TLR5 activation in hepatocytes alleviates the functional suppression of intrahepatic CD8⁺ T cells

The liver is an immune-privileged organ with a tolerogenic environment for maintaining liver homeostasis. This hepatic tolerance limits the intrahepatic CD8⁺ T-cell response for eliminating infections. The tolerant microenvironment in the liver is orchestrated by liver-specific immunoregulatory cells that can be functionally regulated by pathogen-associated molecular patterns (PAMPs). Here, we report that flagellin, a key PAMP of gut bacteria, modulates the intrahepatic CD8⁺ T-cell response by activating the TLR5 signalling pathway of hepatocytes. We found that mice treated with Salmonella-derived recombinant flagellin (SF) by hydrodynamic injection had a significantly elevated IFN-γ production by the intrahepatic lymphocytes in 7 days after injection. This was correlated with a reduced immune suppressive effect of primary mouse hepatocytes (PMHs) in comparison with that of PMHs from mock-injected control mice. In vitro co-culture of SF-treated PMHs with splenocytes revealed that hepatocyte-induced immune suppression is alleviated through activation of the TLR5 but not the NLRC4 signalling pathway, leading to improved activation and function of CD8⁺ T cells during anti-CD3 stimulation or antigen-specific activation. In an acute HBV replication mouse model established by co-administration of SF together with an HBV-replicating plasmid by hydrodynamic injection, SF significantly enhanced the intrahepatic HBV-specific CD8⁺ T-cell response against HBV surface antigen. Our results clearly showed that flagellin plays a role in modulating the intrahepatic CD8⁺ T-cell response by activating the TLR5 pathway in PMHs, which suggests a potential role for gut bacteria in regulating liver immunity.