Auto-necroptosis in macrophages

TNF was first described as a factor from the serum of mice that were primed with BCG and challenged with LPS that could kill tumor cells in vitro and in vivo (Carswell et al, PNAS, 1975). In the intervening 40 years, we have come to understand a lot about the role played by TNF in inflammation during infections, and in inflammatory disorders such as rheumatoid arthritis, IBD and psoriasis. The pro-inflammatory function of TNF is largely attributed to its ability to induce the NF-?B and MAPK pathways, and their induction of pro- inflammatory genes. Despite this progress, the biological function of its first described property, that of killing cells, remains unclear. It is highly unlikely that the cytotoxic function of TNF has evolved to deal with cancer, rather it is more likely that it evolved to fight infections but there is little evidence to support this hypothesized death-inducing function of TNF. Studying the death-inducing function of TNF has been challenging due to the complicated nature of its signaling pathways. Binding of TNF to its main receptor TNFR1 can induce either a survival or death response. Ligation of TNFR1 on normal cells induces cell survival, but not death, due to the presence of two sequential cell death checkpoints. The first checkpoint occurs when RIPK1 undergoes non- degradative ubiquitination, an event not dependent on new gene expression. This restricts RIPK1 from associating with death-signaling molecules. Instead, RIPK1 associates with a survival complex to propagate the survival signal. The second checkpoint occurs with the NF-?B-dependent induction of pro-survival genes, providing a longer-lasting protection against death. Disrupting Checkpoint 1 by blocking the ubiquitination of RIPK1 unleashes its ability to induce either apoptosis or necroptosis. Checkpoint 1 is further sustained by a pro-survival CASP8, which cleaves and degrades CYLD, a deubiquitinase specific for K63-linked ubiquitin. CYLD removal leads to prolonged RIPK1 ubiquitination and protection from death. However when CASP8 is inhibited, CYLD is available to deubiquitinate RIPK1 to initiate death, and by virtue of the CASP8 blockade, the only option available is necroptosis. With this newfound understanding of Checkpoint 1 regulation, we can genetically manipulate it such that death predominates so we can begin to study its biological function. Using an inducible macrophage-restricted deletion of Casp8 (causing necroptosis), Casp8 and Cyld (abrogating necroptosis) or Casp8 and Tnf (to demonstrate TNF dependency), we propose to study the immunological effects of triggering necroptosis in macrophages during infections by extracellular and intracellular bacteria. We hypothesize that necroptosis in these sentinel cells, which are wired to undergo self-inflicted death following the induction of TNF expression, serves to heighten both innate and adaptive immune responses to an infection. These analyses will provide significant new insights into an unresolved enigma of 40 years.