Abstract
The activation marker CD69 is expressed by skin γδ T cells. Here we found that CD69 controlled the aryl hydrocarbon receptor (AhR)-dependent secretion of interleukin 22 (IL-22) by γδ T cells, which contributed to the development of psoriasis induced by IL-23. CD69 associated with the aromatic-amino-acid-transporter complex LAT1-CD98 and regulated its surface expression and uptake of L-tryptophan (L-Trp) and the intracellular quantity of L-Trp-derived activators of AhR. In vivo administration of L-Trp, an inhibitor of AhR or IL-22 abrogated the differences between CD69-deficient mice and wild-type mice in skin inflammation. We also observed LAT1-mediated regulation of AhR activation and IL-22 secretion in circulating Vγ9+ γδ T cells of psoriatic patients. Thus, CD69 serves as a key mediator of the pathogenesis of psoriasis by controlling LAT1-CD98-mediated metabolic cues.
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Change history
11 July 2016
In the version of this article initially published online, the identification of dermal and epidermal γδ T cells in the legend for Figure 3f was reversed; a label was missing above the far left column of Figure 4c; and the red and blue lines were switched in the keys for the far right plots in Figure 6i. The legend for Figure 3f should read "...identified by high expression (top right; epidermal) or low expression (bottom right; dermal) of the γδ TCR." The far left column in Figure 4c should include the label "CD69-KO" above. The correct keys for Figure 6i are as follows: blue line, FIZC (37 °C), and red line, FICZ + BCH (37 °C); and blue line, CD69-KO (37 °C), and red line, WT (37°C). These errors have been corrected for the print, PDF and HTML versions of this article.
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Acknowledgements
We thank D. Rotin (University of Toronto) for the plasmid for the expression of LAT1-mCherry; P. Taylor (University of Dundee) for antiserum to human LAT1; M. Navarro (Universidad Autónoma de Madrid, Spain) for IL-23R-GFP reporter mice; S. Bartlett for English editing; and T. Hernandez and R. Brid Doohan for technical assistance with immunohistochemistry. Supported by the Spanish Ministry of Economy and Competitiveness (SAF2011-25834 and SAF2014-55579-R to F.S.-M.; SAF2011-27330 to P.M.; and SAF2013-42850 to M.F.), Comunidad de Madrid (INDISNET-S2011/BMD-2332 to F.S.-M.; and 2010/BMD-2332 from M.F. and F.S.-M.), Instituto Salud Carlos III (Red Cardiovascular RD 12-0042-0056 to F.S.-M.; BIOIMID to M.F. and F.S.-M.), the European Research Council (ERC-2011-AdG 294340-GENTRIS to F.S.-M.) and the Ramón Areces foundation (M.F. and F.S.-M.).
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D.C. performed mice experimentation, analyzed and interpreted data and wrote the manuscript; M.L.S. collaborated on mice experimentation, data interpretation and writing of the manuscript; H.d.l.F. performed analysis of psoriatic patients; R.S.-D. and O.M.-G. performed quantitative PCR; I.J., A.F. and J.V. performed proteomic and metabolic-mass spectrometry analyses; C.P. contributed expertise in radioactive assays; M.F., M.V.-M. and P.M.F.-S. provided reagents and helped with the revision of the manuscript; E.D. provided biopsies from psoriatic patients and their clinical diagnostics; P.M. helped to design research, provided reagents, collaborated in data interpretation and manuscript writing; and F.S.-M. planned research, discussed results and collaborated in writing the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Resident skin γδ T cell populations are not affected by CD69 expression.
(a) Flow cytometry (left) analyzing the expression of IL-22 and IL-17 by CD69-deficient and wild-type TH17 cells obtained in vitro. Numbers in quadrants indicates percent cells in each (through). Middle, frequency of IL-22+ and IL-17+ cells gated as above. Right, ELISA quantification of IL-22 and IL-17 levels in the supernatants of cells described above. (b) Expression of AhR in TH17 cells as in a. (c) Quantitative PCR analysis of Il22, AhR, Ahrr, Cyp1a1 and Cyp1b1 mRNA in TH17 cells from CD69-deficient and wild-type mice (Key) cultured by 48h as in a; results calculated as in Fig. 2a,b are present relative to those of CD69-deficient cells. (d) Flow cytometry of total or epidermal or dermal skin cells suspensions from ears of wild-type mice (e) Expression of CD69 in epidermal and dermal γδ T cells sorted from the skin of mice. (f) Frequency of CD3+ populations of cells detected in the skin of CD69-deficient and wild-type mice, in steady state. NS, * P < 0.05, ** P < 0.01, *** P < 0.001; data was analyzed with unpaired t-test for a-c; and two way ANOVA with bonferroni multiple comparison test for f. Three independent experiments were performed with similar results for a, b (n=4 mice per group) and for f (n=5 mice per group). Error bars represent the mean + SEM for a-c and f.
Supplementary Figure 2 Dermal γδ T cells are the main target of IL-23-induced psoriasis.
(a) Flow cytometry (left) analyzing IL-22 and IL-17 secretion by wild-type dermal γδ T cells five hours after a single intradermal IL-23 (500 ng) or PBS injection, and with an intraperitoneal injection of Brefeldin A (250 µg/mice). Right, frequency of IL-22+ and IL-17+ cells gated from dermal γδ T cells as above, and total number of cells detected per ear after IL-23 and PBS treatment. (b) Flow cytometry (left) analyzing IL-22 and IL-17 secretion by sorted wild-type dermal γδ T cells stimulated in vitro with IL-23 plus IL-1β from 24h and 48h. Middle, frequency of IL-22+ and IL-17+ cells gated as above. Right, expression of AhR in wild-type dermal γδ T cells in vitro stimulated as above. (c) Expression of RORγt, pSTAT3 and pSTAT5 detected by flow cytometry in sorted dermal γδ T cells from CD69-deficient and wild-type mice after 24h of stimulation with IL-23+IL-1β. (d) Flow cytometry (left) of skin suspensions from wild-type and IL23R-GFP+ reporter mice showing the expression of GFP+ restricted to dermal γδ T cells in homeostatic conditions. Right, expression of IL-23R (GFP) in epidermal and dermal γδ T cells sorted from the skin of wild-type and IL-23R-GFP reporter mice. (e) Flow cytometry analyzing expression of IL-23R in dermal γδ T cells gated on live cells from CD69-deficient and wild-type mice skin suspensions. Right, expression of IL-23R in sorted dermal γδ T cells from CD69-deficient and wild-type mice. NS, * P < 0.05, ** P < 0.01, *** P < 0.001, unpaired t-test was used for panel a, c and e. Two independent experiments were performed with similar results for panels a-e. Data is showed as mean+s.e.m.; n=5 mice per group for panels a, c, e; n=4 mice per group for panel b.
Supplementary Figure 3 AhR regulates skin inflammation induced by IL-23.
(a) Analysis of ear thickness of CD69-deficient and wild-type mice during the administration of eight doses of IL-22 (500ng/dose) (horizontal axis) (left). Quantitative PCR analysis of Il1b, mS100a8 and mS100a9 genes detected after completed study indicated above (right) (presented as in Fig. 2a,b). (b) Flow cytometry (left) of live-gated dermal γδ T cells from AhR-deficient (AhR-KO) and wild-type littermates mice after three doses of IL-23 (500ng/dose) or PBS and sacrificed upon systemic administration of Brefeldin A. Right, percent and total number per ear of IL-22+ and IL-17+ cells gated as above. (c) Flow cytometry (left) of sorted dermal γδ T cells from CD69-deficient and wild-type mice stimulated in vitro with IL-23 plus IL-1β in the presence of CH-223191(3µM) or its vehicle (DMSO) by 24 hours. Right, frequency of IL-22+ and IL-17+ cells gated as above (d) Expression of CD69 in wild-type CD27- γδ T cells sorted from spleen and lymph-nodes suspensions and stimulated in vitro by 24h with IL-23, IL-1β or combination of both. (e) CD69-deficient and wild-type spleen CD27-γδ T cells were stimulated in vitro with IL-23 plus CH-223191 or DMSO. Flow cytometry (left) and frequency (right) of IL-22+ and IL-17+ γδ T cells gated from live cells. US, unstimulated. AU, arbitrary units, NS, not significant; *** P < 0.001. Two way ANOVA with bonferroni multiple comparison test was used for panels a, c, e; and one way ANOVA with newman-keuls multiple comparison test was used for panels b, d. Two independent experiment were performed for each panels; n=6 mice per group for panel a (mean ± s.d), n=5 mice per group for panel b (each symbol represent an individual mice and small horizontal lines indicate the mean ± s.d.) and n=4 mice per group for panels c, d, e (mean+s.e.m.).
Supplementary Figure 4 CD69 binds specifically to LAT1 and CD98.
(a) Extracted ion chromatogram traces of the monoisotopic peaks from the doubly-charged ions corresponding to four selected peptides from LAT1 and CD98 or from two non-specifically binding proteins (DNPEP and KV2A4). (b-c) Quantitative PCR of nutrient transporters were assessed in CD69-deficient and wild-type CD4+ T cells activated by 24 hours with anti-CD3 and anti-CD28 antibodies. NS, * P < 0.05, data was analyzed with two way ANOVA with bonferroni multiple comparison test for panel b, and unpaired t-test for panel c. Three independent proteomic assays were performed and two experiments for panels b, c, (n=5 mice per group, mean + s.e.m.) with similar results.
Supplementary Figure 5 Internalization of CD69 reduces the uptake of amino acids through LAT1.
(a) Uptake of 3H-labeled amino acids (AAs) by CD4+ T cells from CD69-deficient and wild-type mice activated by 24h with anti-CD3 and anti-CD28, at various times (horizontal axes, top row). Uptake of each labeled amino acid after 15 min, in the presence or absence of BCH (horizontal axes, bottom row). (b) Flow cytometry analyzing membrane and total expression of LAT1 and CD98 in CD69-deficient and wild-type CD4+ T cells activated as above and treated with anti-CD69 blocking Ab (2.2.) or its control isotype Ab (2.8). (c) Confocal image of a HEK cell co-transfected with CD69-GFP and LAT1-Cherry expressing plasmids that was incubated with Alexa-647 Zenon labeled anti-CD69 mAb. (d) Uptake of 3H-labeled L-Trp and L-Phe by wild-type and CD69-deficient activated CD4+ T cells treated as in b at various time (horizontal axes, left) and uptake of those amino acids after 15 minutes in the presence or absence of BCH (horizontal axes, right). NS ** P < 0.01; *** P < 0.001. Data were analyzed with two way ANOVA and Bonferroni multiple comparison test. For amino acid uptake curves a linear regression analysis with slopes comparison were conducted. At least three independent experiments were conducted for each panel, with similar results (mean + s.e.m, n = 5 replicates from each group).
Supplementary Figure 6 CD69 controls mTORC and the secretion of IL-22 by TH17 cells.
Immunoblot analysis of mTORC signaling in CD69-deficient and wild-type TH17 cells obtained after 24 hours (a) and 96 hours (b) cultured in RPMI medium. (c) Flow cytometry (above plots) analyzing the expression of IL-22 and IL-17 by CD69-deficient and wild-type TH17 cells polarized in vitro in RPMI medium alone or supplemented with BCH or FICZ, or cultured in IMDM medium. Frequency (bottom row) of IL-22+ and IL-17+ CD4+ T cells gated as above. (d) Quantitative PCR of L-Trp degrading enzymes mRNA expression were tested in the skin of CD69-deficient and wild-type mice that received IL-23 or IL23 plus IL-22, at the end of the studies (presented as in Fig. 2 a.b). NS, * P < 0.05, results were compared using two way ANOVA followed by bonferroni multiple comparison test. Two independent experiments were conducted for each panel, n=3 or 4 mice per group (panels a, b,c), n=5 mice per group (panel d). Error bar mean + s.e.m. for c,d.
Supplementary Figure 7 CD69 controls the uptake of L-Trp, activation of AhR and IL-22.
CD69 expression is required for the optimal expression of LAT1 in the membrane and uptake of aromatic amino acid by activated lymphocytes. L-Trp is photoconverted or catabolized in the cytosol to several AhR ligands, including FICZ, that regulates AhR-mediated signaling and IL-22 secretion.
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Cibrian, D., Saiz, M., de la Fuente, H. et al. CD69 controls the uptake of L-tryptophan through LAT1-CD98 and AhR-dependent secretion of IL-22 in psoriasis. Nat Immunol 17, 985–996 (2016). https://doi.org/10.1038/ni.3504
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DOI: https://doi.org/10.1038/ni.3504