Notch Signaling Contributes to Liver Inflammation by Regulation of Interleukin-22-Producing Cells in Hepatitis B Virus Infection

Abstract

The mechanism of hepatitis B virus (HBV) induced liver inflammation is not fully elucidated. Notch signaling augmented interleukin (IL)-22 secretion in CD4⁺ T cells, and Notch-IL-22 axis fine-tuned inflammatory response. We previously demonstrated a proinflammatory role of IL-22 in HBV infection. Thus, in this study, we analyzed the role of Notch in development of IL-22-producing cells in HBV infection by inhibition of Notch signaling using γ-secretase inhibitor DAPT in both hydrodynamic induced HBV-infected mouse model and in peripheral blood cells isolated from patients with HBV infection. mRNA expressions of Notch1 and Notch2 were significantly increased in livers and CD4⁺ T cells upon HBV infection. Inhibition of Notch signaling in vivo leaded to the reduction in NKp46⁺ innate lymphoid cells 22 (ILC22) and lymphoid tissue inducer 4 (LTi4) cells in the liver. This process was accompanied by downregulating the expressions of IL-22 and related proinflammatory cytokines and chemokines in the liver, as well as blocking the recruitment of antigen-non-specific inflammatory cells into the liver and subsequent liver injury, but did not affect HBV antigens production and IL-22 secretion in the serum. Furthermore, IL-22 production in HBV non-specific cultured CD4⁺ T cells, but not HBV-specific CD4⁺ T cells, was reduced in response to in vitro inhibition of Notch signaling. In conclusion, Notch siganling appears to be an important mediator of the liver inflammation by modulating hepatic ILC22. The potential proinflammatory effect of Notch-mediated ILC22 may be significant for the development of new therapeutic approaches for treatment of hepatitis B.

Keywords: Notch signaling; hepatitis B virus; inflammation; innate lymphoid cells; interleukin-22.

Figure 1

Inhibition of Notch signaling did not affect HBV antigens production but reduced liver injury. Mice (4 per group) were hydrodynamically injected with saline, pHBV1.3 plasmid, or pHBV1.3 plus γ-secretase inhibitor DAPT. Mice were scarified 96 h post injection. Livers and serum were harvested for analysis. mRNA expressions corresponding to Notch1 (A) and Notch2 (B) in the livers were measured by PT-PCR. Results are displayed as fold differences relative to the saline injection group, and normalized to β-actin. HBsAg (C) and HBeAg (D) in the serum were measured by electro-chemiluminescence. (E) Serum ALT levels were measured by Infinity ALT reagent. (F) IL-22 concentrations in the serum were tested by ELISA. All values are presented as the average from each group, and error bars represent SE.

Figure 2

Inhibition of Notch signaling reduced the HBV-induced elevation of IL-22, and related cytokines and chemokines mRNA expressions in the liver. mRNA expressions corresponding to IL-22 (A), IFN-γ (B), TNF-α (C), CXCL9 (D), and CXCL10 (E) in the livers were measured by PT-PCR. Results are displayed as fold differences relative to the saline injection group, and normalized to β-actin.

Figure 3

Inhibition of Notch signaling reduced the intrahepatic NKp46⁺ILC22 and LTi4 cells. (A) Populations in IHLs were discriminated based on expression of CD3, NK1.1, CD127, RORγt, NKp46, and CD4. These included populations of CD3⁺CD4⁺NK1.1⁻RORγt⁺ (mostly Th17 and Th22 cells), CD3⁻NK1.1⁻RORγt⁺CD127⁺NKp46⁺CD4⁻ILC22, and RORγt⁺CD127⁻NKp46⁻ cells (CD4⁺ and CD4⁻). Representative experiments of flow cytometry is shown. Numbers of (B) CD3⁺CD4⁺NK1.1⁻RORγt⁺ cells, (C) NKp46⁺ ILC22, (D) CD4⁻ILC22, and (E) LTi4 cells are shown. All values are presented as the average from each group, and error bars represent SE.

Figure 4

Inhibition of Notch signaling blocked the recruitment of antigen-non-specific cells into the liver. IHLs were isolated and analyzed by flow cytometry. (A) CD3⁺CD4⁺ cells. (B) CD3⁺CD4⁻NK1.1⁻ cells. (C) CD3⁻NK1.1⁺ cells. (D) CD3⁺NK1.1⁺ cells. All values are presented as the average from each group, and error bars represent SE.

Figure 5

Expressions of Notch1 and Notch2 were elevated in peripheral CD4⁺ T cells isolated from patients with HBV infection. mRNA expressions corresponding to Notch1 (A) and Notch2 (B) in CD4⁺ T cells were measured by PT-PCR. Results are displayed as fold differences relative to normal controls, and normalized to 18sRNA. All values are presented as the average from each group, and error bars represent SE.

Figure 6

Inhibition of Notch signaling did not affect IL-22 mRNA expression in isolated CD4⁺ T cells, but reduced the HBV non-specific IL-22 production in the supernatants from cultured CD4⁺ T cells. (A) mRNA expressions corresponding to IL-22 in CD4⁺ T cells were measured by PT-CPR. Results are displayed as fold differences relative to anti-CD3 treatment, and normalized to 18sRNA. (B) IL-22 production in the supernatants were measured by ELISA. All values are presented as the average from each group, and error bars represent SE.