Research ArticleFUNGAL INFECTION

Oral epithelial cells orchestrate innate type 17 responses to Candida albicans through the virulence factor candidalysin

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Science Immunology  03 Nov 2017:
Vol. 2, Issue 17, eaam8834
DOI: 10.1126/sciimmunol.aam8834
  • Fig. 1 Proliferation of oral TCRαβ+ cells after C. albicans infection.

    (A) Il17aeYFP mice (17) were challenged sublingually with phosphate-buffered saline (PBS) (sham) or C. albicans. Homogenates were prepared from pooled tongues (n = 2 to 3). YFP+TCRαβ+ or YFP+TCRγδ+ cells in the CD45+ gate were assessed by flow cytometry. Data show fold increase versus sham, pooled from three to four independent experiments. (B and C) WT mice (C57BL/6J) were infected with C. albicans, and tongue homogenates were prepared on days 1 or 2 after infection (p.i.). Cells were gated on lymphocytes, and staining of CD45 and TCRβ is shown (top). Proliferation of CD45+CD4+TCRβ+ cells was determined by staining for Ki67 (bottom). Data are representative of 10 experiments. Graphs in (B and C) show percent of proliferating TCRαβ+ cells on days 1 and 2. (D) WT mice were infected with C. albicans, and tongue homogenates were prepared on day 2 after infection. Proliferation was determined by PCNA staining. Data are representative of three experiments. (E) WT cLNs were harvested on day 2 after infection. Proliferation of CD45+CD4+TCRβ+ cells was determined by anti-Ki67 staining. Graph shows mean ± SEM of Ki67+ CD4+ cells in cLNs. Data are representative of two experiments. For statistical analyses, Student’s t test or one-way analysis of variance (ANOVA) was used. NS, not significant. ***P < 0.001 and ****P < 0.0001.

  • Fig. 2 CCR6 is dispensable for expansion of innate TCRαβ+ cells in oral candidiasis.

    (A) WT or Ccr6−/− mice were infected with C. albicans, and proliferation of oral TCRαβ+ cells was determined at day 2 after infection. Data are representative of three independent experiments. (B) Indicated mice were infected orally with C. albicans, and fungal burden was assessed by CFU enumeration on day 4 after infection. Bars represent the geometric mean. Each point represents an individual mouse. Dashed line represents the limit of detection (LOD; 30 CFU) (52). Data are compiled from four independent experiments. (C) WT mice were injected with 100 μg of anti-CCL20 antibodies or isotype controls on day −1 relative to infection. Proliferation of TCRαβ+ cells was determined on day 2 after infection. Data are representative of two experiments. For statistical analysis, Student’s t test or ANOVA was used. ****P < 0.0001.

  • Fig. 3 A primary C. albicans infection activates innate TCRαβ+ cells without engaging the TCR.

    (A) Nur77GFP mice were sham-treated (red line) or infected with C. albicans, and tongue homogenates were prepared on days 1 or 2 after infection (blue line). Controls received anti-CD3 antibodies (green line) to stimulate the TCR on all T cells. WT (“non-Tg”) mice were negative controls for GFP staining (gray line). Left: Fluorescence intensity of GFP in oral CD45+CD4+TCRβ+ cells. Right: Relative mean fluorescence intensity (MFI) of GFP in CD45+CD4+TCRβ+ cells was assessed and normalized to sham. Data are from three independent experiments. (B) Nur77GFP mice were infected with C. albicans. Tongue homogenates were prepared 2 days after infection (“1° inf”). To induce C. albicans–specific TCR signaling (“2° inf”), mice were infected orally, rested for 6 weeks, and then rechallenged with a second oral infection (8). Left: GFP fluorescence in oral CD45+CD4+TCRβ+ cells, with the percentage of GFPhi cells indicated. Green line shows staining in mice administered agonistic anti-CD3 antibodies, as in (A). Right: Compiled percentage of GFPhi cells per cohort. Data are representative of three to four independent experiments. Graphs show mean + SEM, as analyzed by Student’s t test or ANOVA. **P < 0.01 and ***P < 0.001.

  • Fig. 4 TLR2 and dectin-1 are dispensable for C. albicans–induced proliferation of innate TCRαβ+ cells.

    (A to C) Indicated mice were infected with C. albicans, and proliferation of CD45+CD4+TCRβ+ cells was determined on day 2 after infection. Data are representative of two to three independent experiments. (D) Indicated mice were infected, and fungal burden was quantified on day 5 after infection. Bars represent the geometric mean. Dashed line represents the LOD. Data are from two independent experiments. Data were analyzed by ANOVA and Mann-Whitney correction. (E) Average percent weight loss is shown. **P < 0.01.

  • Fig. 5 Candidalysin drives the proliferation of innate IL-17–producing TCRαβ+ cells.

    (A) Il17aeYFP mice were infected with C. albicans (ece1Δ/Δ or Rev), and homogenates were prepared 2 days after infection. Staining of CD45 and yellow fluorescent protein (YFP) in lymphocyte gate is shown. Data are representative of two experiments. (B) WT mice were infected with the indicated strains of C. albicans, and expansion (top) and proliferation (bottom) of oral TCRαβ+ cells were analyzed at day 2 after infection. Data are representative of three experiments. (C) Fold expansion of TCRβ+ cells after infection with ece1Δ/Δ or Rev strains. Data are pooled from four experiments. (D) Fungal loads were assessed at day 2 after infection. Bar represents the geometric mean. Data were pooled from two experiments. (E and F) Tongue homogenates were prepared 2 days after infection with the indicated C. albicans strains. Total mRNA was subjected to quantitative real-time polymerase chain reaction (qPCR) normalized to Gapdh. Graphs show mean + SEM normalized to sham. Data are compiled from seven to eight mice per group from two independent experiments. (G) Percentage of CD11b+Ly6Ghi cells in the tongue was assessed at day 2 after infection. Graphs indicate mean + SEM, as compiled from three experiments. Statistics were analyzed by Student’s t test or ANOVA. PMNs, polymorphonuclear leukocytes. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 IL-1 activates innate TCRαβ+ cell proliferation and antifungal immunity in a T cell–intrinsic and –extrinsic manner.

    (A) WT mice were infected with the indicated C. albicans strains, and gene expression was measured on day 2 after infection. Data are means + SEM normalized to sham, from seven to eight mice per group in two experiments. (B) Expansion and proliferation of TCRαβ+ cells in Il1r1−/− mice at day 2 after infection. Data are from three experiments. (C) Fungal burdens in the indicated mice were quantified from two experiments on day 5 after infection. (D) WT mice were administered anti–IL-1α, anti–IL-1β, or isotype control antibodies (1.0 mg per mouse used alone or 0.5 mg each when used together) on day −1 relative to infection. Proliferation of oral TCRαβ+ cells was assessed at day 2 after infection. Data are representative of two experiments. (E) Reciprocal adoptive transfers of femoral BM were performed in WT or Il1r1−/− mice, and proliferation of oral TCRαβ+ cells was determined. Experimental chimera results are representative of two experiments; control chimera data are from one experiment. Data were analyzed by Student’s t test or ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 7 Candidalysin and IL-17 signal synergistically or additively in OECs.

    (A) TR146 OECs were untreated (“U”; gray bars) or stimulated with IL-17 (200 ng/ml; black bars). Cells were infected with WT C. albicans (Bwp17 + CIp30; “Parent”), ece1Δ/Δ, or the Rev for 24 hours. Supernatants were analyzed by Luminex (IL-1β, IL-6, and G-CSF) or enzyme-linked immunosorbent assay (ELISA) (CCL20). Graphs indicate mean + SEM. Data are representative of two experiments. (B) TR146 cells were untreated (U; gray bars) or treated with IL-17 (200 ng/ml; black bars) or candidalysin peptide (Clys; 15 μM) for 24 hours and analyzed as in (A). (C) TR146 cells were incubated with C. albicans ± IL-17 (200 ng/ml). LDH in supernatants was evaluated after 24 hours, representative of three experiments. (D) TR146 cells were treated with TNFα (20 ng/ml), IL-17 (200 ng/ml), or candidalysin (15 μM) for 5 min. Lysates were immunoblotted for phospho-IκBα and total IκBα. (E) TR146 cells were incubated with TNFα (20 ng/ml), IL-17 (200 ng/ml), or candidalysin (15 μM) for 30 min or 2 hours. Lysates were immunoblotted for c-Fos, phospho-MKP1, or Actin. Data are representative of two experiments. (F) TR146 cells were transfected with c-Fos small interfering RNA (siRNA) and stimulated for 24 hours with PBS, Clys, or IL-17. Supernatants were assessed for CCL20 by ELISA. Data are representative of two independent experiments. All data were analyzed by ANOVA and Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/17/eaam8834/DC1

    Fig. S1. TCRαβ+ cell proliferation in knockout mice and gating strategies.

    Fig. S2. Expansion of innate TCRαβ+ cells during OPC.

    Fig. S3. Baseline frequency of innate TCRαβ+ cells is similar in WT and Ccr6−/− mice.

    Fig. S4. Virulence factors and TCRαβ+ cell expansion.

    Fig. S5. Factors that activate TCRαβ+ cell expansion.

    Fig. S6. Candidalysin signals synergistically with IL-17 and TNFα but not with IL-22.

    Fig. S7. Model of first encounter to C. albicans in the oral epithelium.

    Data file (provided as Excel file)

    Source data (provided as pdf file)

  • Supplementary Materials

    Supplementary Material for:

    Oral epithelial cells orchestrate innate type 17 responses to Candida albicans through the virulence factor candidalysin

    Akash H. Verma, Jonathan P. Richardson, Chunsheng Zhou, Bianca M. Coleman, David L. Moyes, Jemima Ho, Anna R. Huppler, Kritika Ramani, Mandy J. McGeachy, Ilgiz A. Mufazalov, Ari Waisman, Lawrence P. Kane, Partha S. Biswas, Bernhard Hube, Julian R. Naglik,* Sarah L. Gaffen*

    *Corresponding author. Email: sarah.gaffen{at}pitt.edu (S.L.G.); julian.naglik{at}kcl.ac.uk (J.R.N.)

    Published 3 November 2017, Sci. Immunol. 2, eaam8834 (2017)
    DOI: 10.1126/sciimmunol.aam8834

    This PDF file includes:

    • Fig. S1. TCRαβ+ cell proliferation in knockout mice and gating strategies.
    • Fig. S2. Expansion of innate TCRαβ+ cells during OPC.
    • Fig. S3. Baseline frequency of innate TCRαβ+ cells is similar in WT and Ccr6−/− mice.
    • Fig. S4. Virulence factors and TCRαβ+ cell expansion.
    • Fig. S5. Factors that activate TCRαβ+ cell expansion.
    • Fig. S6. Candidalysin signals synergistically with IL-17 and TNFα but not with IL-22.
    • Fig. S7. Model of first encounter to C. albicans in the oral epithelium.

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    Other Supplementary Material for this manuscript includes the following:

    Files in this Data Supplement:

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