Identification of Signaling Pathways by Which CD40 Stimulates Autophagy and Antimicrobial Activity against Toxoplasma gondii in Macrophages

CD40 is an important stimulator of autophagy and autophagic killing of Toxoplasma gondii in host cells. In contrast to autophagy induced by nutrient deprivation or pattern recognition receptors, less is known about the effects of cell-mediated immunity on Beclin 1 and ULK1, key regulators of autophagy. Here we studied the molecular mechanisms by which CD40 stimulates autophagy in macrophages. CD40 ligation caused biphasic Jun N-terminal protein kinase (JNK) phosphorylation. The second phase of JNK phosphorylation was dependent on autocrine production of tumor necrosis factor alpha (TNF-α). TNF-α and JNK signaling were required for the CD40-induced increase in autophagy. JNK signaling downstream of CD40 caused Ser-87 phosphorylation of Bcl-2 and dissociation between Bcl-2 and Beclin 1, an event known to stimulate the autophagic function of Beclin 1. However, TNF-α alone was unable to stimulate autophagy. CD40 also stimulated autophagy via a pathway that included calcium/calmodulin-dependent kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and ULK1. CD40 caused AMPK phosphorylation at its activating site, Thr-172. This effect was mediated by CaMKKβ and was not impaired by neutralization of TNF-α. CD40 triggered AMPK-dependent Ser-555 phosphorylation of ULK1. CaMKKβ, AMPK, and ULK1 were required for CD40-induced increase in autophagy. CD40-mediated autophagic killing of Toxoplasma gondii is known to require TNF-α. Knockdown of JNK, CaMKKβ, 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, CaMKKβ and TNF-α are upstream molecules by which CD40 acts on ULK1 and Beclin 1 to stimulate autophagy and killing of T. gondii.

 

INTRODUCTION

Macroautophagy (commonly referred as autophagy) is a conserved cellular mechanism where portions of the cytosol or organelles are encircled by an isolation membrane, leading to the formation of an autophagosome (1). This structure fuses with lysosomes, resulting in an autolysosome and degradation of its cargo (1). The Unc-51-like kinase 1/2 (ULK1/2) complex (analog of the autophagy-related 1 [Atg1] of yeast) and the Beclin 1–phosphatidylinositol 3-kinase catalytic subunit type 3 (PI3KC3; also known as VPS34) complex play a central role in the initiation of autophagy in response to nutrient deprivation in mammals (2,–4). The activation of both the ULK1/2 complex and the Beclin 1-PI3KC3 complex drives the recruitment of Atg proteins to the isolation membrane, promoting autophagosome formation and maturation (1).

ULK1 is regulated by kinases that sense nutrient and energy status: AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1). ULK1 is activated by AMPK in response to falling energy status, and as a result, autophagy is stimulated (5,–7). In contrast, ULK1 is inhibited by mTORC1 under nutrient-rich conditions, leading to the inhibition of autophagy (8). Beclin 1 can be regulated through protein interactions. Beclin 1 binds proteins that can either promote or inhibit autophagy (e.g., Atg14L and Bcl-2 family members, respectively) (9,–11).

Autophagy can be stimulated by pattern recognition receptors, including Toll-like receptor (TLR) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2). TLR4 induces K63-linked ubiquitination of Beclin 1 followed by dissociation of Beclin 1 from Bcl-2 and stimulation of autophagy (12). Immunity-related GTPase M (IRGM) links NOD2 to the autophagy pathway. NOD2 enhances K63-linked ubiquitination of IRGM, 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 immunity through cytokines such as gamma interferon (IFN-γ) and type I interferon as well as CD40. The induction of autophagy by IFN-γ is reported to occur independently of Irgm1 (14). IFN-γ functions through ATF6 and C/EBP-β, transcription factors that upregulate death-associated protein kinase (DAPK) (15), a molecule previously reported to cause Beclin 1–Bcl-X_L dissociation and enhance autophagy (16). The activity of ATF6 is regulated by p38 mitogen-activated protein kinase (MAPK) (17), likely explaining why IFN-γ 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 the function of autophagy proteins. Taken together, less is known about how cellular immunity activates upstream molecules that regulate autophagy, particularly ULK1.

The interaction of CD40 with CD154 (CD40 ligand) increases the formation of autophagosomes and autolysosomes (increased autophagy flux) and increases the conversion of the autophagy protein LC3 I to LC3 II, events that require Atg5, Atg7, and Beclin 1 (19,–24). CD40 triggers autophagy-mediated killing of Toxoplasma gondii (19,–21, 23, 24) and probably of Mycobacterium tuberculosis (25). CD40 ligation in mammalian cells results in the encasement of T. gondii by an LC3-positive (LC3⁺) structure, followed by Rab7-mediated vacuole-lysosome fusion and parasite killing dependent on Atg5, Atg7, Beclin 1, PI3KC3, protein kinase double-stranded RNA-dependent (PKR), and lysosomal enzymes (19,–21, 23, 24). These events are relevant to protection against toxoplasmosis since CD40^−/−, Becn1^+/−, and PKR^−/− mice or mice with an Atg7 deficiency targeted to macrophages/microglia are susceptible to cerebral and ocular toxoplasmosis (20, 21).

Here we report that calcium/calmodulin-dependent kinase kinase β (CaMKKβ) and autocrine tumor necrosis factor alpha (TNF-α) are upstream molecules by which CD40 triggers signaling cascades that act on ULK1 and Beclin 1 and stimulates autophagy. CD40 triggers CaMKKβ-dependent Thr-172 phosphorylation of AMPK and AMPK-dependent Ser-555 phosphorylation of ULK1 and stimulates autophagy via these signaling molecules. In addition, CD40 stimulates autophagy via autocrine TNF-α production followed by Jun N-terminal protein kinase (JNK) phosphorylation, Ser-87 phosphorylation of Bcl-2, and dissociation of Bcl-2 from Beclin 1. CD40 induces T. gondii killing through CaMKKβ, AMPK, ULK1, and JNK. These findings, together with our previous report that TNF-α is required for CD40-induced autophagic killing of T. gondii (22), indicate that CD40 requires both upstream molecules to induce killing of T. gondii.

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MATERIALS AND METHODS

Macrophages.

RAW 264.7 cells that express a chimera of the extracellular domain of human CD40 and the intracytoplasmic domain of mouse CD40 (hmCD40-RAW 264.7) were previously described (22). hmCD40-RAW 264.7 cells were treated with multimeric CD154, a nonfunctional CD154 mutant (T147N) (obtained from Richard Kornbluth, Multimeric Biotherapeutics Inc., La Jolla, CA) (26), or recombinant soluble CD154 (1 μg/ml) plus an enhancer (2 μg/ml) (Enzo Life Sciences). The specificity of CD154 was confirmed by detection of >95% neutralization in response to coincubation with anti-human CD154 monoclonal antibody (MAb) (Ancell Corporation). Bone marrow-derived macrophages (BMM) from C57BL/6 and TNF-α^−/− mice (both from Jackson Laboratories) were incubated with mouse CD154 (obtained from Richard Kornbluth). In certain experiments, cells were incubated with the JNK inhibitor SP600125 (50 μM; Sigma), the CaMKKβ inhibitor STO-609 (1 μM; Tocris), the AMPK inhibitor compound C (10 μM; Tocris), a neutralizing anti-TNF-α MAb, or an isotype control MAb (10 μg/ml) (eBioscience). Studies involving mice were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University.

1. gondiiinfection.

Tachyzoites (RH strain) were maintained in human foreskin fibroblasts. Macrophages were cultured on eight-chamber tissue culture glass slides (Falcon; Becton-Dickinson Labware, Franklin Lakes, NJ), followed by challenge for 1 h with T. gondii tachyzoites. Monolayers were washed to remove extracellular parasites. At the indicated time points, monolayers were fixed and stained with Diff-Quick (Dade Diagnostics, Aguada, Puerto Rico). The percentages of infected macrophages and the numbers of parasites per 100 cells in triplicate monolayers were determined by light microscopy by counting at least 200 cells per monolayer (19, 21).

Transfections.

hmCD40-RAW 264.7 cells were transfected with JNK1/2 small interfering RNA (siRNA) (Dharmacon), ULK1 siRNA (Life Technologies), CaMKKβ siRNA (27), AMPKα1 siRNA (27), AMPKα2 siRNA (27), or control siRNA by using an Amaxa Nucleofector kit. Cells were subsequently transfected with a plasmid encoding tandem monomeric red fluorescent protein (RFP)-green fluorescent protein (GFP)-tagged LC3 (tfLC3) (28) (gift from T. Yoshimori, National Institute for Basic Biology, Okazaki, Japan).

Immunofluorescence.

To assess autophagy flux, hmCD40-RAW 264.7 cells expressing tfLC3 were cultured with or without CD154 for 6 h and fixed with 4% paraformaldehyde. Slides were analyzed by fluorescence microscopy for distinct LC3-positive structures (20).

Immunoblotting.

Samples were probed with antibodies (Abs) to total JNK, phospho-JNK (Thr183/Tyr185), total ULK1, phospho-ULK1 (Ser555), total AMPK, phospho-AMPK (Thr172), CaMKKβ, total raptor, or phospho-raptor (Ser792) (all from Cell Signaling); total Bcl-2, phospho-Bcl-2 (Ser87), or actin (Santa Cruz Biotechnologies); or p62/SQSTM1 (Proteintech Group), followed by incubation with the corresponding secondary Ab conjugated to horseradish peroxidase (Santa Cruz Biotechnologies). Bands were visualized by using a chemiluminescence kit (Pierce Bioscience). Densitometric analysis of band intensities was conducted by using ImageJ software (NIH). Both the 46- and 54-kDa bands of JNK were used for densitometry.

Immunoprecipitation.

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

Cytokine enzyme-linked immunosorbent assay (ELISA).

Cells were incubated with or without CD154. Supernatants were collected at 4 h and used to determine concentrations of TNF-α (eBioscience, San Diego, CA).

Statistical analyses.

Statistical significance was assessed by 2-tailed Student's t test and analysis of variance (ANOVA). Differences were considered statistically significant when the P value was <0.05.

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RESULTS

JNK signaling is required for CD40-induced autophagy.

JNK signaling stimulates autophagy induced by starvation or ceramide (29, 30). We examined the role of JNK in autophagy triggered by CD40. RAW 264.7 cells that express chimeric CD40, which consists of the extracellular domain of human CD40 and the intracellular domain of mouse CD40, were incubated with or without human CD154. CD40 stimulation caused strong Thr-183/Tyr-185 phosphorylation of JNK at 15 min (Fig. 1A). This was followed by a second phase of JNK phosphorylation detected at 4 h. Similar biphasic JNK phosphorylation was observed when BMM from C57BL/6 mice were incubated with mouse CD154 (Fig. 1B). Next, we determined whether CD40 ligation required JNK signaling to stimulate autophagy. Incubation of hmCD40 RAW 264.7 cells with the JNK inhibitor SP600125 diminished JNK phosphorylation after incubation with CD154 (Fig. 1C). Cells were transfected with a plasmid that encodes tandem-fluorescence-labeled LC3 (tfLC3) (28). This plasmid allows the detection of autophagosomes (expressing both green and red fluorescence) and autolysosomes (expressing only red fluorescence since acidic pH quenches green fluorescence) (28). SP600125 markedly impaired the increase in the number of LC3⁺ structures compatible with autophagosomes (LC3-GFP⁺ and LC3-RFP⁺) and autolysosomes (only LC3-RFP⁺) observed in cells incubated with CD154 (Fig. 1C). The levels of p62/SQSTM1 are inversely correlated with autophagic activity (31). CD154 stimulation decreased p62/SQSTM1 expression (Fig. 1D). This effect was prevented by SP600125 (Fig. 1D). JNK1/2 knockdown also markedly impaired the increase in the number of autophagosomes/autolysosomes in cells incubated with CD154 (Fig. 1E). Thus, JNK signaling is required for the upregulation of autophagy in response to CD40 ligation.

 

 

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 of phospho-JNK and total JNK expression by immunoblotting. Densitometries of bands from CD154-treated cells were compared to those of 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. 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 described above. (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 of phospho-Thr183/Tyr185 JNK and total JNK was assessed by immunoblotting. The average numbers of autophagosomes (arrows) or autolysosomes (arrowheads) per cell 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 transfected with control or JNK siRNA, followed by transfection with tfLC3. Total JNK and actin expression levels were assessed by immunoblotting. Average numbers of autophagosomes and autolysosomes per cell were determined as described above after 6 h of incubation with or without CD154. Results are shown as means ± standard errors of the means and are representative of data from 3 independent experiments. ***, P < 0.001.

TNF-α mediates the second phase of JNK phosphorylation and is required for CD40-induced autophagy.

CD40 ligation in hmCD40 RAW 264.7 cells caused autocrine production of TNF-α that was detected 4 h after incubation with CD154 (Fig. 2A). The CD40-driven increase in the numbers of autophagosomes and autolysosomes was markedly impaired by neutralization of TNF-α (Fig. 2B). Incubation with a neutralizing anti-TNF-α MAb ablated the second phase of JNK phosphorylation observed in hmCD40 RAW 264.7 cells stimulated with CD154 (Fig. 2C). In contrast, TNF-α neutralization had no appreciable effect on the early phase of JNK phosphorylation. In agreement with these results, BMM from C57BL/6 mice but not from TNF-α^−/− animals exhibited the second phase of JNK phosphorylation after incubation with mouse CD154 (Fig. 2D). Thus, autocrine production of TNF-α is required for the second phase of JNK phosphorylation and the increased autophagy triggered by CD40 ligation.

 

 

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FIG 2

TNF-α mediates the second phase of JNK phosphorylation after CD40 ligation and is required for CD40-induced autophagy. (A) hmCD40 RAW 264.7 cells were incubated with or without CD154 for 4 h. TNF-α production was examined by an ELISA. (B) hmCD40 RAW 264.7 cells transfected with tfLC3 were preincubated with an anti-TNF-α or isotype control MAb for 1 h, followed by the addition of CD154. The average numbers of autophagosomes and autolysosomes per cell were determined by fluorescence microscopy at 6 h. (C) hmCD40 RAW 264.7 cells were incubated with an anti-TNF-α 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 macrophages from C57BL/6 (B6) and TNF-α^−/− 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 each control band were given a value of 1. Densitometry data represent means ± standard errors of the means of results from 3 experiments. Results shown are representative of data from 3 independent experiments. ***, P < 0.001.

CD40 ligation causes Bcl-2 phosphorylation that is dependent on TNF-α and JNK signaling.

JNK-mediated Bcl-2 phosphorylation at Ser-87 results in the dissociation of Bcl-2 from Beclin 1, enabling the binding of Beclin 1 to PI3KC3 and the initiation of autophagy (29). Incubation of hmCD40 RAW 264.7 cells with CD154 increased Bcl-2 phosphorylation at Ser-87, which was detected at 4 h (Fig. 3A). A neutralizing anti-TNF-α MAb or SP600125 prevented Bcl-2 phosphorylation in hmCD40 RAW264.7 cells treated with CD154 (Fig. 3B and ​andC).C). Thus, CD40 triggered the phosphorylation of Bcl-2 at Ser-87 that was mediated by TNF-α and JNK signaling.

 

FIG 3

CD40 ligation causes phosphorylation of Bcl-2 that is dependent on TNF-α and JNK. (A) hmCD40 RAW 264.7 cells were incubated with or without CD154. Phospho-Ser87 Bcl-2 and total Bcl-2 levels were assessed by immunoblotting. Densitometries of bands from CD154-treated cells were compared to those of 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. (B and C) hmCD40 RAW 264.7 cells were incubated with anti-TNF-α versus an isotype control MAb (B) or SP600125 versus vehicle (C), followed by the addition of 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 means of results from 3 experiments. Results are representative of data from 3 independent experiments.

CD40 ligation diminishes Bcl-2–Beclin 1 association via JNK signaling.

hmCD40 RAW 264.7 cells were incubated with or without CD154 to examine the effects of CD40 stimulation on the association of Bcl-2 with Beclin 1. Beclin 1 was immunoprecipitated with Bcl-2 under basal conditions (Fig. 4A). However, CD40 stimulation reduced the immunoprecipitation of Beclin 1 with Bcl-2. Next, we examined the effects of JNK signaling on the association between Bcl-2 and Beclin 1. In the presence of SP600125, incubation with CD154 no longer diminished the immunoprecipitation of Beclin 1 with Bcl-2 (Fig. 4B). Thus, CD40 stimulation appears to decrease the association of Bcl-2 with Beclin 1 in a manner that is largely dependent on JNK signaling.

 

 

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 without CD154 for 4 h. Lysates were immunoprecipitated by incubation with an anti-Bcl-2 antibody and immunoblotted as indicated. Results are representative of data from 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.

CD40 ligation triggers CaMKKβ-, AMPK-, and ULK1-dependent autophagy.

While TNF-α is required for CD40-induced autophagy, TNF-α alone was unable to stimulate this process (Fig. 5A). These results were observed even at concentrations of TNF-α that were 7 times higher than those detected in cells incubated with CD154. Thus, we explored whether CD40 ligation activates other events that are crucial for the stimulation of autophagy.

 

 

FIG 5

ULK1 is required for CD40-induced autophagy. (A) hmCD40 RAW 264.7 cells transfected with the tfLC3 plasmid were incubated with increasing concentrations of mouse recombinant TNF-α. 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 by immunoblotting. (C) hmCD40-RAW 264.7 cells were transfected with control or ULK1 siRNA, followed by transfection with tfLC3. ULK1 and actin expression levels were assessed by immunoblotting. The average numbers of autophagosomes and autolysosomes per cell were determined after 6 h of incubation with or without CD154. Expression levels of p62/SQSTM1 and actin were assessed by immunoblotting of cells transfected with control or ULK1 siRNA and incubated with 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.

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

ULK1 is activated by AMPK under conditions of nutrient deprivation (5,–7). We examined the role of AMPK in CD40-induced ULK1 phosphorylation and autophagy. After 4 h of incubation with CD154, hmCD40 RAW 264.7 cells exhibited increased AMPK phosphorylation at Thr-172 (Fig. 6A). Phosphorylation of this residue causes marked AMPK activation (33). The addition of a neutralizing anti-TNF-α MAb failed to impair AMPK phosphorylation, indicating that, in contrast to the second phase of JNK phosphorylation, CD40 enhanced Thr-172 phosphorylation of AMPK independently of autocrine TNF-α production (Fig. 6B). Ser-555 phosphorylation of ULK1 was dependent on AMPK, since treatment of hmCD40 RAW 264.7 cells with the AMPK inhibitor compound C impaired the ability of CD154 to cause Ser-555 ULK1 phosphorylation (Fig. 6C). Thus, CD40 causes AMPK-dependent Ser-555 phosphorylation of ULK1 and triggers ULK1-dependent autophagy.

 

FIG 6

CaMKKβ-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 errors of the means of results from 3 experiments. (B) hmCD40 RAW 264.7 cells were incubated with or without anti-TNF-α MAb, followed by stimulation with CD154. Total AMPK and phospho-Thr1174 AMPK levels were assessed by immunoblotting at 4 h. Densitometry data represent means ± standard errors of the means of results from 3 experiments. (C) hmCD40 RAW 264.7 cells were incubated with or without compound C (CC), followed by stimulation with human CD154. Expression levels of phospho-Ser555 ULK1 and total ULK1 were assessed by immunoblotting at 4 h. Densitometry data represent means ± standard errors of the means of results from 3 experiments. (D) hmCD40 RAW 264.7 cells were incubated with or without compound C, followed by stimulation with CD154. Expression levels of phospho-Ser 792 raptor and total raptor were assessed by immunoblotting at 4 h. (E) hmCD40-RAW 264.7 cells were transfected with the control or CaMKKβ. CaMKKβ and actin expression levels were assessed by immunoblotting. Total AMPK and phospho-Thr172 AMPK levels were assessed 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 and actin were assessed by immunoblotting at 24 h. (G) hmCD40-RAW 264.7 cells were transfected with control, CaMKKβ, or AMPK1/2 siRNA, followed by transfection with tfLC3. Total AMPK and actin expression levels were assessed by immunoblotting. Numbers of autophagosomes and autolysosomes were determined as described above after 6 h of incubation with or without CD154. Results are shown as means ± standard errors of the means and are representative of data from 3 independent experiments. ***, P < 0.001.

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

CD40 ligation increases intracytoplasmic calcium concentrations (35, 36). We examined whether CD40 causes AMPK phosphorylation via CaMKKβ since this signaling molecule is activated by increased Ca²⁺ concentrations and CaMKKβ can activate AMPK (37, 38). Figure 6E shows that the knockdown of CaMKKβ ablated AMPK phosphorylation at Thr-172 induced by CD40 ligation. Next, we determined whether CD40 requires CaMKKβ and AMPK to stimulate autophagy. Cells were incubated with the selective CaMKKβ inhibitor STO-609 at 1 μM, a concentration that does not impair AMPK activation induced by liver kinase B1 (LKB1), the other major stimulator of AMPK (37). Treatment of hmCD40 RAW 264.7 cells with STO-609 or the AMPK inhibitor compound C impaired the ability of CD154 to increase the numbers of autophagosomes and autolysosomes as well as diminish p62/SQSTM1 expression (Fig. 6F). Moreover, knockdown of CaMKKβ or AMPK1/2 ablated the ability of CD154 to increase the numbers of autophagosomes and autolysosomes (Fig. 6G). Taken together, a pathway that includes CaMKKβ, AMPK, and ULK1 is also required for CD40 to stimulate autophagy.

JNK, CaMKKβ, 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 of the parasites by LC3 and parasite killing dependent on Atg5, Atg7, Beclin 1, PI3KC3, and lysosomal enzymes (19, 21, 23, 24). We examined the role of JNK, CaMKKβ, AMPK, and ULK in CD40-induced parasite killing. CD40 ligation in hmCD40 RAW 264.7 cells transfected with control siRNA reduced the percentage of infected cells and the number of parasites per 100 macrophages (Fig. 7A). Knockdown of JNK1/2, CaMKKβ, AMPK1/2, or ULK1 ablated the killing of T. gondii (Fig. 7A). Studies with BMM revealed that CD40-induced killing of T. gondii is dependent on autocrine production of TNF-α (22). Similarly, pharmacological inhibition of CaMKKβ, the molecule upstream of AMPK and ULK1, prevented CD40-induced parasite killing in BMM from C57BL/6 mice (Fig. 7B). Of note, the CaMKKβ inhibitor STO-609 did not affect the initial percentage of infected BMM (2 h postchallenge) (not shown). Taken together, in addition to TNF-α (22), JNK, CaMKKβ, AMPK, and ULK1 are required for the killing of T. gondii triggered by CD40.

 

FIG 7

JNK, CaMKKβ, AMPK, and ULK1 are required for CD40-induced killing of T. gondii. (A) hmCD40-RAW 264.7 cells were transfected with control siRNA or siRNA against JNK1/2, CaMKKβ, AMPK1/2, or ULK1, followed by incubation with or without CD154 and challenge with T. gondii. Percentages of infected macrophages and numbers of tachyzoites/100 macrophages were determined by light microscopy. (B) BMM from C57BL/6 mice were pretreated with STO-609 or 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 are representative of data from 2 to 3 independent experiments. **, P < 0.01.

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DISCUSSION

Initiation of autophagy requires both ULK1 and the Beclin 1-PI3KC3 complex. This study identifies biochemical mechanisms triggered by CD40 that act upstream of ULK1 and Beclin 1. CD40 induces CaMKKβ-mediated Thr-172 AMPK phosphorylation, a marker of AMPK activation. In turn, AMPK signaling causes Ser-555 ULK1 phosphorylation and ULK1-mediated autophagy. The second mechanism is dependent on CD40-induced autocrine production of TNF-α that causes JNK-dependent phosphorylation of Bcl-2 at Ser-87 and dissociation of Bcl-2 from Beclin 1 (Fig. 8). The release of Beclin 1 from Bcl-2 was reported to allow the binding of Beclin 1 to PI3KC3 and the initiation of autophagy (11). Interestingly, these mechanisms occur at approximately the same time after CD40 engagement. This synchronization likely optimizes the ability of CD40 to stimulate autophagy. The roles of these molecules are relevant to the killing of T. gondii induced by CD40, a process mediated by autophagy (19, 21, 23, 24). Not only TNF-α (22) but also JNK, CaMKKβ, AMPK, and ULK1 are required for T. gondii killing induced by CD40.

 

 



FIG 8

Effects of CD40 signaling on ULK1 and Beclin 1. Shown is a schematic diagram illustrating the signaling pathways activated by CD40 that act on ULK1 and Beclin 1. Studies reported previously (21) revealed that CD40 ligation upregulates Beclin 1 protein levels through the reduction of p21 expression. Ser-555 ULK1 phosphorylation, Ser-87 Bcl-2 phosphorylation, and Beclin 1 upregulation appear to occur simultaneously. CD40 may activate additional mechanisms that act on ULK1 and Beclin 1 (see Discussion).

ULK1 is key for the stimulation of canonical autophagy in mammalian cells (2,–4). CD40 causes AMPK-dependent phosphorylation of ULK1 at Ser-555. This residue is a major site phosphorylated by AMPK (5, 7, 32). Moreover, AMPK-mediated ULK1 phosphorylation activates ULK1 kinase activity (6), an effect that appears essential for autophagosome formation (39). Indeed, the stimulation of autophagy in CD40-activated macrophages requires both AMPK and ULK1. In addition to ULK1, ULK2 is another mammalian homologue that closely resembles Atg1 (40). Both ULK1 and ULK2 regulate autophagy, and their effect can be redundant under certain conditions (mouse embryonal fibroblasts subjected to starvation) (41, 42). However, similarly to our studies, a deficiency of ULK1 was sufficient to impair autophagy in human embryonal kidney 293 cells subjected to amino acid starvation or treated with rapamycin and in cerebellar granule neurons exposed to low-potassium and serum-free conditions (42, 43).

In addition to direct effects on the ULK1/2 complex, AMPK can stimulate autophagy through effects on mTORC1 and Beclin 1. Evidence that CD40 ligation causes Ser-792 phosphorylation of raptor raises the possibility that AMPK-mediated inhibition of mTORC1 may be another mechanism for the stimulation of autophagy after CD40 ligation. AMPK also stimulates autophagy through the phosphorylation of Beclin 1 at Ser-91 and Ser-94 following glucose starvation (44).

AMPK is an energy sensor and a key regulator of autophagy (5,–7). The kinases LKB and CaMKKβ activate AMPK in response to low AMP levels and an increase in the cytoplasmic Ca²⁺ concentration, respectively (37, 45). Our work identified CD40 as an activator of AMPK, an effect that requires CaMKKβ. Of relevance to these findings, CD40 has been reported to increase cytoplasmic Ca²⁺ concentrations (35, 36), and experimental evidence indicates that Ca²⁺ modulates autophagy. It has been proposed that an increase in the cytoplasmic Ca²⁺ concentration would enhance autophagy if it occurs under conditions of cellular stress (46) or if it takes place in nonexcitable cells (47). CaMKKβ may also promote autophagy in CD40-activated macrophages because of accompanying events such as Beclin 1–Bcl-2 dissociation. The coexistence of Beclin 1–Bcl-2 dissociation may also explain why CaMKKβ downstream of CD40 signaling triggers rapid induction of autophagy (5 to 6 h after CD40 ligation), whereas CaMKKβ signaling triggered by vitamin D stimulates autophagy after 1 to 3 days (48, 49).

The Beclin 1-PI3KC3 complex is central to the formation of autophagosomes. This complex can be regulated through interactions with various proteins. Under normal conditions, Bcl-2 binds to the BH3 domain of Beclin 1 (11). This prevents the binding of Beclin 1 to PI3KC3 and the initiation of autophagy (11). JNK1 signaling triggered by starvation mediates Bcl-2 phosphorylation at Thr-69, Ser-70, and Ser-87 (29). In turn, this releases Beclin 1 from Bcl-2 and stimulates autophagy (29). Our studies revealed that CD40 activates this cascade of events through JNK signaling mediated by CD40-induced autocrine production of TNF-α. Additional mechanisms that stimulate autophagy through Beclin 1-protein interactions include TRAF6-mediated ubiquitination of Lys-117 in the BH3 domain of Beclin 1 with subsequent release from Bcl-2 (12), DAPK-mediated phosphorylation of Thr-199 in the BH3 domain of Beclin 1 followed by dissociation from Bcl-X_L (16), and ULK1-mediated phosphorylation of activating molecule in Beclin 1-regulated autophagy 1 (AMBRA 1) causing the release of Beclin 1 from the Dynein motor complex (50). The fact that the inhibition of JNK signaling prevented CD40 from decreasing the Beclin 1–Bcl-2 association suggests that CD40 modulates the Beclin 1–Bcl-2 interaction largely through JNK signaling.

We previously reported that CD40 increases Beclin 1 protein levels through downregulation of p21, a protein that degrades Beclin 1 (21) (Fig. 8). This event is functionally relevant since Beclin 1 overexpression enables suboptimal CD40 ligation to trigger autophagic killing of T. gondii, and CD40 fails to trigger parasite killing when Beclin 1 upregulation is prevented (21). The timing of Beclin 1 upregulation (3 to 4 h post-CD40 ligation) corresponds to the timing of Bcl-2 phosphorylation and Beclin 1–Bcl-2 dissociation. This synchronization of events together with evidence that CD40 also activates PKR (20), an important promoter of autophagy, may assist in optimizing the ability of CD40 ligation to stimulate autophagy and the induction of toxoplasmacidal activity.

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

The studies presented here represent a significant step toward understanding how cell-mediated immunity stimulates autophagy and triggers anti-T. gondii activity in macrophages. Studies that further explore the molecular events responsible for CD40-driven stimulation of autophagy may have implications for the development of novel therapeutic modalities against toxoplasmosis given the importance of CD40 and autophagy in the activation of toxoplasmacidal activity and protection against ocular and cerebral toxoplasmosis (20, 21).

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ACKNOWLEDGMENTS

We thank Richard Kornbluth and Tamotsu Yoshimori for providing reagents. We thank Scott Howell for image collection.

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FUNDING STATEMENT

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

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