Cytokines in Salmonellosis

PIETRO MASTROENI^* AND CLARE BRYANT^*

[SECTION EDITORS: FERRIC FANG AND MARTIN KAGNOFF]

Posted December 29, 2004

Bacterial Infection Group, Centre for Veterinary Science, Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, United Kingdom

Corresponding authors. Pietro Mastroeni: Phone, 44 1223 765800; Fax, 44 1223 337610; E-mail, This e-mail address is being protected from spambots. You need JavaScript enabled to view it . Clare Bryant: Phone, 44 1223766232; Fax, 44 1223 337610; E-mail, This e-mail address is being protected from spambots. You need JavaScript enabled to view it .

INTRODUCTION

Salmonella enterica is a facultative intracellular pathogen that resides within phagocytes and dendritic cells and can sometimes be seen in B cells. The bacteria also interact with M cells and epithelial cells in the gut. The recruitment and activation of phagocytic cells in infected tissues and the induction of T-cell- and B-cell-dependent acquired immunity are crucial for the control and resolution of Salmonella infections. These complex processes require the interaction of bacteria with a multitude of cell surface receptors and the controlled production of soluble mediators. The mechanisms of cytokine induction in response to Salmonella and the role of cytokine networks in Salmonella infections are the main foci of this chapter.

RECEPTORS AND SIGNALING EVENTS INVOLVED IN CYTOKINE PRODUCTION IN RESPONSE TO SALMONELLA

Toll-like Receptors Are Stimulated by Salmonella, Activate Cytokine Production, and Mediate Host Resistance to the Bacterium

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).

Currently 10 different human TLRs have been sequenced (82, 86), and functional ligands have been described for TLR-1, -2, -3, -4, -5, -7, and -9. TLRs may act in synergy with each other. For example, costimulation of TLR-2 and TLR-4 by their respective ligands results in a synergistic stimulation of host responses (10). The TLRs most likely to be involved in recognition of salmonellae by the host include TLR-1, -2, -4, -5, -6, and -9.

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).

TLR-2 responds to bacterial proteins (4, 11, 31) and peptidoglycan (21, 88, 93, 95, 110) and activation of this receptor has principally been associated with recognition of gram-positive bacterial ligands and resistance to infections with Mycobacterium leprae (36) and staphylococci (93). Functionally TLR-2 receptors can dimerize with other TLRs such as TLR-1 or TLR-6. This can explain how host cells are able to respond discretely to a broad range of pathogen ligands specifically (24, 28, 75, 96, 97). A further complexity in the function of TLR-2 is its ability to heterodimerize with receptors other than TLRs. For example, TLR-2 interaction with the dectin receptor allows TLR-2 to recognize fungal components. TLR-2 is up-regulated during Salmonella infection in vivo although the functional significance of this is currently unclear (100). The involvement of TLR-2 in cytokine production has been shown in cultured macrophages and in mice exposed to Salmonella. In fact, TNF-α and IL-6 responses are reduced in mice lacking both TLR-2 and TLR-4 as compared with mice lacking only TLR-4 (41). TLR4^–/– TLR2^–/– mice and macrophages harvested from these animals can still produce small amounts of TNF-α and IL-6 in response to Salmonella, indicating a possible role for other receptors in cytokine production in response to whole Salmonella microorganisms (41).

The ability of TLR-4-deficient mice to produce TNF-α in response to Salmonella can explain also the observation that C3H/HeJ mice develop acquired immunity and become resistant to challenge with virulent salmonellae if they have previously been immunized with a live attenuated Salmonella vaccine (18). The expression of this acquired resistance requires TNF-α production, further indicating that this cytokine can be produced in vivo in the absence of functional TLR-4.

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).

Signaling Events: Activation of TLRs and Production of Cytokines

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.

CYTOKINES AND HOST RESISTANCE TO SALMONELLA INFECTIONS

Invasion of the Gut Triggers the Production of Inflammatory Cytokines

Salmonella infects animals and humans by the oral route. After ingestion, a proportion of the organisms survives the low pH of the stomach and reaches the distal ileum and the cecum. Invasion of epithelial cells and M cells in the Peyer’s patches is one of the mechanisms used by Salmonella to gain access to the systemic compartment. During this process, the SPI-1-encoded SipB protein triggers the activation of intracellular caspase-1 within resident macrophages. Caspase-1, a member of a family of cysteine proteases, induces apoptosis in the infected macrophages, resulting in the escape of Salmonella from these cells. Caspase-1 also directly cleaves the proinflammatory cytokines IL-1β and IL-18 to produce bioactive cytokines that enhance local inflammation and infiltration of polymorphonuclear phagocytes (PMNs). SipB-mediated activation of caspase-1 results in enhanced colonization of the Peyer's patches and mesenteric lymph nodes (65). Salmonella infection can elicit the production of inflammatory CC and CXC chemokines in the gut (111). Chemokine production appears to be positively and negatively modulated by defined bacterial effector proteins. Experiments with bovine ligated ileal loops show that the expression of the chemokines GRO-α, GRO-γ, IL-8, and GCP-2 is enhanced by products of the Salmonella sipA, sopABDE2 genes (111). In vitro experiments in intestinal cells also suggest that the Salmonella SspH1 and SptP proteins can inhibit NF-κB gene expression, resulting in the down-regulation of IL-8 production (25).

Cytokines as Mediators of Innate Immunity to Salmonella

General.

Once the invasion process has been completed, Salmonella reaches the blood and is transported to an intracellular location within the reticuloendothelial system (RES). Salmonella can also invade the gut and distribute to the RES by being transported in the blood by CD18-expressing cells (105). In the RES the bacteria initially associate with resident macrophages. The early interaction of the bacteria with resident macrophages is followed by the migration of inflammatory PMNs in the tissues that form discrete pathological lesions surrounded by normal tissue. The PMN-rich lesions are later infiltrated by mononuclear phagocytes and acquire the appearance of macrophage-rich granulomas.

Phagocytic cells within pathological lesions control the growth of Salmonella by using reactive oxygen intermediates (ROI [generated via the phagocyte NADPH-oxidase]) and reactive nitrogen intermediates (RNI [generated via the inducible nitric oxide synthase, iNOS, encoded by the NOS2 gene]) (43, 49).

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).

Fig. 1Cytokine networks in innate immunity to Salmonella infections. Red arrows and red text indicate activation pathways. Black arrows and black text indicate inhibitory pathways. Salmonellae infect resident macrophages (RM) and induce production of TNF-α, IL-12, and IL-18 from these cells. TNF-α mediates the recruitment of inflammatory macrophages (IM) in the tissues and the formation of granulomas. IL-12 and IL-18 trigger IFN-γ production from NK cells. IFN-γ activates macrophages, increasing their antibacterial functions. IL-4 production by NK1.1⁺ TCR⁺ cells as a consequence of interactions with macrophages (probably via CD1 molecules) can exert a negative effect on macrophage functions. IL-10 can also potentially counteract macrophage activation by IFN-γ. IL-10 and IL-4 production is down-regulated by IL-12 and IFN-γ.

TNF-α Mediates the Formation of Granulomas and Enhances Salmonella Killing by Macrophages.

TNF-α is an essential mediator in immunity to Salmonella infection. Mice treated with anti-TNF-α antibodies prior to Salmonella infection or gene-targeted mice lacking TNF receptor 55 (TNFR55^–/– mice) become extremely susceptible to salmonellosis, and bacterial growth in their tissues proceeds unrestrained. TNF-α is dispensable for the control of bacterial growth in the first few days of the infection and for the formation of early PMN-rich pathological lesions in the infected tissues. However, TNF-α becomes essential for host resistance later in infection when it is required for the formation and persistence of mature macrophage-rich granulomas. In the absence of biologically active TNF-α or functional TNFR55, poorly organized granulomas and reduced numbers of inflammatory mononuclear cells are seen in the infected tissues (20, 44, 48), resulting in necrosis and widespread dissemination of salmonellae extracellularly and to nonphagocytic cells (48). TNF-α is constantly required for the persistence of the granulomas once these have formed in the tissues (53).

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).

IFN-γ and Macrophage Activation in Salmonella Infections.

IFN-γ is a crucial mediator of host resistance to Salmonella infection. Experiments in IFNγ ^–/– mice, IFNγ ^–/– receptor (IFNγR^–/–) mice, or in mice injected with neutralizing anti-IFN-γ antibodies have shown the absolute requirement for this cytokine in immunity to Salmonella (29, 68, 71).

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).

IFN-γ activates mouse macrophages to kill intracellular Salmonella, probably by enhancing phagosome-lysosome fusion and increasing the production of nitric oxide derivatives (35). IFN-γ is also needed for the formation of discrete granulomas in the tissues (35, 47, 48, 52). In the absence of biologically active IL-12 or IL-18, IFN-γ levels in serum and tissues are reduced and macrophage activation is severely impaired. The lack of phagocyte activation results in overwhelming bacterial growth leading to death of the host (47). Under these experimental conditions there is widespread infiltration of macrophages in the tissues that are not organized in discrete localized lesions (47).

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-4 and IL-10 produced during a primary Salmonella infection and could potentially exert detrimental effects on the infected host because of their ability to suppress macrophage functions (78). IL-4 is produced by NK1.1⁺ TCR-αβ cells at the early phase of the disease, probably as a consequence of CD1-mediated cell interactions with phagocytic or dendritic cells (70). Expression of the murine IL-4 gene in Salmonella exacerbates the course of the infection and impairs macrophage killing of the bacteria (15). Conversely, the absence of IL-4 confers increased resistance to Salmonella infection as indicated by the delayed time to death and reduced abscess formation seen in IL4^–/– mice compared with IL4^+/+ mice (19). Furthermore, macrophage functions and IL-12 production are enhanced following IL-4 neutralization by specific antibodies and are suppressed by exposure to rIL-4 (34, 70).

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).

It appears that during a Salmonella infection there is a fine balance between the phagocyte activation systems involving IL-12, IL-18, IL-15, and IFN-γ and the inhibitory signals delivered by IL-10 and IL-4. Whenever IL-12 is deficient, the levels of IL-10 and IL-4 rise, resulting in the inhibition of the antibacterial functions of phagocytes (47). Conversely, neutralization of IL-4 and IL-10 by specific antibodies results in increased production of IL-12, TNF-α, and IFN-γ, increased numbers of γ δ T cells, and enhancement of macrophage functions (6, 70).

Cytokines and Cytokine Receptors in Immunosuppression

Animals that are infected with Salmonella and carry high bacterial loads in the tissues transiently show reduced ability to mount effective antibody responses to several antigens (e.g., tetanus toxoid, sheep red blood cells) and have impaired B- and T-cell responses to mitogens. The transient immunosuppression is mediated by macrophages through the production of nitric oxide, requires IFN-γ and IL-12, and can be reversed by administration of rIL-4 (3, 42, 87).

Salmonella can directly inhibit T-cell proliferation by suppressing the expression of the β- and γ-chains of the IL-2 receptor on T cells. The inhibition is mediated by a 745-amino-acid protein designated STI (Salmonella typhimurium-derived inhibitor) (54, 55, 56, 57, 58).

Clearance of a Primary Infection

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 (CD44^high, CD62L^low) (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.

Cytokines and Acquired Immunity to Salmonella

The Development of IL-2- and IFN-γ-Producing Antigen-Specific T Cells.

Primary infections with live Salmonella induce the activation of T-cell immunity that has predominantly a Th1-type profile. During infection there is an increase of CD4⁺ and CD8⁺ T cells that produce IL-2 and IFN-γ. These cells persist long after clearance of a primary infection and can be reactivated by exposure to Salmonella antigens. Conversely, exposure to killed Salmonella or some bacterial components (e.g., porins) leads to the activation of IL-4-dominated T helper 2 (Th2)-type responses (23, 98).

Macrophages and dendritic cells exposed to Salmonella produce an array of cytokines (IL-1, IL-6, IL-12, IL-18, and TNF-α) capable of contributing to the development and expression of acquired immune responses. However, the in vivo relevance of T-cell stimulatory cytokines in the initiation and amplification of IL-2- and IFN-γ-dominated antigen-specific acquired T-cell responses has been difficult to study because mice lacking these cytokines are extremely susceptible to primary Salmonella infections (46, 52).

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.

In secondary infections, both TNF-α and IFN-γ are needed for host resistance to infection and for the formation of discrete granulomas. IFN-γ and IL-12 are required for macrophage activation (47, 52). The histological picture occurring in mice lacking biologically active TNF-α and IFN-γ is similar in primary and secondary infections (52).

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).

MODULATION OF HOST RESISTANCE BY EXPRESSION OF CYTOKINES IN RECOMBINANT SALMONELLA

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).

Delivery of cytokines by attenuated Salmonella can be a feasible therapeutic treatment in infectious diseases or tumors. Recombinant Salmonella expressing IL-2, IFN-γ, and macrophage migration inhibitory factor can enhance host resistance in mice challenged with Leishmania major (109). Administration of salmonellae carrying eukaryotic plasmids that express IL-4 or IL-18 can enhance the antibody response against Echinococcus granulosus. The IL-4-expressing plasmid can also reduce in vivo growth of the parasite (84). Salmonellae carrying the IL-4- or IL-18-expressing plasmids can prolong the survival time of mice inoculated with B16F1 melanoma cells (84).

Thus, expression of cytokines in Salmonella can be an attractive tool to study the function and relative importance of cytokine networks at the sites of bacterial infection and may have a potential for the treatment of infection and cancer.

CYTOKINES, LPS TOXICITY, AND DEATH

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).

It is not clear whether the death of animals infected with Salmonella is due to endotoxic mediated septic shock. This is a syndrome characterized by bacteremia, clinical signs of infection, hypoxia, hypoperfusion, and disseminated intravascular coagulation leading to multiorgan failure. Endotoxic shock is usually caused by extracellular gram-negative bacteria, such as Escherichia coli, Haemophilus, and Neisseria, and is mediated by the endotoxic effect of bacterial LPS. Cytokines such as TNF-α, IL-1β, IL-6, IL-12, and IFN-γ, platelet-activating factor, chemokines, and eicosanoids are produced by host cells in response to LPS and are involved in the pathogenesis of endotoxic shock. These factors mediate tissue inflammation, intravascular aggregation of neutrophils, endothelial damage with increased permeability, vascular dilation, and coagulation disorders leading to circulatory shock and death (103).

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.

The lethal effects induced by Salmonella LPS are mediated by mechanisms more complex that those involved in endotoxic shock induced by other gram-negative bacteria such as E. coli. In fact, TNF-α and lymphotoxin-α play a central role in lethality due to E. coli LPS, while the lethal effect of Salmonella LPS also involves other cytokines such as IFN-γ, IL-12, IL-1, and IL-18 (47, 72). For example, mice treated with anti-IL-12 antibodies during Salmonella infections have reduced levels of IFN-γ, can carry very high bacterial loads (10 to 100 times greater than the ones usually seen in premortem animals), and yet show very mild symptoms of infection (47). Furthermore, mice infected with Salmonella become hypersusceptible to LPS, and this increased susceptibility can be reversed by administration of anti-IL-12 or anti-TNF-α antibodies (47, 51).

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.