Natural Killer Cells Exhibit a Peculiar Phenotypic Profile in Systemic Sclerosis and Are Potent Inducers of Endothelial Microparticles Release

Abstract

The pathophysiology of systemic sclerosis (SSc) involves early endothelial and immune activation, both preceding the onset of fibrosis. We previously identified soluble fractalkine and circulating endothelial microparticles (EMPs) as biomarkers of endothelial inflammatory activation in SSc. Fractalkine plays a dual role as a membrane-bound adhesion molecule expressed in inflamed endothelial cells (ECs) and as a chemokine involved in the recruitment, transmigration, and cytotoxic activation of immune cells that express CX3CR1, the receptor of fractalkine, namely CD8 and γδ T cells and natural killer (NK) cells. We aimed to quantify circulating cytotoxic immune cells and their expression of CX3CR1. We further investigated the expression profile of NK cells chemokine receptors and activation markers and the potential of NK cells to induce EC activation in SSc. We performed a monocentric study (NCT 02636127) enrolling 15 SSc patients [15 females, median age of 55 years (39-63), 11 limited cutaneous form and 4 diffuse] and 15 healthy controls. Serum fractalkine levels were significantly increased in SSc patients. Circulating CD8 T cells numbers were decreased in SSc patients with no difference in their CX3CR1 expression. Circulating γδ T cells and NK cells numbers were preserved. CX3CR1 expression in CD8 and γδ T cells did not differ between SSc patients and controls. The percentage and level of CX3CR1 expression in NK cells were significantly lowered in SSc patients. Percentages of CXCR4, NKG2D, CD69-expressing NK cells, and their expression levels were decreased in NK cells. Conversely, CD16 level expression and percentages of CD16⁺ NK cells were preserved. The exposure of human microvascular dermic EC line (HMVEC-d) to peripheral blood mononuclear cells resulted in similar NK cells degranulation activity in SSc patients and controls. We further showed that NK cells purified from the blood of SSc patients induced enhanced release of EMPs than NK cells from controls. This study evidenced a peculiar NK cells phenotype in SSc characterized by decreased chemokine and activation receptors expression, that might reflect NK cells involvement in the pathogenic process. It also highlighted the role of NK cells as a potent mechanism inducing endothelial activation through enhanced EMPs release.

Keywords: CX3CR1; endothelial microparticles; fractalkine; natural killer cells; systemic sclerosis.

Figure 1

Number of circulating cytotoxic immune cells in systemic sclerosis (SSc) patients. Percentages of circulating immune cells were assessed in the peripheral blood of SSc patients (n = 15) with flow cytometry and compared with healthy controls (HCtl) (n = 14). Absolute numbers of circulating cells including CD8 T cells (A); natural killer (NK) cells (B); NK cells CD56^dim (C); γδ T cells (D); Vδ1 (E); and Vδ2 T cells (F) were obtained based on the white blood lymphocyte count and expressed as number of cells per mm³. Results were expressed as median ± interquartile range. Statistical difference was established using Mann–Whitney U test. *p < 0.05.

Figure 2

Soluble fractalkine levels in systemic sclerosis (SSc) patients. Soluble fractalkine levels were quantified with ELISA, in the serum of SSc patients (n = 15) and healthy controls (HCtl) (n = 15). Results were expressed as median ± interquartile range and statistical significance was established using the non-parametric Mann–Whitney U test. ***p < 0.001.

Figure 3

CX3CR1 expression in circulating cytotoxic immune cells from systemic sclerosis (SSc) patients. Percentages of CX3CR1 expressing immune cells were evaluated with flow cytometry analysis on the surface of CD8 T cells (A); γδ T cells (B); Vδ1 (C); and Vδ2 T cells (D) from SSc patients (n = 15) in comparison with healthy controls (HCtl) (n = 15). Results were expressed as median percentages and median ± interquartile range.

Figure 4

Chemokine receptors expression in natural killer (NK) cells in systemic sclerosis (SSc) patients. CX3CR1 (A,B) and CXCR4 expression levels and percentages (C,D) were assessed with flow cytometry analysis in whole NK cell from SSc patients (n = 15) in comparison with healthy controls (HCtl) (n = 15). Results were expressed as percentages of positive cells among NK cells (A,C) and median fluorescence intensity (B,D). Results were depicted as median ± interquartile range. Statistical difference was established using Mann–Whitney U test. **p < 0.005.

Figure 5

Activation receptors and markers in natural killer (NK) cells of systemic sclerosis (SSc) patients. NKG2D (A), DNAM-1 (B), CD69 (C), and CD16 (D) expression levels were evaluated with flow cytometry analysis of NK cells among peripheral blood mononuclear cells from SSc patients and healthy controls (HCtl). Results were expressed as median fluorescence intensity (MFI) and median ± interquartile range. Statistical difference was established using Mann–Whitney U test. *p < 0.05; ***p < 0.001.

Figure 6

Natural killer (NK) cells degranulation of systemic sclerosis (SSc) patients toward microvascular endothelial cell (EC) target. Cytolytic degranulation of NK cells was assessed by flow cytometry analysis of CD107a/b expression of NK cells among peripheral blood mononuclear cells (PBMCs) after a 4-h coculture with human microvascular dermal ECs (HMVEC-d) with an effector/target ratio of 2/1. (A) PBMCs from patients with SSc (n = 6) and healthy controls (HCtl) (n = 6) were cultured with HMVEC-d with or without thymoglobulin (ATG). (B) PBMCs from SSc patients (n = 6) and HCtl (n = 6) were cultured with HMVEC-d and either SSc serum or HCtl serum and conversely. Cumulative data from five independent experiments. Results were expressed as median percentages ± interquartile range. Statistical significance was established using the non-parametric paired Wilcoxon U-test. *p < 0.05.

Figure 7

Endothelial microparticles (EMPs) release from microvascular endothelial cells (ECs) induced by natural killer (NK) cells from systemic sclerosis (SSc) patients. EMPs were obtained from the supernatant of overnight-cultured human microvascular dermal EC (HMVEC-d) line. (A) HMVEC-d were used as ECs targets and cultured with medium (med), i.e., EBM2 + 25% FCS ± IFNγ (50 ng/ml)/TNFα (20 ng/ml) or serum of SSc patients or healthy controls (HCtl) ± their autologous peripheral blood mononuclear cells (PBMCs) (n = 10 and n = 9, respectively) with a PBMCs/HMVEC-d target ratio of 1/1. (B) HMVEC-d were cultured with EBM2 medium with serum from SSc patients or healthy controls and purified NK cells (pur. NK) from SSc patients (n = 10) or healthy controls (n = 9–10) with a NK cells/HMVEC-d ratio of 2/1. Results were expressed as median number of EMPs per microliter ± interquartile range. Cumulative data from 10 independent experiments. Statistical significance was established using the non-parametric paired Wilcoxon U-test. *p < 0.05, **p < 0.005.