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Protection against epithelial damage during Candida albicans infection is mediated by PI3K/Akt and mammalian target of rapamycin signaling.

Moyes DL, Shen C, Murciano C, Runglall M, Richardson JP, Arno M, Aldecoa-Otalora E, Naglik JR - J. Infect. Dis. (2013)

Bottom Line: Using reconstituted oral epithelium, we assessed epithelial gene transcriptional responses to Candida albicans infection by microarray.Transcript profiling demonstrated early upregulation of epithelial genes involved in immune responses.Many of these genes constituted components of signaling pathways, but only NF-κB, MAPK, and PI3K/Akt pathways were functionally activated.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Immunology, King's College London Dental Institute.

ABSTRACT

Background: The ability of epithelial cells (ECs) to discriminate between commensal and pathogenic microbes is essential for healthy living. Key to these interactions are mucosal epithelial responses to pathogen-induced damage.

Methods: Using reconstituted oral epithelium, we assessed epithelial gene transcriptional responses to Candida albicans infection by microarray. Signal pathway activation was monitored by Western blotting and transcription factor enzyme-linked immunosorbent assay, and the role of these pathways in C. albicans-induced damage protection was determined using chemical inhibitors.

Results: Transcript profiling demonstrated early upregulation of epithelial genes involved in immune responses. Many of these genes constituted components of signaling pathways, but only NF-κB, MAPK, and PI3K/Akt pathways were functionally activated. We demonstrate that PI3K/Akt signaling is independent of NF-κB and MAPK signaling and plays a key role in epithelial immune activation and damage protection via mammalian target of rapamycin (mTOR) activation.

Conclusions: PI3K/Akt/mTOR signaling may play a critical role in protecting epithelial cells from damage during mucosal fungal infections independent of NF-κB or MAPK signaling.

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Inhibition of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling induction in TR146 cells infected with Candida albicans. A, Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on cytokine production after 24 hours (multiplicity of infection [MOI] = 0.01) shown as percentage of the dimethyl sulfoxide (DMSO) vehicle control. B, Effect of inhibition of PI3K/Akt (wortmannin [1 µM] and LY294002 [50 µM]) or p38 (SB203580 [10 µM]) signaling on cell damage (lactate dehydrogenase [LDH] release) after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). C, Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cell damage (LDH release) after 24 hours (MOI] = 0.01) shown as percentage of DMSO vehicle control. D, Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cytokine production after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). E, Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of IκBα 2 hours postinfection (MOI = 10). F, Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of MKP1 and c-Jun and on the production of c-Fos 2 hours postinfection (MOI = 10). Candida albicans was added as 100% yeast, which switched to hyphal growth by 2 hours postinfection. Data are the mean (A–D) or representative (E and F) of at least 3 independent experiments. *P < .05, **P < .01, ***P < .001. Abbreviations: G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin.
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JIT824F4: Inhibition of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling induction in TR146 cells infected with Candida albicans. A, Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on cytokine production after 24 hours (multiplicity of infection [MOI] = 0.01) shown as percentage of the dimethyl sulfoxide (DMSO) vehicle control. B, Effect of inhibition of PI3K/Akt (wortmannin [1 µM] and LY294002 [50 µM]) or p38 (SB203580 [10 µM]) signaling on cell damage (lactate dehydrogenase [LDH] release) after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). C, Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cell damage (LDH release) after 24 hours (MOI] = 0.01) shown as percentage of DMSO vehicle control. D, Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cytokine production after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). E, Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of IκBα 2 hours postinfection (MOI = 10). F, Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of MKP1 and c-Jun and on the production of c-Fos 2 hours postinfection (MOI = 10). Candida albicans was added as 100% yeast, which switched to hyphal growth by 2 hours postinfection. Data are the mean (A–D) or representative (E and F) of at least 3 independent experiments. *P < .05, **P < .01, ***P < .001. Abbreviations: G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin.

Mentions: Because PI3K/Akt signaling plays a role in modulating TLR-induced cytokine production in ECs [22], we inhibited this pathway with 2 separate PI3K inhibitors, wortmannin (1 µM) and LY294002 (50 µM), to determine its functional significance in EC cytokine responses. Both inhibitors significantly reduced GM-CSF levels (P < .05 and P < .001; Figure 4A), whereas LY294002 additionally reduced G-CSF levels (P < .001). In contrast, IL-1α production was increased, although not significantly (Figure 4A), whereas IL-6 production was unaffected. Given that IL-1α production is associated with damage [23], these data suggest a role for PI3K/Akt signaling in protection/prevention of damage by C. albicans. Therefore, we analyzed 24 hours postinfection supernatant LDH levels with or without inhibition, finding significantly increased LDH release (Figure 4B). Inhibition of p38 signaling had no effect on LDH release (Figure 4B), indicating that despite mediating discrimination between C. albicans yeast and hyphae via c-Fos [11], p38 plays no role in EC damage protection in fungal infection.Figure 4.


Protection against epithelial damage during Candida albicans infection is mediated by PI3K/Akt and mammalian target of rapamycin signaling.

Moyes DL, Shen C, Murciano C, Runglall M, Richardson JP, Arno M, Aldecoa-Otalora E, Naglik JR - J. Infect. Dis. (2013)

Inhibition of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling induction in TR146 cells infected with Candida albicans. A, Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on cytokine production after 24 hours (multiplicity of infection [MOI] = 0.01) shown as percentage of the dimethyl sulfoxide (DMSO) vehicle control. B, Effect of inhibition of PI3K/Akt (wortmannin [1 µM] and LY294002 [50 µM]) or p38 (SB203580 [10 µM]) signaling on cell damage (lactate dehydrogenase [LDH] release) after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). C, Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cell damage (LDH release) after 24 hours (MOI] = 0.01) shown as percentage of DMSO vehicle control. D, Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cytokine production after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). E, Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of IκBα 2 hours postinfection (MOI = 10). F, Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of MKP1 and c-Jun and on the production of c-Fos 2 hours postinfection (MOI = 10). Candida albicans was added as 100% yeast, which switched to hyphal growth by 2 hours postinfection. Data are the mean (A–D) or representative (E and F) of at least 3 independent experiments. *P < .05, **P < .01, ***P < .001. Abbreviations: G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin.
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JIT824F4: Inhibition of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling induction in TR146 cells infected with Candida albicans. A, Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on cytokine production after 24 hours (multiplicity of infection [MOI] = 0.01) shown as percentage of the dimethyl sulfoxide (DMSO) vehicle control. B, Effect of inhibition of PI3K/Akt (wortmannin [1 µM] and LY294002 [50 µM]) or p38 (SB203580 [10 µM]) signaling on cell damage (lactate dehydrogenase [LDH] release) after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). C, Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cell damage (LDH release) after 24 hours (MOI] = 0.01) shown as percentage of DMSO vehicle control. D, Effect of inhibition of mTOR activity with 10 μM Ku-63794 on cytokine production after 24 hours (MOI = 0.01) shown as percentage of the vehicle control (DMSO). E, Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of IκBα 2 hours postinfection (MOI = 10). F, Effect of inhibition of PI3K/Akt signaling by 1 µM wortmannin or 50 µM LY294002 on phosphorylation of MKP1 and c-Jun and on the production of c-Fos 2 hours postinfection (MOI = 10). Candida albicans was added as 100% yeast, which switched to hyphal growth by 2 hours postinfection. Data are the mean (A–D) or representative (E and F) of at least 3 independent experiments. *P < .05, **P < .01, ***P < .001. Abbreviations: G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin.
Mentions: Because PI3K/Akt signaling plays a role in modulating TLR-induced cytokine production in ECs [22], we inhibited this pathway with 2 separate PI3K inhibitors, wortmannin (1 µM) and LY294002 (50 µM), to determine its functional significance in EC cytokine responses. Both inhibitors significantly reduced GM-CSF levels (P < .05 and P < .001; Figure 4A), whereas LY294002 additionally reduced G-CSF levels (P < .001). In contrast, IL-1α production was increased, although not significantly (Figure 4A), whereas IL-6 production was unaffected. Given that IL-1α production is associated with damage [23], these data suggest a role for PI3K/Akt signaling in protection/prevention of damage by C. albicans. Therefore, we analyzed 24 hours postinfection supernatant LDH levels with or without inhibition, finding significantly increased LDH release (Figure 4B). Inhibition of p38 signaling had no effect on LDH release (Figure 4B), indicating that despite mediating discrimination between C. albicans yeast and hyphae via c-Fos [11], p38 plays no role in EC damage protection in fungal infection.Figure 4.

Bottom Line: Using reconstituted oral epithelium, we assessed epithelial gene transcriptional responses to Candida albicans infection by microarray.Transcript profiling demonstrated early upregulation of epithelial genes involved in immune responses.Many of these genes constituted components of signaling pathways, but only NF-κB, MAPK, and PI3K/Akt pathways were functionally activated.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Immunology, King's College London Dental Institute.

ABSTRACT

Background: The ability of epithelial cells (ECs) to discriminate between commensal and pathogenic microbes is essential for healthy living. Key to these interactions are mucosal epithelial responses to pathogen-induced damage.

Methods: Using reconstituted oral epithelium, we assessed epithelial gene transcriptional responses to Candida albicans infection by microarray. Signal pathway activation was monitored by Western blotting and transcription factor enzyme-linked immunosorbent assay, and the role of these pathways in C. albicans-induced damage protection was determined using chemical inhibitors.

Results: Transcript profiling demonstrated early upregulation of epithelial genes involved in immune responses. Many of these genes constituted components of signaling pathways, but only NF-κB, MAPK, and PI3K/Akt pathways were functionally activated. We demonstrate that PI3K/Akt signaling is independent of NF-κB and MAPK signaling and plays a key role in epithelial immune activation and damage protection via mammalian target of rapamycin (mTOR) activation.

Conclusions: PI3K/Akt/mTOR signaling may play a critical role in protecting epithelial cells from damage during mucosal fungal infections independent of NF-κB or MAPK signaling.

Show MeSH
Related in: MedlinePlus