Interleukin-17 Contributes to the Pathogenesis of Autoimmune Hepatitis through Inducing Hepatic Interleukin-6 Expression

T helper cells that produce IL-17 (Th17 cells) have recently been identified as the third distinct subset of effector T cells. Emerging data suggests that Th17 cells play an important role in the pathogenesis of many liver diseases by regulating innate immunity, adaptive immunity, and autoimmunity. In this study, we examine the role and mechanism of Th17 cells in the pathogenesis of autoimmune hepatitis (AIH). The serum levels of IL-17 and IL-23, as well as the frequency of IL-17+ cells in the liver, were significantly elevated in patients with AIH, compared to other chronic hepatitis and healthy controls. The hepatic expressions of IL-17, IL-23, ROR-γt, IL-6 and IL-1β in patients with AIH were also significantly increased and were associated with increased inflammation and fibrosis. IL-17 induces IL-6 expression via the MAPK signaling pathway in hepatocytes, which, in turn, may further stimulate Th17 cells and forms a positive feedback loop. In conclusion, Th17 cells are key effector T cells that regulate the pathogenesis of AIH, via induction of MAPK dependent hepatic IL-6 expression. Blocking the signaling pathway and interrupting the positive feedback loop are potential therapeutic targets for autoimmune hepatitis

The Ectopic Expression of CaRop1 Modulates the Response of Tobacco Plants to Ralstonia solanacearum and Aphids

In plants, Rho-related GTPases (Rops) are versatile molecular switches that regulate various biological processes, although their exact roles are not fully understood. Herein, we provide evidence that the ectopic expression of a Rop derived from Capsicum annuum, designated CaRop1, in tobacco plants modulates the response of these plants to Ralstonia solanacearum or aphid attack. The deduced amino acid sequence of CaRop1 harbors a conserved Rho domain and is highly homologous to Rops of other plant species. Transient expression of a CaRop1-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed localization of the GFP signal to the plasma membrane, cytoplasm, and nucleus. Overexpression (OE) of the wild-type CaRop1 or its dominant-negative mutant (DN-CaRop1) conferred substantial resistance to R. solanacearum infection and aphid attack, and this effect was accompanied by enhanced transcriptional expression of the hypersensitive-reaction marker gene HSR201; the jasmonic-acid (JA)-responsive PR1b and LOX1; the insect resistance-associated NtPI-I, NtPI-II, and NtTPI; the ethylene (ET) production-associated NtACS1; and NPK1, a mitogen-activated protein kinase kinase kinase (MAPKKK) that interferes with N-, Bs2-, and Rx-mediated disease resistance. In contrast, OE of the constitutively active mutant of CaRop1(CA-CaRop1) enhanced susceptibility of the transgenic tobacco plants to R. solanacearum infection and aphid attack and downregulated or sustained the expression of HSR201, PR1b, NPK1, NtACS1, NtPI-I, NtPI-II and NtTPI. These results collectively suggest that CaRop1 acts as a signaling switch in the crosstalk between Solanaceaes’s response to R. solanacearum infection and aphid attack possibly via JA/ET-mediated signaling machinery

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

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

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

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