The pathogenicity of many organisms resides in an ability to resist, escape, or neutralize host defense mechanisms. Pathogens stimulate innate and adaptive immune responses but also can use them to their own ends (1, 2). In this review, we focus on the strategies used by pathogens to take advantage of or to modify host defense mechanisms, emphasizing effects on innate and adaptive immune responses. Streptococcus pneumoniae and Pseudomonas aeruginosa have been chosen as primary examples because the gram-positive coccus S. pneumoniae is a common causative microorganism in community-acquired pneumonia, and the gram-negative bacterium P. aeruginosa is frequently involved in nosocomial pneumonia.
Virulence is related to the intrinsic characteristics of the pathogen and is manifested as an ability to evade host defense mechanisms and to replicate in the host. Pathogenicity, in contrast, is an ability to create characteristic tissue lesions secondary to the inflammatory response triggered by the release and activation of various pathogen components. In host–pathogen interaction, bacterial, viral, or fungal components modulate the inflammatory and immune responses. This modulating effect varies not only across species but also across classes or even strains of bacteria. Genetic polymorphisms of pathogens, as well as of humans, and the distinctive features of each infection site in the body conspire to complicate the study of the pathophysiological mechanisms involved in sepsis.
Virulence is multifactorial
Many factors contribute to the virulence and pathogenicity of S. pneumoniae (3–23), including the polysaccharide capsule, cell wall components (such as lipoteichoic acid and peptidoglycan), pneumolysin, autolysins, neuraminidases, CbpA adhesins, IgA1 proteases, surface protein A (PspA), surface adhesin A (PsaA), and hyaluronidase. These components allow pathogenic strains to evade host defense mechanisms, to divide, and to exhibit local or systemic invasiveness as well as to trigger the inflammatory cascade (4–23) (Table 1).
The situation is similar for P. aeruginosa, whose virulence factors fall into three main groups (24–30) (Table 2): (a) parietal virulence factors [pili, flagella, adhesions, alginate/biofilm, and lipopolysaccharide (LPS)]; (b) intracellular factors [exoenzyme S (ExoS), ExoT, ExoU, ExoY] secreted and translocated directly to the cytosol of the host eukaryote cells via the contact-dependent type III secretion system; and (c) extracellular factors secreted by the type I and type II secretion systems, which act as soluble toxins for target cells. The secretion of this arsenal of extracellular virulence factors is controlled by a complex system called quorum sensing. In quorum sensing, the bacterial population density regulates intercellular signals that orchestrate and increase the production of extracellular factors by the bacteria (24–27, 30).
Infection site and virulence
By inducing successive mutations in the S. pneumoniae genome, Polissi et al. recently identified 126 genes (among the 2236 known S. pneumoniae genes) that affect virulence (31), confirming the multifactorial nature of this characteristic. The roles or functions of 63 of these genes remain unknown. Studies of mutated strains in murine models of pneumonia or peritonitis showed variations in the effects of virulence factors across infection sites. For instance, autolysin LytA, hyaluronidase (Hyl), and neuraminidase (NanA) were associated with increased virulence in pneumonia models but not in peritonitis models (31).
More recently, Hava and Camilli identified 261 new mutations involved in the virulence of S. pneumoniae (32). Thus, 387 virulence genes in all have been found, although their functions are not completely known. In the same study (32), the site specificity of virulence factors was clearly apparent. Thus, four classes of S. pneumoniae strains were differentiated in various murine models of pneumonia, bacteremia, or nasopharyngeal carriage. Class I was specific for the lungs, class II for the lungs and blood, class III for the lungs and nasopharynx, and class IV for the lungs, blood, and nasopharynx (32). Among the site-specific virulence factors incriminated in site specificity, many transcriptional regulators are found in each of the four classes, reinforcing the idea that tissue-specific regulation of virulence factors is important for pneumococcal pathogenesis. Bacterial transcription regulation factors that differentially modulate virulence genes across tissues may allow S. pneumoniae to adapt to the various environments in the body.
It has been shown that the Pseudomonas aeruginosa genome is highly conserved and that there are a core set of genes, including nearly all known virulence factors, that are present in all strains regardless of disease source (33). The same genes are conserved among environmental isolates. The remarkable conservation (∼97%) of genes encoding proteins associated with virulence suggests that most Pseudomonas aeruginosa strains, regardless of source, possess the basic pathogenic mechanisms necessary to cause a wide variety of human infections. Nevertheless, in the study by Wolfgang et al. (33), the gene content of individual strains and whether detected genes were functional could not be predicted.
Synergy among virulence factors
The effects of virulence factors are additive or even synergistic (34). In a murine model of intraperitoneal infection, isogenic S. pneumoniae strains deficient in pneumolysin (Ply−/−), autolysin (LytA−/−), and surface protein A (PspA−/−) were associated with survival gains of 6, 6, and 3 days, respectively, as compared with the parent strain. In contrast, deficiency in hyaluronidase (Hyl−/−), neuraminidase (NanA−/−), and adhesins (Cbp−/−) did not influence survival in this model. Synergy between virulence factors was demonstrated in this animal model using isogenic S. pneumoniae strains deficient in two different virulence factors. Thus, S. pneumoniae strains deficient in both Ply−/− and NanA−/−, Hyl−/−, or CbpA−/− showed survival gains of 10, >21, and >21 days, as compared with the parent strain (34).
S. pneumoniae undergoes spontaneous phase variation in colony morphology between a transparent and an opaque colony phenotype (5, 35–40) (Table 3). Transparent variants, which have more cell wall teichoic acid than opaque S. pneumoniae, demonstrate an increased ability to adhere to human lung epithelial cells and are selected for during nasopharyngeal colonization in rodent models but are unable to generate systemic infection. In contrast, opaque variants have more capsular polysaccharide than transparent S. pneumoniae and are characteristically more virulent and associated with invasive disease. Because populations of S. pneumoniae are a heterogeneous mixture of opaque and transparent organisms, phase variation provides S. pneumoniae with a unique advantage in vivo (39). Each phase has characteristics that provide a selective advantage for either carriage or systemic infection. These phenotype variations are associated with quantitative differences in virulence factor expression (35, 37, 40) (Table 3). Thus, it has been recently shown that the ability of S. pneumoniae opacity phase variants to induce proinflammatory gene expressions in vivo was different (38). The transparent variant appears to be a more potent inducer of the expression and production of inflammatory mediators (TNF-α, IL-1α, IL-1β, IL-6, iNOS) in vivo, with the result being the accumulation of more inflammatory cells.
Up-regulation and differential expression in vivo
Ogunniyi et al. (41) recently compared the levels of five well-characterized virulence protein genes (Ply, PspA, PsaA, CbpA, and cps2A) in pneumococci grown in broth culture or isolated from the blood of mice at different times after intraperitoneal infection. Twelve hours after intraperitoneal infection, the mRNA species of Ply, PspA, and PsaA were up-regulated, unequivocally, demonstrating an altered pattern of gene expression in vivo versus in vitro (41). Moreover, the pattern was not constant for the virulence genes during the course of infection. At 24 h, expression of Ply and, significantly, PspA was further increased, but the expression of the other virulence factors was not greatly dissimilar to 12-h levels. Thus, up-regulation of virulence gene expression in pneumococci in the blood of infected mice appears to be largely attributable to host factors. During the course of an infection, the pneumococcus must adapt to a range of environments, and optimal survival in any one niche may require expression of a distinct subset of the organism’s armory of potential virulence factors.
Effect of inoculum size or bacterial population density
The size of the bacterial inoculum or bacterial population density also plays a role in the expression and effects of virulence factors (25, 34). A hierarchy among virulence factors has been shown in a murine model of S. pneumoniae intraperitoneal infection by injecting increasingly large inocula of isogenic strains deficient in various virulence factors (34). Variations in virulence that were subtle with small inocula were readily detected with large inocula. However, Ogunniyi et al. reported that the expression of S. pneumoniae virulence genes (Ply, PspA, PsaA, and Cbs2A) was not subjected to coordination by a global regulatory factor (41).
In P. aeruginosa, extracellular virulence factors are regulated by a global control system closely dependent on bacterial cell density and responsible for cell-to-cell signaling within the bacterial population (24–27, 42). This cell signaling system, called quorum sensing (QS), allows the pathogens to react to the environment as a population rather than as individual bacterial cells, thereby increasing their chances of overwhelming host defense mechanisms. QS is a mechanism whereby bacteria are able to sense the environment and, as a population, regulate the expression of various genes. Isolated production of extracellular virulence factors by a small number of bacteria would probably generate a host response capable of neutralizing the pathogen. In contrast, coordinated gene activation and expression of high concentrations of extracellular virulence factors by the entire bacterial population allows the pathogen to overwhelm the host defense mechanisms. A similar quorum sensing system has been found for many other human gram-negative pathogens, including Serratia marcescens, S. liquefaciens, Escherichia coli, Aeromonas hydrophila, Vibrio cholerae, V. parahaemoliticus, Yersinia enterolitica, Y. pseudotuberculosis, Y. pestis, Enterobacter agglomerans, Citrobacter freundii, Brucella melinentis, Hafnia alvei, and Rhanella aquatis (26). Equivalent systems have been described for gram-positive bacteria (Bacillus subtilus, S. pneumoniae, Enterococcusfaecali, Streptococcus spp., and Staphylococcus aureus) (26, 43, 44). S. pneumoniae has a well-characterized quorum sensing system involved in regulation of competence for genetic transformation and in virulence (45, 46).
Pseudomonas aeruginosa has at least two quorum sensing systems (lasR/lasI and rhlR/rhlI), which interact with each other. As the bacterial population increases, the extracellular concentrations of autoinducer signal molecules by P. aeruginosa, including N-(3-oxododecanoyl)-l-homoserine lactone (3O-C12-HSL) and N-butyryl homoserine lactone (C4-HSL), increase, and as soon as an intracellular threshold concentration is achieved, these molecules bind to and activate their cognate transcriptional regulators (24, 27, 42). Both the las and rhl systems have been found to regulate the production of multiple virulence factors. There is increasing evidence that quorum sensing is functionally active during P. aeruginosa infections (27, 42). For example, the formation of a normally dense biofilm by P. aeruginosa has been shown to be dependent on the synthesis of the signal proteins for quorum sensing HSL. Bacteria lacking the lasI gene produce a loose and easily disrupted biofilm. Bacterial strains isolated from urinary catheters and contact lenses can be shown to produce HSL when they are grown on agar plates. Sputum samples from cystic fibrosis patients chronically colonized with P. aeruginosa contain mRNA transcripts for the QS genes lasR and lasI. HSL were also directly extracted and measured in the sputum of these patients. Finally, quorum sensing has been shown relevant to the outcome of pseudomonal burn infections in mice, pseudomonal bacteremia in mice, and pseudomonal pneumonia in rats and mice (27, 42).
Bacterial avoidance of phagocytosis: antiphagocytosis
Phagocytosis constitutes the primary line of host innate and adaptative defense against incoming microbial pathogens, providing an efficient means for their removal and destruction. Whereas intracellular pathogens have developed sophisticated strategies to enter and survive within host cells, some bacteria prevent their own uptake. To achieve this goal, another means of subverting host cell processes is by the injection of bacterial proteins directly into the host cell. Several gram-negative pathogenic bacteria use specialized type III secretion systems that are dedicated to the delivery of these bacterial proteins into host cells to subvert host cell processes (28). Microbial pathogens recognize phagocytosis as either an opportunity or an obstacle to their own survival and replication and respond accordingly. Blocking phagocytosis allows the pathogen to avoid destruction through the degradative endocytic pathway and, in some instances, paralyze phagocytic responses and subsequently impair the development of cellular immunity. Several bacterial pathogens—i.e., enteropathogenic E. coli, P. aeruginosa, Yersinia spp., Helicobacter pylori—have antiphagocytic capabilities (47). Pseudomonas aeruginosa mediates antiphagocytic effects by using its type III secretion system to deliver at least two effectors, ExoS and ExoT, into host cells that are capable of disrupting essential regulatory proteins of phagocytic signaling processes (47–52).
Bacterial pathogens have a broad repertoire of strategies to block phagocytosis (50, 51), including (a) surface antigenic variations to avoid recognition by specific antibodies and thus avoid phagocytosis through Fcγ receptors, (b) interference with antibacterial antibodies (such strategy is exemplified by protein A from Staphylococcus aureus, which binds the Fc region of IgG and prevents a normal interaction with Fcγ receptors), (c) interference with complement opsonisation and deposition (Streptococcus pneumoniae, E. coli, K. pneumoniae, S. aureus, N. meningitidis), and (d) interaction with factor H or FH-like protein 1 (Streptococcus pneumoniae, S. pyogenes, Yersinia enterolitica, Neisseria gonorrhoeae, Borrelia burgdorferi).
VIRULENCE FACTORS AND IMMUNE AND INFLAMMATORY RESPONSES: INDUCTION OF IMMUNE AND INFLAMMATORY RESPONSES IS MULTIFACTORIAL
Lipoteichoic acid and peptidoglycan, two components of the S. pneumoniae cell wall, together with pneumolysin play a central role in inducing an inflammatory response to S. pneumoniae infection (Table 1). In vitro, these substances activate monocytes to release the proinflammatory cytokines including TNF-α (6–8, 52), interleukin (IL)-1β (6–9, 53), IL-6 (7), IL-8 (10, 54), and interferon (IFN)-γ (55). Their effects are clearly dose dependent (8). The level at which TNF-α and other inflammatory mediators are expressed is directly correlated to the size of the bacterial inoculum. In contrast, the virulence of S. pneumoniae does not seem to affect the expression of TNF-α in a quantitative manner (8). These results suggest that the virulence of a S. pneumoniae strain is not related only to an ability to stimulate TNF-α secretion by macrophages (8). However, of the 387 virulence genes identified to date, very few have been studied in terms of their ability to induce immune and inflammatory responses. Recently, the genes in the human monocytic cell line THP-1 that are pneumolysin responsive have been identified (54). Of 4133 genes evaluated, 142 were found to be responsive in a pneumolysin-dependent fashion, whereas 40 were found to be responsive independent of pneumolysin. Of the 142 pneumolysin-dependent genes, 116 were found to be up-regulated, and 26 were found to be down-regulated, in response to the presence of pneumolysin. Among those up-regulated were genes for lysozyme, prostaglandin E synthase, mannose-binding lectin 1, IL-1-receptor antagonist (IL-1Ra), α-catenin, cadherin 17, cell division cycle 25B, caspases 4 and 6, macrophage inflammatory protein 1β (MIP-1β), monocyte chemotactic protein 3 (MCP-3), IL-8, IL-2 receptor β (IL-2Rβ), IL-15 receptor α (IL-15Rα), and interferon receptor 2 (54). These results indicate clearly that the interaction between host and pneumococcus are very complex, relying on many factors in the pathogen to elicit such proinflammatory responses.
The multifactorial nature of immune and inflammatory response induction by pathogens is shown even more clearly by studies of P. aeruginosa. Many factors capable of inducing these responses have been identified. In addition to the classic LPS-dependent activation of immune and inflammatory responses, flagellins (TNF-α, IL-8, IL-6, NF-κB), type IV pili (IL-8, NF-κB), nitrate reductases (TNF-α), porins (TNF-α, NF-κB), pyocyanin (IL-8), extracellular slime glycoprotein (TNF-α, NF-κB, AP1), phospholipase C (TNF-α, IL-1, IL-6, IFN-γ, MIP-1α, and MIP-1β), 3O-C12-HSL (IL-8, IL-1α, Il-1β, IL-6, IFN-γ, MIF, MIP-1α, MIP-1β, MIP-2, MCP-1, PGE2, NF-κB, and Cox-2), exoenzyme U (IL-6, AP1), and exoenzyme S (TNF-α, IL-1, IL-6, IFN-γ, IL-2, TGF-β, IL-1ra, IL-10, IL-8, MIP-1α, MIP-1β, and MCP-1) are virulence factors capable of inducing immune and inflammatory responses (27, 56–65). This list is undoubtedly incomplete, and the effects of these substances have not been fully characterized.
Whereas LPS of gram-negative bacteria has been extensively studied, less research has been done on other virulence factors for these bacteria. Exoenzyme S (ExoS) expressed by P. aeruginosa is a toxin with both extra- and intracellular effects. ExoS can be directly translocated to the cytosol of target host cells, where it inactivates cell functions, exerting a cytotoxic effect. In addition, ExoS can act as a soluble extracellular toxin that interacts with host cells (24, 57), inducing expression in monocytes of biologically active pro- and antiinflammatory cytokines (TNF-α, IL-1, IL-6, IFN-γ, IL-2, TGF-β, IL-1ra, IL-10) and chemokines (IL-8, MIP-1α, MIP-1β, MCP-1). This effect of ExoS is dose dependent. Moreover, the ability of ExoS to induce proinflammatory cytokines is similar to that of the P. aeruginosa LPS. Finally, ExoS directly activates T lymphocytes, causing them to undergo apoptosis (56). The relative roles of LPS and ExoS in inducing immune and inflammatory responses have not been investigated.
Inhibition of immune and inflammatory responses
The cytokines TNF-α, IL-8, IL-1, and IL-12 play a major role in innate and adaptive immune responses. They enhance the bactericidal capabilities of phagocytes, induce accumulation of innate-response cells at the infection site, induce dendritic cell maturation, and orchestrate a secondary adaptive immune response specific to the invasive pathogen. In contrast, the cytokines IL-10, IL-6, and TGF-β suppress immune responses. IL-10 inhibits macrophage activation, proinflammatory cytokine expression, free radical production, generation of reactive nitrogen species, and expression of mediators involved in specific immune responses, such as HLA-Dr and CD86. Some pathogens can induce the expression of antiinflammatory and immunosuppressive mediators, namely, IL-10, IL-6, and TGF-β, thereby modulating the host immune response. Examples include mycobacteria, Yersinia enterocolitica, and Bordetella pertussis (1). LPS and also ExoS produced by P. aeruginosa, as discussed above, induce the expression of both proinflammatory and antiinflammatory mediators. The impact of pathogen-induced antiinflammatory mediator release on modulation of the host immune response remains unclear. Moreover, cytokines have also been shown to be susceptible to degradation by bacterial proteases (66–69). It has been demonstrated that alkaline protease and elastase of P. aeruginosa are able to degrade and inactivate IL-2, IFN-γ, and TNF-α (66–68).
Pathogens can modulate host immune responses by directly inhibiting the expression of proinflammatory mediators (51, 70). The role for P. aeruginosa exotoxin A (ExoA) remains controversial (57, 71) because studies have shown either activation or inhibition of the expression of TNF-α, IL-8, IL-6, and IL-10. Nevertheless, all clinical P. aeruginosa strains express ExoA. This exotoxin profoundly inhibits the expression of TNF, IL-6, IL-8, and IL-10 but does not affect the expression of IL-12 or IFN-γ (71). This effect allows P. aeruginosa to interfere with host defenses in lower respiratory tract infections by impairing local bacterial clearance. Similarly, the P. aeruginosa signal protein for quorum sensing, N-(3-oxododecanoyl)-l-homoserine lactone (HSL), which diffuses passively in the extracellular medium, inhibits immune and inflammatory responses (27, 72). HSL prevents activation and proliferation of T cells as well as LPS-dependent expression of TNF-α and IL-12 by monocytes. IL-12 is instrumental in the stimulation of IFN-γ production by T cells, which is important for the activation of macrophages and the induction of proinflammatory responses. Such inhibition by IL-12 could potentially alter the activation of T cells and production of IFN-γ. Furthermore, apoptosis induction by ExoS, exotoxin pyocyanin, ExoA, porins, or HSL inhibits the secretion of immune and proinflammatory mediators by target cells (57, 73–75).
S. pneumoniae induces two distinct pathways of apoptosis in dendritic cells (11). Apoptosis produced by the first pathway is rapid (significant DNA fragmentation as early as 3 h of culture) and caspase independent and requires live S. pneumoniae that release pneumolysin. A second distinct pathway of dendritic cell apoptosis in response to intact S. pneumoniae has also been demonstrated. This pneumolysin-independent caspase-dependent apoptosis is delayed (> 24 h), associated with terminal DC maturation, and is mediated by the interaction between subcapsular components in the bacteria and death receptors on the DC surface linked to MyD88 and likely a TLR.
Active modulation of immune and inflammatory responses by the pathogen has been clearly documented in a recent study by Boldrick et al. (76). Human peripheral blood monocytes showed different immune responses to killed Bordetella pertussis cells and to live cells from the same B. pertussis strain. For instance, with both killed and live bacteria, the genes encoding TNF-α, MIP-1, IL-1α, and IL-1β were rapidly activated. This effect persisted after exposure to killed B. pertussis cells but underwent prompt inhibition in monocytes exposed to live B. pertussis, suggesting an active mechanism used by the pathogen to influence host responses.
Variability of immune and inflammatory responses
The structure of the proinflammatory component of LPS, lipid A, varies among gram-negative bacteria of different species (77). Gram-negative bacteria, including Pseudomonas aeruginosa, can also modulate the structure of their LPS after invasion of host tissues to resist killing by the innate immune system and to maintain outer membrane integrity (78, 79). It has been reported that P. aeruginosa bacteria taken directly from the lungs of patients with cystic fibrosis (CF) show differences in the structure of the lipid moiety of their LPS compared with bacteria grown in culture (78). LPS from environmental isolates and from laboratory-adaptated strains of P. aeruginosa grown in conventional bacteria culture media has a penta-acylated structure (80). In contrast, isolates from the airways of CF-affected individuals synthesize hexa-acylated LPS (80). The LPS from the CF isolates is more effective at stimulating production of proinflammatory mediators. Human TLR4 responds to a low concentration of hexa-acylated LPS but requires much higher concentrations of penta-acylated LPS (80). Nevertheless, this adaptation is not found in P. aeruginosa LPS from bloodstream isolates or urinary tract isolates or from individuals with bronchiectasis associated with chonic lung disease (78).
Differences between gram-positive cocci and gram-negative bacteria in inducing expression profiles for immune and inflammatory response mediators have been convincingly documented (81–85), and differences have also been found across classes within a given bacterial species (86–91). In a murine model of pneumonia induced by a 100% lethal inoculum of S. pneumoniae (100% mortality within 3–4 days after direct intratracheal injection of the smallest inoculum used), expression by the lungs of the proinflammatory cytokines TNF-α, IL-1, and IL-6 as well as of the antiinflammatory cytokine IL-10 differed significantly across five clinical strains (two serotype 3 strains, two serotype 6 strains, and one serotype 19 strain) in terms of maximal concentrations achieved, time from inoculation to maximum cytokine expression, and total quantitative expression of cytokines (as measured by the area under the curve) (92). These differences were not dependent on the S. pneumoniae serotype: significant differences in cytokine expression profiles were seen both between the two serotype 3 strains and between the two serotype 6 strains used. These data and the studies discussed above suggest that immune and inflammatory responses induced by the pathogen or its components are dependent not only on the bacterial genus or species but also on the bacterial strain within a given species.
Pulendran et al. showed recently that LPS produced by two different gram-negative bacteria, E. coli and Porphyromonas gingivalis, induced different specific adaptive immune responses: with E. coli, there was a Th1 response with high levels of expression of IL-12 and IFN-γ, whereas P. gingivalis induced a Th2 response with increased expression of IL-5, IL-10, and IL-13 (91). Agrawal et al. demonstrated that activation of dendritic cells via TLRs did not always result in Th1 responses but could also induce a skew toward Th2 responses (93). These data suggested that although TLR4 and TLR5 ligands (E. coli LPS and flagellin, respectively) favored Th1 responses, TLR2 ligands clearly tilt the balance toward Th2 responses.
Huang et al. reported a study in which they used microarrays to compare in vitro gene expression profiles of human dendritic cells exposed to fungal infection by Candida albicans, bacterial infection by E. coli, or viral infection by the influenza A virus (an RNA virus) (94). The objectives of the study were to determine how dendritic cells discriminated among these three infection types and whether this discrimination reflected activation of genes specific for a given infection type. Of the 6800 genes on the microarray, 685 were modulated by E. coli, 531 by influenza A, and only 289 by C. albicans. These three pathogens from radically different species modulated a common set of 166 genes that included those involved in pathogen recognition, phagocytosis, encoding cytokines, chemokines, and receptors involved in recruiting dendritic cells and macrophages to infected sites, cytoskeleton modulation, encoding nuclear transcription factors (most notably NF-κB), intracellular signaling pathways, and the regulation of T cells, apoptosis, stress responses, free radical homeostasis, and antigen presentation. Although these data suggest the existence of shared pathways for pathogen recognition, expression of these genes varied across pathogens in terms of intensity and time from infection. For instance, the changes induced by C. albicans occurred later and were less marked overall than those induced by E. coli. A number of genes were activated by E. coli and inhibited by influenza A. In addition, examination of the various genes involved in innate and specific adaptive immune responses showed noticeable differences among the three pathogens. The innate immune response to influenza A was quantitatively modest as compared with that to E. coli and did not include neutrophil-activating genes. The specific adaptive immune response induced by influenza A was also clearly different from the response induced by E. coli. Thus, strong activation of antiviral genes (encoding IFN-α, IFN-β, and IFN-dependent chemokines) occurred in response to influenza A but not to E. coli. Finally, although a set of 166 genes was modulated by all three pathogens, 118 genes were specifically affected by E. coli, and 58 by influenza A. Surprisingly, C. albicans did not specifically modulate any genes in human dendritic cells. The role for the genes specifically modulated by E. coli and influenza A in the pathogenicity of these organisms remains undefined. Nevertheless, these findings are consistent with activation of pathogen-specific pathways.
Interactions between cell populations as well as the tissue site and cell type studied exert a major influence on gene expression in sepsis (76). Arcaroli et al. showed that modulation of gene expression by cultured neutrophils stimulated by LPS in vitro were modest compared with those observed in pulmonary neutrophils in a murine model of endotoxinemia (95). In vivo, 423 different genes were activated (at least twofold increase in expression as compared with controls) and 401 were inhibited (at least twofold decrease in expression as compared with controls). In contrast, activation in vitro was noted for only eight genes (also activated in vivo), and no genes were inhibited. These results document the importance of modulation by cell populations present in the lung. Finally, it remains to be determined whether this “single” recognition response truly benefits the host rather than the pathogens.
The role of cytokines in the innate immune response to respiratory tract infections differs in models in which different pathogens are used. In experimental pneumonia caused by the gram-negative bacterium Klebsiella pneumoniae or by the gram-positive bacterium S. pneumoniae, proinflammatory cytokines such as TNF-α and/or IL-1 are important for the clearance of bacteria from the lungs, whereas the antiinflammatory cytokine IL-10 impairs host defenses in these models (96–99). In contrast, the proinflammatory cytokines induced by P. aeruginosa in models of subacute and acute pneumonia impair bacterial clearance from the pulmonary compartment (100, 101).
Effect of cytokines on growth of bacteria
A number of studies have also reported the ability of cytokines to bind to bacteria, which can, in some cases, affect growth of the organism (69, 102–106). This binding may occur when the organism is either outside or inside a host cell. It has been shown that TNF-α, IL-1β, IL-2, GM-CSF, TGF-β, IL-6, IL-3, and epidermal growth factor (EGF) not only bind to bacteria but also stimulate their growth (72, 102–106). For example, IL-1β at concentrations as low as 10 ng/mL stimulated the growth of virulent strains of E. coli, and this effect could be abolished by IL-1ra (103, 104). IL-1 was found to bind to virulent, but not avirulent, strains of E. coli. Antiinflammatory cytokines or receptor antagonists, such as IL-4, IL-10, and IL-1ra, have also been reported to promote bacterial growth (105).
MODULATION OF THE NF-κB TRANSCRIPTION FACTOR SYSTEM
The Rel/NF-κB transcription factor system is central to the activation of numerous genes involved in immune responses, most notably those reflecting innate immunity, and in inflammatory responses such as those involving proinflammatory cytokines and their receptors, chemokines, adhesion molecules, or antiapoptosis proteins (107–112). A recent study by Huang et al. comparing in vitro gene expression profiles of dendritic cells exposed to three types of infection (C. albicans, E. coli, or influenza A) established a pivotal role for NF-κB (94). Of the 166 genes modulated by all three pathogens, those involved in encoding the Rel/NF-κB transcription factor system were consistently activated, and most of the other activated genes were regulated by Rel/NF-κB. Quantitative differences in expression of such genes in dendritic cells stimulated by E. coli versus C. albicans may contribute to the lower level of Rel/NF-κB activation by C. albicans (94).
Rapid translocation of Rel/NF-κB complexes to the nucleus occurs in response to a variety of bacterial components [such as lipopolysaccharide (LPS), peptidoglycan, pneumolysin, HSL, flagellin, slime glycoprotein, bacterial DNA] that bind to toll-like receptors (TLRs) (63, 65, 113–116). There are multiple Rel/NF-κB and inhibitory IκB-like proteins [Rel/NF-κB proteins: p65 (RelA), c-Rel, RelB, NFKB1 (p50 and its precursor p105), NFKB2 (p52 and its precursor p100); IκB proteins: IκBα, IκBβ, IκBγ, IκBε; IκB-like proteins: Bcl-3, p100, and p105]. The broad range of possible associations of such proteins into homodimers or heterodimers, the diversity of possibilities for inhibition of these dimers by the IκB-like proteins, and variability in their affinity for κB regulatory elements (the specific binding site for the Rel/NF-κB complexes) result in a vast array of possibilities for modulation of the Rel/NF-κB activation system. In addition, the affinity of the NF-κB complex for κB regulatory elements increases (as does transcriptional activity) with the degree of phosphorylation of the NF-κB units, most notably p65.
The importance of NF-κB for resistance to infection is illustrated by experiments showing that mice deficient in one of the Rel/NF-κB proteins are particularly vulnerable to viral (c-Rel–deficient mice/influenza virus), parasitic (c-Rel–deficient mice/Leishmania major; c-Rel, RelB, or NF-κB2–deficient mice/T. gondii), or bacterial (c-Rel, RelB, NF-κB1, or NF-κB2–deficient mice/L. monocytogenes; c-Rel or NF-κB1–deficient mice /S. pneumoniae) infections (117). Nevertheless, responses to infection with four different bacteria—L. monocytogenes, S. pneumoniae, H. influenzae, and E. coli—revealed striking differences in outcome between p50−/− and control mice (118). When infected with L. monocytogenes, p50−/− mice eradicated extracellular bacteria but were impaired in elimination of intracellular bacteria. Although p50−/− mice eliminated extracellular L. monocytogenes, control of the replication of an aggressive, extracellular, and gram-positive pathogen, S. pneumoniae, was compromised. Mice died of overwhelming sepsis. The defect in control of S. pneumoniae did not extend to the gram-negative pathogens H. influenzae and E. coli. These results highlight the complex involvement of p50 and NF-κB in transcriptional responses to infection. Of note, in the same model, enhanced susceptibility to L. monocytogenes and S. pneumoniae contrasted with the greater resistance of p50−/− mice to infection with the murine encephalomyocarditis (EMC) virus, a cytopathic picornavirus that can lead to a fatal encephalopathy.
Some microorganisms can affect signaling pathways that result in NF-κB activation and inhibit NF-κB activation. Inhibition of NF-κB activation allows the pathogen to interfere with the development of immune and inflammatory responses in a way that promotes its growth and dissemination (116) (Table 4). In this setting, the expression by lymphocytes and macrophage/monocytes of inflammatory mediators (TNF-α, IL-8, IL-12) and antiapoptotic proteins is profoundly depressed. Consequently, cellular apoptosis is enhanced, and release of proinflammatory cytokines is decreased, both of which can shield bacteria from the immune response (51, 117). Moreover, neutrophil apoptosis is important in the normal resolution phase of inflammation because it leads to functional down-regulation (119) and to the recognition and clearance of apoptotic neutrophils by macrophages (120). Ingestion of apoptotic neutrophils triggers macrophages to produce antiinflammatory cytokines and suppresses the generation of proinflammatory mediators (121, 122). Because apoptotic death is less proinflammatory, accelerated or inappropriate induction of neutrophil apoptosis could confer a further advantage on an invading pathogen. The pathogens induce cell death by a variety of mechanisms and include (a) pore-forming toxins, which interact with the host cell membrane and permit the leakage of cellular components (S. aureus α toxin, L. monocytogene listeriolysin O, E. coli α-hemolysin), (b) protein synthesis inhibitors (P. aeruginosa exoA, C. diphtheriae A-B toxin, Shigella dysenteriae Shiga and Shiga-like toxins, enterohemorrhagic E. coli verotoxin), (c) effector proteins delivered directly into host cells by a highly specialized type-III secretory system (Shigella spp., Salmonella spp., Yersinia spp., P. aeruginosa), (d) superantigens (S. aureus exotoxins A, B, D, and E, staphylococcal toxic shock syndrome toxin 1, Streptococcus pyogenes exotoxin A, B, and C), and (e) other modulators of host cell death (123, 124). In Pseudomonas-induced apoptosis, ExoS, ExoT, exotoxin pyocyanin, ExoA, cell surface porin, and HSL have been reported to manipulate the host apoptosis cascade (57, 73–75, 125). P. aeruginosa has been shown to induce apoptosis in macrophages, lymphocytes, neutrophils, mouse airway epithelial cells, human respiratory epithelial cells, and human endothelial cells. In Streptococcus pneumoniae–induced apoptosis, pneumolysin and H2O2 have been reported to manipulate the host apoptosis cascade (126).
Pathogens can use NF-κB activation to their own advantage (117). Although the activation of NF-κB in response to microbial stimuli is normally associated with the initiation of protective immune responses, some pathogens use the NF-κB system to enhance their own proliferation, survival, and dissemination within the host. Through NF-κB activation, these pathogens antagonize apoptotic signals. The importance of inhibition of apoptosis has been documented in a murine model of P. aeruginosa involving the induction of pneumonia in transgenic mice unable to express the CD95 or CD95 ligands. In these knockout mice, unlike control mice, lung epithelial cells do not undergo apoptosis, and susceptibility to P. aeruginosa is particularly marked, with 100% mortality within 48–72 h as compared with 0% in the controls (127). Such results indicate that activation of apoptotic mechanisms in infected cells may protect the host. Pathogens can be associated with apoptotic bodies, which are then internalized in the phagosomes and lysosomes of other cells, so that the pathogen is digested. In contrast, a pathogen that is internalized in the absence of concomitant apoptosis can block the maturation of the phagosome. Thus protected from the host immune system, the pathogen can survive or even divide within the host cell.
A number of additional pathogens use NF-κB activation to their own ends. The intracellular pathogen Chlamydia pneumoniae activates NF-κB, inhibiting apoptotis and increasing the survival of infected cells (128, 129). This manipulation or exacerbation of the inflammatory response by the pathogen results in increased monocyte recruitment to the infected site, an effect that promotes dissemination of the pathogen in the host. Of note, Listeria monocytogenes can infect newly recruited monocytes, causing the infection to become systemic (130).
The role of the pathogen in host–pathogen interactions is important but has received only limited attention. Multiple microbial virulence factors modulate host immune and inflammatory responses. These factors vary not only across genus but also across species or even across strains. In the future, sequencing of pathogen genomes will provide new information on virulence factors, their effects, and their respective importance. Pathogens can modulate virulence factor expression, both quantitatively and qualitatively, thereby affecting their pathogenicity. This modulation varies with the density of the bacterial population, the microenvironment, and the site of infection. Host immune and inflammatory responses induced by pathogens include shared responses and specific responses. Shared pathways for pathogen recognition show differences, however, in the intensity of, and time to, modulation of genes involved in innate and adaptive immune responses. It remains to be determined whether this shared pathogen recognition pathway truly benefits the host rather than the pathogen. Pathogen-specific activation pathways have also been identified. The complexity and specificity of the expression of virulence and pathogenicity by microorganisms, together with genetic polymorphisms in human hosts and differences in host responses between infection sites, conspire to complicate the unraveling of pathophysiological mechanisms underlying sepsis-induced organ failure and shock.
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