N4BP1 coordinates ubiquitin-dependent crosstalk within the IκB kinase family to limit Toll-like receptor signaling and inflammation

Abstract

The ubiquitin-binding endoribonuclease N4BP1 potently suppresses cytokine production by Toll-like receptors (TLRs) that signal through the adaptor MyD88 but is inactivated via caspase-8-mediated cleavage downstream of death receptors, TLR3, or TLR4. Here, we examined the mechanism whereby N4BP1 limits inflammatory responses. In macrophages, deletion of N4BP1 prolonged activation of inflammatory gene transcription at late time points after TRIF-independent TLR activation. Optimal suppression of inflammatory cytokines by N4BP1 depended on its ability to bind polyubiquitin chains, as macrophages and mice-bearing inactivating mutations in a ubiquitin-binding motif in N4BP1 displayed increased TLR-induced cytokine production. Deletion of the noncanonical IκB kinases (ncIKKs), Tbk1 and Ikke, or their adaptor Tank phenocopied N4bp1 deficiency and enhanced macrophage responses to TLR1/2, TLR7, or TLR9 stimulation. Mechanistically, N4BP1 acted in concert with the ncIKKs to limit the duration of canonical IκB kinase (IKKα/β) signaling. Thus, N4BP1 and the ncIKKs serve as an important checkpoint against over-exuberant innate immune responses.

Keywords: Toll-like receptors; cytokines; inflammation; innate immunity; signal transduction.

Figure 1.. RNAse activity and regulated mRNA decay appear to play little or no role in anti-inflammatory function of N4BP1.

(A) 293T cells were co-transfected with plasmids encoding HIV-1 (pNL4-3) and wild-type or mutant human (h) or mouse (m) N4BP1, as indicated. Plot depicts HIV-1 P24 protein levels in supernatants of transfected 293T cells, as measured by ELISA. Dots represent biological replicates; lines indicate mean ± SD. The ΔUb abbreviation refers to mouse N4BP1(I861A,F862A,P863A). (B) Immunoblots of BMDMs of indicated genotypes showing protein levels of N4BP1. Lanes represent BMDMs from different mice. (C) BMDMs of indicated genotypes were electroporated with Cas9 and control or N4bp1-targeting guide RNAs. At 7 days after electroporation, BMDMs were stimulated with R837 for 24 h and IL-6 secretion was measured in cell culture supernatants. Dots represent biological replicates; bars indicate the mean. (D) Schematic for experimental workflow in panel E. Wild-type or N4bp1^−/− BMDMs were stimulated with R837 for 3 hours, at which point Actinomycin D was added to the cultures. RNA-Seq was performed at 3 hours post-R837 stimulation and after an additional 30 min, 1 h, 2, and 4 h of Actinomycin D treatment. (E) Graphs indicate the transcript level in BMDMs of indicated genotypes after actinomycin D treatment relative to that at 3 h after R837 treatment. Dots represent biological replicates. Lines indicate the mean. (F) Transcript levels in BMDMs of indicated genotypes after stimulation with R837. RPKM, reads per kilobase per million reads. Symbols represent biological replicates. Lines indicate the mean.

Figure 2.. N4BP1 controls late-phase inflammatory gene transcription.

(A) Scheme for co-culturing BMDMs, created with BioRender.com. Wild-type eGFP+ BMDMs were co-cultured with either N4bp1^+/+ or N4bp1^−/− BMDMs and stimulated with R837 followed by single-cell (sc)RNA sequencing. Data for this experiment is displayed in panels B and C. (B) UMAP plots of single-cell (sc)RNA sequencing data from co-cultured BMDMs, as depicted in panel A. (C) Dot plots of Tnf, Il6, Cxcl1, and Csf3 expression from indicated cell populations of co-cultured BMDMs. Dot color represents average expression; size of dot represents percent expression. (D) RNA Polymerase II ChIP-Seq profiles for Cxcl1 and Cxcl2 in N4bp1^+/+ and N4bp1^−/− BMDMs at baseline and stimulated with R837 for 2, 4, or 6 h. (E) Plot shows the number of significantly differential ChIP-Seq peaks genome-wide among N4bp1^−/− versus N4bp1^+/+ BMDMs stimulated with R837. These data are part of the same experiment in panel D. Cutoffs for determining differential peaks were log₂FC > 0 or log₂FC < 0 and FDR < 0.05, using Wald test. (F) Gene sets enriched in upregulated RNA Polymerase II ChIP-Seq peaks among N4bp1^−/− BMDMs stimulated with R837 for 4 h in panel E. See also Figure S1.

Figure 3.. Deletion of TANK or the ncIKKs phenocopies the effect of deleting N4BP1.

(A, B) Plots depict cytokine and chemokine secretion in cell culture supernatants of BMDMs electroporated with Cas9 plus control or N4bp1- or Tank-targeted guide RNAs and stimulated with LPS, R837, Pam3csk4 (Pam3), or CpG for 24 h. (C) Plots depict IL-6 and G-CSF secretion in cell culture supernatants of Ripk3^−/− or Ripk3^−/− Casp8^−/− BMDMs that were electroporated with Cas9 plus plus control or N4bp1- or Tank-targeted guide RNAs and stimulated with LPS for 24 h. (D) Plot depicts IL-6 secretion from N4bp1^+/+ and N4bp1^−/− BMDMs electroporated with Cas9 plus control or Tank-targeted guide RNAs and stimulated with R837 at 2, 0.5, or 0.125 ug/ml for 24 h. (E) Plot depicts cytokine and chemokine secretion in cell culture supernatants of BMDMs electroporated with Cas9 plus control, Tbk1, Ikke or both Tbk1 and Ikke-targeted guide RNAs and stimulated with R837 for 24 h. (F) Plot depicts cytokine and chemokine secretion in cell culture supernatants of unstimulated Ripk1^+/+ and Ripk1^D138N/D138N BMDMs electroporated with Cas9 plus control, Tbk1, Ikke or both Tbk1 and Ikke-targeted guide RNAs. (G) Plots depict IL-6 secretion in cell culture supernatants of Ripk3^−/− Casp8^−/−, Ripk1^D138N/D138N, and Tnf^−/− BMDMs electroporated with Cas9 plus control or Tbk1 and Ikke-targeted guide RNAs and stimulated for 24 h with R837. In all panels, symbols represent biological replicates; bars indicate the mean. Where indicated, p-values determined by Dunnett’s multiple comparisons test. See also Figure S2.

Figure 4.. Role of TBK1 kinase activity and overlap among ncIKK- and N4BP1-regulated transcriptional profiles.

(A) Plot shows IFNβ secreted from Tbk1^+/+ R26^CreERT2/+ and Tbk1^cKD/cKD R26^CreERT2/+ BMDMs treated with 4OHT and stimulated with 2’3’-cGAMP. Biological replicates shown as dots; lines indicate the mean. (B) Plots show secretion of G-CSF (left) or IL-6 (right) from BMDMs of indicated genotypes that had been electroporated with Cas9 plus control or Ikke-targeted guide RNAs and stimulated for 24 h with poly(I:C) (p(I:C)), LPS, R837, Pam3csk4 (Pam3), or CpG. Biological replicates shown as dots; bars represent mean values. (C) Four-way plot comparing differential gene expression among N4bp1^+/+ and N4bp1^−/− BMDMs stimulated with R837 (x-axis) versus differential gene expression among 4OHT-treated Tbk1^cKD/cKD R26^CreERT2/+ ΔIkke and Tbk1^WT/WT R26^CreERT2/+ ΔControl BMDMs (y axis) stimulated with R837. (D) Pie charts show the proportion of N4BP1-regulated R837-induced genes whose expression is also upregulated in R837-stimulated Tbk1^cKD/cKD R26^CreERT2/+ ΔIkke BMDMs (upper), and the proportion of genes upregulated in R837-stimulated Tbk1^cKD/cKD R26^CreERT2/+ ΔIkke BMDMs that are also upregulated in R837-stimulated N4bp1^−/− BMDMs (lower). See also Figure S3.

Figure 5.. ncIKK-mediated suppression of TLR7-dependent cytokine production.

(A) Immunoblots of BMDMs electroporated with Cas9 plus the indicated guide RNAs. (B) IL-6 secretion from N4bp1^+/+ and N4bp1^−/− BMDMs electroporated with control or Rela/Nfkb1-, or Nfkb2/Relb-targeted guide RNAs and stimulated with R837 for 24 h. Symbols represent biological replicates. Bars indicate the mean. (C) Immunoblots of BMDMs electroporated with control or Tank-targeted guide RNAs and stimulated with R837 or LPS for indicated lengths of time. (D) Immunoblots of N4bp1^+/+ and N4bp1^−/− BMDMs untreated or stimulated with R837 for 3 h. (E) Immunoblots of N4bp1^+/+ and N4bp1^−/− BMDMs either untreated or stimulated with R837 after pre-treatment with TPCA-1 or DMSO as control. Immunoblots representative of 2 or 3 independent experiments.

Figure 6.. Negative regulation of persistent canonical IKK activation by N4BP1 and the ncIKKs.

(A) Immunoblots of NEMO/IKKβ immunoprecipitates and input lysates from N4bp1^+/+ and N4bp1^−/− BMDMs stimulated with R837 or LPS as indicated. (B) Immunoblots of NEMO/IKKβ immunoprecipitates from N4bp1^+/+ and N4bp1^−/− BMDMs stimulated with R837 as indicated. (C) Immunoblots of NEMO/IKKβ immunoprecipitates from 4OHT-treated Tbk1^+/+ R26^CreERT2/+ and Tbk1^cKD/cKD R26^CreERT2/+ BMDMs electroporated with Cas9 plus control or Ikke-targeted guide RNAs and treated with R837 as indicated. Each blot is representative of 2-3 independent experiments.

Figure 7.. K63/Linear polyubiquitin-binding by N4BP1 facilitates cytokine suppression and enables co-association with noncanonical and canonical IKK complexes.

(A) Co-crystal structure of linear di-ubiquitin (grey with blue and yellow) bound by the CUE-like domain of N4BP1 (green). (B) Immunoblots of input lysates and Flag immunoprecipitates of 293T cells transfected with the plasmids indicated. (C) Immunoblots of N4bp1^+/+ and N4bp1^ΔUb/ΔUb BMDMs treated with LPS or TNF for indicated lengths of time. (D) IL-6 and G-CSF secretion from N4bp1^+/+, N4bp1^ΔUb/ΔUb, and N4bp1^−/− BMDMs stimulated with R837 for 24 h. Symbols represent biological replicates. Bars indicate the mean. (E) Plots depict serum cytokine levels among N4bp1^+/+ and N4bp1^ΔUb/ΔUb mice at baseline or 6 h after intraperitoneal injection of Pam3csk4. Dots represent individual mice. Lines indicate the mean. (F) Immunoblots of input lysates and Flag immunoprecipitates from 293T cells transfected with indicated plasmids. Results representative of 2-3 independent experiments. See also Figures S4 and S5 and Table S1.