Regulation of Proinflammatory Cytokine Responses by a Caspase-8-N4BP1 Axis

Project Summary/Abstract Rare monogenic immune disorders have illuminated key aspects of inflammation, but many of the underlying mechanisms remain poorly understood. For example, autoimmune lymphoproliferative syndrome (ALPS), a disorder in which T cells fail to undergo apoptosis, is most often caused by genetic defects in the death receptor FAS or its ligand FASL. However, mutations in caspase-8 or its adaptor FADD – which mediate cell death downstream of FAS – cause a combination of ALPS plus severe immunodeficiency. Since immunodeficiency is not generally observed in patients with FAS or FASL mutations, I hypothesized that FADD-caspase-8 must have an apoptosis-independent function downstream of an immune receptor other than FAS. Indeed, I recently discovered that activation of multiple immune receptors elicits the caspase-8-mediated cleavage of Nedd4- binding protein 1 (N4BP1), a novel cytokine suppressor. This represents a critical point of regulation during inflammation. Notably, deletion of N4BP1 does not ordinarily affect the TRIF-dependent subset of toll-like receptors (TLRs) that activate caspase-8 (e.g., TLR3 and TLR4). However, the impaired cytokine production of caspase-8-deficient macrophages stimulated with a TLR4 agonist is restored to normal by co-deletion of N4BP1. In contrast, N4BP1 deletion leads to exorbitant cytokine responses by the TRIF-independent TLRs (e.g., TLR1/2, TLR7 and TLR9) that do not directly activate caspase-8. Thus, N4BP1 cleavage by caspase-8 inactivates the anti-inflammatory activity of intact, un-cleaved N4BP1. These findings offer a novel mechanistic explanation for immunodeficiency caused by FADD-caspase-8 mutations, whereby the inability to cleave N4BP1 results in its aberrant persistence and constriction of cytokine responses. Like TLR3 and TLR4 agonists, tumor necrosis factor (TNF) also leads to caspase-8 cleavage of N4BP1, endowing TNF with the ability to inactivate N4BP1 and thereby license cytokine production by the TRIF-independent TLRs. This latter finding highlights a key point of molecular crosstalk between the TNF and TLR systems that converges on caspase-8 cleavage of N4BP1. In the current proposal, I have linked the mechanism by which N4BP1 suppresses cytokine production to a series of proteins with both previously recognized and heretofore unknown roles in inflammation. In Aim 1, I will attempt to decipher the mechanism by which N4BP1 controls the activity of this novel kinase-dependent pathway that suppresses inflammation. In Aim 2, I will dissect how N4BP1 suppresses late phase inflammatory gene expression using genome-scale technologies. In Aim 3, I will explore the mechanisms and in vivo consequences of signal integration by the TNF-caspase-8-N4BP1 axis. Together, these aims will provide novel mechanistic insights explaining a key regulatory circuit underlying inflammation. They also will serve to launch my independent research career.