Surfactant and its role in the pathobiology of pulmonary infection

1.1. Surfactant and its role in lung homeostasis

It has been established that a primary role of surfactant is to lower surface tension in the alveoli thereby stabilizing these structures to prevent alveolar collapse [3–12]; in this manner, surfactant reduces the work associated with breathing [1, 3, 7]. This surface material also serves as a barrier to pathogens [7], improves mucociliary transport [8], helps limit evolution of high-surface-tension pulmonary edema [8], and inhibits leakage of serum components into the airway [3]. The biological role of surfactant, however, as an integral component of the host defense system that exerts immunomodulatory activity has now emerged as an intriguing focus of several investigations using animal models of pulmonary infection and consideration in surfactant replacement therapies [6, 13–15]. Taken together, the demonstration of key physiological and biological activities of this material has kept investigations of surfactant as a major focus in both basic as well as translational studies.

1.2. Surfactant composition, synthesis, and secretion

Pulmonary surfactant is a complex mixture of lipids and proteins that forms a monomolecular film that lines the alveolar air-surface interface [4–6]. Surfactant is comprised of roughly 90% lipids; 80–85% of which are phospholipids, 5–10% are neutral lipids [16–20], and 10% are proteins (Figure 1) [4, 7, 8]. Phosphatidylcholine (PC) is the most predominant phospholipid in surfactant, accounting for ~75–80% of total phospholipid weight; this fraction of lipid is highly conserved across mammalian species [8, 11, 16, 18]. The major surface-active species of PC, termed dipalmitoylphosphatidylcholine (DPPC), is a saturated phospholipid comprising ~ 50% of PC that largely provides for the surface-tension lowering ability of this material. Phosphatidylglycerol (PG) and phosphatidylinositol (PI) account for 8–15% of the phospholipid portion [8, 11, 18]. Other minor phospholipids and neutral lipids are also detectable [8, 19, 21]. Although proteins make up a small fraction of surfactant, the four surfactant-associated proteins, surfactant protein (SP)-A, SP-B, SP-C, and SP-D [2, 6], all perform important roles in regulating surfactant function.

Surfactant is synthesized within the alveolar type II epithelial cells [6–9, 22]. After its biosynthesis, the surfactant components are packaged within lamellar bodies prior to secretion into the alveolar surface via calcium-dependent exocytosis (Figure 2) [21–23]. β-adrenergic agents, and changes in ventilatory pattern are physiological triggers for surfactant secretion from alveolar epithelia, in addition to several biochemical mediators [24]. Once secreted, the components of lamellar bodies transform into tubular myelin [21], a lattice-like structure that is believed to be the precursor to the surfactant monolayer that forms and spreads over the air-liquid interface [4, 6, 22]. The surfactant film is formed by the rapid adsorption of the tubular myelin on the alveolar surface [4, 11, 21]. The film compresses, thus facilitating reduction in surface tension and is also purified during the breathing cycle as certain protein components are squeezed out of the film [21]. As surfactant is actively being secreted, materials are constantly being exchanged from the film and are recycled into the type II epithelial cells to maintain constant surfactant pool size [4, 11, 21]. Thus, many recycled alveolar surfactant components traffic back into newly formed lamellar bodies (Figure 2)[21].

1.3. Lipids in Surfactant Behavior

The lipid portion of surfactant is primarily responsible for the surface activity of the film [4, 7, 19], though it is not the sole component contributing to this function. The film is enriched in DPPC [11], the main component conferring reduced surface activity although other saturated molecular species of PC also may exert similar properties [25]; DPPC becomes a highly packed monomolecular film with a squeeze out of other unsaturated lipids during compression phases of the respiratory cycle to achieve the low surface tensions needed to protect alveoli from atelectasis during end-expiration [7, 19]. Other phospholipids including the anionic phospholipids, PG and PI, are believed to enhance adsorption into the film [11]. These phospholipids may also interact with the hydrophobic surfactant proteins, SP-B and SP-C, to stabilize the film [6, 8]. Optimal surface tension-reducing capabilities are observed when these components in surfactant act together, specifically the phospholipids, neutral lipids, SP-B, and SP-C [1]. Cholesterol is the predominant surfactant neutral lipid and addition of cholesterol and the other neutral lipids into DPPC or DPPC-PG films increases adsorption rates [8, 11, 17]. This process could result in increased fluidity of the film and regulate its re-distribution [7, 11, 17, 18]. Improper amounts of surfactant cholesterol may also disrupt surface tension lowering ability [8, 17, 18].

1.4. Surfactant Proteins

Surfactant contains four associated proteins, SP-A, SP-B, SP-C, and SP-D, all of which have been well studied and contribute importantly to surfactant behavior. SP-A and SP-D are larger, hydrophilic glycoproteins [4, 8, 16, 23] that play a significant role in the host defense system [1, 2, 6, 22]. SP-B and SP-C on the other hand are much smaller polypeptides, highly hydrophobic, and often co-isolate with lipids [4, 8, 16, 18]. These hydrophobic proteins play key roles in alveolar stability to assist in reducing surface tension thereby preventing alveolar collapse [1, 26], aiding in phospholipid adsorption and redistribution in surfactant films [2, 4, 7, 8, 20], and regulating surfactant production [1]. PLUNC (Palate, Lung, Nasal Epithelial Clone) appears to be a relatively new surfactant-like protein found in the upper airways [27]. PLUNC behaves similarly to SP-B and SP-C through its surface tension reducing properties but also resembles SP-A ad SP-D via its role in immunity [27]. PLUNC impairs Pseudomonas aeruginosa ability to form biofilms. In addition, related family members of PLUNC, specifically bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide binding protein (LBP), react to gram negative bacteria to perhaps exert antimicrobial activity [27].

1.5. Hydrophobic surfactant proteins

Mutations in the gene encoding SP-B within chromosome 2 or reduced amounts of SP-B protein cause severe or even fatal respiratory failure in newborns [7, 8]. SP-B is synthesized as a dimeric structure, stored, and secreted from the lamellar bodies along with the surfactant phospholipids [7, 20]. This dimer is integral in fostering stability of compressed films and enhancing surface tension-reducing properties [7, 20]. Some studies suggest that SP-B may be involved in host defense by helping to initiate the clustering and eventual killing of bacteria, including Pseudomonas aeruginosa and Staphylococcus aureus [8]. Hence, SP-B is crucial for survival [18].

SP-C has many similar functions to SP-B. SP-C is a very small protein (~4.2 kDa) whose gene is localized to chromosome 8 [1, 4]. Like SP-B, it is stored and secreted from the lamellar bodies in conjunction with the phospholipids [7]. SP-C is a membrane protein harboring a transmembrane fragment that is capable of operating as a signal peptide [7, 8]. This transmembrane portion assists SP-C to link the surfactant monolayer at the interface with the lipid bilayer [2, 7]. SP-C promotes adsorption and recycling of phospholipids and the spreading of lipids at the interface to optimize surfactant activity [2, 7, 8, 21]. Along with its roles in regulating surface activity, SP-C may be involved in host defense as it interacts with both lipopolysaccharide (LPS) and the pattern recognition molecule CD-14 found on phagocytes to reduce LPS-elicited responses [1, 8, 28]. Studies have demonstrated that SP-C binding to LPS may depend on the structure of LPS as well as its glycolipid components [1]. The ability of SP-C to associate with CD-14 suggests a role for this protein to contribute to the removal of pathogens although this requires further study [1]. Interestingly, targeted disruption of SP-C in mice is not lethal, but the absence of SP-C or the presence of misfolded SP-C proteins may be associated with interstitial lung disease [29, 30]. These results demonstrate that even though SP-C may not be as critical for some activities ascribed to surfactant, its deficiency may be linked to a broad range of regulatory events as an integral component of surfactant.

1.6. Hydrophilic surfactant proteins

SP-A and SP-D are the large, hydrophilic proteins found in surfactant. Both proteins are members of the collectin family of proteins, which are calcium-dependent carbohydrate binding lectins [2, 8, 21]. Encoded in rodents from genes located at the same locus on chromosome 10 [4, 13], SP-A and SP-D monomers entail four core domains: the NH₂-terminal domain, a collagen domain, the neck region, and the C-terminal domain known as the carbohydrate recognition domain (CRD) (Figure 3) [2, 14, 16, 31]. These monomers come together as a trimer [16], at which point SP-A forms an octadecamer or bouquet-like structure [2, 4, 13, 15] and SP-D forms a dodecamer, or crucible-like structure (Figure 3)[2, 15]. SP-A has been shown to facilitate the formation of tubular myelin from lamellar bodies, specifically when SP-B, SP-C and calcium are also present [2, 4, 22, 32]. Mice devoid of SP-A lack tubular myelin [2]. Additionally, similar experiments have demonstrated that lack of SP-D causes disruptions in surfactant homeostasis [2]. In addition to the important roles played by SP-A and SP-D in surfactant structure and metabolism, these collectins are critical components within the host defense system.

2.1. Responding to pathogens

2.2. Regulating inflammatory responses

The collectin proteins can also modulate the host inflammatory response independent of activities against microbial agents. For example, SP-A and SP-D play a major role in regulating inflammation by hastening the clearance of apoptotic cells and impeding the release of cytokines and other pro-inflammatory products [2, 6]. Multiple mechanisms exist by which cells remove dying cells. Apoptotic neutrophils are cleared via the interaction of SP-A and SP-D with myeloperoxidase, which is present on the surface of these cells [41]. SP-D has also been found to interact with immunoglobulin M to enhance phagocytic removal of late apoptotic cells [42]. When not interacting with pathogens, SP-A and SP-D bind to the signal inhibitory peptide (SIRP-alpha) on macrophages to attenuate release of pro- inflammatory products, specifically cytokines [2]. In some experiments using SP-A or SP-D knockout mice, the presence of pro-inflammatory cytokines is increased and inflammatory response resulting from infection is accentuated [13]. Further, these collectins are prone to nitrosylation, a modification that perturbs their tertiary structure. For example, S-nitrosylation of SP-D is chemoattractive for macrophages and induces pro-inflammatory signaling [43].

SP-A regulates signaling pathways in macrophages in response to microbial recognition that controls inflammation. In type II pneumocytes, SP-A can bind to various receptors, including P63 [44] and SPAR [45], to activate the phosphatidylinositol 3-kinase (PI-3K) pathway triggering phosphorylation of the downsteam kinase, Akt. Activation of the PI-3K-Akt pathway is pro-survival as Akt normally inactivates downstream pro-apoptotic signals such as glycogen synthase kinase -3β and forkhead transcription factor FKHR [45]. Akt activation leads to phosphorylation of IκBα leading to nuclear translocation of the transcription factor, NFκB, that induces expression of pro-inflammatory genes. However, in macrophages, SP-A increases the expression of Toll-like receptor (TLR) 2, and yet impairs its activity resulting in decreased Akt activation and nuclear NFκB [46]. Thus, SP-A differentially regulates critical signaling events within pulmonary cells that in turn controls inflammatory gene expression [46].

3.1. Immunosuppression by phospholipids

Recently, anionic phospholipids, such as palmitoyl-oleoyl-phosphatidylglycerol (POPG) and phosphatidylinositol (PI), relatively minor components of pulmonary surfactant, appear to exhibit immunomodulatory activity. POPG and PI were observed to inhibit LPS-induced nitric oxide and tumor necrosis factor-alpha responses in lung macrophages by interfering with Toll-like receptor interactions with adaptor molecules, CD-14 and MD-2, involved in NF-κb induced cytokine signaling [49]. The same group demonstrated that anionic phospholipids impair pro-inflammatory signaling induced by M. pneumoniae and RSV [50]. Another anionic phospholipid and very minor component of surfactant, cardiolipin, was also found to be immunosuppressive after release from injured host cells in pneumonia [51]. Cardiolipin in this study was observed to potently inhibit surfactant activity.

3.2. Lipid inhibition of viral infectivity

Viruses are common pathogens that interact with surfactant phospholipids. To date, dipalmitoylphosphatidylglycerol (DPPG) has been the only phospholipid member of surfactant to inhibit viral infection, though others may also play a role [52]. Specifically, DPPG interacts with Vaccinia virus, an orthopoxvirus. Small vesicles containing DPPG inhibit viral infection by blocking attachment of the virions to host cells [52]. DPPG likely impairs interaction with cell membranes by obstructing viral ligand binding with cell surface receptors [52]. Future studies will need to determine if other phospholipids have similar effects on viral infectivity.

3.3. Facilitating viral host cell entry by lipids

A significant body of data also suggests that surfactant phospholipids facilitate the entry of viruses into host cells [53–56]. DPPC, the predominant surface-active lipid, binds and enhances adenoviral entry into alveolar epithelium [52, 53]. As surfactant DPPC is reutilized after alveolar secretion by reincorporation into alveolar cells during recycling, it would appear that adenovirus exploits this pathway for cell entry. These effects on adenoviral trafficking with lipids appear to be specific to saturated lipids (e.g. DPPC), as monounsaturated forms of PC bind the virus but impede particle entry [53].

Phosphatidylserine (PS) also enhances viral infection by increasing fusion of the virus to cell membranes [54] by masquerading as an apoptotic signal [55, 56]. In cells undergoing apoptosis, PS transmigrates from the inner to outer membrane of dying host cells as a signal for efferocytosis, a form of phagocytosis [56]. In viruses, PS is associated mainly with enveloped forms [54]. Vaccinia virus, particularly the mature viral form, by either expressing PS on its envelope or interacting with surfactant PS, is recognized by many cell types as an efferocytotic signal for internalization [55]. By exploiting this mechanism for host cell entry, which inhibits the initiation of the inflammatory response, the virus may go undetected by the immune system enabling it to spread to surrounding cells [55]. However, this ability of PS to facilitate some viruses to enter cells can also be utilized as a translational tool by using enveloped viral vectors for therapeutic gene transfer [54].