Necroptotic and apoptotic signaling during development and immune homeostasis
Receptor interacting protein kinase 1 (RIP1) was originally identified as a protein associated with Fas and tumor necrosis factor receptor 1 (TNFR1), which are death receptors (DRs) that initiate programmed cell death (PCD). Specifically, RIP1 is a part of the death inducing signaling complex (DISC) along with Fas-associated death domain protein (FADD), caspase 8, and a related kinase, RIP3. The DISC functions as a signaling platform to trigger programmed cell death (PCD) downstream of the DRs. While the DISC primarily induces apoptosis via cysteine-specific proteolytic enzymes called caspases, RIP1 has been shown to be dispensable for apoptotic signaling. Instead, RIP1 initiates an alternative form of cell death called necroptosis (programmed necrosis) by binding to and phosphorylating RIP3. Necroptosis primarily occurs when the apoptotic pathway is blocked due to lack of caspase activity and results in cell swelling, membrane rupture, and release of intracellular components. RIP1-/- mice die within one day of birth from an unknown cause. Therefore, the in vivo role for RIP1 has been poorly studied due to a lack of available mouse models. To circumvent this issue, this thesis utilizes both a kinase-dead RIP1 knock-in mouse model and a RIP1 conditional knockout (KO) mouse model to dissect the function of RIP1 in neonates as well as T and B lymphocytes, which are particularly reliant on cell death for development, homeostasis, and function. I show that mice with a T cell-specific deletion of RIP1 (RIP1t-/- mice) display no defect in T cell development in the thymus, but have severely decreased T cell numbers in the periphery compared to wild type mice. The T lymphopenic condition and impaired proliferative responses of RIP1-/- T cells are due to increased caspase activity after TNFα treatment or TCR stimulation that leads to apoptosis. Furthermore, inactivation of the kinase domain has no effect on T cell homeostasis or proliferative responses. Therefore, my data reveal a novel function of RIP1 in T cells, where RIP1 protects them from uncontrolled apoptosis in a kinase-independent manner rather than inducing necroptosis, as was previously understood. In contrast, mice with a B cell-specific deletion of RIP1 (RIP1b-/- mice) have peripheral B cell populations comparable to wild type mice and RIP1-/- B cells display normal proliferative responses upon BCR activation. However, lack of RIP1 sensitizes B cells to necroptosis mediated by Toll-like receptor (TLR)3 and TLR4, and results in impaired proliferative responses and antibody production. Blocking the necroptosis pathway by RIP3 ablation rescues this sensitivity to TLR4-induced cell death. Therefore, in B cells, RIP1 is dispensable for development and homeostasis but is essential to protect against uncontrolled necroptosis induced by innate immune receptors TLR3 and TLR4. Lastly, this thesis investigates the cause of RIP1-/- perinatal lethality, which has been unsolved since the initial observation almost 20 years ago. My data demonstrate that ablation of RIP3 mildly improved the survival of RIP1-/- mice to seven days after birth. However, blocking both apoptosis via FADD deletion and necroptosis via RIP3 deletion resulted in survival of RIP1-/- mice into adulthood. Furthermore, since the extrinsic death pathways in RIP1-/-FADD-/- RIP3-/- mice were blocked, their lymphocytes were unable to undergo normal homeostatic turnover and accumulated in the peripheral lymphoid organs, displaying a lymphoproliferative disease reminiscent of the classic lpr mouse model. Overall, this thesis reveals a novel mechanism by which RIP1 regulates the immune system and perinatal development. The primary function for RIP1 in lymphocytes is not to induce necroptosis but to protect cells against uncontrolled cell death. RIP1 plays differential roles in protecting against cell death in lymphocytes, by protecting against apoptosis in T cells and necroptosis in B cells. However, it is necessary to block not only apoptosis by ablation of FADD, but also necroptosis by ablation of RIP3 in order for RIP1-/- mice to survive perinatal development. These results reveal important, novel functions of RIP1 that are of great interest to the cell death field and can ultimately be applied to treat a wide variety of diseases like cancers, autoimmune disease, and inflammatory conditions in which cell death dysregulation is a primary cause.
Dowling, John P, "Necroptotic and apoptotic signaling during development and immune homeostasis" (2016). ETD Collection for Thomas Jefferson University. AAI10252312.