IRF8 Directs Stress-Induced Autophagy in Macrophages and Promotes Clearance of Listeria Monocytogenes

ARTICLE

Received 21 May 2014 | Accepted 23 Jan 2015 | Published 16 Mar 2015 DOI: 10.1038/ncomms7379 IRF8 directs stress-induced autophagy in macrophages and promotes clearance of Listeria monocytogenes

Monica Gupta1, Dong-Mi Shin2,3, Lakshmi Ramakrishna1, Dennis J. Goussetis4, Leonidas C. Platanias4,5, Huabao Xiong6, Herbert C. Morse III2 & Keiko Ozato1

Autophagy, activated by many stresses, plays a critical role in innate immune responses. Here we show that interferon regulatory factor 8 (IRF8) is required for the expression of autophagy-related genes in dendritic cells. Furthermore in macrophages, IRF8 is induced by multiple autophagy-inducing stresses, including IFNg and Toll-like receptor stimulation, bacterial infection, starvation and by macrophage colony-stimulating factor. IRF8 directly activates many genes involved in various steps of autophagy, promoting autophagosome formation and lysosomal fusion. Consequently, Irf8 À / À macrophages are deﬁcient in autophagic activity, and excessively accumulate SQSTM1 and ubiquitin-bound proteins. We show that clearance of Listeria monocytogenes in macrophages requires IRF8-dependent activation of autophagy genes and subsequent autophagic capturing and degradation of Listeria antigens. These processes are defective in Irf8 À / À macrophages where uninhibited bacterial growth ensues. Together these data suggest that IRF8 is a major autophagy regulator in macrophages, essential for macrophage maturation, survival and innate immune responses.

1 Program in Genomics of Differentiation, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA. 2 Laboratory of Immunopathology, NIAID, National Institutes of Health, 5640 Fishers Lane, Room 1421, Rockville, Maryland 20852, USA. 3 Department of Food and Nutrition, Seoul National University, Seoul 151-742, Korea. 4 Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, Illinois 60611, USA. 5 Division of Hematology-Oncology, Jesse Brown VA Medical Center, Chicago, Illinois 60612, USA. 6 Immunology Institute, Mount Sinai School of Medicine, New York, New York 10029, USA. Correspondence and requests for materials should be addressed to K.O. (email: [email protected]).

NATURE COMMUNICATIONS | 6:6379 | DOI: 10.1038/ncomms7379 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7379

utophagy is a catabolic process by which misfolded self- WT and Irf8 À / À mice on stimulation by TLR ligands, proteins and damaged organelles are captured and lipopolysaccharide (LPS) and CpG. With a cutoff line of 42 Â Aenzymatically degraded1,2. Autophagy is activated by a with Pr0.05 (identified by one-way analysis of variance), 326 variety of stress signals such as starvation and inflammation1.In and 713 genes were expressed higher in WT DCs than in Irf8 À / À macrophages (M^s) and dendritic cells (DCs), autophagy is also DCs in untreated (UT) and TLR-stimulated DCs, respectively triggered by inflammatory cytokines such as interferong (IFNg), (Fig. 1a, left), whereas the expressions of 350 and 648 genes were ligands for Toll-like receptors (TLRs) and other pathogen lower in WT DCs than in Irf8 À / À DCs (Fig. 1a, right). Thus, recognition receptors and is required for innate clearance of IRF8 regulates many constitutive and TLR-stimulated genes in invading pathogens3–6. The absence of autophagy genes such as DCs either positively or negatively, as reported before for other Atg5 and Atg7 impairs pathogen recognition activity, type I IFN cell types21,27,28. Gene ontology (GO) analysis of positively production and increases susceptibility to pathogens, including regulated genes showed significant enrichment for immune Mycobacterium (M) tuberculosis and Listeria monocytogenes7–10. system processes, inflammatory responses, lysosome functions, Recent studies indicate that autophagy is involved in M-CSF- while genes negatively regulated by IRF8 were enriched with cell induced differentiation of monocytes to M^s11,12. cycle, cell division and DNA replication (Supplementary Autophagy follows a well-ordered sequence of events where the Fig. 1a,b). A large number of TLR-stimulated genes were activation of ULK1 and Beclin1 leads to LC3 lipidation and upregulated by IRF8, consistent with previous reports that IRF8 autophagosome formation. Autophagosomes then fuse with is critical for TLR activation of DCs (Supplementary Fig. 1c and lysosomes to allow the hydrolysis of captured materials. Supplementary Table 1)18,20,29. Eleven percent of those were Autophagic degradation is linked to the ubiquitin conjugation found in the Interferome, confirming a functional link between pathway, as some ubiquitinated proteins are escorted to IRF8- and IFN-related regulation (http://interferome.org/)30 autolysosomes through ubiquitin adaptors such as SQSTM1 (Supplementary Fig. 1d). (p62) to allow proteasome independent degradation10,13,14. Inspection of IRF8-stimulated genes belonging to the category IFNg-induced GTPases, such as IRGM1, are recruited to the of immune system processes and lysosome functions autolysosomes and facilitate autophagic clearance of bacteria15. (Supplementary Fig. 1a) revealed that a number of genes in the It is thought that autophagic activation and the subsequent autophagy pathway are downregulated in Irf8 À / À DCs (Fig. 1b elimination of captured molecules are directed by a mechanism and Supplementary Table 2). These genes, except for Atg2a, were that unifies and coordinates complex autophagic processes1. induced after TLR, as confirmed by quantitative reverse Although our understanding on this aspect of autophagy is transcription-PCR (qRT–PCR) analysis (Fig. 1c). Because IRF8 limited, there are a few examples where autophagic events are regulates shared sets of genes in DCs and M^s and autophagy joined together as a defined network: in these cases, transcription has been extensively investigated in M^s, we hereafter studied factors, such as TEFB and FOXO3 act as autophagy master the role of IRF8 in M^ autophagy. The expressions of 24 regulators in a cell type- and signal-dependent manner16,17. autophagy genes were first tested in BM-derived M^s from WT Interferon regulatory factor 8 (IRF8) is a transcription factor and Irf8 À / À mice stimulated with IFNg overnight followed by that promotes the differentiation of M^s and DC subsets18,19. TLR ligands, LPS and CpG: IFNg and TLR ligands provide IRF8 is induced by IFNg plus TLRs in M^s and stimulates genes priming and triggering signals, respectively, which together boost important for host defense, including type I IFNs, promoting the autophagic activity in M^s31,32. Data in Fig. 1d showed that 17 production of reactive oxygen species and nitric oxide20,21. IRF8 autophagy genes were downregulated in Irf8 À / À M^s relative to is essential for innate resistance against intracellular bacteria, WT M^s and that all but Atg7 were induced by IFNg plus TLR including M. tuberculosis, M. Bovis (BCG), Salmonella and (IFNg/TLR) stimulation. Atg7 expression, which fell after Listeria, although underlying mechanisms have not been fully stimulation, was also consistently lower in Irf8 À / À M^s than understood22–26. WT cells. These genes represent essentially all phases of We show here that IRF8 is induced in M^s and DCs by diverse autophagy (see Fig. 1d, left). As reported previously, IFNg/TLR stresses that activate autophagy and stimulates transcription of treatment also stimulated Irf8 expression18,19. The remaining many autophagy genes, thereby facilitating the entire autophagic seven genes were not induced by IFNg/TLR and did not differ in processes. Accordingly, Irf8 À / À M^s are defective in various WT and Irf8 À / À M^s (Supplementary Fig. 2). These results steps of autophagy induced by IFNg/TLR stimulation, Listeria indicate that many autophagy genes are induced by IFNg/TLR infection, starvation and macrophage colony-stimulating factor stimulation in an IRF8-dependent manner. (M-CSF). As a result, ubiquitin-linked SQSTM1 accumulates in Rescue experiments in Fig. 1e showed that transfer of the Irf8 greater amounts in Irf8 À / À M^s than wild-type (WT) M^s. gene into Irf8 À / À M^s restored the expression of 10 autophagy Further, infection by Listeria monocytogenes leads to a dramati- genes on IFNg/TLR stimulation. As expected, Irf8 transfer did not cally induced IRF8 that coincides with marked activation of restore the expressions of these genes in unstimulated M^s. The multiple autophagy genes, which results in autophagic control of transcriptionally defective mutant, Irf8K79E, in contrast, failed to bacterial growth. Irf8 À / À M^s are, however, unable to activate rescue these autophagy genes. It is of note that Irf8 transfer did autophagy genes and succumb to uninhibited Listeria growth. not rescue all 17 genes, which may be attributed to insufficient Transfer of the Irf8 gene into Irf8 À / À M^s partially rescues the levels of IRF8 expression, insufficient post-translational changes expression of autophagy genes and autophagic activity in Listeria in IRF8 proteins or other mechanisms. infected M^s. Together, IRF8 is an autophagy master regulator that acts in MFs to meet diverse stresses. IRF8 binds to and stimulates autophagy genes in M^s. Seven of the 17 autophagy genes upregulated by IRF8 carried Results IRF8-binding motifs within the 3.5-kb upstream promoter Microarray analyses reveal a role of IRF8 in autophagy. region (Fig. 2a)19,33. We performed qPCR-based chromatin Previous genome-wide studies reported that IRF8 regulates more immunoprecipitation (ChIP) analysis to test the binding of IRF8 than 1,500 genes in monocytes, M^s and B cells21,27,28. To gain to these genes in M^s. As shown in Fig. 2b, IRF8 bound to all genome-wide information on IRF8 in DCs, we performed seven genes in WT M^, but not in Irf8 À / À M^s. IRF8 binding microarray analyses with bone marrow (BM)-derived DCs from increased after IFNg/TLR stimulation for most of the genes,

2 NATURE COMMUNICATIONS | 6:6379 | DOI: 10.1038/ncomms7379 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7379 ARTICLE consistent with enhanced mRNA expression after stimulation fluorescent protein (EGFP)-LC3B (Fig. 3a). This vector detects (Fig. 1d). Consistent with the expression data, Atg7 showed high acid-sensitive (EGFP) and -resistant (mCherry) LC3, enabling us IRF8 binding in UT WT M^s, and the expression fell slightly to assess the formation of autophagosomes and the subsequent after stimulation. Figure 2c summarizes the data for mRNA fusion with lysosomes34. Before stimulation, GFP and mCherry expression, rescue by IRF8 and ChIP assay, illustrating that IRF8 signals were diffusely distributed over the cytoplasm both in stimulates transcription of many autophagy genes constitutively WT and Irf8 À / À M^s. After IFNg/TLR stimulation, GFP and after IFNg/TLR stimulation. and mCherry signals relocalized to form prominent punctate structures representing autophagosomal vesicles in WT cells (see Irf8 À / À M^s are defective in IFNc/TLR-induced autophagy. arrows in Fig. 3a). In contrast, few fluorescent vesicles were seen To test whether defective gene expression in Irf8 À / À M^s in Irf8 À / À M^s. Quantification in Fig. 3a (lower panel) affects autophagic functions, we next examined the autophago- confirmed that the number of cells with fluorescent vesicles was some formation in M^s expressing mCherry-enhanced green much fewer in Irf8 À / À M^s. Deficiency in Irf8 À / À M^sto

a Positively regulated Negatively regulated UT TLR UT TLR N=326 N=713 N=350 N=648

239 87 626 274 76 572

b WT Irf8–/– c –/– UT TLR UT TLR WT Irf8 Irf8 UT TLR UT TLR Atg16l1 Irf8 3.2E–03 8.3E–02 1.3E–05 9.4E–06 Atg16l1 1.5E–02 3E–02 5E–03 6E–03 Atg2a Atg2a 4E–03 1E–03 2E–03 1E–03 Atg4b Atg4b 6E–03 1E–02 2E–03 6E–03 Atg4d Atg4d 5E–03 2E–02 5E–03 2E–03 Becn1 1.5E–01 3E–01 8E–03 1E–01 Becn1 Ctsc 1.2E–01 2.5E–01 7E–02 1.2E–01 Ctsc Rab8b 2E–01 7E–01 4E–02 1E–01 Rab8b Min Mid Max 1.5 1:1 1.5 P-0.05 deWT Irf8 –/– Irf8 –/– UT 3 h 6 h 10 h UT 3 h 6 h 10 h Untreated IFNγ/TLR Irf8 1 4.5 188.8 0 0 0 0 Ulk1 1 0.7 2.2 11 0.5 0.6 1 2 Initial activation Atg13 1 3.3 0.5 0.4 0.1 0.10 0.1 Control IRF8 IRF8K79EControl IRF8 IRF8K79E Becn1 1 13 8.9 8.6 0.7 444.5 Irf8 000.3 0.2 0.5 0.5 Atg12 conjugation Atg16l1 1 2.6 3.2 6 0.6 0.9 2 1.3 ll12b 1 2 1.2 1 4.6 1.1 Atg7 1 0.4 0.2 0.4 0.5 0.2 0 0.1 Ulk1 1 2.5 1.5 1 5.1 2 Map1lc3b 1 1.7 2.8 0.4 0.3 0.2 1 0.8 Atg13 1 0.7 0.6 1 6.7 1.7 Gabarap 1 2 1.3 1 0.6 1 1 0.2 Atg16l1 1 1.3 1.4 1 7.1 1.8 Atg8/LC 3 conjugation Gabarap12 1 1.6 0.6 0.6 0.5 0.4 0 0.3 Atg7 1 1.1 1.2 1 7.2 2 Atg4d 1 7.7 12 17 1.8 2.6 4 2 Gabarap12 1 0.7 0.9 1 2.1 1.2 Rab5a 1 2.6 14 10 0.5 2.9 31.8 Gabarap 1 0.8 0.7 1 2.6 0.8 Rab9a 1 3.1 2.6 1.9 1.1 1.4 1 0.5 Atg4d 1 0.2 0.6 1 5 0.8 Rab10 1 3.7 4.1 3 0.8 1.2 1 0.9 lrgm1 1 0.7 1.2 1 5.5 1.2 Formation and maturation Rab8b 1 7.2 3.8 3.4 0.6 0.6 0 0.3 Lamp2 1 0.4 0.5 1 3.2 1.1 of autolysosome Ctsc 1 1.2 1 1 6.2 1 lrgm1 1 18 10 5.9 1.8 5.9 6 1.6 Hprt 110.5 0.4 0.6 0.5 Lamp2 1 1.4 5.2 0.9 0.3 0.3 1 0.6 Cst3 1 12 4.4 4 0.8 1.3 1 0.9 Min Mid Max Lysosomal enzyme Ctsc 13.42.6 1.9 1.1 1.4 1 0.5

Min Mid Max Figure 1 | Microarray analyses reveal a role of IRF8 in autophagy. (a) The Venn diagrams depict the number of genes positively and negatively regulated by IRF8 in UT DCs and those treated with TLR ligands for 6 h identified by microarray analysis. Overlapped regions represent the number of genes positively or negatively regulated both in UT and TLR-stimulated DCs. See GO classification in Supplementary Fig. 1a,b. (b) IRF8-dependent autophagy genes identified by microarray analysis. Differentially expressed genes were identified by one-way analysis of variance (Pr0.05 and fold change Z2). Colour gradients indicate average signal intensities of genes in log2 scale. Normalization was performed by GeneChip Operating Software (GCOS) and Expression Console software’s relative to the UT. (c) qRT–PCR analysis of IRF8-dependent autophagy genes in DCs. WTand Irf8 À / À DCs were stimulated with the TLR ligands for 6 h. The numbers represent transcript levels normalized by gapdh levels. Data represent the average of three independent assays. Pr0.01, Student’s t-test. (d) qRT–PCR analysis of IRF8-dependent autophagy genes in MFs. WT and Irf8 À / À MFs were treated overnight with IFNg and stimulated with TLR ligands for the indicated times. The number in each box represents transcript levels normalized by the value of UT WTcells. Values are the average of three independent assays. Pr0.01 (Student’s t-test). See autophagy genes not affected by IRF8 in Supplementary Fig. 2. (e) Irf8 À / À M^s were transduced with pMSCV retroviral vector containing WT Irf8 or mutant Irf8 (K79E), and stimulated with IFNg/TLR for 8 h. Relative expression of indicated autophagy genes was detected by qRT–PCR. The numbers represent transcript levels normalized by those of cells transduced with empty vector. Irf8 expression was normalized by gapdh. Values are the average of three experiments. Pr0.05 (Student’s t-test). Il12b and Hprt were tested as controls.

NATURE COMMUNICATIONS | 6:6379 | DOI: 10.1038/ncomms7379 | www.nature.com/naturecommunications 3 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7379

a b WT lrf8 –/– 6 Map1lc3b * 6 Gabarap Gene Location from transcription start site (TSS) Motif 5 5 ** Atg7 –1,240 –1,220 IECS 4 4 Map1lc3b –1,680 –1,677 IECS 3 3 Gabarap –525 –503 EICE:IECS 2 2 Atg4d –1,245 –1,211 EICE:IECS 1 1 Cst3 –360 –330 IECS –280 –268 EICE 0 0 Ctsc UT IFNγ/TLR UT IFNγ/TLR Lamp2 –68 –53 EICE 70 Ctsc ** 10 Cst3 60 8 * 50 40 6 c Summary of autophagy genes controlled by IRF8 30 4 Gene IRF8 Rescue Binding 20 dependent (ChlP) 2 10 expression Ctsc + + + 0 0 UT IFNγ/TLR UT IFNγ/TLR Atg4d + + + Atg7 + + + 12 Lamp2 12 Atg4d ** Gabarap + + + 10 ** 10 Lamp2 + + + Relative ChIP binding 8 8 Cst3 + – + Map1lc3b + – + 6 6 Becn1 + + – 4 4 Atg16l1 + + – 2 2 Ulk1 + + – 0 0 Atg13 + + – UT IFNγ/TLR UT IFNγ/TLR Gabarap12 + + – 14 Atg7 10 -globin Irgm1 + + – 9 12 Rab8b + – – 8 Rab10 + – – 10 ** 7 8 6 Rab5 + – – 5 Rab9a + – – 6 4 4 3 2 2 1 0 0 UT IFNγ/TLR UT IFNγ/TLR

Figure 2 | IRF8 binds to the promoters of autophagy genes. (a) Consensus IRF8-binding motifs are shown in bold on indicated autophagy gene promoters. (b) IRF8 binding to the above motifs was detected by ChIP for WTand Irf8 À / À M^s (UTor treated with IFNg/TLR) normalized by normal IgG binding, and with b-globin as a negative control. Values represent the average of five independent experiments. *Pr0.05 and **Pr0.01. (Student’s t-test). (c) Summary of qRT-PCR analysis rescue experiment and ChIP assays. form LC3 vesicles was also evident with endogenous LC3 autophagosome formation, the amounts of Atg5–Atg12 complex (Supplementary Fig. 3a). Electron microscopy analysis increased in WT M^s, but not in Irf8 À / À M^s on IFNg/TLR additionally showed a noticeable increase in autophagic vesicles stimulation (Fig. 3e). in stimulated WT M^s, but not in Irf8 À / À M^s (Fig. 3b, right We noted that although autophagy is linked to the down- panel)35. Immunoblot analysis was performed to assess regulation of mammalian target of rapamycin (mTOR) kinase in phosphatidylethalamine conjugation of LC3 as detected by a starvation model1, IFNg/TLR stimulation did not downregulate changes in LC3I and LC3II levels36. Cells were treated with phospho-mTOR expression, but it rather upregulated it in bafilomycin A1 to block the fusion of autophagosomes to WT and Irf8 À / À M^s (Supplementary Fig. 4), indicating that lysosomes36. Data in Fig. 3c (right panel) showed the amounts IFNg/TLR-induced autophagy may not be directly controlled by of LC3II increased in WT M^s after IFNg/TLR stimulation. mTOR activity, consistent with the recent report5. LC3II levels were significantly lower in Irf8 À / À M^s before As autophagy proceeds further, autophagosomes fuse stimulation and did not measurably increase after simulation, as with lysosomes to become autolysosomes that carry LC3 and confirmed by quantification (Fig. 3c, right panel). Flow various lysosome-associated components2. LAMP2, a lysosomal cytometric analysis to detect membrane-bound LC3 further membrane protein, is involved in the autophagosome–lysosome validate these results, in that LC3 signals increased after fusion38. Immunostaining and immunoblot data in Fig. 4a,b stimulation in WT M^s. However, LC3 levels were lower in found that LAMP2 expression was lower in Irf8 À / À M^s than Irf8 À / À M^s before stimulation and remained low after in the WT cells, consistent with reduced Lamp2 mRNA stimulation (Fig. 3d)37. To ascertain whether IRF8 has a role in expression in Irf8 À / À cells (Fig. 1d). To test whether IRF8 autophagosome–lysosome fusion and lysosomal clearance, affects autolysosome formation, we examined the co-localization immunoblot and flow cytometry assays were performed in the of endogenous LC3 and lysosomes as detected by LysoTracker- absence of bafilomycin A1 (Supplementary Fig. 3b,c)36.InWT red by immunostaining36. Immunostaining data in Fig. 4c M^s, the amounts of LC3 hardly increased after stimulation, showed that the majority of LC3 vesicles merged with suggesting lysosomal turnover of LC3. On the other hand, LC3 LysoTracker-red staining in WT M^s on IFNg/TLR levels were again lower in Irf8 À / À M^s and the amounts were stimulation (arrow heads). However, in Irf8 À / À M^s, LC3 unchanged after stimulation. These data support the notion that and LysoTracker-red double positive vesicles were much fewer IRF8 plays a role in autophagosome formation, and subsequent (Fig. 4c). These results indicate that through transcriptional autophagolysosome formation and function in IFNg/TLR- control, IRF8 indirectly facilitates maturation of autophagosomes induced autophagy. Further supporting the role of IRF8 in to autolysosomes in response to IFNg/TLR stimulation.

4 NATURE COMMUNICATIONS | 6:6379 | DOI: 10.1038/ncomms7379 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7379 ARTICLE

abmCherry LC3 GFP LC3 Merged

WT UT BA1

WT IFNγ /TLR

WT IFNγ/TLR BA1

Irf8 –/– UT BA1

–/– Irf8 IFNγ /TLR

Irf8 –/– IFNγ/TLR BA1 1.8 WT 100 Bafilomycin A1 (200 nM) –/– ** WT c 1.6 Irf8 –/– ** –/– Irf8 WT Irf8 1.4 80 γ/ UTIFNγ/TLR UT IFN TLR 0.2 60 16 LC3 1 14 40 0.8 β-Tubulin 0.6 20 50 LC3 II/Tubulin

Cells bearing LC3+ kDa 0.4 autophagosomes (%) 0 0.2 UT IFNγ/TLR 0 deUT IFNγ/TLR WT UT BA1 WT IFNγ/TLR BA1 WT Irf8 –/– 300 Irf8 –/– UT BA1 UT γ /TLR UT γ /TLR Irf8 –/– IFNγ/TLR BA1 97

200 64

Count 51 Atg5–Atg12 complex (56 kDa) 39 100

28 0 19 0 1 2 3 4 10 10 10 10 10 14 LC3 PE

50 β-Tubulin kDa Figure 3 | Defective autophagosome formation in Irf8 À / À M^s. (a) LC3 vesicles were visualized in WT and Irf8 À / À M^s expressing mCherry-EGFP- LC3 vector, without (UT) or with IFNg/TLR stimulation for 8 h. Bafilomycin A1 (BA1; 200 nM) was added for the final 2 h. Cells were counterstained for DNA (blue). Scale bar, 20 mm. Below: the number of cells with more than five mCherry-positive vesicles was counted by microscopic inspection of more than 200 cells. The values represent the percentage of cells with fluorescent vesicles. **Pr0.01 (Student’s t-test). See Supplementary Fig. 3a for endogenous staining of LC3. (b) WTand Irf8 À / À M^s treated with IFNg overnight followed by TLR ligands for 8 h was inspected by transmission electron microscopy. The bracketed region in the left panel was enlarged in the right panel. Arrows indicate autophagic vacuoles. Scale bar, 0.5 mm. (c) Reduced LC3I to LC3II conversion in Irf8 À / À M^s. WT and Irf8 À / À M^s were treated with IFNg/TLR as above with BA1 (200 nM) treatment for the final 2 h. Immunoblot analysis was performed with 10 mg of extracts with b-Tubulin as a control. Right panel: the amounts of LC3II in three independent samples were quantified using the ImageJ software. *Pr0.05 and **Pr0.01 (Student’s t-test). See Supplementary Fig. 3b for LC3 amount in the absence of BA1. (d) Membrane-bound LC3 in WT and Irf8 À / À MFs treated with IFNg/TLR was detected by flow cytometry. BA1 (200 nM) was added for the final 2 h. The histogram is a representative of three independent experiments. See Supplementary Fig. 3c for LC3 amount in the absence of BA1. (e) Immunoblot detection of the ATG5–ATG12 conjugate. WT and Irf8 À / À M^s were treated as above and immunoblot detection of the ATG5–ATG12 conjugate proteins was performed. Ten micrograms of the extracts were tested with antibody against ATG5 or b-Tubulin.

Irf8 À / À M^s accumulate SQSTM1/ubiquitinated proteins. the immunostaining results (Fig. 5a, right panel). Immunoblot Misfolded self-proteins and invading pathogens are often analysis further confirmed that SQSTM1 accumulates in conjugated to ubiquitin and escorted to autophagosomes to be greater amounts in Irf8 À / À than WT M^s with and without degraded in lysosomes. This process is partly mediated by the bafilomycin A1 treatment (Fig. 5b upper panel and ubiquitin adaptor SQSTM1 (p62)13,14. Some autophagy-deficient Supplementary Fig. 3b). In addition, the amounts of ubiquitin- cells form aggregates, called aggresomes or inclusion bodies that positive proteins increased markedly after stimulation and to a contain SQSTM1 and ubiquitin14. In light of defective autophagy, greater extent in Irf8 À / À M^s than WT cells (Fig. 5b, left and it was possible that Irf8 À / À M^s abnormally accumulate right panels). In agreement with the increased ubiquitin-positive SQSTM1 and ubiquitin-containing proteins. Immunostaining of proteins, we previously noted that IFNg/TLR stimulation ubiquitin and SQSTM1 showed extensive deposition of SQSTM1 increases ubiquitin-conjugated proteins in M^s39. Next, we that co-localized with ubiquitin-positive proteins in Irf8 À / À tested whether the larger increase in SQSTM1 protein expression M^s after IFNg/TLR stimulation, in contrast to WT M^s that in Irf8 À / À cells was due to higher Sqstm1 transcription in Irf8 À / showed little deposition of these proteins (Fig. 5a, left panel). À M^s. Relevant to this question, we previously showed that Quantification of SQSTM1 and ubiquitin-positive cells confirmed Sqstm1 mRNA expression increases after IFNg/TLR stimulation

NATURE COMMUNICATIONS | 6:6379 | DOI: 10.1038/ncomms7379 | www.nature.com/naturecommunications 5 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7379

a LAMP2 Merged c Lysotracker LC3 Merged

70 ** UT WT UT 60 WT UT IFNγ/TLR 50

WT IFNγ/TLR WT IFNγ/TLR 30

20 positive structures (%)

Cells bearing LC3/Lysotracker 10

Irf8 –/– UT 0 –/– Irf8 UT WT Irf8–/–

Irf8–/– IFNγ/TLR Irf8 –/– IFNγ/TLR

LAMP2 b 1.6 WT Irf8–/– 1.4 ** 1.2

LAMP2 1 100 0.8

β 50 -Tubulin 0.6 LAMP2/Tubulin kDa 0.4

0.2

0 WT Irf8–/– Figure 4 | Defective autophagosome maturation in Irf8 À / À M^s. (a) Immunostaining of LAMP2 in WT and Irf8 À / À M^s stimulated with IFNg overnight and TLR ligands for 8 h. Cells were counterstained for DNA (blue). Scale bar, 20 mm. (b) Immunoblot detection of LAMP2 in WT and Irf8 À / À M^s. Ten micrograms of extracts were tested with antibody against LAMP2 or b-Tubulin. Below: the amounts of LAMP2 in three independent experiments were quantified using the ImageJ software. **Pr0.01. (c) WT and Irf8 À / À M^s were immunostained for LC3 antibody and LysoTracker-red to detect LC3-positive and lysosome-positive structures. Right: cells with double-positive vesicles were quantified as in Fig. 3a. Data represent the averageof three independent experiments. Pr0.01 (**). Scale bar, 20 mm. in WT M^s40. qRT–PCR data showed that the levels of Sqstm1 Listeria transcripts rose sharply in Irf8 À / À M^s during 48 h of mRNA were comparable in WT and Irf8 À / À M^s, indicating infection, while remained very low in WT M^s, including ActA that SQSTM1 proteins aberrantly accumulate in Irf8 À / À cells shown to bypass host cell autophagy41. On the other hand, after stimulation, due to deficiency in autophagic degradation Listeria infection markedly increased expression of many (Supplementary Fig. 5). To ascertain the role of autophagic autophagy genes in WT M^s, in some cases by nearly degradation in the elimination of ubiquitin-conjugated 100-folds (Fig. 6b). Among induced genes was Nod1, important proteins, in addition to proteasome mediated degradation, for autophagy-mediated control of Listeria43. Interestingly, on immunoblot analysis was performed for cells treated with a Listeria infection, Irf8 expression was also dramatically increased. proteasome inhibitor, MG132. As seen in Fig. 5c left and right In contrast, none of these autophagy genes were induced in panels, Irf8 À / À M^s accumulated greater amounts of ubiquitin- Irf8 À / À M^s at comparable levels (Fig. 6b). Accordingly, while bound proteins than WT M^s in the presence of MG132, WT M^s restricted bacterial growth to a minimum, Irf8 À / À suggesting that autophagy partly accounted for excess M^s relented to the logarithmic growth of Listeria, as verified by accumulation of SQSTM1 and ubiquitin-bound proteins in bacterial accumulation in the cytoplasm (Fig. 6c). Further Irf8 À / À M^s. Further supporting the accumulation of supporting IRF8-dependent autophagic activation, LC3II levels unprocessed proteins in Irf8 À / À M^s, MitoTracker-positive increased during Listeria infection in WT M^s but not in Irf8 À / materials that co-localized with monodansylcadaverine staining À M^s, as observed in the presence of bafilomycin A1 (Fig. 6d). were more abundant in Irf8 À / À M^s than WT cells In contrast, LC3II amounts remained much lower in Irf8 À / À (Supplementary Fig. 6)36. M^s during infection. In the absence of bafilomycin A1, LC3II amounts increased at 24 h, then gradually decreased afterwards up to 48 h (Supplementary Fig. 7a) in WT M^s. In Irf8 À / À IRF8 is required for autophagic clearance of Listeria. M^s, LC3II amounts also increased at 24 h, but remained high Autophagy is a major mechanism by which M^s eliminate throughout 48 h of infection, suggesting inefficient LC3 turnover intracellular bacteria, such as M. tuberculosis, Salmonella and in lysosomes36,44. In agreement, SQSTM1 and SQSTM1-positive Listeria6,10,41. Previous studies showed that IRF8 is essential for proteins accumulated in Irf8 À / À M^s in greater amounts controlling infection of the above bacteria22–26. However, it has than WT cells with and without bafilomycin A1 (Fig. 6d, not been clear whether IRF8 employs autophagy to confer Supplementary Fig. 7a). Together these results reinforce the view protection. We first examined whether Listeria infection activates that during Listeria infection, IRF8 plays a major role in autophagy genes in M^s: Listeria monocytogenes is a food-borne promoting both autophagosome formation and the subsequent pathogen that causes listeriosis and is widely studied in mouse autolysosomal functions. Importantly, immunostaining analysis models42. qRT–PCR data in Fig. 6a showed that the expression of in Fig. 6e revealed that Listeria antigens co-localized with LC3

6 NATURE COMMUNICATIONS | 6:6379 | DOI: 10.1038/ncomms7379 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7379 ARTICLE

SQSTM1 Ubiquitin Merged UB+ and SQSTM1+ aggregates 80 **

and 70 + 60 50 WT IFNγ/TLR BA1

aggregates 40 + 30 20 10 SQSTM1

% Of cells bearing UB 0 WT UT WT Irf8 –/– Irf8 –/– Irf8 –/– IFNγ/TLR BA1 IFNγ/TLR UT IFNγ/TLR

Bafilomycin A1 (200 nM) SQSTM1 0.8 WT Irf8 –/– 0.7 ** UT IFNγ IFNγ/TLR UT IFNγ IFNγ/TLR 0.6 64 0.5 SQSTM1 51 0.4 0.3 ** 191 0.2 SQSTM1/tubulin 0.1 97 0 UT IFNγ IFNγ/ UT IFNγ IFNγ/ 64 TLR TLR 51 WT Irf8 –/– 39 Ubiquitin Total ubiquitin 8 ** 28 7 6 ** 19 5 14 4 3 6 2 Ubiquitin/tubulin 64 1 β 49 -Tubulin 0 UT IFNγ IFNγ/ UT IFNγ IFNγ/ kDa TLR TLR WT Irf8 –/–

WT Irf8 –/– – – + + – – + + MG132 Total ubiquitin ––++––++IFNγ/TLR 5 ** 191 4.5 ** 97 4 64 3.5 * 51 3 2.5 39 Ubiquitin 2 28 Ubiquitin/tubulin 1.5 19 1 14 0.5 0 6 UT IFNγ/ UTIFNγ/ UT IFNγ/ UT IFNγ/ 64 TLR TLR TLR TLR β-Tubulin UTMG132 UT MG132 49 –/– kDa WT Irf8

Figure 5 | SQSTM1 and ubiquitin-conjugated proteins form aggregates in Irf8 À / À MUs. (a) SQSTM1- and ubiquitin-positive proteins (arrow heads) in WTand Irf8 À / À M^s stimulated with IFNg overnight and TLR ligands for 8 h were visualized by immunostaining. Bafilomycin A1 (200 nM) was added for the final 2 h. Cells were counterstained for DNA (blue). Right panel: the percentage of cells with SQSTM1- and ubiquitin-positive aggregates. Data represent the average of three independent experiments ±s.d. with **Pr0.01 (Student’s t-test). (b) Immunoblot detection of SQSTM1- and ubiquitin-positive proteins in WT and Irf8 À / À M^s stimulated with IFNg or IFNg/TLR for 8 h. Bafilomycin A1 (200 nM) was added for the final 2 h. Right panel: relative amounts of ubiquitin-bound proteins. Data represent the average of three independent experiments ±s.d. with **Pr0.01 (Student’s t-test). See Supplementary Fig. 3b for immunoblot detection of SQSTM1 in the absence of bafilomycin A1. (c) Immunoblot detection of ubiquitin-bound proteins in the presence of MG132. WT and Irf8 À / À M^s were stimulated with IFNg/TLR in the presence of 10 mM of MG132. Right panel: relative amounts of ubiquitin-bound proteins. Data represent the average of three independent experiments ±s.d. with *Pr0.05 and **Pr0.01 (Student’s t-test).

NATURE COMMUNICATIONS | 6:6379 | DOI: 10.1038/ncomms7379 | www.nature.com/naturecommunications 7 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7379 and formed autophagosomal vesicles in WT cells, indicative of by baﬁlomycin A1 led to logarithmic growth of Listeria in WT autophagic capturing of bacterial antigens. However, the Listeria M^s, comparable to that in Irf8 À / À M^s, supporting antigens were more abundant in Irf8 À / À M^s than in WT cells autophagic control of Listeria infection (Fig. 6c). To ascertain and the co-localization with LC3 was hardly observed (Fig. 6e). whether autophagic control of Listeria relies on de novo We also found that blocking of autophagosome–lysosome fusion transcription of autophagy genes, infected WT and Irf8 À / À

WT Irf8 –/– 0.25 Hly WT 0.06 Prfa WT Irf8 –/– Irf8 –/– UT 36 h 48 h UT 36 h 48 h 0.2 0.05 Irf8 1 30 80 0 0 0 0.04 0.15 Ulk1 1 70 69 0.7 10 8 0.03 Atg13 1 70 100 1 20 10 0.1 0.02 Becn1 1 35 60 0.5 5 0.5 Atg16l1 1 80 80 0.3 15 8 0.05 0.01 Atg7 1 40 20 0.8 5 3 0 0 Gabarap 1 14 15 0.8 3 1.5 UT 2 4 6 8 2436 48 UT2468243648 Gabarap12 1 8 15 1.5 1.5 4 0.6 2 lap WT Acta WT –/– 1.8 Rab10 1 3 4 0.7 0.2 0.9 0.5 Irf8 Irf8 –/– 1.6 Rab8b 1 4 10 1 1 5 0.4 1.4 Irgm1 1 10 20 1 2 0

Relative mRNA expression Relative 1.2 0.3 1 Lamp2 1 30 2.5 1 10 2 0.8 Cst3 1 5 4.5 0.5 2.5 2 0.2 0.6 Ctsc 1 20 5 1 6 3 0.1 0.4 0.2 Nod1 1 27 15 1 1 2.6 0 0 UT2468243648 UT 2 4 6 8 243648 Min Mid Max

Listeria infection (h) Bafilomycin A1 (200 nM) WT Irf8 –/– UT 24 h 36 h 48 h UT 24 h 36 h 48 h 6 Extracellular Listeria LC3l 16 Short exposure LC3II 14 5 LC3I 16

–1 Long exposure 4 LC3II 14

3 WT 39 WT BA1 (200 nM) GAPDH Irf8 –/– 191 2 97 log10 c.f.u. ml log10 c.f.u. SQSTM1 1 64

0 GAPDH 0824 36 48 60 39 Time (h) kDa LC3 SQSTM1 1.6 WT 18 –/– WT 1.4 Irf8 ** –/– Listeria LC3 Merged ** ** 15 Irf8 1.2 ** 1 12 0.8 9 ** 0.6 6 0.4 LC3II/GAPDH 3

0.2 SQSTM1/GAPDH WT 0 0 UT243648 UT 24 36 48 Time (h)

Irf8 –/– Untreated Listeria ControlIRF8IRF8K79E Control IRF8 IRF8K79E Irf8 –/– Irf8 0 0.3 0.2 0 0.5 0.5 Ulk1 1 1 1 1 19 6 Listeria-autophagosome Extracellular Listeria co-localization Atg13 1 0.6 0.3 1 5.3 3 60 4.5 ** Becn1 1 0.7 0.6 1 28 0.3 50 4 WT UT WT ActD Atg16l1 1 0.8 0.7 1 16.3 4.5 3.5 –/– 40 –1 Irf8 UT Atg7 1 0.7 1 1 10 2 3 Irf8 –/– ActD Gabarap 1 0.9 0.4 1 11 4.5 30 2.5 2 Gabarap12 1 0.8 0.7 1 6.4 6 % Of cells 20 1.5 Rab8b 1 1.2 0.5 1 13 5.2 log10 c.f.u. ml log10 c.f.u. 1 Rab10 1 0.7 0.4 1 8.3 3.7 10 0.5 Irgm1 1 0.6 0.8 1 6.4 1.2 0 0 Cst3 1 1.1 0.3 1 2.5 0.5 WT Irf8 –/– 0246 Lamp2 1 1 0.4 1 4 4 Time (h) Hprt 1 0.5 0.21 0.2 0.4

1.8 Min Mid Max Control IRF8 IRF8K79E 1.6 ** UT Listeria UT Lm UT Lm UT Lm 1.4 LC3I 1.2 LC3II 1 14 0.8 GAPDH 39 0.6 LC3II/GAPDH kDa 0.4 0.2 Control Irf8 Irf8K79E 0 Control IRF8 IRF8K79E

8 NATURE COMMUNICATIONS | 6:6379 | DOI: 10.1038/ncomms7379 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7379 ARTICLE

M^s were briefly treated with Actinomycin D (Act D) and tested examined mCherry-EGFP-LC3 fluorescent signals after starvation for bacterial growth. As shown in Fig. 6f, bacterial counts sharply and found many fluorescence-positive vesicles in WT M^s, while increased after 2 h of Act D treatment both in WT and Irf8 À / À only diffuse signals in Irf8 À / À M^s, indicating that starvation M^s. Act D also inhibited autophagosome formation and induces autophagosome formation in WT M^s, but this process expression of autophagy genes on IFNg/TLR stimulation is severely impaired in Irf8 À / À M^s (Fig. 7b, see quantification (Supplementary Fig. 8). These results are analogous to the below). LC3 flux analysis in Fig. 7c showed that the amount of previous report where muscle cell autophagy depends on LC3II fell 8 and 24 h after starvation in WT M^s. Whereas, the FOXO3-mediated transcription of autophagy genes17. amounts of LC3 remained similar in Irf8 À / À M^s under these Rescue experiments in Fig. 6g showed that transfer of the WT conditions, suggesting that LC3 was autophagically processed in Irf8 gene into Irf8 À / À M^s restored expression of most of the WT M^s as reported for other cells, and this process was autophagy genes induced by Listeria shown in Fig. 6b. In contrast, defective in Irf8 À / À M^s44 (Fig. 7c top and bottom). Consistent none of these genes were rescued by the mutant Irf8K79E with the immunoblot data, flow cytometry analysis in Fig. 7d (Fig. 6g). Furthermore, transfer of WT Irf8, but not the mutant, found that amounts of membrane-bound LC3 were reduced after restored the ability to increase LC3II protein expression after starvation in WT M^s, but not in Irf8 À / À M^s. Cell viability Listeria infection (Fig. 6h, left and middle panels). Consequently, assay confirmed that the reduced LC3 amounts in WT M^s Irf8 gene transfer led to a marked increase in LC3-positive during starvation were not due to toxicity of nutrient deprivation vesicles (Fig. 6h, right panel and Supplementary Fig. 7b). These (Supplementary Fig. 10b). Above assays were performed in the data provide strong evidence that IRF8-dependent transcription absence of bafilomycin A1. To assess the role of IRF8 in plays a major role in autophagic control of Listeria infection autophagosome formation, we performed similar assays in the in M^s. presence of bafilomycin A1. In immunoblot assays, amounts of LC3II appeared similar in WT M^s and Irf8 À / À M^s after starvation (Supplementary Fig. 10c). These data support IRF8 regulates starvation and M-CSF-induced autophagy. lysosomal processing of LC3 in WT M^s as suggested above. Starvation is one of the best-studied autophagy models1. Because Nonetheless, the results differed from those seen by all other starvation is seemingly unrelated to innate immunity, and have stresses tested, including M-CSF (below), in which LC3II levels not been studied extensively in M^s, we asked whether starvation were lower in Irf8 À / À M^s than WT M^s in the presence of induces autophagy in M^s and if so, whether IRF8 plays a role. bafilomycin A1. This difference may be due to differential We found that many autophagy genes were induced in WT M^s influence of IRF8 on LC3II protein levels under different stresses. on starvation, although the pattern of induction differed from Importantly, however, EGFP-positive punctae as well as that seen by IFNg/TLR or Listeria infection (Fig. 7a). Notably, the membrane-bound LC3 were markedly lower in Irf8 À / À M^s genes active in early stages of autophagy were induced after relative to WT M^s in the presence of bafilomycin A1 (see starvation, whereas those involved in later stages were not. microscopy and flow cytometry data in Supplementary Moreover, Irf8 itself was also induced after starvation in WT cells, Fig. 10d,e). Overall, these results point to a deficiency in although modestly. In contrast, these genes were not induced forming intact autophagosomes in Irf8 À / À M^s in addition to in Irf8 À / À M^s at comparable levels. Basal expression of many defects in the subsequent steps such as lysosomal fusion and autophagy genes was also lower in Irf8 À / À M^s than WT cells. protein processing. Further, we found that the amounts of Interestingly, Tfeb, a master regulator of starvation-induced SQSTM1 were higher in Irf8 À / À M^s than WT M^s during 8 h autophagy, involved in lysosomal biogenesis, was induced in M^s of starvation both with or without bafilomycin A1 (Fig. 7c and in an IRF8-dependent manner16. On the other hand, Foxo3, the Supplementary Fig. 10c). We also found that starvation transcription factor that regulates autophagy in atrophying downregulated phospho-mTOR in WT and Irf8 À / À M^s muscle cells was expressed at similar levels in WT and Irf8 À / À (Supplementary Fig. 10f), as shown in other cell types1 M^s, and was not induced by any of the stimuli tested in this (Supplementary Fig. 4). Thus, Irf8 À / À M^s, although capable study (Supplementary Fig. 9)17. of downregulating mTOR on starvation, are nevertheless Rescue experiments in Supplementary Fig. 10a showed that defective in starvation-induced autophagy. transfer of WT Irf8, but not Irf8K79E mutant into Irf8 À / À M^s Recent reports document that autophagy promotes M-CSF- restored the expression of five autophagy genes during starvation mediated monocyte to M^ differentiation in vitro11,12. In view of condition. Thus, starvation activates transcription of multiple the fact that IRF8 drives M^ differentiation, it was of interest to autophagy genes in M^s in an IRF8-dependent manner. We next test whether IRF8 affects M-CSF-mediated autophagy during M^

Figure 6 | IRF8 is required for the autophagic clearance of Listeria. (a) WT and Irf8 À / À M^s (106) were infected with Listeria (5 Â 107) and bacterial transcripts were detected by qRT–PCR, normalized by Gapdh. Data represent the average of three independent experiments. Pr0.05 (Student’s t-test). (b) Autophagy gene expression in indicated M^s was detected as above and normalized to those in uninfected WT MFs. Data represent the average of three independent experiments. P r0.05 (Student’s t-test). (c) Bacterial yields tested by the colony-forming unit (c.f.u.) assay. Some WT M^s were treated with bafilomycin A1 (200 nM). Values represent the average of three determinations ±s.d. (d) Immunoblot analysis of LC3 and SQSTM1 in Listeria- infected WT and Irf8 À / À M^s in the presence of bafilomycin A1 (200 nM). Below: amounts of LC3 and SQSTM1 from three independent samples were quantified by ImageJ software. **Pr0.01 (Student’s t-test). See Supplementary Fig. 7a for LC3 and SQSTM1 amounts in the absence of bafilomycin A1. (e) Distribution of Listeria antigens and LC3 after 36 h of Listeria infection in immunostaining. Arrow heads: LC3-associated Listeria. Scale bar, 20 mm. Below: the percentage of cells showing the co-localization of LC3 and Listeria antigens. Values represent the average of three independent experiments ±s.d. with **Pr0.01 (Student’s t-test). (f) Bacterial yields in M^s infected with Listeria for 24 h and treated with Act D (2 mgmlÀ 1). Data represent the average of three determinations ±s.d. See Supplementary Fig. 8 for details of Act D effects. (g) Irf8 À / À M^s transduced with Irf8 vector were infected with Listeria for 36 h and autophagy gene expression was detected as above. The numbers represent transcript levels normalized by those with empty vector. Irf8 transcripts were normalized by gapdh. Values are the average of three experiments. Pr0.05 (Student’s t-test). Hprt: a negative control. (h) Left and middle: Irf8 À / À M^s transduced as above were tested for LC3II using GAPDH as a control. The amounts of LC3II in two independent experiments were quantified using ImageJ software. **Pr0.01 (Student’s t-test). Right: Irf8 À / À M^s expressing mCherry-EGFP-LC3 were transduced as above and infected with Listeria for 36 h and fluorescent LC3 signals was visualized by confocal microscopy. See Supplementary Fig. 7b for microscopy image with controls.

NATURE COMMUNICATIONS | 6:6379 | DOI: 10.1038/ncomms7379 | www.nature.com/naturecommunications 9 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7379 maturation19,21. qRT–PCR analysis in Fig. 7e showed that cells with LC3 vesicles was noticeably higher in WT cells than expression of various autophagy genes increased during 5 days Irf8 À / À cells. In accordance, immunoblot analysis showed that of WT BM cell cultures with M-CSF. In contrast, none of these the levels of LC3II increased in WT cells during M-CSF, but in genes were induced in Irf8 À / À cells. Irf8 was strongly induced in Irf8 À / À cells LC3 levels only meagerly increased with or without WT cells cultured in M-CSF33. Flow cytometric analysis showed baﬁlomycin A1, indicating defective autophagosome formation that membrane-bound LC3 signals increased in WT cell cultures, and fusion with lysosomes (Supplementary Fig. 11b). The but only modestly in Irf8 À / À cell cultures (Fig. 7f and amounts of SQSTM1 were higher in M-CSF treated Irf8 À / À Supplementary Fig. 11a). LC3 signals in UT cells were also cells as compared with WT cells also with and without lower in Irf8 À / À cells than WT cells. Furthermore, baﬁlomycin A1, suggesting greater accumulation of SQSTM1 immunostaining analysis in Fig. 7g showed that the number of in Irf8 À / À cells than WT cells (Supplementary Fig. 11b).

Starvation-induced autophagy mCherry LC3 GFP LC3 Merged WT Irf8 –/– UT 4 h 8 h 10 h 24 h UT 4 h 8 h 10 h 24 h Irf8 122.4 1.4 2.4 0 0 0 00 UIk1 1 1.8 2.1 1.3 2.2 1 1.3 1 0.9 0.7 WT UT Atg13 1 1.2 1.71.3 1.7 0.5 1.8 1.5 1.5 1.5 Becn1 110.7 1.1 1.3 0.3 1 0.6 0.5 0.8 1 0.8 2.4 1.4 1.5 0.9 0.9 1 0.9 0.9 Atg16I1 WT Starvation Atg7 1 0.6 0.9 0.5 0.7 0.5 0.5 1 0.6 0.8 Map1Ic3b 1 0.3 1.3 0.9 0.4 0.5 0.5 0.9 0.7 0.5 Gabarap 1 0.9 3.1 2.2 2.2 0.4 0.9 0.8 1.1 0.7 Gabarap12 1 0.9 1.4 0.7 1.7 0.7 0.8 0.6 0.7 1.1 Irf8–/– UT Atg4d 1 1.7 3.2 1.6 2.9 1 1.3 1.6 1 1 Rab5a 1 1.3 2 2 2.1 0.2 0.7 0.9 0.9 0.9 Rab9a 1 1.7 3.1 3.1 1.2 1 1.2 1.3 1.1 1.1 –/– Rab10 1 0.7 1.1 1 0.8 0.1 0.5 0.5 0.5 0.7 Irf8 Starvation Rab8b 1 0.7 1.5 1.3 0.8 0.1 0.6 1 1 0.8 100 90 ** Irgm1 1 0.7 0.9 0.7 0.9 0.6 0.8 0.5 0.6 0.7 80 Lamp2 1 0.8 1.5 0.9 1 0.4 0.9 0.9 0.8 0.8 70 60 Cst3 1 0.3 0.3 0.3 0.3 0.1 0.4 0.4 0.5 0.2 50 Ctsc 1 1.4 1.4 1 1 1 1.8 0.9 1 1 40 30 ** Tfeb 1 1.2 1.4 2.2 2.1 0.1 1.1 0.8 0.3 0.3 20 10

Mid punctuate bearing cells 0 Min Max % Of mCherry GFP LC3 –/– UT

WT Irf8 –/– WT UT Irf8 starvation WT Irf8 –/– starvation UT 4 h 8 h 24 h UT 4 h 8 h 24 h 100 LC3I 300 WT UT UT WT starvation Starvation (2 h) LC3II 14 Irf8–/– UT 80 ** Irf8–/– starvation 200 64 60 SQSTM1 51

Count 100 40 GAPDH 39 20 kDa 0 0 1 2 3 4 10 10 10 10 10 0

LC3II SQSTM1 Mean fluorescence intensity (%)

LC3 PE –/– 1.6 WT WT 1.8 WT Irf8 1.4 ** –/– ** Irf8–/– 1.6 Irf8 1.2 * 1.4 M-CSF-induced autophagy 1 ** 1.2 1 WT Irf8 –/– 0.8 0.8 0 3 D 4 D 5 D 0 3 D 4 D 5 D 0.6 0.6 Irf8 1 1.1 3.5 6 0000 UT samples 0.4 0.4 Amount of LC3 0.2 0.2 UIk1 1 0.8 2.2 5.5 0.6 0.5 0.7 0.9 SQSTM1/GAPDH 0 0 Atg16l1 1 0.6 1.1 1.1 0.4 0.2 0.6 0.5

normalized with respective 4824 UT 4824 Atg7 1 0.8 0.6 1.7 0.60.2 0.6 0.4 Starvation (h) Starvation (h) Atg13 1 0.6 0.4 2.2 0.4 0.4 0.2 0.2 Rab8b 1 2 1.6 1.6 0.5 0.2 1.1 0.2 Rab10 1 0.6 0.5 0.6 0.5 0.2 0.5 0.2 WT UT 300 WT M-CSF Min Mid Max Irf8–/– UT Irf8–/– M-CSF 200

Count 100

0 100 101 102 103 104 –/– LC3 PE UT WT M-CSF Irf8 M-CSF

50 WT M-CSF WT M-CSF CQ (50 μM) Irf8–/– M-CSF 104 104 4 40 ** Q1 Q2 Q1 Q2 10 Q1 Q2 0.28 5.83 0.34 1.60 4.89E-3 0.28 103 103 103 30 102 102 102 20 1 1 1

10 10 10 bearing cells FL3-H::PerCP Q3 FL3-H::PerCP 10 Q4 FL3-H::PerCP Q4 Q3 Q4 Q3 37.6 PerCP-F4/80 0 56.3 0 71.9 26.2 0 7.62 92.1 10 10 10 % Of LC3 punctuate 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 –/– FL1-H::FITC FL1-H::FITC FL1-H::FITC UT WT Irf8 M-CSF M-CSF FITC-CD11b

10 NATURE COMMUNICATIONS | 6:6379 | DOI: 10.1038/ncomms7379 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7379 ARTICLE

As expected, WT cell cultures generated many more cells genes regulated by IRF8 ranged widely, covering essentially the expressing authentic M^ markers, F4/80 and CD11b, compared entire autophagic pathways, from the initial autophagosome with Irf8 À / À cell cultures (Fig. 7h). Treatment with Chloroquine activation to the final lysosomal degradation of captured targets, to inhibit autolysosome function led to a marked reduction in F4/ indicating that IRF8 influences autophagic events from the 80 and CD11b-positive cells (Fig. 7h). These results support a role beginning to the end. Because many of autophagy genes were for autophagy in M-CSF-mediated M^ differentiation in which transcriptionally activated on stress in M^s, efficient autophagic IRF8 plays an important part. execution in these cells must require transcriptional input. Our data show that IRF8 activates transcription of autophagy genes Discussion directly or in cooperation with other factors, since (a) IRF8 bound In this study, we show that IRF8 stimulates a series of autophagy to the promoter of multiple autophagy genes and (b) expression genes on various stresses known to activate autophagy in M^s, of these and additional autophagy genes was rescued by Irf8 gene including IFNg/TLR, Listeria infection, starvation and M-CSF transfer in Irf8 À / À M^s. Consequently, Irf8 À / À M^s were stimulation. Consequently, IRF8 was found to play a critical role impaired in many autophagic functions, ranging from defective in autophagic progression, from autophagosome formation to autophagosome formation to autophagic degradation of cellular autolysosomes and degradation of cellular components. More- components, including ubiquitin-bound proteins such as over, on Listeria infection, IRF8 critically contributed to the SQSTM1. Most importantly, Irf8 À / À M^s were unable to elimination of the intracellular bacteria. It is clear that IRF8, control the growth of Listeria. although not an autophagy effector by itself, is indispensable for On the basis of the central role that IRF8 plays in all autophagy the execution of autophagy in M^s. In most cases, autophagy settings we tested, IRF8 may be regarded as one of the master

Autophagy inducer • IFNγ/TLR stimulation • Intracellular Listeria ULK1 complex • Starvation IRF8 • M-CSF stimulation

ATG12 complex

Phagophore LC3 LAMP2 Lysosome IRGM1 Rab GTPases Fusion Proteolysis

Autophagosome

Autolysosome

Figure 8 | A model for IRF8-dependent autophagy in MUs. On various stresses (IFNg/TLR stimulation, Listeria infection, starvation and M-CSF stimulation), IRF8 is activated and promotes the expression of a series of autophagy genes. These genes encode factors active at various stages of autophagy, largely covering the whole autophagic cascade. Irf8 À / À MFs are defective in autophagic activation and fail to degrade target ubiquitin- conjugated factors, organelle and intracellular pathogens. Together, IRF8 acts as an autophagy master regulator in MFs to coordinate stress responses critical for innate immunity.

Figure 7 | IRF8 stimulates starvation and M-CSF-induced autophagy. (a) Autophagy gene expression in WT and Irf8 À / À M^s after starvation was detected by qRT–PCR and normalized by transcript values in UT WT cells. The number represents the average of three independent experiments. Pr0.05 (Student’s t-test). (b) WT and Irf8 À / À M^s expressing mCherry-EGFP-LC3 after starvation for 6 h and LC3 fluorescence signals (arrow heads) were visualized. Below: the percentage of cells with double-positive LC3 signals. Data represent the average of three independent experiments. **Pr0.01 (Student’s t-test). Scale bar, 20 mm. See Supplementary Fig. 10d for LC3 fluorescence signals in the presence of bafilomycin A1. (c) Immunoblot detection of LC3II and SQSTM1 in starved WT and Irf8 À / À M^s. Below: the amounts of total LC3 and SQSTM1 in three independent samples quantified by ImageJ software. *Pr0.05 and **Pr0.01 (Student’s t-test). See Supplementary Fig. 10c for LC3 and SQSTM1 amounts in the presence of bafilomycin A1. (d) Left panel: membrane-bound LC3 in WT and Irf8 À / À M^s after 2 h starvation was detected by flow cytometry. The histogram is a typical example of three independent experiments. Right panel: mean fluorescence intensity of membrane-bound LC3. Values represent the mean of three independent experiments. **Pr0.01 (Student’s t-test). See Supplementary Fig. 10e for fluorescence-activated cell sorting detection in the presence of bafilomycin A1. (e) Autophagy gene expression in WT and Irf8 À / À BM cells cultured in M-CSF was detected by qRT–PCR, and transcripts were normalized by values in WTcells on day 0. The number represents the average of three independent experiments. Pr0.05 (Student’s t-test). (f) Membrane-bound LC3 in WTand Irf8 À / À MFs cultured in M-CSF for 4 days was detected by flow cytometry. The histogram is a typical example of three independent experiments. Similar results were observed with MFs on days 3 and 5 (See Supplementary Fig. 11a). (g) LC3 vesicles in WTand Irf8 À / À MFs cultured in M-CSF for 3 days was detected by immunostaining. Below: the percentage of cells bearing LC3 vesicles. **Pr0.01 (Student’s t-test). Scale bar, 20 mm. (h) Expression of F4/80 and CD11b was detected in WTand Irf8 À / À MFs cultured in M-CSF for 3 days by flow cytometry. Chloroquine (50 mM) was added in the middle panel for the final 18 h. Similar results were observed in three independent experiments.

NATURE COMMUNICATIONS | 6:6379 | DOI: 10.1038/ncomms7379 | www.nature.com/naturecommunications 11 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7379 regulators of MF autophagy. In many cases, autophagy is likely to transcription critically contributes to the control of Listeria be initiated by the preexisting cytoplasmic autophagy effectors. infection in MFs. Nevertheless, some autophagy components are presumably It is noteworthy that IRF8 has been known to play a crucial turned over to be replaced by newly synthesized components. role in combating Listeria infection since 1997 (ref. 23). Our There are a few examples where proteins required for autophagy results are consistent with this early study and provide deeper are synthesized during autophagic events by distinct transcription insight into the mechanism by which IRF8 confers resistance to factors16,17. For example, muscle atrophy-associated autophagy the bacteria. In a similar context, it has been shown that IRF8 is requires FOXO3, a transcription factor of the forkhead family17. essential for controlling infection by other bacteria, including FOXO3 is activated by starvation and it then induces a number of M. tuberculosis: mutations in Irf8 are associated with increased autophagy genes to stimulate lysosomal proteolysis, which leads susceptibility to Mycobacteria infection in mice and humans24–26. to the degradation of cellular components and muscle cell However, underlying molecular mechanisms have not been fully atrophy. Second, TFEB, a transcription factor of the bHLH clarified. In light of recent reports that autophagic clearance is family, regulates genes important for biogenesis and functions of critically required for controlling Mycobacteria infection, it seems lysosomes. Through this action, TFEB coordinates autophagy- likely that autophagy is an important means by which IRF8 exerts mediated lipid metabolism16. In these cases, FOXO3 and TFEB antimycobacterium activity6,7. IRF8 may also augment other provide a mechanism to coordinate autophagic processes. The aspects of innate immunity through autophagy, such as major function of IRF8 in M^ autophagy found in this study is histocompatibility complex II-mediated antigen presentation to comparable to those of FOXO3 and TFEB, although these factors TLR-dependent type I IFN induction4,9. act on different aspects of autophagy in different cell types. The In conclusion, IRF8 activates autophagic cascades in M^sat above reports, combined with our results, lead us to a line of the level of transcription, and orchestrates capturing and thinking that activities of individual autophagy genes are elimination of endogenous targets and infectious pathogens. organized as a network by a central regulator that integrates separate autophagic events to achieve orderly elimination of Methods target factors. FOXO3 and TFEB may thus represent the founding Mice and cells. WT and Irf8 À / À mice on C57BL/6 background18 were members of autophagy master regulators. It is not surprising that maintained in the NICHD animal facility. All animal work conformed to the autophagy master regulators so far identified are transcription NICHD animal care and use committee guidelines. BM mononuclear cells isolated factors, given that they regulate multiple genes in a signal- from age-matched mice (female, 6–10 weeks old) were cultured as described previously20. In brief, BM mononuclear cells were cultured in complete RPMI dependent manner. medium (RPMI, 10% fetal bovine serum, 1 mM sodium pyruvate, 0.1 mM NEAA, À 1 While IRF8 was important for the execution of autophagy after 0.05 mM b-mercaptoethanol, 2 mM L-glutamine, 100 U ml streptomycin/ varying stresses, we noted that the patterns of autophagy gene penicillin) in the presence of Flt3L (100 ng ml À 1) or M-CSF (20 ng ml À 1) for 5 to ^ induction varied under different stress conditions. For example, 6 days to generate DCs and M s, respectively. DCs were stimulated with 100 ng ml À 1 of LPS (Sigma) and 1 mgmlÀ 1 of CpG 1826 (Lofstrand Labs) for Atg7, downregulated by IFNg/TLR and starvation was, however, 4–6 h for microarray analysis. MFs were treated with IFNg (100 U ml À 1) upregulated by Listeria infection. In addition, the range of overnight followed by stimulation with LPS (100 ng ml À 1) and CpG 1826 autophagy genes rescued by Irf8 gene transfer varied among (1 mgmlÀ 1) for indicated periods. For starvation, M^s were washed with PBS and different stresses. Moreover, IRF8 influenced LC3II levels in incubated in Earle’s balanced salt solution at 37 °C for indicated times. In some cases, M^s were treated with Act D (Sigma, cat. no. A1410) at 2 mgmlÀ 1 for starvation differently from other stresses. indicated times. For bacterial infection, MFs were stimulated with IFNg Autophagic competence is a vital requirement for the (100 U ml À 1) in antibiotics free media for 20 h followed by infection with Listeria function and survival of M^s and DCs, since these cells, monocytogenes 10403S at a bacteria to cell ratio of 50:1 and maintained in complete acting as sentinels, must detect and eliminate invading media for indicated times46. Bacterial yields were measured by colony-forming unit ^ assay after plating serially diluted culture supernatants of infected cells on BHI agar pathogens rapidly. M s and DCs thus produce a large amount (Sigma, cat. no. 70138). For M-CSF treatment, BM-derived mononuclear cells were of reactive oxygen species and nitric oxide as part of the incubated in media containing M^-colony-stimulating factor (M-CSF (Peprotech), antimicrobial defense. These compounds, however, as free 20 ng ml À 1)at37°C for indicated days. radicals, likely damage the integrity of cellular components, which need to be processed in a timely manner. IRF8 was Microarray analysis. Total RNA from unstimulated and TLR-stimulated DCs was induced by classic stresses, IFNg/TLR, starvation and Listeria processed through RNeasy column (Qiagen) to remove possible DNA con- infection. Although not regarded as a classic stress factor, taminants. Two independent RNA samples prepared from UT and TLR-stimulated À / À M-CSF too may involve some types of stress, given that it DCs of WT and Irf8 mice were subjected to microarray analysis using the 12 Affymetrix Genechip Mouse 430 A 2.0 through Cogenics Inc. In brief, 1 mg of total activates the stress kinase JNK . Thus, one could envisage that RNA was converted to double-stranded complementary DNA with the Bioarray IRF8 may be adopted by M^s and DCs to meet heightened Single-Round RNA Amplification and Labeling Kit (Enzo Life Sciences) and requirement for responding to stresses by augmenting autophagic multiple copies of biotinylated cRNA were synthesized by in vitro transcription functions. with the Bioarray HighYield RNA Transcript Labeling Kit (Enzo Life Sciences), and assessed for quality by an Agilent Bioanalyzer. Six and a half mg of biotinylated It was striking that Listeria infection caused a dramatic and cRNA spiked with bioB, bioC, bioD was hybridized to the array for 16 h at 45 °C. prolonged expression of many autophagy genes along with that of Arrays were washed, stained in an Affymetrix GeneChip Fluidics Station and Irf8 itself. The high, sustained expression of autophagy genes scanned with an Affymetrix GeneChip Scanner 3000. Quality checks and data indicate that many autophagy factors were newly synthesized and analyses were carried out using Affymetrix GeneChip Operating Software (GCOS) replenished in M^s during infection in an IRF8-dependent and Expression Console. All arrays have passed Cogenics’ internal QC metrics. Values with Pr0.05 and two-fold cutoff were considered significant. Analysis of manner to support autophagic control of bacterial growth. Our variance (including t-tests), was used to identify differentially expressed genes28. observation that a brief Act D treatment gave way to rapid Raw data files have been deposited in the NCBI Gene Expression Omnibus under bacterial growth in WT M^s may further support the view that the accession number GSE64666. A complete list of IRF8 depended differentially continuous autophagy gene transcription is required for sustained expressed genes is provided in Supplementary Table 2. GO analyses were performed using Database for Annotation, Visualization and resistance to Listeria infection. Furthermore, our results that LC3 Integrated Discovery (DAVID; http://david.abcc.ncifcrf.gov/)47. co-localized with Listeria antigens are consistent with the report that autophagy plays a critical role in combating Listeria infection 45 Immunofluorescence staining and flow cytometry analysis. MFs grown on by linking ubiquitin pathways . Moreover, our observations that coverslips were stimulated with required treatment as above. Some cells were Irf8 gene transfer led to a partial rescue of autophagosome treated with 200 nM bafilomycin A1 (Invivogen, cat. no. tlrl-baf) for 2 h, fixed with formation provide compelling evidence that IRF8-dependent 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100 for 5 min and

12 NATURE COMMUNICATIONS | 6:6379 | DOI: 10.1038/ncomms7379 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7379 ARTICLE

blocked by 5% bovine serum albumin with 0.1% Tween 20. Cells were incubated 7. Castillo, E. F. et al. Autophagy protects against active tuberculosis by for 5 h with antibody against LC3 (MBL, cat. no. M152-3) or LAMP2 (ProSci, cat. suppressing bacterial burden and inflammation. Proc. Natl Acad. Sci. USA 109, no. 3627), SQSTM1 (MBL, cat. no. PM045), Ubiquitin (Santacruz, cat. no. sc8017) 3168–3176 (2012). or Listeria (Abcam, cat. no. ab35132) diluted at 1:1,000 followed by 1 h incubation 8. Henault, J. et al. Noncanonical autophagy is required for type I interferon with goat Alexa-488/Alexa-592 anti-rabbit or anti-mouse antibody (Molecular secretion in response to DNA-immune complexes. Immunity 37, 986–997 Probes). Cells were counterstained with DAPI for DNA. To detect autophagic (2012). m vacuoles, mitochondria and lysosomes, cells were incubated with 10 M 9. Lee, H. K. et al. In vivo requirement for Atg5 in antigen presentation by Monodansylcadaverine (Sigma 30432), 50 nM MitoTracker red (MT, Invitrogen dendritic cells. Immunity 32, 227–239 (2010). M22426) or 50 nM LysoTracker-Red (Invitrogen L7528). Stained cells were viewed 10. Mostowy, S. et al. p62 and NDP52 proteins target intracytosolic Shigella and on a confocal microscope (Leica, SP2) with a Â 63 oil immersion objective. Listeria to different autophagy pathways. J. Biol. Chem. 286, 26987–26995 Membrane-bound LC3 was detected by the flow cytometry method as described37. In brief, the cells were washed with PBS containing 0.05% saponin and incubated (2011). with mouse anti-LC3 and then with PE-conjugated goat anti-mouse antibody 11. Jacquel, A. et al. Autophagy is required for CSF-1-induced macrophagic (Biolegend, cat. no. 405307). Data were analysed using the FlowJo software. differentiation and acquisition of phagocytic functions. Blood 119, 4527–4531 (2012). 12. Zhang, Y., Morgan, M. J., Chen, K., Choksi, S. & Liu, Z. G. Induction of Electron microscopy. MFs were fixed in 0.1-M sodium cacodylate buffer pH 7.3 autophagy is essential for monocyte-macrophage differentiation. Blood 119, containing 2% paraformaldehyde, 2.5% glutaraldehyde (EMS, 15949) and post 2895–2905 (2012). fixed with 2% osmium tetroxide in 0.1-M sodium cacodylate buffer and processed 13. Itakura, E. & Mizushima, N. p62 Targeting to the autophagosome formation 35 as described . Samples were sectioned on a Leica Ultracut UC6 ultramicrotome. site requires self-oligomerization but not LC3 binding. J. Cell Biol. 192, 17–27 Thin sections (70 nm) were post stained with 3% uranyl acetate and Reynolds lead (2011). citrate and examined on FEI Tecnai Spirit G2 TEM. Digital images were captured 14. Komatsu, M. et al. Homeostatic levels of p62 control cytoplasmic inclusion on an FEI Eagle camera. Samples were processed for TEM by the Cell Imaging body formation in autophagy-deficient mice. 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