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.