Distinct metabolic profiles of circulating plasmacytoid dendritic cells in systemic sclerosis patients stratified by clinical phenotypes

Abstract

Background: Plasmacytoid dendritic cells (pDCs) play a key role in systemic sclerosis (SSc) pathophysiology. However, despite the recognised importance of metabolic reprogramming for pDC function, their metabolic profile in SSc remains to be elucidated. Thus, our study aimed to explore the metabolic profile of pDCs in SSc and their potential contribution to the disease.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors and SSc patients. SCENITH™, a single-cell flow cytometry-based method, was applied to infer the metabolic profile of circulating pDCs from patients with SSc. pDCs (CD304⁺ Lin^-) at steady-state or stimulated with CpG A were analysed. Toll-like receptor (TLR)9 activation was confirmed by ribosomal protein S6 phosphorylation.

Results: Circulating pDCs from ten healthy donors and fourteen SSc patients were analysed. pDCs from anti-centromere antibody-positive (ACA⁺) patients displayed higher mitochondrial dependence and lower glycolytic capacity than those from anti-topoisomerase I antibody-positive (ATA⁺) patients. Furthermore, cells from both ACA⁺ patients and limited cutaneous SSc (lcSSc) patients showed a stronger response towards TLR9 activation than cells from ATA⁺, anti-RNA polymerase III antibody-positive (ARA⁺) or diffuse cutaneous SSc (dcSSc) patients.

Conclusions: An innovative single cell flow cytometry-based methodology was applied to analyse the metabolic profile of pDCs from SSc patients. Our results suggest that pDCs from ACA⁺ patients rely more on oxidative phosphorylation (OXPHOS) and are more responsive to external stimuli, whereas pDCs from ATA⁺ patients may exhibit a more activated or exhausted profile.

Keywords: Dendritic cells; Immunometabolism; Plasmacytoid dendritic cells; Scleroderma; Systemic sclerosis.

Fig. 1

Scheme representing the key steps of the methods used. PBMCs isolated from the blood of HCs and SSc patients were seeded in 96-well plates and treated with CpG A for 3 h, before 40-minute incubation with the SCENITH™ drugs – control (C), 2-deoxy-glucose (2-DG), oligomycin (O), harringtonine (H) and puromycin. Finally, stainings were performed and cells were analysed by flow cytometry

Fig. 2

The percentage of circulating pDCs in SSc patients changes with progression of the disease. (A) Identification of CD304⁺ Lin⁻ pDCs by flow cytometry. (B-K) The percentage of pDCs was obtained by flow cytometry analysis and comparisons were performed between patient groups. Data are expressed as median and IQR and each dot represents data from one donor. One-way ANOVA followed by Tukey’s multiple comparisons test (B, K) or unpaired t test (C-J), except for (E) where Mann-Whitney was performed. *p ≤ 0.05

Fig. 3

pDCs from HCs and SSc patients have no major differences in their metabolic profile. (A, B) Translation levels in pDCs after treatment with 2-deoxy-glucose (2-DG), oligomycin (O), harringtonine (H) and CpG A. Representative histograms for cells not TLR9-activated. (C) Translation and (D) S6 phosphorylation (MFI) levels. (E, F) SCENITH™ metabolic profiles. Data expressed as median and IQR; dots represent independent donors. Kruskal-Wallis test followed by Dunnet’s multiple comparisons test (A, B), one-way ANOVA followed by Sidak’s multiple comparisons test (C, D) and two-way ANOVA followed by Sidak’s multiple comparisons test (E, F) were used. **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001

Fig. 4

pDCs from lcSSc patients are more reactive to CpG A than cells from dsSSc. (A) p-S6 levels and (B) translation levels at basal and TLR9-activated conditions were assessed in pDC from SSc patients by flow cytometry. (C) Metabolic profiles. Dash line corresponds to 1. Data are expressed as median and IQR and each dot represents data from one donor. Statistical significance was tested with unpaired t test (A, B-right, C-mitochondrial dependence and glycolytic capacity) or Mann-Witney test (B-left, C-glucose dependence and FA/AA oxidation capacity). *p ≤ 0.05

Fig. 5

pDCs from ACA⁺ patients are more dependent on mitochondrial metabolism and have lower glycolytic capacity than cells from ATA⁺ patients. (A) SCENITH™ metabolic profiles, (B) translation levels, and (C) p-S6 levels were determined by flow cytometry for pDCs from patients with different patterns of SSc autoantibodies. Data are expressed as median and IQR and each dot represents data from one donor. Kruskal-Wallis test followed by Dunnet’s multiple comparisons test was used for (A), one-way ANOVA followed by Tukey’s multiple comparisons test for (B) and one-way ANOVA followed by Sidak’s multiple comparisons test for (C). *p ≤ 0.05, **p ≤ 0.01