Oropharyngeal candidiasis is an opportunistic infection caused primarily by Candida albicans (C. albicans), a ubiquitous fungal organism that is part of the normal oral microflora in up to 80% of healthy individuals (Ref. Reference Odds1). The transition from commensalism to infection in the oral mucosa is caused by changes in the local oral microenvironment, or by an inadequate host defence, which results in C. albicans overgrowth (Ref. Reference Scully, el Kabir and Samaranayake2). The prevalence of oropharyngeal candidiasis continues to be high because of an increasing population of immunocompromised patients, including individuals with diabetes, HIV, transplant recipients and patients with solid tumors or haematological malignancies receiving stem cell therapy (Refs Reference Rubin3, Reference Ohmit4, Reference Redding5, Reference Hermann6, Reference Penk and Pittrow7). In general, epidemiological studies suggest a key role of an intact immune system in protection against this infection, and indicate a specific role for T cell immunosuppression in susceptibility to infection (Refs Reference Rubin3, Reference Ohmit4, Reference Redding5, Reference Hermann6, Reference Penk and Pittrow7). Oral lesions are generally associated with local discomfort, altered taste sensation and dysphagia, resulting in poor nutrition (Ref. Reference Neville8). Moreover, oropharyngeal candidiasis is associated with esophageal candidiasis, a more invasive form of infection with the potential for systemic spread (Refs Reference Anaissie and Bodey9, Reference Richet10, Reference Samonis and Bafaloukos11). Immune defence against opportunist infection with C. albicans depends on proper host recognition, followed by activation of antifungal mechanisms in effector cells. This review discusses the current understanding of recognition of C. albicans by the host and activation of anti-Candida mechanisms in mucosal structural and immune cells. We also discuss how to apply this knowledge to the development of novel treatment strategies against this fungal organism.

Candida recognition by structural mucosal and innate immune cells

Activation of protective mechanisms in innate immune and nonimmune effector cells depends on appropriate pathogen recognition. Rapid recognition of pathogens is mediated by the expression of pattern-recognition receptors (PRRs) on host cells, which recognise conserved pathogen-associated molecular patterns (PAMPs) (Ref. Reference Mukhopadhyay12). These recognition receptors are expressed by numerous cells, including polymorphonuclear leukocytes (PMNLs), dendritic cells, monocytes, macrophages, B cells, T cells, endothelial and epithelial cells. Recognition of PAMPs by PRRs induces intracellular signalling pathways that modulate protective and inflammatory responses, such as activation of antimicrobial mechanisms, expression of costimulatory molecules, release of proinflammatory cytokines and modulation of acquired immunity.

C. albicans possesses a variety of PAMPs, including chitin (a cellulose-like biopolymer consisting predominantly of N-acetyl-D-glucosamine), glucan (a β-linked branched-chain polymer of glucose), mannan (an α-linked polymer of mannose), mannoproteins and glycolipids. Differential recognition of these components on the C. albicans cell wall is mediated through numerous PRRs, such as Toll-like receptors (TLRs), mannose receptors, Dectin-1, complement receptor 3 (CR3) and mannose-binding lectins (Refs Reference Brown13, Reference Forsyth, Plow and Zhang14, Reference Underhill15) (Fig. 1).

Figure 1.

Innate immune recognition of Candida albicans. PRRs are expressed in oral mucosal surveillance cells including: (a) structural oral epithelial cells, (b) dendritic cells and (c) professional phagocytes, such as macrophages and neutrophils. Recognition of C. albicans by PRRs induces intracellular signalling pathways that modulate protective, inflammatory and regulatory responses, including activation of antimicrobial mechanisms, and release of proinflammatory and immune-instructive cytokines. Abbreviations: CR3, complement receptor 3; IL-8, interleukin-8; NO, nitric oxide; PMNL, polymorphonuclear leukocyte; PRR, pattern-recognition receptor; ROS, reactive oxygen species; TLR, Toll-like receptor; TNF, tumour necrosis factor.

Immune and nonimmune antifungal effector cells in the oral mucosa

Multiple immune and nonimmune cells contribute directly or indirectly to protection against mucosal infection by recognising C. albicans, modulating protective and inflammatory responses or having direct antifungal effector functions. One of the consequences of C. albicans recognition by the host is induction of inflammatory and immune regulatory cytokines such as IL-1, TNF-α, IFNγ, IL-4, IL-5, IL-6, IL-8, IL-13, IL-17 and several chemokines (Refs Reference Lilly63, Reference Dongari-Bagtzoglou and Kashleva64, Reference Li, Kashleva and Dongari-Bagtzoglou65, Reference Li and Dongari-Bagtzoglou66, Reference Djeu67, Reference Djeu, Serbousek and Blanchard68) (Fig. 1). Antimicrobial effector functions of structural and immune cells are highly inducible via these signalling molecules. Direct candidacidal activity has been demonstrated for four cell types: PMNLs, monocytes/macrophages, non-MHC-restricted CD8⁺ T cells and epithelial cells (Refs Reference Marquis69, Reference Romani70, Reference Steele71, Reference Steele72) (Fig. 1).

Role of regulatory T cells in oral mucosal protection

T-cell-deficient mice have been used to demonstrate that CD4⁺ T cells play a key regulatory role in the immune response against oral candidal infection. Even though the number and function of neutrophils and monocytes in these mice were normal, they could not clear C. albicans oral mucosal infection (Refs Reference Farah98, Reference Cantorna and Balish114). Moreover, reconstitution of these immunodeficient mice with naive CD4⁺ T cells (but not CD8⁺ T cells) significantly decreased oral colonisation compared with that in controls. Similarly, depletion of CD4⁺ T cells in the oral mucosa of BALB/c mice has been shown to increase the severity of oral infection with C. albicans (Ref. Reference Farah98). Interestingly, high levels of IL-12 and IFN-γ are produced by lymphocytes derived from the lymph nodes of resistant mice following C. albicans infection, suggesting that CD4⁺ T cells may augment phagocytic cell function via secretion of Th1 cytokines (Ref. Reference Farah98). However, there appears to be no protective role of IFN-γ against Candida in the oral mucosa (Ref. Reference Farah and Ashman115).

More recently, the role of CD4⁺ CD25⁺ regulatory T (Treg) cells in host protection against C. albicans has also been investigated in animal models of candidiasis. Two different types of Treg cells participate in the immune response against C. albicans – naturally occurring Treg cells and inducible Treg cells (Ref. Reference De Luca116). Naturally occurring Treg cells migrate to inflamed sites where they modulate the effector function of PMNLs and limit early inflammatory responses. Although migration of these Treg cells into infected sites requires activation of the TRIF-signalling pathway, their anti-inflammatory activity is suppressed by the MyD88-signalling pathway (Ref. Reference De Luca116). Later, inducible populations of Treg cells expand in both disseminated and gastrointestinal infection with C. albicans (Ref. Reference De Luca116). The induction of these cells during C. albicans infection is determined by the level of expression of costimulatory molecules, such as CD28 and CD86, and by signals provided by TLR2- and TRIF-signalling pathways (Refs Reference Netea24, Reference De Luca116, Reference Montagnoli117, Reference Sutmuller118). During infection, inducible Treg cells producing IL-4, IL-10 and transforming growth factor-beta (TGF-β) restrict inflammatory Th1 and Th17 responses, which on one hand limits the ability of the immune system to control fungal growth (Refs Reference Netea24, Reference Montagnoli117, Reference Zelante119), but on the other hand increases host resistance to reinfection (Ref. Reference Montagnoli117).

Reports from systemic infection models have suggested that memory CD4⁺ T cells producing IL-17 (Th17 cells) are generated as part of the immune response to C. albicans (Ref. Reference LeibundGut-Landmann120). Generation of Th17 cells is triggered by exposure of newly primed CD4⁺ T cells to IFN-γ, TGF-β, IL-6 and IL-23 (Refs Reference Veldhoen121, Reference Mangan122, Reference Bettelli123). Exposure of CD4⁺ T cells to these cytokines induces the expression of transcription factor retinoic acid receptor (RAR)-related orphan receptor gamma (RORγ), which controls the expression of IL-23 receptors on CD4⁺ T cells (Ref. Reference Ivanov124). Increased expression of IL-23 receptors on CD4⁺ T cells in turn renders these cells susceptible to IL-23 – a cytokine that promotes proliferation and differentiation of T-helper cells into Th17 cells (Refs Reference Aggarwal125, Reference Langrish126). C. albicans hyphal cells stimulate the production of IL-23 by monocyte-derived dendritic cells (Ref. Reference Acosta-Rodriguez127). Activation of Dectin-1 signalling through the Syk-CARD9 pathway induces maturation of dendritic cells and secretion of IL-6, IL-23, and TNF-α by these cells, which in turn, induces differentiation of naive T cells into Th17 cells (Ref. Reference LeibundGut-Landmann120). This Dectin-1–Syk–CARD9 signalling pathway has been shown to be required for the development of a potent Th17 response during haematogenouly disseminated candidiasis (Ref. Reference LeibundGut-Landmann120).

Production of IL-17 by Th17 cells in response to C. albicans (Ref. Reference LeibundGut-Landmann120) can potentially induce the recruitment and activation of neutrophils, and production of proinflammatory cytokines by epithelial cells, endothelial cells and macrophages (Ref. Reference Weaver128). In fact, defective mobilisation and influx of neutrophils into infected sites, increased fungal burden and impaired survival have all been shown in IL-17-receptor-knockout mice after systemic challenge with C. albicans (Ref. Reference Huang129). However, the increased susceptibility of IL-17-deficient mice to candidal infection was not confirmed in subsequent studies using animal models of systemic and gastrointestinal infections. By contrast, IL-17-knockout mice were more resistant to candidal infection, and the increased resistance was associated with a greater number of IFN-γ-producing Th1 cells (Ref. Reference Zelante119). These recent data suggest that a crossregulation exists between IFNγ/Th1 and IL-23/Th17 pathways. Th17 responses have not been characterised in oral models of infection; however, it is speculated that Th17 cells, which express chemokine receptors involved in mucosal homing (Ref. Reference Acosta-Rodriguez127), may play a role in the immune response to oropharyngeal candidiasis.

Cytokines as adjuncts to traditional antifungal therapies in the oral mucosa

Most cases of oropharyngeal candidiasis are treated with antifungals, such as azoles or nystatin; however, prolonged use of antifungals is associated with emergence of resistant strains (Ref. Reference Rogers130). Moreover, severely immunocompromised patients often relapse after antifungal treatment (Ref. Reference Rogers130), indicating the necessity for more effective long-term therapeutic approaches. Growth factors and cytokines are signalling molecules that regulate the differentiation, proliferation and function of immune cells. In this context, presence of these bioactive molecules in the infected tissues dictates the outcome of the host defence against microbial infection. Increased levels of GM-CSF, IFN-γ, TNF-α IL-8, IL-18 and IL-1α have been identified in the oral mucosa and saliva of subjects with oral candidiasis and in experimental models of oral candidiasis (Refs Reference Lilly63, Reference Schaller131, Reference Tardif132). Since antimicrobial effector functions of immune cells are highly inducible by these signalling molecules, these molecules are expected to play a central role in the protection against C. albicans. Few cytokine-based therapies have been investigated in both murine models and human cases of oropharyngeal candidiasis (Table 1).

Table 1.

Cytokines with potential therapeutic applications in oropharyngeal candidiasis

Abbreviations: G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte monocyte colony-stimulating factor; HIV, human immunodeficiency virus; IFN-γ, interferon gamma; IL-8, interleukin-8; OPC, oropharyngeal candidiasis; PMNL, polymorphonuclear leukocyte; ROS, reactive oxygen species; TNF-α, tumour necrosis factor alpha.

GM-CSF has been shown to accelerate haemopoiesis in the early phases of differentiation of myeloid cells, resulting in elevated production of monocytes, neutrophils and eosinophils. In addition to its ability to increase cell numbers, GM-CSF elicits the expression of chemotaxis receptors in PMNLs, stimulates chemotaxis, phagocytic activity and the release of ROS by PMNLs (Refs Reference Gadish85, Reference Ziege133), and prolongs their survival by delaying apoptosis (Ref. Reference Richardson, Brownlie and Shankland134). GM-CSF also stimulates TLR2 expression in PMNLs (Ref. Reference Kurt-Jones135) and Dectin-1 expression in macrophages (Ref. Reference Willment136), which can potentially contribute to the process of pathogen recognition. Furthermore, GM-CSF may increase antifungal resistance by inducing the expression of chitotriosidase by professional phagocytes – an enzyme that attacks chitin-containing fungal cell walls (Ref. Reference van Eijk137).

Owing to its potential ability to promote granulocyte and monocyte activation and influx into infected sites (Ref. Reference Richardson, Brownlie and Shankland134), the therapeutic use of GM-CSF has been investigated for oropharyngeal candidiasis. In one human pilot study, GM-CSF, in association with either fluconazole or amphotericin B, resulted in modest clinical and mycological improvements in patients with acquired immunodeficiency syndrome (AIDS) with oropharyngeal candidiasis that was resistant to antifungals (Refs Reference Vazquez, Gupta and Villanueva138, Reference Vazquez, Hidalgo and De Bono139). Although adjunctive GM-CSF therapy somewhat promoted resolution of oropharyngeal and esophageal lesions, patients relapsed within 2 weeks after discontinuation of GM-CSF (Refs Reference Vazquez, Gupta and Villanueva138, Reference Vazquez, Hidalgo and De Bono139), suggesting that an intact immune response is important for sustained fungal clearance and that fungal eradication in patients with advanced AIDS might be a difficult task even when those patients are treated with adjunctive immunoregulatory agents. Moreover, G-CSF in combination with fluconazole showed no additional benefit over fluconazole monotherapy in reducing fungal burden in a murine model of chronic orogastrointestinal candidiasis (Ref. Reference Clemons and Stevens140).

IFN-γ acts as a major regulator of the Th1/Th2 response, enhancing differentiation of Th1-cell-mediated immunity, which is considered to be protective against C. albicans infection (Ref. Reference Romani70). IFN-γ has been shown to stimulate the activation of several host effector cells, including macrophages, monocytes, natural killer cells and neutrophils (Refs Reference Gallin141, Reference Murray142, Reference Torrico143). Moreover, it induces the expression of defensin-3, TLR-2 and TLR-4 in epithelial cells (Refs Reference Uehara, Sugawara and Takada29, Reference Garcia109). As a key activator of Th1-cell-mediated immunity (Ref. Reference Romani70), IFN-γ represents a promising treatment for Candida infection. Subcutaneous administration of IFN-γ resulted in a drastic improvement of clinical signs and symptoms in a HIV-infected patient with azole-resistant oropharyngeal candidiasis (Ref. Reference Bodasing144). However, this cytokine failed to improve the efficacy of fluconazole in the treatment of oropharyngeal candidiasis in severe combined immunodeficiency (SCID) mice (Ref. Reference Clemons and Stevens140).

Finally, reduced levels of C. albicans were found on the tongues of infected mice treated by oral administration of TNF-α, supporting a protective role of TNF-α during oral candidiasis (Ref. Reference Ohta145). TNF-α is a proinflammatory cytokine required for the development of effective innate and adaptive immunity to fungal infection. First, TNF-α plays a key role in the process of transmigration of neutrophils into infected sites by inducing the expression of leukocyte and endothelial cell-adhesion molecules (Ref. Reference Gamble146). Second, this cytokine has been shown to increase the microbicidal activity of neutrophils via increased secretion of ROS (Refs Reference Diamond, Lyman and Wysong82, Reference Le and Vilcek83, Reference Tan84). Third, TNF-α induces secondary production of other cytokines, such as IFN-γ, IL-1, IL-6 and IL-12 (Ref. Reference Netea, Kullberg and Van der Meer94), which further amplify the protective immune response to C. albicans infection. Although data from in vitro and animal studies suggest that TNF-α plays a central role in host protection against C. albicans infection, the effects of TNF-α treatment on human cases of oropharyngeal candidiasis are not known.

Future approaches for antifungal discovery

Treatment of oropharyngeal candidiasis has relied strongly on the use of antimicrobial drugs. However, unsatisfactory results with drug monotherapy and emergence of resistant strains in extremely immunocompromised patients have prompted investigations on the use of adjunctive immunotherapies. The major promise of new adjunctive immunoregulatory therapies lies in their potential to generate and/or augment an appropriate host immune response that is required for control of the microorganisms. This would be particularly helpful in immunocompromised patients who suffer from persistent or recurrent episodes of oropharyngeal candidiasis.

A small spectrum of immunomodulators has been tested in animal models of oropharyngeal candidiasis. Clinical experience with immunoregulatory cytokines is limited, and controlled multicentre clinical trials will be necessary to validate the initial results from small studies or case reports. For example, although GM-CSF was shown to temporarily improve the immune response and positively influenced resolution of oropharyngeal candidiasis in a very small group of HIV-infected patients (Refs Reference Vazquez, Gupta and Villanueva138, Reference Vazquez, Hidalgo and De Bono139), larger-scale human clinical trials are still required to establish its therapeutic potential. Moreover, the role of other cytokines in the treatment or prevention of oropharyngeal candidiasis still needs to be assessed. Owing to its positive effects on the migration and activation of PMNLs (Refs Reference Baggiolini, Walz and Kunkel76, Reference Casale and Carolan77, Reference Schroder and Christophers86), IL-8 might represent a potential candidate cytokine for the treatment of chronic oropharyngeal candidiasis. Finally, because of the high potential for toxicity, data on the safety of the clinical use of cytokines administered locally or systemically, need to be fully disclosed before large-scale incorporation in therapeutic regimens. One of the advantages of treating fungal infection in the oral mucosa is the accessibility of this tissue to drug delivery, which allows local as opposed to systemic therapeutic interventions. Thus, as long as local delivery is accompanied by adequate substantivity, locally applied cytokines may find a therapeutic application in oropharyngeal candidiasis. Nevertheless, the design of an appropriate delivery system that keeps the immunomodulator in place for a sufficient period will be as important as the choice of the immunomodulator itself, for clinical success.

The identification of new PRR-signalling agonists that can amplify a protective inflammatory response to invasive fungal infection may also provide novel therapeutic targets for the treatment of oropharyngeal candidiasis in the immunocompromised host. For example, oral delivery and gastrointestinal absorption of soluble glucans has been shown to modulate the expression of PRRs, such as Dectin-1 and TLR2, and to increase systemic levels of IL-12, which in turn promotes protection against C. albicans infection (Ref. Reference Rice147).

To date, most TLR-signalling-based therapeutics that have been developed are low molecular mass inhibitors that suppress inflammatory cytokines in chronic inflammatory disorders, agonists used as adjuvants in vaccine development or agonists used as adjuvants in cancer chemotherapy (Refs Reference Verstak, Hertzog and Mansell148, Reference Garay149). The design of novel pathogen (ligand)-based or adaptor-molecule-based mimetics that can trigger the TLR-mediated protective immune response to pathogens is still in its infancy. TLR2 and TLR4 agonists show promise as molecules for treatment of fungal infection because they can trigger transitory increases in pro- and anti-inflammatory cytokines, as well as activating an array of antifungal functions of neutrophils and macrophages. The kinetics and array of cytokines triggered by TLR2 agonists versus TLR4 agonists differ; thus, the outcome of an inflammatory response to infection can be selectively manipulated using one or a combination of such agonists. For example, it is known that the transcription of several cytokine genes remains active for extended periods in cells stimulated via TLR2, but rapidly declines in cells stimulated by TLR4 (Ref. Reference Re and Strominger150). In addition, the release of the anti-inflammatory cytokine IL-10 is preferentially activated via TLR2 signalling (Ref. Reference Netea24). Therefore, by administering a TLR2-signalling agonist during the later stages of infection, it may be possible to limit host damage from a proinflammatory cytokine response triggered by a TLR4 agonist.

Several chemically pure TLR agonists that have been tested in cancer immunotherapy (Ref. Reference Garay149) could have potential applications as adjuvants in the treatment of drug-resistant or recurrent fungal infections. For example, the muramyl dipeptide-Lys18 (L18), which is a TLR2 agonist, restores PMNL function in immunosuppressed hosts (Ref. Reference Namba151), and given the importance of this cell type in oral infection they could provide an attractive therapeutic target. Another TLR agonist with potential application in fungal infections is a bacterial lipid-A derivative (OM-174), which stimulates both TLR2- and TLR4-mediated pathways. In addition to being well tolerated in human clinical trials, this TLR2/4 agonist triggers a biphasic cytokine response, with an early proinflammatory response followed by a delayed IL-10 response, which limits inflammatory toxicity and host damage (Ref. Reference Garay149). Other properties of this molecule, which may enhance its potential as an antifungal, are the ability to induce iNOS expression and the activation of professional phagocytic cells (Ref. Reference Onier152). It is expected that the field of immunoenhancement strategies will grow in this direction in future years, and might revolutionise the therapy of recurrent oropharyngeal candidiasis or other deep-seated fungal infections in immunocompromised hosts.

In addition to much-needed progress in investigations focusing on the therapeutic use of cytokines and PRR agonists in the treatment of candidiasis, new strategies for the development of effective vaccines in high-risk populations are also needed. Vaccines developed so far are based on C. albicans adhesins Als1p and Als3p. Experiments with Als1p and Als3p vaccines were first conducted in animal models of disseminated candidiasis (Refs Reference Spellberg153, Reference Spellberg154, Reference Ibrahim155, Reference Ibrahim156). Both Als1p and Als3p vaccines caused a marked improvement in the fungal burden and survival of both immunocompromised and immunocompetent mice infected with C. albicans (Refs Reference Spellberg153, Reference Spellberg154, Reference Ibrahim155, Reference Ibrahim156). Positive results in the systemic models of infection prompted the evaluation of the efficacy of Als1p and Als3p vaccines in murine models of oropharyngeal candidiasis. These vaccination strategies have shown promising results in mucosal infection, because they reduce both the tongue fungal burden and the severity of lesions (Refs Reference Spellberg154, Reference Ibrahim156). Although these vaccines increase Candida resistance in animal models of infection, their clinical success remains to be established in human trials.