The Journal ofJournal Infectious of DiseasesInfectious Diseases Advance Access published March 1, 2016 BRIEF REPORT Mannose Receptor Is Required for type 17 (Th17) responses in a C. albicans infection model [5–7]. Although Th17 cells play a requisite role in antifungal Optimal Induction of Vaccine-Induced responses [8], the role of PRRs that recognize mannan and in- T-Helper Type 17 Cells and Resistance duce differentiation of Th17 cells have not been fully elucidated. to Blastomyces dermatitidis Infection MR was reported to trigger Th17 cells in response to C. albi- cans [6, 7]. The design of that study involved stimulation of Huafeng Wang,1 Vanessa LeBert,1 Mengyi Li,1 Tassanee Lerksuthirat,1 Kevin Galles,1 C. albicans, Bruce Klein,1,2,3 and Marcel Wüthrich1 human mononuclear cells in vitro in the presence of 1Departments of Pediatrics, 2Department of Internal Medicine, and 3Department of Medical its components, and blocking antibody against MR. Despite Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, strong MR-dependent responses in that study, human memory – University of Wisconsin Madison T cells (CD44hi) and not naive T cells were the main source of Downloaded from interleukin 17 (IL-17) produced, owing to the high frequency of We investigated how innate sensing by the mannose receptor exposure to Candida in the general population. Since cytokines (MR) influences the development of antifungal immunity. We required to prime naive T cells differ from those that propagate demonstrate that MR senses mannan on the surface of attenu- −/− ated Blastomyces dermatitidis vaccine yeast and that MR already primed memory T cells, the experimental design of that mice demonstrate impaired vaccine immunity against lethal study did not address whether MR induces Th17 cell differen- http://jid.oxfordjournals.org/ experimental blastomycosis, compared with wild-type con- tiation of naive T cells. Here, we took advantage of an adoptive trol mice. Using naive Blastomyces-specifictransgenicCD4+ transfer system in which we can transfer naive antifungal T cells T cells, we found that MR regulates differentiation of naive into recipient mice to investigate the role of host factors or re- T cells into T-helper type 17 (Th17) effector cells, which are ceptors. We studied how naive T cell differentiation into Th17 essential in vaccine immunity against systemic dimorphic cells is influenced by development in MR-deficient hosts. We fungi. Thus, MR regulates differentiation of Th17 cells and is report that MR is required not only for recall of memory re- required to induce vaccine immunity against lethal pulmonary sponses, but also for differentiation of naive T cells into Th17 at University of Wisconsin-Madison Libraries on March 8, 2016 blastomycosis. effector cells and optimal induction of vaccine resistance against Keywords. mannose receptor; T-cell differentiation; Blastomyces dermatitidis infection. vaccine immunity; fungi. MATERIALS AND METHODS Fungal Cultures The cell wall of medically important fungi contains 3 major B. dermatitidis strains used were ATCC 26199, a wild-type vir- polysaccharides: β-glucan, chitin, and mannan [1]. β-(1,3)- ulent strain, and the isogenic, attenuated mutant lacking BAD1, glucans containing covalent links to β-(1,6)-glucan and chitin designated strain 55 [9]. Isolates of B. dermatitidis were main- form the core structural component. Mannoproteins that are tained as yeast on Middlebrook 7H10 agar with oleic acid- glycosylated via N and O linkages are attached to this skeleton. albumin complex (Sigma) at 39°C. Whereas mammalian proteins rarely have exposed mannose Mouse Strains residues, fungi use mannose as their preferred sugar [2]. Highly Inbred wild-type C57BL/6 were obtained from Jackson Labora- mannosylated polysaccharides are referred to as mannans, and tories (Bar Harbor, Maine). Blastomyces-specific T-cell receptor in the cell wall of Candida albicans, chains comprising up to (TCR) transgenic (Tg) 1807 mice were generated in our labora- 200 mannose groups can be found [1, 2]. Importantly, mannans tory and were backcrossed to congenic Thy1.1+ mice as de- tend to be on the outer fungal cell wall, whereas β-glucans are −/− scribed elsewhere [10]. MR mice were a generous gift from largely on the inside. Recognition of mannan through a combi- Dr Stuart Levitz at the University of Massachusetts, who sent nation of pattern-recognition receptors (PRRs), including C- us the mice with the permission of Dr Michele Nussenzweig type lectin receptors, promotes antifungal immune responses [11]. All mice were 7–8 weeks old at the time of experiments. [3, 4]. Notably, mannan has been shown to induce T-helper Mice were housed and cared for according to guidelines of the University of Wisconsin Animal Care Committee, who ap- Received 29 October 2015; accepted 31 December 2015. proved all aspects of this work. Correspondence: M. Wüthrich, University of Wisconsin, Microbial Sciences Bldg, 1550 Linden Dr, Madison, WI 53706 ([email protected]). FITC-ConA Staining of Vaccine Yeast The Journal of Infectious Diseases® Heat-killed strain 55 yeast were incubated with 15 µg/mL © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail [email protected]. FITC-ConA (Sigma) in binding buffer (20 mM Tris [pH 7.6], DOI: 10.1093/infdis/jiw010 0.3 M NaCl, 1 mM MnCl2,1mMMgCl2,and1mMCaCl2) BRIEF REPORT • JID • 1 in the dark for 30 minutes. Stained cells were washed twice 17 expression were measured in cell culture supernatants by to remove residual FITC-ConA, fixed with 2% PFA, and in- ELISA (R&D Systems). spected for fluorescence, using an Olympus BX60 fluorescent Real-Time Polymerase Chain Reaction (PCR) Analysis for IFN-γ and microscope. IL-17 Transcripts In Vitro Coculture for Cytokine Protein Measurement Total RNA was isolated from lung homogenates at day 4 after Bone marrow–derived dendritic cells (BMDCs; 105 cells/well) infection, using the Qiagen RNeasy kit. Genomic DNA was re- were cocultured with 3 × 105 heat-killed vaccine yeast in com- moved using Turbo DNase (Ambion), the RNA was cleaned plete Roswell Park Memorial Institute (RPMI) 1640 medium using Qiagen RNeasy columns, and complementary DNA overnight. Soluble mannan (a generous gift from Dr Jim Cutler) (cDNA) was synthesized using the iScript cDNA Synthesis was generated from C. albicans ATCC strain 3153A [12]and Kit (Bio-Rad). Real-time PCR was performed with iQ SYBR added at indicated concentrations. Twenty-four hours later, Green Supermix (Bio-Rad), and the reactions were run on a magnetic-bead-purified naive CD4+ T cells from 1807 mice MyiQ real-time PCR detection system (Bio-Rad). The n-fold −/− were added at a concentration of 2 × 105 cells/well. Supernatants change of gene expression for MR versus wild-type controls Downloaded from were collected 3 days later, and levels of T-cell–expressed cyto- was calculated using the comparative cycle threshold method. kines were determined by an enzyme-linked immunosorbent Statistical Analysis assay (ELISA). To measure antigen uptake and cytokine pro- Differences in the numbers and percentages of activated or cy- duction by BMDCs, mCherry-expressing yeast were cocultured tokine-producing T cells were analyzed using the Wilcoxon with BMDCs. To distinguish extracellular from intracellular http://jid.oxfordjournals.org/ rank test for nonparametric data or the t test (using GraphPad yeast, we stained extracellular yeast with 1 µg/mL Uvitex for Prism) when data were normally distributed [14]. Factorial 20 minutes on ice (intracellular yeast were shielded from the analysis of variance using SAF software was used to compare dye). BMDCs were stained with CD11b to quantify the frequen- − the vaccine-induced reduction in lung CFUs between different cies of ingested (mCherry+, Uvitex ) and attached (mCherry+, strains of mice. P < .05 is considered statistically significant. Uvitex+) yeast among CD11b+ BMDCs, using fluorescence- activated cell-sorting (FACS) analysis. RESULTS AND DISCUSSION at University of Wisconsin-Madison Libraries on March 8, 2016 Vaccination and Experimental Infection MannanontheB. dermatitidis Cell Wall Drives IL-17 Production Mice were vaccinated subcutaneously with 106–107 strain 55 Many fungal cell wall proteins are modified by N-linked man- yeast dorsally twice over 4 weeks and were infected intratra- nosylation, O-linked mannosylation, and phosphomannosyla- cheally with 2 × 103 or 2 × 104 isogenic, wild-type B. dermatiti- tion [7]. To investigate whether the cell wall of B. dermatitidis dis strain 26199 yeast as described previously [8]. At day 4 after harbors mannan on its surface, we stained yeast with ConA infection, lung T cells were analyzed by FACS analysis. The bur- FITC. Abundant mannan was found to decorate the surface den of lung infection was determined by plating lung homoge- of the yeast (Figure 1A). Since MR recognizes fungal mannan nates on brain-heart infusion (Difco) agar, followed by and induces IL-17 by human memory T cells in response to enumeration of colony-forming units (CFUs). C. albicans in vitro [6], we investigated whether soluble mannan affects the production of IL-17 by naive B. dermatitidis–specific Adoptive Transfer of Transgenic 1807T Cells and Intracellular Cytokine 1807 T cells induced in response to the yeast. Soluble mannan Staining inhibited IL-17 production by 1807 T cells in a concentration- A total of 106 magnetic bead-purified CD4+ cells from TCR Tg dependent manner (Figure 1B). Surface-exposed mannans are 1807 (Thy1.1+) mice were injected intravenously into Thy1.2+ recognized by multiple PRRs, including MR. To investigate C57BL/6 recipients prior to vaccination. At day 4 after infec- whether IL-17 production requires the presence of MR on an- tion, lung T cells were enumerated as described previously −/− tigen-presenting cells, we cocultured MR BMDCs with Blas- [8]. Anti-CD3 and anti-CD28 monoclonal antibody (mAb)– −/− tomyces yeast and naive 1807 T cells. MR BMDCs induced stimulated cells were stained with anti–interferon γ (IFN-γ) less IL-17 than BMDCs from wild-type controls (Figure 1C), and anti–IL-17-mAbs as described previously [8].