Pathogen-associated molecular pattern receptors play an important role in recognition of bacteria by the host. Toll-like receptors (TLRs) are a group of pathogen-associated molecular pattern receptors found in plants, insects, birds, and mammals and recognize bacterial components, including lipopolysaccharide (LPS), peptidoglycan, lipoproteins, flagella proteins, and DNA. Other receptors for bacterial ligands include the Nod proteins and other peptidoglycan recognition receptors. Ligand activation of TLRs induces proinflammatory cytokines such as interleukin 1 (IL-1), IL-6, and IL-8 and tumor necrosis factor alpha (TNF-α) (61).
TLR-4 is involved in the recognition of the lipid A portion of bacterial LPS and plays critical roles in the host response to Salmonella (38, 94). LPS activation of TLR-4 mediates the inflammatory response of macrophages to Salmonella enterica serovar Typhimurium infection (83, 85). Recognition of LPS by TLR-4 is highly complex, requiring at least three other proteins (CD-14, LPS binding protein [LBP], and MD-2) for full activation. TLR-4 recognition of LPS depends on MD-2, whereas the sensitivity of the receptor complex to LPS is markedly increased by CD14 and LBP (91, 102). In addition to LPS recognition, MD-2 is also important in LPS signaling and translocation of the TLR-4 to the cell surface as a MD-2 TLR-4 complex (37, 106).
C3H/HeJ and C57BL/10ScCr mice are endotoxin resistant and are very useful for research into gram-negative infections because they do not respond to many of the effects of LPS. Genetic studies have shown that C3H/HeJ mice have a Pro712His dominant-negative mutation in TLR-4, whereas C57BL/10ScCr mice have a TLR-4 gene deletion (79, 80, 107). TLR-4-deficient mice are impaired in their ability to mount innate immune responses to Salmonella infections, including the production of TNF-α and other inflammatory cytokines. In C3H/HeJ mice, TNF-α synthesis is regulated both at the transcriptional and posttranscriptional level, with transcription of the TNF-α gene being reduced and mRNA translation completely inhibited (8). Surprisingly, the inability of TLR-4-deficient mice to respond to purified LPS is not absolute and can be bypassed by pretreating cultured macrophages with gamma interferon (IFN-γ) prior to LPS stimulation (8, 9). This observation can explain why C3H/HeJ mice become susceptible to the TNF-α-mediated toxic effects of purified LPS when they are previously infected with live salmonellae (59). In fact, the presence of IFN-γ has been documented in sera and tissues of mice during systemic salmonellosis, thus enabling the infected mice to produce TNF-α in response to LPS. Macrophages from endotoxin nonresponder C3H/HeJ mice, C57BL/10ScCr mice, and TLR4–/– mice produce TNF-α and IL-6 in response to whole Salmonella microorganisms. Injection of Salmonella microorganisms into TLR4–/– also results in the presence of TNF-α and IL-6 in plasma (41). These results suggest the involvement of receptors other than TLR-4 in the induction of cytokines in response to Salmonella (22, 41).
The role of the other TLRs in infection, such as TLR-9 and TLR-5, is unclear. TLR-9 mediates the host inflammatory response to bacterial DNA (7, 27, 92). No information is yet available to suggest that animals or patients with a functionally defective TLR-9 have altered susceptibility to Salmonella infection. TLR-5 is involved in the host response to Salmonella infections by responding to bacterial flagellin protein (26, 66, 89). TLR-5 recognizes a region of flagellin that is conserved in S. enterica serovar Typhimurium, Pseudomonas aeruginosa, and Listeria monocytogenes (33). This region forms a binding site that spans amino acids 386 through 407 (64). TLR-5 is expressed on the surface of epithelial cells (1) and is likely to be one of the primary detection mechanisms for enteric infections with bacteria such as S. enterica serovar Typhimurium. MOLF/Ei mice have defective TLR-5 function that might be at the basis of their increased susceptibility to Salmonella infections (89).
TLR activation recruits adapter proteins that facilitate the activation of the transcription factor NfκB, phosphoinositide 3-kinase, and the mitogen-activated protein kinases JNK, p38, and ERK. These adapter molecules contain a TIR domain that interacts with the TIR domain of the TLRs. Five adapter molecules have been identified so far: MyD88, TIRAP/Mal, TRIF/TICAM, TRAM, and SARM. TLR recognition of bacterial pathogens, by the formation of both homo- and heterodimers and the use of different combinations of adapter molecules recruited to the receptor complex may diversify the repertoire of TLR-mediated responses. Activation of different signaling pathways will result in a range of transcriptional consequences and may alter the profile of cytokines produced in response to Salmonella infection.
Avoidance of phagocyte killing and escape from infected cells and from pathological lesions is a key feature of Salmonella growth in the tissues and is likely to represent one of the mechanisms used by the bacteria to counteract and evade the host immune response.
Effective immunity against the bacterium therefore relies on the ability of the host to recruit phagocytes in the tissues and to enhance the antibacterial functions of these inflammatory cells. Both these processes require the concerted actions of cytokine networks throughout the infection. TNF-α, IFN-γ, IL-12, IL-15, and IL-18 are needed for the full expression of innate host resistance to Salmonella (Fig. 1) (20, 29, 30, 39, 45, 46, 47, 48, 50, 53, 67, 71, 99).
In addition to its involvement in the formation of macrophage-rich pathological lesions, TNF-α binding to TNFR55 is needed for the localization of vesicles containing NADPH oxidase to vacuoles containing Salmonella. This process is inefficient in macrophages lacking TNFR55, leading to impairment in bacterial killing (104).
Evidence for a role of TNF-α in resistance to human typhoid fever comes from genetic studies performed in South Vietnam, where the polymorphic allele variant of the human TNF-α gene TNFA*2 (–308) was associated with susceptibility to typhoid fever, whereas TNFA1 (–308) was associated with resistance to the disease (17).
IL-12, IL-18, and IL-15 are produced mainly by macrophages and dendritic cells after exposure to Salmonella and positively modulate IFN-γ production. NK cells, macrophages, and neutrophils are the main source of IFN-γ during the early stages of a Salmonella infection (40) ( Fig. 1). IFN-γ can be produced in response to IL-12 and IL-18 by rag1 –/– mice and scid mice that lack T cells (45, 46, 47, 81). IL-15 mediates IFN-γ production by NK cells and γ δ T cells and is necessary for resistance to salmonellosis (30, 73).
Genetic mutations that result in partial or total loss of function of IL-12, IFN-γ, or their respective receptors and signaling molecules lead to increased susceptibility to salmonellosis in humans. Complete IFNγR and complete STAT-1 deficiencies result in severe infections with a poor outcome, whereas complete IL-12R, complete IL-12p40, partial IFNγR, and partial STAT-1 deficiency all have a milder clinical phenotype (16). Salmonella infections in these immunocompromised individuals are extraintestinal, ranging from gastroenteritis to near-fatal septicemia. These infections are recurrent and difficult to treat with conventional antibiotic therapy. In some patients with IL-12 component defects, combining IFN-γ therapy with antimicrobial therapy can lead to bacteriological cure (5, 14, 77).
IL-4 and IL-10 and the Balance between Activation and Suppression Signals.
IL-10 is produced during salmonellosis and it continuously down-regulates the anti-Salmonella functions of phagocytes in the RES (6, 78). In fact, in vivo neutralization of IL-10 enhances host resistance to Salmonella (6).
Clearance of the organisms from the RES requires the CD28-dependent activation of CD4+ TCR-αβ + T cells (60, 63, 74). In this phase of the infection there is an increase in IFN-γ-producing CD4+ and CD8+ T cells with an activated phenotype (CD44high, CD62Llow) (62). This could lead to the assumption that production of IFN-γ by T cells increases the activation of phagocytic cells and mediates the elimination of intracellular bacteria from the tissues. However, administration of anti-IFN-γ-neutralizing antibodies in the later stages of salmonellosis does not inhibit the elimination of the bacteria from the tissues (69). Similarly, TNF-α does not appear to be needed for bacterial clearance (53). Therefore, the mechanisms used by CD4+ TCR-αβ + T cells to clear the infection remain elusive.
Other key mediators in the development of Th1 T-cell responses have been identified with gene-targeted mice that survive and clear infections with salmonellae of low virulence. For example, the absence of B cells in Igh-6 –/– mice results in reduced activation, frequency and long-term persistence of IFN-γ- and IL-2-producing CD4+ and CD8+ T cells that is paralleled by increased production of IL-4 by T cells (101).
Cytokines as Effectors of Acquired Resistance in Secondary Infections.
After salmonellae are cleared from the tissues in the late stages of primary infections, long-lasting immunity persists in the host, resulting in resistance to reinfection with virulent Salmonella. Acquired immunity coincides with the presence of Salmonella-specific T cells able to produce Th1-type cytokines (IL-2 and IFN-γ) upon antigen stimulation. Similarly to what is seen in primary infections, cytokine networks are essential in the expression of acquired resistance.
TNF-α appears to be required throughout the course of a secondary infection in immunized mice. In fact, administration of anti-TNF-α antibodies after the secondary infection has been brought under control induces the relapse of bacterial growth and can lead to the death of the infected animals (53). Conversely, neutralization of IFN-γ in the late stages of a secondary Salmonella infection does not affect the course of the disease (69).
The genes for mammalian cytokines can be cloned into suitable vectors and expressed in Salmonella as functional proteins. The in vivo production of cytokines by Salmonella carriers can have therapeutic applications and can modulate immune functions in the host. For example, Salmonella expressing human IL-β can protect mice against lethal gamma irradiation (12); oral administration of Salmonella expressing transforming growth factor β (TGF-β) reduces carrageenin-induced inflammation in mice, reduces IL-2 and IFN-γ production in the lymph nodes that drain the sites of inflammation, and enhances IL-10 secretion (32). Expression of cytokines in Salmonella can also affect the ability of the host to control the infection. For example, salmonellae expressing IL-4 show enhanced growth and survival in mice and are killed less efficiently by macrophages (15). Conversely, expression of IL-2 in Salmonella results in enhanced bacterial clearance from the host tissues (2).
The possibility to modulate antigen-specific immune responses by expressing cytokines in Salmonella is illustrated by the increase in Salmonella-specific IgA responses induced by administration of IL-5-expressing bacteria (108).
In addition to playing a crucial role in host defense, cytokines may also mediate the events leading to the death of animals infected with Salmonella.
Rapidly lethal systemic infections can be induced experimentally in mice by using a combination of innately susceptible animals and virulent salmonellae, administering high initial doses of bacteria to innately resistant mice, or suppressing the immune system of the animal by genetic or immunological manipulations. Death occurs when the microbial load in the liver and spleen reaches approximately 108 to 109 live bacteria (13).
The same cytokines that are responsible for endotoxic shock are elevated in the late stages of lethal Salmonella infections, indicating that the toxicity of Salmonella LPS may actually be contributing to the death of the host. The observation that Salmonella waaN mutants, which biosynthesize LPS molecules lacking a fatty acyl chain, can grow to unusually high numbers with most animals surviving the infection seems to support these views (38). The waaN mutants induce lower levels of TNF-α, IL-1, and iNOS responses than wild-type salmonellae. This may further suggest that death in systemic salmonellosis directly depends on the production of some cytokines known to be involved in LPS-induced shock.
There is also experimental evidence that questions the involvement of LPS and cytokines in death due to enteric salmonellosis. First, TNF-α, one of the principal mediators of endotoxic shock, is undetectable in the late stages of lethal Salmonella infections in mice and calves (44, 76). Second, mice infected with Mycobacterium tuberculosis BCG are hypersusceptible to the lethal effects of bacterial LPS or TNF-α; such BCG-sensitized mice die of a Salmonella infection with the same terminal bacterial numbers in the RES as unsensitized controls (90). Third, the premortem bacterial loads in LPS nonresponder and LPS responder mice are similar (P. Mastroeni, unpublished work).
Taken together, the contrasting experimental evidence available so far seems to indicate that LPS toxicity and cytokine networks may contribute to the lethal effects of high bacterial loads in Salmonella infections. However, the LPS-mediated events that cause the death of animals infected with Salmonella are probably distinct from the events seen in the classical septicemic models of endotoxic shock.
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