This study is the first investigation to demonstrate GLK overexpression in patients with active AOSD relative to HCs. The advent of flow cytometry analysis of intracellular signaling molecules [24
] has greatly expanded the opportunities to study a single cell in heterogeneous cell populations. In the present study, CD3+
T-cells, including CD4 and CD8 subsets, demonstrated increased GLK expression in patients with active AOSD. Our results also showed significantly elevated frequencies of circulating GLK-expressing T-cells, which correlated with disease activity, including clinical activity scores and serum ferritin levels, in patients with AOSD. Moreover, a parallel decrease in GLK production with disease remission was found in these patients. These data concerning patients with AOSD were similar to the results of our recent study showing elevated levels of circulating GLK-expressing T-cells correlating with activity index in patients with SLE [12
], suggesting that GLK overexpression plays an important role in AOSD pathogenesis, and is thus a potential activity marker of this disease. However, a large prospective study should be conducted to confirm the findings presented here.
To verify GLK expression at the protein and transcript levels in patients with AOSD, western blotting and qPCR for GLK expression were performed in peripheral blood lymphocytes from our patients with active untreated AOSD. We have demonstrated that the relative expression levels of GLK proteins and transcripts were significantly higher in our patients than in HCs. Moreover, the positive correlations between the frequencies of circulating GLK-expressing T-cells and the expression levels of GLK proteins in our study are consistent with the findings of previous studies showing that intracellular flow cytometry and western blotting are equivalent assays for measuring MAPK signaling status [25
]. In addition, the expression levels of GLK proteins as well as transcripts were significantly correlated with clinical activity scores in our patients with AOSD. These data provide the first direct and robust evidence of GLK overexpression in the T-cells of patients with AOSD.
Accumulating evidence indicates that Th17 cells play an important role in the pathogenesis of both AOSD and SLE [20
]. IL-6 synergizes with IL-1β to enhance the differentiation and generation of Th17 cells [29
]. Th17 cells can secrete IL-17, a pleiotropic cytokine which participates in tissue inflammation by inducing the expression of proinflammatory cytokines and chemokines [30
]. Our recent study showed that GLK-deficient mice are resistant to the development of EAE and showed decreased Th17 responses [12
]. The results from in vitro
T-cell differentiation assays also indicate that GLK plays a positive role in Th17 cell differentiation [12
]. In the present study, the results revealed elevated serum levels of Th17-related cytokines, IL-6 and IL-17A, which were correlated with the expression levels of GLK in T-cells from patients with AOSD. Our data also support previous findings showing that MAPK pathway plays an important role in the regulation of Th17 cell function [33
], and that IL-17 production is mediated by a MAPK-dependent mechanism [34
]. In addition, inhibition of MAPK could suppress IL-17 production in Vogt-Koyanagi-Harada syndrome [35
], and attenuate the Th17-mediated autoimmune disease EAE [36
]. These observations suggest either GLK overexpression or MAPK signaling can participate in the production of Th17-related cytokines. However, there still exists the possibility that GLK upregulation might represent an epiphenomenon of inflammation rather than a primary event in the pathogenesis of AOSD.
Our longitudinal follow-up of patients with AOSD showed a significant decrease in the levels of circulating GLK-expressing T-cells as well as the expression levels of GLK protein and transcript, paralleling the clinical remission and the decrease in inflammatory parameters after effective therapy (Figure ). Our results support the hypothesis that inhibitors of more upstream MAPK signaling pathways, such as MAP2K (MKK3 or MKK6) and MAP3K (transforming growth factor activated kinase 1), can be a promising therapeutic modality for rheumatic diseases [37
]. As an upstream MAPK, GLK could also be targeted as a potential therapeutic strategy by broadly inhibiting downstream MAPKs or several p38 isoforms [39
]. Furthermore, upstream signaling molecules might be better targets than downstream molecules such as p38MAPK, blockade of which could result in considerable toxic effects [37
Figure 4 Changes in the levels of circulating GLK-expressing T-cells, the expression levels of GLK proteins as well as transcripts, and serum levels of soluble interleukin-2 receptor in 12 patients with adult-onset Still's disease after effective therapy. Data (more ...)
There were some limitations in this study. Because biopsy tissue is difficult to obtain, we did not investigate the GLK expression in lesion specimens from patients with AOSD. Although some studies reported elevated IL-1β levels in AOSD and a substantial benefit of IL-1β receptor antagonist (anakinra) for treatment of inflammatory diseases [43
], our results showed no significant difference in IL-1β levels between patients with AOSD and healthy volunteers. This discrepancy may result from differences in the detection methods or other unknown confounding factors undetected in this study. The lack of any significant association of GLK expression with clinical features may be owing to the small sample size in this clinically heterogeneous and uncommon disease.