Myeloid STAT3 inhibits T cell-mediated hepatitis by regulating T helper 1 cytokine and interleukin-17 production

Abstract

Background & aims: T cell-mediated hepatitis is a leading cause of acute liver failure; there is no effective treatment, and the mechanisms underlying its pathogenesis are obscure. The aim of this study was to investigate the immune cell-signaling pathways involved-specifically the role of signal transducer and activator of transcription 3 (STAT3)-in T cell-mediated hepatitis in mice.

Methods: T cell-mediated hepatitis was induced in mice by injection of concanavalin A (Con A). Mice with myeloid cell-specific and T-cell-specific deletion of STAT3 were generated.

Results: STAT3 was activated in myeloid and T cells following Con A injection. Deletion of STAT3 specifically from myeloid cells exacerbated T-cell hepatitis and induced STAT1-dependent production of a T helper cell (Th)1 cytokine (interferon [IFN]-gamma) and to a lesser extent of Th17 cytokines (interleukin [IL]-17 and IL-22) in a STAT1-independent manner. In contrast, deletion of STAT3 in T cells reduced T cell-mediated hepatitis and IL-17 production. Furthermore, deletion of IFN-gamma completely abolished Con A-induced T-cell hepatitis, whereas deletion of IL-17 slightly but significantly reduced such injury. In vitro experiments indicated that IL-17 promoted liver inflammation but inhibited hepatocyte apoptosis.

Conclusions: Myeloid STAT3 activation inhibits T cell-mediated hepatitis via suppression of a Th1 cytokine (IFN-gamma) in a STAT1-dependent manner, whereas STAT3 activation in T cells promotes T-cell hepatitis to a lesser extent, via induction of IL-17. Therefore, activation of STAT3 in myeloid cells could be a novel therapeutic strategy for patients with T-cell hepatitis.

Fig. 1. Activation of STAT3 and STAT1 in myeloid and T cells in Con A-induced hepatitis

C57BL/6 mice were injected with vehicle (saline) or Con A for various time points. A, Phospho-STAT3 immunostaining on liver tissues from control and Con A-treated mice. Blue and red arrows depict pSTAT3 in hepatocytes and small cells in the sinusoids, respectively. B, Western blot analyses of the spleen tissues. C, Flow cytometry analyses of CD11b⁺ myeloid cells and CD3⁺ T cells from the spleen of mice treated with Con A for 2 hrs with pSTAT3 or pSTAT1 antibodies. D, Absolute number of CD11b⁺ and CD3⁺ cells stained with pSTAT3 or pSTAT1. *P<0.05 and **P<0.01.

Fig. 2. Myeloid STAT3 depletion exacerbates Con A-induced hepatitis and promotes preferentially innate inflammatory, Th1 (IFN-γ) cytokines and Th17 cytokines to a lesser extent without affecting Th2 (IL-4) cytokine

A, Serum ALT levels. B, H & E staining of liver sections 12 hrs post Con A injection. Arrows indicate necrotic areas. C, Western blot analyses of liver protein extracts from mice 3 hrs post Con A injection. D, Serum pro-inflammatory cytokines. E, Relative induction of cytokines 2 hrs post Con A injection. The values from wild-type mice were set as 1. F, Myeloperoxidase immunostaining of liver tissues 12 hrs post Con A injection. *P<0.05, **P<0.01, and ***P<0.005 (n=5-8), in comparison with the corresponding WT groups. ND for Not Detected.

Fig. 3. Myeloid-cell specific STAT3 deficiency promotes T-cell STAT3 activation and IL-17 response

A, STAT3 activation (pSTAT3⁺ cells) in hepatic CD11b⁺ and CD3⁺ T cells 2 hrs post Con A treatment analyzed by flow cytometry. B-C, Real-time PCR analyses of mRNAs from spleen CD4⁺ T cells (B) and liver tissues (C) of mice treated with Con A. D, E, Serum levels of IL-17 and IL-22. *P<0.05, **P<0.01, and ***P<0.005 (n=4-8), in comparison with the corresponding WT groups.

Fig. 4. Deletion of STAT3 in T cells reduces IL-17 production and liver injury in Con A-induced hepatitis

A, Phosphorylated STAT3 detection in CD3+ T cells in liver mononuclear cells (MNCs) and splenocytes from mice treated with Con A for 2 hrs by flow cytometry. B, Serum inflammatory cytokines. C, Serum ALT levels. D, Liver tissues stained by Hematoxylin and eosin 12 hrs post Con A injection. Arrows indicate necrotic areas. E, Myeloperoxidase immunostaining of liver tissues 12 hrs post-Con A injection. *P<0.05 and **P<0.01 (n=6-12). The number of mice used in the all groups in panel B was the same. ND for Not Detected.

Fig. 5. IFN-γ plays an essential role, while IL-17 plays a minor but significant role in T-cell-mediated liver injury: IL-17 stimulates Kupffer cells to produce cytokines but prevents IFN-γ-induced hepatocyte apoptosis

A, Serum ALT levels 12 hrs post Con A injection. B, H & E staining of liver sections 12 hrs post Con A injection. C, Serum inflammatory cytokines. D, Western blot analyses of phospho-P65 and phospho-STAT3 in IL-17-treated liver macrophages. E, Inflammatory cytokines from IL-17-treated liver macrophage culture medium 24 hrs later. F, Isolated hepatocytes were cultured for 3 days without or with IFN-γ (10ng/ml), IL-17 (10ng/ml), or both cytokines. Lactate dehydrogenase (LDH) activity and caspase 3 were measured. *P<0.05, **P<0.01, ***P<0.001. ND for Not Detected.

Fig. 6. Deletion of IL-17 in STAT3^Mye-/- mice does not reduce Con A-induced hepatitis

A, Serum ALT levels 12 hrs post Con A injection. B, H & E staining of liver sections 12 hrs post Con A injection. C, Serum inflammatory cytokines 12 hrs post Con A injection.

Fig. 7. Deletion of STAT1 in STAT3^Mye-/- mice ameliorates liver injury and abolishes innate immune and Th1 but not Th2/Th17 cytokine production during Con A-induced hepatitis

A, Activation of pSTAT1 in WT and STAT3^Mye-/- splenocytes 2 hrs post Con A injection analysed by Western blotting. B-C, Serum levels of cytokines. D, Serum ALT levels. *P<0.05, **P<0.01, and ***P<0.005 (n=3-6). ND for Not Detected.

Fig. 8. A model depicting the hepatoprotection of myeloid cell STAT3 in T-cell hepatitis

During T-cell hepatitis, myeloid cell STAT3 inhibits STAT1 signaling in these cells, followed by preventing IL-12/IL-27 production and subsequently inhibiting IL12/IL-27 stimulation of IFN-γ production by Th1 cells. Myeloid cell STAT3 inhibits STAT1 and NF-κB activation, followed by reducing production of inflammatory cytokines (IL-6 and TNF-α) and subsequently inhibiting IL-6 stimulation of IL-17 production by Th17 cells. IFN-γ plays an essential role in T-cell hepatitis via induction of inflammation and hepatocyte death, while IL-17 only stimulates weakly liver inflammation but prevents hepatocyte death, thereby playing a double-edged sword role in T-cell hepatitis. Myeloid cell STAT3 also inhibits IL-22 production via unknown mechanisms.