Emerging IL-12 family cytokines in the fight against fungal infections

Highlights • IL-12 and IL-23 have established roles during anti-fungal immunity.• IL-27 promotes regulatory effector responses during fungal infections.• IL-35 drives T cell differentiation to produce anti-inflammatory responses.• Increasing evidence for IL-12 family cytokines in maintaining anti-fungal immune homeostasis.

Aspergillus fumigatus is an airborne fungus that causes various types of disease including invasive pulmonary aspergillosis (IPA) and allergic bronchopulmonary aspergillosis (ABPA). An important role of the host immune response is to limit spore germination and restrict hyphal invasion. Spore swelling, the first step of germination, exposes fungal ligands that bind to CLRs and TLRs resulting in inflammatory cytokine production [44,45]. This then induces a Th1 response and a much smaller Th17 response in a pulmonary infection model [25,46] whereas a Th2 response is induced in an allergic model of disease [47,48].
The T cell responses mediated by these different pathogens (Candida and Aspergillus) are likely controlled by the ligands exposed on their cell surface and by the environment (e.g. systemic, mucosal, pulmonary) of the infection. The yeast form of C. albicans expresses mannans on the cell surface and β-glucans are rapidly exposed at bud scars whereas dormant Aspergillus conidia are covered in a rodlet layer that initially hides β-glucans from the immune system [44,49,50]. Mannans present on the yeast form of C. albicans have been shown to induce IL-1β and IL-23 in a Dectin-2 dependent manner [37]. Additionally, both yeast and hyphal forms of C. albicans induced Dectin-2-dependent Th17 differentiation in vitro and Il17a deficient mice were more susceptible to systemic infection with C. albicans [37]. Further to this, dendritic cells (DCs) activated by Dectin-1 engagement by curdlan, a β-glucan preparation, demonstrated Th17 cell differentiation of CD4 + T cells in vitro, and the use of curdlan as an adjuvant in vivo, promoted both Th17 and Th1 responses [34]. We have previously demonstrated differences in T cell differentiation in vitro and in vivo. Heat killed yeast predominantly induced Th17 differentiation in vitro, whereas, mice systemically infected with C. albicans displayed a Th1-biased response in vivo [19], therefore the environment is also important for determining T cell bias. In the context of A. fumigatus infections, Th1 and Th17 CD4 + T cells are driven via TLR/MyD88 and Dectin-1 respectively. TLR2 and TLR4 recognise A. fumigatus conidia [51], whereas Dectin-1 recognises later morphological forms such as swollen conidia and early germlings [44]. It is possible that the TLRs initiate a Th1 response to A. fumigatus and then Dectin-1 induces the Th17 response once germination of A. fumigatus is further underway. As C. albicans and A. fumigatus engage different combinations of PRRs, interactions between these PRRs likely dictate the balance between Th1 and Th17 responses to these two pathogens.
IL-27 signals through IL-27Rα (WSX-1/TCCR) paired with glycoprotein (gp)130 [84] (Fig. 1). IL-27 can bind WSX-1 with low affinity in the absence of gp130, however both IL-27R subunits are required for effective signal transduction [10,84]. IL-27 signaling activates various JAK and STAT family members in a cell-specific manner. For example, IL-27 activates both STAT1 and STAT3 in naïve CD4 + T cells but in fully active T cells only STAT3 activation is retained [85]. IL-35 signaling is unconventional as it utilizes different receptor combinations including IL-12Rβ2 and gp130 homodimers and heterodimers (Fig. 2). STAT1 and/or STAT4 are activated depending on which receptor homodimer/heterodimer is engaged. In B cells IL-35 signals through IL-12Rβ2 and WSX-1 to activate STAT1 and STAT4. More work is required to fully understand IL-35 signaling downstream of the different homodimers or heterodimers [17,86].
3. IL-12 family cytokines in anti-fungal host defense

IL-12
IL-12 is induced by microbial products in monocytes, macrophages and DCs and acts on NK and T cells to induce IFN-γ. IFN-γ in turn activates monocytes and macrophages to induce further IL-12 production [87]. This positive feedback mechanism helps protect against certain pathogens that induce low IL-12 levels [88]. Deficiency of IL-12 or IL-12R genes leads to impaired cell mediated immunity and enhanced disease susceptibility [89][90][91][92]. We will only briefly discuss the role of IL-12 during Candida infections as this has been reviewed elsewhere [22,93].
Several C. albicans infection experiments have been performed in mice deficient in different components of the IL-12 family (Table 1). However, due to subunit sharing between different family members, interpretation of these results is rather complex. Overall, IL-12 appears to be important during systemic infection but less important during mucocutaneous infections. Il12p40 -/mice were susceptible to oral infection with C. albicans, however, this was mainly attributed to IL-23 and Th17 responses [29]. Interestingly, Il12p35 -/mice demonstrated disseminated infection following oral infection with C. albicans suggesting that while IL-12 is not overly important in the local response during an oral infection, it controls systemic dissemination [29]. In addition, Il12p35 -/and IFN-γ -/mice displayed enhanced susceptibility to systemic infection with C. albicans [7,[39][40][41], indicating an important role for IL-12/Th1 responses during systemic infection. Il12p40 -/mice displayed variable susceptibility to systemic infection in different studies [7,26,94]. Additionally, in a mouse model where mannoproteins from the fungal pathogen Cryptococcus neoformans were shown to protect against systemic C. albicans infection, this protective response was reversed upon IL-12 blockade [95]. Further, when IL-12 was fused to enolase (a C. albicans antigen) and administered during systemic infection with C. albicans, mice demonstrated increased survival and decreased fungal burden in their kidneys [27]. However, administration of IL-12 during systemic infection increased disease severity due to IFN-γ mediated pathology [96]. These data suggest that while IL-12 and IFN-γ are important for controlling systemic infections with C. albicans, this response needs to be finely tuned for optimal fungal clearance while minimising pathology.
The importance of IL-12 has also been demonstrated during A. fumigatus infections. Human DCs infected with A. fumigatus induced IL-12p70 production and promoted IFN-γ production from CD4 + T cells [20]. Rivera et al. [25] showed that Th1 and Th17 responses are induced during an in vivo model of A. fumigatus lung infection. The authors showed that TLR and Dectin-1 pathways modulate the balance between Th1 and Th17 populations by controlling production of IL-12 family members. In addition, IL-12 has been shown to induce human monocytes to damage A. fumigatus hyphae via an oxidative burst response in an IFN-γ-independent manner [97].
During an in vivo infection model with A. fumigatus, Cenci et al. [98] compared the immune response of resistant (intact DBA/2 mice) and susceptible (leukopenic DBA/2 mice) mice. Interestingly, the authors demonstrated that resistant mice produced high levels of IL-12, TNF and IFN-γ while susceptible mice produced an IL-10 and IL-4 skewed response. In addition, immunosuppressed Il12p40 -/and Ifng -/mice were more susceptible to invasive pulmonary aspergillosis [99]. In agreement with this, Nagai et al. [100] showed that administration of IFN-γ protected mice from invasive aspergillosis. Furthermore, Delsing et al. [101] showed that adjunctive therapy with IFN-γ and an antifungal drug partially restored immune function in patients with invasive Candida and/or Aspergillus infections indicating that further clinical studies are warranted to fully determine the potential clinical benefits of IFN-γ therapy during invasive fungal infections.

IL-23
IL-23 is involved in the maintenance of Th17 cells and the activation of memory T-cells [78,102]. It induces CD4 + Th17 cells to produce IL-17 and IL-22 [80,[103][104][105]. IL-17A is a proinflammatory cytokine that promotes neutrophil recruitment via upregulation of other cytokines and chemokines [106]. While the IL-23-IL-17 pathway is important for protective responses against infection, it can also contribute to autoimmunity and increased pathology [107,108]. IL-23 is also important for regulation of innate lymphocyte cells (e.g. innate lymphoid, NK, NKT and γδ T-cells [109]).
C. albicans induces IL-23p19 and/or IL-23 in cultured macrophages and DCs and at sites of inflammation during infection [19,24]. Interestingly, human monocyte derived DCs produced IL-23 when challenged with C. albicans hyphae, whereas C. albicans yeast induced IL-12 production and IL-23 was only produced with high concentrations of yeast [110]. This suggests that the hyphal form of C. albicans triggers the Th17 response in vivo. During systemic infection with C. albicans, IL-23 was dispensable, however the IL-23/Th17 pathway is important during chronic muccocutaneous candidiasis (CMC) [7]. Il23p19 -/-, and Il17ra -/mice were more susceptible to oral C. albicans infection [29]. However, Yano et al. (2012) found that fungal burden and S100A8/S100A9 alarmin-mediated neutrophil recruitment was normal in Il23p19 -/-, Il17ra -/and Il22 -/mice in a mouse vaginal infection model suggesting that the vaginal alarmin S100 response is independent of the IL-23/Th17 axis [111]. In contrast, several other studies have found important roles for Th17 cells, IL-17 and IL-22 in protecting patients against CMC [29,43,112]. Patients with impaired Th17 and Th17-associated cytokines due to mutations in genes such as STAT3 (hyper IgE syndrome, [113,114]), IL-17RA [115], IL-17F [115], ACT1 [116], STAT1 [117,118], AIRE [119,120], IRF8 [121], CARD9 and DECTIN-1 [122][123][124] are prone to CMC. In addition, two Mexican patients with a mutation in IL12RB1, suffered from infections with Baccille Calmette-Guérin (BCG) and C. albicans and they both died early at ages 4 and 16 [125]. Furthermore, in an international study of 141 patients, 23% of patients lacking IL12RB1were found to suffer from CMC [126]. However, as both IL-12 and IL-23 signal through IL-12Rβ1, this could be due to a lack of responsiveness to either cytokine. IL-23 and IL-6 were detected in vaginal secretions from women infected with vaginal candidiasis [24,127]. Additionally, healthy PBMCs stimulated with C. albicans induced a Th17 response demonstrating that Th17 cells are involved in the defense against C. albicans [128]. Furthermore, reduced . gp130 couples with IL-12Rβ2 to form the IL-35 receptor complex and signals through JAK1 and JAK2 to activate STAT1, 3 and 4. STAT-1-STAT4 form a heterodimer to induce nuclear translocation and induction of IL-12α/p35 and EBI3. Additionally, homodimers of gp130 or IL-12Rβ2 have been shown to elicit a partial IL-35 induced response in T cells [86], and IL-12Rβ2 can also couple with IL-27Rα to also form an IL-35 receptor complex in B cells [17]. Table 1 Effects of different Candida and Aspergillus infections on Il12p40 -/-, Il12p35 -/-, Il23p19 -/and Il27ra -/mice. Th17 responses have been reported in human PBMCs stimulated with C. albicans from patients with hyper IgE syndrome, demonstrating that Th17 cells play an important role in the defense against C. albicans [113]. These data indicate a protective role for the IL-23/Th17 pathway during C. albicans infection, however, heightened IL-17 and IL-23 responses can also negatively regulate Th1 responses to C. albicans resulting in increased inflammation and susceptibility to candidiasis [7]. Therefore, similar to IL-12 and IFN-γ, the IL-23/Th17 response needs to be tightly controlled. Th17 cells express IL-10Rα and IL-10 signalling has been shown to control IL-17A producing CD4 + T cells in a colitis model [129], suggesting that IL-10 may be an important regulator of IL-23/Th17 responses during anti-fungal immunity. Studies have demonstrated a role for the IL-23/Th17/IL-17 pathway during A. fumigatus infection [7,130]. Il12p40 -/-, Il12p35 -/and Il23p19 -/mice displayed reduced fungal burden in the lungs of A. fumigatus infected mice, indicating that a heightened IL-23/Th17 response is associated with susceptibility [7]. However, Dectin-1 and TLR9 deficient mice displayed reduced IL-23, IL-17A and/or IL-22 responses and increased fungal burden in the lungs of A. fumigatus challenged mice, indicating that the IL-17 pathway is involved in a protective response to A. fumigatus [131,132]. In contrast, an immunopathogenic role for Dectin-1 and IL-22 has been shown during chronic fungal allergy [104], once again demonstrating the importance for tight regulation of these pathways. IL-17 has been shown to inhibit neutrophil mediated killing of A. fumigatus and in vivo clearance [7]. Th1 cells, Treg cells, and Th17 cells are all important in producing both an inflammatory and anti-inflammatory response against A. fumigatus [7,48,133]. The balance between Th17 cells and Treg cells has been suggested to be important in determining whether commensal C. albicans causes infection and the same could be true for Aspergillus infections [134,135].

IL-27
The ability of IL-27 to regulate T cell responses has been reviewed elsewhere [136,137]. Briefly, IL-27 was shown to enhance in vitro Th1 cell differentiation [138]. However, more recently, anti-inflammatory properties of IL-27 have been discovered. IL-27 has been shown to inhibit T cell responses by negatively regulating IL-2 signaling and by inducing IL-10 [11,12,66,[139][140][141][142]. IL-10 has been shown to inhibit both CD4 + and CD8 + T cell responses [134,143]. Additionally, a role in inhibiting Th17 cell differentiation has been demonstrated to protect against Th17 associated disease, such as encephalomyelitis [11][12][13][14]. Th17 cells in encephalomyelitis promotes the development of ectopic lymphoid structures in the central nervous system [144]. Interestingly, IL-27 inhibits the development of ectopic lymphoid structures in inflamed tissues [145]. These functions of IL-27 help protect against Tcell driven pathology. IL-27 has shown contrasting effects on regulatory T cells. It has been shown to promote Treg growth and survival at sites of infection [16] but also to indirectly inhibit Treg development by inhibiting IL-2 production [15,16].
IL-27 has been reported to be involved in the immune response to various pathogens including Trypanosoma cruzi [146], Leishmania major [147][148][149], Mycobacteria tuberculosis [11] and Toxoplasma gondii [12,14,150]. In addition, a limited number of studies reviewed here, have recently examined the role of IL-27 in response to fungal pathogens. The first report in a fungal model demonstrated that EBI3 levels were significantly induced in human monocyte derived DCs following stimulation with A. fumigatus while IL27 levels only showed a slight increase, demonstrating that A. fumigatus-induced IL-27 production is minimal [20]. Indeed, it is possible that the increased EBI3 levels could actually reflect IL-35 induction rather than IL-27 production, as human monocyte derived tolerogenic DCs express and produce IL12A and EBI3 [151], although this remains to be determined in a fungal model. However, in a later study these authors went on to show that IFN-β treated DCs challenged with A. fumigatus resulted in increased IL27 mRNA levels, IL-12p70 production and increased expression of CD86 and CD83 [152]. While it is well established that IFN-β induces IL-27 [153], this report describes a new role for A. fumigatus in IL-27 production [152]. Further, DCs stimulated with IFN-β and challenged with A. fumigatus resulted in increased Th1-mediated IFN-γ production [152]. This suggests that IFN-β could be used as a possible therapy to enhance a beneficial Th1 response during infection with A. fumigatus.
Type 1 regulatory T cells (Tr1) are instrumental in protecting against Th1/Th17-mediated autoimmunity in mice and reactions to common allergens in humans [154,155], and may also play an important role during A. fumigatus infections [156]. Tr1 cells are Foxp3regulatory T cells that produce high IL-10 levels and suppress T cell expansion in vitro and in vivo [157]. Bedke et al. [156] identified Crf-1/ p41-specific latent associated peptide + and IL-10 + Tr1 cells in healthy humans and in mice following vaccination with an Aspergillus peptide (Crf-1/p41) and zymosan. In agreement with previous studies [158,159], Tr1 cell differentiation in vivo was found to be dependent on the aryl hydrocarbon receptor, c-Maf and IL-27. These data indicate that IL-27 plays a role in maintaining anti-fungal immune homeostasis via induction of Tr1 cell differentiation [156].
Several inflammatory cytokines/molecules, such as lipopolysaccharide (LPS) [10,22,160,161], IL-1β [162], Poly(I:C) [161] and IFN-β [152,153,163], have been reported to induce IL-27 as a protective response to prevent excess inflammation. Interestingly, increased expression of IL27 and EBI3 has been shown in THP-1 cells challenged with heat killed C. albicans compared with LPS [22]. In contrast, IL-27 production was reduced in the RAW264.7 macrophage cell line when treated with bakers yeast (saccharomyces cerevisiae)-derived β-glucan (BBG) [160]. In addition, we showed that β-glucan alone does not induce IL-27 in bone marrow derived macrophages (BMDMs) [30]. We also showed that select Candida spp. induce IL-27 production, such as C. parapsilosis, C. glabrata and C. tropicalis but not C. albicans. IL-27 production by C. parapsilosis was dependent on phagocytosis, TLR7/MyD88 and nucleotide-binding oligomerisation domain-containing protein (NOD)2 signaling. Further, C. parapsilosis induced IFN-β production, which then signaled through the IFN-α/β receptor (IFNAR) and STAT1/ 2 to induce IL-27 (Fig. 3). Interestingly, while we found that C. albicans did not induce IL-27, likely due to the absence of IFN-β induction, it also actively blocked C. parapsilosis-induced IL-27 via a soluble mediator [30]. This soluble mediator has yet to be identified, but it is tempting to speculate that BBG might induce the same soluble mediator to block IL-27 production [160]. However, it is interesting to note that live C. albicans blocked C. parapsilosis-induced IL-27 in mouse BMDMs [30] while heat killed C. albicans enhanced LPS-induced IL-27 in the human THP-1 cell line [22]. These discrepancies could be due to differences in mouse vs human IL-27/cells or due to exposure of different ligands on live vs heat killed C. albicans and requires further study to unravel these differences.
Similar to infections with other pathogens such as M. tuberculosis, P. berghei and T. gondii [11,164], we demonstrated improved clearance of systemic C. parapsilosis infection in WSX-1 (Il27ra) deficient mice. This was accompanied by increased IFN-γ and IL-17 T cell responses in addition to increased serum pro-inflammatory cytokine levels [30]. While we observed improved fungal clearance and only a modest increase in inflammation, other groups have shown improved pathogen clearance in WSX-1 deficient mice accompanied by fatal organ pathogenesis due to highly elevated pro-inflammatory responses [11,12,164]. Interestingly, some studies showed that organ pathology could be prevented by depleting CD4 + T cells but not CD8 + T cells [12,164]. As C. parapsilosis induced a CD8 + T cell biased response rather than a CD4 + T cell response, this could account for the improved fungal clearance without inducing fatal organ pathology [30]. These data suggest that blocking IL-27 during infection with C. parapsilosis could have therapeutic benefits.
In this review, it has already been mentioned that Th17 cells, IL-17, IL-22 and IL-23 cytokines have important role in protecting patients against CMC [7,24,29,43,112]. Patients with a gain of function mutation in STAT1 have higher susceptibility to Candida infections [165]. Further, increased STAT1 function showed increased IL-27, IFN-γ and IFN-α production but reduced IL-17 responses [165][166][167]. In a separate study, it has been reported that reduced IFN-γ and IL-27 responses from impaired STAT1-expression was observed in two unrelated patients (from Japan and Saudi Arabia) with heterozygous missense mutations in the STAT1 SH2 domain [168]. Further studies in both mice and humans are required to fully unravel the role of IL-27 during anti-fungal immunity.

IL-35
IL-35 is mainly produced by Bregs and Tregs [169]. Stimulation of naïve effector T cells with recombinant IL-35 suppressed T cell proliferation in a murine model and ectopic expression of IL-35 on naïve T cells conferred regulatory activity [71]. IL-35 has been shown to suppress immune responses important for the induction of inflammatory disease in murine models such as collagen-induced arthritis [69,71]. However, human T regulatory cells do not express IL-35 and as result a role for IL-35 in humans remains unclear [72]. As cytokine subunits are shared between IL and 35, IL-12 and IL-27, mechanistic studies to determine specific roles for IL-35 in the regulation of anti-fungal immunity are challenging.
Il12p35 -/mice have been shown to have lower fungal burden during oral candidiasis. As IL-12p35 is a subunit of IL-35, it is possible that IL-35 may play an important role in reducing tissue damage during oral infection [29]. Recently, heat killed C. albicans was shown to suppress LPS-induced IL-12p70 production in M2 macrophages due to increased Ebi3 expression, a subunit of IL-35. Further, the authors demonstrated that C. albicans induced IL-35 production, which inhibited an LPS-induced M2 (anti-inflammatory) to M1 (inflammatory) phenotype change in BMDMs, thereby supressing an inflammatory response.
These results demonstrate a possible mechanism for how C. albicans is able to evade immune detection [23].   [30].