Autophagy Regulates IL-23 Secretion and Innate T Cell Responses through Effects on IL-1 Secretion This information is current as Celia Peral de Castro, Sarah A. Jones, Clíona Ní Cheallaigh, of September 24, 2021. Claire A. Hearnden, Laura Williams, Jan Winter, Ed C. Lavelle, Kingston H. G. Mills and James Harris J Immunol 2012; 189:4144-4153; Prepublished online 12 September 2012; doi: 10.4049/jimmunol.1201946 Downloaded from http://www.jimmunol.org/content/189/8/4144 Supplementary http://www.jimmunol.org/content/suppl/2012/09/12/jimmunol.120194 Material 6.DC1 http://www.jimmunol.org/ References This article cites 48 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/189/8/4144.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Autophagy Regulates IL-23 Secretion and Innate T Cell Responses through Effects on IL-1 Secretion Celia Peral de Castro,*,† Sarah A. Jones,*,† Clı´ona Nı´ Cheallaigh,‡ Claire A. Hearnden,‡ Laura Williams,* Jan Winter,‡ Ed C. Lavelle,†,‡ Kingston H. G. Mills,*,† and James Harris*,† Autophagy controls IL-1b secretion by regulating inflammasome activation and by targeting pro–IL-1b for degradation. In this article, we show that inhibition of autophagy, either with the PI3K inhibitors 3-methyladenine, wortmannin, and LY294002 or with small interfering RNA against autophagy proteins augmented the secretion of IL-23 by human and mouse macrophages and dendritic cells in response to specific TLR agonists. This process occurred at the transcriptional level and was dependent on reactive oxygen species and IL-1R signaling; it was abrogated with an IL-1R antagonist or with IL-1–neutralizing Abs, whereas treatment with either rIL-1a or IL-1b induced IL-23 secretion. Dendritic cells treated with LPS and 3-methyladenine secreted Downloaded from enhanced levels of both IL-1b and IL-23, and supernatants from these cells stimulated the innate secretion of IL-17, IFN-g, and IL-22 by gd T cells. These data demonstrate that autophagy has a potentially pivotal role to play in the induction and regulation of inflammatory responses by innate immune cells, largely driven by IL-1 and its consequential effects on IL-23 secretion. The Journal of Immunology, 2012, 189: 4144–4153. utophagy is a highly conserved homeostatic mechanism oxygen species (ROS), and mitochondrial DNA (10–13). In ad- http://www.jimmunol.org/ that orchestrates the sequestration and degradation of dition, inhibition of autophagy enhances the secretion of IL-1a A damaged or noxious cytosolic constituents, including or- and IL-18 in response to LPS (10). Conversely, autophagosomes ganelles, and regulates the catabolism of macromolecules during have been shown to target both pro–IL-1b and the inflammasome nutrient deprivation. More recently, autophagy has been shown components ASC and NLRP3 within cells, thus directly regulating to regulate inflammation and immune responses to infection. In the production and availability of IL-1b (10, 14). Moreover, both particular, autophagy can mediate the intracellular killing of some IL-1b and IL-1a induce autophagy in macrophages (15), sug- bacteria by macrophages (1) and is involved in the presentation of gesting that these cytokines might regulate their own secretion. Ags via MHC class I and class II molecules (2). Autophagy is These data, together with studies showing that autophagy alters by guest on September 24, 2021 regulated by cytokines; in macrophages, it is induced by IFN-g disease outcomes in animal models of sepsis (10, 11) and colitis and TNF-a (3, 4) and inhibited by IL-4, IL-13, and IL-10 (5‑8). (12), suggest that autophagy is a potent regulator of inflammatory Numerous studies have also demonstrated a role for autophagy responses. Supporting this, polymorphisms in human ATG16L1, in regulating the secretion of cytokines (reviewed in Ref. 9), in- a protein involved in autophagosome formation, have been asso- cluding members of the IL-1 family. ciated with an increased risk of Crohn’s disease (16, 17). Inhibition of autophagy allows the processing and secretion Both IL-1b and IL-1a, as well as IL-18, can drive IL-17 se- of the proinflammatory cytokine IL-1b in response to TLR3 and cretion by Th17 and gd T cells, but only in the presence of IL- TLR4 agonists, and this process may be dependent on TIR do- 23 (18–20). This pathway can drive inflammatory pathologies in main-containing adaptor protein inducing IFN-b (TRIF), reactive multiple autoimmune diseases, including multiple sclerosis and rheumatoid arthritis (21–24), and is important in immunity to in- fection (18, 25, 26). In this study, we demonstrate that inhibition of *Immune Regulation Research Group, School of Biochemistry and Immunology, autophagy promotes the secretion of IL-1a, IL-1b, and IL-23 by Trinity Biomedical Sciences Institute, Trinity College, Dublin 2, Ireland; †Immunology macrophages and dendritic cells (DC), which in turn influences the Research Centre, Trinity College, Dublin 2, Ireland; and ‡Adjuvant Research Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity innate secretion of IL-17, IFN-g, and IL-22 by gd T cells. More- College, Dublin 2, Ireland over, this is dependent on IL-1 and NF-kB signaling, and both IL- Received for publication July 12, 2012. Accepted for publication August 10, 2012. 1a and IL-1b augment IL-23 production. These data demonstrate This work was supported by Science Foundation Ireland as part of the Immunology a potentially pivotal role for autophagy in regulating inflammatory Research Centre, Science Foundation Ireland Strategic Research Cluster (Grant cytokines and innate T cell-driven immune responses. 07/SRC/B1144). Address correspondence and reprint requests to Dr. James Harris, School of Bio- chemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College, Materials and Methods Dublin 2, Ireland. E-mail address: [email protected] Materials and reagents The online version of this article contains supplemental material. Wortmannin, LY294002, and rapamycin were from Sigma-Aldrich (Poole, Abbreviations used in this article: BMDC, bone marrow-derived dendritic cell; DC, U.K.), and 3-methyladenine (3-MA) was from Sigma-Aldrich or Merck dendritic cell; iBMM, immortalized bone marrow-derived macrophage; IL-1Ra, IL- Millipore (Billerica, MA). Hydrocinnamoyl-L-valyl pyrrolidine (IL-1R an- 1R antagonist; 3-MA, 3-methyladenine; mTOR, mammalian target of rapamycin; poly(I:C), polyinosinic:polycytidylic acid; rh, recombinant human; ROS, reactive tagonist [IL-1Ra]) and BAY 11-7082 (NF-kB inhibitor) were from Merck oxygen species; siRNA, small interfering RNA; TRIF, TIR domain-containing adap- Millipore. LPS from Escherichia coli serotype R515 was from Enzo Life tor protein inducing IFN-b. Sciences (Exeter, U.K.), and polyinosinic:polycytidylic acid [poly(I:C)], R837, and CpG were from InvivoGen (San Diego, CA). PP242 was from Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 Chemdea (Ridgewood, NJ). Recombinant human (rh)IL-1b was from www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201946 The Journal of Immunology 4145 Immunotools (Friesoythe, Germany). Granzyme B-cleaved and full-length propidium iodide (1 mg/ml) for 5 min. A CyAN (DakoCytomation, Dublin, murine IL-1a were gifts from Prof. S. Martin (Trinity College Dublin, Ireland) flow cytometer was used to analyze fluorescence, and the data Ireland). were analyzed using FlowJo software (Tree Star, Ashland, OR). Cell culture Quantitative PCR C57BL/6 mice were obtained from Harlan Olac (Bicester, U.K.) and were Mouse BMDC were stimulated for 4 h with 3-MA (2.5–10 mM) in the used at 8–16 wk of age. Animals were maintained according to the reg- presence or absence of LPS (10 ng/ml). RNA was purified with the TRIzol ulations of the European Union and the Irish Department of Health. Bone (Invitrogen)/chloroform method; this process was followed by tran- marrow-derived DC (BMDC) were generated as previously described (27) scription into cDNA with a High-Capacity cDNA Reverse Transcription Kit and grown in RPMI 1640 medium (Biosera, East Sussex, U.K.) with 10% (Applied Biosystems). Real-time PCR for the detection of IL-23p19 (il23a) FCS, L-glutamine (2 mM), and penicillin and streptomycin (50 U/ml (Mm00518984_m1) and IL-1b (il1b) (Mm00434228_m1) mRNA was and 50 mg/ml, respectively) (complete medium). The medium was sup- performed with predesigned TaqMan gene expression assays (Applied plemented with GM-CSF (40 ng/ml). Cells were plated out on day 10 Biosystems) using a 7500 Fast Real-Time PCR machine (Applied Bio- and stimulated on day 11. BMDC from TRIF+/+ and TRIF2/2 mice were systems). Expression was normalized to 18s rRNA. provided by Prof. K. Fitzgerald (University of Massachusetts Medical Center, Worcester, MA). Confocal microscopy + CD3+ T cell isolation and culture Immortalized GFP-LC3 iBMM were grown and stimulated on nitric acid- treated glass coverslips (19 mm diameter) in 12-well plates. Cells were CD3+ T cells were purified from spleens of C57BL/6 mice using magnetic fixed with 2% paraformaldehyde for 30 min at room temperature and beads for negative selection (Pan T Cell Isolation Kit II, mouse; Miltenyi permeabilized for 10 min with Triton X-100 (0.1% in PBS).