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

> **NIH NIH DP5** · SLOAN-KETTERING INST CAN RESEARCH · 2022 · $442,500

## Abstract

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.

## Key facts

- **NIH application ID:** 10479473
- **Project number:** 1DP5OD033360-01
- **Recipient organization:** SLOAN-KETTERING INST CAN RESEARCH
- **Principal Investigator:** Alexander Gitlin
- **Activity code:** DP5 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $442,500
- **Award type:** 1
- **Project period:** 2022-09-13 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10479473

## Citation

> US National Institutes of Health, RePORTER application 10479473, Regulation of Proinflammatory Cytokine Responses by a Caspase-8-N4BP1 Axis (1DP5OD033360-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10479473. Licensed CC0.

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