A Framework for Understanding the Evasion of Host Immunity by Candida Biofilms

Abstract

Candida biofilms are a major cause of nosocomial morbidity and mortality. The mechanism by which Candida biofilms evade the immune system remains unknown. In this perspective, we develop a theoretical framework of the three, not mutually exclusive, models, which could explain biofilm evasion of host immunity. First, biofilms may exhibit properties of immunological silence, preventing immune activation. Second, biofilms may produce immune-deviating factors, converting effective immunity into ineffective immunity. Third, biofilms may resist host immunity, which would otherwise be effective. Using a murine subcutaneous biofilm model, we found that mice infected with biofilms developed sterilizing immunity effective when challenged with yeast form Candida. Despite the induction of effective anti-Candida immunity, no spontaneous clearance of the biofilm was observed. These results support the immune resistance model of biofilm immune evasion and demonstrate an asymmetric relationship between the host and biofilms, with biofilms eliciting effective immune responses yet being resistant to immunological clearance.

Keywords: Candida; biofilms; cytokines; immune resistance; trained immunity.

Figure 1

A theoretical framework for understanding Candida biofilm immune evasion. (A) In a healthy host, infection with the yeast form of Candida albicans causes pathogen-associated molecular pattern (PAMPs) release and drives the induction of Th17 cells. Th17 cells, in turn, coordinate an effective host anti-pathogen response, clearing the infection. This process is seemingly inoperative in a Candida biofilm growth, for unknown reasons. (B) The first model capable of explaining the immune evasion of C. albicans biofilms is one of immunological silence. In this model, biofilm structure prevents the release of PAMPs and thereby prevents the initiation of T cell activation and polarization into the effective Th17 lineage. (C) The second model to potentially explain biofilm immune evasion is that of immunological deviation. In this model, while the biofilm causes the release of PAMPs and thus the activation of a T cell response against C. albicans, additional factors are produced which deviate the responding T cells from an effective Th17 program into an ineffective (e.g., Th2) program. The resulting host immunity is therefore unable to clear the infection. (D) The final model capable of explaining the persistence of C. albicans biofilms is that of immune resistance. In this model, even in cases where an effective Th17 anti-Candida response is initiated, the biofilm remains resistant to the host immunity (e.g., exclusion of effector cells from the biofilm).

Figure 2

Candida albicans biofilms are robustly maintained in C57BL/6 mice without immunosuppression. (A) Catheter pieces were incubated overnight in mouse serum and for 90 min in C. albicans culture at day 0, following by subcutaneous implantation. In the dexamethasone-treated (DEX) group, dexamethasone (1 mg/L) was added in the drinking water from day 2 and maintained over the course of the experiment. Biofilm colony-forming units (CFUs) were read out at indicated days postimplantation. (B) Log₁₀ of biofilm CFUs compared between immunocompetent (IC) and DEX mice (n = 3) at 2 and 6 days. Mean ± SEM are shown. (C) Log₁₀ of biofilm CFUs followed over time (n = 3–6/group), day 0 = before implantation of catheter pieces. Mean and SEM are shown. One-way ANOVA with multiple comparisons was used for comparison of the different time points (*p < 0.05). (D) Representative scanning electron microscopy images of preimplantation (top), 2 days after implantation (middle), and day 15 after implantation (bottom). (E) Confocal laser scanning microscopy images of catheter pieces before implantation (top) and 15 days postimplantation (bottom). Scale bar = 20 µm.

Figure 3

Candida albicans biofilm promotes sterilizing immunity against infection lethal dose of yeast form C. albicans. (A) Mice were surgically implanted with sterile catheters (“naïve” group, n = 24), with catheters seeded with 5 × 10⁴ Candida for biofilm growth (“biofilm immunized” group, n = 25) or i.v. injected with 5 × 10⁴ Candida (“yeast-form immunized” group, n = 25) on day 0. On day 14, each group was challenged with a 10⁷ colony forming unit (CFU) Candida i.v. Mice were monitored for cytokine expression on days 0, 7, 14, 21, and 28. (B) Serum samples from all the time points were analyzed for IL-2, (C) IFNγ, (D) IL-6, (E) TNFα, (F) muCXCL1, and (G) IL-10. Kruskal–Wallis test was used to compare the different groups at the same time point and to different time points. Average ± SEM. (H) Survival of all mice groups post high-dose challenge (1 × 10⁷ CFU). (I) Kidney colonization, showing the average count (Log₁₀ CFU) per mouse on both kidneys after high-dose challenge for naïve mice (date of death, n = 12), biofilm immunized mice (n = 10, day 14 post-challenge), and yeast-form immunized mice (n = 12, day 14 post-challenge). Cytokine and survival data are pooled from two independent experiments (**p < 0.005, ***p < 0.0001, and ****p < 0.00001).