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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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Levels of DNA-binding activity of STAT1 (A) and NFAT (B) in TR146 cells 30 minutes and 3 hours postinfection with Candida albicans. C, Production of granulocyte colony-stimulating factor by TR146 cells in response to TLR3 (poly I:C, 25 μg/mL) and RIG-I (Poly dA:dT, 1 μg/mL) agonists. D, Increasing phosphorylation of PDK1, Akt, and GSK3β from 5 minutes up to 2 hours postinfection. In all cases, multiplicity of infection = 10. Candida albicans was added as 100% yeast that switched to hyphal growth by 2 hours after infection. Results shown are the mean (A–C) or representative (D) of 3 independent experiments. Abbreviation: PBS, phosphate-buffered saline.
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JIT824F3: Levels of DNA-binding activity of STAT1 (A) and NFAT (B) in TR146 cells 30 minutes and 3 hours postinfection with Candida albicans. C, Production of granulocyte colony-stimulating factor by TR146 cells in response to TLR3 (poly I:C, 25 μg/mL) and RIG-I (Poly dA:dT, 1 μg/mL) agonists. D, Increasing phosphorylation of PDK1, Akt, and GSK3β from 5 minutes up to 2 hours postinfection. In all cases, multiplicity of infection = 10. Candida albicans was added as 100% yeast that switched to hyphal growth by 2 hours after infection. Results shown are the mean (A–C) or representative (D) of 3 independent experiments. Abbreviation: PBS, phosphate-buffered saline.

Mentions: As well as NF-κB and MAPK signaling [11], the microarray data indicated increased IRF3 and Janus kinase/signal transducer and activator of transcription (JAK/STAT)–regulated genes, as well as increases in NFAT expression (Table 2). Therefore, we determined whether these pathways and their downstream TFs were functionally activated (phosphorylated) in response to C. albicans at early time points (optimum 2 hours [11]) in monolayer TR146 ECs, which comprise the ROE model. TR146 monolayers were used to maximize the signal, and times up to 2 hours were optimum to identify signal pathways driving 6-hour gene expression. Despite the well-documented link between MAPK, NF-κB, and IRF3 signaling after PRR stimulation [3], IRF3 phosphorylation was not detected up to 2 hours postinfection (data not shown), even though NF-κB and MAPK signaling is activated [11]. To confirm that this lack of response was not a result of a defect in the TLR3 or RIG-I pathways, we stimulated TR146 monolayers with the TLR3 (Poly I:C) and IPS (Poly dA:dT) agonists. Both these agonists increased the production of G-CSF from TR146 cells (Figure 3C), indicating that these pathways are functional in TR146 cells. STAT3 phosphorylation was not detected up to 2 hours postinfection (data not shown), and STAT1 DNA binding and transcriptional activity showed no increase at 30 minutes or 3 hours postinfection (Figure 3A). Finally, although NFAT signaling is associated with myeloid cell responses to C. albicans [4], we found no NFAT DNA-binding activity at 30 minutes or 3 hours postinfection (Figure 3B). Together, these data suggest that IRF3, JAK/STAT, and NFAT signaling play no role in EC responses to C. albicans infection.Figure 3.


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)

Levels of DNA-binding activity of STAT1 (A) and NFAT (B) in TR146 cells 30 minutes and 3 hours postinfection with Candida albicans. C, Production of granulocyte colony-stimulating factor by TR146 cells in response to TLR3 (poly I:C, 25 μg/mL) and RIG-I (Poly dA:dT, 1 μg/mL) agonists. D, Increasing phosphorylation of PDK1, Akt, and GSK3β from 5 minutes up to 2 hours postinfection. In all cases, multiplicity of infection = 10. Candida albicans was added as 100% yeast that switched to hyphal growth by 2 hours after infection. Results shown are the mean (A–C) or representative (D) of 3 independent experiments. Abbreviation: PBS, phosphate-buffered saline.
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JIT824F3: Levels of DNA-binding activity of STAT1 (A) and NFAT (B) in TR146 cells 30 minutes and 3 hours postinfection with Candida albicans. C, Production of granulocyte colony-stimulating factor by TR146 cells in response to TLR3 (poly I:C, 25 μg/mL) and RIG-I (Poly dA:dT, 1 μg/mL) agonists. D, Increasing phosphorylation of PDK1, Akt, and GSK3β from 5 minutes up to 2 hours postinfection. In all cases, multiplicity of infection = 10. Candida albicans was added as 100% yeast that switched to hyphal growth by 2 hours after infection. Results shown are the mean (A–C) or representative (D) of 3 independent experiments. Abbreviation: PBS, phosphate-buffered saline.
Mentions: As well as NF-κB and MAPK signaling [11], the microarray data indicated increased IRF3 and Janus kinase/signal transducer and activator of transcription (JAK/STAT)–regulated genes, as well as increases in NFAT expression (Table 2). Therefore, we determined whether these pathways and their downstream TFs were functionally activated (phosphorylated) in response to C. albicans at early time points (optimum 2 hours [11]) in monolayer TR146 ECs, which comprise the ROE model. TR146 monolayers were used to maximize the signal, and times up to 2 hours were optimum to identify signal pathways driving 6-hour gene expression. Despite the well-documented link between MAPK, NF-κB, and IRF3 signaling after PRR stimulation [3], IRF3 phosphorylation was not detected up to 2 hours postinfection (data not shown), even though NF-κB and MAPK signaling is activated [11]. To confirm that this lack of response was not a result of a defect in the TLR3 or RIG-I pathways, we stimulated TR146 monolayers with the TLR3 (Poly I:C) and IPS (Poly dA:dT) agonists. Both these agonists increased the production of G-CSF from TR146 cells (Figure 3C), indicating that these pathways are functional in TR146 cells. STAT3 phosphorylation was not detected up to 2 hours postinfection (data not shown), and STAT1 DNA binding and transcriptional activity showed no increase at 30 minutes or 3 hours postinfection (Figure 3A). Finally, although NFAT signaling is associated with myeloid cell responses to C. albicans [4], we found no NFAT DNA-binding activity at 30 minutes or 3 hours postinfection (Figure 3B). Together, these data suggest that IRF3, JAK/STAT, and NFAT signaling play no role in EC responses to C. albicans infection.Figure 3.

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