Kluyveromyces marxianus and Saccharomyces boulardii Induce Distinct Levels of Dendritic Cell Cytokine Secretion and Significantly Different T Cell Responses In Vitro

Abstract

Interactions between members of the intestinal microbiota and the mucosal immune system can significantly impact human health, and in this context, fungi and food-related yeasts are known to influence intestinal inflammation through direct interactions with specialized immune cells in vivo. The aim of the present study was to characterize the immune modulating properties of the food-related yeast Kluyveromyces marxianus in terms of adaptive immune responses indicating inflammation versus tolerance and to explore the mechanisms behind the observed responses. Benchmarking against a Saccharomyces boulardii strain with probiotic effects documented in clinical trials, we evaluated the ability of K. marxianus to modulate human dendritic cell (DC) function in vitro. Further, we assessed yeast induced DC modulation of naive T cells toward effector responses dominated by secretion of IFNγ and IL-17 versus induction of a Treg response characterized by robust IL-10 secretion. In addition, we blocked relevant DC surface receptors and investigated the stimulating properties of β-glucan containing yeast cell wall extracts. K. marxianus and S. boulardii induced distinct levels of DC cytokine secretion, primarily driven by Dectin-1 recognition of β-glucan components in their cell walls. Upon co-incubation of yeast exposed DCs and naive T cells, S. boulardii induced a potent IFNγ response indicating TH1 mobilization. In contrast, K. marxianus induced a response dominated by Foxp3+ Treg cells, a characteristic that may benefit human health in conditions characterized by excessive inflammation and positions K. marxianus as a strong candidate for further development as a novel yeast probiotic.

Fig 1. K. marxianus and S. boulardii induce distinct DC modulation.

A. Levels of IL-12, IL-1β, IL-6, and IL-10 secreted by human monocyte-derived DCs following 20 h incubation with DC media (unstimulated), a probiotic reference strain (S. boulardii), or K. marxianus at a yeast:DC ratio of 10:1 (MOI 10). Data are representative of at least five independent experiments, error bars represent SEM. B. Levels of DC expression of CD80, CD86, CCR6, and CCR7 following 20 h incubation with DC media (unstimulated), a probiotic reference strain (S. boulardii), or K. marxianus at a yeast:DC ratio of 10:1 (MOI 10). Data are expressed as mean ± SEM of at least five independent experiments (five donors). One-way ANOVA, Tukey’s multiple comparisons test, indicating significant differences as follows: *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant.

Fig 2. K. marxianus induces a strong T_reg cell response whereas S. boulardii induces a T cell response comprised of IFNγ, IL-17, and IL-10.

T cell responses following 3 days co-incubation of yeast stimulated DCs and naive autologous T cells. For each yeast, DC stimulation was performed at a yeast:DC ratio of 10:1 (MOI 10), and the subsequent co-incubation of DCs and naive T cells was performed at a DC:T cell ratio of 1:20. A. T cell secretion levels of IFNγ, IL-17, and IL-10. Data are representative of at least five independent experiments, error bars represent SEM. B. Quantification of the T_reg subset as CD4⁺CD25⁺Foxp3⁺ cells. Dotplots gated on the CD4⁺ cell population from a single representative donor displaying the percentage of cells in each quadrant. C. Data from three independent experiments (three donors) expressed as the percentage of CD25⁺Foxp3⁺ cells in the CD4⁺ cell population, error bars represent SEM. One-way ANOVA, Tukey’s multiple comparisons test, indicating significant differences as follows: *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant.

Fig 3. Yeast induced DC cytokine secretion relies on Dectin-1 mediated phagocytosis.

DC secretion levels of IL-12, IL-1β, IL-6, and IL-10 following 20 h stimulation with DC media (unstimulated), a probiotic reference strain (S. boulardii), or K. marxianus at a yeast:DC ratio of 10:1 (MOI 10). A. For each yeast strain, DC stimulation was performed following a 30 min DC pre-incubation with HBSS (no treatment), a nonspecific isotype matched control antibody (control mAb) or monoclonal blocking antibodies specific for Dectin-1, TLR2, or DC-SIGN. Data are expressed as percent cytokine secretion relative to pre-incubation with HBSS (no treatment) and displayed as mean ± SEM of four independent experiments (four donors). B. For each yeast strain, DC stimulation was performed following a 30 min DC pre-incubation with HBSS (no treatment) or 20μM Cytochalasin D, a potent inhibitor of actin polymerization. Data are expressed as percent cytokine secretion relative to pre-incubation with HBSS (no treatment) and displayed as mean ± SEM of three independent experiments (three donors). Two-way ANOVA, Tukey’s multiple comparisons test. *, P<0.05; **, P<0.01; ***, P<0.001.

Fig 4. β-glucan containing yeast cell wall extracts are poor IL-12 inducers but potent inducers of DC IL-1β, IL-6, and IL-10 secretion.

A. DC secretion levels of IL-12, IL-1β, IL-6, and IL-10 following 20 h stimulation with live S. boulardii or S. boulardii cell wall extract corresponding to a yeast:DC ratio of 10:1 (MOI 10). B. DC secretion levels of IL-12, IL-1β, IL-6, and IL-10 following 20 h stimulation with live K. marxianus or K. marxianus cell wall extract corresponding to a yeast:DC ratio of 10:1 (MOI 10). Data are representative of three independent experiments, error bars represent SEM. One-way ANOVA, Tukey’s multiple comparisons test. *, P<0.05; **, P<0.01; ***, P<0.001.

Fig 5. Yeast cell wall extract induced cytokine secretion involves multiple PRRs and is entirely dependent on DC uptake.

DC secretion levels of IL-12, IL-1β, IL-6, and IL-10 following 20 h stimulation with DC media (unstimulated) or yeast cell wall extracts prepared from a probiotic reference strain (S. boulardii) or K. marxianus corresponding to a yeast:DC ratio of 10:1 (MOI 10). A. DC stimulation was performed following a 30 min DC pre-incubation with HBSS (no treatment), a nonspecific isotype matched control antibody (control mAb) or monoclonal blocking antibodies specific for Dectin-1, TLR2, or DC-SIGN. Data are expressed as percent cytokine secretion relative to pre-incubation with HBSS (no treatment) and displayed as mean ± SEM of two independent experiments. Two-way ANOVA, Dunnett’s multiple comparisons test. *, P<0.05; **, P<0.01; ***, P<0.001. ND, not detected. B. DC stimulation was performed following a 30 min DC pre-incubation with HBSS (no treatment) or 20μM Cytochalasin D, a potent inhibitor of actin polymerization. Data are expressed as percent cytokine secretion relative to pre-incubation with HBSS (no treatment) and displayed as mean ± SEM of two independent experiments. Two-way ANOVA, Tukey’s multiple comparisons test. *, P<0.05; **, P<0.01; ***, P<0.001. ND, not detected.