CX3CR1 deficiency exacerbates immune-mediated hepatitis by increasing NF-κB-mediated cytokine production in macrophage and T cell

Abstract

Immune-mediated hepatitis is marked by liver inflammation characterized by immune cell infiltration, chemokine/cytokine production, and hepatocyte injury. C-X3C motif receptor 1 (CX3CR1), as the receptor of chemokine C-X3C motif ligand 1 (CX3CL1)/fractalkine, is mainly expressed on immune cells including monocytes and T cells. Previous studies have shown that CX3CR1 protects against liver fibrosis, but the exact role of CX3CL1/CX3CR1 in acute immune-mediated hepatitis remains unknown. Here, we investigate the role of the CX3CL1/CX3CR1 axis in immune-mediated hepatitis using concanavalin A (ConA)-induced liver injury model in CX3CR1-deficient (Cx3cr1^-/-) mice. We observed that Cx3cr1^-/- mice had severe liver injury and increased pro-inflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interferon-gamma [IFN-γ], interleukin-1 beta [IL-1β], and IL-6) in serum and liver compared to wild-type (Cx3cr1^+/+) mice after ConA injection. The deficiency of CX3CR1 did not affect ConA-induced immune cell infiltration in liver but led to elevated production of TNF-α in macrophages as well as IFN-γ in T cells after ConA treatment. On the contrary, exogenous CX3CL1 attenuated ConA-induced cytokine production in wild type, but not CX3CR1-deficient macrophages and T cells. Furthermore, in vitro results showed that CX3CR1 deficiency promoted the pro-inflammatory cytokine expression by increasing the phosphorylation of nuclear factor kappa B (NF-κB) p65 (p-NF-κB p65). Finally, pre-treatment of p-NF-κB p65 inhibitor, resveratrol, attenuated ConA-induced liver injury and inflammatory responses, especially in Cx3cr1^-/- mice. In conclusion, our data show that the deficiency of CX3CR1 promotes pro-inflammatory cytokine production in macrophages and T cells by enhancing the phosphorylation of NF-κB p65, which exacerbates liver injury in ConA-induced hepatitis.

Keywords: CX3CR1; NF-κB p65; T cell; immune-mediated hepatitis; macrophage.

Figure 1.

Increased Con A-induced liver injury in Cx3cr1^−/− mice compared to wild-type Cx3cr1^+/+ mice. Immune-mediated hepatitis was induced by intravenous injection with 15 mg/kg body weight of ConA in Cx3cr1^+/+ and Cx3cr1^−/− mice. (A) Kaplan-Meier survival curves of Cx3cr1^+/+ (n = 12) and Cx3cr1^−/− (n = 12) mice. (B) Cx3cr1^+/+ (n = 12) and Cx3cr1^−/− (n = 8) mice were weighed before ConA injection (initial body weight), and before euthanasia (final body weight). The data of A and B were combined from three independent experiments. (C, D) Serum AST and ALT were measured at 8 and 24 h after ConA treatment (Cx3cr1^+/+ n = 4; Cx3cr1^−/− n = 4). (E) Left: Representative H&E staining in the liver of ConA-treated Cx3cr1^+/+ mice and Cx3cr1^−/− mice. (original magnification: 40× and 200×). The scale bars are 500 μm (40×) and 100 μm (200×), respectively. Right: The quantification of liver necrosis area. (A color version of this figure is available in the online journal.)

Figure 2.

CX3CR1 deficiency does not affect ConA-induced immune cells infiltration in liver. (A, B) ConA-induced immune cell infiltration in the liver of Cx3cr1^−/− (n = 4) and Cx3cr1^+/+ (n = 4) mice were analyzed by flow cytometry. Liver samples were collected at 8 h (A) and 14 h (B) after ConA treatment. (C, D) Representative immunofluorescence staining of F4/80 (C) and CD3 (D) and quantification in the liver of Cx3cr1^−/− and Cx3cr1^+/+ mice at 24 h after ConA (The scale bars are 100 μm). (A color version of this figure is available in the online journal.)

Figure 3.

CX3CR1 deficiency leads to increased cytokine and chemokine levels in serum and liver. (A) Serum was collected from Cx3cr1^−/− (n = 12) and Cx3cr1^+/+ (n = 12) mice at 8 h after ConA treatment, serum cytokines and chemokines were detected by LEGENDplex™ Mouse Inflammation kit. Data were combined from three independent experiments. (B) Hepatic mRNA expressions of TNF-α, IL-1β, IL-6, IFN-γ, CCL2, and CX3CL1 were measured by real-time PCR. Liver samples were harvested from Cx3cr1^−/− (n = 4) and Cx3cr1^+/+ (n = 4) mice at 3 h after ConA injection. (A color version of this figure is available in the online journal.)

Figure 4.

Disruption of CX3CL1/CX3CR1 axis leads to enhanced pro-inflammatory properties of macrophages and T cells. (A, B) TNF-α and IFN-γ levels in the supernatant of ConA-treated Cx3cr1^+/+ and Cx3cr1^−/− BMDMs (10 μg/mL). (C) BMDMs were pretreated with 100 ng/mL full-length CX3CL1 (CX3CL1-FF), 100 ng/mL the domain of CX3CL1 (CX3CL1-CD) or 100 ng/mL BSA, then incubated with ConA (10 μg/mL) for 48 h to detect TNF-α level. (D) IFN-γ levels of ConA-treated spleen cells (10 μg/mL) were detected by ELISA assay. (E) T cells and non-T cells in spleen cells were separated by pan T cells isolation kit, then total spleen cells, T cells, and non-T cells incubated with ConA for 48 h. IFN-γ levels of each group were measured by ELISA assay. (F) IFN-γ levels of the supernatant of T cells were detected after co-incubation with ConA + BSA, ConA + CX3CL1-CD and ConA + CX3CL1-FF. (A color version of this figure is available in the online journal.)

Figure 5.

The deficiency of CX3CR1 increased the inflammation by increasing the phosphorylation of NF-κB-p65 in macrophages and T cells. BMDMs and T cells were isolated from Cx3cr1^+/+ and Cx3cr1^−/− mice and treated with ConA or PBS. (A and B, D and E) The phosphorylation level of NF-κB-p65 in BMDMs (A) and T cells (D) was detected by Western blotting, their quantification is shown in B and E. (C) BMDMs were incubated with PBS + vehicle (dimethyl sulfoxide), ConA + vehicle and ConA + resveratrol for 48 h, and their TNF-α level was measured by ELISA. (F) T cells were incubated with the same combination and IFN-γ level was measured by ELISA. (A color version of this figure is available in the online journal.)

Figure 6.

P-NF-κB-p65 inhibitor resveratrol can protect the liver from CX3CR1 deficiency–enhanced ConA-induced damage. Cx3cr1^+/+ and Cx3cr1^−/− mice were pre-treated with 30 mg/kg body weight of resveratrol or vehicle (DMSO) seven times by intragastric administration, followed by intravenous injection with 13 mg/kg body weight of ConA. (A, B) Serum AST and ALT were measured at 8 and 24 h after ConA injection. (C, D) Serum TNF-α, IFN-γ, IL-6, IL-1β levels at 8 h (C) and 24 h (D) after ConA injection. (E) Liver necrosis was evaluated by H&E staining (original magnification: 40× and 200×). The scale bars are 500 μm (40×) and 100 μm (200×), respectively. (F) The quantification of liver necrosis area. (A color version of this figure is available in the online journal.)