale bar = 2 µm. (N) Quantification of the Euclidean distance between the TLR7/8 locus and the edge of the nearest X-chromosome territory. n = 109 XIST+ nuclei across five fields of view with no, mono-, or biallelic expression of TLR7. Error bars represent ±SEM, statistical differences between groups were calculated using a one-way ANOVA with Sidack’s multiple comparison test. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Evidence for enhanced reactivation of TLR7 from the inactive X chromosome in pDCs of SSc women. (A and B) Representative RNA FISH analysis of pDCs from SSc women and HDs. Z-projection of 3D RNA FISH confocal microscopy planes of cell nuclei hybridized with fluorescent probes for transcripts arising from (A) TLR7 (red) and (B) TLR8 (green). Nuclei were counterstained with DAPI (gray). The arrowheads indicate TLR7 or TLR8 transcript foci occurring on a single X chromosome (monoallelic cells; left panels) or on both X chromosomes (biallelic cells; right panels). Scale bar, 1 µm. (C and D) Quantification of nuclei with one or two RNA FISH signals (mono + biallelic) in pDCs obtained from age-matched control HD (n = 16) and SSc women (n = 21) from four independent experiments (P#01 to P#04); see Table S1. (E) Frequency of TLR7 biallelic nuclei with reference to the total number of positive nuclei (mono + biallelic) in control HD and SSc women from P#01 to P#04 as described in C and D. Statistical differences were analyzed using a paired Student’s t test and indicated as **P < 0.01. (F) Quantification of allelic expression for TLR7 and TLR8 primary transcripts in pooled pDC nuclei from HDs and SSc patients from experiments P#01 to P#04 (C and D). Statistical differences were analyzed using a Fisher’s exact test and indicated as *P < 0.05. (G)TLR7 mRNA relative expression from pooled single-cell sorted pDCs from HDs (n = 500 cells, mean cell number/donor = 100, n = 5) and SSc patients (n = 732, mean cell number/donor = 134, n = 5). Statistical analysis was performed using a Mann–Whitney test and indicated as ***P < 0.001. (H) pDCs from HDs (n = 3) were single-cell sorted before and after overnight incubation with IFN-β (1 ng/ml) and TLR7 expression level was quantified by RT-qPCR as described (Abbas et al., 2022). Statistical differences were analyzed using a paired Student’s t test and is indicated as *P < 0.05, ***P < 0.001. (I–K) pDCs were purified from frozen PBMCs and cultured for 2 days with IL-3 (5 ng/ml) and then incubated with IFN-β (1 ng/ml) for 2 h and processed for RNA FISH analysis with TLR7 (red) and XIST (cyan) specific probes. Representative images showing TLR7 RNA signals colocalized (expressed from the Xi) or far from the XIST cloud (expressed from the Xa), in cells without (left panel) or with IFN-β stimuli (right panel). (J and K) (J) Quantification of TLR7 primary transcripts expression (mono + biallelic cells) and (K) robust XIST RNA cloud formation (Types I–II). (L and M) Sequential RNA-DNA FISH of TLR7 expression from the active (Xa) and/or XIST-coated inactive (Xi) chromosomes (RNA FISH) and localization within the X territory (DNA FISH) in female human pDCs. The left panels show single confocal sections of RNA FISH for TLR7 primary transcripts (red) and XIST RNA (cyan) and right panels show DNA FISH for the TLR7/8 locus (red) and the X chromosomes (green) in the same nuclei. Top row: example of a nucleus with TLR7 expressed from both the Xa (arrowhead) and XIST-coated Xi (white arrow) (biallelic). Bottom row: example of a nucleus with a TLR7 RNA signal from the Xa (arrowhead) but not from the Xi (yellow arrow) (monoallelic). DAPI is shown in gray. Scale bar = 2 µm. (N) Quantification of the Euclidean distance between the TLR7/8 locus and the edge of the nearest X-chromosome territory. n = 109 XIST+ nuclei across five fields of view with no, mono-, or biallelic expression of TLR7. Error bars represent ±SEM, statistical differences between groups were calculated using a one-way ANOVA with Sidack’s multiple comparison test. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

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