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Despite the development of vaccines and antibiotics, Streptococcus pneumoniae (the pneumococcus) continues to be a major cause of human morbidity and mortality throughout the world. In recent years our understanding of how the host innate immune system recognizes and responds to pneumococcal infection has advanced significantly. Herein, we highlight some of the key features of the innate response to the pneumococcus.
The Gram-positive bacterium Streptococcus pneumoniae, the pneumococcus, is a common commensal of the human nasopharynx. However, it is also a significant pathogen, able to spread from the site of carriage to cause a range of infections, most notably: otitis media, pneumonia, bacteremia, and meningitis. The significance of S. pneumoniae as a human pathogen is highlighted by the estimate that among children under 5 years old there were 14.5 million episodes of serious pneumococcal disease in 2000 with 826,000 deaths 1. Other estimates suggest the pneumococcus to be responsible for a total of 1.6 million deaths annually 2. Shortcomings with the currently available vaccines (which cover only selected capsular serotypes) and the spread of antibiotic resistance suggest that the pneumococcus will remain an important human pathogen for some time to come.
The immune system consists of a diverse array of defense mechanisms including complement, mucociliary clearance, phagocytes, antibodies and effector T-cells. The non-specific early defenses of the innate immune system are critical for the prevention and slowing of infections; allowing time for the adaptive immune response to develop if needed. For a recent review on the adaptive immune response and vaccine development in the context of the pneumococcus see Malley3. Herein, we discuss recent advances in our understanding of the host innate immune response to the pneumococcus.
The complement system comprises over thirty serum and membrane proteins which, when activated, contribute to host defense by serving as chemotactic factors, opsonins, and bactericidal agents. Briefly, three pathways of complement activation exist: 1) The classical pathway which is activated by antibody binding to an antigen or other immune factors such as acute phase proteins. 2) The alternative pathway which is continuously activated at low levels but is only amplified on foreign surfaces due the absence of inhibitors present on host cells. 3) The lectin pathway, which is activated by mannose-binding lectin recognition of carbohydrates on microbial surfaces. Regardless of the activation pathway, the key functions of complement are: the opsonization of the microbial surface promoting phagocytosis, activation of neutrophil chemotaxis, and the direct killing of the microbe via formation of the membrane attack complex.
The importance of complement to host defense against the pneumococcus is shown by the severe infections suffered by those with genetic complement deficiencies. For instance patients genetically deficient for C3, the central complement component common to all three activation pathways, suffer recurrent pneumococcal (and other encapsulated bacterial) infections throughout their life. The essential role of complement is further supported by work in gene knock-out mice. Comparisons of mice lacking complement components specific to each activation pathway suggest that it is the classical pathway which is the dominant pathway for protection against the pneumococcus 4. Natural antibodies and the acute phase proteins, serum amyloid protein and C-reactive protein, have been found to be important in the activation of the classical pathway during pneumococcal infection 4–6. The alternative pathway also contributed to protection in these studies although its contribution was generally weaker in comparison to the classical pathway 4.
The importance of complement is further substantiated by the fact the pneumococcus processes a variety of complement-evasion mechanisms. All invasive clinical isolates of S. pneumoniae are encapsulated and the polysaccharide capsule is recognized to be the sine qua non virulence determinant. Capsule contributes to disease pathogenesis by protecting the pneumococcus from complement-mediated opsonophagocytosis, but also by preventing entrapment in mucus7 and neutrophil extracellular traps8 (see below). Capsule prevents opsonophagocytosis by multiple mechanisms including decreasing classical pathway activation by impairing C-reactive protein and IgG binding to the bacterial surface. Capsule also reduces alternative pathway activation, although the mechanism for this remains unclear, and decreases the degradation of C3b to iC3b on the bacterial surface. As a result of these combined effects the pneumococcal capsule impedes phagocytosis by Fcγ receptors, complement receptors, with evidence that it also protects against nonopsonic receptors 9.
Today, over 90 serologically distinct capsule serotypes are recognized and most have been isolated from individuals with disease. Importantly several serotypes, such as 1, 4, 5, 7 and 14, are overrepresented among invasive isolates while other serotypes less frequently cause disease. It is believed that the chemical composition and net charge of each serotype is central to these differences in virulence. However, considerable genetic diversity exists between strains thus making it difficult to assess the significance of capsule type versus other virulence factors. Recent studies using capsule swap pneumococcal mutants now clearly show that protection against complement-mediated immunity varies between capsular serotypes even when expressed in an otherwise isogenic background and that this effect correlates to observed differences in colonization and invasive potential 10, 11.
The capsule is not the only strategy the pneumococcus employs to avoid complement and phagocytosis. For example, the cytolysin pneumolysin is a key virulence factor produced by clinical isolates. It possesses a wide range of biological activities that contribute to carriage and disease (for review see 12, 13). With regards to complement, pneumolysin activates the classical pathway in the absence of antibodies specific to the toxin 14, 15. Deletion of the gene encoding pneumolysin results in increased opsonophagocytosis of pneumococci via the classical pathway and therefore suggests that complement activation by released toxin occurs distally leaving less available to deposit on the bacterial surface 16. In support of the latter, the contribution of pneumolysin to bacterial virulence in complement knockout mice is diminished 16.
The pneumococal surface protein CpbA (often referred to as PspC) recruits human but not mouse serum factor H, a negative regulator of the alternative pathway, to the bacterial surface 17. Likewise, certain CbpA alleles recruit the classical pathway inhibitor, C4-binding protein to the bacterial surface 18. PspA, the pneumococcal histidine triad proteins (PhtA, B, D and E), neuraminidase A, β-galactosidase and N-acetylglucosaminidase are additional pneumococcal surface proteins that act to reduce complement deposition on the bacterial surface and protect against opsonophagocytosis 19–21.
Pathogen recognition receptors (PPR) are a diverse collection of germ line-encoded host molecules that function to provide early recognition and initiate the inflammatory response to infection 22. This occurs via the recognition of conserved microbial features, so-called pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) from Gram-negative bacteria, lipotechoic acids from Gram-positive bacteria, mannan from fungi, and double-stranded viral RNA. It is worth noting that host products may also be recognized and stimulate inflammation via PRRs. The toll-like receptor (TLR) family are one group of PRRs that have received much attention due to their central role in a variety of infectious and inflammatory diseases (for recent review see 23). There are at least 10 TLRs in humans and 13 in mice each recognizing a distinct repertoire of ligands with several implicated in the host response to the pneumococcus.
Among the diverse ligands recognized by TLR2 is bacterial, including pneumococcal, lipoteichoic acid (LTA) 24–27. This interaction involves LPS binding-protein and CD14 26. TLR2 −/− mice show heighted susceptibility in models of pneumococcal meningitis with increased disease severity and higher bacterial counts compared to wild type controls 28, 29. In models of pneumonia and sepsis TLR2 −/− mice show only a modest increase in susceptibility 30, 31 probably due to the compensatory affects of TLR4 recognition of pneumolysin (see below) 32. Finally, in a nasopharyngeal carriage model, TLR2−/− mice show impaired clearance of pneumococci 33. Structural differences in LTA between pneumococcal strains alter their ability to stimulate TLR2 in vitro which may contribute to differences in virulence 24.
TLR4 was the first TLR to be recognized and is widely studied due to its role in the response to LPS 34, but also recognizes other ligands including pneumolysin 35, 36. Although the structural basis of this interaction is unclear, it is independent of the pore-forming and complement-activating activity of pneumolysin 35. The role of pneumolysin-TLR4 recognition during nasopharyngeal carriage is ambiguous with one study finding TLR4 −/− mice to have increased colonization levels and heightened risk of invasive disease 35. In contrast other work described no difference between wild type and TLR4 −/− mice 33. The reason(s) for such differences remain unresolved but may relate to different experimental approaches including the use of different pneumococcal strains. Finally, TLR4 plays only a limited role in pneumonia, systemic infection or meningitis 37–40. As indicated, this is probably due in part to redundancy between different TLRs 32, 41.
Bacterial DNA has inflammatory activity due to the presence of unmethylated cytosine-phosphate-guanosine (CpG) motifs that are recognized by TLR9 42, 43. TLR 9 −/− mice display enhanced susceptibility to pneumococcal pneumonia, a phenotype more severe than that seen with mice lacking TLR2 or TLR4 40. Unlike TLRs 2 and 4, which are on the cell surface, TLR 9 is expressed within endosomal compartments, thus it may serve to amplify/modulate responses following phagocytosis. TLR1 and TLR6 are both implicated in the innate recognition of pneumococci in vitro although their significance has not yet been tested in an infection model 25, 44. Both of these TLRs dimerize with TLR2 and TLR2/6 heterodimers recognize diacylated lipoproteins, whereas TLR1/2 heterodimers recognize triacylated lipopeptides 45. Myeloid differentiation factor 88 (Myd88) is a key signaling adaptor protein during TLR as well as the Interleukin (IL)-1 family receptor signaling. In line with a role for different TLRs during pneumococcal recognition, Myd88 −/− mice show an increase in susceptibility in various infection models, although the contribution of IL-1 and IL-18 signaling may also contribute to this affect 31, 46, 47
Finally, supporting a role for TLRs and their signaling pathways during human infection, various polymorphisms in TLRs and their signaling molecules have been associated with susceptibility or resistance to pneumococal infection 48–52. Of particular note is the signaling adaptor Mal (also known as TIRAP), which functions following TLR2 and TLR4 activation. Interestingly, heterozygous carriage of a S180L variant of Mal is associated with resistance to invasive pneumococcal disease, malaria and tuberculosis 48. It is thought that the protective effect of S180L heterogeneity is mediated through the production of a reduced yet sufficient inflammatory response that confers protection while not inducing immunopathology. In contrast, a L181L variant of Mal which lacks signaling activity confers what appeared to be increased susceptibility to infection. However, the mutation was found so infrequently that any association could not be robustly assessed. The relative scarcity of L181L homozygote, especially in areas of high endemic disease is consistent with increased disease susceptibility and a strong selective pressure against such a genotype. This example highlights that while TLR signaling in humans is required for protection against pneumococcal disease it may also be detrimental if not properly regulated.
The elderly have an increased incidence of pneumococcal pneumonia that is associated with rapid-onset of severe disease with high mortality. Although there are likely to be several mechanisms underlying this heighted susceptibility, recent data from aged mice suggests reduced TLR function plays an important role. Compared to young mice, aged mice had reduced TLR1, 2 and 4 protein levels in their lungs, and were more susceptible to pneumococcal pneumonia 53. Consistent with decreased TLR function, aged mice showed a reduction in NFkB activation during infection and reduced pro-inflammatory cytokine production following intratracheal challenge with purified pneumococcal components 53.
In humans, elderly patients with pneumonia often present without one or more of the three classic symptoms of pneumonia (cough, fever and dyspnea) with ~10% showing no signs of infection other than confusion or delirium 54. Reduced TLR function is a possible explanation for such atypical presentations in the elderly. Prior to pneumococcal infection, age-associated inflammation also increases the expression of the pneumococcal ligands polymeric immunoglobulin receptor and platelet aggregating factor receptor53. Bacterial interaction with these proteins has been shown to initiate their uptake and translocation through the cell and binding to these proteins is thought to be important for development of bacteremia and meningitis 55, 56. TLR dysfunction and inflammation-mediated lung priming probably work simultaneously to heighten the susceptibility to pneumonia of aged animals and possibly human subjects as well.
NOD-like receptors (NLR) are intracellular PRRs acting in the cytosol to recognize and respond to microbial products. NOD1 and NOD2 recognize bacterial cell wall components containing D-glutamyl-meso-diaminopimelic acid and muramyl dipeptide, respectively. They function via activation of the inflammasome, a cytosolic multiprotein complex which activates caspase-1 which in turn promotes the cleavage and secretion of IL-1β and IL-18. They have also been found to induce pyroptosis, a caspase-1-dependent form of cell death that is highly inflammatory 57, 58. Although the pneumococcus stimulates the NLR NOD2 59 and activates caspase-1 in vitro 60 the significance in vivo is not clear given that caspase-1 −/− mice show no altered susceptibility to infection 40. On the other hand, Nod1−/− mice are more susceptible to pneumococcal sepsis due to the role of Nod1 recognition of the intestinal microbiota in priming the innate immune system 61.
Whilst the pneumococcal capsule is a key target of the adaptive immune system, forming the basis of current vaccines, its recognition by the innate immune system should not be overlooked. The C-type lectin SIGN-related 1 (SIGN-R1) is expressed by macrophages and binds purified capsular polysaccharide of different serotypes as well as to whole cells thus promoting their uptake 62. The significance of this recognition is shown by the increased susceptibly of SIGN-R1 knockout mice to systemic and pulmonary pneumococcal infection 63, 64. Absence of SIGN-R1 also reduces the levels of natural antibodies against the pneumococcus which most likely also impedes host defense Finally, SIGN-R1 activates the classical complement pathway by binding C1q and promoting complement disposition on SIGN-R1-bound pneumococci 65.
Scavenger receptors (SR) represent a large, diverse family of surface glycoproteins expressed predominantly on macrophages, dendritic cells and endothelial cells 66. Although first studied for their role in the clearance of oxidized low-density lipoproteins their function as PRRs is now appreciated, in particular as phagocytic receptors mediating direct non-opsonic phagocytosis of microbes 67. The scavenger receptors SR-A and MARCO both bind pneumococci, although the bacterial features that are recognized are not yet known, however, mice lacking either receptor have increased susceptibility to pneumococcal pneumonia 68, 69. In addition down-regulation of MARCO following influenza infection has been implicated in the the severity of secondary pneumococcal infections 70.
Pulmonary surfactant is a mixture of proteins and lipids that prevent alveoli from collapsing during expiration due to surface tension. In addition, the surfactant proteins (SPs) SPA and SP-D play a role in host defense against infection by binding to and opsonizing microbes and by modulating immune cell function 71, 72. In the case of S. pneumoniae, SP-D knockout mice show increased susceptibility to infection 73 while SP-A promotes phagocytosis of pneumococci in vitro 74. This latter effect is seemingly due to the up-regulation of SR-A (on macrophages) by SP-A rather than direct opsonization of the pneumococcus 75.
While the role of neutrophils in the phagocytotic uptake and intracellular killing of pathogens has long been recognized, more recently they have been shown to produce so-called neutrophil extracellular traps (NETs) to entrap and kill pathogens in the extracellular environment 76, 77. NETs consist of extruded DNA to which antimicrobial components, such as elastase, lactoferrin, bactericidal permeability increasing protein and myeloperoxide are attached. The pneumococcus is relatively resistant to NET-mediated killing due to D-alanylation of lipoteichoic acid 8. This results in a positive charge in the bacterial surface which generates an electrochemical repulsion against the antimicrobial peptides in the NETs. Furthermore, the pneumococcus produces an extracellular endonuclease, EndA, which facilitates pneumococcal escape from entrapment 78. Despite these evasion strategies it seems likely that entrapment by NETs may impede bacterial dissemination even if only transiently. Interestingly, strain to strain variation was observed for pneumococal extracellular nuclease activity, and this could contribute to the differences in virulence between strains and serotypes 78.
A variety of cytokines including IL-12 79, IL-17 79, IL-18 80–83 and IL-6 84 have all been shown to be important for the innate response to pneumococci. However, it appears the overlapping affects of TNF-α and IL-1 are of central importance during the early stages of infection being responsible for enhancing cytokine expression, neutrophil recruitment, and resultant bacterial killing via NFκB RelA activation 85–87. The importance of TNF-α during pneumococcal infection is substantiated in numerous experimental studies 87–90 showing that deletion or neutralization of TNF-α was detrimental and by the finding that human patients treated with anti-TNF-α therapies may have increased risk of invasive pneumococcal disease 91. However, while protective, TNF-α may also contribute to tissue damage and the up-regulation of pneumococcal receptors such as during chronic low-grade inflammation in the elderly 92, 53. The latter is substantiated by studies showing that the individuals with elevated levels of TNF-α and IL-6 were more likely to develop community-acquired pneumonia than those with lower levels 93.
The response to pneumococcal infection is multi-faceted and involves almost all aspects of the innate immune system in the lungs, many of which could not be discussed due to space limitations. These interactions are key for preventing severe disease, as is often demonstrated by the severe infections observed in individuals or animals that with deficiencies in various components. It may be possible to augment the host-defense through targeting of specific innate components, however, such an approach carries considerable risk as the immune system can be a double-edged sword that can protect or inflict damage to the host.
This review is supported by NIH grant AG033274.
Gavin Paterson earned his PhD from the University of Glasgow in 2004 for work on pneumococcal-host interactions. Positions at the Defence Science Technology Laboratory (DSTL), Porton Down and the Department of Veterinary Medicine, University of Cambridge followed before his current post of Postdoctoral Fellow in the Orihuela Laboratory.
Carlos Orihuela has worked on pneumococcal host-pathogen interactions since entering graduate school in 1996. He received his PhD from the University of Texas Medical Branch in Galveston in 2001 and completed his postdoctoral training at St. Jude Children’s Research Hospital in 2005. He is currently an Assistant Professor at the University of Texas Health Science Center in San Antonio.
Conflict of interest statement: C. J. O. has received funding for research unrelated to this review from Novartis Pharmaceuticals.