C/EBPβ Promotes Immunity to Oral Candidiasis through Regulation of β-Defensins

Abstract

Humans or mice subjected to immunosuppression, such as corticosteroids or anti-cytokine biologic therapies, are susceptible to mucosal infections by the commensal fungus Candida albicans. Recently it has become evident that the Th17/IL-17 axis is essential for immunity to candidiasis, but the downstream events that control immunity to this fungus are poorly understood. The CCAAT/Enhancer Binding Protein-β (C/EBPβ) transcription factor is important for signaling by multiple inflammatory stimuli, including IL-17. C/EBPβ is regulated in a variety of ways by IL-17, and controls several downstream IL-17 target genes. However, the role of C/EBPβ in vivo is poorly understood, in part because C/EBPβ-deficient mice are challenging to breed and work with. In this study, we sought to understand the role of C/EBPβ in the context of an IL-17-dependent immune response, using C. albicans infection as a model system. Confirming prior findings, we found that C/EBPβ is required for immunity to systemic candidiasis. In contrast, C/EBPβ(-/-) mice were resistant to oropharyngeal candidiasis (OPC), in a manner indistinguishable from immunocompetent WT mice. However, C/EBPβ(-/-) mice experienced more severe OPC than WT mice in the context of cortisone-induced immunosuppression. Expression of the antimicrobial peptide β-defensin (BD)-3 correlated strongly with susceptibility in C/EBPβ(-/-) mice, but no other IL-17-dependent genes were associated with susceptibility. Therefore, C/EBPβ contributes to immunity to mucosal candidiasis during cortisone immunosuppression in a manner linked to β-defensin 3 expression, but is apparently dispensable for the IL-17-dependent response.

Fig 1. C/EBPβ^-/- mice exhibit increased susceptibility to systemic candidiasis but are resistant to oral candidiasis.

(A) The indicated mice (n = 3 per group) were injected with C. albicans in the lateral tail vein. Time to sacrifice is indicated (days). *P<0.05 versus (vs) C/EBPβ^+/- mice using Log-rank (Mantel-Cox). (B) The indicated mice were infected sublingually with C. albicans for 75 mins. After 5 d, fungal loads in tongue were assessed by CFU enumeration of tongue tissue homogenates. Bars indicate geometric mean with 95% CI. C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ (n = 5), C/EBPβ^+/- (n = 9), C/EBPβ^-/- (n = 3), C/EBPβ^+/+ plus 225mg/kg cortisone (n = 3); cortisone acetate was administered by subcutaneous injection on days -1, +1 and +2 relative to infection. P<0.05 by t-test with Mann-Whitney correction: * vs C/EBPβ^+/+ WT, ≠ vs C/EBPβ^+/-, # vs C/EBPβ^-/-, π vs C/EBPβ^+/+ 225mg/kg. C. Weights of mice were assessed daily and graphed as percent of starting weight. * vs C/EBPβ^+/+ WT. P<0.05 by t-test with Mann-Whitney correction. Experiment was performed once.

Fig 2. C/EBPβ^-/- mice exhibit increased susceptibility to oral candidiasis in the context of cortisone-induced immunosuppression.

(A) C65BL/6 mice (“WT”) were treated with the indicated doses of cortisone acetate at days -1, +1 and +2 relative to infection. After 5 d, fungal loads in tongue were assessed by CFU enumeration of tongue tissue homogenates. SHAM (n = 3), No cortisone control (n = 5), 60mg/kg (n = 9), 120/112mg/kg (n = 8), and 225mg/kg (n = 8). Data are pooled from 2 independent experiments. Bars indicate geometric mean with 95% CI. P<0.05 by t-test with Mann-Whitney correction: * vs NO CORT, ≠ vs 60mg/kg, # vs 120/112mg/kg and π vs 225mg/kg. (B) The indicated mice were infected orally as described in panel A. Cortisone acetate was administered subcutaneously on days -1, +1 and +2 relative to infection. C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ (n = 5), C/EBPβ^+/+ 60 mg/kg (n = 16), C/EBPβ^+/- 60mg/kg (n = 16), C/EBPβ^-/- 60mg/kg (n = 10) and C/EBPβ^+/+ 225mg/kg (n = 4). P<0.05 by t-test with Mann-Whitney correction: * vs C/EBPβ^+/+ NO CORT, ≠ vs C/EBPβ^+/+ 60mg/kg, # vs C/EBPβ^+/- 60mg/kg and π vs C/EBPβ^-/- 60mg/kg. Data are pooled from two independent experiments. C. Representative tongue sections from the indicated mice were stained with H&E or Periodic-acid Schiff (PAS). Scale bar indicates 200 μM. White arrows indicate hyphae.

Fig 3. The susceptibility of C/EBPβ^-/- mice to OPC does not correlate with expression of prototypical IL-17-regulated genes.

mRNA from tongue was isolated from the indicated mice 5 days after oral C. albicans infection [C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ NO CORT (n = 3), C/EBPβ^+/+ 60mg/kg (n = 5), C/EBPβ^+/- 60mg/kg (n = 6), and C/EBPβ^-/- 60mg/kg (n = 5)]. Complementary DNA was prepared and subjected to qPCR analysis to detect the indicated genes. Results are presented as fold induction over SHAM treated mice and normalized to expression of Gapdh. Data are pooled from 2 independent experiments. P<0.05 by student unpaired t-test: * vs C/EBPβ^+/+ NO CORT, ≠ vs C/EBPβ^+/+ 60mg/kg, # vs C/EBPβ^+/- 60mg/kg and π vs C/EBPβ^-/- 60mg/kg.

Fig 4. Susceptibility of C/EBPβ^-/- mice to OPC correlates with expression of BD3.

(A) mRNA from tongue was isolated from the indicated mice 5 days after oral C. albicans infection [C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ NO CORT (n = 3), C/EBPβ^+/+ 60mg/kg (n = 5), C/EBPβ^+/- 60mg/kg (n = 6), and C/EBPβ^-/- 60mg/kg (n = 5)]. Complementary DNA was prepared and subjected to qPCR analysis to detect the indicated genes. Results are presented as fold induction over SHAM treated mice and normalized to expression of Gapdh. Data are pooled from two independent experiments. P<0.05 by student unpaired t-test. * vs C/EBPβ^+/+ NO CORT, ≠ vs C/EBPβ^+/+ 60mg/kg, # vs C/EBPβ^+/- 60mg/kg and π vs C/EBPβ^-/- 60mg/kg. (B) mRNA from tongue was isolated from the indicated mice 5 days after oral C. albicans infection and analyzed as in panel A. C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ (n = 2), C/EBPβ^+/- (n = 2), and C/EBPβ^-/- (n = 2). Data are from one experiment. * P<0.05 by student unpaired t-test. vs C/EBPβ^+/+, ≠ vs C/EBPβ^+/- and π vs C/EBPβ^-/-. (C) OKF6/TERT2 human oral keratinocytes were treated with 200 ng/ml IL-17 plus 2ng/ml TNFα or with 2×10⁶ HK C. albicans for 24 h. Complementary DNA was prepared and subjected to qPCR analysis to detect DEFB4A. Data are normalized to expression of GAPDH and represent absolute levels. Data are representative of 2 independent experiments. *P<0.05 compared to unstimulated OKF6/TERT2 cells.