Blood samples were obtained from healthy controls (HC, n = 28), chronic hepatitis B (CHB, n = 18) and autoimmune hepatitis (AIH, n = 29) patients.

<p>Peripheral blood mononuclear cells (PBMCs) were isolated, labeled with fluorescent antibodies against CD4, CD25, CCR4 and CCR6, and analyzed by flow cytometry. (A) Plasma IL-17 and IL-23 levels. (B) Representative dot plots; and (C) Mean (±SD) percentage of Th17 (CD4⁺CD25⁻ CCR4⁺CCR6⁺) cells in PBMC. Panel B and C are gated on CD4⁺CD25⁻ cells. *p<0.05, **p<0.01.</p

Liver tissue were obtained from healthy controls (HC, n = 5), chronic hepatitis B (CHB, n = 12) and autoimmune hepatitis (AIH, n = 28) patients.

<p>Th17 related cytokines RORγt, IL-17, IL-1β, IL-6, IL-21 and IL-23 expressions were determined by quantitative RT-PCR and normalized with GAPDH expression. *p<0.05, **p<0.01.</p

Characterization of the Study Participants.

<p>Data is shown as media and range. ALT: alanine aminotransferase; AST: aspartate aminotransferase. There is no statistical difference between all three groups in sex, age. There is no statistical difference between CHB and AIH groups in serum transaminases and histological findings.</p

Figure 4

<p>(A) IL-17 was added to HepG2 cell culture. IL-6 production by HepG2 cells in the media was measured by ELISA. (B) Total protein extract was made from HepG2 cells after stimulated with IL-17 (100 ng/ml). Western bolts were performed with antibodies specific against phosphorylated ERK1/2, p38 MAPK, JNK. The membranes were then stripped and re-probed with an antibody to total ERK, p38 MAPK and JNK. β-actin expression was determined as loading controls. (C) Specific inhibitors of MAPK signaling pathways (SB203580 for MAPK, PD98059 and U0126 for ERK, SP600125 for JNK, and DMSO as the carrier) were added to HepG2 cell culture before IL-17 stimulation. IL-6 in the media was measured by ELISA. (D) IL-17 was added to HepG2 cell culture. MCP-1 production by HepG2 cells in the media was measured by ELISA.</p

Liver biopsies were obtained from patients with either autoimmune hepatitis (AIH, n = 39) or chronic hepatitis B (CHB, n = 32).

<p>Th17 cells in the liver were evaluated by immunohistochemical staining of IL-17. (A) Representative histology of Th17 cells (IL-17+, brown stained cells, 400×); (B) Mean (±SD) of Th17 cells in AIH and CHB patients; (C) Mean (±SD) of hepatic inflammatory scores of AIH and CHB patients; (D) Confocal staining of CD4 (in green), IL-17 (in red) and DAPI (for nuclei in blue) in the liver of AIH patients. The frequency of Th17 cells in the liver is positively correlated with hepatic inflammatory degrees (E) and fibrosis grades (F) in AIH patients.</p

The primers sequences of Th17 cells related genes and GAPDH.

<p>For each sample, mRNA expression level was normalized to the level of GAPDH housekeeping genes using the ΔΔCt algorithm.</p

Quantitation of the Rank-Rankl Axis in Primary Biliary Cholangitis

<div><p>There is substantial data that suggests an abnormality of innate immunity in patients with primary biliary cholangitis (PBC) which includes the transcription factor nuclear factor-kB (NF-kB) and well as downstream inflammatory signaling pathways. In addition, ImmunoChip analysis has identified a novel PBC-associated locus near the receptor activator of NF-kB ligand (RANKL) gene. Based on these observations, we investigated the role of the RANKL axis in the liver of patients with PBC compared to controls. We used immunohistochemistry to quantitate liver expression of RANKL, its receptor (RANK), and importantly the decoy receptor osteoprotegerin (OPG), including a total of 122 liver samples (PBC = 37, primary sclerosing cholangitis = 20, autoimmune hepatitis = 26, chronic hepatitis B = 32 and unaffected controls = 7). In addition, we studied RANKL-RANK-OPG co-localization in CD4 and CD8 T cells, B cells, dendritic cells, macrophages, NK, NKT cells, hepatocytes, and cholangiocytes. We report herein that RANK is constitutively expressed by cholangiocytes in both unaffected and diseased liver. However, cholangiocytes from PBC express significantly higher levers of RANK than either the unaffected controls or liver diseased controls. CD4, CD8 and CD19 cells with in the portal areas around bile ducts in PBC express significantly higher levels of RANKL compared to controls. Importantly, the overall hepatic RANKL level and the ratio of hepatic RANKL/OPG correlated with disease severity in PBC. In conclusion, our data indicate a role of RANK-RANKL axis in the innate immune activation in PBC and we hypothesize that the damaged cholangiocytes, which express high levels of RANK, lead to the recruitment of RANKL positive cells and ultimately the classic portal tract infiltrates.</p></div

Immunohistochemistry of receptor activator of nuclear factor κB (RANK) in liver.

<p>(A) Representative staining images from patients with PBC, AIH, CHB, PSC and unaffected controls (HC) are shown; (B) Statistical analysis of expressed RANKL in liver. (**, p<0.01; ***, p<0.001).</p

Immunohistochemistry of receptor activator of nuclear factor κB ligand (RANKL) in liver.

<p>(A) Representative staining images from PBC, AIH, CHB, PSC and unaffected controls (HC) are shown; (B) Statistical analysis of expressed RANKL in liver. (*, p<0.05; **, p<0.01).</p

RANK expressed in cholangiocytes was elevated in patients with PBC.

<p>(A) Representative staining images (magnification 400×) from the portal areas in PBC, AIH, CHB, PSC and unaffected controls are shown (BD, bile duct, red arrow); (B) Comparison of cholangiocytic RANK levels in PBC versus unaffected and other chronic liver disease controls. Red arrows indicate bile ducts. (***, P<0.001).</p