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. 2014 Dec;178(3):428-37.
doi: 10.1111/cei.12429.

Interferon regulatory factor 3 as key element of the interferon signature in plasmacytoid dendritic cells from systemic lupus erythematosus patients: novel genetic associations in the Mexican mestizo population

K Santana-de Anda  1 , D Gómez-Martín, A E Monsivais-Urenda, M Salgado-Bustamante, R González-Amaro, J Alcocer-Varela
Affiliations

Interferon regulatory factor 3 as key element of the interferon signature in plasmacytoid dendritic cells from systemic lupus erythematosus patients: novel genetic associations in the Mexican mestizo population

K Santana-de Anda et al. Clin Exp Immunol. 2014 Dec.

Abstract

Many genetic studies have found an association between interferon regulatory factors (IRF) single nucleotide polymorphisms (SNPs) and systemic lupus erythematosus (SLE); however, specific dendritic cell (DC) alterations have not been assessed. The aim of the present study was to address the expression of IRF3 and IRF5 on different DC subsets from SLE patients, as well as their association with interferon (IFN)-α production and novel SNPs. For the genetic association analyses, 156 SLE patients and 272 healthy controls from the Mexican mestizo population were included. From these, 36 patients and 36 controls were included for functional analysis. Two IRF3 SNPs - rs2304206 and rs2304204 - were determined. We found an increased percentage of circulating pDC in SLE patients in comparison to controls (8.04 ± 1.48 versus 3.35 ± 0.8, P = 0.032). We also observed enhanced expression of IRF3 (64 ± 6.36 versus 36.1 ± 5.57, P = 0.004) and IRF5 (40 ± 5.25 versus 22.5 ± 2.6%, P = 0.010) restricted to this circulating pDC subset from SLE patients versus healthy controls. This finding was associated with higher IFN-α serum levels in SLE (160.2 ± 21 versus 106.1 ± 14 pg/ml, P = 0.036). Moreover, the IRF3 rs2304206 polymorphism was associated with increased susceptibility to SLE [odds ratio (OR), 95% confidence interval (CI) = 2.401 (1.187-4.858), P = 0.021] as well as enhanced levels of serum type I IFN in SLE patients who were positive for dsDNA autoantibodies. The IRF3 rs2304204 GG and AG genotypes conferred decreased risk for SLE. Our findings suggest that the predominant IRF3 expression on circulating pDC is a key element for the increased IFN-α activation based on the interplay between the rs2304206 gene variant and the presence of dsDNA autoantibodies in Mexican mestizo SLE patients.

Keywords: IRF3; dendritic cells; interferon; systemic lupus erythematosus.

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Figures

Fig. 1
Fig. 1
Monocyte-derived dendritic cells (moDC) from systemic lupus erythematosus (SLE) patients display no abnormalities in differentiation–maturation processes. CD14+ cells were purified from peripheral blood mononuclear cells from SLE patients and healthy controls by positive selection with magnetic microbeads. Briefly, cells were cultured for 7 days with granulocyte–macrophage colony-stimulating factor (GM-CSF) (50 ng/ml) and interleukin (IL)-4 (15 ng/ml) to induce differentiation of moDC and 2 more days with lipopolysaccharide (LPS) (200 ng/ml) to induce maturation. moDC were defined as CD11c+/human leucocyte antigen D-related (HLA-DR)+. No significant differences in the expression of surface molecules (CD80, CD86, CD11c, HLA-DR, CD40) were observed between in-vitro moDC from SLE patients in comparison to healthy controls.
Fig. 2
Fig. 2
Systemic lupus erythematosus (SLE) patients show expansion of peripheral plasmacytoid dendritic cells (pDC). Multi-parametric flow cytometry was performed on peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy controls and peripheral DC subset were defined as follows. Myeloid DC (mDC): Lin−/human leucocyte antigen D-related (HLA-DR)+/CD11c+/ blood dendritic cell antigens (BDCA1)+ and plasmacytoid DC (pDC): Lin-/HLA-DR+/CD11c−/BDCA4+. Increased percentage of circulating pDC was found in SLE in comparison to healthy controls, while no differences were observed in mDC frequencies. Bars represent the pooled data (mean ± standard deviation) from 36 SLE patients and 36 age- and sex-matched healthy volunteers. *P < 0·05.
Fig. 3
Fig. 3
Increased interferon regulatory factors (IRF)3 and IRF5 expression is restricted to peripheral plasmacytoid dendritic cells (pDC) from systemic lupus erythematosus (SLE). IRF3 and IRF5 expression was assessed by flow cytometry. We observed (a) increased percentage of CD11c−/BDC4+/IRF3+ as well as CD11c−/BDC4+/IRF5+ cells in SLE patients compared to healthy controls. A representative zebra-plot from one patient with SLE and one healthy control is shown. (b) The enhanced expression of IRF3 and IRF5 on SLE pDC was also corroborated by mean intensity fluorescence (MIF). Bars represent the pooled data (mean ± standard deviation) from 36 SLE patients and 36 age- and sex-matched healthy volunteers. *P < 0·05.
Fig. 4
Fig. 4
Increased phosphorylated interferon regulatory factor (IRF3) expression on peripheral plasmacytoid dendritic cells (pDC) from systemic lupus erythematosus (SLE). Phosphorylated IRF3 (Ser396) expression was assessed by flow cytometry. We observed (a) an increased percentage of CD11c−/BDC4+/phospho-IRF3+ cells in SLE patients compared to healthy controls. A representative zebra-plot from one patient with SLE and one healthy control is shown. (b) The enhanced expression of phospho-IRF3 on SLE pDC was also corroborated by mean intensity fluorescence (MIF). Bars represent the pooled data (mean ± standard deviation) from 36 SLE patients and 36 age- and sex-matched healthy volunteers. *P < 0·05.
Fig. 5
Fig. 5
Activation of in-vitro monocyte-derived dendritic cells (moDC) does not induce interferon regulatory factors (IRF)3 and IRF5 expression. Monocyte-derived mature dendritic cells from systemic lupus erythematosus (SLE) patients and controls were stimulated with lipopolysaccharide (LPS), serum from SLE patients and healthy controls (autologous and allogeneic) and analysed IRF3 and IRF5 expression by Western blot. No differences were observed on the expression of these transcription factors IRF3 and IRF5 between study groups upon any of the aforementioned stimuli. (a) Box-plots represent the pooled data (median ± interquartile range) from 36 SLE patients and 36 age- and sex-matched healthy volunteers. (b) A representative WB image from one SLE patient and one healthy control (HC) is shown along with the quantification by densitometry.
Fig. 6
Fig. 6
Interferon regulatory factor (IRF)3 rs2304206 is associated with increased susceptibility to systemic lupus erythematosus (SLE) and higher interferon (IFN)-α serum levels. SLE patients and healthy controls from the Mexican–mestizo population were genotyped for promoter IRF3 single nucleotide polymorphisms (SNPs). The rs2304206 TT and CT genotypes were associated with increased susceptibility to SLE as well as higher levels of serum IFN-α in SLE patients in comparison to healthy controls. *P < 0·05.
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