Innate immune cell response upon Candida albicans infection

abstract

Candida albicans is a polymorphic fungus which is the predominant cause of superficial and deep tissue fungal infections. This microorganism has developed efficient strategies to invade the host and evade host defense systems. However, the host immune system will be prepared for defense against the microbe by recognition of receptors, activation of signal transduction pathways and cooperation of immune cells. As a consequence, C. albicans could either be eliminated by immune cells rapidly or disseminate hematogenously, leading to life-threatening systemic infections. The interplay between Candida albicans and the host is complex, requiring recognition of the invaded pathogens, activation of intricate pathways and collaboration of various immune cells. In this review, we will focus on the effects of innate immunity that emphasize the first line protection of host defense against invaded C. albicans including the basis of receptor-mediated recognition and the mechanisms of cell-mediated immunity.

Keywords:

• Candida albicans
• immune cells
• innate immunity
• receptor-mediated recognition
• signal transduction

Introduction

Candida albicans is an opportunistic pathogen that can not only live as a common benign commensal fungus in immunocompetent individuals but also cause mucocutaneous and systemic infections in immune compromised patients. The determinant of friendly colonization or invasive dissemination is the balance of fungal proliferation and host defense. The imbalanced interplay between the host and C. albicans can lead to mucosal and life-threatening deep-seated fungal infection, especially in immunocompromised individuals caused by cancer chemotherapy, human immunodeficiency virus (HIV) infection, organ transplantation or indwelling medical devices.¹ However, due to the limited effectiveness of existing therapies and increasing resistance of the pathogen to antifungal agents, new therapies are urgently needed to prevent high morbidity and mortality in candidemia. To overcome the increasing emergence of antifungal resistance, it is necessary to gain insights into mechanisms by which Candida species invade the host and the host responds to the invasion.

The innate immune system is typically the first-line defense of host defense encountered by invading pathogens.² The sophisticated pathogen invasion is initiated by impairing the host physical barrier that consists of the skin and mucosa. The morphological transition of the yeast to hyphal form has been considered as a contributing factor to the invasion of C. albicans.³ Breach of the mucosal barrier enables C. albicans to gain access to the underlying host tissues. To access distant tissues and organs, the organism must cross the host endothelium in order to reach the vasculature and disseminate in the blood.^4,5 Although C. albicans hyphae are recognized as a crucial virulence factor for both penetrating epithelium and piercing phagocytes, the yeast form of the fungus is believed to be required for dissemination during systemic infection.⁶

More specifically, host combat against fungal disease requires a series of complex molecular mechanisms involving the recognition of evolutionarily conservative fungal cell wall components, the activation of host immune cell signaling cascades, and the release of cytokines and chemokines.⁷ Being the core of the immune response, professional immune cells act as the most effective weapon for ingesting and killing the pathogens, though complement system and antimicrobial peptides are the other 2 components of innate immune defense mechanisms.⁸ In this review, we will concentrate on the innate immune protection during the process of infection. Therefore, it's useful to depict the recognition of C. albicans by the host, as well as the interactions between C. albicans and versatile host cell types. Understanding these mechanisms will be a great benefit to fungal infectionprevention and new treatment development.

Receptor-mediated recognition of Candida albicans

The interaction between C. albicans and the host immune system is initially achieved by detection of fungal cell wall components, predominantly carbohydrate polymers and proteins. According to the current knowledge, the C. albicans cell wall is a rigid structure consisting of an outer layer of mannoproteins, an inner layer of β-1,3- and β-1,6-glucans, as well as the innermost layer of chitin.^9,10 Pattern recognition receptors (PRRs) are responsible for recognizing these conserved microbial chemical signatures, also called pathogen-associated molecular patterns (PAMPs), which activate intracellular signaling pathways, elicit innate immune responses and help develop adaptive immunity. Groups of PRRs demonstrated to participate in sensing different pathogens include TLRs (Toll-like receptors),¹¹ CLRs (C-type lectin receptors),¹² NLRs (nucleotide-binding domain leucine-rich repeat-containing receptors),¹³ and RLRs (retinoic acid-inducible gene-I (RIG-I) receptors),¹⁴ among which TLRs and CLRs are known to play a central role in the antifungal immune response (Fig. 1). The NLRs and RLRs have not been proved to directly participate in fungal recognition.¹³

Figure 1. Recognition of C. albicans by pattern recognition receptors (PRRs). CLRs and TLRs are major membrane receptors for recognizing C. albicans. (A) Fungal recognition by dectin-1 can activate Syk, NFAT, ERK and NF-κB or trigger reactions through Raf-1 independent of Syk. The activation of dectin-1 results in the production of several inflammatory factors, including IL-2, IL-6, IL-10, TNF-α and COX2. (B) Dectin-1 collaborates with TLR2 in producing ROS and activating NLRP3 inflammasome that facilitates IL-1β maturation. (C) Dectin-2 and dectin-3 form a heterodimer to detect microbial infections and initiate Syk-mediated activation of NF-κB. FcRγ is recruited to help dectin-2 and mincle to activate intracellular signaling cascades. Dectin-3 is also demonstrated to associate with FcRγ. Both TLR2 and TLR4 are vital receptors for pro-inflammatory reactions mediated by MyD88 and Mal. (D) TLR2 binding enhances the amount of IL-10 and TGFβ (Tumor growth factor β) through the ERK-cFos pathway, which will cause immunesupression and the inhibition of pro-inflammatory signals. (E) TLR4 is efficient in mediating pro-inflammatory reactions, releasing IL-8, IL-12 and TNF. Besides individual effects, the combination of TLR1/TLR2 and TLR2/TLR6 will enhance the production of cytokines.

Accumulated evidence shows that TLR2 recognizes PLM (phospholipomannan) components,¹⁵ while TLR4 prefers short linear structures O-bound mannan and α-linked mannose structures.¹⁶ TLR9 is distinct from other TLRs because of its ability to detect intracellular DNA (CpG-oligodeoxynucleotides) of C. albicans.¹⁷ More recently, studies have focused on the synergies of different PRRs. TLR2 is reported to heterodimerize with 2 other family members, TLR1 or TLR6 and to recognize acylated lipoprotein as dimers.¹⁸ The recognition by TLRs is followed by the activation of different pathways, which contributes to the production of cytokines and chemokines. Both the TLR2 and TLR4 bindings contribute to the induction of pro-inflammatory signals in immune cells via MyD88 (myeloid differentiation factor 88), Mal-mediated pathways, and further NF-κB (nuclear factor κB) pathway.¹⁹ The CLRs are another major family of PRRs implicated in C. albicans recognition. The family includes dectin-1, dectin-2, dectin-3, Mincle (macrophage-inducible C-type lectin), MR (mannose receptor), and DC-SIGN (dendritic cell specific intercellular adhesion molecule-3-grabbing non-integrin). Dectin-1 is an important CLR that can recognize β-glucans in the fungal cell wall and initiate a series of cellular responses via the Syk/CARD9 and Raf-1, Syk-independent signaling pathways.^20,21 Owing to substantial differences in the composition and nature of fungal cell walls, dectin-1 has recently been surprisingly demonstrated to be strain-specific during infection in vivo.²² Dectin-2 can recognize α-mannan and high-mannose structures in hyphae.^23,24 However, both yeast and hyphal forms of C. albicans can be recognized by dectin-2.²⁵ Also, dectin-2 can form a heterodimeric PRR with dectin-3, a recently identified CLR, to sense fungal infection and show stronger binding to α-mannan, leading to potent inflammatory responses against fungal infections.²³ Dectin-2 signal transduction was demonstrated to interact with FcγR (Fcγ receptor) chain and activate Syk-CARD9-NF-κB signaling pathways to induce the production of cytokines such as TNF (tumor necrosis factor),^25,26 though the exact mechanism remains to be elucidated. Like dectin-2, mincle also recognizes α-mannose residues of C. albicans but it plays a significant role in the defense against yeast C. albicans.^27,28 Mincle is highly expressed in macrophages and contributes to innate inflammatory response by associating with FcγR chain and recruiting Syk.²⁹ Other CLRs, MR and DC-SIGN recognize N-linked mannan and specifically mediate C. albicans binding and internalization via phagocytes.^30-32 Although MR lacks signaling motifs, it performs endocytosis, mediates internalization of their ligands and also contributes to antigen presentation.³³ Galectin-3, an S-type lectin receptor, recognizes β -(1-2) oligomannan and efficiently helps defend against disseminated C. albicans in a mouse model.^19,34

NLRP1, a subset of NLRs, cytosolic receptors were able to assemble and oligomerize into a common structure which collectively activated the caspase-1 cascade, thereby leading to the production of pro-inflammatory cytokines especially IL-1β and IL-18.³⁵ This NLRP1 multi-molecular complex was referred to as the “inflammasome”.³⁶ NLRP subfamily members contain a central nucleotide-binding domain (NACHT), an N-terminal pyrin domain (PYD) and C-terminal leucine rich repeats (LRR) which function as ligand sensor.^13,36 Inflammasome formation is initiated through activation of a nod-like receptor protein (NLRP1, NLRP3, or NLRC4) and the recruitment of the adaptor molecule ASC (apoptosis-associated speck-like protein containing a CARD). This process is followed by the activation of caspase-1 and the cleavage of pro-IL-1β into active mature IL-1β.³⁷ NLRP3 and NLRC4 inflammasomes have been demonstrated to play an important role in defense against dissemination of mucosal infection and mortality in vivo.³⁸ NLRP10 has no effect on innate immune responses in disseminated C. albicans infection but influences the adaptive responses independent of the NLRP3 inflammasome and the production of IL-1β.¹³

Epithelial cells

The mucosal epithelium is commonly recognized as the first line of host defense after the initial contact with invading pathogens. The interaction between the epithelia and the microorganism either causes commensalism or violation of the superficial barrier on mucosal surfaces. The infection process of C. albicans consists of adhesion, invasion and cell damage. During the initial adherence of C. albicans to human epithelial surfaces, a great number of specialized adhesins are needed to build the attachment to the host, such as Hwp1p,^39-42 Eap1p,^43,44 Iff4p,^45,46 Ssa1p^47,48 and Als proteins mainly involving Als1-7p and Als9p.^39,49-52 Adhesion can only enable C. albicans to develop their virulence. In order to establish the infection and dissemination, deep and fast penetration into the epithelium is necessary.⁵³ C. albicans has been demonstrated to gain entry into host epithelial cells through 2 mechanisms: induced endocytosis and active penetration.^54,55 Endocytosis is mediated by adhesion by forcing epithelial cell actin to aggregate around the invading microorganism to produce pseudopods, which act like nets to catch the pathogen. Research has shown that, during fungal invasion, both Als1 and Als3 induce endocytosis via binding to E-cadherin on oral epithelial cells.^51,52 Active penetration is another way for C. albicans to invade oral epithelial cells. However, invasion into stomach and intestine has only been observed via active penetration. Collectively, invasion by C. albicans may depend on the epithelial cell type and the differentiation stage of epithelial cells, indicating the presence of different susceptibilities between various epithelial cells. Both adhesion and active penetration result in the final damage to epithelial cells. Recent studies have demonstrated that 3 C. albicans gene families, SAP (secreted aspartyl proteinase), PL (phospholipases) and LIP (lipases), play crucial roles in producing a number of extracellular hydrolytic enzymes contributing to the damage to host cells structures. SAPs have been most widely studied among all these hydrolases and the function of SAP5 has already been demonstrated to destroy epithelial cell junctions on oral epithelial cells.⁵⁶ Recently, SAP4-6 were discovered to bind integrins on epithelial cells via their RGD/KGD amino acid motifs.⁵⁷ In summation, C. albicans invades the host epithelial cells via the combined effect of contact-sensing, directed hyphal extension, active penetration and expression of pathogenicity factors to promote inter-epithelial invasion and dissemination, ultimately causing damage to host cells.

Given the contribution of Sap and Als proteins to fungal virulence, researchers have considered them as candidates for vaccine targets. As protein vaccines, Sap2p, Als1p and Als3p not only have defined amino acid sequences and structure, but also relatively safer compared with live attenuated fungal strains.^58,59 Sap2p, a common expressed Sap is proved to be effective against vulvovaginal candidiasis (VVC) in vivo and now has already gone through Phase I clinical trials against C. albicans infection.^60,61 Three Als vaccines, rAls1p-N (recombinant N-terminus of Als1p),⁶² rAls3p-N (recombinant N-terminus of Als3p)^63,64 and NDV-3 (recombinant N-terminus of Als3p formulated with alum adjuvant)^65,66 have been explored to prevent candidal infections. Compared with rAls1p-N, rAls3p-N vaccine showed equal efficacy against disseminated candidiasis but was more superior in treating oropharyngeal or vaginal candidiasis.⁶⁴ NDV-3, as a vaccine candidate against Candida, has proven effective against VVC through both humoral and adaptive immune responses and has already been tested in a Phase I clinical trial.⁶⁶ Thus, fully understanding the interaction between the pathogen and host does provide new ideas for the discovery of new drug targets and the invention of more antifungal therapies.

The recognition of fungal pathogens by epithelial cells depends on the interaction between PAMPs and PRRs. Up to now, only certain TLRs and CLRs expressed on epithelial cells have been demonstrated to detect C. albicans. The exact distribution and composition of PRRs on epithelial cells remain unclear.⁶⁷ Different groups of TLRs such as TLR2, TLR4, TLR6, and TLR9 are functionally expressed on the oral epithelium, highlighting the essential status of TLRs in oral defense.^68,69 TLR2 and TLR4 have been found to be elevated inside the inflamed gingivae.⁷⁰ TLR4 is found to be involved in the vaginal PMN recruitment response initiated by S100A8 alarmin.⁷¹ Also, human epithelial TLR4 has been shown to participate in defense against fungal infection in oral mucosa via a process mediated by (polymorphonuclear leukocytes) PMNs.⁷² As for CLRs, dectin-1 is the only receptor that shows some effects in the recognition of human gingival epithelia.⁶⁹ Ligation of epithelial PRRs by invading pathogens is usually followed by the activation of cascade signaling, and the subsequent production of pro-inflammatory, growth factors and antimicrobial factors. It was found that oral and vaginal epithelial cells activate NF-κB and the biphasic MAPK (mitogen-activated protein kinase) pathway in response to the stimulation caused by C. albicans.^73,74 Once a sufficient fungal burden and hypha-associated surface moieties are detected, the MAPK/MKP1/c-Fos pathway is activated, triggering release of pro-inflammatory cytokines. A new study has demonstrated that PI3K-Akt-mTOR signaling plays a key role in epithelial immunity for the protection against cell damage without the help of MAPK or NF-κB signaling.⁷⁵ However, the pro-inflammatory response in various epithelial cells is quite different for the production of cytokines. C. albicans strongly induces the production of cytokines such as G-CSF (granulocyte colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), TNF-α(tumor necrosis factor-α), IL-6(interlukin-6), IL-8, IL-1α and IL-1β in oral epithelia.^76-79 Vaginal epithelial cells have a low level of IL-6 production and almost no G-CSF or CCL20 (Chemokine (C-C Motif) Ligand 20).⁷⁴ IL-1β is an important cytokine associated with the activation of NLRP3 and NLRC4 inflammasomes, both of which have been demonstrated to protect against C. albicans infection in the oral cavity.^37,38

Additionally, upon recognition of C. albicans, epithelial cells are regulated to secret antimicrobial peptides, such as defensins, cathelicidins, and histatins to clear the invading pathogen directly.⁶ These host defense peptides have been recognized as important antimicrobial effectors in innate immune responses but may result in different respective activities if released into different local microbial environments.⁸⁰ It is well worth mentioning the protective effect of Th17 responses which mediate the production of IL-17 and IL-22. IL-17 and IL-22 both contribute to clear mucosal candidal infections by upregulating the expression the antimicrobial peptides by oral epithelial cells.^81,82 IL-17 has been shown to enhance the production of IL-8 and GM-CSF in oral epithelial infection and afterwards perform the recruitment of neutrophils.⁷⁶ As mentioned above, epithelium plays an important role in the inhibition of C. albicans dissemination by activating pathways to induce the production of cytokines that recruit immune cells.

Endothelial cells

In immune compromised hosts, C. albicans can disseminate hematogenously and migrate from circulation into the tissues to cause extensive organ damage and systemic candidiasis. Blood-borne C. albicans that fails to be eliminated by phagocytes and fungicidal factors must adhere to and penetrate into endothelial cells before they can disseminate in deep-seated organs. As the initiation of hematogenous infections, vascular endothelium acts as a barrier to prevent the pathogen dissemination. To get entry into the tissues successfully, C. albicans must conquer 2 main difficulties: adhering to endothelial cells and gaining access to endothelial layers. In the first step, C. albicans needs to find a way to adhere to the endothelium. There has been controversy over the necessity of morphogenetic change for the yeast form to adhere to the endothelial surface. Collectively, ample evidence indicates that morphogenetic transformation certainly occurs during the adhesion process, but no clear sign has proved it to be a prerequisite so far. Research has demonstrated that both yeast and the hyphal form of C. albicans can adhere under the condition of flowing pressure, which is similar to capillary blood pressure.⁸³ Although PRRs are predominantly involved in the recognition of correspondent cell wall structure during the host immune defense, they are also helpful in mediating the adhesion of C. albicans to endothelial cells of blood vessels. It has been demonstrated that TLR2 and TLR4 are expressed on endothelial cells.^84,85 MR, a mannan receptor, and galectins including galectin-1,-3 and -9 are also found on the endothelium. In addition, C. albicans uses different mediators to help the adhesion process. Recently, Gpm1 (Phosphoglyceratemutase 1), a candida surface protein, has been shown to mediate fungal adhesion to human endothelial cells by binding to vitronectin.⁸⁶ There are 2 other mediators (integrin α_vβ₃ and α_vβ₅-like adhesin) that use vitronectin as a surface ligand. Getting across the endothelial barrier is the second difficulty to overcome. C. albians transmigrates endothelial cell lines by inducing their own endocytosis with the help of the expressed invasins (Als3 and Ssa1) on their surface.⁵⁰ New research has found that N-cadherin expressed on the endothelium binds to C. albicans Als3 and Ssa1 in a complex that also contains an intracellular GTP-binding protein, septin-7.^87,88 This binding process induces endothelial cell microfilaments, thereby producing pseudopods that engulf the organism.

Polymorphonuclear neutrophils

PMNs (Polymorphonuclear neutrophils) are predominant phagocytes in host defense and are the first line of protection during engulfing the Candida pathogen in the innate cellular immune system.^89,90 TLR2, TLR4 and dectin-1 are all participating in the recognition of C. albicans and the highly expressed CR3 (complement receptor 3) and FcγR assist in the process of opsonization.⁹¹ Investigations have clearly shown that several pro-inflammatory cytokines facilitate the migration of PMNs to the site of infection, such as IL-6,⁹² IL-8⁹³ IL-17⁹⁴ and TNF-α.⁹⁵ Also, GM-CSF and G-CSF have been demonstrated to induce the recruitment of PMNs.⁹⁶ As effective phagocytes, PMNs internalize pathogens and clear them by producing ROS (Reactive oxygen species) and lysosomal enzymes.⁹⁷ New evidence shows that PMNs cannot only perform their role of phagocytosis but act as modulators during inflammation. PMNs can weaken pro-inflammatory response after C. albicans stimulation by releasing neutrophil-derived proteases responsible for degrading cytokines such as IL-1β and TNF-α.⁹⁸

As an indispensable defender for the host, neutrophils have both intra- and extracellular antifungal activities. The mechanism of killing the invading fungal pathogen by phagocytes is quite complex involving the production of reactive oxygen and nitrogen intermediates, the expression of antimicrobial peptides, the release of hydrolases, and nutrient limitation as well.² In addition to killing pathogens by phagocytosis, neutrophils can also catch invading pathogens in the extracellular space. In fact, C. albicans can induce neutrophils to release chromatin fibers, also known as NETs (neutrophil extracellular traps), which are capable of killing both yeast-form and hyphal cells⁹⁹ by releasing calprotectin, an antifungal peptide.⁹⁰ Although the comprehension about the function of NETs is growing, factors that orchestrate the formation of NETs remain unclear. Research has confirmed that as an important component of the secretory machinery of azurophilic granule, the Rab family small GTPase, Rab27 in neutrophils plays an important role in NET formation triggered by C. albicans through up-regulating the ROS production mediated by NADPH oxidase.¹⁰⁰

Monocytes and macrophages

Monocytes and macrophages are vital detectors of PAMPs. They participate in the process of defending the host against fungal infection and work together to summon neutrophils to the inflammatory site.¹⁰¹ Vice versa, the influx of neutrophils also has the effect on recruiting monocytes and modulating cytokine release of activated macrophage.¹⁰¹ After being recruited by neutrophils, monocytes differentiate into macrophages and continue to participate in immune responses. New evidence has shown that Candida stimuli induces the differentiation of macrophages from pro-inflammatory phenotype (M1, classically activated macrophages) into a more anti-inflammatory phenotype (M2, alternatively activated macrophages), and thus may enhance fungal survival and reduce the infection damage.¹⁰² As to receptors mediating the recognition of C. albicans, studies have shown high levels of TLRs on the surface of monocytes.¹⁰³ CLRs, such as dectin-1 and mincle, also play an important role in mediating monocytes recognition of C. albicans.²⁸ Recent studies have proposed that monocytes as well as natural killer cells (NK cells) could display innate immune memory, which challenges the dogma of only adaptive immunity being specific and having immunological memory. C. albicans and its cell wall β-glucans can train monocytes to develop an enhanced and lasting response through dectin-1 receptor/Raf-1 pathway, which induces the activation of signaling molecules such as p38.¹⁰⁴ The receptors mediating signaling modulation and epigenetic histone modifications lead to increased production of pro-inflammatory cytokines and antifungal effects.¹⁰⁵ In-depth research of the trained immunity may provide important evidence for vaccine design especially about the strength and duration of induced trained immunity.¹⁰⁶

Macrophages are effective in the host immune defense because they can not only control C. albicans burden but also recruit other immune cells to help clear the pathogens. Pathogen detection triggers several intracellular signaling pathways containing MAPK and NF-κB, which leads to the production of pro-inflammatory cytokines, essential to combat the aggressive pathogen. Dectin-1 has been shown to coordinate the antifungal response in macrophages. However, the protective functions are dependent on fungal morphology. The filamentous morphology is not recognized efficiently due to the lack of exposed β-glucan.¹⁰⁷ Recently, BTK (Bruton's Tyrosine Kinase) and Vav1, 2 new interactors localized to the Candida-containing phagocytic cup, are found to contribute to dectin-1-mediated phagocytosis in macrophages.¹⁰⁸ Although dectin-1 signals are sufficient to trigger phagocytosis, collaboration with TLR2 signals does help up-regulate NF-κB responses and cytokine production (IL-6, TNF-α). The recognition signal of dectin-1, transferred through its ITAM-like motif, usually results in the activation of Src and Syk, and the release of IL-2, IL-10 and IL-1β in macrophages.^109,110 The activated Syk signaling pathway is one of the prerequisites for the production of ROS due to its antifungal activity.^111,112 NFAT (Nuclear Factor of Activated T-cells), a regulator of T cell activation, is also modulated by the collaboration of dectin-1 and TLR or by dectin-1 independently.¹¹³ The activation of NFAT transcription factors in macrophages promotes both pro-inflammatory (IL-2, IL-12 and COX-2) and anti-inflammatory (IL-10) responses.¹⁰⁹ Also, the activation of NFAT in DCs produces a similar result. Mincle is highly expressed on macrophages and shown to mediate macrophage response to yeast C. albicans and induce the production of the inflammatory cytokine TNF-α.²⁸

Upon mediation by several opsonic and nonopsonic receptors on macrophages, phagocytosis of C. albicans occurs. After ingestion, the activation of macrophages will cause the production of antimicrobial effectors, including ROS (reactive oxygen species) and RNS (reactive nitrogen species). Although some C. albicans can be easily killed, many still survive. They have successfully developed escape mechanisms. To cope with these oxidative anti-fungal mechanisms, most pathogens including C. albicans can encode SODs (superoxide dismutases) to detoxify excessive ROS species.¹¹⁴ Some pathogens actively suppress the generation of toxic compounds to protect themselves. New research has revealed that C. albicans can actively block NO (nitric oxide) production of macrophage via a secreted mediator that functions as the inhibitor of iNOS (inducible nitric oxide synthase).¹¹⁵ Related studies have shown that in addition to physically bursting out of macrophages, the hyphae of C. albicans can also initiate pyroptotic cell death in macrophages as an additional means of escape following phagocytosis.^116,117 In the presence of C. albicans, PRRs on the macrophage, such as TLR2, dectin-1 and dectin-2, activate the transcription and translation of NLRP3 inflammasome, pro-IL-1β as well as pro-IL-18.¹¹⁰ The activation of cysteine protease caspase-1, mediated by NLRP3, contributes to cleave pro-IL-1β and pro-IL-18 into their bioactive forms. This process is beneficial to host survival from systemic candidiasis, because the release of IL-1β and IL-18 is important for the recruitment of additional phagocytes and the initiation of Th1 and Th17 adaptive immune responses in the prevention of candidiasis dissemination.^110,118 Knowing that PMNs and macrophages are both professional phagocytes for clearing invading pathogens, a question about which one is more effective arises. Co-culture of PMNs and macrophages showed that macrophages had higher ability of engulfing hyphae while PMNs played a predominant role in clearing yeast form C. albicans.⁸⁹ But when they were incubated separately, PMNs showed lower overall phagocytic capacity but higher susceptibility as compared with macrophages.⁸⁹ Taken together, it is hard to judge which phagocyte is more efficient for the lack of researches especially in vivo. New evidence also reveals that human macrophages may take up apoptotic neutrophils, so that they can own the antimicrobial peptides or acquire neutrophil granules to promote their antimicrobial activity.¹¹⁹

Dendritic cells

It is essential to have a deeper comprehension about the interaction between DCs and invading pathogenic fungi, for DCs maturation is believed to be the link of innate and adaptive immunity. DCs are known to play a central role in a series of processes, such as detecting fungal pathogens in the surroundings through PRRs localized on their surfaces, secreting cytokines, engulfing pathogen particles by phagocytosis, and finally inducing adaptive immune reaction via the presentation of antigens to T cells.¹²⁰ The immune response is equally initiated with the recognition of PAMPs by PRRs expressed on DCs surface or inside them. Because of the great role that they play during the process of antigen presentation, DCs have most of PRRs for the recognition of C. albicans, such as TLRs, CLRs, and FcγR.¹²¹ Among them, MR and DC-SIGN, as 2 subgroups of CLRs, are vitally important in mediating the recognition and internalization of C. albicans by human DCs.¹²² Research also found that the recognition of C. albians by DC-SIGN was a time and concentration-dependent process.¹²³ C. albicans enters DCs via DC-SIGN but not via dectin-1, which results in inhibition of the NADPH oxidative and ROS production.¹²⁴ In contrast to DC-SIGN, dectin-1 plays a role in mediating NADPH oxidase activation which leads to Candida-killing activity of DCs. Dectin-1 can directly activate NF-κB in dendritic cells via the signaling adaptor molecule CARD9 and DCs maturation.¹²⁵ The dectin-1 and TLRs of dendritic cells have a synergistic effect on mediating the production of cytokines such as IL-12 and TNF-α¹²⁶. Finally, after being internalized by DCs, the antigen is delivered into endosomes and lysosomes, and is finally presented to T cells.

Natural killer cells

NK cells are also essential modulators of the innate immune system and have influence on adaptive immune responses. NK cells have been demonstrated as lymphocytes in the innate immune system. They exhibit not only cytotoxicity against viral infection and bacteria invasion but also anti-tumor effects.¹²⁷ Studies have shown the inhibitory effect of NK cells on a variety of fungi including Cryptococcus neoformans,¹²⁸ Candida albicans,¹²⁹ Aspergillus fumigatus¹³⁰ and Rhizopusoryzae.¹³¹ More progress has been made in understanding the function of NK cells in candidiasis. NK cells have been demonstrated to be important in immunosupressed hosts against C. albicans, and to be the cause of hyperinflammation in immunocompetent hosts.¹³² As for the specific action, studies have reported that NK cells in both humans and mice have fungicidal activity against C. albicans and other pathogens mainly through 2 different ways. In a direct and efficient way, NK cells exert antifungal activity through secreting granule content such as perforin. Also, NK cells contribute to the immune response indirectly via the generation of corresponding cytokines (GM-CSF, IFN-γ and TNF-α), which may recruit and activate specialized immune cells such as PMNs, dendritic cells, macrophages and other T cells.¹³³ Conversely, they are also regulated by the cytokines produced by other immune cells, thus forming an interactive process.

The interplay between NK cells and fungus similarly begins with the detection of PAMPs by the receptors on the host cells which may moderate the function and intrinsic stability of NK cells. NK cells have a diverse array of receptors to recognize pathogens and balance the signal transduction. These receptors, include NCRs (natural cytotoxicity receptors), KIRs (killer-cell-Ig-like-receptors), and TLRs.^134,135 Among so many receptors identified, NKp30 receptor, one of the NCRs family members, has been demonstrated to be vital in the recognition of fungal pathogens and antifungal cytotoxicity.^135,136 While, other NK cell receptors mentioned above are mainly involved in killing tumor and virus-infected cells. Although NKp30 is considered to mediate fungal killing, there is no clear evidence regarding the identity of the associated ligands. What is known is that NK cells can be activated via the PI3K/AKT and ERK pathways during fungal infection, leading to the formation of a microbial synapse between the pathogen and NK cell.¹³⁷ It is plausible that the synapse leads to a polarization of the receptors on NK cells, followed by degranulation, the release of perforin, granzyme, granulysin or other effector molecules.^129,136,138 Perforin has been proved to play an important role in direct antifungal process.¹²⁹ Perforin displays its cytotoxicity by perforating the cell membrane and finally inducing lysis of the target cell.¹³⁹ High levels of Granzyme B have been similarly observed in NK cells invaded by C. albicans. Moreover, human NK cells activated by C. albicans can secret GM-CSF, IFN-γ, and TNF-α.^140,141 However, previous studies showed that the production of IFN-γ by murine NK cells was down-regulated by C. albicans.¹⁴² IFN-γ is generally recognized as an important modulator for promoting candidacidal activity of PMNs and macrophages.^143,144 TNF-α and GM-CSF have effects on potentiating antifungal activity of PMNs.¹⁴⁵ Paradoxically, new research results have shown that the activation of NK cells, specifically their contact with pathogen, is inhibited in the presence of PMNs. Meanwhile, the presence of activated NK cells can activate more PMNs and increase PMNs anti-fungal activity by increasing the production of ROS and delaying the apoptosis of PMNs.^146,147 This interplay between NKs and PMNs could be a mechanism to prevent the overreaction of the immune system and the damage of surrounding tissues caused by the overreaction. DCs can also act on NK cells by producing inflammatory mediators, resulting in NK cells releasing another mediator, GM-CSF. Vice versa, DC maturation is largely attributed to IFN-γ and TNF released by activated NK cells.¹⁴⁸ Additionally, NK cells can be activated by soluble factors such as IL-12, IL-15, IL-18 and type I IFNs produced by activated macrophages and dendritic cells. Thus, DCs, macrophages and PMNs in turn form the basis of NK cell immunity initiation.¹⁴⁹ The reciprocal impact of immune cells and cytokines and chemokines helps to highlight the interaction between different immune cells.

Conclusion

During the past decades, significant progress has been made in our understanding of the interplay between C. albicans and the host immune system. The interplay mechanisms include how the host innate immune system recognizes, responds to and clears the invading pathogen. Additionally, fungal pathogens have developed virulence factors to invade the host and contribute to pathogenesis. Moreover, virulence factors have become targets for developing Candida vaccines against disseminated candidiasis.

The recognition between the PRRs and fungal PAMPs is the fundamental step to initiate an immune response. Although lots of PRRs and fungal PAMPs have been identified, new evidence of unexplored interaction between them is still anxiously expected for its importance in new drug invention. As the defender of the host, the innate immune system is a huge complex network equipped with different kinds of immune cells. Epithelial and endothelial cells are not classical immune cells. But they are important barriers for infection agents and have indispensable effects on directing antifungal responses. Professional phagocytes are definitely main components in host defense responses. Moreover, the interaction and cooperation among different phagocytes provide effective protection against disseminated C. albicans (Fig. 2). Since, traditional antifungal therapies are confronted with the emergence of resistance and the rise of toxic side effects, new therapies, such as vaccines are urgently needed. However, as a potential fungal treatment, vaccines still have problems in several aspects and require further clinical validation. In summary, all the perspective antifungal strategies will be based on a better understanding of innate immune cell responses to fungi.

Figure 2. Innate cell response network against C. albicans infection. C. albicans interacts with epithelia by the process: adherence, invasion and cell damage. (A) To clear the invading pathogen, epithelial cells secret antimicrobial agents as the first defending step. In healthy circumstances, PMNs are strictly regulated and controlled by chemokines. (B) Upon Candida infection, the release of several chemoattractants and cytokines recruits PMNs to the site of infection. (C) Immune cells that receive the recruitment signals traverse the endothelial cells to participate in immune defense. (D) The activated PMNs clear the invading pathogen by 2 ways: phagocytosis by forming neutrophil extracellular traps (NETs). (E) They are indispensable for recruiting monocytes and facilitating their differentiation into macrophages. (F) Macrophages and DCs both contribute to the activation of NK cells. (G) Afterwards, the active NK cells can reinforce the immunological function of PMNs through the release of TNF-α, TNF-γ and GM-CSF.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by National Key Basic Research Program of China (No 2013CB531602), National Science Foundation of China (No 81173100, 81273556), Shanghai Science and Technology Major Project (11JC1415400).