NLRP6 deficiency enhances macrophage-mediated phagocytosis via E-Syt1 to inhibit hepatocellular carcinoma progression

Background Current treatments with tyrosine kinase inhibitors and immune checkpoint inhibitors have limited efficacy for hepatocellular carcinoma (HCC) due to drug resistance. Emerging therapies such as chimeric antigen receptor T (CAR-T) and macrophage-based cell therapies are promising but need to be improved. Objectives This study investigates the role of macrophage NOD-like receptor family pyrin domain containing 6 (NLRP6) in HCC progression and its therapeutic potential. Design Immunofluorescence staining was performed in patient samples. Liver tumour models (autonomous, orthotopic, subcutaneous) were developed, and RNA sequencing, flow cytometry and immunohistochemistry were performed in wild-type, Nlrp6 −/− mice, and cell-specific Nlrp6 knockout mice. Phagocytosis was assessed using particles or tumour cells. Multiomics, immunoprecipitation mass spectrometry, western blot and co-immunoprecipitation were performed to examine the interaction between NLRP6’s PYD domain and E-Syt1’s SMP domain. Results CD68 (a macrophage marker) and NLRP6 expression were detected in patient HCC tissues, and patients with lower macrophage NLRP6 expression had longer survival. Compared with their wild-type mice, Nlrp6 −/− mice and macrophage cell-specific Nlrp6 knockout mice showed delayed tumour growth. Adoptive transfer of Nlrp6 −/− macrophages reduced tumour growth in vivo. Macrophages from Nlrp6 −/− mice were more abundant and exhibited enhanced phagocytosis compared with those from wild-type mice. Co-immunoprecipitation and phagocytosis experiments revealed E-Syt1 promoted phagocytosis, which was negatively regulated by NLRP6 through interaction with its PYD domain. Conclusions NLRP6 promotes HCC progression by inhibiting macrophage infiltration and suppressing phagocytosis via the interaction between its PYD domain and E-Syt1’s SMP domain. Transfer of Nlrp6 −/− macrophages is a promising therapeutic strategy for reducing HCC tumour growth.