Periodic Reporting for period 1 - IMPACT (Immune Mechanisms of Necrotic DNA Phagocytosis by Neutrophils: A Role for Integrins)
Berichtszeitraum: 2020-01-01 bis 2021-12-31
Injury is common to all living beings. By performing daily activities and being exposed to harmful agents, we are at constant risk of suffering damage. In humans, that happens most often by mechanical forces (crushing, fractures), extreme temperatures (burns, frostbite) and chemical toxicity (substance abuse, adverse reactions). These situations lead to necrosis, a type of cell death strongly associated to inflammation. One of the consequences of necrosis is the generation of cell debris, which can be deposited in tissues and even reach our bloodstream. Cell debris is a powerful inducer of inflammation, and when accumulated in tissues, can actively delay tissue regeneration and recovery from injury. For this reason, it was paramount that we understood how the necrotic debris is removed from tissues in the first place. It is hypothesized that white blood cells named phagocytes go into the injury sites and clear the cell debris by themselves. The aim of the IMPACT project was to understand how phagocytes are able to do that and to find means to enhance the clearance of cell debris, such as cellular DNA, accelerating tissue recovery after necrotic injuries. To achieve this, necrotic debris clearance were characterized in vitro using a new model of necrotic cell debris phagocytosis and in live tissues using intravital microscopy. This enabled to visualize directly the activity of phagocytes and how they performed their role. By grasping the mechanisms of necrotic debris clearance, we could develop molecules and peptides that improved the function of phagocytes and facilitated the recognition of cell debris in tissues. This provides the basis for the creation of new therapies for a variety of necrotic diseases, including toxic liver injury, atherosclerosis, severe trauma, and even chronic diseases such as systemic lupus erythematosus. The conclusions and products of developing this project were multiple, including: 1) the demonstration and characterization of necrotic debris clearance from injury sites by neutrophils; 2) Establishing the central role of antibodies and the complement system in the recognition and elimination of necrotic cell debris; 3) The identification of a peptide that binds DNA from necrotic cells both in vitro and in vivo, reducing tissue inflammation; 4) Creation of new therapeutic strategies to treat injury, based on the improvement of necrotic cell clearance by antibodies; 5) Training and maturation of the researcher into an independent principal investigator.
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.
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.
The project identified receptors in phagocytes involved in necrotic debris clearance. Moreover, we developed a peptide that binds to and neutralizes the inflammatory properties of necrotic debris. The potential socio-economic impact of these findings/molecule is massive, considering that they may be used to treat a variety of diseases that involve injury and necrosis; this includes acute liver injury, burn injuries, fractures, severe trauma, atherosclerosis, lupus and others.
The research results were published in peer-reviewed journals (Hepatology Communications, International Journal of Molecular Sciences, and JHEP Reports) and are also available in repositories (Lirias, BioRxiv) for open-access to the society. They were also presented orally in multiple international conferences in Europe and the Americas, including the Gordon Research Conferences, the European Chemokine conference and National Immunology meetings in Belgium and Brazil. Lastly, our research results were disseminated to the general public in 2 independent events, the STEM University (2022) and the Dag Van Wetenschap (2023), both of which are focused on children up to 15 years-old. Overall, all the work packages and proposed objectives have been fulfilled with adequate and substantial results and its corresponding deliverables.
The research results were published in peer-reviewed journals (Hepatology Communications, International Journal of Molecular Sciences, and JHEP Reports) and are also available in repositories (Lirias, BioRxiv) for open-access to the society. They were also presented orally in multiple international conferences in Europe and the Americas, including the Gordon Research Conferences, the European Chemokine conference and National Immunology meetings in Belgium and Brazil. Lastly, our research results were disseminated to the general public in 2 independent events, the STEM University (2022) and the Dag Van Wetenschap (2023), both of which are focused on children up to 15 years-old. Overall, all the work packages and proposed objectives have been fulfilled with adequate and substantial results and its corresponding deliverables.