Lung infections and innate host defense

Introduction

Human lungs move around 14,000 liters of air every day. Thus significant amounts of organic and inorganic particulates and microbes inhaled from the environment and aspirated from the posterior pharynx can reach the 150 square meters of alveolar surface. The integrity of the thin alveolar membrane is essential to assure oxygen and CO₂ gas exchange; therefore the recognition and handling of these particulates without causing excessive inflammation are extremely important. Specialized lung innate immune responses play a key role in this process. Recognition of particulates is broad and based on use of Pattern Recognition Receptors (PRRs); however, the immune responses following recognition in the lung are unique enabling dampening of pro-inflammation and thereby limiting damage to the alveolar surface. Alveolar macrophages (AMs) and dendritic cells (DC) are the first cellular line of defense in the alveoli and their surfaces are rich in PRRs. Evidence is accumulating that soluble and cell-associated C-type (Ca²⁺-dependent) lectins play a key role in shaping the innate response in the lung. In addition to the established role for Toll-like receptors (TLRs) in this process, recent evidence indicates that NOD-like proteins (NLR) also play an important role as intracellular sensors that regulate inflammatory responses. Here we will discuss these important cellular and soluble determinants of the lung innate immune response, provide examples of their roles in modifying the host response to specific infectious agents during disease pathogenesis and address potential therapeutic applications.

Surfactant proteins A and D

The alveolar space consists of flat lining cells or type I cells important in gas exchange and type II cells that produce and secrete a mixture of proteins and phospholipids that comprise surfactant. Surfactant proteins B and C have important biological properties that result in lowering surface tension and preventing the alveoli from collapsing. A deficiency of these proteins in premature infants leads to infant respiratory distress syndrome, for which administration of exogenous surfactant is one of the treatments (1). Surfactant proteins A and D (SP-A and SP-D) are secreted collectins that belong to the C-type lectin super family. SP-A and SP-D contain specialized domains, assemble as trimers and form oligomers with specific physicochemical properties. They contain an N-terminal collagen-like region important in the formation of trimers and a carbohydrate recognition domain (CRD) at the C-terminus that is important in the recognition of microorganisms and host determinants. CRDs bind to carbohydrates such as mannose and fucose that are prevalent on the surface of microbial species, but not on the surface of mammalian cells (2) and regulate interactions between microbes and host cellular components.

SP-A and SP-D bind to different types of microbes including Gram positive, and Gram negative bacteria, mycobacteria, yeast and viruses as previously reviewed (3). Models using SP-A knockout mice have shown that there is a decreased clearance of H. influenzae (4), P. aeruginosa (5) and Pneumocystis carinii (6, 7) after tracheal instillation with the concomitant increase in microbial dissemination (8). The binding of these lung collectins with microbes promotes opsonization and growth inhibition. Some microbial surface molecules have been identified as ligands of SP-A and SP-D, among them LPS (9, 10) and lipoarabinomannan (LAM) from M. tuberculosis (11, 12).

SP-A enhances the uptake of microbes through Fc receptors and complement receptor 1 on alveolar macrophages (13, 14). In vitro studies have confirmed that lung collectins increase receptor-mediated uptake of different pathogens. SP-A enhances the phagocytosis of S. aureus, K. pneumoniae, S. pneumoniae and H. influenzae by macrophages (15, 16) (17, 18). There is growing evidence that SP-A and SP-D increase the expression of other phagocytic receptors of macrophages, independent of microbial binding. For example, SP-A and SP-D increase the cell surface localization of the mannose receptor (MR) on macrophages, which enhances M. avium and M. tuberculosis phagocytosis (19, 20). Another example is the increased scavenger receptor A (SR-A) cell surface expression on macrophages mediated by SP-A that enhances the uptake of S. pneumoniae (21). The nature of the macrophage-collectin interactions and mechanisms implicated in the up-regulation of phagocytosis continue to be uncovered (22).

Because uncontrolled inflammation within the lung can be lethal, mechanisms to modulate the inflammatory response are necessary. Such mechanisms are in place to alert the host of the presence of pathogens while avoiding excessive inflammation and compromise of gas exchange. One recently proposed mechanism suggests dual functions for SP-A and SP-D. During basal conditions both SP-A and SP-D bind to the signal-inhibitory protein α (SIRP α) through their CRDs blocking pro-inflammatory mediator production. In contrast, when collectins bind microbes or cell debris, the collagenous tails will interact with calreticulin/CD91 on the cell surface stimulating phagocytosis and pro-inflammatory responses (23). The role of collectins in modulation of inflammation has been demonstrated in SP-A -/- mice which produced more pro-inflammatory cytokines and nitric oxide compared to SP-A +/+ mice after endotracheal instillation of LPS (5, 24). SP-A may interact with TLR2 decreasing the inflammatory response elicited by peptidoglycan (PGN) (25). Finally, SP-A has been shown to down-regulate the oxidative response to agonists in macrophages by decreasing the recruitment of p47^phox to the phagosomal membrane (26).

The genes encoding SP-A and SP-D contain several polymorphic sites. SP-A is encoded by two highly polymorphic genes. Some of the single nucleotide polymorphisms (SNPs) occur with relative frequency in the general population (27). SP-A gene polymorphisms are associated with neonatal respiratory distress syndrome (27-32). SP-A and SP-D polymorphisms are also associated with tuberculosis and respiratory syncytial virus infection (RSV) (33-35), and with chronic cavitary pulmonary aspergillosis (36), supporting their role in innate immunity. Recently, a SP-A2 gene polymorphism in the CRD of SP-A has been associated with increased susceptibility and risk of death in patients with meningococcal disease (37).

In conclusion SP-A and SP-D are essential players of the lung innate immune system. They participate in the agglutination and opsonization of microbes and enhance phagocytosis through different mechanisms, while modulating the immune response to prevent excessive inflammation. They may also play roles in innate immunity outside of the lung (38)

Alveolar Macrophages

Because of its location at the alveolar air tissue interface, the alveolar macrophage (AM) is the first line of cellular defense against inhaled environmental particles and infectious microorganisms that enter the lungs. These cells express several immune receptors, including Fc-γ receptors and complement receptors (e.g. CR1, CR3 and CR4); and particularly high levels of pattern recognition receptors (PRRs) such as the MR, Dectin-1 (β-glucan receptor), scavenger receptors, Toll-like receptors (TLRs) and NOD like receptors (NLRs) (39-44) (Figure 1). During inflammation, immigrating neutrophils also contribute to the inflammatory response. These cells possess their own array of PRRs and express significant quantities of cationic anti-microbial peptides (45).

Figure 1. Cartoon depiction of major immune and pattern recognition monocyte/macrophage membrane receptors.

These receptors play a major role in the recognition of a variety of pathogenic microbes and have been found to dictate early host responses, such as phagocytosis, trafficking, the oxidative response, pro- and anti-inflammatory cytokines, and antigen presentation. The MR, DC-SIGN and Dectin-1 are calcium-dependent (C-type) lectins. Abbreviations; FN = fibronectin, CRD = carbohydrate recognition domain, cys = cysteine, LRR = Leucine-Rich Repeats, TIR = Toll-IL-1 receptor domain, ITAM = immunoreceptor tyrosine-based activation motif, ITIM = immunoreceptor tyrosine-based inhibitory motif, MR (CD206) = mannose receptor, DC-SIGN (CD209) = dendritic cell-specific ICAM-grabbing non-integrin, TLR = Toll-like receptor, FcγRI (CD64), FcγRIIA,BC (CD32), FcγRIIIA,B (CD16), CR1 (CD35) = complement receptor 1, CR3 (Mac-1, CD11b/CD18) = complement receptor 3, CR4 (CD11c/CD18) = complement receptor 4

Despite constant stimulation by inhaled particles and pathogens, AMs display an anti-inflammatory phenotype described as an “alternative activation” state, which includes altered cytokine responses (e.g. increased IL-10 and TGFβ (40), (46, 47), reduced oxidant production in response to stimuli (48), and reduced microbicidal activity (49). Thus, AMs seem best adapted for removal of small airborne particulates with minimal induction of inflammatory immune responses.

Phagocytosis is considered the primordial function of AMs; the uptake of microorganisms is significantly enhanced by the surface receptors for antibody (IgG subtypes) and complement (most prominently the C3 fragments C3b and C3bi) which serve as opsonins. Once the microbe is engulfed by the AM, the formed phagosome fuses with lysosomes; an important step in the destruction of microorganisms. This process is followed by antigen processing and presentation, and subsequent lymphocyte stimulation with initiation of the acquired immune response. While alternatively activated macrophages may be adequate for the efficient clearance of routinely inhaled extracellular pathogens, they may be inadequate for host-adapted intracellular pathogens. For instance M. tuberculosis is highly adapted to survive in the phagosome, interfering with its normal maturation and avoiding its fusion with the lysosome (50, 51). AM can be stimulated by bacterial products, such as LPS or peptidoglycan, which triggers the translocation of NF-κB with the subsequent transcription of pro-inflammatory cytokines such as TNF-α and IL-8. The importance of TNF-α in lung immunity has been confirmed after episodes of M. tuberculosis reactivation and fungal infections that occur among patients receiving TNF-α blocking agents for certain medical conditions (52).

The Mannose Receptor

The mannose receptor (MR) is a C-type lectin that is expressed on tissue macrophages, AMs and DCs but not monocytes (53-55). The MR is a Type I transmembrane protein with a short cytoplasmic tail and an extracellular domain that shares homology with other C-type lectins. The extracellular domain is a PRR that binds with high affinity to mannose- and fucose-containing glycoconjugates frequently found on the surface of a variety of microbes referred to as pathogen-associated molecular patterns (PAMPs) (56). Macrophages contain large intracellular pools of MR within early endosomes, which undergo continual rapid recycling to the cell surface (57). SP-A increases trafficking of preformed MR to the macrophage cell surface (19). Additionally, the MR can serve as a molecular link between innate and adaptive immune responses (55). For example, the MR mediates loading of mycobacterial LAM onto CD1 molecules for LAM presentation to T cells (58).

The MR also plays a role in the internalization of microbial glycoconjugates by receptor-mediated pinocytosis (57) and different organisms including C. albicans, Pneumocystis carinii and M. tuberculosis by phagocytosis (57, 59-63). MR-mediated phagocytosis can direct the fate of the ingested microorganism. Engagement of the MR on human macrophages by M. tuberculosis mannosylated LAM (ManLAM) and phosphatidyl-myo-inositol mannosides (PIMs) initiates a specific phagocytic pathway that results in limited phagosome-lysosome (P-L) fusion for the bacterium (64, 65). Conversely, although the MR facilitates entry of HIV-1 through gp120, the MR-mediated pathway does not lead to a productive HIV-1 infection possibly by the MR facilitating antigen presentation via CD1b and inducing cell-mediated immunity (66).

DC-SIGN

Dendritic cells are a diverse group of myeloid and lymphoid-origin cells that play a key role in the adaptive immune response. One way these cells can regulate this immune response is through the expression of major pattern recognition receptors (67). The dendritic cell (DC)–specific ICAM-grabbing non-integrin (DC-SIGN, CD209) is a C-type lectin that binds to HIV gp120 (68) (69). Like the MR, DC-SIGN has an extracellular CRD. Its cytoplasmatic domain is important for antigen internalization and signal transduction (70). DC-SIGN is expressed by immature and mature DCs (71) but its presence on macrophages is dependent on the tissue type and state of activation. Like other members of the C-type lectin family, DC-SIGN binds pathogens that express mannose- or fucose-containing glycans such as HIV, M. tuberculosis, C. albicans, H. pylori and Schistosoma mansoni (72, 73). The formation of DC-SIGN tetramers facilitates high-affinity ligand binding (74). DC-SIGN also directs antigens to late endosomes or lysosomes for processing and presentation to T cells (75), thus initiating the adaptive immune response. Some organisms can exploit DC-SIGN internalization to avoid normal pathways of lysosomal degradation. For instance, M. tuberculosis ManLAM binding to DC-SIGN prevents DC maturation and induces the secretion of IL-10, an immunosuppressive cytokine (76). A recent study showed that certain DC-SIGN polymorphisms are associated with a reduced risk of tuberculosis (77).

Dectin-1

This C-type lectin is expressed on macrophages, DCs and neutrophils and is primarily a PRR for fungal β-glucan (74, 78). It contains an extracellular CRD and an intracellular immunoreceptor tyrosine-based activation motif (ITAM) required for interactions with TLR2 (74) and the cytoskeletal changes that occur after Dectin-1 mediated phagocytosis (79). A unique feature of Dectin-1 is that it mediates the production of TNF-α in response to C. albicans and Streptomyces cerevisiae (80). The production of pro-inflammatory cytokines such as IL-12 and TNF-α by macrophages and DCs following stimulation of Dectin-1 requires collaboration with TLR2 (80-82). There is recent evidence that Dectin-1 in cooperation with TLR2 mediates the production of TNF-α by murine macrophages infected with avirulent or attenuated mycobacteria strains but not virulent strains (82).

Toll-like receptors

Toll-like receptors (TLRs) are membrane-associated type I receptors that largely function to recognize PAMPs (83). There are 11 mammalian TLRs which vary in function largely with respect to the ligands that they recognize (84). The externalized amino terminus contains variable arrangements of leucine rich repeats (LRRs) which serve to recognize the PAMPs. The cytosolic carboxy terminus of TLRs is highly homologous to the IL-1 receptor and contains a Toll/IL-1R (TIR) domain that forms the nidus for the assembly of signaling intermediates such as MyD88, IRAK1, IRAK4, IRAKM and Mal/Tiram.

The most extensively studied TLRs are TLR4, which senses the endotoxin of Gram negative organisms (85), and TLR2, which has a particular affinity for Gram positive PAMPs, such as lipoteichoic acid (84, 86). In general, TLR signaling drives NFκB activation via phosphorylation of IκBα.

NOD Proteins

Although surface PRRs such as TLRs are widely recognized regulators of immune responses, 23 cytosolic NOD (nucleotide-binding oligomerization domain) like receptors (NLRs) implicated in the innate recognition of intracellular pathogens have been recently described (87-92). They are composed of a C-terminal series of leucine-rich repeats (LRRs) similar to the extracellular domain of TLRs, a central nucleotide-oligomerization domain (NOD) and an amino-terminal protein-protein interaction domain, such as caspase activation and recruitment domain (CARD), baculovirus inhibitor repeat domains, or pyrin domains. NOD1/CARD4 is a ubiquitous protein that recognizes only microbial components of Gram-negative bacteria (93) while NOD2/CARD15 is restricted to antigen presenting cells and has been implicated in the recognition of Gram-negative and Gram-positive bacterial cell wall products (92, 94, 95). NOD1 and NOD2 have been found to recognize the peptidoglycan (PGN)-derived peptides γ-d-Glu-meso-diaminopimelic acid (iE-DAP) and muramyl dipeptide (MDP), respectively (94, 96). As a result of the NOD-PGN interaction, the NOD protein undergoes oligomerization leading to activation of NF-κB and release of pro-inflammatory cytokines (97, 98). This pro-inflammatory response is enhanced by simultaneous stimulation of TLR3, TLR4 and TLR9 (99-101). Recent articles showed that the NOD protein Ipaf/CARD12 senses bacterial flagellin independent of TLR5, while cryopyrin/NALP3 senses bacterial RNA. Upon recognition of their ligands, Ipaf and cryopyrin mediate caspase-1 activation and IL-1β release (102, 103).

The importance of NLRs in the recognition of pathogens intracellularly has been demonstrated in several microbial models (91, 104-110). The etiology of Crohn's disease (a granulomatous disease of the bowel) has been hotly debated for many years, with some researchers advocating M. paratuberculosis as the etiologic agent (111). In recent years the discovery that NOD2 gene mutations are the most common mutations associated with Crohn's disease (112) has spurred the debate again regarding mycobacteria as potential etiologic agents.

Mannose binding lectin (MBL)

The mannose binding lectin (MBL) is a soluble collectin present in serum (113). Like other collectins, it serves as a PRR for microorganisms. There is increasing evidence that polymorphisms in the MBL are associated with different types of infections such as HIV, cryptosporidiosis, meningococcal disease and tuberculosis (114-117).

Examples of lung innate immune responses to infectious agents

M. tuberculosis

M. tuberculosis is an intracellular pathogen of mononuclear phagocytes and highly adapted to the human host. This bacterium enters macrophages by the phagocytic process using a defined subset of receptors, and subsequently multiplies within a unique phagosomal compartment. SP-A and SP-D regulate the early interaction between M. tuberculosis and macrophages. SP-A increases the phagocytosis of M. tuberculosis through a direct interaction of the protein with macrophages (118), which up-regulates MR activity (19). In contrast, SP-D has been shown to decrease M. tuberculosis phagocytosis by macrophages by binding with high avidity to the mannose caps of ManLAM on the bacilli, thereby reducing the interaction of the bacterium with the MR (12). SP-D-opsonized M. tuberculosis bacilli that are phagocytosed undergo increased phagosome-lysosome (P-L) fusion and have reduced intracellular growth (12, 119). Heterogeneity in the genetic background of patients with tuberculosis can be important in the progression of disease. As one example, different SP-A and SP-D alleles may increase or decrease the host susceptibility to tuberculosis (33).

The presence of mannose on the surface of M. tuberculosis aids in host cell recognition and response. The terminal mannose caps of ManLAM bind to the macrophage MR (120, 121). ManLAMs from different M. tuberculosis strains vary in the degree to which they bind to the MR pointing to a potential relationship between the length and/or presentation of the mannose caps and their avidity for the MR (121). ManLAM caps also bind to DC-SIGN on DCs (122, 123). Recent studies show that the MR and DC-SIGN regulate phagosome trafficking differently. As noted earlier, MR, but not DC-SIGN-mediated phagocytosis of M. tuberculosis, is associated with decreased P-L fusion (64). Since macrophages express high MR and low DC-SIGN, it is speculated that this difference may explain why macrophages serve as the major intracellular niche for M. tuberculosis.

Lipomannan and the 19kDa glycolipoprotein from M. tuberculosis have been shown to induce apoptosis through TLR2 (124, 125). In vivo studies showed that TLR4 deficient mice had reduced bacterial clearance and develop a chronic pneumonia (126). Although there is conflicting evidence about the role of TLR2 in M. tuberculosis-infected mice, TLR2^−/− mice had reduced bacterial clearance, a defective granulomatous response and developed chronic pneumonia (127). There is additional evidence that after stimulation with sonicated M. tuberculosis, TLR2 and TLR4 deficient cells produce less TNF-α, however TNF-α production is not completely abolished suggesting the presence of other pathways important in producing pro-inflammatory cytokines (110). Recent evidence supports the importance of NOD2 as an intracellular sensor for M. tuberculosis; a synergistic effect in TNF-α production was observed when TLR2 and NOD2 were simultaneously stimulated by the 19kDa glycoprotein and M. tuberculosis, respectively (110). The effects of the C-type lectins in the function of NODs is unknown, however it is possible that similar to the TLRs, they may act in concert with the NODs to regulate the inflammatory response.

Streptococcus pneumoniae

S. pneumoniae is the principal cause of bacterial pneumonia in the general population. Evidence supports the importance of surfactant in the lung innate immune response to S. pneumoniae. SP-A augments scavenger receptor A (SR-A)-mediated phagocytosis of the bacteria by murine AMs (21). SP-D has been shown to bind to and aggregate three serotypes of S. pneumoniae enhancing their uptake by neutrophils (17). As discussed above, SP-A and SP-D bind carbohydrate structures present on the pneumococcal surface through their CRDs (21, 128). Experiments using SP-D deficient mice showed enhanced colonization and infection of the upper and lower respiratory tract by the pneumococci and an earlier onset and longer persistence of bacteremia (129).

Evidence supports the importance of TLR2 and TLR4 in pneumococcal infection (130-132). The recognition of pneumococci cell wall products by TLRs may cause the up-regulation of NODs, as demonstrated by the increased levels of NOD1 and NOD2 6 hours after infection in mice. In the same study the authors showed that NOD2 but not NOD1 mediated NF-kB activation after exposure to inactivated pneumococci (109).

Pseudomonas aeruginosa

Among immunocompromised patients, patients on mechanical ventilation or those with cystic fibrosis, P. aeruginosa is a major cause of pneumonia. In SP-D -/- mice there is a decrease in bacterial phagocytosis by AMs, that is partially restored after the addition of exogenous recombinant SP-D (133). Studies have shown that SP-A -/- mice have decreased clearance of bacteria after intratracheal instillation (4, 5, 133).

Different cell wall components of P. aeruginosa such as peptidoglycan, LPS, flagellin and CpG DNA, are recognized by TLR2, TLR4, TLR5 and TLR9 (134-137). Although important in P. aeruginosa infection, TLRs are not the only system implicated in the genesis of a pro-inflammatory response. In the absence of TLR2-, TLR4- and TLR5-mediated signaling there is still a significant production of inflammatory cytokines and unimpaired bacterial clearance (137). In this regard, after infection of epithelial cells deficient in TLRs, NOD1 was able to induce NF-kB activation; the same study showed that in mice, the activation of NOD1 was necessary for the production of KC, a CXC chemokine important in the recruitment of neutrophils (138).

Future Directions and Therapeutic Options

For more than 25 years surfactant therapy has been successfully used in neonates with respiratory distress syndrome with the purpose of facilitating alveolar gas interchange. Surfactant therapy has also been used in adults with acute respiratory distress syndrome (ARDS) without benefit (139). However, surfactant replacement therapy has been based on the biomechanical properties of surfactant rather than its biological properties, and neither SP-A nor SP-D are components of artificial surfactant. Since the levels of these collectins are decreased in certain diseases such as ARDS and severe bacterial and viral pneumonias (140, 141), replacement therapy may be useful in these clinical settings. In one study, the use of recombinant human SP-D was associated with the down-regulation of allergic hypersensitivity in mice sensitized to allergens of Aspergillus fumigatus (142). Because of their immunomodulatory and antimicrobial properties, aerosolized SP-A and SP-D may be useful as adjuncts to conventional treatment in patients with selected lung disorders.

Although TLRs play an important role in increasing the pro-inflammatory response to infectious agents, they are not essential as recently demonstrated (143). Thus other surface receptors including C-type lectins may be important in this process. Targeting antigens to C-type lectin surface receptors to enhance the cellular immune response against pathogens deserves some attention. For example, as reviewed above, the MR and DC-SIGN are capable of directing antigen presentation, thus serving as links between innate and adaptive immunity, a property than can be exploited in the development of more effective vaccines. For example, directing mannosylated proteins from Cryptoccocus neoformans into APCs through the MR is necessary for an efficient T cell response (144). The MR can also be used as a target for delivering drugs to macrophages; this approach has been explored to deliver antibiotics into the cytosol (145). On the other hand, since MR-mediated phagocytosis of M. tuberculosis favors the survival of bacilli by inhibition of P-L fusion, therapeutic blockade of this pathway may theoretically decrease the survival of M. tuberculosis in the macrophage. Another potential vaccine strategy is to develop agonists that target NODs, thereby increasing the immunogenicity of vaccine antigens. New antibiotics that target bacterial cell wall PGN would potentially enhance the production of MDP and DAP which would be expected to activate the NODs, thus bolstering the cellular immune response during therapy.

In conclusion, a better understanding of the soluble and cellular determinants underlying lung immune mechanisms will allow for the development of better diagnostic and screening tests, therapies and vaccines. Development of agents to these targets will represent a fundamentally new way to treat lung infections.