DISCUSSION
The transverse expression from innate to adaptive immune lymphocytes makes NKG2D a remarkable activating receptor. Dysregulation of the NKG2D/NKG2DL pathway may lead to inadequate NK and T cell activation, and contributes to initiating or maintaining an inflammatory cascade, resulting in self-reactivity [16]. In this study, we explored the roles of NKG2D in the pathogenesis of different subgroups of JIA and its regulatory roles in the crosstalk between CD4+ T cells and DCs in JIA patients.
NKG2D expression on CD4+ cells in the main three subgroups of active JIA patients was significantly increased. In addition, NKG2DL was markedly increased compared the levels in healthy controls. The findings indicate a role for NKG2D in the activation of CD4+ T cells in JIA patients. This indication is consistent with the previously reported associations between NKG2D/NKG2DL polymorphisms and rheumatoid arthritis. In particular, genetic variation within the NKG2D gene may be involved in disease susceptibility, characteristics, severity, and efficacy of anti-tumor necrosis factor therapy [17-23]. Thus, NKG2D may be a potential biomarker of JIA disease activity.
The marked variation in clinical features among subtypes of JIA is caused by the different immunological characteristics. Activation of CD4+NKG2D+ T cells and NKG2DL were greater in paJIA and poJIA patients than in soJIA patients. Joint disease of articular JIA usually overshadows the general symptomatology. It is generally thought that activation of autoreactive CD4+ T cells is a key manifestation in the pathogenesis of arthritis [24-27]. Dysregulation of local CD4+NKG2D+ T cells results in autoreactive T cell and arthritis. Systemic JIA is mainly characterized as an auto-inflammatory disease and is the most distinct clinical subtype of JIA with its marked extra-articular feature of spiking fevers, which can easily lead to misdiagnosis at the early stage. An NKG2D-dependent regulatory role of NK cells in systemic JIA has been described [3]. We assayed the expression of NKG2DL on DCs, which are the strongest antigen present cells. Articular JIA patients displayed higher levels of MICA and MICB on DCs than healthy individuals and systemic JIA patients. The different roles of NKG2D in various immune cells might contribute to the marked clinical variation among subsets of JIA.
The influence of the regulation of NKG2D on interaction between CD4+T cells and DCs were explored by using blood samples from arthritic JIA patients and healthy controls. NKG2D modulated subgroups of Th1 and Th17, with declines in the Th2 and Treg subgroups by regulation of the secretions of IFN-γ, IL-17, and IL-4 and expressions of transcription factors RORc, Foxp3, T-bet and GATA3 in JIA patients. In addition, NKG2D also promoted higher production of the IL12 proinflammatory cytokine and reduced productions of the TGF-β and IL-10 anti-inflammatory cytokines by CD4+T cells of JIA. These findings indicate the role of the NKG2D-mediated signaling pathway in CD4+ T cell inflammation in JIA pathogenesis. Furthermore, NKG2D had a stronger regulatory effect on CD4+ T cells in JIA patients than in healthy controls. NKG2D typically is not expressed, or is expressed at a low level on healthy CD4+ T cells. NKG2DL is termed a “stress-induced ligand” based on the expression after autoimmune stimulation. Marked overproduction of NKG2DL was observed in local synovial fibroblasts and synovial fluid of rheumatic arthritis patients [28], accounting for the powerful regulation of NKG2D on unhealthy cells of JIA patients. NKG2D/NKG2DL act as a powerful costimulatory signal in the autoimmune condition, and lead to inflammation induced by antigen-specific CD4+ T cells [29]. The collective findings implicate NKG2D as a potential target for the amelioration of Th1- and Th17-mediated chronic inflammatory diseases in JIA.
The bidirectional crosstalk between CD4+ T cells and DCs lead to mutual activation, maturation of DCs, and differentiation of CD4+ T cells. Accordingly, we investigated the effects of genetically NKG2D modified CD4+ T cells on DCs during interactions between the cell types. NKG2D indirectly lowered the productions of TGF-β and IL-10, but not IL-12, by DCs in healthy individuals, and lowered the secretion of IL-10 from DCs in JIA patients. The indirect regulations of NKG2D on DCs is more potent in healthy individuals than in JIA patients, and relies on anti-inflammatory IL-10. IL-10 suppresses the activation of T cells, induces differentiation of T regulatory cells, and also blocks the maturation process and down-regulates costimulatory molecules of DCs. These actions subsequently limit the autoimmune response [30,31]. In addition, NKG2D indirectly prompted the expression of the CD83 DC maturation marker in both healthy and JIA individuals, but had no effects on the expression of the costimulatory molecules CD80 and CD86, and HLA-DR on DCs. MICA and MICB expressions on DCs were also increased dramatically by NKG2D indirectly in both JIA and healthy controls. The collective findings indicate that NKG2D indirectly regulates the activation and maturation of DCs through their crosstalk with CD4+ T cells.
In conclusion, CD4+NKG2D+ T cells and NKG2D ligands MICA and MICB are increased dramatically in JIA patients, particularly paJIA and poJIA patients. NKG2D directly regulates the differentiation and activation of CD4+ T cells, and indirectly regulates the maturation and activation of DCs. The stronger regulations of NKG2D on CD4+ T cells of JIA implicates targeting CD4+ NKG2D+T cells as a potential therapy to treat the inflammation of JIA.