RA is an inflammatory disorder that mainly affects the joints and synovial tissue and leads to pain and physical disability. The pathogenesis of RA is complex and the etiology is unknown. So far, despite many avenues, including antirheumatic drugs and immune-modulate therapies, have pursued, there are still no effective, safe and affordable treatments for the disease. Previous studies have indicated that certain parasitic infections could reduce the incidence and severity scores of RA [15]. In this study, we evaluated whether C.sinensis infection affects RA by using CIA mice models.
To confirm the mice were successfully infected with C.sinensis, the livers of mice were obtained for pathological analysis. The infiltration of several inflammatory cells was found in hepatic portal areas. Besides, the destruction of liver tissue structure caused by fibroblast proliferation and collagen deposition, and the destruction of bile duct structure that characterized with cholangiocyte hyperplasia, narrowing of bile duct lumen and periductal fibrosis were found (Figure1). These results were consistent with the typical pathological of C.sinensis infection as described in our previous research [16].
Current studies show that different parasitic infections have distinct effects on rheumatism. For example, Schistosoma japonicum infection could reduce the severity of rheumatic diseases in the CIA mice [7]. However, Graepel et al. reported that when mice infected with Hymenolepis diminuta, the arthritis was exaggerated, and the mice had more severe clinical symptoms [17]. We assessed the effect of C.sinensis infection on CIA in mice. Obvious joint swelling and significant clinical scores were found in CIA mice in this study. Besides, with pathological analysis, compared to CIA mice, many inflammatory cells infiltration around the joints, fibroblastic proliferation, and pannus formation were found in C.sinensis+CIA mice (Figure2). Based on the results of disease score and pathological changes, our data indicated that C.sinensis infection could increase the severity of the disease.
Collagen is an important component of cartilage. In rheumatoid arthritis, type-Ⅱ collagen is a critical autoantigen, and it could induce the production of specific antibodies. Besides, the anti-type-Ⅱ collagen antibodies could form immune complexes and activate complement in RA patients [18-19]. In the study, we found that the levels of anti-type-Ⅱ collagen antibody were elevated in CIA mice, while C.sinensis infection in mice could not change the levels of anti-type-Ⅱ collagen antibody in the CIA model.
With joint swelling, pain and the development of arthritis, the physical activities of C.sinensis+CIA mice and CIA mice were found to be seriously limited. However, the mice in the CIA group and the C.sinensis+CIA group had the same performance. Interestingly, the RER in the CIA group and C.sinensis+CIA group were higher than that control group. It was reported that mice with chronic inflammation have increased RER [20], and the mice could alter their metabolism as a response to colonic inflammation. In the study, no difference in RER was observed in C.sinensis+CIA mice and CIA mice. These results suggested that the CIA could decrease the physical activities but increase RER, while C.sinensis infection has no significant impacts on the physical activities as well as RER in the mice model we built.
Currently, serveral studies indicated that multiple immune cells were involved in the development of arthritis. For example, neutrophil could be enrolled in the site of inflammation and play an important role in the disease [13]. In RA patients, 90% of cells in the synovial fluid are neutrophils [21]. Besides, neutrophils can also be seen in the pannus and cartilage. Citrullinated autoantigens and neutrophil extracellular traps (NETs) can be taken up by fibroblast-like synoviocytes (FLS), and then presented to T cells. The neutrophil is considered as a bridge connecting FLS to T cells to promote the development of the disease [22]. Furthermore, it has been reported that neutrophil depletion can inhibit the progression of arthritis [23,24]. LY6Chi monocytes is capable of recruiting to the sites of inflammation, and after extravasation, they can differentiate into macrophages and dendritic cells [25,26]. Besides, after migrated to inflammatory joints, monocytes could contribute to Th17 differentiation, local antigen presentation, and osteoclastogenesis, and make a significant contribution during CIA development. Moreover, clinical and experimental studies indicated that the number of Ly6Chi monocytes is associated with the severity of the disease, and Ly6Chi monocytes depleting can reduce the inflammation and bone erosion in CIA [27,28]. B cells and T cells also play important roles in RA. On the one hand, in the early stage of arthritis, B cells, served as antigen-presenting cells, could present antigens to CD4+T cells, and activate CD4+T cells to secrete IL-2 and IFN-γ in the synovial membrane [29,14]. On the other hand, T-B cell interaction also activate B cells to differentiate into plasma cells, and produce antibodies, autoantibodies, and cytokines, to contribute to the pathogenesis of rheumatoid arthritis [30]. Meanwhile, the activation of T and B cells further causes the production of associated cytokines and chemokines and results in the formation of feedback loops between T and B cells [31]. In the study, considering the importance of these immune cells in arthritis; we are interested in detecting whether the C.sinensis infection has an impact on immune response mediated by neutrophils, monocytes, T cells and B cells mediated in ICA.
Based on the data as shown in Figure5, we found that the number of neutrophils and LY6Chi monocytes in both the CIA group and the C.sinensis+CIA group were higher than those in control group. Furthermore, compared to the CIA group, the number of neutrophils and LY6Chi monocytes were higher in the C.sinensis+CIA group. Besides, compared with the control group and the CIA group, the number of B cells and CD4+T cells in the C.sinensis+CIA group were reduced. Taken together, these results suggested that C.sinensis infection could cause the dysfunction of immune response mediated by neutrophils, LY6Chi monocytes, CD4+T cells, and B cells in the CIA.
Cytokines are usually secreted by immune cells, and contribute to the activation, differentiation, migration, and survival of host cells with various types in vivo. It has been shown that several cytokines, including IL-6, TNF-α, IL-17, and IFN-γ play dominant roles in RA [32]. These cytokines could regulate immune response through complex signal pathways and thus affect the pathological process of RA. For example, IL-6 is a cytokine produced by a variety of cells, including B cells, T cells, and macrophages. High level of IL-6 can be detected in RA patients and participates in joint destruction by acting on neutrophils and pre-osteoclasts, through RANKL dependent or RANKL independent mechanisms [3]. The elevation of TNF-α has also been observed in the serum of RA patients. TNF-α can strongly promote osteoclast differentiation with RANKL, and causes pannus formation and inflammatory bone resorption [33]. Besides, blocking the function of TNF-α and IL-6 by monoclonal antibodies could significantly reduce the pathobiology of RA [34,35]. IL-17 is mainly produced by Th17 cells and could activate macrophages and synovial fibroblasts, increase the production of MMP1 and MMP3 as well as inflammatory cytokines, including TNF-α, IL-6, and IL-1. In addition, IL-17 can also recruit neutrophils and monocytes to the site of inflammation and promotes osteoclast differentiation, which leads to cartilage destruction and bone erosion in arthritis [36]. IFN-γ is a Th1-type cytokine that facilitates leukocytes transfer through the endothelial layer by promoting the expression of chemokines. IFN-γ also activates macrophages to increase antigen presentation and inducible nitric oxide synthase (iNOS) during the development of arthritis [37,38]. In the study, we found that, compared to the control group, the cytokines, including IL-6, TNF-α and IFN-γ were elevated in both the CIA group and the C.sinensis+CIA group. But there was no difference between CIA group and C.sinensis+CIA group. These results indicated that C.sinensis infection has no significant effect on the expression of these three kinds of cytokines. In addition, the levels of IL-4 and IL-17 in the C.sinensis+CIA group were found to be significantly higher than those in both the CIA group and the control group. During the acute phase of infection, Clonorchis sinensis is capable of inducing the host immune response towards a strong Th1 immune response, which could induce the expression of IFN-γ. With the development of infection, Th1 immune response could shift to Th2, following the overexpression of IL-4 [10,11]. These results suggested that the immune response in the CIA may be influenced by C.sinensis infection, via increased expression of IL-4 and IL-17 in mice.