Double-Negative T Cells Regulate Hepatic Stellate Cell Activation to Promote Liver Fibrosis Progression via NLRP3

Abstract

Aim: We mainly explored the role and mechanism of double-negative T cells (DNTs) in liver fibrosis.

Methods: DNTs were co-cultured with mouse hepatic stellate cells (HSCs). Later, cell viability was detected by Cell Counting Kit-8 (CCK-8) assay; α-SMA expression was measured through fluorescence staining; TNF-α, IL-6, and MMP-9 levels were measured by ELISA; and the expression of Bcl-2, TGF-β1, NLRP3, ASC, and TNFR1 proteins in HSCs was detected by Western blotting (WB) assay. At the same time, HSC-NLRP3^-/- and HSC-TNFR1^-/- are used to explore the mechanism. In mouse experiments, mice were intraperitoneally injected with DNTs; afterward, the hepatic tissue fibrosis degree was detected by Masson staining, α-SMA expression was measured through immunohistochemistry (IHC) assay, and histopathological changes were detected by sirius-red staining and H&E staining.

Results: The results suggested that DNTs promoted HSC activation and NLRP3 activation. The effect of DNTs on activating HSC-NLRP3^-/- was suppressed, and the difference was significant as compared with HSCs. HSC-TNFR1^-/- activation was also inhibited. To explore the mechanism of DNT-secreted TNF-α in TNFR1-NLRP3 activation, we transfected DNTs with TNF-α siRNA; as a result, DNTs with TNF-α silencing did not significantly affect HSC activation. DNTs promoted hepatic tissue fibrosis progression and HSC activation; after treatment with NLRP3 inhibitor, the effect of DNTs on promoting fibrosis was suppressed.

Conclusion: We discovered that DNTs played an important role in liver fibrosis and that DNTs promoted HSC activation via the TNF-α-TNFR1-NLRP3 signal axis, thus further promoting liver fibrosis progression.

Keywords: NLRP3; TNFR1; double-negative T cells; hepatic stellate cells; liver fibrosis.