Deletion of tumor necrosis factor α receptor 1 leads to increased tissue damage and fibrosis in the chronically inflamed liver

Question:

Chronic hepatic inflammation has several major consequences including cirrhosis, liver failure and cancer. Tumor necrosis factor α receptor-1 (TNFR1) activation is known to induce cell death, inflammation, and fibrosis but also hepatocyte survival and regeneration. In this study we analyzed the role of TNFR1 during chronic liver inflammation. Therefore, we ablated TNFR1 in a mouse model of chronic hepatitis and inflammation induced hepatocellular carcinoma (multidrug resistance protein 2 knockout mice; mdr2 ^-/-) creating tnfr1/mdr2 ^-/- mice.

Methods:

Liver injury was assessed by plasma levels of alanine aminotransferase (ALT) and alkaline phosphatase (ALP). Cytokine concentrations were determined by Legendplex assay. Immune cell composition was determined by flow cytometry. Hepatic immune cells were stimulated ex vivo with PMA & Ionomycin. Fibrosis was analyzed through quantification of the hepatic hydroxyproline content and Sirius Red staining. Hepatic expression levels of targets involved in inflammation, fibrosis, and tissue regeneration were analyzed via real time RT-PCR.

Results:

We observed significantly increased ALT and ALP plasma levels, as well as significantly increased neutrophil and TH17 cell infiltration into the liver of tnfr1/mdr2 ^-/- compared to mdr2 ^-/- mice. Ex vivo stimulated hepatic immune cells of tnfr1/mdr2 ^-/- mice produced significantly more IL-17, which directly correlated with tissue injury. Tnfr1/mdr2 ^-/- mice showed significantly increased hepatic expression of pro-inflammatory cytokines (IL-1β, IL-23, TGF-β, CCL2, CX3CL1), mediators of cell death (Pycard, Rip3), and fibrosis (α-sma, collagen type I and III, MMPs) compared to mdr2 ^-/- mice. The tnfr1/mdr2 ^-/- mice further showed increased hepatic hydroxyproline levels and Sirius Red positive areas in stained tissue sections. The expression of proliferation markers (PCNA, CyclinA2) was up-regulated in the tnfr1/mdr2 ^-/- mice indicating a normal regenerative response.

Conclusion:

Constitutive knockout of TNFR1 did not improve the pathological phenotype of mdr2-/- mice. It instead caused enhanced neutrophil and TH17 cell infiltration, accompanied by higher IL-17 production and exacerbated tissue damage, which resulted in increased fibrosis. Increased inflammation and fibrosis in combination with active regeneration implies an ideal microenvironment for tumor development in the livers of tnfr1/mdr2 ^-/- mice. Long term studies will show whether the tnfr1 knockout alters tumor development and progression in the mdr2^-/- model.