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

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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 augmented the secretion of IL-23 by human and mouse 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, 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 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 involved in 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 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 and rheumatoid (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 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 ; DC, U.K.), and 3-methyladenine (3-MA) was from Sigma-Aldrich or Merck dendritic cell; iBMM, immortalized bone marrow-derived ; 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 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). All subse- Biotec), according to the manufacturer’s instructions. In some experi- quent steps were conducted at room temperature. The cells were blocked ments, cells were cultured with IL-1b (10 ng/ml), IL-23 (10 ng/ml), LPS with 5% goat serum in PBS with 1% BSA (blocking buffer) for 1 h and Downloaded from (10 or 100 ng/ml), 3-MA (5–10 mM), IL-1Ra (5 mg/ml), or combinations stained with primary Ab (1/200 in blocking buffer) for 1 h. After washing thereof. Alternatively, CD3+ T cells were cultured with supernatants (50 with PBS (five times), the cells were stained with Alexa Fluor 568-con- ml/200 ml CD3+ cells) obtained from DCs cultured for 24 h with 3-MA (5– jugated goat anti-rabbit IgG (1/500 in blocking buffer; Life Technologies, 10 mM) in the presence or absence of LPS (10 ng/ml). Similarly, CD3+ Grand Island, NY) for 1 h. In some experiments, cells were stained T cells were cultured with supernatants obtained from immortalized bone with tetramethylrhodamine isothiocyanate–phalloidin (American Peptide, marrow-derived macrophages (iBMM) transfected with control (scram- Sunnyvale, CA) at 1 mg/ml to stain polymerized actin. The coverslips were bled), beclin I, or Atg7 small interfering RNA (siRNA) stimulated for 18 h mounted onto glass slides with fluorescent Mounting medium (DakoCyto- with LPS (10–100 ng/ml). After 72 h, IL-17A, IL-17F, and IFN-g con- mation) and analyzed on an Olympus FV1000 laser scanning confocal mi- http://www.jimmunol.org/ centrations in supernatants were determined by ELISA. croscope. For characterization of gd T cells, lymph node T cells depleted of gd T cells (2 3 105)orgd T cells (2 3 104) enriched from lymph nodes using Immunoblotting g d+ a TCR / T cell MACS Isolation kit, according to manufacturer’s BMDC and immortalized iBMM were washed twice with PBS and lysed, m instructions (Miltenyi Biotec), were cultured for 72 h in 200 l complete according to a previously described protocol (28). Samples were loaded and m medium. Supernatants (50 l) or recombinant cytokines were added to separated on 10, 12, or 15% SDS-polyacrylamide gels and transferred to g each well, as described above. Cytokine (IL-17A, IL-22, and IFN- ) se- a nitrocellulose membrane. The membrane was blocked for 1 h with 5% cretion was measured by ELISA. milk in PBS/Tween 20 (0.1%). After washing with PBS/Tween 20, the iBMM cell lines membrane was probed with primary Ab (1/500 in 3% BSA) overnight at 4˚C, washed three times with PBS/Tween, and probed with HRP-conjugated by guest on September 24, 2021 iBMM from wild-type C57BL/6, generated using J2 transforming retro- secondary Ab (1/2000 in PBS) for 1 h at room temperature. The membrane viruses, were a gift from Prof. D. Golenbock (University of Massachusetts was washed three times with PBS/Tween 20 and developed with freshly Medical Center). iBMM stably expressing EGFP-LC3 (GFP-LC3) were prepared luminol-based detection solution. Densitometric analysis of blots generated as previously described (10) and expanded under constant selec- was conducted using ImageJ software (National Institutes of Health, tion with 10 mg/ml puromycin. All iBMM were grown in complete medium. Bethesda, MD). Mycobacterium tuberculosis culture LPS challenge in vivo M. tuberculosis strain H37Rv was grown in Middlebrook 7H9 broth with Female BALB/c mice were injected i.p. with 200 ml PBS, LPS (10 mg/ 0.2% glycerol and albumin–dextrose catalase supplement. Mycobacteria mouse in 200 ml PBS), or LPS with rapamycin (1.5 mg/kg in 200 ml PBS). were grown to log phase before use, declumped by vortexing with glass After 4 h, mice were bled, and ELISA analysis was performed on plasma beads for 60 s, and resuspended in RPMI 1640 medium with 10% FCS samples. before use. Statistical analysis Macrophage and DC transfection Unless otherwise stated, data sets were analyzed for statistical significance iBMM or BMDC were transfected by nucleoporation with an Amaxa (p , 0.05) using one-way ANOVA, followed by a Bonferroni post hoc test. Nucleofector Device (Lonza, Wokingham, U.K.) as described previously Statistical analysis was conducted using GraphPad Prism software. (5). iBMM were harvested after 2–3 d in culture and resuspended in 100 ml of the appropriate electroporation buffer (Lonza AG) with 10 nM siGE- NOME SMARTpool siRNA against beclin 1, Atg7, or siGENOME non- Results targeting control siRNA (Thermo Scientific, Lafayette, CO). Transfected The autophagy inhibitor 3-MA enhances LPS-induced cells were incubated overnight in complete medium before stimulation secretion of IL-23 with LPS for 24 h. Targeting efficiency was analyzed by Western blot for beclin 1 and Atg7. 3-MA is a PI3K inhibitor that blocks autophagic degradation of proteins (29). Our previous work has demonstrated that treatment ELISA of murine macrophages and DC with 3-MA stimulates the se- Cytokine secretion in cell culture supernatants was measured by ELISA, cretion of IL-1b, IL-1a, and IL-18 in responses to specific TLR according to the manufacturer’s standard protocols (R&D Systems, ligands (10). To determine whether IL-23 secretion was also Abingdon, U.K.; eBioscience, San Diego, CA; and BD Biosciences, San regulated by autophagy, we treated human PMA-differentiated Diego, CA). Absorbance was read on a Multiscan FC plate reader and analyzed with SkanIt for Multiscan FC software (Thermo Scientific). THP-1 /macrophages with 3-MA and LPS overnight and measured cytokines in the supernatants. At concentrations of 2.5 Propidium iodide incorporation assay mM and above, 3-MA stimulated the secretion of both IL-23 and BMDC were grown and stimulated in 96 well U-bottom tissue culture IL-1b in response to LPS (Fig. 1A, 1B). IL-1a secretion by THP-1 plates. After treatment with LPS and 3-MA, cells were incubated with cells was also stimulated under these conditions (Supplemental 4146 AUTOPHAGY REGULATES IL-23 AND T CELL RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. Inhibition of autophagy enhances LPS-induced secretion of IL-23 by macrophages and DC. PMA-differentiated human THP-1 cells (A–C)or murine bone marrow-derived dendritic cells (BMDC) (D–F) were treated with LPS (100 or 10 ng/ml) for 24 h in the presence of 3-MA at different concentrations and levels of IL-23 (A, D), IL-1b (B, E), TNF-a (C), and IL-6 (F) in the supernatants measured by ELISA. BMDC (G) or PMA-differentiated THP-1 cells (H) were treated with heat-killed E. coli (multiplicity of infection 10:1 bacteria:cells) (G)orliveMycobacterium tuberculosis strain H37Rv (multiplicity of infection 10:1) (H) in the presence of different concentrations of 3-MA for 24 h and IL-23 secretion measured by ELISA. (I) Lysates from BMDC treated with bafilomycin (100 nM) and rapamycin or LPS in the presence or absence of 3-MA (10 mM) were stained for LC3 I (cytosolic) and LC3 II (lipidated, membrane-bound) by Western blot analysis. The samples were stained for b-actin as a loading control. (J and K) BMDC were treated with LPS (10 ng/ml) in the presence of wortmannin (J) or LY294002 (K) at different concentrations and IL-23 secretion measured by ELISA of culture supernatants. Data represent mean values + SD (n $ 3); *p , 0.05 compared with LPS only, one-way ANOVA with Bonferroni’s multiple comparison test.

Fig. 1A). In contrast, LPS-induced TNF-a secretion was enhanced in response to live virulent M. tuberculosis H37Rv (Fig. 1H), by 3-MA at 2.5 mM but inhibited by 10 mM (Fig. 1C). In primary whereas TNF-a secretion was either unaffected or inhibited murine BMDC, 3-MA increased LPS-induced secretion of IL-23 (Supplemental Fig. 1H). To confirm the autophagy-inhibiting ac- and IL-1b (Fig. 1D, 1E), as well as IL-1a (Supplemental Fig. 1B). tivity of 3-MA, we treated BMDC with the autophagy inducer Levels of IL-6 secreted by these cells were not significantly af- rapamycin or with LPS for 2 h in the presence or absence of 3-MA fected (Fig. 1F). This effect was also observed in primary human and bafilomycin (to inhibit autophagic flux (30)) and measured monocyte-derived DC in which 3-MA augmented LPS-induced levels of LC3 I (cytosolic) and LC3 II (lipidated autophagosomal IL-23 secretion but inhibited TNF-a secretion (Supplemental membrane-bound) by Western blotting. Both rapamycin and LPS Fig. 1C, 1D). In addition, 3-MA enhanced the secretion of IL-23 increased the amount of LC3 II present, indicating an increase in by BMDC in response to heat-killed E. coli (Fig. 1G). Secretion of autophagosome biogenesis, whereas 3-MA inhibited this effect IL-1a and IL-1b was also increased in response to E. coli with 3- (Fig. 1I). MA (Supplemental Fig. 1E, 1F), whereas IL-6 secretion was ei- Treatment with other broad-range PI3K inhibitors has also been ther unaffected or inhibited (Supplemental Fig. 1G). Similarly, shown to inhibit autophagy (30). In our model, LPS-induced IL-23 stimulation of THP-1 cells with 3-MA enhanced IL-23 secretion secretion by BMDC was increased in the presence of the PI3K The Journal of Immunology 4147 inhibitors wortmannin and LY294002 (Fig. 1J, 1K), and both IL-1b for IL-1b secretion (10, 12), we treated BMDC from wild-type and IL-23 secretion were enhanced by LY294002 in THP-1 cells and TRIF2/2 mice with LPS and 3-MA. Both IL-1b and IL-23 (Supplemental Fig. 1I, 1J). These data demonstrate that autophagy- secretion was inhibited in the TRIF2/2 cells (Fig. 2E, 2F). inhibiting drugs augment IL-23 secretion by macrophages and DC in response to microbial stimuli. Specific knockdown of autophagy proteins enhances IL-1b secretion in the absence of autophagy is dependent on the LPS-induced secretion of IL-23 TLR adaptor molecule TRIF and thus only occurs in response to Knockdown of autophagy-specific , using siRNA targeting TLR3 and TLR4 agonists, which signal through this pathway (10, beclin 1 and Atg7, enhances LPS-induced secretion of IL-1b 12). In this study, we also observed enhanced secretion of IL-23 by macrophages (10, 12). In this study, we found that siRNA de- by 3-MA–treated BMDC in response to TLR3 and TLR4 ligands pletion of beclin 1 in murine iBMM enhanced secretion of IL-1a but not ligands of other TLRs. Both IL-23 and IL-1b secretion and IL-1b but did not affect secretion of IL-12/23 p40 (Fig. were increased in cells treated with 3-MA in combination with 3A‑D). Moreover, knockdown of either beclin 1 or Atg7 enhanced the TLR3 ligand poly(I:C) or the TLR4 ligands LPS and mono- secretion of IL-23, but not TNF-a, in response to LPS (Fig. 3E– phosphoryl lipid A (which preferentially signals through TRIF) H), replicating the effects produced by treatment with 3-MA. In- but not the TLR2 ligand PAM3CysK4, the TLR7 agonist R837 hibition of autophagy with 3-MA has been demonstrated to drive (imiquimod), the TLR7/8 agonist R848, or the TLR9 ligand CpG IL-1b secretion by human PBMC in response to M. tuberculosis (Fig. 2A–D). Both IL-1b and IL-23 secretion were augmented by (31). Similarly, we show in this study that knockdown of beclin 1 3-MA in response to zymosan, a ligand for TLR2, but which also in BMDC, even though only partial, significantly enhanced IL-23 signals through binding to dectin-1 (Fig. 2C, 2D). To determine but not TNF-a secretion in response to irradiated M. tuberculosis Downloaded from whether the effect of 3-MA on IL-23 secretion was dependent on strain H37Rv (Fig. 3I, 3J). These data demonstrate that in microbe- signaling through TRIF, which has been previously demonstrated activated macrophages and DC, the specific inhibition of autophagy enhances IL-23 secretion.

Inhibition of autophagy alters IL-23 expression at the

transcriptional level http://www.jimmunol.org/ The effects of autophagy inhibition on LPS-driven IL-1b secretion have previously been shown to occur at the transcriptional level (31, 32). In this study, we examined cytokine mRNA expression in murine BMDC in response to LPS and 3-MA. After 4-h treatment with LPS, expression of IL-1b mRNA was increased, but this was unaffected by 3-MA (Fig. 4A). However, expression of IL-23 mRNA was enhanced by LPS, and this was further augmented by the addition of 3-MA (Fig. 4B). Expression of IL-10 mRNA was increased by LPS but unaffected by 3-MA (Fig. 4C). Similarly, in by guest on September 24, 2021 iBMM transfected with beclin 1 siRNA, expression of IL-23p19 mRNA in response to LPS was significantly increased after 4 h compared with cells transfected with scrambled siRNA (Fig. 4D). Thus, inhibition of autophagy not only leads to inflammasome activation but also to the upregulation of IL-23 at the transcrip- tional level. Secretion of IL-23 in response to 3-MA and LPS is dependent on IL-1 signaling In human PBMC exposed to wheat gliadin, IL-23 secretion is driven by IL-1 signaling (33). To determine whether IL-23 se- cretion was dependent on IL-1 signaling in our model, we treated BMDC and PMA-differentiated THP-1 cells with LPS and 3-MA in the presence or absence of hydrocinnamoyl-L-valyl pyrrolidine, an IL-1Ra. In BMDC, IL-1Ra inhibited the secretion of both IL-23 and IL-1b in response to LPS and 3-MA but had no effect on IL- 12p40 or IL-12/23p70 secretion (Fig. 5A–D), whereas in THP-1 cells, IL-1Ra inhibited the secretion of IL-23, but not IL-1b,in response to LPS and 3-MA (Fig. 5E, 5F). In THP-1 cells, a neu- tralizing Ab against IL-1b inhibited IL-23 secretion at all con- FIGURE 2. IL-23 secretion in autophagy-deficient cells is dependent on centrations tested, whereas a neutralizing Ab against IL-1a had specific TLRs. Murine BMDC were treated with different TLR ligands a small inhibitory effect only at the lowest concentration tested (10 ng/ml LPS, 1 mg/ml poly(I:C), 1 mg/ml R837, 10 mg/ml CpG, 1 mg/ml (Fig. 6A). Similarly, secretion of IL-23 by THP-1 cells stimulated PAM3CysK4, 500 ng/ml monophosphoryl lipid A (MPLA), 1 mg/ml R848, with heat-killed E. coli and 3-MA was inhibited with neutralizing and 1 mg/ml zymosan) in the presence or absence of 3-MA (10 mM), and Abs against IL-1b but not IL-1a (Fig. 6B). To further assess the culture supernatants were tested by ELISA for IL-1b (A, C) or IL-23 (B, D). (E and F) Murine BMDC from TRIF+/+ and TRIF2/2 mice were treated ability of IL-1 to induce IL-23 secretion, THP-1 cells were treated with LPS in the presence or absence of 3-MA at different concentrations with rhIL-1a or rhIL-1b. At 10 ng/ml, rhIL-1a induced IL-23 and culture supernatants tested by ELISA for IL-1b (E) or IL-23 (F). Data secretion, both in the presence and absence of LPS (Fig. 6C). represent mean values + SD (n $ 3); *p , 0.05 difference between +3-MA Similarly, rhIL-1b stimulated IL-23 secretion by THP-1 cells, and 23-MA, one-way ANOVAwith Bonferroni’s multiple comparison test. regardless of exposure to LPS (Fig. 6D). 4148 AUTOPHAGY REGULATES IL-23 AND T CELL RESPONSES

FIGURE 3. siRNA-targeted inhibition of autophagy enhances LPS-induced IL-23 se- cretion. (A–C) Murine iBMM were transfected with siRNA against beclin 1 or nontargeting (“scrambled”) siRNA and treated with LPS at different concentrations for 24 h. Secretion of IL-1a (A), IL-1b (B), and IL-12/23 p40 (C) were measured from culture supernatants by ELISA. (D) Western blot analysis of beclin 1 levels in transfected iBMM from (A)–(C). (E and F) iBMM were transfected with siRNA against beclin 1, Atg7, or scrambled siRNA and treated with LPS at different concen- trations for 24 h. Secretion of IL-23 (E) and TNF-a (F) were measured from culture super- natants by ELISA. (G) Western blot analysis of Downloaded from beclin 1 and Atg7 levels in transfected iBMM from (E) and (F). (H and I) Murine BMDC were transfected with siRNA against beclin 1 or scrambled siRNA and treated with irradi- ated M. tuberculosis strain H37Rv (multiplic- ity of infection 10:1 bacteria:cells) for 24 h. http://www.jimmunol.org/ Culture supernatants were analyzed by ELISA for IL-23 (H) and TNF-a (I). (J) Western blot analysis of beclin 1 levels in BMDC trans- fected with scrambled or beclin 1 siRNA. Data represent mean values + SD (n $ 3); *p , 0.05 compared with corresponding scrambled control, one-way ANOVA with Bonferroni’s multiple comparison test. by guest on September 24, 2021

Because the IL-1R1 is known to signal through the NF-kB assess whether the same was true for IL-23, we stained LPS- pathway, we assessed the effects of a NF-kB inhibitor, BAY 11- treated GFP-LC3–expressing iBMM with Ab against IL-1b or 7082, on LPS/IL-1b–induced IL-23 secretion. In PMA-differentiated IL-23 and examined the cells by confocal microscopy. As previ- THP-1 cells, the inhibitor abrogated the effects of LPS and rhIL- ously shown, after overnight treatment with LPS, IL-1b colo- 1b, but not LPS alone, on IL-23 secretion (Fig. 6E). In addition, calized with GFP-LC3 (Fig. 7A). However, no IL-23 could be LPS and rhIL-1b enhanced the secretion of TNF-a by THP-1 detected in GFP-LC3+ autophagosomes (Fig. 7B). This would cells, and this was again inhibited by the NF-kB inhibitor (Fig. suggest that unlike IL-1b, intracellular IL-23 is not sequestered by 6F). These data suggest that activation of NF-kB via the IL-1R1 autophagosomes. stimulates the production of IL-23 as well as TNF-a. To determine the effect of autophagy induction on IL-23 pro- Previous reports have suggested that ROS promote activation of duction, we treated BMDC with LPS and PP242, an autophagy- the inflammasome and secretion of IL-1b in autophagy-deficient inducing mammalian target of rapamycin (mTOR) inhibitor. At cells (10, 12). Given the role of IL-1b in promoting IL-23 secretion, all concentrations tested, PP242 inhibited LPS-induced IL-23 we assessed the potential role of ROS on IL-23 secretion by production (Fig. 7C) Moreover, when another mTOR inhibitor, BMDC. The ROS scavenger N-acetyl-L-cysteine completely rapamycin, was administered i.p. in a murine model of endotoxin- abrogated the secretion of IL-23 in response to LPS and 3-MA induced sepsis, it abrogated the stimulatory effects of LPS on in BMDC at all concentrations tested (Supplemental Fig. 2A). In serum IL-23 (Fig. 7D), as we have previously demonstrated for addition, at higher concentrations, N-acetyl-L-cysteine inhibited IL-1b (10). These data would suggest that although blocking the secretion of IL-1b and IL-1a (Supplemental Fig. 2B, 2C). IL-6 autophagy allows IL-23 production through the enhancement of secretion was affected only at the highest concentration (50 mM) IL-1 secretion, the induction of autophagy has the opposite effect (Supplemental Fig. 2D). Taken together, these data demonstrate and downregulates IL-23 secretion. that IL-23 secretion in autophagy-deficient cells is directly regu- Both IL-1b and IL-1a can induce autophagy (15), suggesting lated by IL-1 signaling and is dependent on the generation of ROS. that these cytokines may limit their own secretion. In agreement with this, we found that both IL-1b and IL-1a increased auto- IL-23 is not sequestered by autophagosomes phagosome formation in GFP-LC3–expressing iBMM (Supple- Our previous work demonstrated that in LPS-stimulated iBMM, mental Fig. 3A, 3B). Interestingly, granzyme B-processed IL-1a, autophagosomes sequester pro–IL-1b for degradation (10). To which has recently been shown to have greater proinflammatory The Journal of Immunology 4149 Downloaded from

FIGURE 4. Inhibition of autophagy enhances IL-23 secretion at the transcriptional level. Levels of IL-1b (Il1b)(A), IL-23p19 (Il23a)(B), and IL-10 (Il10)(C) mRNA in murine BMDC treated with LPS (10 ng/ml) and 3-MA (5 or 10 mM) or 3-MA alone for 4 h were measured by quantitative PCR. Data represent mean values + SD. *p , 0.05 compared with LPS http://www.jimmunol.org/ alone, one-way ANOVA with Bonferroni’s multiple comparison test. (D) Murine iBMM were transfected with siRNA against beclin 1 or with nontargeting (“scrambled”) siRNA and treated with LPS (10 and 100 ng/ ml) for 4 h, and levels of Il23a were measured by quantitative PCR. Data FIGURE 5. IL-23 secretion in autophagy-deficient cells is dependent on represent mean values + SD (n $ 3); *p , 0.05, one-way ANOVA with IL-1 signaling. (A–D) Murine BMDC were treated with LPS (10 ng/ml) Bonferroni’s multiple comparison test. and 3-MA at different concentrations in the presence or absence of the IL-1Ra hydrocinnamoyl-L-valyl pyrrolidine (5 mg/ml) for 24 h. Culture A B effects than the full-length form (34), was a more potent auto- supernatants were analyzed by ELISA for IL-23 ( ), IL-1b ( ), IL-12/

23p40 (C), and IL-12p70 (D). Human PMA-differentiated THP-1 cells by guest on September 24, 2021 phagosome inducer (Supplemental Fig. 3B). In addition, IL-23 in- were treated with LPS (100 ng/ml) and 3-MA (5 mM) with the IL-1Ra at creased autophagosome formation in GFP-LC3–expressing iBMM different concentrations for 24 h, and supernatants were tested by ELISA (Supplemental Fig. 3C). These data would indicate that autophagy for IL-23 (E) and IL-1b (F). Data represent mean values + SD (n $ 3); represents a negative feedback mechanism for the control of IL-1b *p , 0.05 difference between +IL-1Ra and 2IL-1Ra, one-way ANOVA secretion and the subsequent production of IL-23. with Bonferroni’s multiple comparison test. Impaired autophagy in activated macrophages and DC stimulates innate cytokine secretion by gd T cells IFN-g secretion (Fig. 8G–L). Thus, our data demonstrate that, Given that IL-1 and IL-23 synergistically stimulate TCR-independent through the regulation of IL-1b and IL-23 secretion, autophagy IL-17 production by Th17 cells (20) and that the inhibition of can influence innate cytokine production through the indirect autophagy in macrophages and DC enhances the secretion of IL- stimulation of gd T cells. 1b, IL-1a, and IL-23, we sought to determine whether this could, in turn, stimulate innate Th17-like cytokine responses in vitro. Discussion Supernatants from BMDC treated with LPS and 3-MA (to induce Numerous cytokines have been shown to regulate autophagy in IL-1b and IL-23 secretion; Supplemental Fig. 4A, 4B) stimulated macrophages and DC (9). In particular, IFN-g, TNF-a, IL-1a, and IL-17A and IL-17F secretion by MACS-purified splenic CD3+ IL-1b increase autophagosome formation in macrophages (3, 4, T cells (Fig. 8A, 8B). These supernatants also enhanced T cell 15), whereas IL-4, IL-13, and IL-10 inhibit autophagy (5–8). More secretion of IFN-g (Fig. 8C). Moreover, LPS and 3-MA had no recent studies have also demonstrated that autophagy can directly direct effect on T cells (Fig. 8D–F), suggesting that this effect was influence the secretion of cytokines, including IL-1b. Inhibition due to soluble factors secreted by the 3-MA–treated BMDC. of autophagy enhances IL-1b production and secretion, because Supernatants from iBMM transfected with siRNA against beclin 1 of the intracellular accumulation of endogenous inflammasome- similarly enhanced secretion of IL-17A from splenic T cells activating factors, such as mitochondrial DNA and ROS (10–13, (Supplemental Fig. 4C‑H), demonstrating that this effect is due 31, 32). Conversely, autophagosomes themselves can directly target to impaired autophagy. Because gd T cells are a key source of in- pro–IL-1b and the inflammasome components ASC and NLRP3 nate cytokines secreted in response to IL-1b and IL-23 in the (10, 14). In addition, autophagy can regulate the secretion of IL-18 absence of TCR stimulation (20), we isolated gd+ and gd2 T cells and IL-1a, although the mechanisms behind the latter may be from lymph nodes and assessed their capacity to produce IL-17, different to those characterized for IL-1b (10, 12). In the current IL-22, and IFN-g when exposed to supernatants from autophagy- study, we have demonstrated that the inhibition of autophagy can deficient DC. When cultured with supernatants from BMDC also enhance IL-23 secretion in response to LPS and whole treated with LPS and 3-MA or with recombinant IL-1b and IL-23, bacteria. A previous study has reported that wortmannin, but not gd T cells were responsible for the majority of IL-17, IL-22, and LY294002, enhances IL-23 secretion by human DC in response to 4150 AUTOPHAGY REGULATES IL-23 AND T CELL RESPONSES Downloaded from http://www.jimmunol.org/

FIGURE 6. IL-23 secretion is driven by IL-1. PMA-differentiated THP- 1 were treated with LPS (100 ng/ml) and 3-MA (5 mM) (A) or heat-killed E. coli (multiplicity of infection 10:1 bacteria:cells) and 3-MA (5 mM) (B) for 24 h with different concentrations of neutralizing Ab against IL-1a or by guest on September 24, 2021 IL-1b. Secretion of IL-23 into culture supernatants was measured by FIGURE 7. IL-23 is sequestered by autophagosomes. (A and B) Murine ELISA. Data represent mean values + SD (n $ 3); *p , 0.05 compared iBMM stably transfected with pEGFP-LC3 were treated with LPS for 24 h with LPS or bacteria alone, one-way ANOVA with Bonferroni’s multiple and stained with Ab against IL-1b (A) or IL-23 (B). Intracellular staining comparison test. (C and D) PMA-differentiated THP-1 cells were treated was visualized with Alexa 568 secondary Ab and analyzed by confocal with rhIL-1a (C) or rhIL-1b (D) in the presence or absence of LPS (100 microscopy. Arrows point to autophagosomes for comparisons between ng/ml) for 24 h, and IL-23 secretion was measured by ELISA. Data rep- stains. Scale bar, 10 mm. (C) BMDC were treated with LPS (10 ng/ml), and resent mean values + SD (n $ 3); *p , 0.05 compared with unstimulated different concentrations of the mTOR inhibitor PP242 for 24 h; IL-23 or LPS alone, one-way ANOVA with Bonferroni’s multiple comparison secretion into the culture supernatant was measured by ELISA. Data test. (E and F) PMA-differentiated THP-1 cells were treated with rhIL-1b represent mean values + SD (n $ 3); *p , 0.05 compared with control, and LPS (100 ng/ml) in the presence or absence of the NF-KB inhibitor one-way ANOVA with Bonferroni’s multiple comparison test. (D) Female BAY 11-7082 for 24 h and IL-23 secretion (E) or TNF-a secretion (F) C57BL/6 mice were injected i.p. with 200 ml PBS, LPS (10 mg/mouse, in measured by ELISA. Data represent mean values + SD (n $ 3); *p , 0.05, PBS), or LPS + rapamycin (1.5 mg/kg in PBS), and serum levels of IL-23 one-way ANOVA with Bonferroni’s multiple comparison test. were measured after 4 h by ELISA. Data represent actual and mean values from four mice; *p , 0.05, Student t test. TLR ligation via a type I PI3K-independent mechanism (35). In this study, we observed a similar effect, although in our system, dependent pathway that occurs independently of TLR2 (37), and LY294002 did enhance IL-23 secretion, as did both 3-MA and a previous study has demonstrated that b-glucans can induce IL- siRNA knockdown of the autophagy proteins beclin 1 and Atg7. 23 via an IL-1–dependent mechanism (33). Our previous study has This effect was specific to IL-23; IL-12p70 secretion was inhibited demonstrated that pro–IL-1b colocalizes with GFP-LC3 in LPS- by 3-MA, whereas IL-12/23p40 levels were unaffected by auto- treated cells, and induction of autophagy, both in vitro and in vivo, phagy inhibition. has been shown to inhibit IL-1b secretion (10). In the current Our data suggest that autophagy has a largely indirect effect on study, we have demonstrated that although IL-23 is not seques- IL-23 through its regulation of IL-1b secretion. Previous studies tered by autophagosomes, induction of autophagy in vitro and have demonstrated that increased IL-1b secretion in autophagy- in vivo does inhibit IL-23 production, perhaps as a result of effects deficient cells is dependent on TRIF and ROS (10, 12). The present on IL-1b secretion. study suggests that increased IL-23 secretion in autophagy- Although neutralizing IL-1a had little or no effect on IL-23 se- deficient cells is similarly dependent on both ROS and TRIF. In- cretion, IL-1a did itself enhance IL-23 secretion. This suggests that terestingly, zymosan, which is not known to signal through TRIF IL-1R signaling is important but perhaps that IL-1a release in re- but signals through TLR2/MyD88 and the b-glucans sponse to 3-MA does not have consequences as significant as those Dectin-1 receptor (36), also acts synergistically with 3-MA to in- of IL-1b. Interestingly, a recent study has suggested that IL-1a se- duce IL-23. However, Dectin-1 also signals through a Syk kinase- cretion may also depend on IL-1b (38), further implicating IL-1b as The Journal of Immunology 4151

FIGURE 8. Autophagy-deficient DC stimulate gd T cells. (A–C) CD3+ T cells from spleens of female C57BL/6 mice were stimulated with supernatants from BMDC treated with LPS (10 ng/ml) and 3- MA (5 or 10 mM) or 3-MA alone for 72 h. Secretion of IL-17A (A), IL-17F (B), and IFN-g (C) was measured by ELISA. Data represent mean values + SEM (n = 6); *p , 0.05 compared with LPS only, one- way ANOVA with Bonferroni’s multiple comparison test. (D–F)SplenicCD3+ T cells from the same experiment were treated with recombinant murine IL-1b and IL-23, LPS (10 ng/ml), LPS + 3-MA (5 or 10 mM), or 3-MA alone. Secretion of IL-17A (D), IL-17F (E), and IFN-g (F) was Downloaded from measured by ELISA. Data represent mean values + SEM (n = 6); *p , 0.05 com- pared with control, one-way ANOVA with Bonferroni’s multiple comparison test. (G–I) Lymph node T cells were separated into gd+ (104 cells/ml; light bars) and gd- http://www.jimmunol.org/ (105 cells/ml; dark bars) populations and treated with the same BMDC supernatants described above. Secretion of IL-17A (G), IL-22 (H), and IFN-g (I) was measured by ELISA. Data represent mean values + SEM (n = 3); *p , 0.05 compared with LPS only, one-way ANOVA with Bonfer- roni’s multiple comparison test. (J–L) gd+ T cells from the same experiment were treated with recombinant murine IL-1b by guest on September 24, 2021 and IL-23 as a positive control. Secretion of IL-17A (J), IL-22F (K), and IFN-g (L) was measured by ELISA. Data represent mean values + SEM (n = 3); *p , 0.05 compared with control, one-way ANOVA with Bonferroni’s multiple comparison test.

a key driver of inflammation. In agreement with our findings, IL-1b Intriguingly, IL-1b, IL-1a, and IL-23 can all induce autophagy, alone induced IL-23 secretion by human PBMC, and secretion of IL- suggesting a potentially important negative feedback mechanism 23 in response to gliadin and b-glucans was also IL-1 dependent for the control of excessive inflammation. IL-23 is important (33). Our data suggest that IL-1 drives IL-23 (and TNF-a)secretion in driving intestinal inflammation in various mouse models of through NF-kB signaling. Indeed, the IL-23 p19 promoter contains inflammatory bowel disease (41–43). In humans, genome-wide a kB site, which is required for the induction of p19 transcription association studies have identified the IL-23R as an important (39), and IL-1R1 signaling activates NF-kB (40). Taken together, the correlate in Crohn’s disease (44). Moreover, polymorphisms in data presented in this article, coupled with previous studies, sug- ATG16L1, which encodes a protein required for autophagy, have gests that in the absence of autophagy, TRIF- and ROS-dependent been associated with susceptibility to Crohn’s disease (16, 45). inflammasome activation, and IL-1b secretion drives IL-23 pro- In a murine model of dextran sodium sulfate-induced colitis, loss duction and secretion in an NF-kB–dependent manner. It is notable of the autophagy protein Atg16L1 results in more severe disease, that in some cases the effects of 3-MA on IL-23 are nonlinear, with increased weight loss, enhanced colonic inflammation, and whereas those on IL-1b are linear at the dose range tested. This could decreased survival (12). The same study also demonstrated greatly be due to conflicting effects of 3-MA on autophagy and other PI3K- enhanced serum IL-1b levels in Atg16L1-deficient animals, al- dependent processes, although further studies would be required to though the effects on IL-23 were not examined. Thus, our data test this. Induction of autophagy, in contrast, leads to the seques- indicate that autophagy and IL-23 might be closely linked through tration and degradation of inflammasome components, pro–IL-1b, the autophagic control of IL-1b secretion in this model. and other inflammatory stimuli, leading to reduced IL-1b secretion gd T cells are a rapid and potent innate source of cytokines, and thus reduced IL-23 production. including IL-17, IL-22, and IFN-g. Although IL-17 and IFN-g are 4152 AUTOPHAGY REGULATES IL-23 AND T CELL RESPONSES thought to be key modulators of inflammation in disease models, 13. Zhou, R., A. S. Yazdi, P. Menu, and J. Tschopp. 2011. 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