Induced Autophagy −Γ Mediate IFN- Tryptophan Depletion and the Kinase GCN2

The Journal of Immunology

Tryptophan Depletion and the Kinase GCN2 Mediate IFN-g–Induced Autophagy

Sophie Fougeray,*,† Iadh Mami,*,† Gildas Bertho,†,‡ Philippe Beaune,*,†,x Eric Thervet,*,†,{ and Nicolas Pallet*,†

IFN-g is a master regulator of the immune responses that occur in the transplanted kidney, acting both on the immune system and on the graft itself. The cellular responses to IFN-g are complex, and emerging evidence suggests that IFN-g may regulate autophagic functions. Conversely, autophagy modulates innate and adaptive immune functions in various contexts. In this study, we identify a novel mechanism by which IFN-g activates autophagy in human kidney epithelial cells and provide new insights into how autophagy regulates immune functions in response to IFN-g. Our results indicate that IFN-g promotes tryptophan depletion, activates the eIF2a kinase general control nonderepressible-2 (GCN2), and leads to an increase in the autophagic flux. Further, tryptophan supplementation and RNA interference directed against GCN2 inhibited IFN-g–induced autophagy. This process is of functional relevance because autophagy regulates the secretion of inflammatory cytokines and growth factors by human kidney epithelial cells in response to IFN-g. These findings assign to IFN-g a novel function in the regulation of autophagy, which, in turn, modulates IFN-g–induced secretion of inflammatory cytokines. The Journal of Immunology, 2012, 189: 2954–2964.

he renal tubular epithelium plays a central role in the de- helps defend organisms against degenerative, inflammatory, in- velopment of kidney allograft structural deterioration, par- fectious, and neoplastic diseases (17). A primordial function of T ticularlybythegenerationofinflammatoryandprofibrogenic autophagy is the lysosomal degradation of cytoplasmic components mediators secreted in response to injury (1–5). The inflamed kidney in response to nutrient shortage. Autophagy also exerts numerous tissue is a highly immunogenic microenvironment that activates effects on the control of immunity and inflammation: autophagy is professional and nonprofessional APCs and triggers recipient T cell triggered by immune signaling molecules, and autophagy proteins activation and proliferation, ultimately leading to rejection, epithe- negatively regulate innate immunity functions such as inflamma- lium dedifferentiation, and fibrosis (6–9). IFN-g is a master regulator some activation, inflammatory cytokine production, and gene tran- of the homeostasis of the kidney transplant during rejection (8, 10). scription. Autophagy proteins also enhance adaptive immunity, IFN-g is a cytokine that is produced mostly by activated T cells and such as the development and homeostasis of the immune system NK cells and has complex effects on immune and nonimmune cells. andAgpresentation(18). IFN-g plays important roles in inflammation, making it particularly Autophagy and IFN-g interfere reciprocally. IFN-g promotes relevant to transplantation, with diverse and potentially contradictory autophagy in immune and nonimmune mice cells by mechanisms effects on organ allograft survival (11). IFN-g coordinates a diverse that involve Beclin-1 expression (19, 20), and autophagy is re- array of cellular programs through the transcriptional regulation of quired for IFN-g–mediated antimicrobial efficacy, a process that hundreds of genes (12). In the transplanted kidney, IFN-g protects involves immunity-related guanosine triphosphatases (21, 22). The epithelial cells from necrosis (13), regulates MHC expression and Ag mechanism by which IFN-g functions to activate autophagy in presentation by epithelial and endothelial cells (14), and induces human cells is not well understood, and whether IFN-g triggers tryptophan (Trp) metabolism (15). Emerging evidence suggests that autophagy in the kidney is currently unknown. In this study, we IFN-g signaling and autophagy interact together (16). provide evidence suggesting that autophagy is activated in human Macroautophagy (autophagy) is a major protective mechanism renal epithelial cells (HRECs) in response to IFN-g. Mechanis- that allows cells to survive in response to multiple stressors and tically, IFN-g induces Trp metabolism, which then activates the general control nonderepressible-2 (GCN2) kinase, leading to the phosphorylation of the eukaryotic translation initiation factor 2a *INSERM U775, Paris, France; †Universite Paris Descartes, Paris Sorbonne Cite, (eIF2a), an activator of autophagy. Conversely, Trp supplemen- Paris, France; ‡Unite´ Mixte de Recherche 8601, Centre National de la Recherche tation reduces the activation of the GCN2–eIF2a pathway and Scientifique, Paris, France; xPole de Biologie, Hopital Europeen Georges Pompidou, Paris, France; and {Service de Nephrologie, Hopital Europeen Georges Pompidou, inhibits autophagy. Further, targeting of GCN2 expression by RNA Paris, France interference also inhibits IFN-g–induced autophagy. The cellular Received for publication April 26, 2012. Accepted for publication July 16, 2012. impact of autophagy in response to IFN-g is due to its capacity to This work was supported by a grant from INSERM. dampen the IFN-g–induced cytokine secretion by HRECs. Address correspondence and reprint requests to Dr. Nicolas Pallet, INSERM U775, Centre Universitaire des Saints Peres, 45 Rue des Saints-Peres, 75006 Paris, France. E-mail address: [email protected] Materials and Methods Abbreviations used in this article: AO, acridine orange; eIF2a, eukaryotic translation Cell culture initiation factor 2a; ER, endoplasmic reticulum; GCN2, general control nonderepres- Normal HRECs were harvested from human nephrectomy specimens re- sible-2; HREC, human renal epithelial cell; NMR, nuclear magnetic resonance; PDGFB, platelet derived growth factor B; RPL13A, ribosomal protein L13A; RT- moved for renal cell carcinoma and were isolated according to previously qPCR, real-time quantitative PCR; siRNA, small interfering RNA; TOCSY, total published methods, with minor modifications (23, 24). Fragments of correlation spectroscopy; Trp, tryptophan. nontumoral renal cortex were minced and digested with collagenase IV (250 IU/ml) for 3 h at 37˚C. Cells were centrifuged, and the pellets were Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 washed three times with PBS. Cells were then cultured in DMEM con- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201214 The Journal of Immunology 2955 taining 5 mg/ml insulin, 10 mg/ml human apotransferrin, 500 ng/ml hy- no. ab50546) were from Abcam; anti–phospho-GCN2 (1:1000, no. AJ1318a), drocortisone, 10 ng/ml EGF, 6.5 ng/ml triiodothyronine, 5 ng/ml sodium anti-GCN2 (1:500, no. AP7130a) were from Abgent; and anti–b-actin selenite, 1% FCS, 25 IU/ml penicillin, 25 mg/ml streptomycin, and (1:1000, no. A2668) was from Sigma-Aldrich. Immunoblots were quan- 10 mmol/L HEPES buffer. Cells were then incubated at 37˚C in 5% CO2 tified using the ImageJ 1.44 software (http://imagej.nih.gov/ij). We used and 95% air. The characterization of this cellular model was performed a graphical method that involves generating lane profile plots, drawing lines by immunocytochemical peroxidase analysis and flow cytometry anal- to enclose peaks of interest, and then measuring peak areas (i.e., definite ysis (data not shown). Immunocytochemical analysis revealed a positive integrals). After background subtraction, a rectangular selection (region of staining for cytokeratin (clone AE1/AE3) and a6integrin(cloneNKI- interest) is made to enclose the first band, and the rectangular region of GoH3), two markers of epithelial cells, by the vast majority of cells, thus interest is moved over the adjacent lanes. Next, lane profiles plots are confirming their epithelial nature. Cytometry analysis showed the ab- generated, and the area measurements are obtained and compared between sence of staining for CD90 (clone 5E10), which is present on fibroblasts, conditions after having indexed them to the loading control. but not on tubular epithelial cells. These results confirmed the proximal descent of the vast majority of the cultured tubular epithelial cells. Immunofluorescence microscopy Experiments were not performed with cells beyond the third passage because it has been shown that no phenotypic changes occur up to this HRECs were cultured on glass coverslips and fixed with 4% paraformal- passage number. dehyde, rinsed with PBS, and blocked with 50 mM NH4Cl. Cells were We have tested various concentrations of IFN-g to define the working permeabilized with Triton X-100 and incubated with primary Abs (anti- concentration of 10 ng/ml (see Fig. 1E), which induced autophagy in LC3B [1:20, no. 2775] was from Cell Signaling Technology; anti–HLA- DM [1:50, no. sc-32248] was from Santa Cruz Biotechnology). Samples a more reproducible manner than 1 ng/ml, and we have tested various were then incubated with a cyanine 3- or FITC-coupled secondary Ab incubation times, which led us to demonstrate that after 48 h of incubation, the LC3II signal was maximal. (Jackson ImmunoResearch). Acridine orange (Sigma) was diluted in water and incubated in cell culture medium at a final concentration of 2.5 ng/ml Small interfering RNA transfections 2 h before the end of the experiment. Lysotracker Probe (Invitrogen) was directly incorporated in cell culture medium from the beginning of the BECN1, GCN2, and scramble (control) synthetic small interfering RNAs experiment at a 1 mM final concentration. Slides were mounted and viewed (siRNAs) were designed and obtained from Qiagen. Transfection using a Nikon Eclipse TE 2000E imaging fluorescence microscope. was performed using HiPerFect (Qiagen) following the manufacturer’s protocol. Nuclear magnetic resonance spectroscopy analysis of culture supernatants Electron microscopy The nuclear magnetic resonance (NMR) experiments were run at 500.13 For electron microscopy, samples were fixed in 2% glutaraldehyde–0.1 M MHz for 1H on a Bruker AVANCE 500 spectrometer with a 5-mm 1H/13C/15N sodium cacodylate, postfixed in 1% OsO4, dehydrated in alcohol, pro- TXI probe equipped with a z-gradient axis. The spectra of the different cessed for flat embedding in Epon 812, and observed with the Zeiss CEM culture media of the epithelial cells were measured after the addition of 5% 902 electron microscope. 2 (v/v) H2O in standard 5-mm sample tubes. All experiments were performed at 300 K using excitation sculpting water suppression (26) to RNA extraction and real-time quantitative PCR 2 eliminate solvent signal in an H2O/ H2O 95:5 solution. One-dimensional Total RNA was extracted using the RNeasy Mini Kit (Qiagen) following the spectra were measured with 512 scans. Two-dimensional NMR spectra manufacturer’s protocol. The yield and purity of the RNA were measured were detected in the phase-sensitive mode using the States–TPPI method using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies). (27). The two-dimensional correlated spectroscopy and the total correla- Transcript expression levels were quantified by SYBR Green real-time tion spectroscopy (TOCSY) spectra were recorded with 128 scans and 128 quantitative PCR (RT-qPCR) using an ABI PRISM 7900 sequence detector points in the indirect dimension. TOCSY spectra used an MLEV-17 spin- system (Applied Biosystems). Vehicle-treated samples were used as the lock sequence (28) with a mixing time (tm) of 70 ms. The heteronuclear spectra 1H-13C HSQC were recorded with 512 scans and 200 increments in controls, and fold changes for each tested gene were normalized to the ri- 13 bosomal protein L13A (RPL13A) housekeeping gene. The relative expres- the F1 dimension using natural C abundance. Chemical shift assignments sion levels were calculated using the 2(2ΔΔC(T)) (threshold cycle number) referred to internal 3-(trimethylsilyl) propionic acid-2,2,3,3-d4, sodium salt. method (25). For RT-qPCR, the following primers were used: Beclin-1 for- The signals of the compounds of interest were assigned and compared with ward (59-AGGTTGAGAAAGGCGAGACA-39), Beclin-1 reverse (59-AA- a sample of pure kynurenine and Trp measured under the same conditions. TTGTGAGGACACCCAAGC-39); CHOP forward (59-TGGAAGCCTGG- TATGAGGAC-39), CHOP reverse (59-TGTGACCTCTGCTGGTTCTG-39); Cytokine arrays GRP78 forward (59-GGTGAAAGACCCCTGACAAA-39), GRP78 reverse Multiple cytokine expression levels from conditioned media were simul- (59-GTCAGGCGATTCTGGTCATT-39); ICAM-1 forward (59-GAGATCA- taneously assayed by the protein array RayBio Human Cytokine Ab Array CCATGGAGCCAAT-39), ICAM-1 reverse (59-CTGACAAGTTGTGGGG- (RayBiotech). Subconfluent cells were grown in 6-well plates. Cytokine GAGT-39); IDO-1 forward (59-GGCACACGCTATGGAAAACT-39), IDO-1 expression was evaluated in the cell culture supernatant using the RayBio reverse (59-CGCTGTGACTTGTGGTCTGT-39); IP-10 forward (59-CCA- Human Inflammation Ab Array 3 (AAH-IFN-3) according to the manu- CGTGTTGAGATCATTGC-39), IP-10 reverse (59-CCTCTGTGTGGTCCA- facturer’s protocol. The signal intensities were quantified by densitometry TCCTT-39); PDGFB forward (59-CCGCCAGCGCCCATTTTTCA-39), PD- after background subtraction, and positive controls were used to normalize GFB reverse (59-CTTTGCAGCGAGGCTGGAGGG-39); RANTES forward the results from the different membranes being compared. Evaluations of (59-GCTGCAGTGAGCTGAGATTG-39), RANTES reverse (59-GCCAG- the relative cytokine expression levels were made by comparing the signal TAAGCTCCTGTGAGG-39); RPL13A forward (59-CCTGGAGGAGAAG- intensities between the different conditions. The following ratios of ex- AGGAAAGAGA-39), RPL13A reverse (59-GAGGACCTCTGTGTATTTG- pression were measured: 1) intensity in IFN-g–treated cells transfected TCAA-39); TNFa forward (59-TCCTTCAGACACCCTCAACC-39), TNFa with control siRNA/intensity in vehicle-treated cells transfected with reverse (59-CAGGGATCAAAGCTGTAGGC-39); sXBP1 forward (59-GCA- control siRNA (IFNg-control siRNA); and 2) intensity in IFN-g–treated GGTGCAGGCCCAGTTGT-39), sXBP1 reverse (59-TGGGTCCAAGTTG- cells transfected with BECN1 siRNA/intensity in vehicle-treated cells TCCAGAATGC-39). transfected with BECN1 siRNA (IFNg-BECN1 siRNA). Protein extraction and Western blot analysis Statistical analysis Total protein lysate from HRECs was separated by SDS-PAGE under de- All data are expressed as the means 6 SEM of three different experiments naturing conditions and transferred to a polyvinylidene fluoride membrane unless otherwise specified. Biological and histological data were compared (GE Healthcare). Primary Abs were visualized using HRP-conjugated poly- using Student test. Statistical analyses were performed using GraphPad clonal secondary Abs (Dako) and detected by ECL reagent (GE Health- Prism software. Calculated p values ,0.05 were considered significant. care). The following Abs were used: anti-LC3B (1:1000, no. 2775), anti-eIF2a (1:1000, no. 9722), anti–phospho-4E-BP1 (1:1000, no. 9459), anti–4E-BP1 (1:1000, no. 9452), anti–phospho-p70S6K (1:1000, no. Results 9204), anti–p70S6K (1:1000, no. 9202) were from Cell Signaling Tech- IFN-g activates autophagy in HRECs nology; anti-p62/SQSTM1 (1:500, no. sc-28359) was from Santa Cruz Biotechnology; anti–Beclin-1 (1:1000, no. NB5000-249) was from Novus HRECs that are exposed to IFN-g for 48 h accumulate cytoplasmic Biologicals; anti–phospho-eIF2a (1:500, no. ab32157), anti-ATF4 (1:500, vacuoles (Fig. 1A). To characterize the nature of these vacuoles, 2956 IFN-g–INDUCED AUTOPHAGY

FIGURE 1. IFN-g activates autophagy in HRECs. (A) IFN-g promotes the cytoplasmic accumulation of vacuoles. HRECs were incubated with 10 ng/ml IFN-g for 48 h or were left untreated and were analyzed by phase contrast microscopy. A representative photomicrograph of three independent experiments is shown. Scale bar, 20 mm. (B) IFN-g promotes the cytoplasmic accumulation of acidic vacuoles, which may be at least partly of a lysosomal nature. HRECs were incubated with 10 ng/ml IFN-g for 48 h or were left untreated. Cells were then stained with 2.5 ng/ml AO and analyzed by confocal microscopy (top) or were stained with 1 mM Lysotracker and analyzed by epifluorescence microscopy (bottom). Scale bar, 20 mm. A representative image of each staining of three independent experiments is shown. *p , 0.05. (C) IFN-g promotes the cytoplasmic accumulation of LC3-positive puncta. HRECs were incubated with 10 ng/ml IFN-g for 48 h or were left untreated and then stained with Abs to LC3. Cells were analyzed by epifluorescence microscopy. Scale bar, 10 mm. A representative image of three independent experiments is shown. *p , 0.05. (D) IFN-g induces HLA-DM expression, and LC3 and HLA-DM colocalize in IFN-g–treated HRECs. HRECs were transfected with siRNAs targeting BECN1 transcripts or control nontargeted (scramble) siRNAs. Twenty-four hours after transfection, HRECs were incubated with 10 ng/ml IFN-g for 48 h or were left untreated and then stained with Abs to LC3, HLA-DM, and DAPI. The cells were analyzed by confocal microscopy. Scale bar, 10 mm. Top, Representative images of three independent experiments are shown. Bottom, The intensity of the green and red fluorescence according to the distance between the two intensities of the fluorescence is represented. (E) IFN-g induces accumulation of LC3II in HRECs. HRECs were incubated with the indicated concentrations of (Figure legend continues) The Journal of Immunology 2957 we stained cells with acridine orange (AO). AO moves freely results demonstrate that the accumulation of autophagosomes in across biological membranes and accumulates in acidic compart- HRECsinresponsetoIFN-g results from an increased autophagic ments, such as lysosomes and autophagolysosomes, where it is flux. visualized as bright orange/red fluorescence. AO vital staining of IFN-g–treated cells shows the cytoplasmic accumulation of acidic IFN-g induces IDO expression and Trp metabolism organelles (Fig. 1B), which may be at least partly of a lysosomal We next attempted to gain mechanistic insights into IFN-g–induced nature because they also retained Lysotracker, a fluorescent dye autophagy in HRECs and reasoned that amino acid deprivation is that specifically stains lysosomes (Fig. 1B). The Atg8/LC3 protein a master inducer of autophagy (17) and that IFN-g promotes Trp associates with the membranes of autophagic structures and is depletion after IDO production (31). IDO is the first and rate- used to monitor autophagy through indirect immunofluorescence limiting enzyme of Trp catabolism through the kynurenine path- microscopy, in which autophagy is measured as an increase in way and causes the depletion of Trp. We confirmed that IFN-g punctate LC3 (29). IFN-g–treated HRECs accumulated LC3 puncta, promotes strong transcriptional induction of the gene encoding in contrast to vehicle-treated cells, suggesting that autophagosomes IDO (Fig. 2A). NMR spectroscopy analysis of the culture media accumulate in HREC cytoplasm under IFN-g exposure (Fig. 1C). of HRECs collected 48 h after IFN-g exposure showed that the Notably, the autophagosome marker LC3 colocalized with HLA- expression of IDO is accompanied by the catabolism of Trp, DM–positive compartments (Fig. 1D), suggesting that MHC class II which is characterized by Trp depletion and the production of loading compartments can obtain input from autophagosomes in kynurenine metabolites (Fig. 2B). Thus, IFN-g promotes Trp de- IFN-g–treated HRECs. IFN-g also increased the accumulation of pletion in HREC culture media. LC3II, the lipidated form of LC3, which is found specifically linked with autophagosome membranes. LC3 is initially synthesized in an IFN-g does not interfere with mTOR signaling unprocessed form, proLC3, which is converted into a form lacking mTOR is a central regulator of autophagic flux and is inhibited amino acids from the C terminus, LC3I, and is converted into the in response to amino acid deprivation (32, 33), which activates phosphatidylethanolamine-conjugated form, LC3II. LC3II is the autophagosome biogenesis (17). To test whether IFN-g inhibits only protein marker that is reliably associated with completed mTOR activity in our model, we analyzed the phosphorylation autophagosomes (Fig. 1E). Electron microscopy analysis confirmed status of two downstream targets of mTOR, p70S6K and 4E-BP1, that autophagosomes, vesicles limited by two parallel membrane in response to IFN-g. Whereas the mTOR inhibitor rapamycin bilayers separated by an electron-lucent cleft and that contain het- inhibited p70S6K phosphorylation and enhanced 4E-BP1 phos- erogenous materials formed by cytosol and organelles, accumulate phorylation, a paradoxical effect observed after long exposures to in the cytoplasm of HRECs exposed to IFN-g (Fig. 1F). Overall, rapamycin (34) (Fig. 3), we did not observe any modification of these data suggest that IFN-g promotes autophagosome and auto- the phosphorylation statuses of 4E-BP1 and p70S6K under IFN-g phagolysosome accumulation in HRECs. Notably, IFN-g did not exposure compared with the vehicle, findings that suggest that promote LC3II accumulation in HUVECs (data not shown), sug- IFN-g does not interfere with mTOR signaling in HRECs. gesting that the autophagic response to IFN-g is cell-specific. The cytoplasmic accumulation of autophagosomes can result IFN-g activates the eIF2a pathway from either an increased autophagic flux (i.e., increased auto- We next focused on other cellular nutrient status sensors that can be phagosome production, accumulation, and destruction) or from the activated in response to amino acid deprivation and can promote inhibition of the fusion between autophagosomes and lysosomes autophagy. The eIF2a signaling pathway is a well-characterized (29). Inhibition of lysosomal proteases by incubating HRECs with regulator of stress-induced translational control programs (35) E64 and pepstatin led to an increased accumulation of LC3II in activated during nutrient starvation (36, 37). The eIF2a signaling IFN-g–treated cells (Fig. 1G), suggesting that lysosomal functions pathway is also involved in the regulation of autophagy (38). In are intact under IFN-g exposure and that IFN-g does not inhibit our model, IFN-g promoted eIF2a phosphorylation (Fig. 4A) and the fusion of autophagosomes with lysosomes or their degrada- ATF4 protein expression (Fig. 4B) and C/EBP homologous pro- tion. We also monitored p62/sequestosome 1 expression as a marker tein (CHOP) transcription (Fig. 4C), suggesting that the eIF2a of increased autophagic flux (30). The p62 protein links unwanted pathway is activated in HRECs in response to IFN-g exposure. cytoplasmic cargos to LC3 and targets them for degradation in Because PERK, which is activated by endoplasmic reticulum (ER) autophagolysosomes, where they are degraded. Whereas p62 ex- stress, can activate the eIF2a pathway, we tested whether IFN-g pression increased when autophagy was inhibited by RNA inter- could promote ER stress in HRECs, as has been reported for ference against BECN1, the gene encoding Beclin-1, a central oligodendrocytes (39). IFN-g induced neither the expression of regulator of autophagy (Fig. 1H; see also Fig. 6A), its expression the spliced form of X-box binding protein-1 (XBP-1) mRNA nor was reduced in IFN-g–treated HREC (Fig. 1H), suggesting that the transcription of the chaperone glucose-related protein 78 IFN-g increases autophagic flux and p62 degradation. Overall, these (GRP78); these results indicate that the unfolded protein response

IFN-g or 100 nM rapamycin (Rapa), a known autophagy inducer, for 48 h (left) or with 10 ng/ml IFN-g for the indicated periods (right), and then whole- cell lysates were run on an SDS-PAGE gel. LC3I and LC3II protein expression was determined by anti-LC3 immunoblots. Actin blots show general protein amounts. Representative immunoblots of three independent experiments are shown. (F) IFN-g induces the accumulation of autophagosomes. HRECs were incubated with 10 ng/ml IFN-g for 48 h or were left untreated and then ﬁxed and embedded in epon. Cells were analyzed by electron microscopy. Black arrows denote autophagosomes. Scale bar, 250 nm. Images representative of three independent experiments are shown. (G) IFN-g activates autophagic ﬂux. HRECs were incubated with 5 mM E64 (cathepsin inhibitor) and 5 mg/ml pepstatin (acid proteases inhibitor) with or without 10 ng/ml IFN-g. Whole-cell lysates were run on a 12% SDS-PAGE gel, and LC3I and LC3II protein expression was determined by anti-LC3 immunoblots. Actin blots show general protein amounts. Left, A representative immunoblot of three independent experiments is shown. Right, The LC3II/b-actin ratio is presented as the mean 6 SEM of three independent experiments. *p , 0.05. (H) IFN-g reduces p62 accumulation. HRECs were transfected with siRNA targeting BECN1 transcripts or control nontargeting (scramble) siRNAs. Twenty-four hours posttransfection, HRECs were incubated with 10 ng/ml IFN-g for 48 h or were left untreated. Whole-cell lysates were run on an SDS-PAGE gel, and p62 protein expression was determined by anti-p62 immunoblots. Actin blots show general protein amounts. A representative immunoblot of three independent experiments is shown. 2958 IFN-g–INDUCED AUTOPHAGY

FIGURE 2. IFN-g induces IDO expression and Trp depletion. (A)IFN-g induces IDO transcript expression. HRECs were incubated with 10 ng/ml IFN-g for 48 h. IDO transcript levels were measured by RT-qPCR and are presented as the mean 6 SEM relative to the levels in untreated cells for three independent experiments. **p , 0.01. (B) Trp is metabolized during IFN-g treatment. HRECs were incubated with 10 ng/ml IFN-g for 48 h or were left untreated. Trp and kynurenines were detected in the culture medium by NMR spectroscopy. Top, A representative spectrum is displayed. Bottom,PurifiedL-Trp and L-kynurenine spectra are shown. The “IFNg-Ctrl” spectrum corresponds with the difference between IFN-g spectrum and Ctrl spectrum presented at the top. is not activated in our model (Fig. 4D). Overall, these findings and this phosphorylation was reversed by Trp supplementation suggest that the eIF2a signaling pathway is activated in response (Fig. 5A, 5B). Further, Trp supplementation reduced LC3II and to IFN-g independently of ER stress. LC3-positive puncta accumulation during IFN-g exposure (Fig. 5C, 5D), which suggests that the accumulation of autophago- Trp depletion mediates IFN-g–induced autophagy and somes in IFN-g–treated HRECs depends on Trp availability and activates the eIF2a kinase GCN2 might implicate the eIF2a kinase GCN2. Of note, L-kynurenine, Given that IFN-g promotes Trp depletion and activates the eIF2a the principal Trp metabolite product of IDO, did not activate pathway independently of ER stress, we tested whether Trp de- autophagy (data not shown). To examine the role of GCN2 in the pletion could activate the GCN2 kinase, an eIF2a kinase that is activation of autophagy in response to IFN-g and Trp depletion, activated by uncharged tRNAs in amino acid-starved cells (40). we inhibited GCN2 expression by siRNA-mediated RNA inter- GCN2 and eIF2a were phosphorylated in IFN-g–treated HRECs, ference (Fig. 5E). Inhibition of GCN2 expression significantly The Journal of Immunology 2959

of cytokines secreted by HRECs in response to IFN-g (the IFN-g– associated secretory phenotype) and compared this profile with that produced when the expression of BECN1 is inhibited. siRNA- mediated RNA interference directed against BECN1 inhibited Beclin-1 expression (Fig. 6A) and reduced LC3-positive puncta accumulation in response to IFN-g (Fig. 6B). The inhibition of BECN1 expression profoundly altered the IFN-g–associated secretory phenotype and resulted in the increased secretion of proinflammatory and immunostimulatory mediators, including IP- 10, MIP-1d, MCP-2, ICAM-1 (the secreted form), TNF-a,and profibrogenic cytokines such as TGF-b1 and platelet-derived FIGURE 3. IFN-g does not interfere with mTOR signaling. HRECs growth factor B (PDGFB) (Fig. 6C). Conversely, BECN1 inhibi- were incubated with 10 ng/ml IFN-g or 100 nM rapamycin or were left tion reduced the secretion of some cytokines, such as sTNFR I and untreated for 24 or 48 h. Whole-cell lysates were run on an SDS-PAGE gel, II, which act as TNF-a antagonists. This observation suggests that and the protein expression levels of phospho-4E-BP1, 4E-BP1, phospho- the activation of autophagy in HRECs in response to IFN-g could p70S6K, and p70S6K were evaluated by immunoblotting. A representative result in a reduction of the amplitude of the cytokinic secretory immunoblot of two independent experiments is shown. phenotype. The inhibition of BECN1 expression did not alter IP10, TNFa,ICAM1or PDGFB mRNA expression (Fig. 6D), which reduced the accumulation of LC3II in response to IFN-g (Fig. 5F), suggests that autophagy may regulate cytokine production at the suggesting that GCN2 regulates autophagy in HRECs in response posttranscriptional level. to IFN-g. Overall, our results demonstrate that the activation of autophagy in response to IFN-g is promoted by Trp depletion Discussion and relies, at least in part, on the activation of the GCN2–eIF2a Accumulating evidence indicates that autophagy plays a critical pathway. role in kidney maintenance, disease, and aging. Ischemic, toxic, immunological, and oxidative insults can cause an induction of Autophagy interferes with IFN-g–associated secretory autophagy in renal epithelial cells, modifying the course of various phenotype kidney diseases (41–44). The effects of autophagy on the reg- We next tested whether autophagy could interfere with relevant ulation of innate and adaptive immunity and cell viability are IFN-g–related immune functions in HRECs. Although they are particularly relevant in transplanted tissue, which faces a great nonprofessional immune cells, kidney epithelial cells can secrete number of stresses that challenge its viability and immunogenicity numerous proinflammatory cytokines when the kidney tissue is (9). In this study, we have identified a new mechanism by which injured (1, 2), and cytokine release can amplify inflammation and IFN-g promotes autophagy in the human kidney epithelium and facilitate adaptive immunity. We generated an expression profile demonstrated that autophagy modulates IFN-g–induced immune

FIGURE 4. IFN-g activates the eIF2a pathway. (A) IFN-g induces eIF2a phosphorylation. HRECs were incubated with 10 ng/ml IFN-g for 24 or 48 h or were left untreated. Whole-cell lysates were run on an SDS- PAGE gel, and the protein expression levels of phospho-eIF2a and eIF2a were evaluated by immunoblotting. Left, A representative immunoblot of three independent experiments is shown. Right,Densitometric analysis of phospho-eIF2a/eIF2a of three independent immunoblots. *p , 0.05. (B)IFN-g increases ATF4 expression. HRECs were incubated with 10 ng/ml IFN-g for 24 or 48 h or were left untreated. Whole-cell lysates were run on an SDS-PAGE gel, and ATF4 expression was evaluated by anti-ATF4 immunoblotting. A representative immunoblot of three independent experiments is shown. (C)IFN-g induces CHOP mRNA expression. HRECs were incubated with 10 ng/ml IFN-g for 24 or 48 h or were left untreated. CHOP transcript levels were measured by RT-qPCR and are presented as the mean 6 SEM relative to the transcript levels in untreated cells for three independent experiments. *p , 0.05. (D)IFN-g does not promote XBP-1 splicing and GRP78 mRNA expression induction. HRECs were incubated with 10 ng/ml IFN-g for 24 and 48 h or with 2 mg/ml tunicamycin (“Tunica”; a protein glycosylation inhibitor known to induce ER stress) for 24 h or were left untreated. Spliced XBP1 and GRP78 transcript levels were measured by qRT-PCR and are presented as the means 6 SEM relative to levels in untreated cells for three independent experiments. 2960 IFN-g–INDUCED AUTOPHAGY

FIGURE 5. Trp depletion mediates IFN-g–induced autophagy and activates the eIF2a kinase GCN2 . (A) Trp supplementation reduces GCN2 phosphorylation. HRECs were incubated with 10 ng/ml IFN-g alone or with 80 mg/l Trp for 48 h or were left untreated. Whole-cell lysates were run on an SDS- PAGE gel, and the protein expression levels of phospho-GCN2 and GCN2 were evaluated by immunoblotting. Left, An immunoblot representative of three independent experiments is shown. Right, Densitometric analysis of phospho-GCN2/GCN2 of three independent immunoblots. *p , 0.05. (B) Trp supplementation reduces eIF2a phosphorylation. HRECs were incubated with 10 ng/ml IFN-g alone or with 80 mg/l Trp for 48 h or were left untreated. Whole-cell lysates were run on an SDS-PAGE gel, and the protein expression levels of phospho-eIF2a and eIF2a were evaluated by immunoblotting. An immunoblot representative of three independent experiments is shown. The phospho-eIF2a/eIF2a ratio is presented as the mean 6 SEM of three independent experiments. *p , 0.05. (C) Trp supplementation reduces LC3II accumulation. HRECs were incubated with 10 ng/ml IFN-g alone or with 80 mg/l Trp for 48 h or were left untreated. Whole-cell lysates were run on an SDS-PAGE gel, and the LC3I and LC3II protein expression levels were determined by anti-LC3 immunoblots. Actin blots show general protein amounts. Left, A representative immunoblot of three independent experiments is shown. Right, The LC3II/b-actin ratio is presented as the mean 6 SEM of three independent experiments. *p , 0.05. (D) Trp supplementation reduces LC3-positive puncta accumulation. HRECs were incubated with 10 ng/ml IFN-g alone or with 80 mg/l Trp for 48 h or were left untreated and then were stained with Abs to LC3. Cells were analyzed by epifluorescence microscopy. Scale bar, 10 mm. Images representative of three independent (Figure legend continues) The Journal of Immunology 2961 responses in HRECs. We provide evidence that the activation of transplant damage, which depend on the nature, the timing of autophagy by IFN-g in human epithelial cells relies on Trp de- occurrence, and the intensity of the injury, remain to be charac- pletion and the activation of the GCN2–eIF2a pathway. Our terized. results also show that autophagy dampens the IFN-g–induced Our findings underscore the importance of amino acid avail- secretion of cytokines by HRECs. ability in the regulation of immune functions. The immunoreg- Our results do not exclude that other mechanisms might be ulatory functions of the local metabolism of Trp, and to a lesser involved in the regulation of autophagy by IFN-g. In mice, one extent L-arginine, are well known (51). Trp depletion is tolero- immunity-related GTPase protein in particular, Irgm1, has been genic, as it inhibits Th cell proliferation and promotes regulatory shown to exert critical functions in IFN-g–induced autophagy. T cell amplification, and kynurenines, the Trp metabolites, pro- However, the human ortholog of Irgm1, IRGM, is not elicited by mote T cell death. IDO-mediated Trp depletion can activate IFN-g. Data regarding the regulation of autophagy in human cells GCN2 in T cells, leading to proliferative arrest, anergy, and are very scarce. A very recent study demonstrated that IFN-g in- regulatory T cell production (52, 53). The fact that IFN-g–induced duces autophagy and does so independently of Irgm1 described Trp depletion promotes autophagy expands the spectrum of the earlier. This novel pathway used JAK1/2 and p38 MAPK signaling immunoregulatory properties of IDO-induced Trp depletion. In- but does not require STAT1 (45). An in vivo model of IFN-g– deed, autophagy proteins function in the inactivation of immune mediated inflammatory renal injury, like allograft rejection, in signaling by negatively regulating inflammasome activation, NF- autophagy defective mice would be helpful to understand better kB signaling, and inflammatory cytokine production (54). In line the interplay between IFN-g and autophagy. with our findings showing that autophagy dampens IFN-g–induced The eIF2a-signaling pathway is an important regulator of inflammatory cytokine secretion, mice kidneys in which ATG5 has autophagy (17). The mechanisms by which eIF2a activates been selectively knocked out in the tubular epithelium develop autophagy involve the isolation of ER membrane and the induc- a more severe inflammation in response to ischemia–reperfusion tion of the expression of the transcription factors ATF4 and CHOP, injury than their wild-type counterparts (55). A particularly im- which in turn promote the expression of the autophagy genes LC3 portant step will be to reconcile the apparently opposite con- and ATG5 (46, 47). There are four eIF2a kinases in mammals: sequences of autophagy and Trp depletion on innate immunity and PERK; PKR, which is activated by dsRNA during viral infection; adaptive immunity, as autophagy also facilitates adaptive functions HRI, which is activated by iron deficiency in erythrocytes (48); including Ag presentation, thymic education, and lymphocyte pro- and GCN2. IFN-g can regulate the expression or the activity of liferation (18). PERK, PKR, and GCN2. IFN-g induces ER stress and activates p62 is an important signaling adapter protein that interacts PERK through ill-defined mechanisms that involve the increased with TNF receptor-associated factor 6 and promotes NF-kBacti- synthesis of membrane-spanning proteins (39). PKR expression vation (30). p62 is targeted to be degraded by autophagy during is induced by IFN-g, but in an inactive form, and requires viral stressful conditions (56). Consequently, autophagy defects result dsRNA to be activated (12). GCN2 is activated when the level of in NF-kB induction. In other words, autophagy is required to sup- any amino acid, including Trp, diminishes sufficiently to cause the press p62 accumulation and inappropriate activation of NF-kB. accumulation of uncharged tRNAs, which are direct activators of This corroborates with our findings, which demonstrate that in the kinase (37, 40). Because ER stress occurs in the transplanted conditions of impaired autophagy, the secretion of cytokines in kidney (49), there is little doubt that the activation of PERK, in response to IFN-g is higher than in condition of nonaltered auto- addition to GCN2, also promotes autophagy in the injured allo- phagy, which indicates that the degradation of p62 by IFN-g–in- graft (44). duced autophagy could reduce NF-kB signaling and dampen The fact that Trp depletion does not interfere with mTOR secretion of cytokines. Conversely, when autophagy is altered (e.g., signaling suggests that mTOR and GCN2 are nutrient availability when Beclin-1 is inhibited by RNA interference), NF-kB could be sensors with different levels of sensitivity and/or specificity. activated and the secretion of cytokines increased. Whereas intracellular amino acid depletion is sensed directly by Recent results indicate a novel role for autophagy in noncon- GCN2, which binds uncharged tRNAs and can thus theoretically ventional secretion pathways of molecules, including cytokines detect deficiencies in any essential amino acid or nonessential (57–60), which does not involve lysosomal degradation of auto- amino acid (50), the regulation of mTORC1 is most responsive phagosomal contents but instead involves their redirection toward to specific individual amino acids such as leucine and arginine the extracellular delivery. Autophagy is involved in mounting (32), which diminish the capacity of Rheb to bind and activate intercellular communication networks, which could be of broad mTOR (33). However, other factors related to kidney trans- immunological importance (57, 60). For example, autophagy is plantation can interfere with mTOR signaling, including the essential for the immunogenic release of ATP from dying cells use of the immunosuppressive drug rapamycin, and one cannot (60), which will activate purinergic P2RX7 receptors and will exclude the possibility that the inhibition of mTOR signaling promote the production of IL-1b by dendritic cells. Conversely, could be an additive process that contributes to macroautophagy our results indicate that autophagy negatively regulates inflamma- in the transplanted kidney. Therefore, the respective contributions tory cytokine secretion in response to IFN-g, which corroborates of the various inducers of macroautophagy occurring during with previous studies demonstrating that autophagy reduces IL-

experiments are shown. *p , 0.05. (E) Inhibition of GCN2 expression by siRNA-mediated RNA interference. HRECs were transfected with siRNA targeting GCN2 transcripts or control nontargeting (scramble) siRNAs. Twenty-four hours posttransfection, the GCN2 and phospho-GCN2 protein levels were measured by anti-GCN2 and anti–phospho-GCN2 immunoblots. A representative immunoblot of three independent experiments is shown. (F) The inhibition of GCN2 expression reduces LC3II accumulation. HRECs were transfected with siRNAs targeting GCN2 or control, nontargeted (scramble) siRNAs. Twenty-four hours after transfection, HRECs were incubated with 10 ng/ml IFN-g for 48 h or were left untreated. Whole-cell lysates were run on an SDS-PAGE gel, and LC3I and LC3II protein expression levels were determined by anti-LC3 immunoblots. Actin blots show general protein amounts. Left, A representative immunoblot of three independent experiments is shown. Right, The LC3II/b-actin ratio is presented as the mean 6 SEM of three independent experiments. *p , 0.05. 2962 IFN-g–INDUCED AUTOPHAGY

FIGURE 6. Autophagy interferes with the IFN-g–associated secretory phenotype. (A) Inhibition of BECN1 expression by siRNA-mediated RNA interference. HRECs were transfected with siRNA targeting BECN1 transcripts or control nontargeting (scramble) siRNAs. Twenty-four hours posttransfection, the Beclin-1 protein level was measured by anti–Beclin-1 immunoblots. A representative immunoblot of three independent experimentsis shown. (B) Inhibition of BECN1 expression reduces the cytoplasmic accumulation of LC3-positive puncta. HRECs were transfected with siRNA targeting BECN1 transcripts or control nontargeting (scramble) siRNAs. Twenty-four hours after transfection, HRECs were incubated with 10 ng/ml IFN-g for 48 h or were left untreated and then were stained with Abs to LC3. Cells were analyzed by epiﬂuorescence microscopy. Scale bar, 5 mm. Images representative of three independent experiments are shown. *p , 0.05. (C) BECN1 expression inhibition modiﬁes the IFN-g–associated secretory phenotype. HRECs were transfected with siRNAs targeting BECN1 or control, nontargeted (scramble) siRNAs. Twenty-four hours after transfection, HRECs were incubated with 10 ng/ml IFN-g for 48 h or were left untreated. Culture media were analyzed by Human Cytokine Ab Arrays. The cytokines for which expression levels were detectable compared with negative controls are shown. For each protein, the ratio between expression levels in IFN-g–treated and vehicle-treated cell was calculated. The baseline represents the averaged signals from vehicle-treated conditions. Signals above the baseline are yellow; signals below the baseline are blue. The heat map key shows fold changes from baseline. One of two representative experiments is shown. *p , 0.05. (D) BECN1 expression inhibition does not modify cytokine transcript expression. HRECs were transfected with siRNAs targeting BECN1 or control, nontargeted (scramble) siRNAs. Twenty-four hours after transfection, HRECs were incubated with 10 ng/ml IFN-g for 48 h or were left untreated. IP10, ICAM1, PDGFB, and TNFa transcript levels were measured by RT-qPCR and are presented as the means 6 SEM relative to the levels in untreated cells for three independent experiments.

1b production, as a consequence by a mechanism that involves of therapeutic targeting of autophagic processes to modulate the the destruction of inflammasomes and altered mitochondria evolution of active kidney allograft deterioration. (61, 62). In conclusion, we have identified a new mechanism by which Acknowledgments IFN-g, a master regulator of kidney allograft injury, activates We thank Dr. Jean-Pierre Denizot (Unite´ de Neurosciences, Information autophagy in kidney epithelial cells, and our study provides new et Complexite´, Centre National de la Recherche Scientifique, Gif-sur- insights into how autophagy regulates immune functions in re- Yvette, France) for technical assistance in performing the electron micros- sponse to IFN-g. It might be useful to investigate the potential copy study. The Journal of Immunology 2963

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