Several constitutively secreted proteins and peptides line the lung inner surface and many of them have shown to exert direct fungicidal activity in vitro. One of the most abundant proteins with fungicidal activity is lysozyme, a small protein (approximately 15 kDa) produced by epithelial cells and alveolar macrophages in the human lung [10 μg/ml in bronchoalveolar lavage fluid (BALF)] (Dubin et al., 2004). Lysozyme is one of the first characterized antimicrobial proteins, discovered by Alexander Fleming in 1922. Its antimicrobial activity involves two distinct mechanisms (Düring et al., 1999). First, the enzymatic cleavage of the N-glycosidic bonds linking polysaccharides and proteins in the bacterial cell wall and chitodextrins in fungal cell walls. Second, the permeabilization of the plasma membrane, due to its cationic properties, which resembles host defense peptide activity, as described below (Marquis et al., 1982; Ibrahim et al., 1994, 1997; Nishiyama et al., 2001). Fungistatic effects have also been attributed to lysozyme, even at low concentrations (approximately 1 μg/ml for H. capsulatum) (Newman et al., 2000). These include: impairment of yeast budding by C. albicans and P. brasiliensis (Nishiyama et al., 2001; Lopera et al., 2008), decreased production of virulence factors such as aspartyl proteinases (SAP) by C. albicans (Wu et al., 1999) and hyphal disruption of A. fumigatus (Diamond et al., 1978).

Antileukoprotease (ALP) is a major serine protease inhibitor secreted by Clara and goblet cells at the bronchial epithelium, and by serous cells and submucosal glands in the bronchi (De Water et al., 1986). This 12 kDa cationic non-glycosylated protein exhibits antifungal activity in vitro against C. albicans and A. fumigatus at concentrations comparable to that of lysozyme (Tomee et al., 1997). It remains unclear whether the antifungal mechanism of action is similar to its antibacterial mechanism, which resides mainly in the N-terminal region of ALP (Miller et al., 1989). Its proteinase inhibitory activity, however, is associated with the C-terminal region, which in itself does not have antibacterial activity (Hiemstra et al., 1996; Tomee et al., 1997). Recently, a study by Curvelo et al. (2014) has shown that ALP can also affect C. albicans cell membrane stability by inducing several structural changes, most notably irregularities along the cytoplasmic membrane. This finding points toward a mechanism similar to the one displayed by other cationic peptides, such as defensins, known for their ability to permeabilize membranes (see below). In vitro, inhibition of C. albicans and A. fumigatus proteases by ALP decreases fungal adhesion to MA104 epithelial cells from monkey kidneys (Tomee et al., 1997; Curvelo et al., 2014).

Host defense peptides (HDPs) are characterized by their high cationic charge, small size (5–50 amino acids) and amphipathicity. There are two major groups of cationic HDPs secreted into the lung lining: defensins and cathelicidins. Defensins are characterized by three conserved disulfide linkages, which induce a characteristic fold containing a high percentage of β-sheets. A structural subdivision is made between α- and β-defensins based on the location of the cysteine linkages. Four human β-defensins (hBD-1, -2, -3, and -4) are produced by the lung epithelium, either constitutively (hBD-1) or induced upon infection (hBD-2, -3, and -4) (Yanagi et al., 2005; Doss et al., 2010).

The mechanism of action of hBDs has been studied in bacteria, where interaction with cell membranes seems to be an important requirement for their antibacterial activity. However, other effects such as inhibition of DNA, RNA, and protein biosynthesis are also observed and might significantly contribute to growth inhibition and killing (Sahl et al., 2005). hBD-1, hBD-2, and hBD-3 are also known to have antifungal activity but these mechanisms are not well understood (Vylkova et al., 2007). For hBD-1 and hBD-2, the activity against C. albicans is dependent on the energy status of the fungal cells, reflecting a requirement for energy-dependent uptake of peptide, but for hBD-3 energy-independent mechanisms are also observed (Vylkova et al., 2006; Krishnakumari et al., 2009). Similar to what is seen for bacteria, all three defensins permeabilize the fungal cell membrane, indicating that membrane destabilization plays an important role in fungal killing (Krishnakumari et al., 2009). Histatin 5, a host defense peptide present in the oral cavity but not in the lungs, binds to specific receptors on the fungal cell membrane, ultimately leading to cell permeabilization. In line with this, Vylkova et al. (2006) have shown that hDB-2 and hBD-3 require Ssa1/-2 surface proteins to kill C. albicans, and it seems likely that other peptides or proteins use membrane bound receptors as well.

The mechanism of action of defensins against fungi other than C. albicans has been less studied. Contact with metabolically active spores of A. fumigatus, for example, increases transcription and secretion of hBD-2 and the recently described hBD-9 by human bronchial epithelial cells. This indicates that several antimicrobial peptides might be released into the lung lining upon fungal infection (Alekseeva et al., 2009; Evans et al., 2010). Levels of α-defensins 1-3 in whole blood seem to increase after exposure to Candida spp. yeast cells (Gácser et al., 2014). However, to our current knowledge, no mechanistic data of α-defensins against fungi are available.

Cathelicidins constitute a structurally diverse family of host defense peptides characterized by sharing a similar (cathelin-like) prosequence. In the lung, the only human cathelicidin, hCAP18 is processed by proteases such as elastase, cathepsin G, and proteinase 3 (Sørensen et al., 2001). The processed product is the active peptide LL-37. This peptide is secreted by alveolar macrophages, neutrophils, bronchial glands and by the epithelium of the lung lining (Bals et al., 1998). In a healthy lung, LL-37 can be found at the lung lining at measurable concentrations (Agerberth et al., 1999). In vitro antifungal activity of LL-37 has been described for C. albicans (den Hertog et al., 2005; López-García et al., 2005; Krishnakumari et al., 2009; Ordonez et al., 2014). Its activity results mainly from permeabilization of the cytoplasmic membrane, although effects on internal organelle membranes have also been described (Ordonez et al., 2014). The only instance in the literature where the role of LL-37 in fungal infections was mentioned is in a study of patients with chronic rhinosinusitis (Lee et al., 2010). In these patients LL-37 levels increased after contact with A. fumigatus.

In addition to the described molecules, several other components present in the lung have antifungal activity. These include mainly peptides present in neutrophils that are released upon degranulation, including human α-defensins, histones, Cathepsin G, bactericidal/permeability-increasing protein (BPI), and azurocidin (Newman et al., 2000; Cederlund et al., 2010). No detailed mechanistic antifungal studies have been performed on these molecules but they possess activity against fungi in vitro, demonstrating their potential involvement in lung defense against fungal pathogens. An overview of antifungal proteins and peptides is provided in Table 1.

Until now immunomodulatory effects of host defense molecules have been strongly underestimated. In the fight against bacterial infections, immune modulation by host defense molecules plays an important role and it is likely that this also accounts for fungal clearance. A broad range of immunomodulatory functions has been described for several of the innate defense molecules in the airway surface liquid (ASL).

Host defense molecules could prepare immune cells for encounters with fungi in the healthy lung. A study by Scott et al. (2002) shows that LL-37 may contribute to the immune response against bacteria by limiting host cell damage and increasing phagocyte recruitment to infection sites. During infection, an increase in secretion of several defense molecules is observed. Human lactoferrin, for example, triggers the transcription of host defense peptides by bovine tracheal epithelial cells (Velliyagounder et al., 2003). Host defense molecules can increase protection in areas near the infection and additionally attract phagocytic cells. For instance, LL-37 secreted by epithelial cells is known to act as a chemoattractant for neutrophil recruitment (De et al., 2000). LL-37 produced by PMNs has also been suggested to activate the epithelium resulting in IL-8 release (Tjabringa et al., 2003). An overview of the activities of HDPs is given in Figure 2.

Collectins can induce macrophage actin changes (Veillette et al., 1998; Gardai et al., 2003), integrin induction (Senft et al., 2007), and increase cellular receptors on phagocytes (Kuroki et al., 2007). These collectin-induced modifications might be essential for proper phagocyte activation. In fact, macrophages alone are not able to clear A. fumigatus in an in vitro cell-based infection model. In vitro, SP-A downregulates the production of pro-inflammatory cytokines by alveolar macrophages evoked by C. albicans (Rosseau et al., 1999). Additionally, SP-A interacts with macrophages affecting their response to LPS, and decreases macrophage TNFα production by interaction with TLR2 (Fisher et al., 2002; Murakami et al., 2002).

Cell receptors and signaling pathways activated by collectins have been studied mainly in macrophages, but they are also known to trigger lymphocyte proliferation and leukocyte recruitment (Madan et al., 1997; Crouch, 1998; Fisher et al., 2002). Several cell receptors have been found to be responsible for SP-A- and SP-D-induced signaling in inflammation. Gardai et al. (2003) showed that the CRD domain of both collectins bind to the inhibitory signal regulatory protein α (SIRPα) at the macrophage surface, while the collagenous domain binds to calreticulin/CD91. Binding to SIRPα blocks pro-inflammatory responses and decreases phagocytosis of apoptotic cells, while binding to calreticulin/CD91 increases pro-inflammatory responses, chemotaxis, and macrophage phagocytosis. Additionally, another pathway for the immunomodulatory function of SP-D has been recently described by Olde Nordkamp et al. (2014), where the collagenous domain of SP-D was found to bind the Leukocyte-Associated Ig-like Receptor 1 (LAIR-1) at the neutrophil surface, inhibiting the production of FcαR-mediated reactive oxygen. These examples show the complexity of the immunomodulatory role of these soluble lung effector molecules, with sometimes seemingly opposing (pro- and anti-inflammatory) effects. It is even likely that the described examples only constitute a fraction of all immunomodulatory routes, which emphasizes again that a better understanding of the interaction between all components is required to predict the outcome of an immune response.

In general, the ASL of a healthy lung is known to have a neutral pH of 6.94 ± 0.03 (Johansson et al., 1998; Nakayama et al., 2002; Olde Nordkamp et al., 2014) and a decrease in pH affects the ability of the ASL to eliminate pathogens (Pezzulo et al., 2012), likely due to a reduced activity of several defense molecules (Berkebile and McCray, 2014). This can happen during infection at specific locations inside the lung, or during certain diseases such as cystic fibrosis. From what is described for bacteria, a low pH may reduce the activity of LL-37 by perturbing its α-helical conformation and interaction with membranes (Johansson et al., 1998). Low pH also decreases HBD-1 secretion by epithelial cells (Nakayama et al., 2002), phagocyte chemotaxis, and ROS production. However, specific effects of pH with respect to their antifungal activities have not been described yet (Lardner, 2001; Ng et al., 2004). On the other hand, the Ca²⁺-binding properties of SP-D and SP-A are mostly retained at pH values as low as 5.0 (Haagsman et al., 1987; Crouch, 1998). It is not known, however, whether pH changes may affect phagocyte interactions with these collectins (Rosseau et al., 1999). Whether such a low pH could exist locally in a healthy lung is debatable, but phagocytic responses might generate acidic microenvironments that could reach low pH values.

The activity of most HDPs is based on their high cationic charge and the ASL ionic concentration can therefore strongly affect their activities. The major component contributing to the ionic strength of the ASL is NaCl, present in a concentration of approximately 100 mM at the ASL of the healthy lung (Jayaraman et al., 2001). In vitro, such salt concentrations significantly diminish the direct antifungal activity of several host defense peptides including defensins and LL-37 (Vylkova et al., 2007; Krishnakumari et al., 2009; Coorens et al., 2017), lysozyme and ALP (Tomee et al., 1997), although the antifungal activity of HBD-3 appears less sensitive as compared to that of other defensins (Turner et al., 1998; Krishnakumari et al., 2009). The high amount of antimicrobials needed to exert adequate antifungal killing at physiological ionic concentrations does not correspond with the concentrations normally found at the ASL. However, there is evidence that at lower than minimum inhibitory concentrations, innate effector molecules can still affect fungal growth. Lysozyme, for example, can still decrease C. albicans growth, morphology transition, and protein production at inflammatory concentrations around 400–800 μg/ml (Lopera et al., 2008). Additionally, lysozyme inhibits C. albicans virulence-related proteins at sub-inhibitory concentrations (10 μg/ml) (Wu et al., 1999).

The collectins SP-A and SP-D are also affected by ionic concentrations but in opposite ways. While purified SP-A precipitates at physiological salt concentrations, SP-D requires physiological salt concentrations to maintain its structure and functionality. This could correlate with their localization in vivo. Inside the lung, SP-A is mainly forming a complex with surfactant lipids whereas most of SP-D locates in the aqueous salt-containing sub-phase (Crouch, 1998). The localization of these two collectins could make the difference for their availability to bind fungal cells and how they modulate presentation to phagocytes. SP-D has shown to be able to bind fungal spores in BALF (Yang et al., 2000). Nevertheless, a fraction of SP-A produced by Clara cells and submucosal glands at the conductive airway may not be bound to lipids (as is the case for SP-A at the alveoli) and therefore could bind to inhaled microorganisms (Khoor et al., 1993). It is unclear though, to what extend SP-A might affect macrophage activation.

The presence of divalent cations at the lung lining, such as Mg²⁺, Fe²⁺, and Ca²⁺, strongly influences ASL antifungal activity. In vitro concentrations of ∼5 μM Ca²⁺ or ∼25 μM Mg²⁺ are sufficient to abrogate the candidacidal activity of defensins, especially HBD-1 and HBD-2 (Krishnakumari et al., 2009). The antifungal activity of LL-37 is also decreased by Ca²⁺ at concentrations as low as 1 μM (Ordonez et al., unpublished data). This is similar to what has been observed for its antibacterial activity where the minimum inhibitory concentration of LL-37 for Escherichia coli increased 10-fold in the presence of 1 mM CaCl₂ (Turner et al., 1998). There are also several chelators present at the ASL that sequester divalent cations as a defense against pathogenic organisms. Lactoferrin, for example, is a monomeric multifunctional glycoprotein of 80 kDa secreted by lung epithelium and submucosal glands into the ASL. There, lactoferrin is found in high concentrations (Dubin et al., 2004; Viejo-Díaz et al., 2004). This protein is best known for its ability to trap iron ions. According to Ding et al. (2014), iron ions are essential for fungal virulence. In line with these observations, Soukka et al. (1992) described candidacidal effects of lactoferrin that were dependent on iron ion sequestering since Fe³⁺-saturated lactoferrin completely lost its ability to kill C. albicans. Transferrin, a siderophore present in the deep lung, is also known to limit iron availability, thus inhibiting microbial growth. Although this siderophore will chelate ions, several pathogenic bacteria such as P. aeruginosa are known for their ability to use them as a source of ions (Cornelis and Dingemans, 2013). Interestingly C. neoformans is also able to use transferrin as a source of iron (Saikia et al., 2014).

Mucus is a complex structure mainly composed of highly glycosylated proteins called mucins. This structure covers the epithelial surface throughout the conductive airways, thereby serving as a barrier against inhaled microorganisms. The regulation of mucus secretion by epithelial cells is tightly linked to pH and ionic concentrations at the ASL. To date, 20 genes coding for mucosal proteins have been described (Rose and Voynow, 2006). A total of 14 of these mucins are expressed in the lung as demonstrated by RNA expression, with MUC5AC and MUC5B being the most abundant components of the respiratory mucus (Hovenberg et al., 1996). Interaction of mucus with cationic peptides is likely to occur since mucins are negatively charged. It is known that LL-37 and HBD-2 bind mucins in the gastrointestinal tract (Felgentreff et al., 2006; Cobo et al., 2015). Mucins are highly glycosylated, and this enables their interaction with lectins, such as SP-D. Although SP-D is produced mainly in the deep lung, its localization in the aqueous phase might enable it to travel throughout the conductive tract. Nevertheless, low expression of this protein was also observed at the level of the trachea (Madsen et al., 2000; Herias et al., 2007). Whether SP-D interactions with mucus at the conductive airway interfere with SP-D binding to pathogens remains to be studied.