Intracellular Bacterial Infection Lysosomal Biogenesis Limits

Published December 14, 2015, doi:10.4049/jimmunol.1501595 The Journal of Immunology

Activator of G-Protein Signaling 3–Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection

Ali Vural,* Souhaila Al-Khodor,†,1 Gordon Y. C. Cheung,‡ Chong-Shan Shi,* Lalitha Srinivasan,x Travis J. McQuiston,{ Il-Young Hwang,* Anthony J. Yeh,‡ Joe B. Blumer,‖ Volker Briken,x Peter R. Williamson,{ Michael Otto,‡ Iain D. C. Fraser,† and John H. Kehrl*

Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Ga subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deﬁcient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deﬁcient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens. The Journal of Immunology, 2016, 196: 000–000.

athogens, especially those that survive and proliferate in Lysosomes maintain cellular homeostasis by acting as a deg- intracellular niches, have developed sophisticated strate- radative and recycling organelle in the cytosol. They recycle P gies to subvert host defense systems (1). Subject to strong macromolecules, reducing the energy-intensive de novo syn- selective pressures, drug-resistant microorganisms have emerged thesis of raw materials, and dysfunction causes inefﬁcient energy that are unresponsive or poorly responsive to current antimicrobial use, diverting energy to synthesis pathways at the expense of by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. agents (2). This provides an incentive to better understand host– energy-storage depots. Lysosomes degrade substrates and met- pathogen interfaces: on the pathogen side to unravel the mecha- abolic waste products by fusing with endocytic, autophagic, and nisms by which pathogens target and hijack host factors for their phagocytic vesicles (4). Both lysosomal mass and function are own beneﬁt and on the host side to delineate how host cells subject to regulation by the cellular physiologic state and in re- marshal intracellular defense systems to combat infections. Such sponse to pathologic conditions, including bacterial infections, understanding may lead to alternative strategies to improve host neurodegenerative disorders, and lysosomal storage diseases defense and enhance cellular resistance (3). Lysosomes occupy a (LSD) (5). The coordinated upregulation of lysosomal volume and crucial position in host–pathogen interactions, by being both tar- function is governed by the transcriptional factor EB (TFEB), geted by pathogens and serving as a major mechanism for killing which controls lysosomal biogenesis by enhancing the expression http://classic.jimmunol.org intracellular invaders. of the CLEAR gene network. Among the induced genes are ly-

*B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National A.V. and J.H.K. designed and analyzed the experiments; A.V. did the experimental Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, work; S.A.-K. performed Burkholderia cenocepacia J2315 infection experiments; MD 20892; †Signaling Systems Unit, Laboratory of Systems Biology, National Institute G.Y.C.C. and A.J.Y. performed methicillin-resistant Staphylococcus aureus infection of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; experiments; L.S. performed Mycobacterium tuberculosis infection experiments; ‡Pathogen Molecular Genetics Section, Laboratory of Human Bacterial Pathogenesis, T.J.M. performed the autophagic proteolysis experiments; I.-Y.H. performed ﬂow Downloaded from National Institute of Allergy and Infectious Diseases, National Institutes of Health, cytometry experiments; J.B.B., C.-S.S., P.R.W., I.D.C.F., M.O., and V.B. provided Bethesda, MD 20892; xDepartment of Cell Biology and Molecular Genetics, University expert advice; and A.V. and J.H.K. wrote and edited the manuscript. of Maryland, College Park, MD 20742; {Translational Mycology Unit, Laboratory of Address correspondence and reprint requests to Dr. John H. Kehrl, B-Cell Molecular Clinical Infectious Disease, National Institute of Allergy and Infectious Diseases, Na- ‖ Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy tional Institutes of Health, Bethesda, MD 20892; and Department of Cell and Molecular and Infectious Diseases, National Institutes of Health, 10 Center Drive, MSC Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 1876, Building 10, Room 11N212, Bethesda, MD 20892-1876. E-mail address: Charleston, SC 29425 [email protected] 1Current address: Infectious Disease Unit, Division of Translational Medicine, Sidra Abbreviations used in this article: AGS3, activator of G-protein signaling 3; BMDM, Medical and Research Center, Doha, Qatar. bone marrow–derived macrophage; GDI, guanine nucleotide dissociation inhibitor; ORCIDs: 0000-0003-1796-6058 (G.Y.C.C.); 0000-0001-7826-8101 (T.J.M.); 0000-0002- GPCR, G-protein–coupled receptor; iBMDM, immortalized BMDM; LDH, lactate 0020-3815 (J.B.B.); 0000-0002-2222-4115 (M.O.); 0000-0002-6526-159X (J.H.K.). dehydrogenase; LSD, lysosomal storage disease; MOI, multiplicity of infection; MRSA, methicillin-resistant S. aureus; mTOR, mammalian target of rapamycin; Received for publication July 15, 2015. Accepted for publication November 14, mTORC1, mTOR complex 1; siRNA, small interfering RNA; S6K1, S6 kinase 1; 2015. TFEB, transcriptional factor EB; TSC, tuberous sclerosis complex; WT, wild-type. This work was supported by the intramural program of the National Institute of Allergy and Infectious Diseases. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$30.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501595 2 MACROPHAGE AGS3 EXPRESSION ENHANCES LYSOSOMAL BIOGENESIS

sosomal hydrolases, lysosomal membrane proteins, and the com- fected into THP-1 cells, as previously described (29), using the human ponents of vacuolar H+-ATPase, which causes lysosomal acidifi- monocyte nucleofector kit (VPA-1007; Lonza). The cells were selected cation (6). Recently, TFEB was shown to be rapidly activated in with G418 (100 mg/ml) and later sorted on the basis of GFP expression into AGS3lo and AGS3hi cells using a FACSAria flow cytometer (Becton murine macrophages upon Staphylococcus aureus infection and Dickinson). The AGS3-GFP stable HeLa cell lines were generated by required for the proper transcriptional induction of several transfection of the AGS3-GFP expression plasmid using Lipofectamine proinflammatory cytokines and chemokines (7). Yet, the precise 2000. The cells were selected with 1000 mg/ml G418 and sorted based on lo hi hi signaling pathways that govern TFEB activity and lysosome GFP expression into HeLa AGS3 and AGS3 cell lines. The AGS3 cells were periodically resorted to maintain high levels of GFP expression. biogenesis in macrophages are largely unknown. In this article, we show the level of activator of G-protein sig- Burkholderia cenocepacia infection naling 3 (AGS3), also referred to as G-protein signaling modulator B. cenocepacia J2315 bacteria were grown on blood agar plates at 37˚C for (Gpsm1), in macrophages helps to govern lysosomal biogenesis 3 d. Single colonies were picked and grown overnight at 37˚C in 10 ml and function by affecting the translocation of TFEB into the nu- Luria-Bertani broth while shaking. The OD of bacterial suspensions was cleus through the regulation of the mammalian target of rapa- measured at an absorbance of 600 nm, and the CFU/ml was calculated 3 8 mycin (mTOR) pathway. AGS3 was originally identified as a based on the previously established equation: CFU/ml/OD600 = 2.7 10 . guanine nucleotide dissociation inhibitor (GDI) in a yeast-based In addition, formalin-killed bacteria were prepared using 3.65% paraformaldehyde in PBS solution for 30 min and then washed with 150 mM functional screen of mammalian cDNA libraries for proteins that NaCl. The infections were performed at a multiplicity of infection (MOI) activate G-protein signaling independent of G-protein–coupled of 1 into THP-1 and BMDM cell cultures, which were seeded at a density receptors (GPCR) (8). As a GDI, AGS3 binds to the Gi/Go/ of 2.0 3 105 cells/ml 2 d earlier. The plates were centrifuged at 1200 rpm transducin family of G proteins via its G-protein regulatory for 5 min to synchronize the infection, and this step corresponded to the zero time point. Next, the infected cells were incubated for an additional (GoLoco) motif and stabilizes the Ga subunit in its GDP-bound hour in 5% CO2 at 37˚C. The cells were washed three times with culture conformation. AGS3 was implicated in a surprisingly broad medium and incubated for 2 h with an antibiotic mixture (gentamicin 250 array of intracellular functions, including neuronal asymmetric mg/ml, ceftazidime 500 mg/ml) to eradicate the remaining extracellular cell divisions (9), adaptive responses to addiction and craving bacteria (30). At the end of 3- and 8-h infections, the supernatant was behavior (10–13), autophagy (14–18), membrane protein traf- aspirated, and the macrophages were lysed by adding 200 ml sterile water and shaking for 15–20 min at room temperature. For 24-h infection, the ficking (19, 20), metabolism, cardiovascular function (21), poly- cell supernatant and lysates were combined in a 1:1 ratio to include the cystic kidney disease (22–24), and leukocyte migration (25–27). In bacteria escaped from the cells at later stages of infection. The lysates were macrophages, LPS stimulation raised AGS3 levels, which led transferred to 96-well plates, and serial dilutions were performed. The to lysosomal enrichment and a decreased susceptibility to three aliquots were plated on blood agar plates to determine the number of bacteria (CFU/ml). antibiotic-resistant bacteria. Mycobacterium tuberculosis infection Materials and Methods M. tuberculosis H37Rv was grown in 7H9 medium supplemented with Mice albumin, dextrose, catalase, glycerol, and Tween-80 to mid-log phase (OD 0.6–0.8). Bacteria were washed and resuspended in PBS containing 0.05% The animal experiments and protocols were performed according to the Tween-80 prior to infection. THP-1 monocytes were maintained in RPMI regulations of the National Institute of Allergy and Infectious Diseases 1640 supplemented with 10% heat-inactivated FBS. Cells were differen- by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. Animal Care and Use Committee at the National Institutes of Health. All tiated with 20 ng/ml PMA for 20–24 h and washed with PBS, followed by animals were bred and housed under pathogen-free conditions. Wild-type the addition of infection media containing 5% human serum. Bacteria were (WT) C57BL/6 mice were purchased from The Jackson Laboratory. AGS3- added to cells at an MOI of 0.5 for 4 h at 37˚C, after which extracellular deficient (Gpsm12/2) mice were described previously (21). All experi- bacteria were removed by washing with PBS, and chase medium without ments were performed using sex- and age-matched animals, typically antibiotics was added. At the indicated time points postinfection, cells between 6 and 10 wk old. were lysed with 0.1% Triton X-100 in PBS, and appropriate dilutions were plated on 7H11 medium in triplicate. Abs and reagents S. aureus infection The AGS3 polyclonal Ab was described previously and was kindly provided Dr. D. Ma (Department of Molecular, Cellular, and Developmental Biol- The clinical community-associated methicillin-resistant S. aureus (MRSA) http://classic.jimmunol.org ogy, University of California Santa Barbara, Santa Barbara, CA) (15). The isolates USA300 clone LAC and MRSA252 were grown in tryptic soy following commercial Abs were used: LC3B Ab (L7543), b-actin–per- broth at 37˚C with shaking at 180 rpm. For all experiments, 50-ml cultures oxidase Ab (A3854) (Sigma-Aldrich); LAMP-1 (1D4B), LAMP-2 (H4B4), were inoculated with 1/100 dilution of an overnight bacterial culture and and CD-63/LAMP-3 (H5C6) Abs (Developmental Studies Hybridoma grown for 2 h in 125-ml baffled flasks. The bacteria were harvested and Bank); TGN-46 (ab16059) and LAMP-1 (ab25630) (Abcam); cathepsin D washed with sterile PBS and adjusted to the desired bacterial concentration (sc-6486) (Santa Cruz); Akt (11E7), p-Akt (Ser473) (D9E), tuberous scle- in RPMI 1640 without phenol red and 20 mM HEPES (assay media). rosis complex (TSC)2 (D93F12), p-TSC2 (Thr1462) (5B12), 4E-BP1 Bacteria were added to THP-1 cells or BMDMs, which had been seeded (53H11), p–4E-BP1 (Thr37/46) (236B4), p70 S6 kinase (49D7), p-p70 S6 into 24-well plates (3.5 3 105 cells/ml) with assay media. Synchronized Downloaded from kinase (Thr389) (108D2), and GAPDH (14C10) (Cell Signaling); and phagocytosis was initiated by centrifugation at 500 3 g for 5 min. For TFEB Ab (A303-672A) (Bethyl Laboratories). The following reagents BMDM infections, S. aureus USA300 clone LAC and MRSA252 were were used: PMA (Sigma-Aldrich), bafilomycin A1 and glycogen synthase used at an MOI of 6 and 30, respectively. For THP-1 infections, S. aureus kinase 3 inhibitor VIII (Calbiochem), G418 sulfate (Cellgro), LPS (Enzo USA300 clone LAC and MRSA252 were used at an MOI of 2 and 4, Life Sciences), and S. aureus (Wood strain without protein A) BioParticles, respectively. Plates were incubated at 37˚C for 1 h to allow phagocytosis of Escherichia coli (K-12 strain) BioParticles, and DQ-Red BSA (Molecular S. aureus. After the incubation period, THP-1 cells, but not BMDMs, were Probes). washed to remove extracellular bacteria and then replaced with RPMI 1640 without phenol red and 20 mM HEPES and incubated further at Cell isolation and culture 37˚C. Supernatant was collected from assay wells every hour for lactate Bone marrow–derived macrophage (BMDM) cell cultures were prepared dehydrogenase (LDH) detection using the Cytotoxicity Detection Kit as described (18). WT, MyD88-deficient, and TRIF-deficient immortalized (Roche). Cells treated with 1% (v/v) Triton X-100 (Sigma-Aldrich) served BMDM (iBMDM) were provided by James Harris (Monash University, as the 100% positive control. Images were taken using an EVOS FL mi- Melbourne, Australia) (28). THP-1 cells (American Type Culture Collec- croscope (Life Technologies). tion) were maintained in RPMI 1640 medium containing 10% (v/v) FBS Small interfering RNA knockdown along with 50 mM 2-ME. THP-1 cells were treated with 50 nM PMA for $3 h to promote their differentiation into macrophages. To generate Control small interfering RNA (siRNA), AGS3 siRNA, and GFP siRNA AGS3-expressing THP-1 cell lines, a AGS3-GFP plasmid was nucleo- were purchased from Santa Cruz. HeLa cells were seeded at a density of 3.0 The Journal of Immunology 3

3 105 cells/well into six-well plate 1 d prior to transfection. HeLa and CAGCTTCCTGTTCTGG-39 and reverse: 59-GTGTCCGTTCACCAA- HeLa AGS3lo/hi cells were transfected with 10–20 nM of scrambled, CAGGAAG-39; SQSTM1 forward: 59-GCACCCCAATGTGATCTGC-39 AGS3, and GFP siRNAs for 72 h using Lipofectamine 2000 reagent, and reverse: 59-CGCTACACAAGTCGTAGTCTGG-39; ATP6V1H according to the manufacturer’s instructions. The knockdown results were forward: 59-GGAAGTGTCAGATGATCCCCA-39 and reverse: 59- confirmed by Western blot for each experiment. CCGTTTGCCTCGTGGATAAT-39; CTSD forward: 59-AACTGCTGGA- CATCGCTTGCT-39 and reverse: 59-CATTCTTCACGTAGGTGCTGGA- Immunoblotting and nuclear fractionation 39; LAMP1 forward: 59-ACGTTACAGCGTCCAGCTCAT-39 and reverse: 59-TCTTTGGAGCTCGCATTGG-39; and GAPDH forward: 59-TGCAC- The immunoblot analysis of BMDM and THP-1 cell lysates was performed CACCAACTGCTTAGC-39 and reverse: 59-GGCATGGACTGTGGT- 3 5 as described (18). HeLa cells were seeded in a six-well plate at 3.0 10 CATGAG-39. cells/well 36–40 h prior to collection and preparation of cell lysates. The results were normalized to the expression levels of GAPDH mRNA, Following the indicated treatments, HeLa cells were lysed with a buffer and relative mRNA levels were calculated using the 22DDCt method. consisting of 25 mM HEPES (pH 7.4), 4% glycerol, 0.5% (v/v) Non- idet P-40, 150 mM NaCl, 2 mM CaCl2, protease inhibitor mixture, and Statistical analysis a PhosSTOP inhibitor mixture (Roche Applied Sciences). Lysates were shaken at 4˚C for 30 min, transferred to Eppendorf tubes, and centri- Data are expressed as mean 6 SEM. All results were confirmed in at least fuged at 10,000 rpm for 10 min at 4˚C. The supernatant corresponds to three independent experiments. Data were analyzed with Prism software the cytosolic fraction. Using the same lysis buffer, the pellet was (GraphPad) using the Student t test or ANOVA. The p values ,0.05 were resuspended, washed, and centrifuged twice at 5000 rpm for 5 min. considered statistically significant. Subsequently, the pellet was resuspended with a lysis buffer including 0.5% SDS and incubated for 30 min at 4˚C and this pellet corresponds to the nuclear fraction. The samples were subjected to 4–20% SDS- Results PAGE and analyzed by immunoblotting. Increased AGS3 expression reduces p62 levels without substantially affecting autophagic induction Immunocytochemistry Considerable evidence implicates heterotrimeric G-protein sig- HeLa cells, BMDM, and PMA-differentiated THP-1 macrophages were naling and AGS3 in the regulation of autophagic processes (14, 15, cultured on 35-mm glass-bottom dishes for 18–24 h prior to treatments. The 17, 31). However, in primary macrophages, no essential role for cells were washed with cold PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.25% Triton-X for 5 min, and incubated with 4% AGS3 was found in starvation- or nigericin-induced autophago- normal goat serum for 30 min at room temperature. Subsequently, the fixed some formation or autophagic flux (18). To reconcile this dis- and blocked cells were incubated overnight at 4˚C with the indicated crepancy, we generated two cell lines stably expressing AGS3 as a primary Ab, washed three times the following day, and incubated with GFP fusion protein: THP-1 cells, a monocyte cell line that can be Alexa Fluor–conjugated secondary Abs for 1 h at room temperature. The cells were imaged by confocal microscopy. For the B. cenocepacia differentiated into macrophages, and HeLa cells. The cells lines hi hi lo lo infections, ∼5.0 3 105 cells were grown on circular glass coverslips in were sorted into GFP (AGS3 ) or GFP (AGS3 ) cells. Initially, 24-well plates. At the end of the infections, the cells were fixed, per- we assessed LC3-II protein levels as a marker for autophagosome meabilized with ice-cold methanol for 5 min at 220˚C, and blocked formation and p62 levels as a marker for long-lived protein deg- with 3% BSA for 1 h at room temperature. The same protocol was radation. We found similar LC3-II/actin ratios in the THP-1 followed for primary and secondary Ab incubations, as indicated lo hi above. AGS3 and AGS3 cells and a slight reduction in the ratio in HeLa AGS3hi compared with AGS3lo cells (Fig. 1A, 1B). In Intracellular calcium and flow cytometry by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. contrast, the p62 protein levels were significantly depressed in hi Cells were seeded at 5 3 104 cells/100 ml loading medium (RPMI 1640, both AGS3 cell lines (Fig. 1A, 1B). To better interpret these lo hi 10% FBS) into poly-D-lysine–coated 96-well black-wall, clear-bottom results, we treated the AGS3 and AGS3 THP-1 cell lines with microtiter plates (Nalgene Nunc). An equal volume of assay loading bafilomycin A1, an inhibitor of vacuolar-type H+ ATPases, which buffer (FLIPR Calcium 4 assay kit; Molecular Devices) in HBSS sup- prevents the fusion between autophagosomes and lysosomes. Now plemented with 20 mM HEPES and 2 mM probenecid was added. Cells hi were incubated for 1 h at 37˚C before adding CXCL12 (100 ng/ml; R&D when we immunoblotted for LC3 and p62, THP-1 AGS3 cells lo Systems), and the calcium flux peak was measured using a FlexStation 3 had elevated levels compared with AGS3 cells (Fig. 1C). Con- (Molecular Devices). The data were analyzed with SOFT max Pro 5.2 sistent with this result, the untreated AGS3hi cells also had ele- (Molecular Devices). Data are shown as fluorescent counts with the y-axis vated SQSTM1 and MAP1LC3B mRNA transcripts, which encode labeled as Lm1. For flow cytometry, the cells were immunostained with lo http://classic.jimmunol.org p62 and LC3, respectively, compared with AGS3 cells (Fig. 1D). allophycocyanin mouse anti-human CD184 (clone 12G5; BD Biosci- ences). The background staining was performed with allophycocyanin- Together, these findings suggested that the elevated AGS3 levels conjugated IgG2a,b (BD Biosciences). Data acquisition was done on a had predominantly affected protein degradation but not auto- FACSCanto II (BD) flow cytometer, and the data were analyzed with phagy per se, perhaps by enhancing the lysosomal portion of the FlowJo software (TreeStar). autophagy/lysosome-degradation pathway. Fluorescence confocal microscopy and image preparation Elevated AGS3 expression enhances lysosomal mass Downloaded from The images were captured using the PerkinElmer Ultraview spinning-wheel confocal system mounted on a Zeiss Axiovert 200 equipped with an argon- To examine the lysosome status in the stable cell lines, we assessed krypton laser with a 1003 Plan-Aprochromax oil-immersion objective the cell lysates for the expression of LAMP-1, LAMP-2, and CD63 (numerical aperture 1.4). Volocity 4.1 (PerkinElmer) and Adobe Photoshop (LAMP-3), integral membrane glycoproteins abundant in lyso- 7 (Adobe Systems) were used to process the images. For the colocalization somes, as well as for cathepsin D, a lysosomal hydrolase (32). The ∼ experiments, 100 cells were analyzed using Imaris (Bitplane Scientific THP-1 and HeLa AGS3hi cell lines possessed significantly ele- Software). For each sample, 8–15 1-mm Z sections were captured for lo analysis. vated levels of these proteins compared with the AGS3 coun- terparts (Fig. 2A, 2B). To verify that the changes in LAMP-1 RNA isolation and quantitative RT-PCR expression were not simply due to the overexpression of GFP, RNA was isolated from AGS3lo and AGS3hi THP-1 and HeLa cells using we checked LAMP-1 expression in a GFP-nucleofected stable an RNeasy Mini Kit (QIAGEN), as per the manufacturer’s instructions. THP-1 cell line. THP-1 GFP cells had lower levels of LAMP-1 cDNA was synthesized from 2 mg RNA using an Omniscript RT Kit expression than did AGS3lo cells, arguing that GFP expression did (QIAGEN). Real-time PCR was performed in triplicate using the StepO- not increase LAMP-1 expression (Fig. 2C). Confocal microscopy neTM Real Time PCR System (Applied Biosystems), according to the + instructions in the Quantitect SYBR Green PCR Kit (QIAGEN). The visualized immunostained LAMP-1 lysosomal vesicles in the following primer pairs were used: MAP1LC3B forward: 59-GAGAAG- HeLa (Fig. 2D) and THP-1 (data not shown) cell lines. The ly- 4 MACROPHAGE AGS3 EXPRESSION ENHANCES LYSOSOMAL BIOGENESIS

FIGURE 1. AGS3 stable cell lines exhibit increased proteolysis without an alteration in autophagic induction. Immunoblot of cell lysates prepared from PMA-differentiated AGS3lo and AGS3hi THP-1 cells (A), AGS3lo and AGS3hi HeLa cells (B), or baﬁlomycin A1–treated AGS3lo and AGS3hi THP-1 cells (C) to examine the expression of the indicated proteins. The results obtained from three experiments are shown below and presented as the percentage of THP-1 AGS3lo (A) and HeLa AGS3lo (B) cells. LC3-II and p62 levels were normalized to the actin level in the corresponding lysates. (D) Quantitative RT-PCR of RNA prepared from AGS3lo and AGS3hi THP-1 cell lines for the indicated genes. *p , 0.05, **p , 0.005.

sosomal vesicles were larger and populated the cytosol more ex- revealed that, under basal conditions, TFEB migrated similarly; tensively in AGS3hi cells. To assess lysosomal proteolytic activity, however, following LPS treatment, the mobility of TFEB shifted 2 2 by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. we used DQRedBSA, a substrate whose proteolysis leads to in- in WT cell lysates but not in Gpsm1 / cell lysates (Fig. 3B, creased ﬂuorescence. Based on this analysis, AGS3hi cells had lower left panel, and data not shown). Examination of cell lysates increased lysosomal proteolytic activity (Fig. 2E). We validated from the THP-1 stable cell lines revealed increased TFEB mo- the lysosomal enrichment in the permanent cell lines by tran- bility in AGS3hi cells (Fig. 3B, lower right panel). siently expressing AGS3-GFP in HeLa cells (Fig. 2F). Conversely, Assessment of steady-state mRNA levels of several lysosomal silencing AGS3 mRNA in AGS3hi HeLa cells (Fig. 2G) or re- genes revealed an increase in THP-1 AGS3hi cells (Fig. 3C). To ducing endogenous AGS3 mRNA in HeLa cells (Fig. 2H) demonstrate that increased AGS3 levels could shift TFEB into the decreased LAMP-1 expression. These data indicate that increased nucleus, we coexpressed GFP-TFEB with AGS3-RFP, which AGS3 expression enhances lysosomal biogenesis in macrophages resulted in a prominent nuclear localization of TFEB (Fig. 3D, http://classic.jimmunol.org and HeLa cells. 3E). These results were supported by cytosol/nuclear fractionation (Fig. 3F). The failure to observe increased LAMP-1 in the LPS stimulation upregulates macrophage AGS3 expression, cotransfection experiment is likely secondary to the collection of and increased AGS3 levels trigger the nuclear translocation of cell lysates after overnight transfection. These data show that in- TFEB creased levels of AGS3 in macrophages lead to the nuclear The levels of AGS3 are sensitive to a variety of stimuli in the translocation of TFEB.

Downloaded from cellular environment (25, 26). In this study, we showed that LPS stimulation raised AGS3 and LAMP-1 expression in THP-1 cells AGS3 basal expression and its upregulation by TLR4 and mouse BMDM (Fig. 3A). Consistent with a role for AGS3 in engagement depend upon MyD88 and TRIF the induction of LAMP-1 expression, LPS-stimulated Gpsm12/2 To examine the TLR4 signaling pathway that elevates AGS3 ex- BMDM had lower LAMP-1 levels than did WT BMDM (Fig. 3A). pression in macrophages, we used iBMDM from WT, MyD882/2, LPS stimulation of cardiomyocytes is known to increase LAMP-1 and Ticam12/2 (TRIF) mice. We stimulated the cells or not with and LAMP-2 expression by triggering the nuclear translocation of the TLR4 ligand LPS and immunoblotted for the expression of TFEB (33). Increased TFEB transcriptional activity depends upon AGS3 and LAMP-1. The stimulated immortalized WT macro- TFEB dephosphorylation, release from cytosolic 14-3-3 proteins, phages had higher AGS3 and LAMP-1 levels, as we had noted and nuclear translocation (34, 35). To test whether LPS affected previously for stimulated THP-1 cells and BMDM (Fig. 4A, left TFEB phosphorylation, we stimulated BMDM and THP-1 cells panels). Surprisingly, MyD88-deficient cells had undetectable with LPS and checked the mobility of TFEB via SDS-PAGE. LPS basal levels of AGS3 and no observable increase following LPS treatment altered TFEB mobility in a manner consistent with stimulation (Fig. 4A, middle panels). Furthermore, LPS failed to partial TFEB dephosphorylation (Fig. 3B, upper panels). Immu- significantly upregulate LAMP-1 expression. LPS stimulation of noblotting BMDM lysates from WT and AGS3-deficient mice TRIF-deficient cells also failed to increase AGS3 levels or LAMP-1 The Journal of Immunology 5 by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd.

FIGURE 2. Lysosome levels are regulated by AGS3 expression. Immunoblots of the indicated proteins in lysates from AGS3lo and AGS3hi stable THP-1 cells (A) and AGS3lo and AGS3hi stable HeLa cells (B)(left panels). Lysosome marker/actin band intensity ratios were normalized, and the results are lo http://classic.jimmunol.org shown as the percentage of AGS3 cells in the bar graphs (right panels). (C) Immunoblots of the indicated proteins in THP-1 stable cells expressing GFP, AGS3lo THP-1 cells, or AGS3hi THP-1 cells. (D) Confocal images of lysosomal vesicles in an AGS3lo HeLa cell (marked by asterisks; left panel) and an AGS3hi (GFP+) HeLa cell immunostained for LAMP-1 (middle panel). Scale bar, 10 mm. The lysosomal mass was quantiﬁed by counting LAMP-1+ vesicles in 50–70 cells of each cell type (bar graph; right panel). (E) Confocal images of an AGS3lo cell (** in upper panel, visible in lower panel) and an AGS3hi THP-1 cell incubated with DQ-BSA overnight. Scale bar, 10 mm. (F) Immunoblot of protein levels in cell lysates from HeLa cells transiently expressing AGS3 for 60 h (upper panel). Lysosome marker/actin band intensity ratios were normalized and shown as the percentage of the control transfected cells (lower panel). (G and H) Immunoblots of the indicated proteins in lysates from AGS3hi HeLa cells transfected with scrambled (ctrl) or GFP

Downloaded from siRNAs (20 nM) or from HeLa cells transfected with scrambled or AGS3 siRNA (20 nM) for 72 h (left panels). The results were normalized to actin levels and are shown as a percentage of HeLa AGS3hi cells (G, right panel) or HeLa cells transfected with control siRNAs (H, right panel). *p , 0.05, **p , 0.005, ***p , 0.001.

expression (Fig. 4A, right panels). These results suggest that results suggest that macrophages may upregulate their expression MyD88-dependent signaling maintains the basal levels of AGS3 of AGS3 and LAMP-1 in response to a variety of pathogens. in macrophages and that TLR4 signaling through MyD88 and TRIF-dependent pathways increases AGS3 and LAMP-1 expres- AGS3 regulates TFEB translocation through the AKT/TSC2/ sion in macrophages. mTOR pathway We also assessed whether exposure of the WT iBMDM to Previous reports linked mTOR activity to the nuclear translocation of bacteria increased AGS3 and LAMP-1 expression. We incubated TFEB (34, 35). Under basal conditions, activated mTOR on the ly- the cells with S. aureus or E. coli BioParticles overnight, prepared sosomal surface phosphorylates TFEB, preventing its nuclear trans- lysates, and immunoblotted for LAMP-1 and AGS3. The over- location. Inhibition of mTOR activity reduces TFEB phosphorylation, night exposures increased the expression of AGS3 and LAMP-1 allowing it to translocate. To examine mTOR activity in the cell lines, ∼2-fold compared with the nonexposed cells (Fig. 4B). These we assessed the phosphorylation status of a downstream target S6 6 MACROPHAGE AGS3 EXPRESSION ENHANCES LYSOSOMAL BIOGENESIS

FIGURE 3. AGS3 levels alter lysosomal biogenesis by regulating the nuclear translocation of TFEB. (A) Immunoblot of the indicated proteins in cell lysates from lo hi 2/2 by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. THP-1 cells, AGS3 and AGS3 stable THP-1 cells, and BMDM derived from WT and Gpsm1 mice. In some instances the BMDM were stimulated with LPS overnight (100 ng/ml). Normalized AGS3 and LAMP-1 levels are shown as a percentage of nonstimulated (left and middle panels)orWT(right panel)cells.(B) Immunoblot of TFEB and actin expression in cell lysates prepared from the indicated cells. The LPS-treated cells were stimulated overnight. (C) Quantitative RT-PCR of RNA prepared from AGS3lo and AGS3hi THP-1 cell lines for the indicated genes. (D) Confocal images of HeLa cells expressing TFEB-GFP and AGS3-RFP or TFEB-GFP only. The location of the AGS3lo cell is marked by asterisks (middle panel). Scale bar, 10 mm. (E) Quantiﬁcation of TFEB nuclear localization in HeLa cells, as in (D). Localization was assessed in 500 cells. (F) Immunoblots of the indicated proteins in cytosolic and nuclear fractions isolated from HeLa cells transfected with vector or AGS3 expression plasmids overnight or treated with a glycogen synthase kinase 3b inhibitor (VIII). **p , 0.005, ***p , 0.001.

kinase 1 (S6K1). We found a 50% reduction in p-S6K1 in AGS3hi role of AGS3 in regulating heterotrimeric G-protein signaling http://classic.jimmunol.org cells (Fig. 5A). Rheb, an essential upstream protein in the mTOR remains controversial. Because AGS3 is a GDI, increased AGS3 pathway, is controlled by TSC1/2 (36, 37). TSC2 activity can be as- levels compete with Gbg for GDP-Gai binding. When GDP-Gai is sessed by its phosphorylation status at threonine 1462, and we found sequestered by AGS3 docking, Gbg remains free to signal or to be that p-TSC2 levels were lower in AGS3hi cells. Because the PI3K- degraded. The increased expression of the AGS3-related protein AKT pathway is upstream of TSC2 (38), we also examined p-AKT LGN resulted in the ubiquitination and degradation of Gbg (39). levels and found that they were reduced in AGS3hi THP-1 cells com- Consistent with this scenario, Gb1 and Gb2 levels were lower in lo hi

Downloaded from pared with AGS3 cells (Fig. 5A). We recapitulated these results by THP-1 AGS3 cells (Fig. 6A). To determine whether elevated transiently expressing AGS3 in HeLa cells (Fig. 5B). Moreover, re- AGS3 levels impacted Gai signaling, we examined chemokine- duced AGS3 expression in HeLa cells had the opposite phenotype induced increases in intracellular calcium, which depend upon hi (Fig. 5C). Finally, because LPS increases AGS3 expression in BMDM, Gbg release from Gai. THP-1 AGS3 cells responded less well we examined the AKT/TSC2/mTOR signaling axis in LPS-induced to the chemokine CXCL12 than did AGS3lo cells (Fig. 6B). In 2 2 WT and Gpsm1 / BMDM. Consistent with our previous results, we addition, THP-1 AGS3hi cells expressed less CXCR4, the cognate noted higher levels of p-AKT, p-TSC2, and p-4EBP1, another mTOR receptor for CXC12, possibly because of enhanced lysosomal 2 2 downstream target, in LPS-induced Gpsm1 / BMDM compared with degradation (Fig. 6C). In agreement with our results, over- WT cells (Fig. 5D). These results indicate that elevated AGS3 levels expression of AGS3 in RBL-2H3-CXCR2 cells reduced the in macrophages decrease p-AKT levels and dampen mTOR activity. chemokine-induced increases in intracellular calcium, and AGS3 knockdown enhanced CXCR2 expression (27). Together, these Elevated level of AGS3 affects heterotrimeric G-protein data suggest that elevated levels of AGS3 in macrophages lower signaling in macrophages p-AKT levels, which leads to decreased mTOR activity, the Although a wide variety of upstream signals affect the PI3K-AKT translocation of TFEB into the nucleus, and enhanced lysosome pathway, a major upstream input is GPCR signaling. However, the biogenesis. The Journal of Immunology 7

cells, indicating that they had controlled the infection better (Fig. 7F). Finally, we assessed whether AGS3 expression affected the course of a MRSA infection, a major cause of hospital- acquired infections (43). Representative strains of community- associated MRSA (USA300 clone LAC) and hospital-acquired MRSA252 were used. Although S. aureus produces an arsenal of virulence factors that assists in the escape from phag- olysosomes, leading to host cell death (44), upon infection with MRSA252, AGS3hi cells exhibited greater resistance, displaying fewer signs of cell lysis and less cell death (Fig. 7G). In addition, USA300 clone LAC and MRSA252-infected AGS3hi cells released signiﬁcantly lower levels of LDH than did AGS3lo cells (Fig. 7H). Conversely, we found that Gpsm12/2 BMDM released more LDH than did WT BMDM incubated with both MRSA strains (Fig. 7I). Together, these data indicate that elevated AGS3 levels help macrophages to eliminate dangerous intracellular bacteria.

Discussion Lysosomes degrade and recycle transported cellular compo- nents and internalized material by fusing with autophagosomes, phagosomes, and late endosomes. The resulting lysosomal break- down products are used to generate new macromolecules and to provide energy in response to the nutritional needs of the cell. Recently, lysosome functions were expanded to include roles in nutrient sensing and energy metabolism (4). In this study, we FIGURE 4. MyD88- and TRIF-dependent signaling pathways involved report that the exposure of macrophages to LPS or heat-killed in AGS3 and LAMP-1 upregulation in macrophages. (A) Representative bacteria raises AGS3 levels. The increases in AGS3 reduced immunoblots of cell lysates prepared from WT or MyD88- or TRIF-defi- signaling through the mTOR pathway. However, in a nutrient- cient iBMDM to examine AGS3 and LAMP-1 expression (upper panels). replete state this did not lead to a substantive increase in The results of three separate experiments are shown in the bar graphs as the autophagy, but it did facilitate the nuclear translocation of percentage increase versus nonstimulated cells (lower panels). The cells TFEB, which transcriptionally activates many of the genes in- were exposed to LPS (100 ng/ml) overnight prior to cell lysis and im- volved in lysosomal biogenesis and function. The subsequent munoblotting. (B) Immunoblot of cell lysates from iBMDM exposed or not increase in lysosomal biogenesis/function helped macrophages by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. overnight to S. aureus or E. coli BioParticles (MOI of 2) to examine LAMP-1 and AGS3 expression (left panels). Quantification of three sep- to resist intracellular infections by several strains of antibiotic- arate experiments (right panel). ***p , 0.001. resistant bacteria. Although AGS3 did not materially affect macrophage autophagosome formation (18), previous studies documented increased AGS3-induced lysosomal enrichment mediates resistance intracellular proteolysis, vesiculation, and vesicle acidity follow- against antibiotic-resistant bacterial strains ing AGS3 overexpression in other cell types (14). Although To assess whether AGS3-induced lysosomal enrichment offered a interpreted as increased autophagy, the accepted gold standard selective advantage against invasive pathogens, we challenged LC3-based assay was not available then. Also, the AGS3-induced http://classic.jimmunol.org macrophages expressing different levels of AGS3 with three increases in monodansylcadavarine staining noted previously (14) bacteria. B. cenocepacia J2315 is a Gram-negative bacterium that may have delineated lysosomes rather than autophagosomes (45). causes life-threatening infections in cystic fibrosis patients (40). When we overexpressed AGS3 as a GFP fusion protein in this hi lo The intracellular replication kinetics in THP-1 cells are known study, we found similar LC3-II levels in AGS3 and AGS3 cell (41). Analysis of the cells infected (MOI = 1) with the DsRed- lines, irrespective of the cell line type, but decreased p62 protein hi tagged B. cenocepacia strain J2315 revealed that, at 24 h postin- in AGS3 cell lines. Conversely, we found a relative increase in lo hi

Downloaded from fection, bacterial counts/cell exceeded 15 in 68% of AGS3 cells, p62 mRNA transcripts in AGS3 cells. When we blocked auto- whereas only 11% of AGS3hi cells harbored similar numbers phagic flux in THP-1 cell lines using bafilomycin A1, the LC3-II (Fig. 7A, 7B). The percentage of initially infected cells was and p62 levels increased in AGS3hi cells compared with AGS3lo similar (data not shown). Conversely, at 24 h postinfection, cells. This indicates that the increased level of AGS3 enhanced Gpsm12/2 BMDM had a 60% higher bacterial burden than did p62 degradation via the autophagosomal/lysosomal pathway with- WT BMDM (Fig. 7C). Next, we infected AGS3lo and AGS3hi out altering basal LC3-II levels in the cell. These observations cells and immunostained for the late endosomal/lysosomal protein suggested that the elevated AGS3 levels under nutrient-replete LAMP-2. At 8 h postinfection, only 15% of the bacteria colo- conditions had not triggered autophagy but rather had impacted calized with LAMP-2 in AGS3lo cells, whereas 88% colocalized lysosome function and/or biogenesis. By examining cells with in AGS3hi cells (Fig. 7D, 7E). This argues that fewer bacteria had elevated or reduced AGS3 levels, we found a corresponding in- escaped the endolysosomal pathway in AGS3hi cells. The second crease or decrease in LAMP-1 and other lysosomal markers. bacterium that we tested was M. tuberculosis, which causes .1 Mouse BMDM constitutively express AGS3 and following million deaths/y worldwide (42). We infected the THP-1 cell lines. exposure to LPS further increase their expression level. Using Although we noted little difference in initial CFU counts by 6 d iBMDM from WT and MyD88- or TRIF-deficient mice stimulated postinfection, a marked decrease had occurred in THP-1 AGS3hi or not with LPS, we showed that the basal level of AGS3 depended 8 MACROPHAGE AGS3 EXPRESSION ENHANCES LYSOSOMAL BIOGENESIS

FIGURE 5. AGS3 regulates mTOR complex via the AKT/TSC2/mTOR pathway. Immunoblots of the indicated proteins in cell lysates from AGS3lo and AGS3hi THP-1 cells (A), control or HeLa cells transiently expressing AGS3 for 72 h (B), HeLa cells treated with control or siRNA targeting AGS3 expression for 72 h (C), or BMDM from WT or Gpsm12/2 mice stimulated with LPS overnight (100 ng/ml) (D)(upper panels). Summation of results from at least three experiments normalized to actin and presented as a percentage of AGS3lo, percentage of control, or percentage of WT (lower panels). *p , 0.05, **p , 0.005, ***p , 0.001.

upon constitutive MyD88 signaling and that TRIF and MyD88 crease in AGS3 noted in lymphocytes following AgR cross-

by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. contributed to the increase in AGS3 that followed LPS stimulation. linking (26). The loss of MyD88 or TRIF also impaired the induction of LAMP-1 Different physiological and pathological inputs affect the status expression following LPS stimulation. The combined role of of mTOR complex 1 (mTORC1), thereby affecting autophagy and MyD88 and TRIF in the early induction and sustained activity of controlling TFEB activity (4). Starvation-induced autophagy de- NF-kB activity upon TLR4 signaling (46) suggests that AGS3 pends upon ULK1 dephosphorylation, a consequence of mTORC1 may be an NF-kB target gene in macrophages. An involvement of inactivation and increased activity of the phosphatase PP2A (47). NF-kB in the induction of AGS3 is also consistent with the in- High AGS3 levels in a nutrient-replete state reduced signaling http://classic.jimmunol.org

FIGURE 6. High levels of AGS3 reduce Gb expression and inhibit chemokine receptor signaling. (A) Downloaded from Immunoblot of cell lysates prepared from AGS3lo and hi AGS3 THP-1 cells for the expression of Gai and Gb. The percentage reduction in Gb1 and Gb2 expression compared with AGS3lo cells (lower panel). (B) Intra- cellular calcium levels following exposure of AGS3hi and AGS3lo cells to different concentrations of CXCL12. (C) Flow cytometry was used to examine CXCR4 expression on AGS3lo and AGS3hi THP-1 cells (left panel). The geometric means are shown in the bar graph (right panel). **p , 0.005, ***p , 0.001. The Journal of Immunology 9 by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd.

FIGURE 7. AGS3-induced lysosomal enrichment mediates resistance against antibiotic-resistant bacteria. (A) Confocal microscopy images of THP-1 AGS3lo and AGS3hi cells infected with the DsRed-tagged B. cenocepacia J2315 strain at an MOI of 1 for 24 h. The location of the AGS3lo cell is indicated by asterisks in the GFP panel. Scale bars, 10 mm. (B) Percentage of THP-1 AGS3lo and AGS3hi cells harboring $15 bacteria/cytosol at 24 h postinfection. (C) Quantification of CFU from WT and Gpsm12/2 BMDM infected with B. cenocepacia J2315 at an MOI of 1 at 24 h. Results were normalized to lo hi http://classic.jimmunol.org WT cells. The results obtained from three separate infection experiments are shown. (D) Confocal microscopy images of THP-1 AGS3 and AGS3 cells infected with B. cenocepacia J2315 at an MOI of 1 at 24 h postinfection. The cells were immunostained with B. cenocepacia– and LAMP-2–specific Abs. Due to very weak GFP signal in the AGS3lo cells, a GFP signal was not captured. The red and blue channels are shown in the merged panel. Scale bars, 10 mm. (E) Quantification of the colocalization rate between B. cenocepacia J2315 and LAMP-2. (F) Kinetic analysis of CFU counts upon M. tuberculosis infection. THP-1 AGS3lo and AGS3hi cells were infected with M. tuberculosis at an MOI of 0.5 for 4 h. The cells were lysed at various time points postinfection, and the lysates were plated on 7H11 media for CFU counts. The results are from three independent experiments performed in triplicate. (G) Brightfield microscopy images of THP-1 AGS3lo and AGS3hi cells infected with MRSA252 at an MOI of 4 for 6 h. Scale bar, 400 mm. Quantification of 2 2 Downloaded from LDH release from THP-1 AGS3lo and AGS3hi cells (H) and from WT and Gpsm1 / BMDM (I) 6 h postinfection with MRSA252 or USA300 clone LAC at an MOI of 6 and 30, respectively. Control cells were incubated with a 2% Triton-X solution to determine 100% LDH release. The amounts of LDH release following bacterial infections are shown as a percentage of the Triton-X–triggered release. The results obtained from three separate infection experiments are shown. **p , 0.005, ***p , 0.001.

through the mTOR pathway, reducing TFEB phosphorylation and Ligand-activated GPCR trigger Ga nucleotide exchange and the triggering its nuclear translocation, but evidently they did not functional dissociation of GTP-Ga and Gbg, both of which ac- suppress the pathway sufﬁciently to trigger ULK1 dephosphory- tivate downstream effectors that can lead to activation of the lation and visible increases in autophagosome formation. The PI3K-AKT pathway. Following nucleotide exchange, Ga remains PI3K-AKT pathway is a well-deﬁned input to mTORC1. In HeLa only transiently bound to GTP because its intrinsic GTPase ac- cells, THP-1 cells, and mouse BMDM, we found that increased tivity returns it to its GDP-bound state, which can recombine with AGS3 expression led to decreased p-AKT, p-TSC2, and p-S6K1 Gbg to terminate signaling. However, because AGS3 competes levels. Although many upstream signals affect the PI3K-AKT with Gbg for binding to GDP-Gai, AGS3 can potentially prolong pathway, a major input is signals generated by GPCR (48). Gbg signaling; conversely, it can trigger Gbg degradation because 10 MACROPHAGE AGS3 EXPRESSION ENHANCES LYSOSOMAL BIOGENESIS

free Gbg in the absence of Ga is unstable. Consistent with the Acknowledgments latter scenario, we found a reduction in Gb1 and Gb2, decreased We thank Vee Yang Tang for technical support and Dr. Anthony Fauci for CXCR4 expression, and a reduced intracellular calcium response long-standing support. in THP-1 AGS3hi cells compared with THP-1 AGS3lo cells. These results suggest that increasing levels of AGS3 in macrophages Disclosures reduced the availability of Gi heterotrimers, which limits GPCR The authors have no financial conflicts of interest. signaling through Gi-linked pathways, thereby decreasing an important input into the PI3K-AKT pathway. In contrast, low AGS3 References levels likely boost the availability of Gi. Similar to our results, the overexpression of AGS3 in RBL-2H3-CXCR2 cells reduced 1. Baxt, L. A., A. C. Garza-Mayers, and M. B. Goldberg. 2013. Bacterial sub- version of host innate immune pathways. 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