STAT3 phosphorylation during phagocytosis is required to activate autophagy and prevent macrophage immuno-senescence

Background: Acute liver injury (ALI) is characterised by hepatocyte death and liver inflammation. Macrophages play a pivotal role in ALI by phagocytosing dead cells and producing pro-regenerative signals. We have previously demonstrated that phagocytic cargo-dependent activation of phosphoSTAT3 is required to maintain phagocytosis efficiency in macrophages. In this study, we aim to understand the molecular mechanisms underlying the pro-phagocytic function of phosphoSTAT3.

Methods: Phagocytosing bone marrow derived macrophages (BMDMs) treated with and without a phosphoSTAT3 inhibitor, or from mice deficient for the cargo digestion step of phagocytosis (Gpnmb-), were analysed using flow cytometry, transmission electron microscopy (TEM), and qPCR. We used paracetamol overdose (POD) as mouse model for ALI. We FAC-Sorted and analysed infiltrating macrophages from livers at early stages of POD (10h post-overdose) with the nCounter® technology (Nanostring) and using proteomics.

Results: Gpnmb- mice are unable to regenerate their liver efficiently after POD: infiltrating phagocytosing macrophages FAC-Sorted from these mice showed increased expression of senescence-related genes, such as Tnfrsf1b and Cdkn1a, but not of apoptosis-related genes, such as Card9. This suggests that macrophages unable to process the internalised cargo enter immuno-senescence, a condition characterised by elevated production of inflammatory cytokines and reduced phagocytosis. TEM and qPCR analyses showed that both Gpnmb- and phosphoSTAT3 inhibitor-treated BMDMs develop a senescent phenotype during phagocytosis, suggesting that cargo digestion-dependent phosphorylation of STAT3 prevents immuno-senescence in phagocytosing macrophages. Autophagy is a mechanism widely used by cells to cope with stress: interestingly, TEM and qPCR analyses unveiled a lower activation of autophagy in Gpnmb- and phosphoSTAT3 inhibitor-treated BMDMs.

Conclusions: Phagocytosing macrophages require phosphoSTAT3 activation.

Learning Objectives:

1. Understand the use of flow cytometry-based medium throughput screening assays as an essential tool to investigate macrophage phagocytosis

2. Describe the role of pSTAT3 as a pro-phagocytic pathway in macrophages

3. Explain that other pathways are controlled downstream of pSTAT3, including autophagy and senescence