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. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: J Immunol. 2015 Sep 25;195(9):4406–4414. doi: 10.4049/jimmunol.1500704

CCR5-dependent activation of mTORC1 regulates translation of iNOS and COX-2 during EMCV infection

Zachary R Shaheen 1, Aaron Naatz’ 1, John A Corbett 1
PMCID: PMC4610876  NIHMSID: NIHMS720174  PMID: 26408666

Abstract

Encephalomyocarditis virus (EMCV) infection of macrophages results in the expression of a number of inflammatory and anti-viral genes including inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. EMCV-induced macrophage activation has been shown to require the presence of CCR5 and the activation of phosphoinositide-3 kinase (PI3K)-dependent signaling cascades. The purpose of this study was to determine the role of PI3K in regulating the macrophage responses to EMCV. We show that PI3K regulates EMCV-stimulated iNOS and COX-2 expression by two independent mechanisms. In response to EMCV infection, Akt is activated and regulates the translation of iNOS and COX-2 through the mTORC1 complex. The activation of mTORC1 during EMCV infection is CCR5-dependent and appears to function in a manner that promotes the translation of iNOS and COX-2. CCR5-dependent mTORC1 activation functions as an antiviral response, as mTORC1 inhibition increases the expression of EMCV polymerase. PI3K also regulates the transcriptional induction of iNOS and COX-2 in response to EMCV infection by a mechanism that is independent of Akt and mTORC1 regulation. These findings indicate that macrophage expression of the inflammatory genes iNOS and COX-2 occurs via PI3K- and Akt-dependent translational control of mTORC1 and PI3K-dependent, Akt-independent transcriptional control.

INTRODUCTION

Encephalomyocarditis virus (EMCV) is a positive single stranded RNA picornavirus that causes pancreatic β-cell destruction and the induction of diabetes in genetically susceptible mice (1-4). EMCV induces diabetes by two mechanisms (5). Infections with high doses of EMCV cause diabetes by infecting β-cells and causing their destruction (6-8). At lower doses, EMCV infection induces the development of diabetes by a process that requires macrophage activation and soluble mediator production (9, 10). Mice depleted of macrophages do not develop diabetes during low dose EMCV infection, whereas B-cell or T-cell depletion has little effect on disease incidence (11). The depletion of the cytokines interleukin 1 (IL-1β) or tumor necrosis factor (TNF-α), as well as the inhibition of inducible nitric oxide synthase (iNOS), each attenuates low dose EMCV-mediated diabetes (12-14). These findings indicate that the mechanisms by which macrophages respond to EMCV are likely to be important in elucidating the pathways by which viral infections may initiate diabetes pathogenesis.

Since macrophage soluble mediator production is important for EMCV to induce diabetes, the mechanisms by which this virus activates macrophages have been explored. Nuclear factor (NF)-κB is normally held in the cytoplasm in an inactive complex by the inhibitory protein IκB. IκB is phosphorylated by inhibitory kappa kinase (IKK), causing its degradation in a proteasomal-dependent manner and allowing for NF-κB nuclear translocation (15). We identified nuclear factor (NF-κB) as a primary transcription factor required for EMCV stimulated iNOS, COX-2 and IL-1β expression by macrophages. In addition to NF-κB, at least one additional signaling cascade that is target gene selective is required for EMCV-stimulated inflammatory gene expression by macrophages (16, 17). The secondary signaling cascades include extracellular signal-regulated kinase (ERK) for IL-1β expression (18), p38 and Jun N-terminal Kinase (JNK) for COX-2 expression (19, 20), and the calcium-independent phospholipase (iPLA2), PKA, and cAMP response element binding protein (CREB) for iNOS (21-23).

While dsRNA sensors play a primary role in the activation of the type 1 IFN antiviral response, we have shown that the induction of iNOS, COX-2 and IL-1β expression in response to EMCV infection in macrophages does not require these sensors (24). Macrophages harvested from mice deficient in the cytosolic dsRNA sensors, such as protein kinase R (PKR) or melanoma differentiation-associated protein (mda-5), or the endosomally located dsRNA sensor toll-like receptor-3 (TLR-3), express iNOS and COX-2 to levels similar to levels induced by EMCV in peritoneal macrophages harvested from wild type mice. While inflammatory gene expression is not modified, the absences of TLR-3 and mda-5 decrease EMCV-stimulated type 1 interferon induction in macrophages (24). These findings suggest that the anti-viral activities of infected macrophages include an early dsRNA sensor-independent inflammatory cascade that is followed by the more selective dsRNA sensor-dependent type 1 IFN-mediated antiviral response. Consistent with this interpretation, EMCV and EMCV capsid protein devoid of virus RNA stimulate the rapid activation of NF-κB, MAP kinases, and CREB within 5 min of infection whereas EMCV RNA accumulation is first detectable 3-6 h post infection (25).

CCR5 is a chemokine receptor expressed on cells of hematopoietic origins, including macrophages (26). Many ligands are known to active CCR5-dependent signaling, including the chemokines (Ccl)3/MIP1α, Ccl4/MIP1β, and Ccl5/regulated on activation normal T-cell expressed and secreted (RANTES) (26-28), as well as bacterial heat shock protein (hsp) 70 (29) and mouse parainfluenza and human influenza respiratory virus (30). CCR5 also serves as a viral entry receptor for HIV (31, 32) by interacting with the HIV envelope protein gp120 (33), and decreased CCR5 expression affords resistance to HIV infection (34, 35). We identified a requirement for the presence of CCR5 in the activation of inflammatory gene expression by EMCV infected macrophages (24). While the absence of CCR5 does not modify the type 1 IFN response to EMCV infection, inflammatory gene expression is attenuated in macrophages isolated from CCR5-deficient mice. Since G protein-coupled receptors, including CCR5, can activate both MAP kinase and phosphoinositide 3-kinase (PI3K)–dependent pathways (26, 30), and PI3K is required for EMCV-stimulated gene expression of iNOS, COX-2, and IL-1β (36) we explored the role of PI3K and its substrate in EMCV-induced inflammatory gene expression by macrophages. In this report we provide evidence that Akt is activated in response to EMCV infection and regulates the translation of inflammatory gene products iNOS and COX-2 in a mammalian target of rapamycin (mTORC1)-dependent manner.

MATERIALS AND METHODS

Materials and animals

Male C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, Maine) or obtained from the MCW Biomedical Resource Center. CCR5−/− mice were purchased from Jackson Laboratories (Bar Harbor, Maine) or were a gift from Dr. Sam Huang (Medical College of Wisconsin, WI). Dulbecco's modified Eagle's medium (DMEM), CMRL-1066 medium, l-glutamine, penicillin, streptomycin, and fetal calf serum were purchased from Life Technologies (Grand Island, NY). LY294002 was purchased from EMD Chemicals (Billerica, MA). MK-2206 was purchased from Active Biochem (Maplewood, NJ). Rapamycin was purchased from Toronto Research Chemicals (Toronto, Ontario). Primary antibodies used and their sources: rabbit anti-phospho-p38, rabbit anti-phospho-JNK, Promega (Madison, WI); rabbit anti-IκBα (C-21), rabbit anti-STAT1, mouse anti-3D Polymerase (3B7), Santa Cruz Biotechnologies (Santa Cruz, CA); rabbit anti-phospho-CREB (Ser133), rabbit anti-phospho-STAT1 (Y701), EMD Millipore (Billerica, MA); rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho FoxO1 (Ser256), rabbit anti-phospho-p70S6K (Thr389), rabbit anti-phospho-4EBP1 (Ser65), Cell Signaling Technology (Beverly, MA); mouse anti-GAPDH, Life Technologies (Grand Island, NY); rabbit anti-COX-2, rabbit anti-iNOS, Cayman Chemical (Ann Arbor, MI). Horseradish-peroxidase (HRP)-conjugated donkey anti-rabbit, and HRP-conjugated donkey anti-mouse were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). All other reagents were obtained from commercially available sources.

Peritoneal Macrophage Isolation and Cell Lines

Non-elicited primary peritoneal macrophages were harvested from mice of at least 8 weeks of age by lavage as previously described (37). After isolation, 5.0×105 cells per 1.0 ml complete CMRL-1066 were incubated at 37°C under an atmosphere of 5% CO2 for at least 24 hours prior to the initiation of experiments. RAW264.7 macrophages were removed from growth flasks by treatment with 0.05% trypsin and 0.02% EDTA at 37°C, washed with DMEM, and plated at the indicated concentrations.

EMCV Propagation and Infection

The B variant of EMCV was a generous gift from Dr. Ji-Won Yoon (University of Calgary, Calgary, Alta., Canada) and has been previously described (38). EMCV was propagated in L929 cells, supernatants were clarified by centrifugation, and virus titers were determined by plaque assay. Cell monolayers were infected at a multiplicity of infection (MOI) of 10 (10 plaque forming units per cell) by the addition of EMCV to culture medium at 37°C for the indicated times (38).

Nitrite determination

Nitrite production was determined by the addition of 50 μl Greiss reagent to 50 μl of macrophage cell culture supernatant. Absorbance at 540 nm was measured using the BioTek SynergyMx plate reader and nitrite quantified by comparison to a sodium nitrite standard curve.

PGE2 release

PGE2 release from cell culture supernatants was determined using a PGE2 competition enzyme immunosorbent assay according to the manufacturer's instructions (Cayman Chemicals #514040).

Western Blot Analysis

Cells were washed with phosphate-buffered saline (PBS) and lysed with laemmli lysis buffer. Protein samples were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane under semi-dry transfer conditions, and blocked in 3% BSA in TBST for 1 h. Membranes were incubated at 4°C overnight with the following primary antibody dilutions: rabbit anti-phospho-p38, 1:2,000; rabbit anti-phospho-JNK, 1:5,000; mouse anti-GAPDH, 1:10,000; all other primary antibodies were used at 1:1,000. Membranes were incubated with secondary antibodies for 1 h at 1:10,000 dilutions. Antigen was detected by chemiluminescence (39). Akt and GAPDH were shown as loading controls and samples were normalized to their respective loading control for densitometry analysis.

Real Time PCR

Cells were lysed and total RNA was purified using the RNeasy Minikit according to manufacturer's instructions (Qiagen). DNase digestion was performed using Turbo DNA-free procedure (Applied Biosystems). First-strand cDNA synthesis was performed using oligo(dT) and reverse transcriptase Superscript Preamplification System (Invitrogen) per the manufacturer's instructions. Quantitative real-time PCR was performed using SsoFast Evagreen Supermix (BioRad) and the BioRad CFX96 Real-Time detection system per manufacturer's instructions. Each sample was normalized to GAPDH (ΔCT) and expressed as a fold change relative to control samples (2-ΔΔCt). Primers used are as follows: 5’-CGA GAC TTC TGT GAC ACA CAG C-3’ and 5’- CAT CTC CTG GTG GAA CAC AGG G -3’ for iNOS; 5’-TTT GTT GAG TCA TTC ACC AGA CAG AT-3’ and 5’-CAG TAT TGA GGA GAA CAG ATG GGA TT-3’; for COX-2; 5’-TCG GTG TGA ACG GAT TTG GCC G-3’ and 5’-TGA AGG GGT CGT TGA TGG CAA CA-3’ for GAPDH. Primers were purchased from Integrated DNA Technologies.

Statistics

Statistical comparisons made between two groups were made using students T-test. Statistical comparisons made between three or more independent conditions were made using one-way analysis of variance. Significant differences between groups (P < 0.05, *) were determined using the Tukey-Kramer post-hoc test.

RESULTS

Role of Akt in the regulation of iNOS and COX-2 expression in EMCV-infected macrophages

In response to EMCV infection the expression of iNOS and COX-2 occurs in a PI3K-dependent manner (36). Since Akt is a primary substrate of PI3K and is phosphorylated within minutes of EMCV infection (36), we examined its role in the regulation of macrophage expression of iNOS and COX-2 following EMCV infection. Pre-treatment of RAW264.7 macrophages with the selective Akt inhibitor MK-2206 attenuates EMCV-stimulated iNOS and COX-2 expression as determined by Western blot analysis (Fig. 1A, 1B). In accordance with the inhibition of iNOS expression, this Akt inhibitor attenuates EMCV-stimulated nitrite formation (an oxidative product of nitric oxide; Fig. 1C) and PGE2 release (Fig. 1D). To confirm that PI3K is required for macrophage activation the selective inhibitor LY294002 is shown to attenuate iNOS and COX-2 expression (Fig. 1A, 1B), nitrite accumulation (Fig. 1C), and PGE2 release (Fig. 1D) in response to EMCV infection. These findings support a role for PI3K-Akt-dependent signaling in macrophage inflammatory gene expression in response to EMCV infection (36).

Figure 1. Akt regulates the expression of iNOS and COX-2 in EMCV-infected macrophages.

Figure 1

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RAW264.7 macrophages (5.0×105 / 2 ml DMEM) were pretreated with or without 10 μM MK-2206 for 3 hours or LY294002 for 1 h. Twenty-four h post EMCV infection (10 MOI) the cells were harvested and iNOS and COX-2 expression was determined by Western blot analysis (A) and quantified by densitometry analysis (B). Densitometric data were normalized to GAPDH and the expression of iNOS and COX-2 in the presence of inhibitors was compared EMCV-infected condition, which was set at 100%. Nitrite production (C) and PGE2 release (D) were determined on cell culture supernatants. Results are representative (A) or are the average ± SEM (B-D) of three independent experiments.

Inhibition of PI3K was previously shown to attenuate transcription of iNOS and COX-2 in response to EMCV infection (36). To examine the potential role of Akt in the transcription of inflammatory genes, PCR was used to evaluate the effects of PI3K and Akt inhibitors on EMCV-stimulated COX-2 and iNOS mRNA accumulation in macrophages. Six h post EMCV infection iNOS and COX-2 mRNA accumulates in macrophages (Fig 2A) and this mRNA accumulation is attenuated in the presence of LY294002 but is not inhibited by MK-2206 (Fig. 2B). Similarly, MK-2206 fails to attenuate COX-2 expression in primary peritoneal macrophages infected with EMCV (Fig. 2C, 2D). While EMCV or other inflammatory ligands like LPS alone are sufficient to stimulate the expression of iNOS in RAW264.7 macrophages, a second signal, IFN-γ is required for EMCV or LPS to stimulate iNOS expression in naïve primary peritoneal macrophages (17, 23). EMCV+IFN-γ stimulates the accumulation of iNOS mRNA to similar levels in the presence or absence of MK-2206 (Fig. 2E, 2F). For the data presented in Figs. 2B, D, and F, total mRNA accumulation of iNOS and COX-2 in response to EMCV infection was set at 100%, and the percent change in mRNA accumulation in response to the Akt inhibitor MK-2206 is shown. These findings suggest that PI3K activation participates in the regulation of transcription and translation (via Akt) while Akt appears to participate in translational control of inflammatory gene expression.

Figure 2. Effects of Akt inhibition of EMCV-stimulated iNOS and COX-2 expression.

Figure 2

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RAW264.7 macrophages (1.0×105 / 400 μl DMEM were pretreated treated with or without 10 μM MK-2206 for 3 hours or LY294002 for 1 h. Six h post EMCV infection (10 MOI) RT-PCR was used to evaluate iNOS and COX-2 mRNA accumulation (A, agarose electrophoresis) and quantified by real-time PCR (B). Mouse peritoneal macrophages (2.5×105 / 0.5 ml CMRL-1066) were pretreated with or without 10 μM MK-2206 for 3 h and then infected with EMCV (10 MOI) ± IFN-γ (150 U/ml). The cells were harvested 6 h post infection and COX-2 (C, D) and iNOS (E, F) mRNA accumulation was evaluated by RT-PCR by agarose gel electrophoresis (C, E) and quantified by realt-time PCR (D, F). Primary peritoneal macrophages require IFN-γ in addition to EMCV for the expression of INOS. Accumulation of iNOS and COX-2 mRNA in the presence of inhibitors was normalized to GAPDH and compared to EMCV (B, D) or EMCV+IFN-γ conditions (F), which were set at 100%. Results are representative (A, C, E) or are the average ± SEM (B, D, F) of three independent experiments.

The effects of PI3K and Akt inhibition on signaling pathways known to control the expression of iNOS and COX-2 in EMCV infected macrophages

Macrophage expression of iNOS and COX-2 in response to EMCV infection requires activation of the transcription factor NF-κB and a secondary signaling cascade selective for the inflammatory gene; CREB for iNOS and p38 and JNK for COX-2 (16, 17, 19-23). In response to infection there is a rapid activation of each of these targets. EMCV stimulates JNK, p38, and CREB phosphorylation and the degradation of inhibitor protein κB (IκB) 30 min post infection (Fig. 3A). Note that IκB degradation is associated with NF-κB nuclear localization and gene expression (15, 40, 41). Surprisingly, the inhibition of PI3K or Akt does not modify EMCV stimulated p38, JNK, or CREB phosphorylation, or IκB degradation in EMCV infected macrophages (Fig. 3A). To confirm that these inhibitors attenuate PI3K and Akt signaling, we show that LY294002 and MK-2206 attenuate EMCV-stimulated phosphorylation of Akt and the Akt substrate FoxO1 (42, 43) (Fig. 3B). Consistent with RAW264.7 macrophages, LY294002 and MK-2206 do not attenuate the stimulatory effects of EMCV infection on IκB degradation despite attenuating Akt phosphorylation in primary mouse macrophages (Fig. 3C). These findings suggest that signaling cascades responsible for the selective expression of iNOS and COX-2 are not the mechanisms by which PI3K and Akt regulate inflammatory gene expression in EMCV-infected macrophages.

Figure 3. Effects of PI3K & Akt inhibition on EMCV-activated signaling pathways required for iNOS and COX-2 expression by macrophages.

Figure 3

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RAW264.7 macrophages (5.0×105 / 2 ml DMEM) (A, B) or mouse peritoneal macrophages (5.0×105 / 1 ml CMRL-1066) (C) were pretreated with or without 10 μM MK-2206 for 3 hours or LY294002 for 1 hour. The cells were harvested 30 min post EMCV infection and the phosphorylation of p38, JNK, CREB, FOXO1 and Akt, and the degradation of IκB were determined by Western blot analysis. GAPDH levels were used as loading controls. Results are representative of 3 independent experiments.

mTORC1 activation controls iNOS and COX-2 translation in response to EMCV infection

The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of cellular metabolism, growth, and survival, in part by promoting 5’ cap-dependent translation through the stimulation of ribosomal biogenesis and translational initiation complex assembly (44, 45). Phosphorylation of the rapamycin-sensitive mTORC1 substrates p70S6K and 4EBP1 promote cap-dependent translation (46-48), and in response to EMCV infection, both p70S6K and 4EBP1 are phosphorylated. Inhibition of PI3K and Akt attenuates EMCV-stimulated phosphorylation of p70S6K and 4EBP1 (Fig. 4A, 4D), suggesting that mTORC1 is activated and may regulate translation of inflammatory genes in response to EMCV infection. In addition, the mTORC1 inhibitor rapamycin also attenuates EMCV-stimulated p70S6K and 4EBP1 phosphorylation in RAW264.7 macrophages (Fig. 4B and E) and p70S6K activation in primary peritoneal macrophages (Fig. 4C and F).

Figure 4. Activation of mTORC1 in response to EMCV infection.

Figure 4

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RAW264.7 (5.0×105 / 2 ml DMEM; A, B, D, E) or mouse peritoneal macrophages (5.0×105 / 1 ml CMRL-1066; C, F) were pretreated with or without 10 μM MK-2206 for 3 hours or LY294002 for 1 hour (A, D) or 1 μM rapamycin for 1 hour (B, C, E, F). The cells were harvested 30 min post EMCV infection (10 MOI) and p70S6K and 4EBP1 phosphorylation was determined by Western blot analysis (A-C) and quantified by densitometry analysis (D-F). Densitometric data were normalized to GAPDH and the phosphorylation of mTORC1 substrates in the presence of inhibitors was compared to EMCV-infected conditions, which were set at 100%. Results are representative (A-C) or are the average ± SEM (D-F) of three independent experiments.

mTORC1 inhibition attenuates the expression of iNOS and COX-2 by EMCV infected macrophages

Rapamycin was used to evaluate the role of mTORC1 in the translational regulation of iNOS and COX-2 expression in EMCV-infected macrophages. This mTORC1 inhibitor attenuates EMCV-stimulated COX-2 and iNOS accumulation as determined by Western blot analysis (Fig. 5A and B), nitrite accumulation (Fig. 5C), and PGE2 release (Fig. 5D) by RAW264.7 macrophages. mTORC1 promotes 5’-cap dependent translation (44, 45), whereas mRNA molecules containing internal ribosomal entry sites (IRES) are translated independent of mTORC1. Since EMCV has a single positive stranded RNA genome containing an IRES (3), we tested the hypothesis that inhibition of cap-dependent translation using rapamycin would enhance the accumulation of the EMCV protein, specifically the 3B7 polymerase. Consistent with this hypothesis, 3B7 polymerase accumulates to detectable levels in mTORC1 inhibited macrophages infected with EMCV (Fig. 5F). The effects of rapamycin appear to be selective to mTORC1 and translation, as rapamycin does not attenuate the phosphorylation of MAP kinases or CREB, or the degradation of IκBα during EMCV infection (Fig. 6A). Furthermore, rapamycin attenuates basal and EMCV-stimulated p70S6K phosphorylation, but does not modify Akt phosphorylation at serine 473 (Fig. 6B), the phosphorylation site that is dependent on the rapamycin insensitive mTOR complex 2 (44, 49).Similarly, the attenuation in iNOS and COX-2 accumulation occurs in the absence of changes in transcription, as EMCV stimulates a ~90-fold increase in iNOS mRNA and a ~30-fold increase in COX-2 mRNA in the presence or absence of rapamycin (Fig.6 C and D).

Figure 5. The role of mTORC1 activation in the control of iNOS and COX-2 expression following EMCV infection.

Figure 5

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The effects of 1 h pretreatment with rapamycin (1 μM) on EMCV-induced iNOS and COX-2 expression by RAW264.7 (5×105 / 2 ml DMEM) were determined by Western blot analysis (A) and quantified by densitometry analysis (B). Densitometric data were normalized to GAPDH and the expression of iNOS and COX-2 in the presence of rapamycin was compared EMCV-infected condition, which was set at 100%. Nitrite formation (C) and PGE2 release (D) were determined on cell culture supernatants. The effects of 1 h pretreatment with rapamycin (1 μM) on 3B7 polymerase accumulation was determined by Western blot analysis (E). Results are representative of three independent experiments (A, E) or are the average ± SEM of three independent experiments (B-D).

Figure 6. The transcriptional-independent effects of rapamycin on iNOS and COX-2 expression following EMCV infection.

Figure 6

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The effects of the mTORC1 inhibitor rapamycin (1 μM) on MAP kinase and NF-κB signaling and on the phosphorylation of the mTORC1 substrate p70S6K and mTORC2 substrate Akt (S473) following 30 minutes EMCV infection (10 MOI) were determined by Western blot analysis (A, B). The levels of GAPDH and Akt are shown as loading controls. The effects of rapamycin (1 μM) on EMCV (10 MOI) induced iNOS and COX-2 mRNA accumulation was determined by real-time PCR and normalized to GAPDH (C, D). Results are representative of three independent experiments (A, B) or are the average ± SEM of three independent experiments (C, D).

CCR5 is required for mTORC1 activation in response to EMCV

PI3K and Akt can be activated by GPCRs such as CCR5 (26, 30) and we have shown that this chemokine receptor is required for MAP kinase phosphorylation, IκBα degradation, and iNOS and COX-2 expression in EMCV-infected macrophages (24). Therefore, the potential role of CCR5 in the regulation of mTORC1 activation was examined using macrophages isolated from wild type and CCR5-deficient mice. EMCV stimulates the activation of PI3K (assessed by Akt phosphorylation) and mTORC1 (assessed by p70S6K phosphorylation) in wild-type but not CCR5-deficient macrophages as determined by Western blot analysis (Fig. 7A-C). These findings are consistent with a role for CCR5 in the rapid activation of mTORC1 in response to EMCV infection.

Figure 7. Role of CCR5 in EMCV-stimulated PI3K and mTORC1 activation.

Figure 7

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Mouse peritoneal macrophages (5.0×105 / 1 ml CMRL-1066) were isolated from either CCR5+/+ or CCR5−/− mice and infected with EMCV for 30 minutes (10 MOI). Cells were harvested p70S6K and Akt phosphorylation was determined by Western blot analysis and quantified by densitometry. Densitometric data were normalized to Akt. Results are representative (A) or are the mean ± SEM (B, C) of three independent experiments.

IFN-γ overcomes the inhibitory actions of MK-2206 on iNOS and COX-2 translation

Primary peritoneal macrophages were used to confirm a role for Akt and mTORC1 in the regulation of EMCV-stimulated iNOS and COX-2 expression. Inhibition of Akt (Fig. 8A, 8C) and mTORC1 (Fig. 8B, 8D) attenuate EMCV-stimulated COX-2 expression by peritoneal macrophages, consistent with RAW264.7 macrophages. While EMCV infection is sufficient to stimulate COX-2 expression in primary peritoneal macrophages (16), it does not stimulate iNOS expression or nitrite formation. A second signal, IFN-γ, is required in addition to EMCV to stimulates iNOS expression and nitric oxide production by primary macrophages (17). In the presence of IFN-γ, MK-2206 and rapamycin no longer inhibit EMCV-stimulated iNOS expression (Fig. 8E-8H) or nitric oxide production (data not shown). Further, MK2206 no longer inhibits COX-2 expression when IFN-γ is present (Fig. 8E, 8G), and rapamycin only modestly reduces COX-2 expression (by 33%; Fig. 8F, 8H) in primary peritoneal macrophages infected with EMCV and treated with IFN-γ.

Figure 8. The effects of IFN-γ on translation of iNOS and COX-2 in EMCV-infected macrophages.

Figure 8

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Mouse peritoneal macrophages (5.0×105 / 1 ml CMRL-1066) were pretreated with or without 10 μM MK-2206 for 3 h or rapamycin for 1 h and then infected with EMCV (10 MOI) (A-D) or EMCV+IFN-γ (10 MOI + 150 U/ml) (E-H). The cells were harvested 24 h post infection and iNOS and COX-2 accumulation determined by Western blot analysis (A, B, E, F) Densitometric data were normalized to GAPDH and the expression of iNOS and COX-2 in the presence of inhibitors was compared EMCV (C, D) or EMCV+IFN-γ -treated conditions (G, H), which were set at 100%. RAW264.7 macrophages (2.5×105 / 1 ml DMEM) were pretreated with or without 10 μM MK-2206 for 3 h and then infected with EMCV (10 MOI) in the presence or absence of IFN-γ (150 U/ml) for 30 minutes. Phosphorylation of PI3K and mTORC1 substrates, as well as phosphorylation of STAT1, was determined by Western blot analysis (I). Results are representative (A, B, E, F, I) or are the mean ± SEM (C, D, G, H) of three independent experiments.

To test whether IFN-γ promotes translation of iNOS and COX-2 in an mTORC1-dependent manner, macrophages were pre-treated with MK-2206 and then infected with EMCV in the presence or absence of IFN-γ for 30 minutes. EMCV stimulates the phosphorylation of Akt and p70S6K to similar levels in the presence or absence of IFN-γ (Fig. 8I). Alone IFN-γ does not increase Akt or p70S6K phosphorylation or rescue Akt and p70S6K phosphorylation in the presence of MK-2206 (Fig. 8I), yet IFN-γ does stimulate the phosphorylation of STAT1, a positive control (Fig. 8I). These data suggest that signaling cascades in addition to Akt are required for iNOS expression and that mTORC1-independent pathways activated by IFN-γ can overcome the inhibitory actions of Akt or mTORC1 inhibition on iNOS and COX-2 translation.

DISCUSSION

Macrophages respond to EMCV infection with a rapid activation of multiple pro-inflammatory signaling pathways, including MAP kinases, CREB, and NF-κB and these pathways stimulate the expression of inflammatory genes such as iNOS and COX-2 (17, 18, 20, 25). The activation of these pathways temporally precede the accumulation of viral RNA within macrophages (25), does not require cytosolic (PKR or mda-5) or endosomal (TLR-3) dsRNA sensors, or viral dsRNA (24, 25). We have shown that EMCV coat protein, devoid of viral RNA, is sufficient to stimulate the phosphorylation of MAP kinases and CREB, and degradation of IκB (24, 25). The chemokine receptor CCR5 appears to be the signaling receptor required for EMCV-stimulated inflammatory gene expression. Macrophages isolated from CCR5-deficient mice fail to express iNOS or COX-2 following infection with this virus; however, the absence of CCR5 does not affect induction of the type 1 IFN antiviral response (24). Importantly, macrophages harvested from CCR5−/− mice infected with EMCV accumulate 7-fold higher levels of EMCV RNA compared to wild-type control mice (24), indicating that CCR5 regulated inflammatory gene expression functions as an antiviral response.

In this present study, the pathways controlling macrophage expression of iNOS and COX-2 following EMCV infection were examined. CCR5 is a GPCR that has been shown to activate PI3K (26, 30), and we have shown that PI3K regulates iNOS and COX-2 expression in EMCV-infected macrophages (36). The current study was designed to evaluate the role of the PI3K substrate Akt in the regulation of iNOS and COX-2 expression in EMCV infected macrophages. While inhibition of PI3K attenuates iNOS and COX-2 mRNA accumulation and protein expression in macrophages infected with EMCV, inhibitors of Akt do not modify EMCV-stimulated iNOS and COX-2 mRNA accumulation. Furthermore, inhibitors of PI3K and Akt do not prevent the activation of pathways previously shown to regulate the transcription of iNOS (NF-κB and CREB) and COX-2 (NF-κB and the MAP kinases p38 and JNK) (16, 17, 19-23). These findings suggest that PI3K controls macrophage expression of inflammatory genes in response to EMCV infection by two distinct pathways: a PI3K-dependent pathway that controls transcription of iNOS and COX-2; and an Akt-dependent pathway that regulates translation of these genes (summarized in Fig. 9).

Figure 9. Proposed model for the regulation of iNOS and COX-2 expression in response to EMCV infection.

Figure 9

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The mammalian target of rapamycin complex 1 (mTORC1) regulates 5’ cap-dependent translation by stimulating both the assembly of translational initiation complexes and ribosomal biogenesis (44, 45). p70S6K and 4EBP1 are rapamycin-sensitive substrates of mTORC1 that when phosphorylated promote cap-dependent translation (46-48). Consistent with a role for mTORC1 in the regulation of inflammatory gene expression, EMCV stimulates the phosphorylation of mTORC1 substrates p70S6K and 4EBP1 occurs within minutes of infection. Inhibition of PI3K, Akt, and mTORC1 attenuate EMCV-induced p70S6K and 4EBP1 phosphorylation (Fig. 4). The rapid activation of mTORC1 in a PI3K- and Akt-dependent manner in response to EMCV infection is consistent with role of Akt in regulating mTORC1 activation in response to growth factors such as insulin and in cancer models (50-58). Consistent with a role for mTORC1 activation in the regulation of translation (44, 45), we show that rapamycin attenuates EMCV-stimulated iNOS and COX-2 expression without modifying mRNA accumulation of either inflammatory gene.

In the presence of IFN-γ the levels of EMCV-stimulated COX-2 and iNOS expression by RAW264.7 macrophages are enhanced (16, 17), and a second signaling (IFN-γ) is required for EMCV to induce iNOS expression in primary mouse macrophages (17). To determine if Akt and mTORC1 participate in the translation of inflammatory genes in naïve primary macrophages, the effects of Akt and mTORC1 inhibition on EMCV ± IFN-γ induced iNOS and COX-2 expression were evaluated by Western blot analysis. While inhibitors of Akt and mTORC1 attenuate EMCV-stimulated COX-2 expression by mouse peritoneal macrophages, this inhibitory action is overcome by the presence of IFN-γ. Further, Akt and mTORC1 inhibition does not attenuate EMCV + IFN-γ induced iNOS expression by mouse peritoneal macrophages. While both type I and type II (-γ) IFNs have been demonstrated to promote mTORC1 activity and mTORC1-dependent translation in a PI3K-Akt dependent mechanism in fibroblasts (59-63), we show that IFN-γ does not stimulate PI3K or mTORC1 activation, nor does it enhance PI3K or mTORC1 activity in the presence of virus infection in macrophages (Fig. 8I). These findings suggest that Akt promotes inflammatory gene translation; however, under conditions of robust inflammation, or in the presence of IFN-γ, additional, mTORC1-independent pathways are activated to allow for translation of iNOS and COX-2. IRF-1 has been shown to be a required transcription factor for IFN-γ to stimulate iNOS transcription in mouse peritoneal macrophages (64). Whether IRF-1 mediates the mTORC1-independent effects of IFN-γ on translation will be the focus of future studies.

PI3K can be activated by GPCR signaling, and CCR5 is known to signal via GPCR activation (26, 30). Furthermore, CCR5 has been shown to participate in the macrophage response to viral infections (30, 36). PI3K is rapidly activated (as measured by accumulation of phosphorylated inositides and the phosphorylation of Akt) within minutes of EMCV infection in macrophages (36) and this temporally coincides with activation of other CCR5-dependent signaling pathways such as MAP kinases and NF-κB in EMCV-infected macrophages (24). Consistent with our previous findings EMCV-induced PI3K (Akt phosphorylation) and mTORC1 activation requires the presence of functional CCR5 (Fig. 7). The CCR5 controlled signaling cascades that activate mTORC1 appear to limit virus propagation, as the essential viral protein 3B7 polymerase accumulates to higher levels in macrophages treated with the mTORC1 inhibitor rapamycin (Fig. 5E). This observation is consistent with previous studies showing that EMCV RNA accumulates to levels that are 7-fold higher in CCR5-deficient macrophages as compared to the levels that accumulate in macrophages harvested from EMCV-infected wild type mice (24). Additionally, rapamycin has been shown to decrease total host cell translation while allowing a rapid and robust increase in EMCV protein synthesis and increased viral yield (65, 66). Indeed, EMCV can promote the dephosphorylation (and thus activation) of the translational repressor 4EBP1, which temporally coincides with the inhibition of host translation (67). The ability of macrophages to sense EMCV coat protein using CCR5, and the resulting activation of mTORC1, appears to function as an antiviral response as mTORC1 functions to promote the translation of inflammatory genes such as iNOS and COX-2 and to attenuate EMCV 3B7 polymerase expression (68, 69). These findings begin to define a pathway by which macrophages sense danger in an indiscriminant manner. In this CCR5-dependent pathway, PI3K and Akt activate mTORC1 resulting in the promotion of inflammatory gene translation while limiting the translation of the EMCV 3B7 polymerase, providing time for the macrophage to determine the type of infection (virus, bacterial) and the more selective response, in this case the dsRNA-activated type I IFN response.

ACKNOWLEDGEMENTS

We thank Jennifer A. McGraw for technical assistance. We thank Dr. Sam Huang and Dr. Xuesong Wu for providing CCR5−/− mice, and the Medical College of Wisconsin Biomedical Resource Center for providing C57BL/6J mice.

This work was supported by grants from the NIH: DK-052194 and AI-44458 (to J.A.C), and F30 Fellowship DK102363-01A1 and was partially supported by a training grant from NIGMS T32-GM080202.

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