6-Sulfo LacNAc monocytes are quantitatively and functionally disturbed in systemic sclerosis patients

Abstract

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis, microangiopathy, and autoantibodies. We previously reported that circulating follicular helper T (cTfh) cells are increased in SSc and induce plasmablast differentiation. However, mechanisms leading to cTfh cell expansion and activation in SSc remain to be established. Tfh cells require IL-12 for their expansion and differentiation. 6-Sulfo LacNAc monocytes (slanMo), a subset of monocytes, have a higher capacity to produce IL-12 and to induce CD4+ T cell proliferation in comparison with dendritic cells (DC) or classical monocytes. The aim of this study was to perform a quantitative and functional analysis of monocytes and DC and to correlate them with cTfh cell expansion and clinical manifestations in SSc. Using flow cytometry, we analyzed different monocyte subsets including slanMo and DC from 36 SSc patients and 26 healthy controls (HC). In vitro culture experiments of sorted slanMo were performed for functional analysis and cytokine production. We observed that slanMo, intermediate and non-classical monocytes were increased in SSc in comparison with HC. Furthermore, the increase in slanMo cells was more potent in patients with diffuse SSc. We observed a significant positive correlation between slanMo and cTfh cell levels in SSc patients but not in HC. Other monocyte subsets did not correlate with cTfh cell expansion. In addition, we observed that in vitro, slanMo cells from SSc patients produced less IL-12 than slanMo from HC. SlanMo are increased in SSc and may participate in the activation of cTfh cells in SSc.

Keywords: 6; LacNAc monocytes; follicular helper T cells; sulfo; systemic sclerosis.

Graphical Abstract

Figure 1

: monocyte and dendritic cell subset frequencies in SSc patients. (a) The gating strategy consisted of selecting CD45 cells. After exclusion of CD3+, CD19+, and CD56+, myeloid cells expressing HLA-DR were selected. Monocyte subsets were then characterized by their CD14/CD16 expression: classical monocytes (CD45⁺CD3⁻CD19⁻CD56⁻HLA-DR⁺CD14⁺CD16⁻), intermediate monocytes (CD45⁺CD3⁻CD19⁻CD56⁻HLA-DR⁺CD14⁺CD16⁺), non-classical monocytes (CD45⁺CD3⁻CD19⁻CD56⁻HLA-DR⁺CD14⁻CD16⁺), and slanMo (CD45⁺CD3⁻CD19⁻CD56⁻HLA-DR⁺CD14⁻MDC8⁺). Conventional dendritic cells (cDC) were characterized by CD1c (CD45⁺CD3⁻CD19⁻CD56⁻HLA-DR⁺CD14⁻CD1c⁺) and plasmacytoid dendritic cells (pDC) were characterized by CD123 and CD303 (CD45⁺CD3⁻CD19⁻CD56⁻HLA-DR⁺CD14⁻CD123⁺CD303⁺). (b, c). Frequencies of plasmacytoid dendritic cells (pDC), conventional DC (cDC), classical monocytes (class mono), intermediate monocytes (int mono), and non-classical monocytes (non-class mono), within PBMC from SSc patients (n = 36) and healthy controls (n = 26). (d). Frequencies of slanMo within PBMC from SSc patients (n = 36) and healthy controls (n = 26). (e). Frequencies of slanMo in SSc patients with limited SSc (n = 22) or diffuse SSc (n = 8). Data represent the mean with SD; *P < .05, **P < .01, ***P < 0.001 by Mann–Whitney U test.

Figure 2:

Slan monocytes from SSc patients correlate with cTfh cells. (a). Frequencies of cTFH CD4⁺CXCR5⁺ within CD3⁺ T cells in SSc patients (n = 36) and HC (n = 24). Data represent the mean with SD; *P < 0.05. Correlation of slanMo and cTfh cell frequencies in SSc patients (n = 34) (b) and in HC (n = 24) (c). The Spearman correlation test was used.

Figure 3:

Slan monocytes from SSc patients secrete less IL-12. Expression of CD86 mean fluorescence intensity (MFI) by monocyte subsets in SSc patients (n = 36) and in HC (n = 24); (a) representative histograms plots for one SSc patients and one HC; (b) data represent the mean with SD. IL-12 concentration (c) or IL-10 concentration (d) in supernatants of slanMo isolated from SSc patients (n = 10) and HC (n = 5). Sorted slanMo were kept for 12 h in culture medium with or without IFN-γ, then cultured for 12 h in the presence or not of R848 as indicated. Data represent the mean with SD; *P < 0.05.