T-helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus?

The 25 patients fulfilling the American College of Rheumatology revised criteria for SLE [28] were recruited at the outpatient clinic of the Immunology Department and the Rheumatology Department of the Erasmus Medical Center Rotterdam. The level of disease activity was assessed by using the SLEDAI [29]. Fifteen healthy controls (HCs), neither with autoimmune diseases nor using corticosteroids, were included. Characteristics of patients and controls are summarized in Table 1. The Medical Ethical Review Committee of the Erasmus MC approved the study, and written informed consent was obtained.

Blood was collected in clotting tubes for serum preparation (stored at -80°C) and in sodium-heparin tubes for peripheral blood mononuclear cell (PBMC) preparation, as described previously [30]. CD14-positive monocytes were isolated as described [30].

Total RNA was isolated from purified monocytes followed by cDNA preparation and real-time quantitative polymerase chain reaction (RQ-PCR) analysis by using predesigned primer/probe sets (Applied Biosystems) [30]. For calculation of relative expression, all samples were normalized against expression of the household gene Abl [31]. Fold-change values were determined from normalized CT values by using the 2^-ΔΔCT method (User Bulletin; Applied Biosystems, Foster City, CA, USA).

PBMCs were restimulated, stained, and measured with flow cytometry, as previously described [32]. For extracellular staining, CD4, CD45RO, and CCR6 monoclonal antibodies were obtained from BD Biosciences (San Diego, CA, USA), and CD25 antibodies from Biolegend Inc. (San Diego, CA, USA). For intracellular staining, IL-17A, IL-17F, IL-21, and IL-22 monoclonal antibodies were obtained from eBioscience, and IL-17A monoclonal antibodies from Biolegend Inc. Samples were measured on a FACScantoII flow cytometer (BD Biosciences). Analysis was performed by using FlowJo v7.6 research software (Tree Star Inc. Ashland, OR, USA).

Statistical analyses were performed by using the SPSS 20.0 package. When data were not normally distributed, values were expressed as medians with interquartile ranges (IQRs), and comparisons were made by using the nonparametric Mann–Whitney U test. In case of more than two samples, the nonparametric Kruskal-Wallis test was performed. Correlations were assessed by using either the Pearson correlation test for normally distributed data or Spearman rho when data were not normally distributed. Differences were considered statistically significant if P < 0.05.

In monocytes of 25 SLE patients and 15 HCs, we assessed the expression levels of 11 IFIGs previously assessed in monocytes from patients with primary Sjögren syndrome (pSS) (IFI27, IFI44L, IFIT3, IFITM1, SERPING1, IFIT1, IFIT2, LY6E, IFI44, XAF1, and MXA) [24], and the expression of which was also found to be increased in SLE patients [2, 3, 33, 34]. To reduce data complexity, expression levels of the 11 genes were submitted to a principal component analysis to identify correlated groups of genes. The results of the principal component analysis identified a subset of four genes (IFI44L, IFITM1, SERPING1, and LY6E) to explain 95% of the total variance of the 11 IFN type I-inducible genes within the SLE cohort. Given that the expression of these four IFN type I-inducible genes was not normally distributed, log transformations of expression values were performed, and IFN scores were calculated as described for pSS [24]. MeanHC and SD_HC of each gene in the HC-group were used to standardize expression levels. IFN scores per subject represent the sum of these standardized scores. When we set the threshold for a positive IFN type I signature at IFN score of 8 [24], 64% of SLE patients displayed an IFN type I signature, and one of the 15 HC subjects (7%) (Figure 1A,B).

Because CCR6 enriches for Th17 cells [35‐37], CCR6⁺ cells were selected after gating on lymphocytes and memory Th cells (CD4⁺CD45RO⁺ cells) within PBMCs and after CD25+ cells were excluded. To investigate whether the IFN type I signature is associated with an increase in Th17 cytokines expressed by memory CCR6⁺ T cells, we measured the percentages of IL-17A, IL-17F, IL-22 and IL-21 producing CCR6⁺ T memory cells in SLE patients positive for the IFN type I signature (IFN⁺) and patients negative for the signature (IFN^-) and HC. The percentages of CCR6⁺ cells within the CD4⁺CD45RO⁺ T-cell population and within total lymphocytes were comparable between the three studied groups (data not shown). Interestingly, the percentages of CCR6⁺IL^-17A⁺ cells were significantly increased in IFN⁺ patients, as compared with IFN^- patients (P = 0.03), and a higher trend was observed compared with HCs (P = 0.07) (Figure 2A). The percentages of CCR6⁺IL^-17F⁺ and, in particular, the IL-17A/IL-17F double producers were significantly increased in the IFN⁺ group compared with HC (P = 0.009) (Figure 2B, and see Additional file 1: Figure S1). The percentages of CCR6⁺IL^-22⁺ cells showed a higher trend for IFN⁺ versus IFN^- patients (^P = 0.06) and IFN⁺ patients versus HC (P = 0.09) (Figure 2C). The percentages of IL-21 expressing CCR6⁺ cells were also significantly increased in IFN⁺ patients compared with IFN^- patients (Figure 2D). These data suggest an association between the presence of the IFN type I signature and the expression of Th17 cytokines in SLE.

In addition, we investigated whether the Th17 cytokine production is associated with disease activity as assessed by SLEDAI scores. No significant correlations were observed between the SLEDAI scores and IL-17A and/or IL-17F expression (data not shown). Except for race, no differences in demographic, clinical, or laboratory data were found between IFN⁺, IFN^- SLE patients and/or HCs (Table 1).

Correlating the expression of IFIGs (as reflected by the total IFN score) with other parameters assessed in this study, we observed a significant positive correlation between the expression of IL-17A and IL-17F within CCR6⁺ cells and IFIG expression (Figure 3A,B). Also in this SLE cohort, IFIG expression correlated strongly with the BAFF mRNA expression in monocytes (r = 0.527; P < 0.0001) (Figure 3C). No correlation was observed between BAFF and IL-17A and/or IL-17F expression. However, we did find a significant correlation between BAFF mRNA and the percentages of IL-21 producing CCR6⁺ cells (r = 0.406; P = 0.010) (Figure 3D). Both BAFF and IL-21 are involved in the selection and activation of B cells, which is crucial in the pathogenesis of SLE, indicating that downstream factors of the IFN type I and Th17 pathways might also be associated.