Integration of innate immune signalling by caspase-8 cleavage of N4BP1

Gitlin, Alexander D.; Heger, Klaus; Schubert, Alexander F; Reja, Rohit; Yan, Donghong; Pham, Victoria C.; Suto, Eric; Zhang, Juan; Kwon, Youngsu; Freund, Emily C.; Kang, Jing‐Qiong; Pham, Anna; Caothien, Roger; Bacarro, Natasha; Hinkle, Trent; Xu, Min; McKenzie, Brent; Haley, Benjamin; Lee, Wyne P.; Lill, Jennie R.; Roose‐Girma, Merone; Dohse, Monika; Webster, Joshua D.; Newton, Kim; Dixit, Vishva M.

doi:10.1038/s41586-020-2796-5

Cited by 79 publications

(65 citation statements)

References 53 publications

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“…While this manuscript was in revision, Gitlin et al 39 reported similar findings showing that N4BP1 negatively regulates cytokine responses induced by TRIF-independent TLRs and that N4BP1 cleavage by caspase-8 contributes to the licensing effect of TNF on cytokine responses to TRIF-independent TLR activation. Beyond this, we show the mechanism by which N4BP1 negatively regulates cytokine responses involves binding to and inhibiting NEMO oligomerization, consequently preventing NF-κB activation.…”

Section: Discussionmentioning

confidence: 62%

N4BP1 negatively regulates NF-κB by binding and inhibiting NEMO oligomerization

et al. 2021

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Many immune responses depend upon activation of NF-κB, an important transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO–NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation-like) domains in N4BP1 mediate interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhances TRIF-independent (TLR2, TLR7, or TLR9-mediated) but not TRIF-dependent (TLR3 or TLR4-mediated) NF-κB activation, leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF causes activation of caspase-8, which cleaves N4BP1 distal to residues D424 and D490 and abolishes its inhibitory effect. N4bp1−/− mice also have diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-κB, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished.

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Section: Discussionmentioning

confidence: 62%

N4BP1 negatively regulates NF-κB by binding and inhibiting NEMO oligomerization

et al. 2021

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“…Card19 lxcn mice showed significantly increased susceptibility to Yersinia infection, with higher systemic burdens and reduced ability to survive infection, consistent with our previous studies demonstrating that the RIPK1-Casp8-dependent cell death pathway enables activation of cytokine production from uninfected bystander cells. Interestingly, Ripk3 -/-Casp8 -/mice were even more susceptible to Yptb infection, which could be due to the fact that caspase-8-deficient cells are completely resistant to Yptb-induced cell death, or that caspase-8 also has cell-death-independent functions in regulating inflammatory cytokine production [89][90][91]. Altogether, our work highlights cell lysis and the release of inflammatory mediators from ruptured cells as a component of inflammatory responses that play an important role in mediating anti-microbial immune defense.…”

Section: Plos Pathogensmentioning

confidence: 99%

Genetic targeting of Card19 is linked to disrupted NINJ1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection

et al. 2021

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Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1β release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as an independently-generated CRISPR/Cas9 Card19 knockout mouse line (Card19Null) showed no defect in macrophage cell lysis. Notably, Card19 is located on chromosome 13, immediately adjacent to Ninj1, which was recently found to regulate cell lysis downstream of GSDMD activation. RNA-seq and western blotting revealed that Card19lxcn BMDMs have significantly reduced NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored their ability to undergo cell lysis in response to caspase-dependent cell death stimuli. Card19lxcn mice exhibited increased susceptibility to Yersinia infection, whereas independently-generated Card19Null mice did not, demonstrating that cell lysis itself plays a key role in protection against bacterial infection, and that the increased infection susceptibility of Card19lxcn mice is attributable to loss of NINJ1. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent gene Ninj1, disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection.

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“…During the process of manuscript submission, Gitlin et al reported that N4BP1 is a negative regulator of cytokine production and its function can be disrupted by FADD/caspase-8-mediated cleavage 49 . They established the N4BP1 knockout mice and examined the response of these mice under IMQ-induced psoriasis model.…”

Section: Discussionmentioning

confidence: 99%

“…They established the N4BP1 knockout mice and examined the response of these mice under IMQ-induced psoriasis model. They observed that the N4BP1-deficient mice developed exacerbated psoriasis with more epidermal hyperplasia, inflammatory cell infiltration, and serum CXCL1 level compared to WT mice 49 . Here, we independently established the N4BP1 knockout mice and performed detailed analysis to uncover the role of N4BP1 in psoriasis.…”

Section: Discussionmentioning

confidence: 99%

The endoribonuclease N4BP1 prevents psoriasis by controlling both keratinocytes proliferation and neutrophil infiltration

Gou

et al. 2021

Cell Death Dis

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Psoriasis is a common chronic skin disease, characterized by abnormal interplay between hyperproliferative epidermal keratinocytes and self-reactive immune cells with not fully addressed molecular mechanism. N4BP1 (NEDD4-binding protein 1) is considered as an immune regulator for a long time but its physiological role is not determined yet. Here, we found that the expression of N4BP1 in skin was highest among all 54 tested tissues, and its expression was further upregulated in psoriatic skin. N4BP1-deficient mice exhibited normal grossly, but developed severe and prolonged IMQ-induced psoriasis-like disease comparing to controls. N4BP1 mainly expressed in keratinocytes and located on nucleus. Up- but not downregulated genes in N4BP1-deficient skin were specifically enriched in keratinocyte proliferation and differentiation. The proliferation of N4BP1-deficient primary keratinocytes was faster compared to that of controls. The upregulated genes upon ablation of N4BP1 were highly enriched in targets of AP-1 transcription factor. Knocking out N4BP1 resulted in upregulation of JunB and FosB, and conversely, overexpression of N4BP1 greatly reduced their expression. Furthermore, N4BP1 binds with JunB and FosB encoding mRNAs and greatly reduces their stability. In addition, with a high expression in neutrophils, N4BP1 limits survival of neutrophils in blood and infiltration of neutrophils in psoriatic skin by targeting CXCL1, CCL20, and S100A8. These findings demonstrate that N4BP1 controls the proper function of keratinocytes and neutrophils by negatively regulating JunB, FosB, and CXCL1, respectively, and that is critical for psoriasis prevention.

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Integration of innate immune signalling by caspase-8 cleavage of N4BP1

Cited by 79 publications

References 53 publications

N4BP1 negatively regulates NF-κB by binding and inhibiting NEMO oligomerization

N4BP1 negatively regulates NF-κB by binding and inhibiting NEMO oligomerization

Genetic targeting of Card19 is linked to disrupted NINJ1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection

The endoribonuclease N4BP1 prevents psoriasis by controlling both keratinocytes proliferation and neutrophil infiltration

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