Identification of Signaling Pathways by Which CD40 StimulatesAutophagy and Antimicrobial Activity against Toxoplasma gondii inMacrophages

Elizabeth Liu,a Yalitza Lopez Corcino,b Jose-Andres C. Portillo,b Yanling Miao,b Carlos S. Subaustea,b,c

Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USAa; Division of Infectious Diseases and HIV Medicine, Department of Medicine, CaseWestern Reserve University, Cleveland, Ohio, USAb; Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio, USAc

CD40 is an important stimulator of autophagy and autophagic killing of Toxoplasma gondii in host cells. In contrast to au-tophagy induced by nutrient deprivation or pattern recognition receptors, less is known about the effects of cell-mediated im-munity on Beclin 1 and ULK1, key regulators of autophagy. Here we studied the molecular mechanisms by which CD40 stimu-lates autophagy in macrophages. CD40 ligation caused biphasic Jun N-terminal protein kinase (JNK) phosphorylation. Thesecond phase of JNK phosphorylation was dependent on autocrine production of tumor necrosis factor alpha (TNF-�). TNF-�and JNK signaling were required for the CD40-induced increase in autophagy. JNK signaling downstream of CD40 caused Ser-87phosphorylation of Bcl-2 and dissociation between Bcl-2 and Beclin 1, an event known to stimulate the autophagic function ofBeclin 1. However, TNF-� alone was unable to stimulate autophagy. CD40 also stimulated autophagy via a pathway that in-cluded calcium/calmodulin-dependent kinase kinase � (CaMKK�), AMP-activated protein kinase (AMPK), and ULK1. CD40caused AMPK phosphorylation at its activating site, Thr-172. This effect was mediated by CaMKK� and was not impaired byneutralization of TNF-�. CD40 triggered AMPK-dependent Ser-555 phosphorylation of ULK1. CaMKK�, AMPK, and ULK1were required for CD40-induced increase in autophagy. CD40-mediated autophagic killing of Toxoplasma gondii is known torequire TNF-�. Knockdown of JNK, CaMKK�, AMPK, or ULK1 prevented T. gondii killing in CD40-activated macrophages.The second phase of JNK phosphorylation—Bcl-2 phosphorylation—Bcl-2–Beclin 1 dissociation and AMPK phosphorylation-ULK1 phosphorylation occurred simultaneously at �4 h post-CD40 stimulation. Thus, CaMKK� and TNF-� are upstream mol-ecules by which CD40 acts on ULK1 and Beclin 1 to stimulate autophagy and killing of T. gondii.

Macroautophagy (commonly referred as autophagy) is a con-served cellular mechanism where portions of the cytosol or

organelles are encircled by an isolation membrane, leading to theformation of an autophagosome (1). This structure fuses withlysosomes, resulting in an autolysosome and degradation of itscargo (1). The Unc-51-like kinase 1/2 (ULK1/2) complex (analogof the autophagy-related 1 [Atg1] of yeast) and the Beclin1–phosphatidylinositol 3-kinase catalytic subunit type 3 (PI3KC3;also known as VPS34) complex play a central role in the initiationof autophagy in response to nutrient deprivation in mammals(2–4). The activation of both the ULK1/2 complex and the Beclin1-PI3KC3 complex drives the recruitment of Atg proteins to theisolation membrane, promoting autophagosome formation andmaturation (1).

ULK1 is regulated by kinases that sense nutrient and energystatus: AMP-activated protein kinase (AMPK) and mechanistictarget of rapamycin complex 1 (mTORC1). ULK1 is activated byAMPK in response to falling energy status, and as a result, au-tophagy is stimulated (5–7). In contrast, ULK1 is inhibited bymTORC1 under nutrient-rich conditions, leading to the inhibi-tion of autophagy (8). Beclin 1 can be regulated through proteininteractions. Beclin 1 binds proteins that can either promote orinhibit autophagy (e.g., Atg14L and Bcl-2 family members, re-spectively) (9–11).

Autophagy can be stimulated by pattern recognition receptors,including Toll-like receptor (TLR) and nucleotide-binding oli-gomerization domain-containing protein 2 (NOD2). TLR4 in-duces K63-linked ubiquitination of Beclin 1 followed by dissoci-ation of Beclin 1 from Bcl-2 and stimulation of autophagy (12).

Immunity-related GTPase M (IRGM) links NOD2 to the au-tophagy pathway. NOD2 enhances K63-linked ubiquitination ofIRGM, enabling IRGM to interact with ULK1 and Beclin 1 (13).IRGM also stimulates autophagy by activating AMPK (13).

Autophagy can be stimulated by cell-mediated immunitythrough cytokines such as gamma interferon (IFN-�) and type Iinterferon as well as CD40. The induction of autophagy by IFN-�is reported to occur independently of Irgm1 (14). IFN-� functionsthrough ATF6 and C/EBP-�, transcription factors that upregulatedeath-associated protein kinase (DAPK) (15), a molecule previ-ously reported to cause Beclin 1–Bcl-XL dissociation and enhanceautophagy (16). The activity of ATF6 is regulated by p38 mitogen-activated protein kinase (MAPK) (17), likely explaining whyIFN-� requires p38 MAPK signaling to stimulate autophagy (14).Type I IFN stimulates autophagy through JAK/STAT signaling(18), although it is not clear how this signaling pathway affects thefunction of autophagy proteins. Taken together, less is known

Received 1 February 2016 Returned for modification 23 March 2016Accepted 21 June 2016

Accepted manuscript posted online 27 June 2016

Citation Liu E, Lopez Corcino Y, Portillo J-AC, Miao Y, Subauste CS. 2016.Identification of signaling pathways by which CD40 stimulates autophagy andantimicrobial activity against Toxoplasma gondii in macrophages. Infect Immun84:2616 –2626. doi:10.1128/IAI.00101-16.

Editor: J. H. Adams, University of South Florida

Address correspondence to Carlos S. Subauste, carlos.subauste@case.edu.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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about how cellular immunity activates upstream molecules thatregulate autophagy, particularly ULK1.

The interaction of CD40 with CD154 (CD40 ligand) increasesthe formation of autophagosomes and autolysosomes (increasedautophagy flux) and increases the conversion of the autophagyprotein LC3 I to LC3 II, events that require Atg5, Atg7, and Beclin1 (19–24). CD40 triggers autophagy-mediated killing of Toxo-plasma gondii (19–21, 23, 24) and probably of Mycobacterium tu-berculosis (25). CD40 ligation in mammalian cells results in theencasement of T. gondii by an LC3-positive (LC3�) structure, fol-lowed by Rab7-mediated vacuole-lysosome fusion and parasitekilling dependent on Atg5, Atg7, Beclin 1, PI3KC3, protein kinasedouble-stranded RNA-dependent (PKR), and lysosomal enzymes(19–21, 23, 24). These events are relevant to protection againsttoxoplasmosis since CD40�/�, Becn1�/�, and PKR�/� mice ormice with an Atg7 deficiency targeted to macrophages/microgliaare susceptible to cerebral and ocular toxoplasmosis (20, 21).

Here we report that calcium/calmodulin-dependent kinase ki-nase � (CaMKK�) and autocrine tumor necrosis factor alpha(TNF-�) are upstream molecules by which CD40 triggers signal-ing cascades that act on ULK1 and Beclin 1 and stimulates au-tophagy. CD40 triggers CaMKK�-dependent Thr-172 phosphor-ylation of AMPK and AMPK-dependent Ser-555 phosphorylationof ULK1 and stimulates autophagy via these signaling molecules.In addition, CD40 stimulates autophagy via autocrine TNF-�production followed by Jun N-terminal protein kinase (JNK)phosphorylation, Ser-87 phosphorylation of Bcl-2, and dissocia-tion of Bcl-2 from Beclin 1. CD40 induces T. gondii killing throughCaMKK�, AMPK, ULK1, and JNK. These findings, together withour previous report that TNF-� is required for CD40-inducedautophagic killing of T. gondii (22), indicate that CD40 requiresboth upstream molecules to induce killing of T. gondii.

MATERIALS AND METHODSMacrophages. RAW 264.7 cells that express a chimera of the extracellulardomain of human CD40 and the intracytoplasmic domain of mouseCD40 (hmCD40-RAW 264.7) were previously described (22). hmCD40-RAW 264.7 cells were treated with multimeric CD154, a nonfunctionalCD154 mutant (T147N) (obtained from Richard Kornbluth, MultimericBiotherapeutics Inc., La Jolla, CA) (26), or recombinant soluble CD154 (1�g/ml) plus an enhancer (2 �g/ml) (Enzo Life Sciences). The specificity ofCD154 was confirmed by detection of 95% neutralization in response tocoincubation with anti-human CD154 monoclonal antibody (MAb)(Ancell Corporation). Bone marrow-derived macrophages (BMM)from C57BL/6 and TNF-��/� mice (both from Jackson Laboratories)were incubated with mouse CD154 (obtained from Richard Korn-bluth). In certain experiments, cells were incubated with the JNK in-hibitor SP600125 (50 �M; Sigma), the CaMKK� inhibitor STO-609 (1�M; Tocris), the AMPK inhibitor compound C (10 �M; Tocris), a neu-tralizing anti-TNF-� MAb, or an isotype control MAb (10 �g/ml) (eBio-science). Studies involving mice were approved by the Institutional Ani-mal Care and Use Committee of Case Western Reserve University.

T. gondii infection. Tachyzoites (RH strain) were maintained in hu-man foreskin fibroblasts. Macrophages were cultured on eight-chambertissue culture glass slides (Falcon; Becton-Dickinson Labware, FranklinLakes, NJ), followed by challenge for 1 h with T. gondii tachyzoites. Mono-layers were washed to remove extracellular parasites. At the indicated timepoints, monolayers were fixed and stained with Diff-Quick (Dade Diag-nostics, Aguada, Puerto Rico). The percentages of infected macrophagesand the numbers of parasites per 100 cells in triplicate monolayers weredetermined by light microscopy by counting at least 200 cells per mono-layer (19, 21).

Transfections. hmCD40-RAW 264.7 cells were transfected withJNK1/2 small interfering RNA (siRNA) (Dharmacon), ULK1 siRNA (LifeTechnologies), CaMKK� siRNA (27), AMPK�1 siRNA (27), AMPK�2siRNA (27), or control siRNA by using an Amaxa Nucleofector kit. Cellswere subsequently transfected with a plasmid encoding tandem mono-meric red fluorescent protein (RFP)-green fluorescent protein (GFP)-tagged LC3 (tfLC3) (28) (gift from T. Yoshimori, National Institute forBasic Biology, Okazaki, Japan).

Immunofluorescence. To assess autophagy flux, hmCD40-RAW264.7 cells expressing tfLC3 were cultured with or without CD154 for 6 hand fixed with 4% paraformaldehyde. Slides were analyzed by fluores-cence microscopy for distinct LC3-positive structures (20).

Immunoblotting. Samples were probed with antibodies (Abs) to totalJNK, phospho-JNK (Thr183/Tyr185), total ULK1, phospho-ULK1(Ser555), total AMPK, phospho-AMPK (Thr172), CaMKK�, total raptor,or phospho-raptor (Ser792) (all from Cell Signaling); total Bcl-2, phos-pho-Bcl-2 (Ser87), or actin (Santa Cruz Biotechnologies); or p62/SQSTM1 (Proteintech Group), followed by incubation with the corre-sponding secondary Ab conjugated to horseradish peroxidase (Santa CruzBiotechnologies). Bands were visualized by using a chemiluminescencekit (Pierce Bioscience). Densitometric analysis of band intensities wasconducted by using ImageJ software (NIH). Both the 46- and 54-kDabands of JNK were used for densitometry.

Immunoprecipitation. Lysates were immunoprecipitated by incuba-tion with an antibody to Bcl-2 (Santa Cruz Biotechnologies) overnight at4°C. Protein complexes were captured by incubation with protein G beads(Sigma) for 2 h at 4°C, followed by washing using a buffer containingprotease and phosphatase inhibitors. Beads were resuspended in samplebuffer and boiled. Immunoprecipitates were immunoblotted for eitherBcl-2 (Santa Cruz Biotechnologies) or Beclin 1 (Cell Signaling).

Cytokine enzyme-linked immunosorbent assay (ELISA). Cells wereincubated with or without CD154. Supernatants were collected at 4 hand used to determine concentrations of TNF-� (eBioscience, SanDiego, CA).

Statistical analyses. Statistical significance was assessed by 2-tailedStudent’s t test and analysis of variance (ANOVA). Differences were con-sidered statistically significant when the P value was 0.05.

RESULTSJNK signaling is required for CD40-induced autophagy. JNKsignaling stimulates autophagy induced by starvation or ceramide(29, 30). We examined the role of JNK in autophagy triggered byCD40. RAW 264.7 cells that express chimeric CD40, which con-sists of the extracellular domain of human CD40 and the intracel-lular domain of mouse CD40, were incubated with or withouthuman CD154. CD40 stimulation caused strong Thr-183/Tyr-185 phosphorylation of JNK at 15 min (Fig. 1A). This was fol-lowed by a second phase of JNK phosphorylation detected at 4 h.Similar biphasic JNK phosphorylation was observed when BMMfrom C57BL/6 mice were incubated with mouse CD154 (Fig. 1B).Next, we determined whether CD40 ligation required JNK signal-ing to stimulate autophagy. Incubation of hmCD40 RAW 264.7cells with the JNK inhibitor SP600125 diminished JNK phosphor-ylation after incubation with CD154 (Fig. 1C). Cells were trans-fected with a plasmid that encodes tandem-fluorescence-labeledLC3 (tfLC3) (28). This plasmid allows the detection of autopha-gosomes (expressing both green and red fluorescence) and au-tolysosomes (expressing only red fluorescence since acidic pHquenches green fluorescence) (28). SP600125 markedly im-paired the increase in the number of LC3� structures compat-ible with autophagosomes (LC3-GFP� and LC3-RFP�) andautolysosomes (only LC3-RFP�) observed in cells incubatedwith CD154 (Fig. 1C). The levels of p62/SQSTM1 are inversely

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FIG 1 JNK signaling is required for CD40-induced autophagy. (A) hmCD40 RAW 264.7 cells were incubated with or without CD154, followed by assessment ofphospho-JNK and total JNK expression by immunoblotting. Densitometries of bands from CD154-treated cells were compared to those of bands from the correspond-ing control cells collected at the same time point. Densitometries for control bands for each time point were given a value of 1. Densitometry data represent means �standard errors of the means of results from 3 experiments. (B) Mouse bone marrow-derived macrophages were incubated with CD154 and examined as describedabove. (C) hmCD40-RAW 264.7 cells transfected with tfLC3 were pretreated with SP600125 or vehicle for 1 h, followed by the addition of CD154. The expression ofphospho-Thr183/Tyr185 JNK and total JNK was assessed by immunoblotting. The average numbers of autophagosomes (arrows) or autolysosomes (arrowheads) percell were determined by fluorescence microscopy at 6 h. DMSO, dimethyl sulfoxide. (D) hmCD40-RAW 264.7 cells were pretreated with SP600125 or vehicle for 1 h,followed by the addition of CD154. Expression levels of p62/SQSTM1 and actin were assessed by immunoblotting at 24 h. (E) hmCD40-RAW 264.7 cells were transfectedwith control or JNK siRNA, followed by transfection with tfLC3. Total JNK and actin expression levels were assessed by immunoblotting. Average numbers ofautophagosomes and autolysosomes per cell were determined as described above after 6 h of incubation with or without CD154. Results are shown as means � standarderrors of the means and are representative of data from 3 independent experiments. ***, P 0.001.

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correlated with autophagic activity (31). CD154 stimulation de-creased p62/SQSTM1 expression (Fig. 1D). This effect was pre-vented by SP600125 (Fig. 1D). JNK1/2 knockdown also markedlyimpaired the increase in the number of autophagosomes/autoly-sosomes in cells incubated with CD154 (Fig. 1E). Thus, JNK sig-naling is required for the upregulation of autophagy in response toCD40 ligation.

TNF-� mediates the second phase of JNK phosphorylationand is required for CD40-induced autophagy. CD40 ligation inhmCD40 RAW 264.7 cells caused autocrine production of TNF-�that was detected 4 h after incubation with CD154 (Fig. 2A). The

CD40-driven increase in the numbers of autophagosomes andautolysosomes was markedly impaired by neutralization ofTNF-� (Fig. 2B). Incubation with a neutralizing anti-TNF-� MAbablated the second phase of JNK phosphorylation observed inhmCD40 RAW 264.7 cells stimulated with CD154 (Fig. 2C). Incontrast, TNF-� neutralization had no appreciable effect onthe early phase of JNK phosphorylation. In agreement withthese results, BMM from C57BL/6 mice but not from TNF-��/� animals exhibited the second phase of JNK phosphoryla-tion after incubation with mouse CD154 (Fig. 2D). Thus, au-tocrine production of TNF-� is required for the second phase

FIG 2 TNF-� mediates the second phase of JNK phosphorylation after CD40 ligation and is required for CD40-induced autophagy. (A) hmCD40 RAW 264.7cells were incubated with or without CD154 for 4 h. TNF-� production was examined by an ELISA. (B) hmCD40 RAW 264.7 cells transfected with tfLC3 werepreincubated with an anti-TNF-� or isotype control MAb for 1 h, followed by the addition of CD154. The average numbers of autophagosomes and autolyso-somes per cell were determined by fluorescence microscopy at 6 h. (C) hmCD40 RAW 264.7 cells were incubated with an anti-TNF-� or isotype control MAb,followed by the addition of CD154. Phospho-Thr183/Tyr185 JNK and total JNK were assessed by immunoblotting. (D) Bone marrow-derived macrophagesfrom C57BL/6 (B6) and TNF-��/� mice were incubated with CD154. Phospho-Thr183/Tyr185 JNK and total JNK levels were assessed by immunoblotting.Densitometries of bands from CD154-treated macrophages were compared to bands from their corresponding control macrophages. Densitometries for eachcontrol band were given a value of 1. Densitometry data represent means � standard errors of the means of results from 3 experiments. Results shown arerepresentative of data from 3 independent experiments. ***, P 0.001.

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of JNK phosphorylation and the increased autophagy triggeredby CD40 ligation.

CD40 ligation causes Bcl-2 phosphorylation that is depen-dent on TNF-� and JNK signaling. JNK-mediated Bcl-2 phos-phorylation at Ser-87 results in the dissociation of Bcl-2 fromBeclin 1, enabling the binding of Beclin 1 to PI3KC3 and the ini-tiation of autophagy (29). Incubation of hmCD40 RAW 264.7cells with CD154 increased Bcl-2 phosphorylation at Ser-87,which was detected at 4 h (Fig. 3A). A neutralizing anti-TNF-�MAb or SP600125 prevented Bcl-2 phosphorylation in hmCD40RAW264.7 cells treated with CD154 (Fig. 3B and C). Thus, CD40triggered the phosphorylation of Bcl-2 at Ser-87 that was mediatedby TNF-� and JNK signaling.

CD40 ligation diminishes Bcl-2–Beclin 1 association via JNKsignaling. hmCD40 RAW 264.7 cells were incubated with or with-out CD154 to examine the effects of CD40 stimulation on theassociation of Bcl-2 with Beclin 1. Beclin 1 was immunoprecipi-tated with Bcl-2 under basal conditions (Fig. 4A). However, CD40stimulation reduced the immunoprecipitation of Beclin 1 withBcl-2. Next, we examined the effects of JNK signaling on the asso-

ciation between Bcl-2 and Beclin 1. In the presence of SP600125,incubation with CD154 no longer diminished the immunopre-cipitation of Beclin 1 with Bcl-2 (Fig. 4B). Thus, CD40 stimulationappears to decrease the association of Bcl-2 with Beclin 1 in amanner that is largely dependent on JNK signaling.

CD40 ligation triggers CaMKK�-, AMPK-, and ULK1-de-pendent autophagy. While TNF-� is required for CD40-inducedautophagy, TNF-� alone was unable to stimulate this process(Fig. 5A). These results were observed even at concentrations ofTNF-� that were 7 times higher than those detected in cells incu-bated with CD154. Thus, we explored whether CD40 ligation ac-tivates other events that are crucial for the stimulation of au-tophagy.

ULK1 is a key initiator of autophagy (3–7). Indeed, stimulationwith CD154 caused the phosphorylation of ULK1 at Ser-555, aresidue that becomes phosphorylated in activated ULK1 (5, 7, 32)(Fig. 5B). Next, we determined whether CD40 ligation requiredULK1 to stimulate autophagy. ULK1 knockdown preventedCD154 from increasing the numbers of autophagosomes and au-tolysosomes and diminishing p62/SQSTM1 expression (Fig. 5C).

FIG 3 CD40 ligation causes phosphorylation of Bcl-2 that is dependent on TNF-� and JNK. (A) hmCD40 RAW 264.7 cells were incubated with or withoutCD154. Phospho-Ser87 Bcl-2 and total Bcl-2 levels were assessed by immunoblotting. Densitometries of bands from CD154-treated cells were compared to thoseof bands from the corresponding control cells collected at the same time point. Densitometries for control bands for each time point were given a value of 1. (Band C) hmCD40 RAW 264.7 cells were incubated with anti-TNF-� versus an isotype control MAb (B) or SP600125 versus vehicle (C), followed by the additionof CD154. Phospho-Ser87 Bcl-2 and total Bcl-2 levels were assessed by immunoblotting at 4 h. Densitometry data represent means � standard errors of the meansof results from 3 experiments. Results are representative of data from 3 independent experiments.

FIG 4 CD40 ligation causes Bcl-2–Beclin 1 dissociation that is dependent on JNK signaling. (A) hmCD40-RAW 264.7 cells were incubated with or withoutCD154 for 4 h. Lysates were immunoprecipitated by incubation with an anti-Bcl-2 antibody and immunoblotted as indicated. Results are representative of datafrom 3 independent experiments. (B) hmCD40-RAW 264.7 cells were pretreated with SP600125 or vehicle for 1 h, followed by the addition of CD154 for 4 h.Lysates were subjected to immunoprecipitation (IP) and immunoblotting as described above. Results are representative of data from 3 independent experiments.WCL, whole-cell lysate; WB, Western blotting.

Liu et al.

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ULK1 is activated by AMPK under conditions of nutrient de-privation (5–7). We examined the role of AMPK in CD40-in-duced ULK1 phosphorylation and autophagy. After 4 h of incu-bation with CD154, hmCD40 RAW 264.7 cells exhibitedincreased AMPK phosphorylation at Thr-172 (Fig. 6A). Phos-phorylation of this residue causes marked AMPK activation (33).The addition of a neutralizing anti-TNF-� MAb failed to impairAMPK phosphorylation, indicating that, in contrast to the secondphase of JNK phosphorylation, CD40 enhanced Thr-172 phos-phorylation of AMPK independently of autocrine TNF-� produc-tion (Fig. 6B). Ser-555 phosphorylation of ULK1 was dependenton AMPK, since treatment of hmCD40 RAW 264.7 cells with theAMPK inhibitor compound C impaired the ability of CD154 tocause Ser-555 ULK1 phosphorylation (Fig. 6C). Thus, CD40causes AMPK-dependent Ser-555 phosphorylation of ULK1 andtriggers ULK1-dependent autophagy.

AMPK also stimulates autophagy through effects on mTORC1,an inhibitor of this process. AMPK causes Ser-792 phosphoryla-tion of raptor, an mTORC1 binding partner, and inhibitsmTORC1 (34). CD40 ligation stimulated Ser-792 phosphoryla-tion of raptor, and this effect was impaired by compound C (Fig.6D). These findings provide additional evidence of CD40-inducedAMPK signaling.

CD40 ligation increases intracytoplasmic calcium concentra-tions (35, 36). We examined whether CD40 causes AMPK phos-phorylation via CaMKK� since this signaling molecule is activatedby increased Ca2� concentrations and CaMKK� can activateAMPK (37, 38). Figure 6E shows that the knockdown of CaMKK�ablated AMPK phosphorylation at Thr-172 induced by CD40 li-gation. Next, we determined whether CD40 requires CaMKK�

and AMPK to stimulate autophagy. Cells were incubated withthe selective CaMKK� inhibitor STO-609 at 1 �M, a concen-tration that does not impair AMPK activation induced by liverkinase B1 (LKB1), the other major stimulator of AMPK (37).Treatment of hmCD40 RAW 264.7 cells with STO-609 or theAMPK inhibitor compound C impaired the ability of CD154 toincrease the numbers of autophagosomes and autolysosomes aswell as diminish p62/SQSTM1 expression (Fig. 6F). Moreover,knockdown of CaMKK� or AMPK1/2 ablated the ability ofCD154 to increase the numbers of autophagosomes and autoly-sosomes (Fig. 6G). Taken together, a pathway that includesCaMKK�, AMPK, and ULK1 is also required for CD40 to stimu-late autophagy.

JNK, CaMKK�, AMPK, and ULK1 are required for CD40-induced killing of T. gondii. CD40 signaling results in autophagy-dependent killing of T. gondii characterized by the encasement ofthe parasites by LC3 and parasite killing dependent on Atg5, Atg7,Beclin 1, PI3KC3, and lysosomal enzymes (19, 21, 23, 24). Weexamined the role of JNK, CaMKK�, AMPK, and ULK in CD40-induced parasite killing. CD40 ligation in hmCD40 RAW 264.7cells transfected with control siRNA reduced the percentage ofinfected cells and the number of parasites per 100 macrophages(Fig. 7A). Knockdown of JNK1/2, CaMKK�, AMPK1/2, or ULK1ablated the killing of T. gondii (Fig. 7A). Studies with BMM re-vealed that CD40-induced killing of T. gondii is dependent onautocrine production of TNF-� (22). Similarly, pharmacologicalinhibition of CaMKK�, the molecule upstream of AMPK andULK1, prevented CD40-induced parasite killing in BMM fromC57BL/6 mice (Fig. 7B). Of note, the CaMKK� inhibitor STO-609did not affect the initial percentage of infected BMM (2 h post-

FIG 5 ULK1 is required for CD40-induced autophagy. (A) hmCD40 RAW 264.7 cells transfected with the tfLC3 plasmid were incubated with increasingconcentrations of mouse recombinant TNF-�. The numbers of autophagosomes or autolysosomes were determined by fluorescence microscopy at 6 h. (B)hmCD40 RAW 264.7 cells were incubated with or without CD154, followed by assessment of phospho-Ser555 ULK1 and total ULK1 expression levels byimmunoblotting. (C) hmCD40-RAW 264.7 cells were transfected with control or ULK1 siRNA, followed by transfection with tfLC3. ULK1 and actin expressionlevels were assessed by immunoblotting. The average numbers of autophagosomes and autolysosomes per cell were determined after 6 h of incubation with orwithout CD154. Expression levels of p62/SQSTM1 and actin were assessed by immunoblotting of cells transfected with control or ULK1 siRNA and incubatedwith or without CD154 for 24 h. Results are shown as means � standard errors of the means and are representative of data from 3 independent experiments. ***,P 0.001.

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FIG 6 CaMKK�-dependent AMPK signaling is required for CD40-induced autophagy. (A) hmCD40 RAW 264.7 cells were incubated with or without CD154,followed by assessment of phospho-Thr172 AMPK and total AMPK expression levels by immunoblotting. Densitometry data represent means � standard errorsof the means of results from 3 experiments. (B) hmCD40 RAW 264.7 cells were incubated with or without anti-TNF-� MAb, followed by stimulation withCD154. Total AMPK and phospho-Thr1174 AMPK levels were assessed by immunoblotting at 4 h. Densitometry data represent means � standard errors of themeans of results from 3 experiments. (C) hmCD40 RAW 264.7 cells were incubated with or without compound C (CC), followed by stimulation with humanCD154. Expression levels of phospho-Ser555 ULK1 and total ULK1 were assessed by immunoblotting at 4 h. Densitometry data represent means � standarderrors of the means of results from 3 experiments. (D) hmCD40 RAW 264.7 cells were incubated with or without compound C, followed by stimulation withCD154. Expression levels of phospho-Ser 792 raptor and total raptor were assessed by immunoblotting at 4 h. (E) hmCD40-RAW 264.7 cells were transfectedwith the control or CaMKK�. CaMKK� and actin expression levels were assessed by immunoblotting. Total AMPK and phospho-Thr172 AMPK levels wereassessed by immunoblotting 4 h after incubation with CD154. Densitometry data represent means � standard errors of the means of results from 3 experiments.(F) hmCD40-RAW 264.7 cells transfected with tfLC3 were pretreated with STO-609, compound C, or vehicle for 1 h, followed by the addition of human CD154.The average numbers of autophagosomes or autolysosomes per cell were determined by fluorescence microscopy at 6 h. Expression levels of p62/SQSTM1 andactin were assessed by immunoblotting at 24 h. (G) hmCD40-RAW 264.7 cells were transfected with control, CaMKK�, or AMPK1/2 siRNA, followed bytransfection with tfLC3. Total AMPK and actin expression levels were assessed by immunoblotting. Numbers of autophagosomes and autolysosomes weredetermined as described above after 6 h of incubation with or without CD154. Results are shown as means � standard errors of the means and are representativeof data from 3 independent experiments. ***, P 0.001.

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challenge) (not shown). Taken together, in addition to TNF-�(22), JNK, CaMKK�, AMPK, and ULK1 are required for the kill-ing of T. gondii triggered by CD40.

DISCUSSION

Initiation of autophagy requires both ULK1 and the Beclin1-PI3KC3 complex. This study identifies biochemical mecha-nisms triggered by CD40 that act upstream of ULK1 and Beclin 1.CD40 induces CaMKK�-mediated Thr-172 AMPK phosphoryla-tion, a marker of AMPK activation. In turn, AMPK signalingcauses Ser-555 ULK1 phosphorylation and ULK1-mediated au-tophagy. The second mechanism is dependent on CD40-inducedautocrine production of TNF-� that causes JNK-dependent phos-phorylation of Bcl-2 at Ser-87 and dissociation of Bcl-2 from Be-clin 1 (Fig. 8). The release of Beclin 1 from Bcl-2 was reported toallow the binding of Beclin 1 to PI3KC3 and the initiation ofautophagy (11). Interestingly, these mechanisms occur at approx-imately the same time after CD40 engagement. This synchroniza-tion likely optimizes the ability of CD40 to stimulate autophagy.The roles of these molecules are relevant to the killing of T. gondiiinduced by CD40, a process mediated by autophagy (19, 21, 23,24). Not only TNF-� (22) but also JNK, CaMKK�, AMPK, andULK1 are required for T. gondii killing induced by CD40.

ULK1 is key for the stimulation of canonical autophagy inmammalian cells (2–4). CD40 causes AMPK-dependent phos-phorylation of ULK1 at Ser-555. This residue is a major site phos-phorylated by AMPK (5, 7, 32). Moreover, AMPK-mediatedULK1 phosphorylation activates ULK1 kinase activity (6), an ef-fect that appears essential for autophagosome formation (39). In-deed, the stimulation of autophagy in CD40-activated macro-phages requires both AMPK and ULK1. In addition to ULK1,ULK2 is another mammalian homologue that closely resemblesAtg1 (40). Both ULK1 and ULK2 regulate autophagy, and theireffect can be redundant under certain conditions (mouse embry-onal fibroblasts subjected to starvation) (41, 42). However, simi-larly to our studies, a deficiency of ULK1 was sufficient to impairautophagy in human embryonal kidney 293 cells subjected toamino acid starvation or treated with rapamycin and in cerebellargranule neurons exposed to low-potassium and serum-free con-ditions (42, 43).

In addition to direct effects on the ULK1/2 complex, AMPK

can stimulate autophagy through effects on mTORC1 and Beclin1. Evidence that CD40 ligation causes Ser-792 phosphorylation ofraptor raises the possibility that AMPK-mediated inhibition ofmTORC1 may be another mechanism for the stimulation of au-tophagy after CD40 ligation. AMPK also stimulates autophagythrough the phosphorylation of Beclin 1 at Ser-91 and Ser-94 fol-lowing glucose starvation (44).

AMPK is an energy sensor and a key regulator of autophagy(5–7). The kinases LKB and CaMKK� activate AMPK in responseto low AMP levels and an increase in the cytoplasmic Ca2� con-centration, respectively (37, 45). Our work identified CD40 as anactivator of AMPK, an effect that requires CaMKK�. Of relevanceto these findings, CD40 has been reported to increase cytoplasmicCa2� concentrations (35, 36), and experimental evidence indi-cates that Ca2� modulates autophagy. It has been proposed thatan increase in the cytoplasmic Ca2� concentration would enhanceautophagy if it occurs under conditions of cellular stress (46) or ifit takes place in nonexcitable cells (47). CaMKK� may also pro-mote autophagy in CD40-activated macrophages because of ac-companying events such as Beclin 1–Bcl-2 dissociation. The coex-istence of Beclin 1–Bcl-2 dissociation may also explain whyCaMKK� downstream of CD40 signaling triggers rapid inductionof autophagy (5 to 6 h after CD40 ligation), whereas CaMKK�signaling triggered by vitamin D stimulates autophagy after 1 to 3days (48, 49).

The Beclin 1-PI3KC3 complex is central to the formation ofautophagosomes. This complex can be regulated through interac-tions with various proteins. Under normal conditions, Bcl-2 bindsto the BH3 domain of Beclin 1 (11). This prevents the binding ofBeclin 1 to PI3KC3 and the initiation of autophagy (11). JNK1signaling triggered by starvation mediates Bcl-2 phosphorylationat Thr-69, Ser-70, and Ser-87 (29). In turn, this releases Beclin 1from Bcl-2 and stimulates autophagy (29). Our studies revealedthat CD40 activates this cascade of events through JNK signalingmediated by CD40-induced autocrine production of TNF-�. Ad-ditional mechanisms that stimulate autophagy through Beclin1-protein interactions include TRAF6-mediated ubiquitina-tion of Lys-117 in the BH3 domain of Beclin 1 with subsequentrelease from Bcl-2 (12), DAPK-mediated phosphorylation ofThr-199 in the BH3 domain of Beclin 1 followed by dissocia-tion from Bcl-XL (16), and ULK1-mediated phosphorylation

FIG 7 JNK, CaMKK�, AMPK, and ULK1 are required for CD40-induced killing of T. gondii. (A) hmCD40-RAW 264.7 cells were transfected with control siRNAor siRNA against JNK1/2, CaMKK�, AMPK1/2, or ULK1, followed by incubation with or without CD154 and challenge with T. gondii. Percentages of infectedmacrophages and numbers of tachyzoites/100 macrophages were determined by light microscopy. (B) BMM from C57BL/6 mice were pretreated with STO-609or vehicle for 1 h, followed by the addition of CD154 and challenge with T. gondii. Results are shown as means � standard errors of the means and arerepresentative of data from 2 to 3 independent experiments. **, P 0.01.

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of activating molecule in Beclin 1-regulated autophagy 1(AMBRA 1) causing the release of Beclin 1 from the Dyneinmotor complex (50). The fact that the inhibition of JNK sig-naling prevented CD40 from decreasing the Beclin 1–Bcl-2 as-sociation suggests that CD40 modulates the Beclin 1–Bcl-2 in-teraction largely through JNK signaling.

We previously reported that CD40 increases Beclin 1 proteinlevels through downregulation of p21, a protein that degradesBeclin 1 (21) (Fig. 8). This event is functionally relevant sinceBeclin 1 overexpression enables suboptimal CD40 ligation to trig-ger autophagic killing of T. gondii, and CD40 fails to trigger par-asite killing when Beclin 1 upregulation is prevented (21). Thetiming of Beclin 1 upregulation (3 to 4 h post-CD40 ligation)corresponds to the timing of Bcl-2 phosphorylation and Beclin1–Bcl-2 dissociation. This synchronization of events together withevidence that CD40 also activates PKR (20), an important pro-moter of autophagy, may assist in optimizing the ability of CD40ligation to stimulate autophagy and the induction of toxoplas-macidal activity.

TNF-� has been reported to cause autophagy. However, thiseffect required silencing of NF-�B (51), prolonged (48-h) in-cubation with TNF-� (52), and high TNF-� concentrations (10to 200 ng/ml) (52–54); it was restricted to certain cell types(rhabdomyosarcoma) or occurred in the presence of a co-stimulant (receptor activator of nuclear factor kappa-� ligand[RANKL]) (53). While CD40 requires TNF-� to induce au-tophagy, this cytokine alone cannot supplant the effect of CD40ligation. Indeed, we found that TNF-� alone was unable torapidly induce autophagy in macrophages. Moreover, whereasCD40 ligation caused autophagic killing of T. gondii in macro-phages, TNF-� alone was unable to induce anti-T. gondii activity(22).

The studies presented here represent a significant step towardunderstanding how cell-mediated immunity stimulates au-tophagy and triggers anti-T. gondii activity in macrophages. Stud-ies that further explore the molecular events responsible forCD40-driven stimulation of autophagy may have implications for

the development of novel therapeutic modalities against toxoplas-mosis given the importance of CD40 and autophagy in the activa-tion of toxoplasmacidal activity and protection against ocular andcerebral toxoplasmosis (20, 21).

ACKNOWLEDGMENTS

We thank Richard Kornbluth and Tamotsu Yoshimori for providing re-agents. We thank Scott Howell for image collection.

FUNDING INFORMATIONThis work, including the efforts of Carlos S. Subauste, was funded by HHS| National Institutes of Health (NIH) (EY018341). This work, includingthe efforts of Elizabeth Liu, was funded by HHS | National Institutes ofHealth (NIH) (T32 EY007157).

The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

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