The project was initiated with the application for ethical permits and preparation of infrastructure and reagents to perform the optimization experiments. Initially, two assays were created to investigate necrotic debris clearance and retention in vitro: the DNA deposit model and the DNA binding assay. Using these novel models, the biochemical properties of DNA were investigated, including the identification of DNA-binding proteins. Once the ethical permits were granted, the optimization of the in vivo model of drug-induced liver injury was started. Dose-response and time-response curves were performed to find the best conditions to investigate the formation, deposition and clearance of debris. Preliminary data indicates that most parameters should be investigated within 24 to 48 hours of the induction of necrotic injury, in this case, induced by an overdose of acetaminophen (paracetamol) at the dose of 600 mg/kg.
A substantial component of the project was the investigation of neutrophil-mediated phagocytosis of necrotic cell in liver injury. In this part, the main aims were to understand the molecular composition of necrotic cell debris and the mechanisms required for its clearance from injury sites. By combining the drug-induced liver injury model and intravital microscopy, we determined that necrotic debris is largely composed of DNA and an intact f-actin cytoskeleton. In vivo, necrotic debris were rapidly opsonized by IgM, IgG, C1q and C3b. Opsonization of necrotic cells by IgM and IgG occurred via auto-reactive natural antibodies, which were required for phagocytosis of necrotic debris in vitro, however, opsonization was dispensable when apoptotic bodies were used, pointing to a mechanistic difference between necrotic debris clearance and phosphatidylserine-dependent efferocytosis. Moreover, opsonization with complement C1q and C3 via the antibody-dependent classical pathway was also required for necrotic debris clearance in vitro. Clearance of necrotic cell debris in vivo was central to drive tissue recovery after drug-induced liver injury, since mice deficient in antibody production or complement presented accumulated cell debris in injury sites and a significant delay in tissue regeneration, as shown by the size of injured areas and decreased hepatocellular proliferation. In addition, phagocytosis of necrotic debris in vivo was completely rescued in Rag2-knockout mice by replenishing them with total IgM and IgG, rescuing also the capacity of the liver to regenerate. Importantly, normal wild-type mice presented an improved recovery post liver injury when supplemented with total IgM and IgG, showing that this strategy has therapeutic potential in immunocompetent individuals. In conclusion, necrotic debris clearance requires both natural antibodies and the complement system, and it is central for the recovery from tissue injury.
In parallel, peptide synthesis was initiated. We opted for production of CXCL9(74-103), a highly positively-charged peptide from the C-terminus of a chemokine. Intravital microscopy showed that this peptide had a high affinity for necrotic cells present in injured livers, specifically for the abundant DNA exposed after their death. Testing of this peptide in the drug-induced liver injury and liver ischemia-reperfusion models yielded interesting discoveries, especially a clear anti-inflammatory and protective effect against liver injury. Neutrophil activation and recruitment were also clearly inhibited by the peptide, showing its capacity to restrain leukocyte activity and confirming its therapeutic value in liver injury.
