Patient characteristics
The clinical characteristics of patients in all groups are shown in Table 1. The PSC group included 47 patients, of whom 19 were men and 28 were women; the median age at diagnosis was 48 years (range, 14–82). We evaluated the disease stage at the time of diagnosis using Ludwig’s classification for histological liver fibrosis. Approximately half of patients were diagnosed at an early stage (stage 1, N = 11; stage 2, N = 11; stage 3, N = 20; and stage 4, N = 5). We found that the prevalence of inflammatory bowel disease (IBD) was 36% (17/47), comparable to that in a recent study in Japan (13). The PBC group included 6 men and 14 women; the median age at diagnosis was 51 years (range, 35–67). According to Ludwig’s classification, most patients were at early stages (stage 1, N = 5; stage 2, N = 12; stage 3, N = 3; stage 4, N = 0). The control group included 7 men and 5 women who had undergone surgery for intrahepatic cholangiocarcinoma (N = 9), intraductal papillary neoplasm of the bile duct (N = 1), or liver metastasis of colon cancer (N = 2). None of them had viral hepatitis. Their resected livers were histologically evaluated using the New Inuyama classification (14); they were all classified as F0, A1, or A2. We found that serum alkaline phosphatase and gamma-glutamyl transpeptidase levels in the PSC group were significantly higher than those in the control group and equivalent to those in the PBC group. In all three groups, most patients (> 90%) were classified as Child–Pugh class A.
Table 1. Clinical characteristics of patients in PSC, PBC and control group
Values are expressed as N (%) otherwise indicated.
* Values are expressed as median (range).
UC, ulcerative colitis;
ICC, Intrahepatic cholangiocarcinoma; IPNB, Intraductal papillary neoplasm of the bile duct; LM-CRC, Liver metastasis of Colorectal adenocarcinoma
AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; gGTP, g-glutamyl transpeptidase; T-Bil, total bilirubin; Alb, albumin; PT-INR, prothrombin time-international normalized ratio; Cre, creatinine; CRP, C-reactive protein
Altered expression of risk genes identified in GWASs in the livers of patients with PSC
To screen for potential candidate genes, we performed RT-qPCR on liver biopsy samples from the PSC group and resected liver samples from the control group. We chose 9 candidate genes from a previous GWAS study for analysis (11): ATXN2, CCL20, FOXP1, HHEX, IL2RA, MST1, NFKB1, PRDX5, and TNFRSF14. We accordingly collected 10 samples from the PSC group that met the inclusion criteria: (1) confirmed expression of the housekeeping gene β-actin, and (2) quantitation of the expression of each gene using a standard curve. We found that all 12 control samples satisfied both criteria. As shown in Fig. 1, the expression of ATXN2, HHEX, PRDX5, and TNFRSF14 was significantly upregulated, whereas that of MST1 was downregulated in the PSC group compared with that of controls. However, we did not detect any significant difference in the expression of CCL20, FOXP1, NFKB1, or IL2RA between the two groups (Fig. 1).
Biliary expression of TNFRSF14 was increased in PSC but not in PBC
Among the five candidate genes identified, we focused on TNFRSF14 because it has also been reported to be a disease susceptibility gene in ulcerative colitis, the most common comorbidity of PSC (5, 15). TNFRSF14 encodes tumor necrosis factor (TNF) receptor superfamily member 14 (TNFRSF14), also known as herpesvirus entry mediator (HVEM). It is expressed in stromal, myeloid, lymphoid, and epithelial cells, and is involved in the inflammatory signal transduction through its interactions with multiple TNF-related ligands and immunoglobulin-superfamily proteins (16).
To validate the TNFRSF14 expression profiles, we performed immunohistochemical staining of liver samples from control patients and patients with PSC. We classified expression levels using staining intensity as TNFRSF14-high, -low, and -negative (Fig. 2A). We assessed a total of well-preserved 45 liver biopsy samples from patients with PSC. We also evaluated the levels of expression in hepatocytes and compared the results with those of liver biopsy samples from patients with PBC.
Consistent with the results of the initial gene expression analysis, the fraction of TNFRSF14-positive (TNFRSF14-high and -low) biliary epithelial cells in patients in the PSC group (96% [43/45]) was significantly higher than that in controls (42% [5/12], P < 0.001) (Fig. 2B). Moreover, it was significantly higher than that in the PBC group (55% [11/20], P < 0.001). Notably, we observed TNFRSF14-high cells only in patients with PSC (Fig. 2B). We obtained similar results with hepatocytes; positivity was significantly higher in the PSC group than in the PBC and control groups (PSC 96% [43/45], PBC 65% [13/20], P = 0.003; control 58% [7/12], P = 0.003) (Fig. 2B). Similar to biliary epithelial cells, we detected TNFRSF14-high hepatocytes only in patients with PSC (Fig. 2B). These findings suggested that TNFRSF14 was upregulated in the biliary epithelial cells and hepatocytes of patients with PSC, whereas this upregulation did not occur in patients with PBC.
Biliary expression of LIGHT is increased in PSC
Recent studies have revealed that the TNF family member LIGHT (lymphotoxin-like, which exhibits inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes), an activating ligand of TNFRSF14, is involved in fibrosis in many tissues, including the lungs, skin, and liver (17–19). The expression of both TNFRSF14 and LIGHT was increased in the synovial tissues of patients with other autoimmune disorders, such as rheumatoid arthritis (20). As the expression of LIGHT was significantly elevated in PSC (Fig. 3A), we next measured the LIGHT expression.
As in the case of TNRSF14, we performed immunohistochemistry in liver tissues from control patients, patients with PSC, and patients with PBC using antibodies against LIGHT. We classified the levels of expression of biliary epithelial cells and hepatocytes based on their staining intensity as LIGHT-high, -low, and -negative (Fig. 3B). We found that the fraction of LIGHT-positive (LIGHT-high and -low) biliary epithelial cells in patients in the PSC group was significantly higher than that in the control group (100% [45/45] vs. 83% [10/12], P = 0.041) (Fig. 3C). However, we did not detect any significant differences in staining between the PSC and PBC groups (95% [19/20], P = 0.308) (Fig. 3C). Interestingly, we observed that LIGHT-high cells were more common in PSC (53% [24/45]) than in PBC (15% [3/20], P = 0.006), whereas they were not observed in the control group (Fig. 3C). Likewise, we found that the proportion of LIGHT-positive hepatocytes was also significantly higher in PSC than in controls (PSC 100% [45/45], control 67% [8/12], P = 0.001) (Fig. 3C). We did not observe any significant differences in positivity between PSC and PBC (100% [20/20], P = 1) (Fig. 3C). We noticed that LIGHT-high hepatocytes were equally common in PSC and PBC (PSC: 64% [29/45], PBC: 55% [11/20], P = 0.655), whereas they were not found in control tissues (Fig. 3C). These observations suggested that the expression of LIGHT was increased in the biliary epithelial cells and hepatocytes of patients with PSC. We more frequently detected high expression of LIGHT in PSC than in PBC biliary tissues, although the levels of expression in hepatocytes were comparable between these groups.
High expression of LIGHT in the bile duct is correlated with fibrotic progression of PSC
We found that patients with PSC showed various expression patterns of TNFRSF14 and LIGHT (Supplementary Fig. 1). Therefore, we determined whether there was a relationship between increased expression of TNFRSF14/LIGHT and clinical characteristics or laboratory data in patients with PSC (Tables 2 and 3).
Table 2
Relations between TNFRSF14 expressions and clinical characteristics and laboratory data
|
Biliary epithelial cells
|
Hepatocytes
|
|
H (N = 19)
|
L/N (N = 26)
|
P value
|
H (N = 21)
|
L/N (N = 24)
|
P value
|
Clinical characteristics
|
Young (N = 20)
|
8 (42%)
|
12 (46%)
|
1
|
9 (43%)
|
11 (46%)
|
1
|
IBD (+) (N = 16)
|
6 (32%)
|
10 (38%)
|
0.872
|
6 (29%)
|
10 (42%)
|
0.546
|
Female (N = 26)
|
13 (68%)
|
13 (50%)
|
0.352
|
15 (71%)
|
11 (46%)
|
0.152
|
Ludwig stage
3 or 4 (N = 24)
|
12 (63%)
|
12 (46%)
|
0.408
|
11 (52%)
|
13 (54%)
|
1
|
Laboratory data
|
Alb (g/dL)
|
3.80 (2.8–4.2)
|
3.95 (2.9–4.7)
|
0.155
|
3.9 (2.9–4.7)
|
3.9 (2.8–4.7)
|
0.784
|
ALT (U/L)
|
43 (12–274)
|
46 (11–240)
|
0.306
|
40 (11–274)
|
53 (11–240)
|
0.909
|
ALP (U/L)
|
648 (216–2002)
|
434 (157–1613)
|
0.707
|
599 (157–2002)
|
588 (188–1590)
|
0.884
|
T-Bil (mg/dL)
|
0.8 (0.5–6.5)
|
0.7 (0.3–1.9)
|
0.116
|
0.7 (0.3–6.5)
|
0.8 (0.3–3.4)
|
0.615
|
PT-INR
|
0.94 (0.87–1.07)
|
0.99 (0.05–1.35)
|
0.279
|
0.93 (0.87–1.20)
|
1.03 (0.05–1.35)
|
0.023*
|
CRP (mg/dL)
|
0.23 (0.02–4.54)
|
0.26 (0.01–6.40)
|
0.558
|
0.21 (0.02–2.93)
|
0.29 (0.01–6.40)
|
0.991
|
Table 3
Relations between LIGHT expressions and clinical characteristics and laboratory data
|
Bile epithelial cells
|
Hepatocytes
|
|
H (N = 24)
|
L/N (N = 21)
|
P value
|
H (N = 29)
|
L/N (N = 16)
|
P value
|
Clinical characteristics
|
Young (N = 20)
|
12 (50%)
|
8 (38%)
|
0.616
|
13 (45%)
|
7 (44%)
|
1
|
IBD (+) (N = 16)
|
9 (38%)
|
7 (33%)
|
1
|
9 (31%)
|
7 (44%)
|
0.598
|
Female (N = 26)
|
13 (54%)
|
13 (62%)
|
0.824
|
15 (52%)
|
11 (69%)
|
0.429
|
Ludwig stage
3 or 4 (N = 24)
|
17 (71%)
|
7 (33%)
|
0.027*
|
20 (69%)
|
4 (25%)
|
0.012*
|
Laboratory data
|
Alb (g/dL)
|
3.9 (2.8–4.3)
|
4.0 (3.4–4.7)
|
0.042*
|
3.9 (2.8–4.6)
|
4.0 (3.4–4.7)
|
0.060
|
ALT (U/L)
|
64 (12–246)
|
35 (11–274)
|
0.099
|
57 (11–246)
|
40 (11–274)
|
0.393
|
ALP (U/L)
|
724 (243–2002)
|
388 (157–1729)
|
0.078
|
660 (157–2002)
|
476 (188–1729)
|
0.391
|
T-Bil (mg/dL)
|
0.8 (0.3–6.5)
|
0.7 (0.4–1.9)
|
0.170
|
0.9 (0.3–6.5)
|
0.6 (0.4–1.1)
|
0.008*
|
PT-INR
|
1.01 (0.88–1.35)
|
0.97 (0.05–1.34)
|
0.368
|
1.02 (0.85–1.35)
|
0.93 (0.05–1.17)
|
0.168
|
CRP (mg/dL)
|
0.46 (0.02–4.54)
|
0.18 (0.01–6.40)
|
0.078
|
0.31 (0.01–4.54)
|
0.22 (0.02–6.40)
|
0.499
|
Regarding the relationship with laboratory data, the following results were obtained with statistical significance. We found that patients with TNFRSF14-high hepatocytes tended to have lower levels of PT-INR (P = 0.023) (Table 2). In addition, we more often detected LIGHT-high biliary epithelial cells or hepatocytes in patients with lower levels of albumin (P = 0.042) or higher levels of total bilirubin (P = 0.008), respectively (Table 3).