Abstract
Tumour necrosis factor α receptor 1 (TNFR1) activation is known to induce cell death, inflammation, and fibrosis but also hepatocyte survival and regeneration. The multidrug resistance protein 2 knockout (Mdr2−/) mice are a model for chronic hepatitis and inflammation-associated hepatocellular carcinoma (HCC) development. This study analysed how the absence of TNFR1 mediated signalling shapes cytokine and chemokine production, immune cell recruitment and ultimately influences liver injury and fibrotic tissue remodelling in the Mdr2−/− mouse model. We show that Tnfr1−/−/Mdr2−/− mice displayed increased plasma levels of ALT, ALP, and bilirubin as well as a significantly higher collagen content, and markers of fibrosis than Mdr2−/− mice. The expression profile of inflammatory cytokines (Il1b, Il23, Tgfb1, Il17a), chemokines (Ccl2, Cxcl1, Cx3cl1) and chemokine receptors (Ccr6, Cxcr6, Cx3cr1) in livers of Tnfr1−/−/Mdr2−/− mice indicated TH17 cell infiltration. Flow cytometric analysis confirmed that the aggravated tissue injury in Tnfr1−/−/Mdr2−/− mice strongly correlated with increased hepatic recruitment of TH17 cells and enhanced IL-17 production in the injured liver. Moreover, we observed increased hepatic activation of RIPK3 in Tnfr1−/−/Mdr2−/− mice, which was not related to necroptotic cell death. Rather, frequencies of infiltrating CX3CR1+ monocytes increased over time in livers of Tnfr1−/−/Mdr2−/− mice, which expressed significantly higher levels of Ripk3 than those of Mdr2−/− mice. Overall, we conclude that the absence of TNFR1-mediated signalling did not improve the pathological phenotype of Mdr2−/− mice. It instead caused enhanced infiltration of TH17 cells and CX3CR1+ monocytes into the injured tissue, which was accompanied by increased RIPK3 activation and IL-17 production.
Highlights
It has been shown that acute and chronic hepatic inflammation, fibrotic tissue remodelling, and potential tumorigenesis is in part promoted by tumour necrosis factor α (TNFα) signalling through TNFα receptor 1 (TNFR1), and to a lesser degree through activation of TNFR26,7 The signalling pathways of both TNF receptors have considerable overlap, with TNFR1 being expressed ubiquitously and responsible for most of the pro-inflammatory, cytotoxic and apoptotic effects of TNFα, while TNFR2 is primarily found on the hematopoietic cell compartment and lacks the intracellular death domain which induces TNFR1-dependent cell death[8]
In order to evaluate how the absence of TNFR1 signalling during chronic liver inflammation influences tissue injury and subsequent fibrosis in der Mdr2−/− mouse model, we used 12-week-old female mice of the respective genotypes
We observed significantly increased gene expression of various markers of fibrosis, including genes for α-smooth muscle actin (Acta2), collagen type 1 (Col1a1) and type 3 (Col3a1), matrix metalloproteinases (Mmp) 2 and Mmp[9] as well as tissue inhibitors of MMPs (Timp) 1 and Timp[2] in the livers of Tnfr1−/−/Mdr2−/− mice compared to Mdr2−/− mice (Fig. 1D)
Summary
In line with the observation that the absence of TNFR1 leads to an altered microenvironment rich in cytokines and chemokines known to be involved in the recruitment and activation of TH17 cells, flow cytometric analysis revealed an increased frequency of IL-17A-expressing TH17 cells in the livers of Tnfr1−/−/Mdr2−/− mice (Fig. 3A,B, gating strategy in Supplementary Fig. 2A). Several genes encoding for proliferation markers including proliferating cell nuclear antigen (PCNA), Cyclin A2 (CCNA2) and cyclin-dependent kinase 1 (CDK1) were significantly increased in livers of Tnfr1−/−/Mdr2−/− mice (Supplementary Fig. 3B).
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