This study is the first attempt to characterize the expression of TLR7 on mDCs and TLR7 MyD88-dependent signaling molecules on PBMCs in AOSD patients. In order to avoid the effects of immunosuppressive agents on our results, new-onset untreated AOSD patients were enrolled. Our results showed significantly elevated frequencies of TLR7-expressing mDCs and upregulated levels of TLR7 transcript and protein on PBMCs. The expression levels of TLR7 were positively correlated with disease activity in AOSD patients. Moreover, a parallel decrease in TLR7 expression levels with disease remission was found in our AOSD patients. Our observations indicate that TLR7 overexpression is involved in the pathogenesis of AOSD. However, a large prospective study should be conducted to confirm our findings.
Similar to AOSD patients, our SLE patients had significantly elevated frequencies of circulating TLR7-expressing mDCs and upregulated levels of TLR7 expression, which were correlated with SLEDAI scores. Our results were consistent with the findings of recent studies showing elevated expression levels of circulating TLR7 transcript using the qPCR method , and were similar to the results of recent studies showing a role for TLR7 genes in the predisposition of Asian populations to SLE [33, 34]. In addition, Christensen et al. revealed that TLR7-deficient lupus-prone mice had ameliorated disease and decreased lymphocyte activation . These findings suggest that TLR7 expression is involved in the pathogenesis of SLE, and elevated frequencies of TLR7-expressing pre-mDCs and mDCs may be a common characteristic of systemic inflammatory diseases including AOSD and SLE.
TLR7 ligation triggers activation of a group of cytosolic adaptor molecules . MyD88 acts as an adaptor that recruits the serine-threonine kinase IRAK and TRAF6 to the TLR7 signaling pathway. MyD88-mediated signaling lies at the center of the TLR-driven immune response [2, 11, 36], and leads to production of proinflammatory cytokines and type I IFN [10, 11, 37]. Our results showed elevated transcript and protein levels of TLR7 MyD88-dependent signaling molecules, including MyD88, IRAK4 and TRAF6 on PBMCs from both AOSD patients and SLE patients. Moreover, a positive correlation between disease activity and the expression levels of TLR7 MyD88-dependent signaling molecules were observed in both AOSD patients and SLE patients (Table 2). In concordance with the findings of previous studies showing that TLR7 activation triggers production of proinflammatory cytokines [10, 11, 38], our results showed elevated levels of serum IL-1β, IL-6, IL-18, and IFN-α positively correlated with the expression levels of TLR7 signaling molecules in AOSD patients. These observations suggest the pathogenic role of the TLR7 MyD88-dependent signaling pathway in AOSD. Although IRF5 is important for regulation of IFN-α after TLR activation , the absence of a significant increase in IRF5 expression in our patients may be related to the enrolled patients' characteristics, differences in experimental procedures and/or the small sample size in our study.
Accumulating evidence shows that IFN-α, a type I IFN, plays a pivotal role in triggering and sustaining inflammatory diseases [40, 41]. Previous studies have identified type I IFN gene expression in PBMCs from patients with active lupus [42, 43], and overproduction of IFN-α, which correlates with disease exacerbation in SLE [43, 44]. Although there are no data on IFN-α in AOSD, elevated levels of IFN-α, which correlated with disease activity in our AOSD patients, suggest the potential role of IFN-α in AOSD pathogenesis. Moreover, we observed a positive correlation between expression levels of TLR7-signaling molecules and IFN-α level in AOSD patients and SLE patients, consistent with the findings of a previous study showing the concordant overexpression of TLR7 and IFN-α in SLE patients  and IFN-α production requiring TLR7/MyD88 signaling in experimental mouse lupus . Our results support the observation that TLR7 inhibitors have a therapeutic application in autoimmune dermatitis with a prominent IFN-α signature .
Given a positive association of TLR7 expression with levels of proinflammatory cytokines, we further investigated the functional relation between TLR7 ligation and the downstream mediators. Our results showed that TLR7 ligand (imiquimod) stimulation of PBMCs induced greater-fold increases in IL-1β levels (up to around 22-fold), IL-6 (up to around 28-fold), IL-18 (up to around15-fold), and IFN-α (up to around 17-fold) in AOSD patients compared to those in healthy controls, indicating that the upregulation of TLR7 is functional. Moreover, we revealed that the cytokine pattern induced by TLR7 ligand stimulation overlaps with a similar serum cytokine panel observed in both AOSD and SLE (Figure 3), suggesting that TLR7 triggering has an important contribution to the inflammatory response in both diseases. Our results were consistent with the findings of a study showing that imiquimod induced production of proinflammatory cytokines and IFN-α . Our data also showed enhanced production of IL-18 after TLR7 ligand stimulation, supporting the findings of sharing of the TLR7 with IL-18 receptor signaling [2, 47]. However, our results are different from the findings of a recent study showing no significant difference in the induction of all measured cytokines between SLE patients and controls . This discrepancy may be related to the difference in the TLR7 ligand used (R837 vs. imiquimod in our study) and in disease activity in SLE patients in our study (SLEDAI, mean ± SD, 3.1 ± 3.0 vs. 7.8 ± 2.3).
Our longitudinal follow-up of AOSD patients showed that the expression levels of TLR7 MyD88-dependent signaling molecules, including TLR7, MyD88, IRAK4, TRAF6, and IFN-α, decreased significantly, paralleling the clinical remission and a decrease in inflammatory parameters after therapy (Figure 4). Our results support the hypothesis that inhibitors of TLR7 signaling and anti-IFN-α therapy, can be a promising therapeutic modality for systemic inflammatory diseases [9, 48–51].
There were some limitations in our study. Because it was difficult to obtain biopsy tissue, we could not investigate the expression of TLR7 signaling molecules on lesion specimens in AOSD patients. To prove that TLR7 signaling is active in AOSD in vivo, further study to investigate phosphorylation of signaling molecules in freshly isolated cells is needed. The lack of significant correlations between expression levels of TLR7 signaling and clinical features of AOSD may be due to the small sample size in this clinically heterogeneous disease. In addition, it is likely that more than one TLR pathway is needed for the initiation of inflammatory response in AOSD. Therefore, a previous investigation suggested that simultaneous or sequential triggering of different TLR pathways is needed to develop an inflammatory disease .