The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rajaram, M. V. S.
Right arrow Articles by Tridandapani, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rajaram, M. V. S.
Right arrow Articles by Tridandapani, S.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH
The Journal of Immunology, 2006, 177: 6317-6324.
Copyright © 2006 by The American Association of Immunologists, Inc.

Akt/Protein Kinase B Modulates Macrophage Inflammatory Response to Francisella Infection and Confers a Survival Advantage in Mice1

Murugesan V. S. Rajaram*, Latha P. Ganesan*, Kishore V. L. Parsa{dagger}, Jonathan P. Butchar*, John S. Gunn{ddagger} and Susheela Tridandapani2,*,{dagger}

* Dorothy M. Davis Heart and Lung Research Institute, {dagger} Ohio State Biochemistry Program, and {ddagger} Department of Molecular Virology, Immunology and Medical Genetics and Center for Microbial Interface Biology, Ohio State University, Columbus, OH 43210


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Gram-negative bacterium Francisella novicida infects primarily monocytes/macrophages and is highly virulent in mice. Macrophages respond by producing inflammatory cytokines that confer immunity against the infection. However, the molecular details of host cell response to Francisella infection are poorly understood. In this study, we demonstrate that F. novicida infection of murine macrophages induces the activation of Akt. Inhibition of Akt significantly decreases proinflammatory cytokine production in infected macrophages, whereas production of the anti-inflammatory cytokine IL-10 is enhanced. Analysis of the mechanism of Akt influence on cytokine response demonstrated that Akt promotes NF-{kappa}B activation. We have extended these findings to show that Akt activation may be regulated by bacterial genes associated with phagosomal escape. Infection with mglA mutants of F. novicida elicited sustained activation of Akt in comparison to cells infected with wild-type F. novicida. Concomitantly, there was significantly higher proinflammatory cytokine production and lower IL-10 production in cells infected with the mglA mutant. Finally, transgenic animals expressing constitutively active Akt displayed a survival advantage over their wild-type littermates when challenged with lethal doses of F. novicida. Together, these observations indicate that Akt promotes proinflammatory cytokine production by F. novicida-infected macrophages through its influence on NF-{kappa}B, thereby contributing to immunity against F. novicida infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The bacterium Francisella tularensis is a Gram-negative intracellular pathogen that primarily infects monocytes and macrophages and causes the disease tularemia (1). Subsequent to the invasion of the host cell, the bacteria escape from the phagosome to replicate in the cytoplasm and cause apoptosis of the host cell. This phagosomal escape is dependent on the Francisella pathogenicity island protein IglC and its transcriptional regulator MglA (2, 3, 4).

Macrophages respond to Francisella infection by producing a variety of inflammatory cytokines including TNF-{alpha}, IL-12, and IL-1beta (5, 6, 7, 8, 9). Among the proinflammatory cytokines produced by the infected host, IL-12 has been reported to be critical for immunity against F. tularensis infection. The importance of IL-12 in immunity against F. tularensis infection is clearly indicated by studies demonstrating that IL-12 knockout animals, or animals treated with IL-12 neutralizing Abs, are unable to clear the bacteria (10, 11). IL-12 stimulates NK cells to produce IFN-{gamma}, which has been shown to inhibit intramacrophage growth of F. tularensis (12, 13, 14). Recent studies have also indicated a critical role for IL-1beta in macrophage response to infection. Animals deficient in the processing and release of IL-1beta succumb sooner to F. tularensis infection when compared with their wild-type littermates (5, 9). Although activation of NF-{kappa}B has been reported during F. tularensis infection, intracellular signaling mechanisms involved in macrophage response are not well defined (7).

Akt is a serine/threonine kinase that is recruited to the plasma membrane in cells stimulated with a variety of stimulants including growth factors and cytokines. Recruitment of Akt to the membrane requires association of the PH domain of Akt with phosphatidylinositol 3,4,5-trisphosphate (PIP3),3 a product of the enzyme PI3K. This results in the phosphorylation and activation of Akt (15, 16, 17). Ectopic expression of membrane-targeted Akt results in constitutive Akt activation (16, 18). Akt plays critical roles in multiple cellular processes such as cell cycle regulation, cell survival, NF-{kappa}B-dependent gene transcription, actin remodeling, and cell migration (19, 20, 21). Interestingly, Akt appears to act as a positive as well a negative regulator of NF-{kappa}B activation and inflammatory cytokine production, depending on the nature of the stimulus. Earlier studies indicated that Akt promotes NF-{kappa}B activation by directly phosphorylating I{kappa}B kinase {alpha} in response to stimuli such as platelet-derived growth factor (PDGF) and TNF-{alpha} (22, 23), but recent studies show that Akt dampens LPS-induced NF-{kappa}B activation by phosphorylating and inactivating glycogen synthase kinase 3beta, which can in turn negatively regulate NF-{kappa}B (24, 25, 26). In this study we investigated the role of Akt in murine macrophage inflammatory response to F. tularensis novicida. This subspecies is highly virulent in mice and has an intracellular lifecycle similar to those of holarctica and tularensis, which are virulent in humans (12).

Our data indicate that Akt is serine- and threonine-phosphorylated in infected macrophages and that Akt promotes proinflammatory cytokine production while down-regulating IL-10 production. We have investigated the molecular mechanism of Akt influence on F. novicida-induced inflammatory cytokine production and found that Akt is an upstream positive regulator of NF-{kappa}B activation. Interestingly, Akt activity appears to be regulated by the pathogenicity island regulator MglA. Thus, in macrophages infected with the mglA mutant (FN mglA) Akt activation is enhanced and sustained when compared with cells infected with wild-type F. novicida. Further, enhanced Akt activation in the mglA mutant-infected cells is accompanied by enhanced NF-{kappa}B activation and inflammatory cytokine production. These data suggest that Akt activation plays a critical role in macrophage inflammatory response to F. novicida infection. Consistent with this, transgenic animals expressing a myristoylated (Myr), constitutively active form of Akt (Myr-Akt) in their macrophages displayed a significant survival advantage over their wild-type littermates when challenged with lethal doses of F. novicida.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells, Abs, and reagents

Raw 264.7 murine macrophage cells were obtained from American Type Culture Collection and maintained in RPMI 1640 with 3.5% heat-inactivated FBS. Abs specific for phosphorylated Erk, p38, JNK, Ser-Akt, and Thr-Akt were purchased from Cell Signaling Technology. Actin and Akt Abs were from Santa Cruz Biotechnology. Akt inhibitor X was purchased from Calbiochem. F. novicida U112 (JSG1819), and an mglA mutant of F. novicida U112 (JSG2250), were used in all experiments. Bacteria were grown on Chocolate II agar plates at 37°C.

Culture of murine bone marrow macrophages (BMM)

BMM were derived from C57/BL6 animals, by methods previously described (27). Briefly, bone marrow cells were cultured in RPMI 1640 containing 10% FBS plus 10 µg/ml polymyxin B and supplemented with 10 ng/ml CSF-1 for 7 days before they were used in experiments. BMM derived in this manner were >99% positive for Mac-1, as determined by flow cytometry.

Cell stimulation, lysis, and Western blotting

Macrophages were infected with a multiplicity of infection of 100 F. novicida or FN mglA that were scraped from plates, resuspended, and diluted in RPMI 1640 (9). Uninfected and infected cells were lysed in TN1 buffer (50 mM Tris (pH 8.0), 10 mM EDTA, 10 mM Na4P2O7, 10 mM NaF, 1% Triton X-100, 125 mM NaCl, 10 mM Na3VO4, 10 µg/ml each aprotinin and leupeptin). Postnuclear lysates were boiled in Laemmli sample buffer and were separated by SDS-PAGE, transferred to nitrocellulose filters, probed with the Ab of interest, and developed by ECL.

Preparation of heat-aggregated IgG

Heat-aggregated IgG was prepared according to methods previously described (28). In brief, Chromopure mouse IgG at a concentration of 350 µg/ml was heated at 62°C for 30 min, then cooled on ice immediately.

Western blot data quantitation

The ECL signal was quantitated using a scanner and a densitometry program (Scion Image), as previously described (29). To quantitate the phospho-specific signal in the activated samples, we first subtracted background, normalized the signal to the amount of actin or total target protein in the lysate, and plotted the values as previously described.

ELISA measurement of cytokine production

BMM were infected with F. novicida or FN mglA for varying time points. Cell supernatants were harvested, centrifuged to remove dead cells, and analyzed by ELISA using cytokine-specific kits from R&D Systems. A paired student’s t test was used for each statistical comparison and a value of p ≤ 0.05 was considered significant.

Transfection and luciferase assays

Raw 264.7 cells were transfected with a plasmid encoding a luciferase gene driven by a NF-{kappa}B binding element (NF-{kappa}B-luc) as well as with the appropriate plasmid DNA using the Amaxa Nucleofector apparatus (Amaxa Biosystems) as previously described (28). Briefly, 5 x 106 cells were resuspended in 100 µl of Nucleofector Solution V and were nucleofected with 1 µg of NF-{kappa}B-luc alone or with 5 µg of empty vector or plasmid encoding wild-type Akt. These plasmids were provided by Dr. P. Tsichlis (Fox Chase Cancer Center, Philadelphia, PA). Immediately postnucleofection, 500 µl of prewarmed RPMI 1640 was added to the transfection mix before transferring to 12-well plates containing 1.5 ml of prewarmed RPMI 1640 per well. Plates were incubated for 12 h at 37°C. Transfected cells were either left uninfected or were infected for 5 h with F. novicida or FN mglA. Cells were lysed in 100 µl of Luciferase Cell Culture Lysis Reagent (Promega). Luciferase activity was then measured using Luciferase Assay Reagent (Promega), as previously described (27, 30). Data are expressed as a percentage of increase in luciferase activity for infected samples over uninfected samples.

Survival assays

Transgenic mice with macrophage-specific expression of Myr-Akt were generated by methods previously described (28). Age-matched wild-type and Myr-Akt mice were injected i.p. with F. novicida (~200 CFU) and returned to their cages. Time of death was recorded, and survival curves were plotted using Sigma Plot software. All mouse experiments were performed with Institutional Animal Care and Use Committee approved protocols.

Statistical analysis

The "survival" package in resting, uninfected cells (R) was used to generate Kaplan-Meier survival curves and to test for group differences using a log rank test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
F. novicida infection of murine macrophages induces activation of Akt

To address whether Akt is phosphorylated on serine and threonine residues in macrophages infected with Francisella, primary murine BMM and Raw 264.7 cells were infected with F. novicida. The cells were lysed at varying time points ranging from 5 to 30 min, and protein-matched lysates were analyzed by Western blotting with Abs specific for serine or threonine phosphorylated Akt (Fig. 1, A and B). All membranes were reprobed with Akt Ab to ensure equal loading of Akt in all lanes. Results show that F. novicida infection of murine macrophages induces robust phosphorylation of Akt on both serine and threonine residues. As a positive control for serine and threonine phosphorylation of Akt, BMM were stimulated with heat-aggregated IgG to cluster Fc{gamma}R as previously reported (28), and the cell lysates were analyzed by Western blotting (Fig. 1C).


Figure 1
View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 1. Activation of Akt by F. novicida infection in macrophages. BMM (A) and murine macrophage cells Raw 264.7 (B) were infected with F. novicida for the time points indicated. Phosphorylation of Akt was detected using phosphorylated Ser-Akt and Thr-Akt Abs. The same membranes were reprobed with Akt Ab. C, BMM were stimulated by clustering Fc{gamma}R using heat-aggregated IgG for 5 min. R, resting, uninfected cells. These results are representative of five independent experiments.

 
Akt promotes proinflammatory cytokine response in F. novicida-infected murine macrophages

We next examined the functional consequence of Akt activation on inflammatory response to F. novicida infection. In this model, BMM were incubated with the pharmacologic inhibitor of Akt, Akt inhibitor X, for 1 h before infection with F. novicida. Cell supernatants were harvested 8 h postinfection and assayed by ELISA for IL-12, IL-6, and TNF-{alpha}. Cell lysates were assayed for IL-1beta because macrophages are severely deficient in their ability to process and secrete this cytokine (31). The data indicate that proinflammatory cytokine production is significantly reduced in cells treated with the Akt inhibitor (Fig. 2, A–D). Similar results were obtained from Raw 264.7 cells (Fig. 2, E–G). Of note, there was no detectable IL-12 production by the Raw 264.7 cell line under any condition. To ensure that the inhibitor did indeed block activation of Akt, protein-matched cell lysates were analyzed by Western blotting with phospho-serine Akt Ab, followed by total Akt Ab (Fig. 2H). To also ensure that the Akt inhibitor was not toxic to the cells, cells were examined for viability at 8 h using trypan blue. No difference in cell viability was seen between the vehicle-treated cells or the Akt inhibitor-treated cells (data not shown).


Figure 2
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 2. Akt promotes proinflammatory cytokine production in murine macrophages infected with F. novicida. BMM (A–D) and Raw 264.7 cells (E–G) were pretreated for 1 h with 10 µM Akt inhibitor (Akt X) or with vehicle control (distilled water (dH2O)) and subsequently infected with F. novicida. Cell supernatants were analyzed by ELISA for IL-12, IL-6, and TNF-{alpha}, and the lysates were assayed for IL-1beta. R, resting, uninfected cells. Data were obtained from three independent experiments. *, p ≤ 0.05. H, The effect of the Akt inhibitor was verified by Western blotting with anti-phosphorylated Ser-Akt Ab (top), and the same membrane was reprobed with Akt Ab (bottom).

 
Akt dampens anti-inflammatory cytokine response in F. novicida-infected murine macrophages

We, and others, have previously demonstrated that Akt can exert opposing effects on pro- and anti-inflammatory cytokine production (24, 25, 26). Thus, we examined cell supernatants from the previous experiments for the presence of IL-10. Interestingly, IL-10 production was significantly elevated in both BMM and Raw 264.7 cells treated with the Akt inhibitor (Fig. 3). Together these data indicate that in F. novicida-infected cells, Akt promotes proinflammatory cytokine production and suppresses anti-inflammatory cytokine production.


Figure 3
View larger version (8K):
[in this window]
[in a new window]
  		FIGURE 3. Akt negatively regulates IL-10 production in macrophages infected with F. novicida. BMM (A) and Raw 264.7 cells (B) were pretreated for 1 h with 10 µM Akt inhibitor (Akt X) or with vehicle control (distilled water (dH2O)) and subsequently infected with F. novicida. Cell supernatants were analyzed by ELISA for IL-10. R, resting, uninfected cells. Data were obtained from three independent experiments. *, p ≤ 0.05.

 
Akt promotes NF-{kappa}B-driven gene transcription in F. novicida-infected macrophages

To examine the molecular mechanism of Akt influence on F. novicida-induced inflammatory cytokine production, we next examined NF-{kappa}B activation, which is upstream of cytokine production. Akt has been previously shown to act as a positive regulator as well as a negative regulator of NF-{kappa}B activation depending on the specific signal received by the cell. For example, in cells stimulated with Escherichia coli LPS, Akt has been shown to inactivate glycogen synthase kinase 3beta, which in turn results in the inactivation of NF-{kappa}B (24, 25); in PDGF- and TNF-{alpha}-stimulated cells, Akt is thought to promote NF-{kappa}B-driven gene transcription, perhaps by directly phosphorylating I{kappa}B kinase {alpha} (22, 23).

In this model, we first transfected Raw 264.7 cells with the plasmid NF-{kappa}B-luc. Transfectants were pretreated for 1 h with Akt inhibitor and infected with F. novicida. Cells were lysed at different time points and assayed for luciferase activity as a measure of NF-{kappa}B activation. The results shown in Fig. 4A demonstrate that inhibition of Akt results in a significant decrease in F. novicida-induced NF-{kappa}B activation. Shown in Fig. 4B is a Western blot demonstrating that Akt phosphorylation was indeed inhibited, with equal levels of total Akt in each sample.


Figure 4
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 4. Akt promotes NF-{kappa}B activation in F. novicida-infected macrophages. A, Raw 264.7 cells were transfected with plasmids encoding the luciferase gene driven by an NF-{kappa}B binding element (NF-{kappa}B-luc). The transfectants were pretreated for 1 h with Akt inhibitor (Akt X, 10 µM) or with vehicle control (distilled water (dH2O)) and subsequently infected with F. novicida. NF-{kappa}B activation was assessed by measuring luciferase activity. B, The effect of the Akt inhibitor was verified by Western blotting with anti-phosphorylated Ser-Akt Ab (top). The same membrane was reprobed with Akt Ab (bottom). R, resting, uninfected cells. C, Raw 264.7 cells were cotransfected with wild-type Akt or empty vector along with plasmids encoding NF-{kappa}B-luc and subsequently infected with F. novicida. Data are obtained from three independent experiments. *, p ≤ 0.05. D, Protein-matched lysates from the transfectants were probed with phosphorylated Ser-Akt (upper), and subsequently reprobed with total Akt (middle) and actin (lower) sequentially.

 
As a second approach to test whether Akt is upstream of NF-{kappa}B activation, Raw 264.7 cells were cotransfected with the NF-{kappa}B-luc plasmid along with either empty vector or a plasmid encoding wild-type Akt. The transfectants were infected with F. novicida and assayed for luciferase activity. Results indicate that overexpression of wild-type Akt enhances F. novicida-induced NF-{kappa}B activation (Fig. 4C). Overexpression of wild-type Akt was confirmed by Western blotting protein-matched lysates from the transfectants (Fig. 4D). Taken together, these data demonstrate that Akt promotes NF-{kappa}B activation in F. novicida-infected macrophages.

Akt activation is sustained in macrophages infected with FN mglA

We next examined whether a mglA mutant of Francisella had an influence on Akt activation. MglA is a critical transcription factor regulating the IglC operon of Francisella, and IglC is important for phagosomal escape (4). For this, Raw 264.7 cells were infected with either F. novicida or FN mglA that is deficient in phagosomal escape. Western blotting for serine and threonine phosphorylation of Akt was done (Fig. 5, upper blots), and the same membranes were reprobed with Akt Ab to ensure equal loading in all lanes (Fig. 5, middle blots). Band intensities were quantitated and data from three independent experiments are shown (Fig. 5, lower graphs). Results indicate that Akt activation is significantly higher in cells infected with FN mglA than with F. novicida. In parallel experiments, MAPK (Erk, p38, and JNK) phosphorylation was examined. Phosphorylation of all three MAPKs was potently induced by both F. novicida and FN mglA at 5 min and declined after 30 min. No significant differences were observed in the activation profiles of the MAPKs by F. novicida or FN mglA (Fig. 6). This observation suggests that Akt activity is specifically and preferentially regulated by a factor or event associated with phagosomal escape.


Figure 5
View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 5. Activation of Akt by F. novicida and FN mglA. Raw 264.7 cells were infected with F. novicida (FN) or FN mglA for the time points indicated. Phosphorylation of Akt was detected using phosphorylated Ser-Akt (A) and phosphorylated Thr-Akt (B) Abs. The same membranes were reprobed with Akt Ab (middle panels). The phospho-Akt signals were normalized to total Akt in each lane. Data graphed represents mean and SD of densitometric values obtained from three independent experiments (lower panels).

 

Figure 6
View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 6. Activation of MAPKs by F. novicida and FN mglA. Raw 264.7 cells were infected with F. novicida (FN) or FN mglA for the time points indicated. Phosphorylation of Erk, p38, and JNK was detected using phosphorylated Erk (A), p38 (B), and JNK (C) Abs. The same membranes were reprobed with actin Ab (middle panels). Phosphorylation signals were normalized to actin in each lane. R, resting, uninfected cells. Data represents mean and SD of densitometric values obtained from three independent experiments (lower panels).

 
NF-{kappa}B activation and inflammatory cytokine production are sustained in macrophages infected with FN mglA

The results described indicate that Akt activation is sustained in macrophages infected with FN mglA when compared with cells infected with F. novicida. Hence, we next examined whether enhanced activation of Akt correlated with enhanced NF-{kappa}B activation and proinflammatory cytokine production. Raw 264.7 cells were transfected with the NF-{kappa}B-luc plasmid and subsequently infected with either F. novicida or FN mglA. As seen in Fig. 7A, infection with FN mglA led to a significant enhancement of NF-{kappa}B activation compared with cells infected with F. novicida.


Figure 7
View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 7. Host cell NF-{kappa}B and cytokine response in macrophages infected with FN or FN mglA. A, Raw 264.7 cells were transfected with NF-{kappa}B-luc plasmid. Transfectants were subsequently infected with FN or FN mglA for varying time points and analyzed for luciferase activity as a measure of NF-{kappa}B activation. B–E, Raw 264.7 cells were infected with FN and FN mglA (multiplicity of infection, MOI = 100) for the points shown. Cell supernatants from infected cells were analyzed by ELISA for IL-6, TNF-{alpha}, IL-10, and lysates were analyzed for IL-1beta. Data are obtained from three independent experiments. *, p ≤ 0.05.

 
We also examined cytokine production in Raw 264.7 cells infected with either F. novicida or FN mglA. ELISA results from three independent experiments showed that cells infected with FN mglA produced significantly higher levels of the proinflammatory cytokines IL-6, TNF-{alpha}, and IL-1beta compared with cells infected with F. novicida (Fig. 7, B–D). In contrast, production of the anti-inflammatory cytokine IL-10 was significantly lower in cells infected with FN mglA (Fig. 7E). Collectively, these data support the notion that elevated Akt activation results in enhanced NF-{kappa}B activation and proinflammatory cytokine production.

Expression of Myr-Akt confers a survival advantage in mice challenged with F. novicida

Because our data indicated that Akt promotes macrophage proinflammatory response to F. novicida, we next asked whether expression of constitutively active Akt in mice can confer protection against lethal doses of F. novicida. In this experiment, 10 pairs of age-matched wild-type and transgenic animals expressing a macrophage-specific Myr-Akt were challenged i.p. with 200 CFU of F. novicida. The animals were monitored for survival every 12 h. As shown in Fig. 8 Myr-Akt-expressing animals display a statistically significant survival advantage over their wild-type littermates.


Figure 8
View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 8. Myr-Akt-expressing animals challenged with F. novicida display a survival advantage over their wild-type littermates. Wild-type Akt- (WT-Akt) and Myr-Akt-expressing transgenic animals (10 mice/group) were challenged with 200 CFU of F. novicida. The animals were monitored every 12 h for survival. Results are expressed as Kaplan-Meier curves. Differences in survival between the two groups were tested using the log rank test. Wild-type showed poorer survival than the Myr-Akt mice, p = 0.00886.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have recently reported that the PI3K pathway is activated in macrophages infected with F. novicida and that it plays an important role in proinflammatory gene expression (32). There are several PH domain-containing downstream effectors of PI3K that are activated by its product PIP3, and in this study we have examined the role of one, namely Akt, in macrophage response to Francisella infection. Our data indicate that Akt promotes the production of proinflammatory cytokines in murine macrophages infected with F. novicida. Interestingly, Akt dampened the production of the anti-inflammatory cytokine IL-10 under the same conditions. Our findings also demonstrate that Akt promotes NF-{kappa}B activation upstream of proinflammatory cytokine production. Although this positive regulatory effect of Akt on NF-{kappa}B activation is consistent with previous findings in PDGF and TNF-{alpha} stimulated cells (22, 23), it is in contrast to the recently reported effect of Akt in cells stimulated with bacterial products such as E. coli LPS (24, 25). In the latter case, Akt dampens NF-{kappa}B activation and the subsequent production of proinflammatory cytokines. It is currently unclear how Akt can mediate these distinctly opposing effects on NF-{kappa}B activation. Perhaps distinct downstream effectors of Akt, bridging signal transduction between Akt and NF-{kappa}B, are activated depending on the specific signal received by the cell. Additional studies are required to identify these intermediary molecules.

Our current studies suggest that NF-{kappa}B is a negative regulator of IL-10 production. In support of this notion, recent studies by Katz et al. (33) have reported enhanced production of IL-10 in F. tularensis live vaccine strain (LVS)-infected macrophages that were treated with NF-{kappa}B inhibitors. We speculate that NF-{kappa}B may compete with an unidentified transcription factor that may be important for IL-10 gene transcription. A shift of the balance toward NF-{kappa}B may promote proinflammatory gene expression, whereas a skewing toward the other transcription factor may lead to enhanced IL-10 production. Studies are underway to identify other transcription factors involved in F. novicida-induced inflammatory response, particularly those important for IL-10 production.

Among the proinflammatory cytokines positively regulated by Akt are IL-12, IL-6, TNF-{alpha}, and IL-1beta (Fig. 2). Of note, in these studies the p40 subunit of IL-12 was specifically measured. We were unable to detect any induction of IL-12 p70 (the disulphide-linked p35 and p40 subunits) by ELISA, likely due to the low levels of production of this cytokine. The role of IL-12 p35 remains controversial and appears to depend upon the infection model. Although earlier studies in mice using F. tularensis LVS indicated no role for the p35 subunit of IL-12 (10), recent studies by Duckett et al. (11) clearly demonstrate that IL-12 p35 knockout animals succumb to LVS doses that are not lethal in wild-type mice. In addition, it has been postulated that during Francisella infection the IL-12 p40 subunit may combine with the p19 subunit of IL-23 to form bioactive IL-23 (10, 11). IL-23 is a newly identified cytokine, which potently induces IFN-{gamma} production by NK cells and shares many biological properties with IL-12 (34).

Akt activation in macrophages occurs early during infection with F. novicida and decreases after 30 min (Fig. 1). A similar pattern of activation is seen for the MAPKs Erk, p38, and JNK. Interestingly, in macrophages infected with FN mglA defective for phagosomal escape, there is a significantly enhanced and sustained activation of Akt but not of the MAPKs (Figs. 5 and 6). This enhanced activation of Akt is accompanied by significantly enhanced NF-{kappa}B activity and the production of proinflammatory cytokines (Fig. 7). It has been previously reported that macrophages infected with mutants of F. tularensis LVS, which are defective for phagosomal escape, produce enhanced levels of TNF-{alpha} compared with cells infected with the wild-type bacteria (7). Our data with FN mglA are consistent with these latter findings. However, it must be noted that the iglC mutant used in the former studies is specifically inactivated for phagosomal escape, whereas FN mglA used in our studies is defective not only in phagosomal escape but also in other pathogenicity island genes that are regulated by MglA. These data suggest that either phagosomal escape or the expression of one or more of the pathogenicity island genes may specifically regulate Akt activation, perhaps through interference with host cell phosphoinositide signaling. Indeed, previous reports have shown that the intracellular pathogen Salmonella produces the effector protein SigD/SopB, which can hydrolyze phosphoinositides such as PIP3 (35). It is tempting to speculate that F. novicida may express a similar molecule that can regulate host cell phospholipid metabolism.

These studies define a central role for Akt in the macrophage inflammatory response to F. novicida infection.


   Acknowledgments
 
We thank Dr. Michael Ostrowski for kindly providing the transgenic mice expressing myristoylated Akt, and Huiqing Fang and Christie Newland for assistance with transgenic mouse breeding colonies.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants U54-AI-057153, R01 AI059406, and P01 CA095426 from the National Institutes of Health. J.P.B. is supported by National Institutes of Health Training Grant T32 CA090223. Back

2 Address correspondence and reprint requests to Dr. Susheela Tridandapani, Dorothy M. Davis Heart and Lung Research Institute, Room 405B, 473 West 12th Avenue, Columbus, OH 43210. E-mail address: tridandapani.2{at}osu.edu Back

3 Abbreviations used in this paper used: PIP3, phosphatidylinositol 3,4,5-trisphosphate; BMM, bone marrow macrophage; LVS, live vaccine strain; PDGF, platelet-derived growth factor; Myr, myristoylated. Back

Received for publication May 26, 2006. Accepted for publication August 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Fortier, A. H., S. J. Green, T. Polsinelli, T. R. Jones, R. M. Crawford, D. A. Leiby, K. L. Elkins, M. S. Meltzer, C. A. Nacy. 1994. Life and death of an intracellular pathogen: Francisella tularensis and the macrophage. Immunol. Ser. 60: 349-361. [Medline]
  2. Santic, M., M. Molmeret, K. E. Klose, S. Jones, Y. A. Kwaik. 2005. The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm. Cell. Microbiol. 7: 969-979. [Medline]
  3. Lauriano, C. M., J. R. Barker, F. E. Nano, B. P. Arulanandam, K. E. Klose. 2003. Allelic exchange in Francisella tularensis using PCR products. FEMS Microbiol. Lett. 229: 195-202. [Medline]
  4. Lauriano, C. M., J. R. Barker, S. S. Yoon, F. E. Nano, B. P. Arulanandam, D. J. Hassett, K. E. Klose. 2004. MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival. Proc. Natl. Acad. Sci. USA 101: 4246-4249. [Abstract/Free Full Text]
  5. Mariathasan, S., D. S. Weiss, V. M. Dixit, D. M. Monack. 2005. Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis. J. Exp. Med. 202: 1043-1049. [Abstract/Free Full Text]
  6. Bolger, C. E., C. A. Forestal, J. K. Italo, J. L. Benach, M. B. Furie. 2005. The live vaccine strain of Francisella tularensis replicates in human and murine macrophages but induces only the human cells to secrete proinflammatory cytokines. J. Leukocyte Biol. 77: 893-897. [Abstract/Free Full Text]
  7. Telepnev, M., I. Golovliov, A. Sjostedt. 2005. Francisella tularensis LVS initially activates but subsequently down-regulates intracellular signaling and cytokine secretion in mouse monocytic and human peripheral blood mononuclear cells. Microb. Pathog. 38: 239-247. [Medline]
  8. Stenmark, S., D. Sunnemark, A. Bucht, A. Sjostedt. 1999. Rapid local expression of interleukin-12, tumor necrosis factor {alpha}, and {gamma} interferon after cutaneous Francisella tularensis infection in tularemia-immune mice. Infect. Immun. 67: 1789-1797. [Abstract/Free Full Text]
  9. Gavrilin, M. A., I. J. Bouakl, N. L. Knatz, M. D. Duncan, M. W. Hall, J. S. Gunn, M. D. Wewers. 2005. Internalization and phagosome escape required for Francisella to induce human monocyte IL-1beta processing and release. Proc. Natl. Acad. Sci. USA 103: 141-146.
  10. Elkins, K. L., A. Cooper, S. M. Colombini, S. C. Cowley, T. L. Kieffer. 2002. In vivo clearance of an intracellular bacterium, Francisella tularensis LVS, is dependent on the p40 subunit of interleukin-12 (IL-12) but not on IL-12 p70. Infect. Immun. 70: 1936-1948. [Abstract/Free Full Text]
  11. Duckett, N. S., S. Olmos, D. M. Durrant, D. W. Metzger. 2005. Intranasal interleukin-12 treatment for protection against respiratory infection with the Francisella tularensis live vaccine strain. Infect. Immun. 73: 2306-2311. [Abstract/Free Full Text]
  12. Santic, M., M. Molmeret, K. Y. Abu. 2005. Modulation of biogenesis of the Francisella tularensis subsp. novicida-containing phagosome in quiescent human macrophages and its maturation into a phagolysosome upon activation by IFN-{gamma}. Cell. Microbiol. 7: 957-967. [Medline]
  13. Anthony, L. S., E. Ghadirian, F. P. Nestel, P. A. Kongshavn. 1989. The requirement for {gamma} interferon in resistance of mice to experimental tularemia. Microb. Pathog. 7: 421-428. [Medline]
  14. Golovliov, I., K. Kuoppa, A. Sjostedt, A. Tarnvik, G. Sandstrom. 1996. Cytokine expression in the liver of mice infected with a highly virulent strain of Francisella tularensis. FEMS Immunol. Med. Microbiol. 13: 239-244. [Medline]
  15. Stokoe, D., L. R. Stephens, T. Copeland, P. R. J. Gaffney, C. B. Reese, G. F. Painter, A. B. Holmes, F. McCormick, P. T. Hawkins. 1997. Dual role phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277: 567-570. [Abstract/Free Full Text]
  16. Bellacosa, A., T. O. Chan, N. N. Ahmed, K. Datta, S. Malstrom, D. Stokoe, F. McCormick, J. Feng, P. Tsichlis. 1998. Akt activation by growth factors is a multiple-step process: the role of the PH domain. Oncogene 17: 313-325. [Medline]
  17. Toker, A., L. C. Cantley. 1997. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature 387: 673-676. [Medline]
  18. Burgering, B. M., P. J. Coffer. 1995. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376: 599-602. [Medline]
  19. Datta, S. R., A. Brunet, M. E. Greenberg. 1999. Cellular survival: a play in three Akts. Genes Dev. 13: 2905-2927. [Free Full Text]
  20. Brazil, D. P., J. Park, B. A. Hemmings. 2002. PKB binding proteins: getting in on the Akt. Cell 111: 293-303. [Medline]
  21. Scheid, M. P., J. R. Woodgett. 2001. PKB/AKT: functional insights from genetic models. Nat. Rev. Mol. Cell Biol. 2: 760-768. [Medline]
  22. Ozes, O. N., L. D. Mayo, J. A. Gustin, S. R. Pfeffer, L. M. Pfeffer, D. B. Donner. 1999. NF-{kappa}B activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401: 82-85. [Medline]
  23. Romashkova, J. A., S. S. Makarov. 1999. NF-{kappa}B is a target of AKT in anti-apoptotic PDGF signalling. Nature 401: 86-90. [Medline]
  24. Guha, M., N. Mackman. 2002. The phosphatidylinositol 3-kinase-Akt pathway limits lipopolysaccharide activation of signaling pathways and expression of inflammatory mediators in human monocytic cells. J. Biol. Chem. 277: 32124-32132. [Abstract/Free Full Text]
  25. Martin, M., K. Rehani, R. S. Jope, S. M. Michalek. 2005. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nat. Immunol. 6: 777-784. [Medline]
  26. Pengal, R. A., L. P. Ganesan, G. Wei, H. Fang, M. C. Ostrowski, S. Tridandapani. 2005. Lipopolysaccharide-induced production of interleukin-10 is promoted by the serine/threonine kinase Akt. Mol. Immunol. 43: 1557-1564.
  27. Fang, H., R. A. Pengal, X. Cao, L. P. Ganesan, M. D. Wewers, C. B. Marsh, S. Tridandapani. 2004. Lipopolysaccharide-induced macrophage inflammatory response is regulated by SHIP. J. Immunol. 173: 360-366. [Abstract/Free Full Text]
  28. Ganesan, L. P., G. Wei, R. A. Pengal, L. Moldovan, N. Moldovan, M. C. Ostrowski, S. Tridandapani. 2004. The serine/threonine kinase Akt promotes Fc{gamma} receptor-mediated phagocytosis in murine macrophages through the activation of p70S6 kinase. J. Biol. Chem. 279: 54416-54425. [Abstract/Free Full Text]
  29. Ganesan, L. P., H. Fang, C. B. Marsh, S. Tridandapani. 2003. The protein-tyrosine phosphatase SHP-1 associates with the phosphorylated immunoreceptor tyrosine-based activation motif of Fc{gamma}RIIa to modulate signaling events in myeloid cells. J. Biol. Chem. 278: 35710-35717. [Abstract/Free Full Text]
  30. Pengal, R. A., L. P. Ganesan, H. Fang, C. B. Marsh, C. L. Anderson, S. Tridandapani. 2003. SHIP-2 inositol phosphatase is inducibly expressed in human monocytes and serves to regulate Fc{gamma} receptor-mediated signaling. J. Biol. Chem. 278: 22657-22663. [Abstract/Free Full Text]
  31. Wewers, M. D., H. A. Dare, A. V. Winnard, J. M. Parker, D. K. Miller. 1997. IL-1beta-converting enzyme (ICE) is present and functional in human alveolar macrophages: macrophage IL-1beta release limitation is ICE independent. J. Immunol. 159: 5964-5972. [Abstract]
  32. Parsa, K. V., L. P. Ganesan, M. V. Rajaram, M. A. Gavrilin, A. Balagopal, N. P. Mohapatra, M. D. Wewers, L. S. Schlesinger, J. S. Gunn, S. Tridandapani. 2006. Macrophage pro-inflammatory response to Francisella novicida infection is regulated by SHIP. PLoS Pathog. 2: e71[Medline]
  33. Katz, J., P. Zhang, M. Martin, S. N. Vogel, S. M. Michalek. 2006. Toll-like receptor 2 is required for inflammatory responses to Francisella tularensis LVS. Infect. Immun. 74: 2809-2816. [Abstract/Free Full Text]
  34. Oppmann, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega, N. Yu, J. Wang, K. Singh, et al 2000. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13: 715-725. [Medline]
  35. Drecktrah, D., L. A. Knodler, O. Steele-Mortimer. 2004. Modulation and utilization of host cell phosphoinositides by Salmonella spp. Infect. Immun. 72: 4331-4335. [Free Full Text]



This article has been cited by other articles:


Home page
 Vet PatholHome page
N. A. Twenhafel, D. A. Alves, and B. K. Purcell
Pathology of Inhalational Francisella tularensis spp. tularensis SCHU S4 Infection in African Green Monkeys (Chlorocebus aethiops)
Vet. Pathol., July 1, 2009; 46(4): 698 - 706.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Nishida, A. Andoh, O. Inatomi, and Y. Fujiyama
Interleukin-32 Expression in the Pancreas
J. Biol. Chem., June 26, 2009; 284(26): 17868 - 17876.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. N. Henning, A. K. Azad, K. V. L. Parsa, J. E. Crowther, S. Tridandapani, and L. S. Schlesinger
Pulmonary Surfactant Protein A Regulates TLR Expression and Activity in Human Macrophages
J. Immunol., June 15, 2008; 180(12): 7847 - 7858.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
T. J. Wiles, B. K. Dhakal, D. S. Eto, and M. A. Mulvey
Inactivation of Host Akt/Protein Kinase B Signaling by Bacterial Pore-forming Toxins
Mol. Biol. Cell, April 1, 2008; 19(4): 1427 - 1438.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
A. Nishida, A. Andoh, M. Shioya, S. Kim-Mitsuyama, A. Takayanagi, and Y. Fujiyama
Phosphatidylinositol 3-kinase/Akt signaling mediates interleukin-32{alpha} induction in human pancreatic periacinar myofibroblasts
Am J Physiol Gastrointest Liver Physiol, March 1, 2008; 294(3): G831 - G838.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. P. Butchar, M. V. S. Rajaram, L. P. Ganesan, K. V. L. Parsa, C. D. Clay, L. S. Schlesinger, and S. Tridandapani
Francisella tularensis Induces IL-23 Production in Human Monocytes
J. Immunol., April 1, 2007; 178(7): 4445 - 4454.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
W.-J. Zhang, H. Wei, T. Hagen, and B. Frei
{alpha}-Lipoic acid attenuates LPS-induced inflammatory responses by activating the phosphoinositide 3-kinase/Akt signaling pathway
PNAS, March 6, 2007; 104(10): 4077 - 4082.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rajaram, M. V. S.
Right arrow Articles by Tridandapani, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rajaram, M. V. S.
Right arrow Articles by Tridandapani, S.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS