Systematic analysis of the IL‐17 receptor signalosome reveals a robust regulatory feedback loop

Abstract IL‐17 mediates immune protection from fungi and bacteria, as well as it promotes autoimmune pathologies. However, the regulation of the signal transduction from the IL‐17 receptor (IL‐17R) remained elusive. We developed a novel mass spectrometry‐based approach to identify components of the IL‐17R complex followed by analysis of their roles using reverse genetics. Besides the identification of linear ubiquitin chain assembly complex (LUBAC) as an important signal transducing component of IL‐17R, we established that IL‐17 signaling is regulated by a robust negative feedback loop mediated by TBK1 and IKKε. These kinases terminate IL‐17 signaling by phosphorylating the adaptor ACT1 leading to the release of the essential ubiquitin ligase TRAF6 from the complex. NEMO recruits both kinases to the IL‐17R complex, documenting that NEMO has an unprecedented negative function in IL‐17 signaling, distinct from its role in NF‐κB activation. Our study provides a comprehensive view of the molecular events of the IL‐17 signal transduction and its regulation.

A The schematic representation of recombinant Strep-Flag-IL-17 (SF-IL-17) construct used in this study. Murine or human IL-17 coding sequence lacking leader peptide was used. B The purity and assembly of SF-IL-17 was analyzed by SDS-PAGE followed by Coomassie staining. Samples were either left untreated or reduced with dithiothreitol (DTT) to disrupt the covalent IL-17 dimers. C ST2 cells were incubated on ice in the presence or absence of SF-IL-17. Subsequently, the cells were stained with fluorescently labeled anti-Flag antibody and analyzed by FACS. D ST2 cells were stimulated with SF-IL-17 (500 ng/ml) for indicated time points or were left unstimulated and IL-17 was added post-lysis. Lysates were subjected to anti-Flag immunoprecitation to isolate IL-17RSC. The samples were analyzed by immunoblotting. E HeLa cells were stimulated with IL-17 (500 ng/ml) or TNF (500 ng/ml) for indicated time points and analyzed via immunoblotting.
Source data are available online for this figure.

Helena Draberova et al
The   A ST2 cells were incubated with or without TBK1/IKKe inhibitor MRT67307 (2 lM) for 30 min and subsequently were left untreated or stimulated for 2 h with IL-17 (500 ng/ml). mRNA was isolated and subjected to RNA sequencing. The principal component analysis from three independent experiments is shown. B Analysis of transcriptional response induced by treatment of ST2 cells with TBK1/IKKe inhibitor only. In red are transcripts considered to be significantly changed (log 2 fold change > 1 or < À1, Àlog 10 Benjamini-Hochberg adjusted P-value > 2, based on analysis of three independent experiments). Names of several significantly upregulated transcripts are indicated. C The Venn diagram representing the number of significantly changed transcripts upon treatment of cells with TBK1/IKKe inhibitor alone or with IL-17 alone. D ST2 cells pretreated or not with TBK1/IKKe inhibitor MRT67307 (2 lM) were left unstimulated or stimulated with IL-17 (500 ng/ml) for 4 or 8 h, and induction of mRNA for selected genes was analyzed by real-time PCR. Mean + SEM from four independent experiments is shown, and statistical significance was determined using unpaired two-tailed Student's t-test. E ST2 wild types or TBK1/IKKe DKO cells were stimulated with IL-17 (500 ng/ml) or TNF (50 ng/ml) for indicated time points and lysates were analyzed by immunoblotting. F ST2 wild types or TBK1/IKKe DKO cells were stimulated with indicated concentration of IL-17 for 15 min and lysates were analyzed by immunoblotting. G HeLa cells pretreated or not with TBK1/IKKe inhibitor MRT67307 (2 lM) were stimulated with IL17 (500 ng/ml) for indicated time points and lysates were analyzed by immunoblotting. H HeLla wild types or TBK1/IKKe DKO cells were stimulated with indicated concentration of IL-17 for 15 min and lysates were analyzed by immunoblotting.  iBAQ Intensity Ratio to IL-17RC iBAQ Intensity Ratio to IL-17RC The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from five independent experiments is shown, and statistical significance was determined by twotailed Mann-Whitney test. C, D ST2 TBK1/IKKe DKO cells were reconstituted with Flag-tagged TBK1 (C) IKKe (D) or GFP that serves as negative control. Cell lysates were subjected to anti-Flag immunoprecipitation and analyzed via immunoblotting. E HOIP KO cells reconstituted with HOIP(WT) or empty vector were pretreated with TBK1/IKKe inhibitor MRT67307 (2 lM), stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting. *indicates nonspecific band. F ST2 wild types or TBK1/IKKe DKO were stimulated with IL-17 (500 ng/ml) for 2 h and induction of mRNA for selected genes was analyzed by real-time PCR. Mean + SEM from five independent experiments is shown, and statistical significance was determined using unpaired two-tailed Student's t-test. inh.
IKKα/β inh. (500 ng/ml) for 15 min or were left unstimulated and IL-17 was added post-lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC (C) or tested for the activation of signaling pathways (D) and samples were analyzed by immunoblotting. E NEMO-deficient ST2 cells were reconstituted with NEMO(WT) or empty vector, treated or not with TBK1/IKKe inhibitor MRT67307 (2 lM) and stimulated with SF-IL-17 (500 ng/ml) as indicated. Activation of signaling pathways was analyzed upon cell lysis by immunoblotting. F, G HeLa wild-type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) for 15 min or were left unstimulated and IL-17 was added post-lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC (F) or tested for the activation of signaling pathways (G) and samples were analyzed by immunoblotting. H NEMO-deficient HeLa cells were reconstituted with NEMO(WT) or empty vector and stimulated with IL-17 (500 ng/ml) as indicated. Cells were solubilized and analyzed by immunoblotting.   Figure EV6. Activity of TBK1 and IKKe kinases promotes release of TRAF6 from the IL-17RSC.
A HeLa wild-type or TBK1/IKKe DKO cells were stimulated with SF-IL-17 as indicated, solubilized, and subjected to anti-Flag immunoprecipitation to isolate IL-17RSC. Samples were analyzed by immunoblotting. B HeLa cells were treated or not with TBK1/IKKe inhibitor MRT67307 (2 lM) and stimulated with SF-IL-17 (500 ng/ml) as indicated. IL-17RSC was isolated by anti-Flag immunoprecipitation and analyzed by immunoblotting. C Cullin1 KO ST2 cells were prepared by CRISPR/ Cas9 and verified by sequencing. Biallelic deletion of 2 base pairs leads to frameshift in Cullin1 coding sequence and premature STOP codon. D Cullin1 KO ST2 cells were stimulated for 15 min with IL-17 (500 ng/ml), and lysates were analyzed by immunoblotting. E ACT1-deficient ST2 cells were reconstituted with ACT1(WT) or ACT1 harboring indicated deletions or empty vector. Cells were stimulated for 15 min with IL-17 (500 ng/ml), and lysates were analyzed by immunoblotting.
Data information: Data are representative of two (A, B, E) or four (D) independent experiments. Source data are available online for this figure.