4. Discussion
Primary biliary cholangitis is considered as a typically liver-specific autoimmune disease due to the well-established autoantigen and its homogeneous clinical expression [19]. The pathogenesis of small bile duct destruction occurs through the interaction between immune response and biliary epithelial cells [19, 20]. The autoimmune nature of PBC involves the loss of tolerance to a series of mitochondrial autoantigens, including the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2) [21, 22]. This response to PDC-E2 contributes to bile duct loss through lymphocytic cholangitis, which progresses to bile duct injury followed by chronic cholestasis and liver fibrosis.
PBC has diverse abnormalities in either immunological responses or regulations, which includes the presence of autoreactive T cells [6, 7], B cells [23], and AMAs [24]. Many autoreactive T cells are found within the portal tracts. They distribute around the damaged bile ducts in PBC patients [25] and secrete multiple cytokines and chemokines. The mechanism through which infiltrating T cells participate in biliary injury remains controversial. Therefore, evaluating the phenotype and function of T cells in PBC patients is highly important for understanding the immunological interactions and excessive immune responses of the disease.
CD4+ T cells play a pivotal role in PBC pathogenesis [26]. Recent studies demonstrated that the IL-12/Th1 signaling pathway is a central mechanism in the etiopathogenesis of PBC. For PBC patients, IL-12A and IL-12RB2 variants were highly correlated with PBC in three independent genome-wide association studies [27-29]. For murine models of PBC, namely dnTGFbRII mice, IL-12p40 KO-dnTGFbRII mice showed a significant improvement in histological cholangitis and dramatically decreased levels of pro-inflammatory cytokines [30].
In the present study, we provide evidence of an increased level of CD4+ T cell activation in the peripheral blood of PBC patients. The HLA-DR antigen, indicating chronic T cell activation [31], had higher expression in CD4+ T cells from PBC patients. As a mid-activation marker, the costimulatory protein ICOS is important for intact immune pathways [32], Th2 responses [33], and Th17 development [34] and regulation. Our results also suggest relatively high ICOS activation in the peripheral CD4+ T cells of PBC patients compared to those of HCs. In accordance with literature supporting the increased levels of IFN-γ, a Th1 cytokine promoting cytotoxic T cell activity; IL-4, a Th2 cytokine promoting B cell activation and antibody production; and IL-17, a Th17 cytokine promoting T cell activation and inflammation [35, 36], our results showed profoundly increased frequencies of Th1, Th2, and Th17 cells in PBC patients. However, the molecular mechanisms occurring in CD4+ T cell hyperactivity during PBC pathogenesis is yet unclear.
Some studies reported that LAMP-2 functions in delivering proteins from the cytoplasm to lysosomes [9, 10] and plays a crucial role in immunocyte responses. It is reported that LAMP-2 influenced the repertoire of peptides presented by MHC class II molecules on B cells, which affected the balance between endogenous and exogenous antigen presentation [37]. Moreover, LAMP-2A was also demonstrated to contribute to immunological recognition and intracellular antigen presentation through forming the lysosomal LAMP-2A-HSC70 complex [38]. On the contrary, LAMP-2C was reported to skew MHC II presentation of cytoplasmic antigens [39]. LAMP-2A has also been shown to participate in CD4+ T cell homeostasis, which was increased in response to TCR engagement. Deletion of the gene encoding LAMP-2A in murine CD4+ T cells showed a reduced activation-induced response in vitro, and T cell-specific LAMP-2A-deficient mice showed deficient responses to immunization as well as infection with Listeria monocytogenes [11].
The findings of our earlier research revealed an increase of LAMP-2 in PBC patients serum, which gradually decreased along with the therapy by ursodesoxycholic acid (UDCA). And the deline of LAMP-2 might help to predict the response to UDCA [40]. Due to the important role of LAMP-2 in PBC and the function of T cell, we designed this study.
Consistent with the results found in mice, our study also suggested that the level of LAMP-2A expression was higher in HLA-DR+/ICOS+ activated CD4+ T cells than in their non-activated counterparts. Interestingly, we found that LAMP-2A expression was upregulated in non-activated and naïve CD4+ T cells of PBC patients compared to those of HCs. Due to the crucial role of LAMP-2A in the process of CD4+ T cell activation, we hypothesized that naïve CD4+ T cells of PBC patients are more liable to be activated. To test this idea, we isolated and stimulated T cells from the PBMCs of PBC patients and HCs in vitro. As expected, naïve CD4+ T cells of PBC patients showed higher capabilities of proliferation and activation-induced cytokine production, whereas the difference in apoptosis was almost undetectable. To investigate the pathological role of LAMP-2A in naïve CD4+ T cells in PBC, we deleted the gene encoding LAMP-2A in naïve CD4+ T cells from PBC patients, and the excessive activation responses were reversed.
Our study potentially provide a novel biomarker to monitor PBC activity, levels of LAMP-2A in naïve CD4+ T cells. Interestingly, there is a high expression level of LAMP-2A in patients with high pathological grade, suggesting that LAMP-2A expression level in naïve CD4+ T cells may more easily and conveniently predict severity of the disease. However, further studies must be done to investigate the relationship between LAMP-2A expression levels in naïve CD4+ T cells and PBC clinical features, including UDCA response and prognosis.
In conclusion, the results of the present study support the concept that increased LAMP-2A expression in the naïve CD4+ T cells of PBC patients may lead to a tendency for increased activation, which may also indirectly reflect activity of the disease as liver biopsy is inconvenient. To reverse the hyperactivity of CD4+ T cells and reduce the resulting biliary injury, LAMP-2A could be a novel therapeutic target for the treatment of PBC.