A genome-wide siRNA screen reveals positive and negative regulators of the NOD2 and NF-κB signaling pathways

Abstract

The cytoplasmic receptor NOD2 (nucleotide-binding oligomerization domain 2) senses peptidoglycan fragments and triggers host defense pathways, including activation of nuclear factor κB (NF-κB) signaling, which lead to inflammatory immune responses. Dysregulation of NOD2 signaling is associated with inflammatory diseases, such as Crohn's disease and Blau syndrome. We used a genome-wide small interfering RNA screen to identify regulators of the NOD2 signaling pathway. Several genes associated with Crohn's disease risk were identified in the screen. A comparison of candidates from this screen with other "omics" data sets revealed interconnected networks of genes implicated in NF-κB signaling, thus supporting a role for NOD2 and NF-κB pathways in the pathogenesis of Crohn's disease. Many of these regulators were validated in secondary assays, such as measurement of interleukin-8 secretion, which is partially dependent on NF-κB. Knockdown of putative regulators in human embryonic kidney 293 cells followed by stimulation with tumor necrosis factor-α revealed that most of the genes identified were general regulators of NF-κB signaling. Overall, the genes identified here provide a resource to facilitate the elucidation of the molecular mechanisms that regulate NOD2- and NF-κB-mediated inflammation.

Fig. 1

A genome-wide siRNA screen identifies regulators of NOD2 signaling. (A) Schematic representation of the reporter cell line engineered to quantitatively assess MDP-induced NOD2 signaling together with known components of the NOD2 signaling pathway. (B) The screen was carried out in triplicate with pools of four distinct siRNAs specific for each gene. Pie charts summarize the percentages of genes whose silencing affected cell viability and the percentages of genes not affecting viability that either decreased (red) or increased (blue) luminescence. (C) Rank order plot of the average normalized NF-κB luciferase reporter activity displayed relative to positive (RIPK2) and negative (non-targeting, NT) siRNA controls present on each assay plate. (D) Rank order plot of the average percentage cell viability displayed relative to NT siRNA and lysed cell controls present on each assay plate. (E) siRNA-mediated silencing of known components of the NOD2 signaling pathway led to decreased (red) or increased (blue) NF-κB luciferase activity relative to that of NT control siRNA cells (upper panel) without major effects on cell viability (lower panel). All luminescence values were statistically significant with I < 0.001. The asterisk indicates viability measurements with P < 0.001.

Fig. 2

Genome-wide screen identifies known regulators of NOD2 and NF-κB signaling pathways. (A) Numerous positive (red) and negative (blue) regulators identified in the siRNA screen interact with core components of the NOD2 and NF-κB signaling pathways (larger circles labeled in bold). Protein-protein interactions depicted by black lines were taken from publically available databases, such as STRING, with a confidence score of ≥ 0.4. (B) siRNA-mediated gene silencing led to decreased (red) or increased (blue) NF-κB luciferase activity upon stimulation with MDP relative to that of cells treated with NT control siRNA, without major effects on cell viability. (C to E) In addition, several components of protein complexes known to affect NF-κB signaling by mediating the proteasomal degradation of (C) IκB or (D) the nuclear translocation of RelA were significantly enriched (table S3) among (E) positive regulators in the screen. All luminescence values were statistically significant with P < 0.001.

Fig. 3

Genome-wide screen identifies networks of positive and negative regulators of the NOD2 and NF-κB signaling pathways. (A) Schematic depiction of selected protein-protein interactions among hits from the siRNA screen identified with a confidence score of ≥ 0.4 from the STRING database. Published interactions are depicted by black lines, whereas dashed lines indicate interactions with NOD2 from a NOD2 pull-down (fig. S9). (B) The relative effect on MDP-induced NF-κB signaling is shown for each gene in the network with positive and negative regulators shown in red or blue, respectively. All luminescence values were statistically significant; P < 0.001.

Fig. 4

Many genes associated with Crohn’s disease risk affect NOD2 signaling. (A) Fifteen genes (labeled in bold) whose silencing either diminished (red) or enhanced (blue) NOD2 signaling in our siRNA screen have been identified as risk factors in the development of Crohn’s disease by GWAS (table S6). Black lines indicate published protein-protein interactions, taken from the STRING database, with other putative NOD2 regulators identified in the primary siRNA screen. A putative link between the Crohn’s disease risk factors VAMP3 and NOD2 is provided through their mutual interacting protein CENTB1 (62), which was not identified as a hit in our screen (gray). (B) siRNA-mediated silencing led to decreased (red) or increased (blue) NOD2-dependent NF-κB luciferase activity relative to that of cells treated with NT control siRNA, without major effects on cell viability. All luminescence values were statistically significant; P < 0.001.

Fig. 5

Secondary validation of hits with independent siRNA pools. (A) Workflow summarizing the secondary validation screen with alternative sets of ON-TARGETplus siRNA pools consisting of four distinct siRNA sequences for each gene. (B) Summary of the secondary validation rate of positive and negative regulators tested for MDP-induced NF-κB luciferase activity. (C) Comparison of relative NF-κB luciferase activity after stimulation with MDP or TNF-α. Positive controls (RIPK2, RIPK1, and RelA) are highlighted along with two selected validated hits. (D) Epistasis analysis reveals that RNF31 is a general regulator of NF-κB signaling. HEK 293 cells were transfected with the indicated siRNA pools. After 48 hours, a second transfection with an NF-κB luciferase reporter and a Renilla reporter (for normalization) was used to monitor NF-κB pathway activation with (i) a constitutively active NOD2 construct (NOD2 CA), (ii) an inducibly active (IA) RIPK2 construct (RIPK2-IA), and (iii) an inducibly active RIPK1 construct (RIPK1-IA) relative to a pcDNA3 empty vector (EV) control. The dimerization agent AP1510 (ARIAD) was added as indicated to induce signaling. Experiments were performed in triplicate, of which one representative example is shown. *, P < 0.05 by a one way ANOVA and Tukey multiple comparison test performed using GraphPad Prism software.

Fig. 6

Components of the LUBAC complex positively regulate NOD2 signaling. Two components of the LUBAC complex (RNF31 and SHARPIN) and many of their associated proteins were revealed as positive components of MDP-induced NOD2 signaling in the primary siRNA screen. (A) Protein-protein interactions are depicted by black lines, whereas a dashed blue line shows that PARK2 is a protein homolog of RBCK1 and that they share a similar domain structure. Factors required for MDP-induced activation of the NF-κB pathway are shaded in red, whereas those genes having no statistically significant effect are gray. Underlined names indicate genes tested by secondary screening, and bold circles indicate genes that were validated. (B) Induction of MDP-induced NF-κB luciferase activity relative to that of cells treated with NT siRNA from the primary screen is shown for each component of the protein-protein interaction network. All luminescence values were statistically significant; P < 0.001. (C) Induction of NF-κB luciferase reporter activity (upper panel) or IL-8 secretion (lower panel) after knockdown with ON-TARGETplus siRNA pools in response to stimulation with MDP (left) or TNF-α (right) or (D) infection with two gram positive bacteria, Enterococcus faecalis (left) or Staphylococcus aureus (right). Bars shaded in red and blue indicate P < 0.05.