Mannose Receptor Is Required for Optimal Induction of Vaccine

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. albicans [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]. FACS data suggesting that MR contributes to the differentiation of Th17 were gathered with an LSRII flow cytometer, and data were an- cells. Since the reduction in IL-17 production is more pro- alyzed with FlowJo software (Tree Star). nounced in wild-type BMDCs treated with soluble mannan −/− Ex Vivo Coculture for Cytokine Protein Measurement than in cocultures with MR , it is likely that other receptors, Splenocytes were harvested from mice at day 34 after vaccina- such as dectin-2 [15], or receptor collaboration with dectin-1 tion, washed, stimulated in complete RPMI 1640 medium con- and Toll-like receptor 2 [6] are involved in the recognition of taining 10 µg/mL yeast cell-wall-membrane antigen [13], and mannose-like structures on B. dermatitidis. plated in 24-well plates at a concentration of 3 × 106 cells/well. Reduced IL-17 production by 1807 T cells could be due to Five days later, supernatants were collected, and IFN-γ and IL- either skewed cytokine responses by BMDCs exposed to yeast

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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, at University of Wisconsin-Madison Libraries on March 8, 2016 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.

−/− or reduced antigen uptake in the absence of MR. To test these we adoptively transferred naive 1807 cells into MR and possibilities, we cocultured BMDCs and mCherry-expressing wild-type mice prior to vaccination. At day 4 after infection, vac- −/− vaccine yeast and measured cytokine production in the cell- cinated MR mice recruited fewer primed (CD44+)fungus- culture supernatant and BMDC ingestion of yeast. Expression specific 1807 cells to the lungs, compared with vaccinated of the Th17 cell–priming cytokines interleukin 1β and interleu- wild-type mice (Figure 2A and 2B). Although not all of the re- −/− kin 6 was significantly reduced from MR BMDCs, compared cruited endogenous CD4+ T cells were likely fungus specific, the +/+ with MR BMDCs (Figure 1D). The frequencies of ingested, numbers of endogenous CD44+ T cells followed a similar trend. − intracellular yeast (mCherry+ Uvitex ) were not significantly Since Th1 and Th17 cells are the key players in mediating −/− +/+ different between MR BMDCs and MR BMDCs (Fig- vaccine-induced immunity to fungi, we examined whether −/− ure 1D). These data indicate that yeast uptake by MR skews MR mice have impaired responses. At day 4 after infection, −/− thecytokineprofile of activated BMDCs but not the rate of vaccinated MR mice recruited 4-fold fewer IL-17–producing yeast uptake. 1807 cells to the lungs than did wild-type controls (Figure 2A and 2B). The number of IFN-γ–producing 1807 cells was re- MR Is Required for the Differentiation of Th17 Cells and Protection In duced 3-fold, but that difference was found to be statistically in- Vivo significant. Lung transcripts for IL-17 but not those for IFN-γ −/− To investigate whether MR regulates the activation of vaccine- were also reduced in vaccinated (and unvaccinated) MR −/− induced T-cell responses in vivo, we vaccinated MR mice mice, compared with wild-type mice, at day 4 after infection and monitored the recruitment of T cells to the lung upon pul- (Figure 2C). IL-17 and IFN-γ protein levels were reduced by monary challenge. To enumerate fungus-specific CD4+ T cells, splenic CD4+ T cells that were stimulated ex vivo with cell-

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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 (CD44hi) 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. wall-membrane antigen (Figure 2C). Thus, MR is required for 1807 T cells, we can definitively conclude that MR regulates dif- the development and recruitment of antifungal Th17 cells to the ferentiation of naive antifungal T cells into effector Th17 cells. lungs. Since we adoptively transferred naive fungus-specific Our observation therefore significantly extends the findings of

4 • JID • BRIEF REPORT other work in which MR was the main receptor that triggered Financial support. This work was supported by the National Institutes the production of IL-17 in response to Candida mannan by mem- of Health (grants R01 AI093553 [to M. W.] and R01 AI035681 and + AI040996 [to B. K.]). ory CD4 T cells that were already committed Th17 cells [6]. Potential conflicts of interest. All authors: No reported conflicts. All To test whether MR is required for vaccine-induced protec- authors have submitted the ICMJE Form for Disclosure of Potential Con- −/− fl fl tion against B. dermatitidis infection, we immunized MR icts of Interest. Con icts that the editors consider relevant to the content of the manuscript have been disclosed. mice and wild-type controls and enumerated lung CFUs at day 4 and day 14 after challenge. At both time points, unvacci- References −/− nated MR mice had a burden of lung infection similar to that 1. Netea MG, Brown GD, Kullberg BJ, Gow NA. An integrated model of the recog- Candida albicans 2008 of unvaccinated wild-type controls, suggesting that MR does nition of by the innate immune system. Nat Rev Microbiol ; 6:67–78. not contribute to innate defense (Figure 2D). However, vacci- 2. Levitz SM, Specht CA. The molecular basis for the immunogenicity of Cryptococ- −/− nated MR mice showed 84- and 53-fold higher lung CFUs cus neoformans mannoproteins. FEMS Yeast Res 2006; 6:513–24. 3. Hoving JC, Wilson GJ, Brown GD. Signalling C-type lectin receptors, microbial than vaccinated wild-type controls at days 4 and 14, respectively recognition and immunity. Cell Microbiol 2014; 16:185–94. (Figure 2D). Thus, MR is required for optimal protection in- 4. Hardison SE, Brown GD. C-type lectin receptors orchestrate antifungal immunity. Nat Immunol 2012; 13:817–22. Downloaded from duced by the vaccine. Since we previously demonstrated that 5. Saijo S, Ikeda S, Yamabe K, et al. Dectin-2 recognition of alpha-mannans and in- Th17 cells are indispensable for mediating vaccine immunity duction of Th17 cell differentiation is essential for host defense against Candida albicans 2010 – to B. dermatitidis and other systemic dimorphic fungi, it is likely . Immunity ; 32:681 91. −/− 6. van de Veerdonk FL, Marijnissen RJ, Kullberg BJ, et al. The macrophage mannose that the reduced numbers of Th17 cells in MR mice were re- receptor induces IL-17 in response to Candida albicans. Cell Host Microbe 2009; 5:329–40. sponsible for the impaired resistance. 2009 7. Levitz SM. Th17 cells bounce off the fungal wall. Cell Host Microbe ; http://jid.oxfordjournals.org/ Taken together, our findings indicate that MR recognizes a 5:311–3. mannan-like structure on the B. dermatitidis cell wall that 8. Wüthrich M, Gern B, Hung CY, et al. Vaccine-induced protection against 3 systemic mycoses endemic to North America requires Th17 cells in mice. J Clin In- leads to the differentiation of naive antifungal T cells into vest 2011; 121:554–68. Th17 effector cells mediating vaccine-induced resistance. An in- 9. Brandhorst TT, Wüthrich M, Warner T, Klein B. Targeted gene disruption reveals an adhesin indispensable for pathogenicity of Blastomyces dermatitidis. J Exp Med triguing idea is that mannoproteins that contain immunodomi- 1999; 189:1207–16. nant T-cell epitopes could be the source of mannans that skew 10. Wüthrich M, Ersland K, Sullivan T, Galles K, Klein BS. Fungi subvert vaccine T fl cell priming at the respiratory mucosa by preventing chemokine-induced influx of cytokine induction by antigen-presenting cells and in uence fl 2012 –

in ammatory monocytes. Immunity ; 36:680 92. at University of Wisconsin-Madison Libraries on March 8, 2016 Th17 cell differentiation. Thus, MR ligands could be harnessed 11. Lee SJ, Evers S, Roeder D, et al. Mannose receptor-mediated regulation of serum 2002 – as adjuvants and beneﬁt the rational design of new T-cell–based glycoprotein homeostasis. Science ; 295:1898 01. 12. Kanbe T, Han Y, Redgrave B, Riesselman MH, Cutler JE. Evidence that mannans antifungal vaccines. of Candida albicans are responsible for adherence of yeast forms to spleen and lymph node tissue. Infect Immun 1993; 61:2578–84. Notes 13. Wüthrich M, Filutowicz HI, Klein BS. Mutation of the WI-1 gene yields an atten- Blastomyces dermatitidis Acknowledgments. We thank Dr Jens Eickhoff from the Department of uated strain that induces host resistance. J Clin Invest 2000; 106:1381–9. Biostatistics and Medical Informatics at the University of Wisconsin– 14. FisherLD,vanBelleG.Biostatistics:amethodologyforthehealthsciences. Madison for performing the statistical analysis. New York: John Wiley & Sons, 1993:611–3. ﬁ All authors have seen and approved the content and contributed signi - 15. Wang H, Lebert V, Hung CY, et al. C-type lectin receptors differentially induce cantly to the work. No writing assistance was provided in the preparation of Th17 cells and vaccine immunity to the endemic mycosis of North America. J Im- the manuscript. munol 2014; 192:1107–19.

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