Mannose Receptor Is Required for Optimal Induction of Vaccine-Induced T-Helper Type 17 Cells and Resistance to Blastomyces dermatitidis Infection

Abstract

We investigated how innate sensing by the mannose receptor (MR) influences the development of antifungal immunity. We demonstrate that MR senses mannan on the surface of attenuated Blastomyces dermatitidis vaccine yeast and that MR^−/− mice demonstrate impaired vaccine immunity against lethal experimental blastomycosis, compared with wild-type control mice. Using naive Blastomyces-specific transgenic CD4⁺ T cells, we found that MR regulates differentiation of naive T cells into T-helper type 17 (Th17) effector cells, which are essential in vaccine immunity against systemic dimorphic fungi. Thus, MR regulates differentiation of Th17 cells and is required to induce vaccine immunity against lethal pulmonary blastomycosis.

The cell wall of medically important fungi contains 3 major polysaccharides: β-glucan, chitin, and mannan [1]. β-(1,3)-glucans containing covalent links to β-(1,6)-glucan and chitin form the core structural component. Mannoproteins that are glycosylated via N and O linkages are attached to this skeleton. Whereas mammalian proteins rarely have exposed mannose residues, fungi use mannose as their preferred sugar [2]. Highly mannosylated polysaccharides are referred to as mannans, and in the cell wall of Candida albicans, chains comprising up to 200 mannose groups can be found [1, 2]. Importantly, mannans tend to be on the outer fungal cell wall, whereas β-glucans are largely on the inside. Recognition of mannan through a combination of pattern-recognition receptors (PRRs), including C-type lectin receptors, promotes antifungal immune responses [3, 4]. Notably, mannan has been shown to induce T-helper type 17 (Th17) responses in a C. albicans infection model [5–7]. Although Th17 cells play a requisite role in antifungal responses [8], the role of PRRs that recognize mannan and induce differentiation of Th17 cells have not been fully elucidated.

MR was reported to trigger Th17 cells in response to C. albicans [6, 7]. The design of that study involved stimulation of human mononuclear cells in vitro in the presence of C. albicans, its components, and blocking antibody against MR. Despite strong MR-dependent responses in that study, human memory T cells (CD44^hi) and not naive T cells were the main source of interleukin 17 (IL-17) produced, owing to the high frequency of exposure to Candida in the general population. Since cytokines required to prime naive T cells differ from those that propagate already primed memory T cells, the experimental design of that study did not address whether MR induces Th17 cell differentiation of naive T cells. Here, we took advantage of an adoptive transfer system in which we can transfer naive antifungal T cells into recipient mice to investigate the role of host factors or receptors. We studied how naive T cell differentiation into Th17 cells is influenced by development in MR-deficient hosts. We report that MR is required not only for recall of memory responses, but also for differentiation of naive T cells into Th17 effector cells and optimal induction of vaccine resistance against Blastomyces dermatitidis infection.

MATERIALS AND METHODS

Fungal Cultures

B. dermatitidis strains used were ATCC 26199, a wild-type virulent strain, and the isogenic, attenuated mutant lacking BAD1, designated strain 55 [9]. Isolates of B. dermatitidis were maintained as yeast on Middlebrook 7H10 agar with oleic acid-albumin complex (Sigma) at 39°C.

Mouse Strains

Inbred wild-type C57BL/6 were obtained from Jackson Laboratories (Bar Harbor, Maine). Blastomyces-specific T-cell receptor (TCR) transgenic (Tg) 1807 mice were generated in our laboratory and were backcrossed to congenic Thy1.1⁺ mice as described elsewhere [10]. MR^−/− mice were a generous gift from Dr Stuart Levitz at the University of Massachusetts, who sent us the mice with the permission of Dr Michele Nussenzweig [11]. All mice were 7–8 weeks old at the time of experiments. Mice were housed and cared for according to guidelines of the University of Wisconsin Animal Care Committee, who approved all aspects of this work.

FITC-ConA Staining of Vaccine Yeast

Heat-killed strain 55 yeast were incubated with 15 µg/mL FITC-ConA (Sigma) in binding buffer (20 mM Tris [pH 7.6], 0.3 M NaCl, 1 mM MnCl₂, 1 mM MgCl₂, and 1 mM CaCl₂) in the dark for 30 minutes. Stained cells were washed twice to remove residual FITC-ConA, fixed with 2% PFA, and inspected for fluorescence, using an Olympus BX60 fluorescent microscope.

In Vitro Coculture for Cytokine Protein Measurement

Bone marrow–derived dendritic cells (BMDCs; 10⁵ cells/well) were cocultured with 3 × 10⁵ heat-killed vaccine yeast in complete Roswell Park Memorial Institute (RPMI) 1640 medium overnight. Soluble mannan (a generous gift from Dr Jim Cutler) was generated from C. albicans ATCC strain 3153A [12] and added at indicated concentrations. Twenty-four hours later, magnetic-bead-purified naive CD4⁺ T cells from 1807 mice were added at a concentration of 2 × 10⁵ cells/well. Supernatants were collected 3 days later, and levels of T-cell–expressed cytokines were determined by an enzyme-linked immunosorbent assay (ELISA). To measure antigen uptake and cytokine production by BMDCs, mCherry-expressing yeast were cocultured with BMDCs. To distinguish extracellular from intracellular yeast, we stained extracellular yeast with 1 µg/mL Uvitex for 20 minutes on ice (intracellular yeast were shielded from the dye). BMDCs were stained with CD11b to quantify the frequencies of ingested (mCherry⁺, Uvitex⁻) and attached (mCherry⁺, Uvitex⁺) yeast among CD11b⁺ BMDCs, using fluorescence-activated cell-sorting (FACS) analysis.

Vaccination and Experimental Infection

Mice were vaccinated subcutaneously with 10⁶–10⁷ strain 55 yeast dorsally twice over 4 weeks and were infected intratracheally with 2 × 10³ or 2 × 10⁴ isogenic, wild-type B. dermatitidis strain 26199 yeast as described previously [8]. At day 4 after infection, lung T cells were analyzed by FACS analysis. The burden of lung infection was determined by plating lung homogenates on brain-heart infusion (Difco) agar, followed by enumeration of colony-forming units (CFUs).

Adoptive Transfer of Transgenic 1807T Cells and Intracellular Cytokine Staining

A total of 10⁶ magnetic bead-purified CD4⁺ cells from TCR Tg 1807 (Thy1.1⁺) mice were injected intravenously into Thy1.2⁺ C57BL/6 recipients prior to vaccination. At day 4 after infection, lung T cells were enumerated as described previously [8]. Anti-CD3 and anti-CD28 monoclonal antibody (mAb)–stimulated cells were stained with anti–interferon γ (IFN-γ) and anti–IL-17-mAbs as described previously [8]. FACS data were gathered with an LSRII flow cytometer, and data were analyzed with FlowJo software (Tree Star).

Ex Vivo Coculture for Cytokine Protein Measurement

Splenocytes were harvested from mice at day 34 after vaccination, washed, stimulated in complete RPMI 1640 medium containing 10 µg/mL yeast cell-wall-membrane antigen [13], and plated in 24-well plates at a concentration of 3 × 10⁶ cells/well. Five days later, supernatants were collected, and IFN-γ and IL-17 expression were measured in cell culture supernatants by ELISA (R&D Systems).

Real-Time Polymerase Chain Reaction (PCR) Analysis for IFN-γ and IL-17 Transcripts

Total RNA was isolated from lung homogenates at day 4 after infection, using the Qiagen RNeasy kit. Genomic DNA was removed using Turbo DNase (Ambion), the RNA was cleaned using Qiagen RNeasy columns, and complementary DNA (cDNA) was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was performed with iQ SYBR Green Supermix (Bio-Rad), and the reactions were run on a MyiQ real-time PCR detection system (Bio-Rad). The n-fold change of gene expression for MR^−/− versus wild-type controls was calculated using the comparative cycle threshold method.

Statistical Analysis

Differences in the numbers and percentages of activated or cytokine-producing T cells were analyzed using the Wilcoxon rank test for nonparametric data or the t test (using GraphPad Prism) when data were normally distributed [14]. Factorial analysis of variance using SAF software was used to compare the vaccine-induced reduction in lung CFUs between different strains of mice. P < .05 is considered statistically significant.

RESULTS AND DISCUSSION

Mannan on the B. dermatitidis Cell Wall Drives IL-17 Production

Many fungal cell wall proteins are modified by N-linked mannosylation, O-linked mannosylation, and phosphomannosylation [7]. To investigate whether the cell wall of B. dermatitidis harbors mannan on its surface, we stained yeast with ConA FITC. Abundant mannan was found to decorate the surface of the yeast (Figure 1A). Since MR recognizes fungal mannan and induces IL-17 by human memory T cells in response to C. albicans in vitro [6], we investigated whether soluble mannan affects the production of IL-17 by naive B. dermatitidis–specific 1807 T cells induced in response to the yeast. Soluble mannan inhibited IL-17 production by 1807 T cells in a concentration-dependent manner (Figure 1B). Surface-exposed mannans are recognized by multiple PRRs, including MR. To investigate whether IL-17 production requires the presence of MR on antigen-presenting cells, we cocultured MR^−/− BMDCs with Blastomyces yeast and naive 1807 T cells. MR^−/− BMDCs induced less IL-17 than BMDCs from wild-type controls (Figure 1C), suggesting that MR contributes to the differentiation of Th17 cells. Since the reduction in IL-17 production is more pronounced in wild-type BMDCs treated with soluble mannan than in cocultures with MR^−/−, it is likely that other receptors, such as dectin-2 [15], or receptor collaboration with dectin-1 and Toll-like receptor 2 [6] are involved in the recognition of mannose-like structures on B. dermatitidis.

Figure 1.

Fungal mannan drives T-helper type 17 (Th17) responses through mannose receptor (MR). A, ConA-FITC staining of Blastomyces dermatitidis yeast. B, Soluble mannan derived from Candida albicans blocks vaccine yeast–induced interleukin 17 (IL-17) production by 1807 cells in a dose-dependent manner. In the absence of yeast, mannan did not trigger an IL-17 response by 1807 cells (data not shown). *P < .05 vs non–mannan-treated controls. C, Naive 1807 cells primed by bone marrow–derived dendritic cells (BMDCs) to become Th17 cells. Wild-type and MR^−/− dendritic cells (DCs), yeast, and naive 1807 cells were cocultured for 3 days, and supernatants were analyzed by an enzyme-linked immunosorbent assay. *P < .05 vs wild-type control. Data are representative of 3 independent experiments. D, mCherry-expressing yeast were cocultured with BMDCs at a multiplicity of infection of 1:1 (yeast to BMDCs; for cytokine production) and 1:10 (for antigen uptake). Cytokine levels in the cell culture supernatant were measured at 16 hours, and yeast ingestion was measured at 5 hours and 48 hours. To distinguish intracellular yeast from extracellular yeast, cocultures were stained with Uvitex. CD11b⁺ events were gated on and assessed for the frequencies of mCherry- and Uvitex-positive events. Data are expressed as mean values ± standard errors of the mean (n = cells raised from 4 mice/group) and are representative of 2 independent experiments. *P < .05 vs wild-type controls.

Reduced IL-17 production by 1807 T cells could be due to either skewed cytokine responses by BMDCs exposed to yeast or reduced antigen uptake in the absence of MR. To test these possibilities, we cocultured BMDCs and mCherry-expressing vaccine yeast and measured cytokine production in the cell-culture supernatant and BMDC ingestion of yeast. Expression of the Th17 cell–priming cytokines interleukin 1β and interleukin 6 was significantly reduced from MR^−/− BMDCs, compared with MR^+/+ BMDCs (Figure 1D). The frequencies of ingested, intracellular yeast (mCherry⁺ Uvitex⁻) were not significantly different between MR^−/− BMDCs and MR^+/+ BMDCs (Figure 1D). These data indicate that yeast uptake by MR skews the cytokine profile of activated BMDCs but not the rate of yeast uptake.

MR Is Required for the Differentiation of Th17 Cells and Protection In Vivo

To investigate whether MR regulates the activation of vaccine-induced T-cell responses in vivo, we vaccinated MR^−/− mice and monitored the recruitment of T cells to the lung upon pulmonary challenge. To enumerate fungus-specific CD4⁺ T cells, we adoptively transferred naive 1807 cells into MR^−/− and wild-type mice prior to vaccination. At day 4 after infection, vaccinated MR^−/− mice recruited fewer primed (CD44⁺) fungus-specific 1807 cells to the lungs, compared with vaccinated wild-type mice (Figure 2A and 2B). Although not all of the recruited endogenous CD4⁺ T cells were likely fungus specific, the numbers of endogenous CD44⁺ T cells followed a similar trend.

Figure 2.

Mannose receptor (MR) drives protective T-helper type 1 (Th1) and Th17 cells and vaccine-induced resistance. A and B, Wild-type and MR^−/− mice adoptively received 10⁶ CD4⁺ purified, naive 1807 transgenic (Tg) cells (Thy1.1⁺) and were or were not vaccinated with 10⁶ heat-killed vaccine yeast. Four weeks after vaccination, mice were challenged intratracheally with 2 × 10⁴ strain 26199 yeast, and the number of activated (CD44⁺) and cytokine-producing lung CD4⁺ T cells were enumerated at day 4 after infection. Dot plots show concatenated samples of 4–6 mice/group. The numbers indicate mean values ± standard errors of the mean (SEMs) of activated (CD44^hi) endogenous CD4⁺ and transferred 1807 Tg (Thy1.1⁺) T cells. Data are representative of 2 independent experiments. *P < .05 vs yeast-stimulated, non–mannan-treated controls. C, Cytokine transcripts in lung homogenates were analyzed at day 4 after infection, and cytokine levels were measured in ex vivo–stimulated splenocytes. D, The lung burden was assessed at day 4 and 2 weeks after infection. Data are the average and SEMs of 3 independent experiments. *P < .05 vs infected wild-type controls. Abbreviations: CFUs, colony-forming units; IFN-γ, interferon γ; IL-17, interleukin 17.

Since Th1 and Th17 cells are the key players in mediating vaccine-induced immunity to fungi, we examined whether MR^−/− mice have impaired responses. At day 4 after infection, vaccinated MR^−/− mice recruited 4-fold fewer IL-17–producing 1807 cells to the lungs than did wild-type controls (Figure 2A and 2B). The number of IFN-γ–producing 1807 cells was reduced 3-fold, but that difference was found to be statistically insignificant. Lung transcripts for IL-17 but not those for IFN-γ were also reduced in vaccinated (and unvaccinated) MR^−/− mice, compared with wild-type mice, at day 4 after infection (Figure 2C). IL-17 and IFN-γ protein levels were reduced by splenic CD4⁺ T cells that were stimulated ex vivo with cell-wall-membrane antigen (Figure 2C). Thus, MR is required for the development and recruitment of antifungal Th17 cells to the lungs. Since we adoptively transferred naive fungus-specific 1807 T cells, we can definitively conclude that MR regulates differentiation of naive antifungal T cells into effector Th17 cells. Our observation therefore significantly extends the findings of other work in which MR was the main receptor that triggered the production of IL-17 in response to Candida mannan by memory CD4⁺ T cells that were already committed Th17 cells [6].

To test whether MR is required for vaccine-induced protection against B. dermatitidis infection, we immunized MR^−/− mice and wild-type controls and enumerated lung CFUs at day 4 and day 14 after challenge. At both time points, unvaccinated MR^−/− mice had a burden of lung infection similar to that of unvaccinated wild-type controls, suggesting that MR does not contribute to innate defense (Figure 2D). However, vaccinated MR^−/− mice showed 84- and 53-fold higher lung CFUs than vaccinated wild-type controls at days 4 and 14, respectively (Figure 2D). Thus, MR is required for optimal protection induced by the vaccine. Since we previously demonstrated that Th17 cells are indispensable for mediating vaccine immunity to B. dermatitidis and other systemic dimorphic fungi, it is likely that the reduced numbers of Th17 cells in MR^−/− mice were responsible for the impaired resistance.

Taken together, our findings indicate that MR recognizes a mannan-like structure on the B. dermatitidis cell wall that leads to the differentiation of naive antifungal T cells into Th17 effector cells mediating vaccine-induced resistance. An intriguing idea is that mannoproteins that contain immunodominant T-cell epitopes could be the source of mannans that skew cytokine induction by antigen-presenting cells and influence Th17 cell differentiation. Thus, MR ligands could be harnessed as adjuvants and benefit the rational design of new T-cell–based antifungal vaccines.