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. 2014 Nov 7;9(11):e112472.
doi: 10.1371/journal.pone.0112472. eCollection 2014.

Intestinal colonization by Candida albicans alters inflammatory responses in Bruton's tyrosine kinase-deficient mice

Karin Strijbis  1 , Omer H Yilmaz  1 , Stephanie K Dougan  1 , Alexandre Esteban  1 , Andrea Gröne  2 , Carol A Kumamoto  3 , Hidde L Ploegh  1
Affiliations

Intestinal colonization by Candida albicans alters inflammatory responses in Bruton's tyrosine kinase-deficient mice

Karin Strijbis et al. PLoS One. .

Abstract

The commensal yeast Candida albicans is part of the human intestinal microflora and is considered a "pathobiont", a resident microbe with pathogenic potential yet harmless under normal conditions. The aim of this study was to investigate the effect of C. albicans on inflammation of the intestinal tract and the role of Bruton's tyrosine kinase (Btk). Btk is an enzyme that modulates downstream signaling of multiple receptors involved in innate and adaptive immunity, including the major anti-fungal receptor Dectin-1. Colitis was induced in wild type and Btk-/- mice by treatment with dextran sodium sulfate (DSS) and the gastrointestinal tract of selected treatment groups were then colonized with C. albicans. Colonization by C. albicans neither dampened nor exacerbated inflammation in wild type mice, but colon length and spleen weight were improved in Btk-deficient mice colonized with C. albicans. Neutrophil infiltration was comparable between wild type and Btk-/- mice, but the knockout mice displayed severely reduced numbers of macrophages in the colon during both DSS and DSS/Candida treatment. Smaller numbers and reduced responsiveness of Btk-/- macrophages might partially explain the improved colon length of Btk-/- mice as a result of Candida colonization. Surprisingly, DSS/Candida-treated Btk-/- animals had higher levels of certain pro-inflammatory cytokines and levels of the anti-inflammatory cytokine TGF-β were reduced compared to wild type. A clustering and correlation analysis showed that for wild type animals, spleen TGF-β and colon IL-10 and for Btk-/- spleen and colon levels of IL-17A best correlated with the inflammatory parameters. We conclude that in Btk-/- immunocompromised animals, colonization of the gastrointestinal tract by the commensal yeast C. albicans alters inflammatory symptoms associated with colitis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Morphology and β1,3-glucan exposure of C. albicans adapted to the gastrointestinal tract.
A: C. albicans isolated from mouse cecum 4 days after inoculation and stained with Dectin1-CRD-Alexa647. After 4 days of gastrointestinal adaptation, the majority of C. albicans display short (pseudo)hyphal morphology in which β1,3-glucan exposure is limimited. B: C. albicans isolated from cecum 18 days after inoculation and stained with Dectin1-CRD-Alexa647. The majority of C. albicans display short (pseudo)hyphal morphology. After 18 days of gastrointestinal adaptation, the majority of C. albicans display long hyphal morphology with extensive β1,3-glucan exposure. Experiments were performed twice with two animals in each group, representative images are shown.
Figure 2
Figure 2. Colonization by C. albicans reduces DSS-induced inflammation in Btk-/- mice.
A: Experimental design of DSS-induced colitis in combination with C. albicans colonization of the gastrointestinal tract. DSS was administered in the drinking water starting on day 1. On day 3, 1×107 CFU of Candida albicans was administered to the DSS/Candida treatment groups by oral gavage. Control groups included 3, DSS groups 5 and DSS/Candida groups included 10-11 animals. Animals were sacrificed on day 8 when the first animals reached 80% of initial body weight. B: Weight loss curve of the indicated groups of mice during DSS- or DSS/Candida treatment. C: Gomori Methenamine Silver (GMS) staining of wild type and Btk-/- colon after DSS/Candida treatment. Arrows indicate Candida hyphae. D: C. albicans colony forming units (CFUs) in cecum content in wild type and Btk-/- mice during DSS/Candida treatment. E: Spleen weights of all mice and treatment groups in DSS/Candida experiment. F: Colon lengths of all mice and treatment groups in DSS/Candida experiment. All graphs show individual data points, the means of the indicated groups and error bars depict standard deviations. *: p<0.05, **: p<0.01, ***: p<0.005, ****: p<0.001.
Figure 3
Figure 3. Btk-/- animals have reduced colonic macrophages, but not neutrophils.
Mice were treated with DSS or the DSS/Candida combination as described in the legend of Figure 2. Sample size (number of animals): control groups (1), DSS groups (3), DSS/Candida groups (3). Animals were sacrificed on day 8 and the distal colons were fixed and prepared for histology. A: H&E staining of indicated mouse distal colon sections. Right: H&E stainings were blindly graded based on the following scheme: 1. Normal mucosa. 2. Cryptitis. 3. Crypt abscesses. 4. Focal ulcerations. 5. Multifocal ulcerations. B: Immunohistochemical staining using MPO antibody shows neutrophil infiltration in the indicated mouse distal colon sections. Right: Quantification of infiltrating neutrophils in DSS and DSS/Candida conditions. C: Immunohistochemical analysis with CD68 antibody showing macrophages in the indicated mouse distal colon sections. Right: Quantification of macrophages in DSS and DSS/Candida conditions. ***: p<0.005, ****: p<0.001. Representative pictures of each genotype and staining are shown.
Figure 4
Figure 4. TGF-β, IL-10 and IL17A in colon and spleen are correlated with inflammatory parameters.
Mice were treated with DSS or the DSS/Candida combination as described in the legend of Figure 2. Sample size (number of animals): control groups (3), DSS groups (5), DSS/Candida groups (10-11). Cytokine levels were determined in lysates of spleen and proximal colon. IL10, IL-17A, IL-6, TNF, IFN-γ, IL-1β and TGF-β cytokine levels in spleen lysates (A) and lysates of the proximal colon (B). Bars and error bars show the mean and standard deviation of the indicated groups and data points resembling individual mice are plotted. A one-way ANOVA test with Holm-Sidak correction for multiple comparisons was applied to determine statistical significance. *: p<0.05, **: p<0.01, ***: p<0.005, ****: p<0.001, C: Clustering analysis of all cytokine data. Each vertical column represents one animal and its respective cytokine values. The average value of the wild type and Btk-/- control groups was set at 0 and DSS and DSS/Candida data were expressed as relative log values. Color codes of relative log values ranging from -10 to 10 are indicated in the legend. D, E: Significantly correlated data after Pearson analysis of cytokine data versus colon length or spleen weight. For wild type animals, spleen TGF-β correlated with colon length and spleen weight and colon IL-10 correlated with colon length (D). For Btk-/- animals, spleen IL-17A correlated with both colon length and spleen weight, while colon IL-17A was correlated with colon length (E).
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