The Aryl Hydrocarbon Receptor Governs Epithelial Cell Invasion during Oropharyngeal Candidiasis

doi:10.1128/mBio.00025-17

. 2017 Mar 21;8(2):e00025-17.

doi: 10.1128/mBio.00025-17.

The Aryl Hydrocarbon Receptor Governs Epithelial Cell Invasion during Oropharyngeal Candidiasis

Norma V Solis¹, Marc Swidergall¹, Vincent M Bruno², Sarah L Gaffen³, Scott G Filler^{4

5}

Affiliations

¹ Division of Infectious Diseases, Department of Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, USA.
² Institute for Genome Sciences and Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
³ Division of Rheumatology and Clinical Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
⁴ Division of Infectious Diseases, Department of Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, USA [email protected].
⁵ Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

PMID: 28325761
PMCID: PMC5362030
DOI: 10.1128/mBio.00025-17

The Aryl Hydrocarbon Receptor Governs Epithelial Cell Invasion during Oropharyngeal Candidiasis

Norma V Solis et al. mBio. 2017.

. 2017 Mar 21;8(2):e00025-17.

doi: 10.1128/mBio.00025-17.

Authors

Norma V Solis¹, Marc Swidergall¹, Vincent M Bruno², Sarah L Gaffen³, Scott G Filler^{4

5}

Affiliations

¹ Division of Infectious Diseases, Department of Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, USA.
² Institute for Genome Sciences and Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
³ Division of Rheumatology and Clinical Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
⁴ Division of Infectious Diseases, Department of Medicine, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, USA [email protected].
⁵ Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

PMID: 28325761
PMCID: PMC5362030
DOI: 10.1128/mBio.00025-17

Abstract

Oropharyngeal candidiasis (OPC), caused predominantly by Candida albicans, is a prevalent infection in patients with advanced AIDS, defects in Th17 immunity, and head and neck cancer. A characteristic feature of OPC is fungal invasion of the oral epithelial cells. One mechanism by which C. albicans hyphae can invade oral epithelial cells is by expressing the Als3 and Ssa1 invasins that interact with the epidermal growth factor receptor (EGFR) on epithelial cells and stimulate endocytosis of the organism. However, the signaling pathways that function downstream of EGFR and mediate C. albicans endocytosis are poorly defined. Here, we report that C. albicans infection activates the aryl hydrocarbon receptor (AhR), leading to activation of Src family kinases (SFKs), which in turn phosphorylate EGFR and induce endocytosis of the fungus. Furthermore, treatment of oral epithelial cells with interferon gamma inhibits fungal endocytosis by inducing the synthesis of kynurenines, which cause prolonged activation of AhR and SFKs, thereby interfering with C. albicans-induced EGFR signaling. Treatment of both immunosuppressed and immunocompetent mice with an AhR inhibitor decreases phosphorylation of SFKs and EGFR in the oral mucosa, reduces fungal invasion, and lessens the severity of OPC. Thus, our data indicate that AhR plays a central role in governing the pathogenic interactions of C. albicans with oral epithelial cells during OPC and suggest that this receptor is a potential therapeutic target.IMPORTANCE OPC is caused predominantly by the fungus C. albicans, which can invade the oral epithelium by several mechanisms. One of these mechanisms is induced endocytosis, which is stimulated when fungal invasins bind to epithelial cell receptors such as EGFR. Receptor binding causes rearrangement of epithelial cell microfilaments, leading to the formation of pseudopods that engulf the fungus and pull it into the epithelial cell. We discovered AhR acts via SFKs to phosphorylate EGFR and induce the endocytosis of C. albicans Our finding that a small molecule inhibitor of AhR ameliorates OPC in mice suggests that a strategy of targeting host cell signaling pathways that govern epithelial cell endocytosis of C. albicans holds promise as a new approach to preventing or treating OPC.

Keywords: Candida albicans; aryl hydrocarbon receptor; epithelial cells; host cell invasion; interferon-gamma.

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Figures

**FIG 1**
Interferon gamma (IFN-γ) inhibits endocytosis of *C. albicans* by oral epithelial cells. The OKF6/TERT-2 oral epithelial cell line was incubated with IFN-γ for 24 h in the absence and presence of an anti-IFN-γ monoclonal antibody (mAb). The cells were infected with *C. albicans* SC5314 for 2.5 h, after which the number of endocytosed organisms was determined using a differential fluorescence assay. Results are means ± standard deviations (SD) from three experiments, each performed in triplicate. Orgs/HPF, organisms per high-power field; ctrl, control. Statistical significance was determined using the unpaired Student’s t test (P ≤ 0.05). (B) RNA-seq analysis of the effects of IFN-γ on the transcriptional response of oral epithelial cells to *C. albicans*. OKF6/TERT-2 epithelial cells were incubated in the presence or absence of IFN-γ for 24 h and then infected with *C. albicans* for 5 h. RNA was extracted and analyzed by RNA-seq. The heat map shows normalized, log-transformed RPKM values of the top 40 IFN-γ-responsive genes. The red arrow indicates the IDO1 gene. (C) Effects of IDO inhibition with l-1-methyl-tryptophan (l-1MT) on OKF6/TERT-2 oral epithelial cell endocytosis of *C. albicans*. Results are means ± SD from three experiments, each performed in triplicate. Statistical significance was determined using the unpaired Student’s t test (P ≤ 0.05).

**FIG 2**
The tryptophan metabolite kynurenine inhibits the endocytosis of *C. albicans*. (A) Effects of IFN-γ and the indicated compounds on epithelial cell endocytosis of *C. albicans*. OKF6/TERT-2 oral epithelial cells were incubated for 24 h with tryptophan (Trp), either alone or in combination with IFN-γ, and then infected with *C. albicans* for 2.5 h. Results are means ± SD from three experiments, each performed in triplicate. (B) Kynurenine production by epithelial cells after incubation with the indicated compounds for 24 h. Results are means ± SD from three experiments. (C) Effects of 24 h of exposure to l-kynurenine (L-Kyn) or the kynurenine analog N-(3,4-dimethoxycinnamoyl)-anthranilic acid (3,4-DAA) on epithelial cell endocytosis of *C. albicans*. Results are means ± SD from three experiments, each performed in triplicate. Statistical significance was determined using the unpaired Student’s t test (P ≤ 0.05). NS, not significant; ctrl, control.

**FIG 3**
IFN-γ activates the aryl hydrocarbon receptor (AhR) and Src family kinases (SFKs), which govern the endocytosis of *C. albicans*. (A) Confocal micrographs of OKF6/TERT-2 oral epithelial cells incubated in the presence and absence of IFN-γ for 24 h. The cells were stained for AhR (green), and the nuclei were stained with DAPI (blue). The perimeters of the cells were determined by differential interference contrast and are indicated by the dashed lines. Scale bar, 20 µm. (B) Endocytosis of *C. albicans* by oral epithelial cells treated with IFN-γ for 24 h or the AhR inhibitor for 1 h. (C) Endocytosis of *C. albicans* by oral epithelial cells transfected with either control siRNA or AhR siRNA. The inset is a representative immunoblot showing knockdown of AhR. (D) Effects of 1 h of exposure to the indicated SFK inhibitors on the endocytosis of *C. albicans*. All endocytosis data are means ± SD from three experiments, each performed in triplicate. (E and F) Effects of *C. albicans* and the AhR inhibitor on SFK phosphorylation. OKF6/TERT-2 cells were pretreated for 1 h with the indicated inhibitor and then infected with *C. albicans* for 1 h, after which the extent of SFK phosphorylation was determined by immunoblotting. (E) Representative immunoblot. (F) Densitometric analysis of 3 immunoblots such as the one shown in panel E. Results are means ± SD from 3 experiments. Statistical significance was determined using the unpaired Student’s t test (P ≤ 0.05). ctrl, control; INH, inhibitor; Ca, *C. albicans*; UNINF, uninfected.

**FIG 4**
IFN-γ inhibits EGFR phosphorylation. (A) Effects of IFN-γ (24 h) and/or the EGFR inhibitor gefitinib (1 h) on the endocytosis of *C. albicans* by OKF6/TERT-2 oral epithelial cells. Results are the means ± SD from three experiments, each performed in triplicate. (B to E) Effects of IFN-γ on *C. albicans*-induced autophosphorylation of the indicated tyrosine residues of EGFR. The oral epithelial cells were incubated in the presence and absence of IFN-γ for 24 and then infected with *C. albicans* for the indicated time points. The phosphorylation of the specific EGFR tyrosine residues was determined by immunoblotting with specific monoclonal antibodies. (B) Representative immunoblots. The images above the blot show the *C. albicans* morphology at the indicated time points. Scale bar, 20 µm. (C to E) Densitometric analysis of the immunoblots. Results are means ± SD from 3 experiments. (F to H) Effects of IFN-γ on SFK-dependent phosphorylation of the indicated tyrosine residues of EGFR. (F) Representative immunoblots. (G and H) Densitometric analysis of the immunoblots. Results are means ± SD from 3 experiments. Statistical significance was determined using the unpaired Student’s t test (P ≤ 0.05). NS, not significant; ctrl, control; p.i., postinfection.

**FIG 5**
*C. albicans*-induced phosphorylation of EGFR depends on AhR and SFK activity. (A to C). Effects of inhibition of AhR and SFKs on *C. albicans*-induced phosphorylation of EGFR. OKF6/TERT-2 epithelial cells were pretreated for 1 h with the indicated inhibitor and then infected with *C. albicans* for 1 h. (A and B) Representative immunoblots showing EGFR phosphorylation at Y1068 (A) and Y1101 (B). (C) Densitometric analysis of the immunoblots in panels A and B. Results are means ± SD from 3 experiments. (D and E) Effects of IFN-γ on SFK phosphorylation. (D) Representative immunoblot. (E) Densitometric analysis of the immunoblots in panel D. Results are means ± SD from 3 experiments. (F, left panel) Proposed model of how IFN-γ inhibits the endocytosis of *C. albicans* by activating IDO, leading to the production of kynurenines that induce prolonged activation of AhR and SFKs, thereby preventing *C. albicans*-induced activation of EGFR and inhibiting endocytosis of the organism. (Right panel) Proposed model in which *C. albicans* activates AhR, stimulating SFKs that phosphorylate EGFR, leading to the endocytosis of the fungus. (G to J) Effects of the epidermal growth factor (EGF) and the AhR inhibitor on endocytosis (G) and EGFR phosphorylation (H to J). Results are means ± SD from three experiments, each performed in triplicate. Statistical significance was determined using the unpaired Student’s t test (P ≤ 0.05). Ca, *C. albicans*; INH, inhibitor; UNINF, uninfected; ctrl, control; Kyn, kynurenines; NS, not significant.

**FIG 6**
Effects of IFN-γ, AhR, and SFKs on epithelial cell damage and cytokine release induced by *C. albicans* (A to C). Oral epithelial cells were incubated with IFN-γ (A) or l-kynurenine (B) for 24 h or with the AhR or SFK inhibitor (C) for 1 h and then infected with *C. albicans* for 7 h. The extent of epithelial cell damage was measured using a ⁵¹Cr release assay. (D and E) Oral epithelial cells were incubated with the indicated compounds as in panels A to C and infected with *C. albicans* for 8 h, after which the supernatant was collected and analyzed for the concentration of interleukin-1α (IL-1α [D]), IL-1β (E), and IL-8 (F). Results are means ± SD from three experiments, each performed in triplicate. Statistical significance was determined using the unpaired Student’s t test (P ≤ 0.05). OEC, oral epithelial cells; ctrl, control; NS, not significant; INH, inhibitor; UNINF, uninfected; L-Kyn, l-kynurenine.

**FIG 7**
Inhibition of AhR reduces severity of disease during experimental OPC. Immunosuppressed (I.S.) and immunocompetent (I.C.) mice were treated with either diluent (control [ctrl]) or the AhR inhibitor (INH) and then orally inoculated with *C. albicans*. (A) Daily body weights of the immunosuppressed mice. (B and C) Oral fungal burden of the immunosuppressed mice after 4 days of infection (B) and of the immunocompetent mice after 1 day of infection (C). Results in panels A and B are medians ± interquartile ranges from the combined results of two separate experiments for a total of 9 to 10 mice per experimental group. Results in panel C are medians ± interquartile ranges from a single experiment with 7 mice per experimental group. (D to F) Analysis of the fungal lesions in tongues of immunosuppressed mice after 4 days of infection. (D) Length of the fungal lesions. (E) Depth of maximal fungal invasion. (F) Number of fungal lesions per tongue section. Results in panels D to F are medians ± interquartile ranges from the analysis of 7 to 8 thin sections from two separate experiments using a total of 6 mice per experimental group. (G to J) Inhibition of AhR reduces phosphorylation of SFKs and EGFR in the oral mucosa. Shown are confocal images of thin sections of the tongues of immunosuppressed (G and I) and immunocompetent (H and J) mice that were administered either diluent alone (ctrl [left panels]) or the AhR inhibitor (right panels) and then infected with *C. albicans* as in panels A to C. The thin sections in panels G and H were stained for phospho-SFK Y416 (green), and the thin sections in panels I and J were stained for phospho-EGFR Y1068 (green). All sections were also stained with an anti-*Candida* antiserum (red). Scale bar, 50 µm. Statistical significance was determined using the Mann-Whitney test (P ≤ 0.05).

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References

1. Phelan JA, Saltzman BR, Friedland GH, Klein RS. 1987. Oral findings in patients with acquired immunodeficiency syndrome. Oral Surg Oral Med Oral Pathol 64:50–56. doi:10.1016/0030-4220(87)90116-2. - DOI - PubMed
1. Feigal DW, Katz MH, Greenspan D, Westenhouse J, Winkelstein W Jr, Lang W, Samuel M, Buchbinder SP, Hessol NA, Lifson AR. 1991. The prevalence of oral lesions in HIV-infected homosexual and bisexual men: three San Francisco epidemiological cohorts. AIDS 5:519–525. doi:10.1097/00002030-199105000-00007. - DOI - PubMed
1. Armstrong-James D, Meintjes G, Brown GD. 2014. A neglected epidemic: fungal infections in HIV/AIDS. Trends Microbiol 22:120–127. doi:10.1016/j.tim.2014.01.001. - DOI - PubMed
1. Kerdpon D, Pongsiriwet S, Pangsomboon K, Iamaroon A, Kampoo K, Sretrirutchai S, Geater A, Robison V. 2004. Oral manifestations of HIV infection in relation to clinical and CD4 immunological status in northern and southern Thai patients. Oral Dis 10:138–144. doi:10.1046/j.1601-0825.2003.00990.x. - DOI - PubMed
1. Chidzonga MM. 2003. HIV/AIDS orofacial lesions in 156 Zimbabwean patients at referral oral and maxillofacial surgical clinics. Oral Dis 9:317–322. doi:10.1034/j.1601-0825.2003.00962.x. - DOI - PubMed

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[1] Phelan JA, Saltzman BR, Friedland GH, Klein RS. 1987. Oral findings in patients with acquired immunodeficiency syndrome. Oral Surg Oral Med Oral Pathol 64:50–56. doi:10.1016/0030-4220(87)90116-2. - DOI - PubMed

[2] Phelan JA, Saltzman BR, Friedland GH, Klein RS. 1987. Oral findings in patients with acquired immunodeficiency syndrome. Oral Surg Oral Med Oral Pathol 64:50–56. doi:10.1016/0030-4220(87)90116-2. - DOI - PubMed

[3] Feigal DW, Katz MH, Greenspan D, Westenhouse J, Winkelstein W Jr, Lang W, Samuel M, Buchbinder SP, Hessol NA, Lifson AR. 1991. The prevalence of oral lesions in HIV-infected homosexual and bisexual men: three San Francisco epidemiological cohorts. AIDS 5:519–525. doi:10.1097/00002030-199105000-00007. - DOI - PubMed

[4] Feigal DW, Katz MH, Greenspan D, Westenhouse J, Winkelstein W Jr, Lang W, Samuel M, Buchbinder SP, Hessol NA, Lifson AR. 1991. The prevalence of oral lesions in HIV-infected homosexual and bisexual men: three San Francisco epidemiological cohorts. AIDS 5:519–525. doi:10.1097/00002030-199105000-00007. - DOI - PubMed

[5] Armstrong-James D, Meintjes G, Brown GD. 2014. A neglected epidemic: fungal infections in HIV/AIDS. Trends Microbiol 22:120–127. doi:10.1016/j.tim.2014.01.001. - DOI - PubMed

[6] Armstrong-James D, Meintjes G, Brown GD. 2014. A neglected epidemic: fungal infections in HIV/AIDS. Trends Microbiol 22:120–127. doi:10.1016/j.tim.2014.01.001. - DOI - PubMed

[7] Kerdpon D, Pongsiriwet S, Pangsomboon K, Iamaroon A, Kampoo K, Sretrirutchai S, Geater A, Robison V. 2004. Oral manifestations of HIV infection in relation to clinical and CD4 immunological status in northern and southern Thai patients. Oral Dis 10:138–144. doi:10.1046/j.1601-0825.2003.00990.x. - DOI - PubMed

[8] Kerdpon D, Pongsiriwet S, Pangsomboon K, Iamaroon A, Kampoo K, Sretrirutchai S, Geater A, Robison V. 2004. Oral manifestations of HIV infection in relation to clinical and CD4 immunological status in northern and southern Thai patients. Oral Dis 10:138–144. doi:10.1046/j.1601-0825.2003.00990.x. - DOI - PubMed

[9] Chidzonga MM. 2003. HIV/AIDS orofacial lesions in 156 Zimbabwean patients at referral oral and maxillofacial surgical clinics. Oral Dis 9:317–322. doi:10.1034/j.1601-0825.2003.00962.x. - DOI - PubMed

[10] Chidzonga MM. 2003. HIV/AIDS orofacial lesions in 156 Zimbabwean patients at referral oral and maxillofacial surgical clinics. Oral Dis 9:317–322. doi:10.1034/j.1601-0825.2003.00962.x. - DOI - PubMed

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The Aryl Hydrocarbon Receptor Governs Epithelial Cell Invasion during Oropharyngeal Candidiasis

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The Aryl Hydrocarbon Receptor Governs Epithelial Cell Invasion during Oropharyngeal Candidiasis

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