IL-17 Regulates Systemic Fungal Immunity by Controlling the Functional Competence of NK Cells

Immunity

Article IL-17 Regulates Systemic Fungal Immunity by Controlling the Functional Competence of NK Cells Eva Ba¨r,1 Paul G. Whitney,2 Kathrin Moor,1 Caetano Reis e Sousa,2 and Salome´ LeibundGut-Landmann1,* 1Institute

of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zu¨rich, Switzerland Laboratory, Cancer Research UK, London Research Institute, Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.immuni.2013.12.002 2Immunobiology

SUMMARY

Interleukin 17 (IL-17)-mediated immunity plays a key role in protection from fungal infections in mice and man. Here, we confirmed that mice deficient in the IL-17 receptor or lacking the ability to secrete IL-17 are highly susceptible to systemic candidiasis, but we found that temporary blockade of the IL-17 pathway during infection in wild-type mice did not impact fungal control. Rather, mice lacking IL-17 receptor signaling had a cell-intrinsic impairment in the development of functional NK cells, which accounted for the susceptibility of these mice to systemic fungal infection. NK cells promoted antifungal immunity by secreting GM-CSF, necessary for the fungicidal activity of neutrophils. These data reveal that NK cells are crucial for antifungal defense and indicate a role for IL-17 family cytokines in NK cell development. The IL-17-NK cell axis may impact immunity against not only fungi but also bacteria, viruses, and tumors.

INTRODUCTION Interleukin 17 (IL-17) has emerged as a central player in the mammalian immune system. Proinflammatory members of the IL-17 cytokine family such as IL-17A and IL-17F are involved in the pathogenesis of autoinflammatory and autoimmune disorders, including inflammatory bowel disease, psoriasis, and ankylosing spondylitis (Korn et al., 2009). From an evolutionary perspective, however, the primary function of IL-17 ought to be host protection. Consistent with that notion, IL-17-deficient mice are highly susceptible to infections with bacteria and fungi, in particular at mucocutaneous surfaces (Ouyang et al., 2008). A nonredundant role of the IL-17 pathway in host defense against fungi has also been identified in humans: rare inborn errors of IL-17 immunity are associated with an increased susceptibility to Candida albicans (Cypowyj et al., 2012). T helper cells act as an important source of IL-17 during infection (Korn et al., 2009). In line with the requirement of IL-17 for antifungal defense, fungi are particularly strong inducers of Th17 cell responses. They engage, among others, pattern recognition receptors of the C-type lectin family and induce high levels of IL-6 and IL-23, which in turn skew the differentiation of T

helper cells toward IL-17-producing effector cells (LeibundGutLandmann et al., 2007). Moreover, vaccine-induced Th17 cells can promote host protection from experimental infections (Ba¨r et al., 2012; Chen et al., 2011; Lin et al., 2009; Moffitt et al., 2011; Sellge et al., 2010; Wu¨thrich et al., 2011) and, conversely, patients with chronic mucocutaneous candidiasis display a paucity of Candida-specific Th17 cells (Eyerich et al., 2008). In addition to Th17 cells, innate lymphocytes including gd T and natural killer T (NKT) cells as well as innate lymphoid cells can serve as a source of IL-17 (Cua and Tato, 2010). These cells respond very rapidly to microbial insults without the need to undergo proliferation and differentiation and are thought to be essential for providing IL-17 at the onset of infection, to bridge the gap before Th17 cell-derived IL-17 becomes available (Buonocore et al., 2010; Gladiator et al., 2013). IL-17 has pleiotropic functions. IL-17RA, the common subunit of several heterodimeric IL-17 receptor complexes that bind different IL-17 cytokine family members, is widely expressed on hematopoietic and nonhematopoietic cells, such as endothelial and epithelial cells. IL-17RA signaling is regulated by a complex including Act1 and TNF receptor associated factor 6 (TRAF6), which induces the expression of genes encoding proinflammatory cytokines, chemokines, and antimicrobial peptides (Gaffen, 2009). IL-17 also promotes granulopoiesis and recruits neutrophils to the inflammatory site. Neutrophils, in turn, kill microbes by a broad array of oxidative and nonoxidative mechanisms (Amulic et al., 2012). The antimicrobial activity of neutrophils requires tight regulation to avoid excessive damage to the host. Based on the tissue distribution of IL-17-secreting cells, IL-17 immunity is thought to be most relevant at mucocutaneous surfaces such as the gastrointestinal tract, the airways, and the skin. However, IL-17RA deficiency also renders mice highly susceptible to systemic infections with certain pathogens, including C. albicans (Huang et al., 2004), suggesting that IL-17 has also an important impact on the systemic immune system. The underlying mechanisms of systemic IL-17-mediated protection, though, remain unclear. Natural killer (NK) cells are innate lymphocytes that act as a first line of defense against systemic infections by certain pathogens, notably viruses. They also play a key role in tumor immunosurveillance. NK cells were named after their cytotoxic function, through which they eliminate infected or altered target cells through perforin- and death receptor-ligand-mediated mechanisms. These NK cell functions are controlled by cell-surface receptors that titrate stimulatory and inhibitory signals Immunity 40, 117–127, January 16, 2014 ª2014 Elsevier Inc. 117

Immunity IL-17RA-Dependent NK Cell Functionality

Figure 1. NK Cells, but Not T Cells, Are Essential for Host Protection from Systemic Candidiasis (A) Fungal burden in the kidneys of C. albicansinfected WT mice, Il17ra
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mice, and WT mice treated with anti-IL-17A and anti-IL-17F or with anti-IL-17RA. (B) Fungal burden in the kidneys of C. albicansinfected WT and Rag1
/
mice as well as WT mice treated with anti-AGM1 or anti-NK1.1. (C) The kidneys of C. albicans-infected WT and Il17ra
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mice as well as WT mice treated with antiAGM1 or anti-NK1.1 were analyzed by histology. Scale bars represent 100 mm. (D) Fungal burden in the kidney of C. albicansinfected Il17ra
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mice that were treated with antiAGM1 or left untreated. Each dot represents one mouse (A, B, D). All data shown are representative of two to four independent experiments. See also Figure S1.

(Ho¨glund and Brodin, 2010). NK cells are also an important source of inflammatory cytokines including IFN-g, TNF, and GM-CSF that can exert regulatory effects on other immune cells (Vivier et al., 2008). As such, NK cells can promote the maturation of dendritic cells (Degli-Esposti and Smyth, 2005) and favor the differentiation of CD4+ T cells into Th1 cells (Martı´n-Fontecha et al., 2004; Morandi et al., 2006). Here we found that NK cells are critical for immunity to systemic fungal infection and that they act by providing GM-CSF, which promoted the fungicidal activity of neutrophils during the innate phase of the response. Notably, this activity depended on cell-intrinsic IL-17RA-mediated signals, which conditioned NK cells during their development to respond to subsequent priming. The discovery of a link between the IL-17 pathway and functional competence of NK cells raises questions about whether some of the purported direct functions of IL-17 in immunity might in fact be direct consequences of NK cell deficiency and might extend to immunity to tumors and viruses. RESULTS IL-17RA-Deficient and NK Cell-Depleted Mice Are Highly Susceptible to Systemic Candidiasis In agreement with Huang et al. (2004), we found that Il17ra
/
mice died rapidly in response to systemic infection with Candida albicans and that this was associated with massive fungal burden in the kidneys at 3 days postinfection when compared to C57BL/6 or 129 wild-type controls or heterozygous littermates (Figure 1A, Figure S1A available online, and data not shown). Similarly, Rorc
/
mice, which are unable to produce IL-17A 118 Immunity 40, 117–127, January 16, 2014 ª2014 Elsevier Inc.

and IL-17F because of a deletion in the gene encoding the transcription factor RORgt (Ivanov et al., 2006), were also highly susceptible to infection (Figure S1B). These observations suggest that IL-17 is important for immunity at the onset of fungal infection. However, IL-17A and IL-17F mRNAs were hardly detectable in spleen, liver, and kidneys of infected mice (data not shown). At 3 days postinfection, a small population of IL-17A-expressing CD3ε+TCRb+ T cells could be identified in the kidneys of wild-type mice but not in H2-Ab1
/
mice (Figures S1C–S1E). However, these cytokine-producing T cells were not required for clearing the infection because H2-Ab1
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mice controlled the infection as well as did wild-type controls (Figure S1F). Furthermore, antibody-mediated neutralization of IL17A and IL-17F or blockade of IL-17RA in wild-type mice during the course of infection did not reproduce the severe susceptibility to systemic candidiasis observed in Il17ra
/
mice (Figure 1A). As a positive control, these antibodies strongly impaired fungal control in a model of oropharyngeal candidiasis (Gladiator et al., 2013, and data not shown). We therefore hypothesized that the susceptibility of Il17ra
/
mice to systemic candidiasis is not attributable to their failure to respond to IL-17 upon pathogen challenge but to a different pre-existing defect. In search of the mechanism responsible for controlling acute candidiasis, we tested the role of different immune cell populations for their impact on protection from systemic infection. Although Cd1d
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mice lacking NKT cells, Tcrg
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mice lacking gd T cells, and Rag1
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mice lacking all B and T cells (including NKT, gd T, and Th17 cells) all showed normal fungal control, Rag2
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Il2rg
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mice, which additionally lack NK and other innate lymphoid cells, were highly susceptible and displayed elevated fungal burden at day 3 postinfection (Figures 1B and S1F and data not shown, also see Figures 4C and 7D). The notion that NK cells may be essential for host defense against systemic candidiasis was corroborated by experiments with wild-type mice treated with anti-asialoGM1 (anti-AGM1) or anti-NK1.1

Immunity IL-17RA-Dependent NK Cell Functionality

antibodies before infection. Selective depletion of NK cells abolished fungal control and led to high fungal burden and massive inflammatory infiltrates in the kidneys of these mice (Figures 1B and 1C), very similar to the burden observed in Il17ra
/
mice (Figures 1A and 1C). Depletion of NK cells in Il17ra
/
mice did not lead to a further increase in fungal burden in the kidney of infected mice (Figure 1D). These data indicate that the defects in fungal control in Il17ra
/
and NK cell-deficient mice are epistatic and suggest that they might therefore be linked. IL-17RA-Deficient NK Cells Are Functionally Impaired We found no change in the relative number of CD49b+NKp46+ CD3
NK cells in the spleen of Il17ra
/
mice compared to wild-type controls (Figures S2A and S2B). Any small reduction in the absolute number of NK cells could be explained by the slightly diminished overall cellularity of the spleen of Il17ra
/
mice (Figure S2C). We then analyzed NK cell maturation by determining the fraction of NK cells coexpressing CD11b and CD27 (Hayakawa and Smyth, 2006). The proportion of immature CD27+CD11b
and CD27+CD11b+ NK cells was slightly higher and that of mature CD27
CD11b+ NK cells was slightly lower in Il17ra
/
mice (Figures S2D and S2E), indicating a small delay in NK cell maturation. NK cell expression of the b chain of the IL-2 and IL-15 receptor (CD122), of the activating receptors NKG2D and Ly49H, and of the inhibitory receptors KLRG1, Ly49A, and Ly49C/I was unchanged in Il17ra
/
mice compared to wildtype controls (Figure S2F). To investigate the functionality of Il17ra
/
NK cells, we injected mice with LPS and analyzed the capacity of splenic NK cells ex vivo to produce cytokines and to kill target cells. Although NK cells from Il17ra
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mice upregulated CD69 expression, they produced less IFN-g than did wild-type NK cells (Figure 2A). Similarly, their cytotoxic activity toward YAC-1 cells was strongly impaired (Figure 2B). Moreover, Il17ra
/
mice were unable to clear mouse cytomegalovirus (MCMV), an activity that is strictly dependent on NK cells (Biron et al., 1999). High virus titers were detected in the spleen and liver of Il17ra
/
mice 4 days postinfection whereas wild-type mice had completely cleared the virus at this time point (Figure 2C). NK cells recognize MCMV-infected cells through their activating receptor Ly49H (Brown et al., 2001), which interacts with the MCMV-encoded protein m157 on the surface of infected cells (Arase et al., 2002). However, Ly49H expression by NK cells from Il17ra
/
mice was normal (Figure S2F), thus ruling out diminished recognition of MCMV-infected cells by Il17ra
/
NK cells as the basis for the increased susceptibility. Together these data suggest that the functional competence of NK cells depends on an intact IL17RA pathway, presumably mediating response to IL-17A and IL-17F during NK cell development. In support of this hypothesis, we found that NK cells taken from Rorc
/
mice displayed reduced IFN-g production and cytotoxicity in response to LPS injection in vivo (Figures 2A and 2D). A defect in cytokine production by NK cells was also observed in mice lacking the IL-17 receptor C (the receptor subunit that together with IL-17RA mediates signaling of IL-17A and IL-17F; Figure S2G), whereas NK cells from IL-17A- or IL-17F-deficient mice displayed an intermediate defect, which is indicative of a partial redundancy of these two closely related cytokines (Figure S2H). NK cells from all strains tested were phenotypically normal (Figure S2F and data

not shown). Together, these data from five independent mouse strains confirm that the impaired NK cell function is indeed caused by a deficiency in the IL-17 pathway and cannot be explained by the genetic background of any one out of the five strains. Next, we wondered whether the need for IL-17RA signaling was intrinsic to NK cells. We set up an in vitro coculture system with NK cells and wild-type bone-marrow-derived DCs (BMDCs) stimulated with LPS (Granucci et al., 2004). Whereas wild-type NK cells readily produced IFN-g under these conditions, no production could be detected from Il17ra
/
NK cells (Figure 2E). Activation of NK cells by LPS-stimulated BMDCs in vitro is unlikely to directly involve IL-17, and accordingly we were unable to detect IL-17 in the supernatant of the BMDC-NK cell cocultures (data not shown). Neutralization of IL-17A and IL-17F or blocking of IL-17RA in the cocultures also did not interfere with the cytokine response of wild-type NK cells to LPS-stimulated BMDCs (Figure 2F). Furthermore, blocking IL-17A and IL-17F during MCMV infection did not abolish viral control in wild-type mice (Figure 2C). To confirm that the functional defect of Il17ra
/
NK cells is cell intrinsic, we generated mixed bone marrow chimeras and found that NK cells derived from Il17ra
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donor bone marrow were hyporesponsive to LPS priming when compared to their WT counterparts in the same recipient (Figures 3A and 3B). Further experiments revealed that IL-17RA is expressed on NK cell precursors (NKP) and on immature NK (iNK) cells in the bone marrow (Figure 3C). Moreover, we show that IL-17RA downstream signaling is activated in these cells in response to IL-17AF stimulation (Figure 3D), whereas more mature subsets of NK cells do not express IL-17RA and do not respond to cytokine stimulation (Figure 3C and data not shown). In agreement with a role of IL-17 signaling during the development of NK cells, IL-17A is produced in the bone marrow (Figure S2I). Collectively these data suggest that IL-17 signaling acts directly on NK cell precursors to regulate acquisition of functional competence. Functional NK Cells Restore Antifungal Defense in Il17ra–/–, Rorc–/–, and Rag2–/–Il2rg–/– Mice Given the involvement of IL-17RA in NK cell development, we tested whether the high susceptibility of Il17ra
/
and Rorc
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mice to C. albicans (see Figures 1A, 1C, and S1B) is due to impaired NK cell function. Consistent with that notion, transfer of wild-type NK cells before infection restored the capacity of Il17ra
/
and Rorc
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mice to control the fungus. The fungal load was reduced to that observed in control wild-type mice (Figures 4A and 4B and data not shown), and no inflammatory infiltrates could be observed in the kidneys on day 3 postinfection (Figure 4B). We then tested whether Rag2
/
Il2rg
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mice could also be rendered resistant to fatal candidiasis by restoring the NK cell compartment. We found indeed that adoptive transfer of wild-type NK cells into Rag2
/
Il2rg
/
mice before infection reduced the fungal burden and the inflammatory infiltrates to the levels observed in wild-type mice (Figures 4C and 4D). In contrast, NK cells from IL-17RA-deficient mice were unable to restore fungal control in Rag2
/
Il2rg
/
mice (Figures 4C and 4D), indicating that their defect is programmed and that their functionality cannot be restored even in an IL-17RA-sufficient environment. In conclusion, these data show that acute Immunity 40, 117–127, January 16, 2014 ª2014 Elsevier Inc. 119

Immunity IL-17RA-Dependent NK Cell Functionality

Figure 2. Il17ra–/– NK Cells Are Functionally Impaired

(A) WT, Il17ra
/
, and Rorc
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mice were primed with LPS, and splenic CD49b+NKp46+CD3ε
NK cells were analyzed for CD69 expression and IFN-g production. (B) NK cells from LPS-primed WT and Il17ra
/
mice were incubated with YAC-1 target cells to evaluate their cytotoxicity. Each symbol represents the mean ± SD of three replicates. (C) Viral load in the spleen and liver of MCMV-infected WT mice, Il17ra
/
mice, and WT mice treated with anti-IL-17A and anti-IL-17F. Each symbol represents one mouse. The dashed line depicts the detection limit of the plaque assay. (D) NK cells from LPS-primed WT and Rorc
/
mice were analyzed for their cytotoxic capacity as described in (B). (E) NK cells isolated from naive WT and Il17ra
/
mice were cocultured with WT BMDCs in the presence of LPS. Cytokine production by CD49b+NKp46+CD3ε
NK cells was assessed by flow cytometry. (F) NK cells isolated from naive WT mice were cocultured with WT BMDCs in the presence or absence of LPS alone or in combination with anti-IL-17A and anti-IL17F or anti-IL-17RA. IFN-g production by CD49b+NKp46+CD3ε
NK cells was analyzed as in (E). Representative FACS plots are shown in (A), (D), (E), and (F). All data are representative of two to three independent experiments. See also Figure S2.

protection from systemic candidiasis in mice depends on the presence of functional NK cells and that a functional deficiency in NK cells in Il17ra
/
and Rorc
/
mice underlies their susceptibility to infection. NK Cells Promote the Candidacidal Activity of Neutrophils To investigate the mechanism by which NK cells mediate fungal control during acute systemic candidiasis, we analyzed their impact on neutrophil recruitment and function. No difference in neutrophil infiltration into the kidneys was observed in infected 120 Immunity 40, 117–127, January 16, 2014 ª2014 Elsevier Inc.

Il17ra
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or NK cell-depleted mice compared to control animals at 1 day postinfection (Figure S3). As a consequence of the high fungal load at later time points, neutrophils accumulated excessively in Il17ra
/
mice (Figures 1C and 4B), thus precluding any interpretation on the role of NK cells in neutrophil recruitment. Next, we analyzed the fungicidal activity of neutrophils isolated from the blood of infected mice. These neutrophils were elicited in response to the infection but have most likely not been in direct contact with the fungus because C. albicans is cleared very rapidly from the bloodstream after intravenous injection (Lionakis et al., 2011). When exposed to C. albicans in vitro,

Immunity IL-17RA-Dependent NK Cell Functionality

Figure 3. IL-17RA Signaling Acts NK Cell Intrinsically (A) Mixed chimeras were generated by reconstituting irradiated WT mice with a 1:1 mixture of WT and Il17ra
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bone marrow. IFN-g production by WT and Il17ra
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splenic NK cells in response to LPS priming was analyzed (filled histograms). Black lines represent PBS-injected controls. (B) Summary graph of data shown in (A). Each symbol represents one mouse. (C) CD122+NKG2D+CD3ε
bone marrow cells from WT or Il17ra
/
mice were analyzed for CD94 and CD49b expression to distinguish NKP (CD94
CD49b
), iNK cells (CD94+CD49b
), and mNK cells (CD94+CD49b+). IL-17RA expression was assessed on all three cell subsets. (D) Total bone marrow cells were stimulated with IL-17AF heterodimer or PMA and ionomycin for 30 min, or left untreated, and mNK cells (NKG2D+CD122+CD3ε
CD49b+IL-17RA
) as well as NKP and iNK cells (NKG2D+CD122+ CD3ε
CD49b
IL-17RA+) were analyzed for phospho-ERK1/2. Filled histograms show stimulated cells whereas black lines represent untreated controls. All data are representative of two to three independent experiments.

neutrophils from Il17ra
/
, Rorc
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, or NK cell-depleted infected mice displayed a strongly reduced ability to kill the fungus compared to neutrophils from untreated wild-type mice (Figure 5A and data not shown). Adoptive transfer of wild-type NK cells into Il17ra
/
mice before infection restored the capacity of blood neutrophils to efficiently kill C. albicans in vitro (Figure 5A). To exclude the possibility that neutrophils from Il17ra
/
mice possess an intrinsic fungicidal defect, we compared the candidacidal activity of blood neutrophils from uninfected Il17ra
/
and wild-type mice and found that both were equally efficient in killing opsonized fungus (Figure 5B). These data suggest that functional NK cells instruct the fungicidal activity of neutrophils in response to C. albicans infection. NK Cell-Mediated Instruction of Neutrophils Depends on NK Cell-Derived GM-CSF NK cells can secrete a number of cytokines, most prominently IFN-g. In response to C. albicans, however, they did not produce IFN-g (Figure 6A). NK cells primed in vitro in the presence of BMDCs and the fungal cell wall component b-1,3-glucan also did not produce IFN-g (data not shown). Moreover, Ifngr1
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mice had no defect in controlling systemic candidiasis (Figure S4A), indicating that IFN-g was not involved in host defense against this type of fungal infection. Instead, NK cells produced GM-CSF in response to C. albicans infection (Figures 6A and 6B). NK cells were the major source of GM-CSF secreted in response to C. albicans (data not shown), and depletion of NK cells led to a strong decrease in GM-CSF-producing cells (Figure 6C). Notably, NK cells from Il17ra
/
and Rorc
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mice were unable to secrete GM-CSF (Figures 6A, 6B, and S4B). To gain insight into the impact of NK cell-derived GM-CSF on neutrophils, we made use of an in vitro assay. NK cells from C. albicans-infected mice enhanced the viability and activation of neutrophils as measured by granularity and Annexin V binding

or by CD11b expression and ROS production, respectively (Figure 6D). This effect was independent of direct cell-cell contact between NK cells and neutrophils and could be inhibited by the addition of GM-CSF-neutralizing antibodies (Figure 6D). The supernatant of NK cells primed in vitro by C. albicans-stimulated BMDCs was sufficient to promote neutrophil survival and activation and this was again dependent on GM-CSF (Figure 6E). Furthermore, the stimulation of neutrophils with recombinant GM-CSF alone was sufficient to promote these effects (Figure 6F). To confirm the role of GM-CSF on the candidacidal activity of neutrophils in vivo, we injected recombinant GM-CSF into wildtype, NK cell-depleted wild-type and Il17ra
/
mice prior to and during candidiasis. This treatment was able to compensate for the lack of functional NK cells in Il17ra
/
and NK cell-depleted mice (Figures 7A and 7B). Finally, we tested the requirement of NK cell-derived GM-CSF for immunity against Candida by transferring GM-CSF-sufficient and GM-CSF-deficient NK cells into Rag2
/
Il2rg
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mice prior to infection. Whereas wild-type NK cells restored the protective response to the fungus, GM-CSFdeficient NK cells were unable to do so (Figures 7C and 7D). The candidacidal activity of neutrophils measured by ex vivo killing of C. albicans correlated inversely with the fungal burden in the kidneys of infected mice and was also affected differentially by transfer of GM-CSF-sufficient versus -deficient NK cells (Figures 7C and 7D). Together, these data show an essential role of NK cell-derived GM-CSF for neutrophil-mediated fungal control during innate protection from systemic candidiasis. DISCUSSION The discovery of Th17 cells a decade ago propelled the IL-17 family of cytokines into the limelight of immunological research. They are now well accepted as key players in host defense at Immunity 40, 117–127, January 16, 2014 ª2014 Elsevier Inc. 121

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Figure 4. WT but Not Il17ra–/– NK Cells Protect from Systemic Candidiasis

(A and B) Fungal burden (A) and histology (B) of the kidneys of WT and Il17ra
/
mice as well as Il17ra
/
mice that received WT NK cells prior to infection with C. albicans. (C and D) Fungal burden (C) and histology (D) of the kidneys of Rag2
/
Il2rg
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mice that did or did not receive WT or Il17ra
/
NK cells prior to infection with C. albicans. Data in (A) and (C) are pooled from two to three independent experiments and each dot represents one mouse. Representative histology sections from two to three independent experiments are shown in (B) and (D). Scale bars represent 100 mm.

mucocutaneous surfaces against certain extracellular bacteria and fungi (Herna´ndez-Santos and Gaffen, 2012; Ouyang et al., 2008). IL-17-dependent effector functions are thought to involve the induction of antimicrobial proteins and the production of cytokines and chemokines that recruit and activate neutrophils. Here we provide evidence for a distinct IL-17-dependent mechanism that affects systemic immunity. We show that the IL-17 receptor regulates the developmentally programmed responsiveness of NK cells to microbial stimuli. IL-17 acts directly on NK cells and provides instructive signals during NK cell development that can be separated in time from the infectious offense. As a consequence, NK cell-mediated responses are strongly impaired in animals lacking a functional IL-17 pathway, including Il17ra
/
, Il17rc
/
, Rorc
/
, Il17a
/
, and Il17f
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mice. The inability of Il17ra
/
mice to control acute systemic fungal infection can be corrected by transfusing IL-17RA-sufficient NK cells, whereas the hyporesponsiveness of IL-17RA-deficient NK cells cannot be restored in an otherwise IL-17RA-sufficient environment, indicating that the defect is NK cell intrinsic. We further show that the newly discovered role of NK cells in antifungal defense relies on their nonredundant function to provide GMCSF to promote the fungicidal activity of neutrophils. In summary our data reveal an unprecedented intersection between NK cells and IL-17 immunity, two evolutionary ancient immune pathways, the consequences of which are probably not restricted to antifungal immunity but also impact immunity against viruses and tumors. 122 Immunity 40, 117–127, January 16, 2014 ª2014 Elsevier Inc.

The molecular pathway of IL-17 receptor signaling in NK cell progenitors and its target genes remain to be determined. Moreover, the cellular source of IL-17 acting on NK cell progenitors has not been identified. The commensal microbiota may play a critical role because specific commensal organisms can induce IL-17 production in steady state (Hooper et al., 2012). Many recent reports have provided evidence that the large number of resident microorganisms colonizing mammalian body surfaces carry out functions that are critical for host physiology and that profoundly shape mammalian immunity (Hooper et al., 2012). For instance, they affect NK cell priming by phagocytes through their capacity to facilitate expression of IFN-I and other proinflammatory cytokines (Ganal et al., 2012). Our data add another layer of complexity by revealing how NK cell responsiveness is acquired during development in a manner regulated by tonic IL-17RA signaling. The IL-17-mediated instruction of NK cells by the IL-17 pathway is reminiscent of NK cell education by MHC class I molecules (Ho¨glund and Brodin, 2010). Triggering of NK cell receptors specific for self-MHC molecules provides a quality control checkpoint in the differentiation process of NK cells. Although the precise pathway and protein targets involved remain ill defined, self-MHC class I interactions are important for NK cells to attain full functional competence. The cell surface phenotype, however, is barely affected by this education process. In recent years it has become clear that a fraction of NK cells expresses no receptors for self-MHC class I molecules but can nevertheless

Immunity IL-17RA-Dependent NK Cell Functionality

Figure 5. WT but Not Il17ra–/– NK Cells Promote Candidacidal Activity of Neutrophils (A) WT mice that were treated or not with anti-AGM1 and Il17ra
/
mice that did or did not receive WT NK cells were infected with C. albicans. Neutrophils were isolated from the blood 3 days postinfection and cocultured with C. albicans to assess their candidacidal activity. (B) Naive WT and Il17ra
/
blood neutrophils were cocultured with opsonized C. albicans at a 1:1 ratio for 2 hr before lysing the cells and assessing the surviving yeast cells on YPD agar plates. Each dot represents the mean of duplicate wells of neutrophils from one mouse. Data shown are representative of two independent experiments. See also Figure S3.

acquire functional competence, in particular for secreting cytokines in response to infection (Fernandez et al., 2005). It has thus been proposed that NK cell functions that are independent of self-MHC recognition may be independent of the process of education, or instead require a MHC class I-independent educational process (Di Santo, 2006). The IL-17-dependent conditioning of NK cells described here may provide a mechanism of alternative NK cell education. Our data, which are based on multiple independent lines of evidence from five distinct mouse strains with different defects in IL-17 production or signaling, confirm that the functional competence of NK cells is strictly dependent on the IL-17 pathway. They further indicate that the dependence of NK cell responsiveness on IL-17 signaling is not restricted to one specific effector function. The capacity of NK cells to secrete IFN-g as well as their cytotoxicity toward YAC-1 target cells is strongly impaired in IL-17 pathway-deficient mice in response to bacterial stimuli. Furthermore, the control of MCMV infection, which depends on both NK cell-derived IFNg and perforin-dependent cytotoxicity (Loh et al., 2005), was also completely abolished in Il17ra
/
mice. Hence, our findings are not limited to one specific response, but rather have longreaching consequences for NK cell-mediated immunity against different types of infectious agents and possibly even against tumors. The role of IL-17 in tumor development is controversial and IL-17 may have different roles depending on tumor type and stage. Experiments with IL-17A-deficient mice suggest that IL-17 has tumor-suppressive effects (Kryczek et al., 2009). This observation may now be explained mechanistically by the new link between the IL-17 pathway and NK cell responsiveness. Consistent with this notion, Kryczek et al. (2009) have reported that the defect in tumor control in Il17ra
/
mice correlates with a reduction in the number of IFN-g-producing NK cells. The IL-17 pathway has recently become a major focus for the development of anti-inflammatory therapies against a range of autoinflammatory and autoimmune disorders. Recent clinical

trials of therapeutic antibodies (Brodalumab and Ixekizumab) targeting the IL-17 pathway in psoriasis have had promising results (Leonardi et al., 2012; Papp et al., 2012). However—and not surprisingly—the blockade of the IL-17 pathway also revealed undesired consequences such as the development of fungal infections in some treated individuals (Hueber et al., 2012). Further adverse effects may become apparent in longer trials. Prolonged and systemic blockade of the IL-17 pathway may also entail an increased occurrence of viral infections and tumor formation as a result of defective NK cell maturation in the absence of IL-17 signaling. NK cells have been extensively studied in viral infections, in which their role is well defined. However, little is known about NK cells in the context of fungal infections. It was suggested that NK cells contribute to protection from Aspergillus fumigatus (Bouzani et al., 2011; Schmidt et al., 2011), Cryptococcus neoformans (Marr et al., 2009), and Pneumocystis murina (Kelly et al., 2013) by exhibiting direct activity against these fungi. We show here that NK cells also play a crucial and unprecedented role in host defense against disseminated candidiasis. Whereas NK cell deficiency (as in Rag2
/
Il2rg
/
or NK cell-depleted mice) or hyporesponsiveness (as in Il17ra
/
or Rorc
/
mice) causes high susceptibility to C. albicans within the first days of infection, adoptive transfer of functional NK cells restores the ability of these mice to control the infection. NK cells regulate the host response against C. albicans by promoting the candidacidal activity of neutrophils. Although the role of neutrophils for the control of systemic candidiasis is well established and has recently been confirmed (Maruyama et al., 2012), our data reveal a mechanism of how the activity of neutrophils is controlled. Two in vitro studies suggested previously that cytokine-stimulated NK cells are able to modulate neutrophil function and survival (Bhatnagar et al., 2010; Costantini et al., 2010). Here we provide evidence that this crosstalk takes place in vivo and that it is highly relevant for host defense against fungal infection. The NK cellmediated effect was independent of IFN-g. Instead, NK cells secreted GM-CSF in response to C. albicans infection, which was critical for neutrophil activation and antifungal immunity. Given the large number of different cell types that can potentially secrete GM-CSF, it is surprising that NK cells have such a nonredundant role in providing GM-CSF to neutrophils. Further studies will be required to determine whether this happens as a consequence of the spatiotemporal superposition of the two cell types with respect to the sites of pathogen growth. Notably, GM-CSF expression in the spleen and kidneys of C. albicans-infected mice have been known to correlate with fungal control, in contrast to most other cytokines and chemokines induced during candidiasis, which are associated with immunopathology instead of protection and thought to be a consequence of high fungal load (MacCallum et al., 2009). GM-CSF is a well-known target gene of IL-17. However, during systemic candidiasis its expression appears to be independent of IL-17, because blocking IL-17A and IL-17F cytokines or the receptor subunit IL-17RA during the course of infection had no gross effect on the ability of the mice to control the fungus. In humans the major risk factors for disseminated candidiasis include general immunosuppression, disruption of the physical barriers as is the case during deep tissue surgery, and neutropenia. Whether NK cell deficiency represents an additional risk Immunity 40, 117–127, January 16, 2014 ª2014 Elsevier Inc. 123

Immunity IL-17RA-Dependent NK Cell Functionality

Figure 6. NK Cell-Derived GM-CSF Promotes the Antifungal Activity of Neutrophils (A) WT, Il17ra
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, and Rorc
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mice were infected with C. albicans, and cytokine production by splenic CD49b+NKp46+CD3ε- NK cells was analyzed by flow cytometry 24 hr postinfection. (B) Summary graph of data shown in (A). Data are pooled from two individual experiments. Each symbol represents one mouse. (C) WT mice were treated or not with anti-AGM1 before infection and the frequency of GM-CSF-producing leukocytes (CD45+ cells) in the spleen was assessed by flow cytometry 24 hr postinfection. (D) Naive blood neutrophils were cocultured with WT NK cells isolated from C. albicans-infected mice either in direct contact or separated by a membrane (transwell, TW). Anti-GM-CSF antibody was added as indicated. CD11b and CD18 expression, ROS production, and Annexin V binding by CD45+Ly6G+ neutrophils was analyzed. CD11bhiCD18lo cells represent viable and activated neutrophils, whereas CD11bloCD18hi cells display a lower scatter and bind more Annexin V, indicating that they are less viable. (E) The supernatant from NK cells that were cocultured with C. albicans-infected BMDCs was added to blood neutrophils from naive WT mice together with antiGM-CSF as indicated. Ly6G+ neutrophils were analyzed for CD11b and CD18 expression as described in (D). (F) Blood neutrophils from naive WT mice that were incubated with recombinant GM-CSF were analyzed for CD11b and CD18 expression as described in (D). Representative FACS plots are shown in (A), (D), (E), and (F). All data are representative of two to four independent experiments. See also Figure S4.

factor has not been observed. Isolated NK cell deficiency (without associated defects in other cell types) is extremely rare in humans and the affected individuals suffer primarily from viral infections (Orange, 2002). The possibility of enhanced susceptibility to invasive candidiasis may simply have passed unnoticed so far because NK cell-deficient individuals are unlikely to be exposed to additional risk factors that allow C. albicans to enter the bloodstream. In conclusion, our data provide evidence for an unprecedented role of the IL-17 pathway in the systemic immune system. In contrast to the situation in mucocutaneous tissues, where IL17 is induced promptly in response to infection (Gladiator et al., 2013) and directly acts on neutrophil recruitment and function 124 Immunity 40, 117–127, January 16, 2014 ª2014 Elsevier Inc.

(Conti and Gaffen, 2010), the impact of the IL-17 pathway on the systemic immune system is indirect and independent of an acute infectious or inflammatory insult. Although the latter provides a basis for understanding the underlying mechanism of antifungal defense against systemic candidiasis, it also provides insights into how IL-17 deficiency may be linked to many other infectious and neoplastic diseases. EXPERIMENTAL PROCEDURES Mice All mouse strains used in this study are described in the Supplemental Experimental Procedures. All animal experiments were conducted in accordance

Immunity IL-17RA-Dependent NK Cell Functionality

Figure 7. NK Cell-Derived GM-CSF Plays a Nonredundant Role in Activating Neutrophil Candidacidal Activity and Promoting Fungal Control (A and B) WT mice that were treated or not with anti-AGM1 and Il17ra
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mice were infected with C. albicans and injected concomitantly with recombinant GM-CSF. (C and D) WT or Csf2
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NK cells were adoptively transferred into Rag2
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Il2rg
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mice prior to infection with C. albicans. The killing activity of blood neutrophils on day 3 postinfection was evaluated in (A) and (C). Each dot represents the mean of duplicates wells of neutrophils from one mouse. The fungal burden in the kidneys is shown in (B) and (D). Each dot represents one mouse. Data are pooled from two independent experiments.

with the guidelines of the Swiss Animal Protection Law and were approved by the Veterinary Office of the Canton of Zu¨rich, Switzerland (license number 184/ 2009 and 201/2012). Fungi, Viruses, and Infections Mice were infected with 105 cfu C. albicans strain SC5314 i.v. The fungal burden in the kidneys was analyzed at day 3 postinfection as described (Gladiator et al., 2013). For opsonization, C. albicans was pretreated for 30 min at 37 C with 5% fresh mouse serum in PBS. For viral infection, mice were injected with 5 3 106 pfu BAC-derived MCMV MW97.01 (Walton et al., 2008) and viral titers were determined at day 4 postinfection by standard virus plaque assay. In some experiments, anti-IL-17RA, anti-IL-17A, anti-IL-17F, antiNK1.1, or anti-AGM1 antibodies or recombinant GM-CSF were administered as detailed in the Supplemental Experimental Procedures.

of surviving yeast cells was assessed on YPD agar. Killing activity is expressed as percent of yeast cells surviving in the presence of neutrophils compared to yeast cells surviving in the absence of neutrophils. To analyze the crosstalk of NK cells and neutrophils in vitro, 106 neutrophils from naive mice were cocultured with 106 purified NK cells from naive or infected mice (see above) for 18 hr. Surface marker expression was analyzed by flow cytometry. ROS production was quantified by measuring de-esterification of 20 ,70 -Dichlorofluorescin diacetate. Neutrophil viability was determined by Annexin V staining. In some experiments NK cells were added into a transwell (0.4 mm pore size), or NK cell-neutrophil cocultures were supplemented with recombinant mouse GM-CSF or with anti-GM-CSF.

Histology Tissues were isolated at day 3 postinfection, embedded in OCT, and snapfrozen in liquid nitrogen. Cryo-sections (7 mm) were stained with periodicacid Shiff (PAS) and counterstained with haematoxylin.

Ex Vivo Analysis of Kidney Lymphocytes Kidneys were isolated at day 3 postinfection and enzymatically digested and lymphocytes were purified over a Percoll gradient. Cytokine production was assessed by flow cytometry after stimulating the cells with recombinant IL-23 and IL-1b. Alternatively, kidney lymphocytes were restimulated on plate-bound anti-CD3ε for 24 hr and cytokine production were quantified by sandwich ELISA.

Adoptive Transfer Experiments NK cells were purified with anti-DX5 microbeads (Miltenyi Biotech) and adoptively transferred into recipient mice 1 hr before infection (8 3 106 NK cells pooled from 4 donor mice per 1 recipient).

Statistics Statistical significance was determined by Mann-Whitney U-test or unpaired Student’s t test by Prism (Graphad Software) with *p < 0.05; **p < 0.01; ***p < 0.001.

Ex Vivo Analysis of NK Cell Function Cytokine production by splenic NK cells was determined by intracelluluar cytokine staining 5 hr after i.v. injection of LPS or 24 hr after C. albicans infection. Alternatively, GM-CSF secretion by splenic NK cells from infected mice was detected by cytometric bead array after 24 hr of culture. NK cell cytotoxicity against YAC-1 cells was determined by assessing YAC-1 cell viability by 7-AAD staining and flow cytometry. All antibodies used for flow cytometry are described in the Supplemental Experimental Procedures. In Vitro Analysis of NK Cell Function Splenic NK cells from naive mice were purified with anti-DX5 microbeads (Miltenyi Biotech) and 2.5 3 105 cells were cocultured with 105 bonemarrow-derived DCs in presence or absence of LPS, recombinant GM-CSF, or anti-GM-CSF antibody. Cytokine production was measured after 18 hr by intracellular cytokine staining. To evaluate IL-17 responsiveness of NK cells, total bone marrow cells were stimulated with IL-17AF heterodimer or PMA and ionomycin for 30 min and phospho-ERK1/2 was analyzed by flow cytometry. Neutrophil Isolation and Functional Assays Neutrophils were purified from the blood by density gradient purification. To test neutrophil-killing activity, 104 C. albicans cells were exposed to 104 neutrophils for 2 hr before the neutrophils were lysed with water and the number

SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and four figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.immuni.2013.12.002. AUTHOR CONTRIBUTIONS E.B. and S.L.-L. designed the experiments, E.B. and K.M. performed the experiments, E.B., P.G.W., C.R., and S.L.-L. analyzed the data, and E.B. and S.L.-L. wrote the manuscript. ACKNOWLEDGMENTS The authors would like to thank B. Becher, M. Greter, S. Pantelyushin, and G. de Libero for mice; the staff of the Rodent Center HCI and the Institute of Laboratory Animal Sciences for animal husbandry; S. Mandaric for help with the MCMV infection experiment; A. Zinkernagel for help with setting up the neutrophil killing experiments; and K. Trautwein-Weidner, A. Gladiator, F. Osorio, and A. Oxenius for helpful advice and discussions. This work was supported by the National Science Foundation (grant number PP00P3_123342) and the Swiss Life Anniversary Foundation.

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