Intestinal Flora-Derived Kynurenic Acid Protects Against Intestinal Damage Caused by Candida albicans Infection via Activation of Aryl Hydrocarbon Receptor

Abstract

Colonization of the intestinal tract by Candida albicans (C. albicans) can lead to invasive candidiasis. Therefore, a functional intestinal epithelial barrier is critical for protecting against invasive C. albicans infections. We collected fecal samples from patients with Candida albicans bloodstream infection and healthy people. Through intestinal flora 16sRNA sequencing and intestinal metabolomic analysis, we found that C. albicans infection resulted in a significant decrease in the expression of the metabolite kynurenic acid (KynA). We used a repeated C. albicans intestinal infection mouse model, established following intake of 3% dextran sulfate sodium salt (DSS) for 9 days, and found that KynA, a tryptophan metabolite, inhibited inflammation, promoted expression of intestinal tight junction proteins, and protected from intestinal barrier damage caused by invasive Candida infections. We also demonstrated that KynA activated aryl hydrocarbon receptor (AHR) repressor in vivo and in vitro. Using Caco-2 cells co-cultured with C. albicans, we showed that KynA activated AHR, inhibited the myosin light chain kinase-phospho-myosin light chain (MLCK-pMLC) signaling pathway, and promoted tristetraprolin (TTP) expression to alleviate intestinal inflammation. Our findings suggest that the metabolite KynA which is differently expressed in patients with C. albicans infection and has a protective effect on the intestinal epithelium, via activating AHR, could be explored to provide new potential therapeutic strategies for invasive C. albicans infections.

Keywords: aryl hydrocarbon receptor; intestinal barrier function; intestinal flora; invasive C. albicans infections; kynurenic acid.

FIGURE 1

Characteristics of kynurenic acid. (A) The relative expression of Kynurenic acid in the peripheral blood of patients with invasive C. albican infection is significantly lower than the content of other metabolites (P < 0.05). (B) Kynurenic acid (HMDB0000715) is a kind of phosphatidic acid molecule with the molecular formula of C₁₀H₇NO₃ and a molecular weight of 189.1675 Da. (C) The kynurenine pathway is the primary route for tryptophan metabolism, with one of the biologically active metabolites being kynurenic acid (KynA). T, The Candida albicans infection group; C, The healthy control group.

FIGURE 2

Invasive C. albicans infection disrupts fecal microbiota in humans. Human fecal samples were collected from healthy people (n = 6) and patients with candidemia (n = 6). 16S rRNA gene amplicon sequencing and metabolomic analysis were done to investigate the bacterial composition and intestinal metabolites. (A) The petal diagram of OTU distribution. (B) Analysis of similarities (ANOSIM). (C) The PCA analysis of OTU abundance. (D) Alpha-diversity analysis. (E) At the genus level, invasive C. albicans infection disrupts fecal microbiota diversity. T, The Candida albicans infection group; C, The healthy control group.

FIGURE 3

Invasive C. albicans infection disrupts fecal metabolites in humans. (A) The PCA scatter plots of metabolomic analysis. (B) The volcano plot of differentially expressed metabolites. (C) The KEGG pathway enrichment analysis. (D) Heatmap of the top 50 differentially expressed metabolites. (E) Spearman correlation analysis. T, The Candida albicans infection group; C, The healthy control group.

FIGURE 4

Kynurenic acid alleviates intestinal injury caused by invasive C. albicans infection. (A) Experimental design including KynA administration and Candida gavage. (B) Mice infected with C. albicans exhibited higher mortality than mice treated with KynA. (C) KynA treatment decreased the colonization of intestinal C. albicans. Fungal load in feces collected from untreated and KynA groups at 1, 3, and 5 days after infection. (D) Fungal load in kidneys, spleen, and liver samples from euthanized mice collected immediately in the KynA-treated groups and 10 days post-infection in the untreated groups. (E–G) The levels of D-lactic acid, IL-1β, and TNF-α were significantly decreased in the CA+KynA group compared to those of the CA group (P < 0.05). (H) Expression of ZO-1 and occludin in the intestinal epithelial cells of the CA+KynA group was significantly higher than in the CA group. (I) Chiu’s pathological scoring system revealed that mice from the CA+ KynA group had less intestinal mucosal damage than the mice in the CA group. Statistical significance was evaluated using the Mann–Whitney U test. P-values < 0.05 (*) or < 0.01 (**) were considered statistically significant. CA, Candida albicans infection; KynA, Kynurenic acid.

FIGURE 5

Kynurenic acid activates AHR expression in the intestinal epithelium. (A,B) Western blot analysis and immunohistochemistry show higher expression of AHR in the CA+KynA group than in the CA group. Statistical significance was evaluated using the Mann–Whitney U test. P-values < 0.05 (*) or < 0.01 (**) were considered statistically significant. CA, Candida albicans infection; KynA, Kynurenic acid.

FIGURE 6

KynA activates the suppression of MLCK-pMLC signaling pathway by AHR. The detection of MLCK expression and MLC phosphorylation was carried out to determine the effect of AHR activation on the MLCK-pMLC signaling pathway. In Caco-2 cells, after KynA treatment, the expression of the AHR downstream protein CYP1A1 increased significantly, as did the expression of the tight junction proteins ZO-1 and occludin. The expression of MLCK and pMLC decreased significantly. In the CA-Caco-2 cell model, expression levels of CYP1A1, ZO-1, and occludin were significantly decreased, but the expression levels of MLCK and pMLC increased. After treatment with KynA and the AHR agonist FICZ, expression of CYP1A1 increased significantly, as did the expression of ZO-1 and occludin, but the expression of MLCK and pMLC decreased. Treatment with an AHR inhibitor (CH223191), led to a decrease in the expression of CYP1A1, ZO-1, and occludin, and to an increase in the expression of MLCK and pMLC. Statistical significance was evaluated using the Mann–Whitney U test. P-values < 0.05 (*) or < 0.001 (***) were considered statistically significant. CA, Candida albicans infection; KynA, Kynurenic acid; FICZ, 6-Formylindolo[3,2-b]carbazole.

FIGURE 7

KynA suppressed the MK2/p-MK2 signaling pathway by activating AHR. We investigated the regulation of TTP and expression changes of MK2 and P-MK2 induced by AHR activation at the cellular level. In Caco-2 cells, after treatment with KynA, expression of CYP1A1 increased significantly, as did expression of TTP also (P < 0.05), while expression of MK2 decreased significantly (P < 0.05). In the CA- Caco-2 cell model, the expression of proteins CYP1A1 and TTP decreased significantly, but the expression of p-MK2 increased (P < 0.01). After the treatment of Caco-2 cells with KynA and the AHR agonist FICZ, the expression of CYP1A1 and TTP was significantly increased, but the expression of p-MK2 decreased significantly (P < 0.01). Treatment with an AHR inhibitor (CH223191) led to a decrease in the expression of CYP1A1 and TTP, and an increase in the expression of p-MK2 (P < 0.01). Statistical significance was evaluated using the Mann–Whitney U test. P-values < 0.05 (*) or < 0.01 (**) were considered statistically significant. CA, Candida albicans infection; KynA, Kynurenic acid; FICZ, 6-Formylindolo[3,2-b]carbazole. P-values < 0.001 (***).