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. 2020 Mar 23;18(3):e3000646.
doi: 10.1371/journal.pbio.3000646. eCollection 2020 Mar.

IL-23 Signaling Regulation of Pro-Inflammatory T-cell Migration Uncovered by Phosphoproteomics

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Free PMC article

IL-23 Signaling Regulation of Pro-Inflammatory T-cell Migration Uncovered by Phosphoproteomics

Candelas Álvarez-Salamero et al. PLoS Biol. .
Free PMC article

Abstract

Interleukin 23 (IL-23) triggers pathogenic features in pro-inflammatory, IL-17-secreting T cells (Th17 and Tγδ17) that play a key role in the development of inflammatory diseases. However, the IL-23 signaling cascade remains largely undefined. Here, we used quantitative phosphoproteomics to characterize IL-23 signaling in primary murine Th17 cells. We quantified 6,888 phosphorylation sites in Th17 cells and found 168 phosphorylations regulated upon IL-23 stimulation. IL-23 increased the phosphorylation of the myosin regulatory light chain (RLC), an actomyosin contractibility marker, in Th17 and Tγδ17 cells. IL-23-induced RLC phosphorylation required Janus kinase 2 (JAK2) and Rho-associated protein kinase (ROCK) catalytic activity, and further study of the IL-23/ROCK connection revealed an unexpected role of IL-23 in the migration of Tγδ17 and Th17 cells through ROCK activation. In addition, pharmacological inhibition of ROCK reduced Tγδ17 recruitment to inflamed skin upon challenge with inflammatory agent Imiquimod. This work (i) provides new insights into phosphorylation networks that control Th17 cells, (ii) widely expands the current knowledge on IL-23 signaling, and (iii) contributes to the increasing list of immune cells subsets characterized by global phosphoproteomic approaches.

Conflict of interest statement

"The authors have declared that no competing interests exist."

Figures

Fig 1
Fig 1. Generation of OT-II/Th17 cells.
OT-II naïve CD4 T cells were co-cultured with irradiated splenocytes loaded with ovalbumin-derived peptide OVA323-339 as APCs, in the presence of Th17 polarizing cytokines (TGFβ, IL-6, IL-1β, IL-21, and IL-23). (a) Graphical representation of OT-II/Th17 culture protocol. (b) Graph shows Rorc mRNA expression in the indicated cell cultures, determined by qPCR and normalized to CD4 naïve cells (mean ± sd, n = 3 independent cell cultures). (c) Representative histogram shows RORγt expression in OT-II/Th0 and OT-II/Th17 cultures, determined by flow cytometry (n = 4 independent cell cultures). (d) Graph depicts the number of CD4 T cells at the indicated days of culture (mean ± sd, n = 4 independent cell cultures). (e) OT-II/Th17 cultures were stimulated with PDBu/Io or left unstimulated for 4 h in presence of Golgi-Plug, and IL-17a and IFNγ production was determined by flow cytometry. Representative contour plots show IL-17a and IFNγ production, and graph shows the percentage of cytokine-producing cells (mean ± sd, n = 10–11 independent cell cultures). (f) Graph shows Il23r mRNA expression in the indicated cell cultures, determined by qPCR and normalized to CD4 naïve cells (mean ± sd, n = 3 independent cell cultures). (g) Western blot analysis of STAT3-Y705 phosphorylation in response to IL-23 stimulation. Representative of n = 4 independent cell cultures. Individual numerical values for quantifications presented in Fig 1 can be found in S1 Data. Western blot raw images for Fig 1G can be found in S1 Raw images. APC, antigen presenting cell IFNγ, interferon gamma; IL-23, Interleukin 23; PDBu/Io, Phorbol 12,13-dibutyrate/Ionomycin; qPCR, quantitative polymerase chain reaction.
Fig 2
Fig 2. Global phosphoproteomic analysis of IL-23-stimulated OT-II/Th17 cells.
(a) Experimental workflow for global phosphoproteomic analysis. (b) Graph represents the number of unique p-sites and number of distinct proteins identified and quantified in the three individual biological replicates, the collective analysis (Total), and the p-sites and proteins consistently identified in the three biological replicates. Pie chart represents the number of Serine (pSer), Threonine (pThr), and Tyrosine (pTyr) among consistently identified p-sites. (c) Graph represents the statistical significance (-log(p-value)) of the top 5 molecular and cellular functions overrepresented within the Th17 phosphoproteome. Inset numbers indicate the number of proteins included in each group. (d) Graph represents the number of potential consensus sites for the indicated kinases present in the Th17 phosphoproteome. Multiple kinase assignment to single p-site was allowed. Kinases with at least 100 consensus sites are represented. Individual numerical values for quantifications presented in Fig 2 can be found in S2 Data. IL-23, Interleukin 23; p-site, phosphorylation site.
Fig 3
Fig 3. Impact of IL-23 stimulation on OT-II/Th17 phosphoproteome.
(a) Graphs show the ratio IL-23/Ctrl (linear value) for the indicated STAT3 residues in the three biological replicates (nd, not determined). (b) Western blot analysis show STAT3 phosphorylation in Y705 and S727 residues in the samples used for the phosphoproteomic analysis. (c, d, e) Volcano plots show IL-23/Ctrl ratio distribution (log2 averaged value, AVG) against the statistical significance (–log (p value), multiple t test FDR 5%) for p-sites identified in the three biological replicates. Each dot represents a unique p-site. (c) Colored dots and inset numbers indicate significant IL-23-responsive p-sites (FDR 5%, red = increased and blue = decreased). (d) Colored dots indicate proteins annotated by Perseus software as protein kinases (Protein kinase activity; GO:0004672). (e) Colored dots indicate proteins annotated as chromatin remodelers (Chromatin organization; GO:006325). Individual numerical values for quantifications presented in Fig 3 can be found in S3 Data. Western blot raw images for Fig 3B can be found in S2 Raw images. Ctrl, untreated control; FDR, false discovery rate; IL-23, Interleukin 23; STAT3, Signal Transducer and Activator of Transcription family member 3.
Fig 4
Fig 4. IL-23 regulated signaling pathways in the OT-II/Th17 phosphoproteome.
(a) Graph represents the statistical significance (-log(p-value)) of the top 10 molecular and cellular processes overrepresented among the IL-23-responsive phosphoproteins. Inset numbers indicate the number of proteins included in each group. (b) Graph represents the statistical significance (-log(p-value)) of the top 10 canonical signaling pathways overrepresented within the IL-23-responsive phosphoproteins. (c) Heatmap represents the ratio IL-23/Ctrl (linear value) for the top significantly up-regulated and down-regulated p-sites in the individual biological replicates. (d) Graphs show the ratio IL-23/Ctrl (linear value) for the indicated residues of the RLC in the three biological replicates. (e) Western blot analysis of RLC-S20 phosphorylation in the samples used for the phosphoproteomic analysis. SMC1 was used as the loading control. Individual numerical values for quantifications presented in Fig 4 can be found in S4 Data. Western blot raw images for Fig 4E can be found in S3 Raw images. Ctrl, untreated control; IL-23, Interleukin 23; RLC, myosin regulatory light chain; SMC1, structural maintenance of chromosomes 1A.
Fig 5
Fig 5. IL-23 induces RLC-S20 phosphorylation in TCRγδ IL-23R+ cells.
(a) Il23r-gfp reporter mice were treated with IMQ or left untreated (Ctrl) for 5 days. Lymph node cells were processed for analysis of IL-23R/GFP expression by flow cytometry. Representative contour plots show CD44 and IL-23R/GFP expression in the indicated subpopulations (CD3+CD4+ and CD3+TCRγδ+), and inset numbers represent the percentage of IL-23R+ cells within the indicated gates. Graph represents the percentage of cells expressing the IL-23R (mean ± sd, n = 4–6 mice). (b) TCRγδ cells were isolated from lymph nodes of Il23r-gfp reporter mice and cultured in presence of IL-7 for 5 days. Representative contour plot shows CD44 and IL-23R (GFP) expression in IL-7-expanded TCRγδ+ cells, and inset number represents the percentage of IL-23R+ cells in the indicated gate. IL-7-expanded TCRγδ cells were stimulated with PDBu/Io in presence of Golgi-Plug or left untreated for 4 h, and IL-17a production was determined by flow cytometry. Representative histograms show IL-17a production in Tγδ17 cells (gated as CD3+TCRγδ+CD44hi), and inset number represents the percentage of IL-17a+ cells. Graph shows the percentage of IL-17a-producers among Tγδ17 cells (mean ± sd, n = 5–6 independent cultures). (c) IL-7-expanded Tγδ17 cells were stimulated with IL-23 or left untreated (Ctrl) for different time points before measuring pRLC-S20 by flow cytometry. Representative histogram shows pRLC-S20 after 6 h of IL-23 stimulation. Graph shows the MFI of pRLC-S20 in response to IL-23 at the indicated time points, normalized to MFI of untreated cells (mean ± sd, n = 5 independent cell cultures, **p = 0.0037, ****p < 0.0001). (d) Total lymph node cells obtained from IMQ-sensitized mice were stimulated ex vivo with IL-23 for 18 h or left untreated (Ctrl) before assessing pRLC-S20 by flow cytometry. Representative histogram shows pRLC-S20 in Tγδ17 (gated as CD3+TCRγδ+CD44hi). Graph shows pRLC-S20 MFI in response to IL-23, normalized to untreated controls (mean ± sd, n = 6 mice, *p = 0.0487). (e) IL-7-expanded Tγδ17 cells were stimulated with IL-23, IL-1β, or IL-2 for 6 h, and pRLC-S20 was determined by flow cytometry. Graph shows pRLC-S20 MFI in Tγδ17 (gated as TCRγδ+CD44hi), normalized to untreated controls (mean ± sd, n = 4–6 independent cell cultures, ***p = 0.0006). Statistical analysis: (c, e) One-way ANOVA test with Dunnett´s correction for multiple comparisons. (d) One sample t test. Individual numerical values for quantifications presented in Fig 5 can be found in S5 Data. Ctrl, untreated control; GFP, green fluorescent protein; IL-23, Interleukin 23; IMQ, Imiquimod; MFI, mean of fluorescence intensity; PDBu/Io, Phorbol 12,13-dibutyrate/Ionomycin; pRLC-S20, phospho-RLC-Serine20; RLC, myosin regulatory light chain.
Fig 6
Fig 6. IL-23 induces pRLC-S20 through JAK2 and ROCK dependent mechanisms in Tγδ17 cells.
(a) IL-7-expanded Tγδ17 cells were stimulated with IL-23 in presence or absence of the indicated JAK2 inhibitors or left unstimulated (uns.) for 6 h before assessing pSTAT3-Y705 and pRLC-S20 by flow cytometry. Representative histograms show pSTAT3-Y705 (left) and pRLC-S20 (right) in the indicated conditions. Graphs show the MFI of pSTAT3-Y705 (***p = 0.0002, ****p < 0.0001) and pRLC-S20 (***p = 0.0004, ****p < 0.0001) in TCRγδ+CD44hi cells, normalized to unstimulated controls (mean ± sd, n = 5 independent cultures). (b) IL-7-expanded Tγδ17 cells were stimulated with IL-23 in presence or absence of ML7 (MLCK inhibitor) or Y27632 (ROCK inhibitor) or left untreated for 6 h, before measurement of pRLC-S20 by flow cytometry. Representative histograms show pRLC-S20 in the indicated conditions. Graph shows pRLC-S20 MFI in TCRγδ+CD44hi cells, normalized to unstimulated controls (mean ± sd, n = 5–10 independent cell cultures, ****p < 0.0001). (c) Lymph node cells obtained from IMQ-sensitized mice were stimulated ex vivo with IL-23 for 18 h in presence or absence of ROCK inhibitor Y27632 or left unstimulated. Representative histogram shows pRLC-S20 in Tγδ17 (gated as CD3+TCRγδ+CD44hi). Graph shows pRLC-S20 MFI in response to IL-23 (relative MFI, mean ± sd, n = 7 mice, *p = 0.017, **p = 0.0031). (d, e) NIH3T3 cells were transduced with lentiviral particles LV-GFP, LV-GFP-shROCK1, or not transduced (Ctrl), and 5 days later, processed for analysis by flow cytometry and western blot. (d) Representative histograms show GFP expression (n = 2 independent experiments). (e) Representative western blots of ROCK1 expression in transduced NIH3T3 α Tubulin was used as loading control (n = 2 independent experiments). (f, g) Tγδ17 cells were transduced with LV-GFP, LV-GFP-shROCK1, or not transduced (Ctrl) and 7 days later were analyzed by flow cytometry. (f) Representative histograms show GFP expression (n = 8 independent experiments). (g) Transduced Tγδ17 cells were stimulated with IL-23 in presence or absence of ROCK inhibitor Y27632 or left untreated for 18 h before assessing pRLC-S20 by flow cytometry. Graph shows pRLC-S20 MFI in Tγδ17 GFP+ cells, normalized to the untreated control (mean ± sd, n = 8, ****p < 0.0001, *p = 0.013). Statistical analysis by one way ANOVA with Dunnet correction for multiple comparisons. Individual numerical values for quantifications presented in Fig 6 can be found in S6 Data. Western blot raw images for Fig 6E can be found in S4 Raw images. Ctrl, untreated; IL-23, Interleukin 23; IMQ, Imiquimod; JAK2, Janus kinase 2; LV-GFP, lentiviral construct encoding a GFP reporter marker; LV-GFP-shROCK1, lentiviral construct encoding a GFP reporter marker and shRNAs against ROCK1; MFI, mean of fluorescence intensity; pRLC-S20, phospho-RLC-Serine20; ROCK, Rho-associated protein kinase.
Fig 7
Fig 7. IL-23 induces pRLC-S20 through ROCK dependent mechanisms in Th17 cells.
(a) Total lymph node cells from Il23r-gfp reporter mice were processed for analysis of IL-23R (GFP) expression by flow cytometry. Representative contour plots show CD44 and IL-23R/GFP expression in the CD3+CD4+ population, and inset number represents the percentage of IL-23R+ cells within the indicated gate. Graph represents the percentage of nTh17 cells, gated as CD3+CD4+CD44hiIL-23R+ (mean ± sd, n = 13 mice). (b) nTh17 cells were sorted from lymph nodes of Il23r-gfp reporter mice as CD4+CD44hiIL-23R+ and cultured in presence of IL-7 for 7 days. Right graph shows IL-23R/GFP MFI after 7 days of culture, normalized to CD4 naïve cells (sorted in parallel to nTh17 as CD4+CD44lowIL-23R- and cultured for 7 days in IL-7, mean ± sd, n = 3 independent cultures, p = 0.0314). IL-7-cultured nTh17 were stimulated with IL-23 or left untreated for 18 h before assessing pSTAT3-Y705 by flow cytometry. Left graph shows pSTAT3-Y705 MFI, normalized to untreated cells (mean ± sd, n = 3 independent cultures, p = 0.0132). (c) IL-7-cultured nTh17 were stimulated with IL-23 in presence or absence of Y27632 or left untreated for 18 h before assessing pRLC-S20 by flow cytometry. Left graph shows pRLC-S20 MFI, normalized to untreated cells (n = 3 independent cultures, *p = 0.0121, **p = 0.0012). (d) Il23r-gfp reporter mice were immunized with CFA/MOG to induce EAE; 12 days after EAE induction, total splenic cells were processed for analysis of IL-23R/GFP expression by flow cytometry. Representative contour plots show CD44 and IL-23R/GFP expression in the CD3+CD4+ population, and inset numbers represent the percentage of IL-23R+ cells within the indicated gate. Graph represents the percentage of iTh17 cells, gated as CD3+CD4+CD44hiIL-23R+ (mean ± sd, n = 14 mice from 2 independent experiments). (e) iTh17 cells were sorted from lymph nodes and spleens of EAE-treated Il23r-gfp reporter mice as CD4+CD44hiIL-23R+ and cultured in presence of IL-7 for 7 days. Right graph shows IL-23R/GFP MFI after 7 days of culture, normalized to CD4 naïve cells (sorted as CD4+CD44lowIL-23R- and cultured for 7d in IL-7, mean ± sd, n = 6 independent cultures, p < 0.0001). IL-7-expanded iTh17 were stimulated with IL-23 or left untreated for 18 h before assessing pSTAT3-Y705 by flow cytometry. Left graph shows pSTAT3-Y705 MFI, normalized to untreated cells (mean ± sd, n = 10 independent cultures, p < 0.0001). (f) IL-7-expanded iTh17 were stimulated with IL-23 in presence or absence of Y27632 or left untreated for 18 h before assessing pRLC-S20 by flow cytometry. Left graph shows pRLC-S20 MFI, normalized to untreated cells (mean ± sd, n = 4–8 independent cultures, ****p < 0.0001). Statistical analysis: (b, e) One sample t test. (c, f) One-way ANOVA test with Dunnett’s correction for multiple comparisons. Individual numerical values for quantifications presented in Fig 7 can be found in S7 Data. CFA, Complete Freunds´ Adjuvant; MOG, Myelin oligodendrocyte glycoprotein; EAE, experimental autoimmune encephalomyelitis; GFP, Green Fluorescent Protein; IL-23, Interleukin 23; iTh17, induced Th17; MFI, Mean of fluorescence intensity; nTh17, natural Th17; pRLC-S20, phospho-RLC-Serine20; ROCK, Rho-associated protein kinase.
Fig 8
Fig 8. IL-23-mediated ROCK activation promotes Tγδ17 cell migration.
(a) IL-7-expanded Tγδ17 cells were stimulated with IL-23 for 6 h in the presence or absence of Y27632 inhibitor or left unstimulated (uns.). Representative histogram shows pSTAT3-Y705 measured by flow cytometry. Graph shows pSTAT3-Y705 MFI in TCRγδ+CD44hi cells, relative to unstimulated cells (mean ± sd, n = 3–5 independent cell cultures). (b) IL-7-expanded Tγδ17 cells were stimulated with IL-23 for 18 h in the presence or absence of the indicated inhibitors or left unstimulated. Cytokine production was assessed by flow cytometry. Graphs show the percentage of IL-17a producers (left) and IL-22 (right) among TCRγδ+CD44hi cells (mean ± sd, n = 3–6 independent cell cultures). Left graph, **p = 0.001, ##p = 0.0028. Right graph *p = 0.0106, #p = 0.0157. (c) IL-7-expanded Tγδ17 cells were shorted as CD27 negative and stimulated with IL-23 for 18 h or left unstimulated. Cells were allowed to settle onto fibronectin/poly-L-Lysine-coated wells for 1 h in presence of 20 μM Y27632 inhibitor before imaging. Graph shows the frequency of cells with polarized morphology (***p = 0.0001, ****p < 0.0001). Images and quantification are representative of n = 2 independent cell cultures. (d) IL-7-expanded Tγδ17 cells from Il23r-gfp reporter mice were stimulated with IL-23 for 18 h or left unstimulated. Next day cells were pretreated for 1 h with AZD1480 or Y27632 and placed into a 3-μM transwell insert. Cells were left to migrate into the lower chamber containing medium with the same conditions as the upper chamber (IL-7, ±IL-23, and ±inhibitors) for 6 h. Cell numbers in the lower chamber were determined by flow cytometry. Graph represents migrated cells (IL-23R+/GFP), relative to untreated cells (migration index, mean ± sd, n = 4–9 independent cultures, ****p < 0.0001). (e) IL-7-expanded Tγδ17 cells from Il23r-gfp reporter mice were stimulated with IL-23 for 18 h or left unstimulated. Next day, IL-23-stimulated cells were placed into a 3-μM transwell insert and left to migrate into the lower chamber containing medium with the same conditions as the upper chamber (IL-7 and IL-23). For chemotactic assays, unstimulated cells were placed in transwell inserts and left to migrate for 6 h into the lower chamber containing the indicated concentrations of chemokine ligands CCL2 or CCL20. Cell numbers in the lower chamber were determined by flow cytometry. Graph represents migrated cells (IL-23R+/GFP) relative to untreated cells (migration index, mean ± sd, n = 3–10 independent cultures, **p = 0.0035). (f) TCRγδ cells were isolated from 5 d IMQ-treated mice, labeled with CellTrace Violet, and adoptively transferred into recipient mice that were previously ear-treated with IMQ for 5 days. Recipient animals received 2 intraperitoneal injections of ROCK inhibitor Y27632 or control vehicle (DMSO/PBS) prior to adoptive transfer of CellTrace-labeled TCRγδ cells; 12 h after transfer, ear skin and LN from recipient mice were harvested and processed for analysis by flow cytometry in presence of Accucheck counting beads to determine numbers and frequencies of adoptively transferred Tγδ17. In skin, the adoptively transferred Tγδ17 cells were gated as dermal TCRγδ cells (CD3+TCRγδintCD44hi and CellTrace+). In LN, adoptively transferred Tγδ17 cells were gated as CD3+TCRγδ+CD44hi and CellTrace+. Left graph represents the number of recruited Tγδ17 cells to the inflamed ear skin in IMQ-sensitized mice. Right graph represents the frequency of recruited Tγδ17 cells in inflamed ear skin and inguinal lymph nodes (nondraining) of IMQ-treated mice among total Tγδ17 cells. Data are pooled from 2 experiments (n = 3–5 mice per group, *p = 0.03, **p = 0.002). (g) IL-7-cultured nTh17 cells from Il23r-gfp reporter mice were stimulated with IL-23 for 18 h or left untreated. Next day cells were pretreated for 1 h with Y27632 and placed into a 3-μM transwell insert. Cells were left to migrate into the lower chamber containing medium with the same conditions as the upper chamber (IL-7, ±IL-23, and ±inhibitor) for 2 h. Cell numbers in the lower chamber were analyzed by flow cytometry in presence of Accucheck counting beads. Graph represents migrated cells (CD4+CD44hiIL-23R+/GFP) relative to untreated cells (migration index, mean ± sd, n = 3 independent cultures, *p = 0.013). (h) IL-7-expanded iTh17 from EAE-treated Il23r-gfp reporter mice were stimulated with IL-23 for 18 h or left untreated. Next day cells were pretreated for 1 h with Y27632 and placed into a 3-μM transwell insert. Cells were left to migrate into the lower chamber containing medium with the same conditions as the upper chamber (IL-7, ±IL-23, and ±inhibitor) for 2 h. Cell numbers in the lower chamber were analyzed by flow cytometry in presence of Accucheck counting beads. Graph represents migrated cells (CD4+CD44hiIL-23R+/GFP) relative to untreated cells (migration index, mean ± sd, n = 8 independent cultures, ***p = 0.0002, ****p < 0.0001). (i) Proposed model of the role of IL-23 signaling pathway in the development of inflammatory diseases. Statistical analysis: (b, c, d, g, h) One-way ANOVA test with Dunnett´s correction for multiple comparisons. (e) One-sample t test. (f) t test with Welch´s correction. Individual numerical values for quantifications presented in Fig 8 can be found in S8 Data. EAE, experimental autoimmune encephalomyelitis; GFP, Green Fluorescent Protein; IL-23, Interleukin 23; IMQ, Imiquimod; LN, lymph nodes; nTh17, natural Th17; ROCK, Rho-associated protein kinase.

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Grant support

This work was supported by grants from the Spanish Goverment (Ministry of Economy and Competitiveness). M.N.N, C.A-S, G.P-F, I.R.M and J.P are funded by grants SAF2013-43833-R, SAF2016-78180-R and RYC-2012-10252 to M.N.N. R.C-G and D.C are funded by SAF2014-55579-R to Prof. Sánchez-Madrid. Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO are also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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