Pretreatment with the Gram-positive bacterial cell wall molecule peptidoglycan improves bacterial clearance and decreases inflammation and mortality in mice challenged with Pseudomonas aeruginosa

The objective of this study was to determine if inflammatory tolerance and enhancement of innate immune function could be induced by the Gram-positive cell wall component peptidoglycan (PGN). Male mice (C57BL6/J or C3H/HeJ, 8-12 weeks of age) were given intraperitoneal injections of 1mg PGN on 2 consecutive days. The mice were then challenged with lipopolysaccharide (LPS) or live Pseudomonas aeruginosa (1 x 10(8) colony-forming units) 2 days after the second pretreatment. Mice pretreated with PGN had diminished plasma concentrations of TNFalpha and IFNgamma and elevated concentrations of IL-10 in response to a subsequent LPS or Pseudomonas challenge when compared to untreated controls. Bacterial clearance was improved in mice pretreated with PGN, and mortality in response to a subsequent Pseudomonas challenge was significantly attenuated. PGN pretreatment of LPS-unresponsive mice (C3H/HeJ) verified that the effect of PGN pretreatment was not due to any LPS contamination. We have previously demonstrated that PGN pretreatment induced resistance to a Gram-positive bacterial challenge. The present study extends those results by showing that exposure to the Gram-positive bacterial cell wall component peptidoglycan also induces cross-tolerance to LPS and non-specifically enhances innate immune function in that PGN-pretreated mice had increased resistance to Gram-negative bacterial challenge.     

Feasibility of prophylaxis and therapy against Gram-negative infections by human monoclonal antibodies

Abstract In a murine model of Gram-negative sepsis, we have shown that the prophylactic application of human monoclonal antibodies (HmAbs) with specificity for lipopolysaccharides (LPS) of Pseudomonas aeruginosa protected against bacterial infection. In this paper we show that the therapeutical application of 5 μg of these HmAbs up to 6 h after challenge with a lethal dose of live P. aeruginosa results in a protection rate of 70–90%. Administration 18 h after bacterial challenge, diminished the protection to 43% survival rate. Furthermore, using a mixture of HmAbs recognizing a total of six different P. aeruginosa serotypes, no interference in their protective capacities was found. Finally, these HmAbs also protected galactosamine-sensitized mice against lethal challenge with LPS. Our data show that the described HmAbs confer bactericidal activity as well as anti-endotoxic activity in vivo.     

Restoration of natural IgM production from liver B cells by exogenous IL-18 improves the survival of burn-injured mice infected with Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most common bacterium of postburn infection. In the present study we investigated the immune mechanism of susceptibility to this type of postburn infection and also examined the efficacy of IL-18 treatment. C57BL/6 mice were challenged with P. aeruginosa on day 7 after burn injury. Although the burn-injured mice showed a poor survival rate after bacterial challenge, they retained their IFN-gamma production. The burned mice showed lower serum IgM levels and a poor IgM response following P. aeruginosa challenge in comparison with the sham mice, whereas IL-18 treatment after burn injury (alternate day injections for 1 wk) greatly improved the serum IgM levels, which are P. aeruginosa-independent natural IgM before bacterial challenge, thereby increasing the survival rate after the challenge. IL-18 treatment also induced specific IgM to P. aeruginosa in the sera 5 days after bacterial challenge in the burned mice. Interestingly, CD43(+)CD5(-)CD23(-)B220(dim) cells, namely B-1b cells, increased in the liver after the IL-18 treatment and were found to actively produce IgM in vitro without any additional stimulation. Furthermore, the IL-18 treatment up-regulated the neutrophil count and the C3a levels in the blood as a result of the increased IgM level, which may thus play a critical role in the opsonization and elimination of any invading bacteria. IL-18 treatment for the burned mice and their resultant natural IgM production were thus found to strengthen the host defense against P. aeruginosa infection.     

TLRs 2 and 4 are not involved in hypersusceptibility to acute Pseudomonas aeruginosa lung infections

TLRs are implicated in defense against microorganisms. Animal models have demonstrated that the susceptibility to a number of Gram-negative pathogens is linked to TLR4, and thus LPS of many Gram-negative bacteria have been implicated as virulence factors. To assess the role of this pathogen-associated molecular pattern as it is exposed on intact Pseudomonas aeruginosa, the susceptibility of mice lacking TLR4 or both TLR2 and TLR4 was examined in a model of acute Pseudomonas pneumonia. These mutant mice were not hypersusceptible to the Pseudomonas challenge and mounted an effective innate response that cleared the organism despite low levels of TNF-alpha and KC in the airways. Bacterial and neutrophil counts in the lung were similar in control and TLR-deficient mice at 6 and 24 h after infection. MyD88(-/-) mice were, however, hypersusceptible, with 100% of mice dying within 48 h with a lower dose of P. aeruginosa. Of note there were normal levels of IL-6 and G-CSF in the airways of TLR mutant mice that were absent from the MyD88(-/-) mice. Thus, the susceptibility of mice to P. aeruginosa acute lung infection does not go through TLR2 or TLR4, implying that Pseudomonas LPS is not the most important virulence factor in acute pneumonia caused by this organism. Furthermore, G-CSF treatment of infected MyD88(-/-) mice results in improved clearance and survival. Thus, the resistance to infection in TLR2/TLR4(-/-) mice may be linked to G-CSF and possibly IL-6 production.     

Redundant Toll-like receptor signaling in the pulmonary host response to Pseudomonas aeruginosa

Activation of pulmonary defenses against Pseudomonas aeruginosa requires myeloid differentiation factor 88 (MyD88), an adaptor for Toll-like receptor (TLR) signaling. To determine which TLRs mediate recognition of P. aeruginosa, we measured cytokine responses of bone marrow cells from wild-type mice and mice lacking TLR2 (TLR2(-/-)), TLR4 (TLR4(-/-)), TLR2 and TLR4 (TLR2/4(-/-)), or MyD88 (MyD88(-/-)) to wild-type P. aeruginosa and to fliC P. aeruginosa, which lacks the TLR5 ligand flagellin. Mice also were challenged with aerosolized bacteria to determine cytokine responses, lung inflammation, and bacterial clearance. TNF induction required MyD88 and was absent in TLR2/4(-/-) cells in response to fliC but not wild-type P. aeruginosa, whereas TLR2(-/-) cells exhibited augmented responses. In vivo, TLR4(-/-) mice responded to wild-type P. aeruginosa with reduced cytokine production and inflammation, but intact bacterial clearance, while TLR2(-/-) mice had partially impaired cytokine responses and delayed bacterial killing despite normal inflammation. When challenged with fliC, MyD88(-/-) mice failed to mount early cytokine and inflammatory responses or control bacterial replication, resulting in necrotizing lung injury and lethal disseminated infection. TLR4(-/-) and TLR2/4(-/-) mice responded to fliC infection with severely limited inflammatory and cytokine responses but intact bacterial clearance. TLR2(-/-) mice had partially reduced cytokine responses but augmented inflammation and preserved bacterial killing. These data indicate that TLR4- and flagellin-induced signals mediate most of the acute inflammatory response to Pseudomonas and that TLR2 has a counterregulatory role. However, MyD88-dependent pathways, in addition to those downstream of TLR2, TLR4, and TLR5, are required for pulmonary defense against P. aeruginosa.     

Intrapulmonary TNF gene therapy reverses sepsis-induced suppression of lung antibacterial host defense

Sepsis syndrome is frequently complicated by the development of nosocomial infections, particularly Gram-negative pneumonia. Although TNF-alpha (TNF) has been shown to mediate many of the pathophysiologic events in sepsis, this cytokine is a critical component of innate immune response within the lung. Therefore, we hypothesized that the transient transgenic expression of TNF within the lung during the postseptic period could augment host immunity against nosocomial pathogens. To test this, mice underwent 26-gauge cecal ligation and puncture (CLP) as a model of abdominal sepsis, followed 24 h later by intratracheal (i.t.) administration of PSEUDOMONAS: aeruginosa. In animals undergoing sham surgery followed by bacterial challenge, PSEUDOMONAS: were nearly completely cleared from the lungs by 24 h. In contrast, mice undergoing CLP were unable to clear P. aeruginosa and rapidly developed bacteremia. Alveolar macrophages (AM) recovered from mice 24 h after CLP produced significantly less TNF ex vivo, as compared with AM from sham animals. Furthermore, the adenoviral mediated transgenic expression of TNF within the lung increased survival in CLP animals challenged with PSEUDOMONAS: from 25% in animals receiving control vector to 91% in animals administered recombinant murine TNF adenoviral vector. Improved survival in recombinant murine TNF adenoviral vector-treated mice was associated with enhanced lung bacterial clearance and proinflammatory cytokine expression, as well as enhanced AM phagocytic activity and cytokine expression when cultured ex vivo. These observations suggest that intrapulmonary immunostimulation with TNF can reverse sepsis-induced impairment in antibacterial host defense.     

TNF-alpha increases sensitivity to LPS in chronically catheterized rats

Patients with severe trauma injury are transiently exposed to increased serum concentrations of tumor necrosis factor-alpha (TNF-alpha). These patients are susceptible to the development of multisystem organ failure (MSOF) triggered by subsequent exposure to bacterial toxins either via infection or increased intestinal permeability. We simulated the cytokine response of trauma by infusing 0.8 or 8.0 microg/kg of TNF-alpha (priming dose) into chronically catheterized rats. After 48 h, rats were challenged with endotoxin [lipopolysaccharide (LPS); 10 or 1,000 microg/kg]. Animals primed with either dose of TNF-alpha and then challenged with 1,000 microg/kg of LPS demonstrated significantly increased mortality, mean peak serum concentrations of interferon-gamma (IFN-gamma), and blood lactate concentrations (P < 0.05) compared with nonprimed animals. Mean peak serum concentrations of IFN-gamma and blood lactate concentrations were increased after challenge with 10 microg/kg of LPS only in animals primed with 8.0 microg/kg of TNF-alpha. Priming with TNF-alpha did not increase mortality after challenge with 10 microg/kg of LPS. These data suggest that both TNF-alpha release and the subsequent exposure to bacterial toxins mediate the pathophysiological progression from trauma to subsequent MSOF.     

Enhancement of dendritic cell production by fms-like tyrosine kinase-3 ligand increases the resistance of mice to a burn wound infection

Fms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine that stimulates the production of dendritic cells. This study evaluated the ability of Flt3L-enhanced dendritic cell production to increase the resistance of mice to a burn wound infection with Pseudomonas aeruginosa, a common source of infections in burn patients that have impaired immunity and are susceptible to opportunistic microorganisms. Treatment of mice with Flt3L for 5 days caused a significant increase in dendritic cell numbers in the spleen and significantly increased survival upon a subsequent burn wound infection. Improved survival in Flt3L-treated mice was associated with limited bacterial growth and spread within the burn wounds and a decrease in systemic dissemination of P. aeruginosa. Resistance to burn wound infection could also be conferred to recipient mice by the adoptive transfer of dendritic cells that had been isolated from spleens of Flt3L-treated mice. Adoptive transfer of the same number of splenic dendritic cells from nontreated mice did not confer resistance to burn wound infection. These data indicate that Flt3L can increase the resistance of mice to a P. aeruginosa burn wound infection through both stimulation of dendritic cell production and enhancement of dendritic cell function.     

Dual effect of IL-4 on resistance to systemic gram-negative infection and production of TNF-alpha

To determine the effect of interleukin 4 (IL-4) administration in a live sepsis model characterised by high-level production of tumour necrosis factor a (TNF-alpha), mice infected systemically with lethal or sublethal inocula of Pseudomonas aeruginosa were given the recombinant cytokine at different times before infection. Improved survival and decreased TNF-alpha production were observed in lethally infected mice treated with the cytokine 1 day before challenge. In contrast, increased mortality and overproduction of TNF-alpha were observed in sublethally infected mice given IL-4 at the time of infection.     

Control of Pseudomonas aeruginosa in the lung requires the recognition of either lipopolysaccharide or flagellin

Acute lung infection due to Pseudomonas aeruginosa is an increasingly serious problem that results in high mortality especially in the compromised host. In this study, we set out to ascertain what components of the TLR system are most important for innate immunity to this microorganism. We previously demonstrated that TLR2,4-/- mice were not hypersusceptible to infection by a wild-type P. aeruginosa strain. However, we now find that mice lacking both TLR2 and TLR4 (TLR2,4-/- mice) are hypersusceptible to infection following challenge with a P. aeruginosa mutant devoid of flagellin production. We demonstrate that this hypersusceptibility is largely due to a lack of innate defense by the host that fails to control bacterial replication in the lung. Further evidence that a response to flagellin is a key factor in the failure of TLR2,4-/- mice to control the infection with the mutant strain was obtained by demonstrating that the intrapulmonary administration of flagellin over a 18 h period following infection, saved 100% of TLR2,4-/- mice from death. We conclude that the interactions of either TLR4 with LPS or TLR5 with flagellin can effectively defend the lung from P. aeruginosa infection and the absence of a response by both results in hypersusceptibility to this infection.     

Colonization resistance against potentially pathogenic bacteria in hexaflora-associated gnotobiotic mice.

A study of colonization resistance against potentially pathogenic bacteria (Escherichia coli and Pseudomonas aeruginosa) was conducted in hexaflora-associated gnotobiotic mice. Groups of germfree AKR mice were swabbed with five bacterial and a single gastrointestinal yeast species: Streptococcus faecalis. Lactobacillus brevis. Staphylococcus epidermidis, Enterobacter aerogenes, Bacteroides fragilis var. vulgatus, and Torulopsis sp. All species became established in the gut in 8 weeks. Later these associated mice were divided and challenged by four graded doses of E. coli or P. aeruginosa. The presence of challenge organism was monitored specifically in the freshly voided fecal specimens of the challenged mice. Escherichia coli colonized the gut of each mouse at each level up to 60 days post challenge. Pseudomonas aeruginosa was completely eliminated from each mouse at each dose level after 30 days post challenge. Evidence suggests that all six species were sufficient to prevent the colonization of P. aeruginosa and not of E. coli in the gut of the gnotobiotic mice.     

BCG-induced susceptibility of mice to challenge with Pseudomonas aeruginosa

Mice infected with Mycobacterium bovis, BCG, were shown to be highly susceptible to subsequent challenge with Pseudomonas aeruginosa. The susceptibility was characterized by the enhanced mortality and shortened survival after challenge with P. aeruginosa. BCG-treated mice did not show any enhanced susceptibility to challenge with Gram-positive bacteria such as Staphylococcus aureus or Listeria monocytogenes. BCG-treated mice eliminated P. aeruginosa from their organs in a pattern similar to that in untreated mice. There was no significant difference in the bactericidal activities of polymorphonuclear cells and macrophages between BCG-treated and untreated mice. An equal amount of endotoxin was detected by the Limulus lysate assay in the blood of both BCG-treated and untreated mice after challenge with P. aeruginosa. The enhanced susceptibility induced by BCG pretreatment could be decreased when the mice were rendered LPS-tolerant by injections of small amounts of LPS. These results suggest that BCG-induced susceptibility to P. aeruginosa can be ascribed to an enhanced susceptibility to the lethal effect of LPS produced by challenge bacteria, and not to the impairment of the ability to eliminate infected bacteria.     

Transgenic cystic fibrosis mice exhibit reduced early clearance of Pseudomonas aeruginosa from the respiratory tract

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been proposed to be an epithelial cell receptor for Pseudomonas aeruginosa involved in bacterial internalization and clearance from the lung. We evaluated the role of CFTR in clearing P. aeruginosa from the respiratory tract using transgenic CF mice that carried either the DeltaF508 Cftr allele or an allele with a Cftr stop codon (S489X). Intranasal application achieved P. aeruginosa lung infection in inbred C57BL/6 DeltaF508 Cftr mice, whereas DeltaF508 Cftr and S489X Cftr outbred mice required tracheal application of the inoculum to establish lung infection. CF mice showed significantly less ingestion of LPS-smooth P. aeruginosa by lung cells and significantly greater bacterial lung burdens 4.5 h postinfection than C57BL/6 wild-type mice. Microscopy of infected mouse and rhesus monkey tracheas clearly demonstrated ingestion of P. aeruginosa by epithelial cells in wild-type animals, mostly around injured areas of the epithelium. Desquamating cells loaded with P. aeruginosa could also be seen in these tissues. No difference was found between CF and wild-type mice challenged with an LPS-rough mucoid isolate of P. aeruginosa lacking the CFTR ligand. Thus, transgenic CF mice exhibit decreased clearance of P. aeruginosa and increased bacterial burdens in the lung, substantiating a key role for CFTR-mediated bacterial ingestion in lung clearance of P. aeruginosa.     

Pyocyanin production by Pseudomonas aeruginosa induces neutrophil apoptosis and impairs neutrophil-mediated host defenses in vivo

Clearance of neutrophils from inflamed sites is critical for resolution of inflammation, but pathogen-driven neutrophil apoptosis can impair host defenses. We previously showed that pyocyanin, a phenazine toxic metabolite produced by Pseudomonas aeruginosa, accelerates neutrophil apoptosis in vitro. We compared wild-type and pyocyanin-deficient strains of P. aeruginosa in a murine model of acute pneumonia. Intratracheal instillation of either strain of P. aeruginosa caused a rapid increase in bronchoalveolar lavage neutrophil counts up to 18 h after infection. In wild-type infection, neutrophil numbers then declined steadily, whereas neutrophil numbers increased up to 48 h in mice infected with pyocyanin-deficient P. aeruginosa. In keeping with these differences, pyocyanin production was associated with reduced bacterial clearance from the lungs. Neutrophil apoptosis was increased in mice infected with wild-type compared with the phenazine-deficient strain or two further strains that lack pyocyanin production, but produce other phenazines. Concentrations of potent neutrophil chemokines (MIP-2, KC) and cytokines (IL-6, IL-1beta) were significantly lower in wild-type compared with phenazine-deficient strain-infected mice at 18 h. We conclude that pyocyanin production by P. aeruginosa suppresses the acute inflammatory response by pathogen-driven acceleration of neutrophil apoptosis and by reducing local inflammation, and that this is advantageous for bacterial survival.     

Cecal ligation and puncture-induced impairment of innate immune function does not occur in the absence of caspase-1

Mice that have been subjected to cecal ligation and puncture (CLP) have an impaired ability to clear a subsequent Pseudomonas aeruginosa challenge compared with that of sham CLP controls. We hypothesized that this outcome is dependent upon a caspase-1 mechanism and tested this hypothesis by measuring caspase-1 after CLP and by measuring clearance of a bacterial challenge in caspase-1-deficient mice after CLP. Wild-type mice subjected to CLP had increased caspase-1 activity as well as increased IL-1β and increased IL-18 production in splenocytes stimulated with heat-killed Pseudomonas and had increased plasma concentrations of IL-1β and IL-18 and impaired clearance of a P. aeruginosa challenge compared with sham controls. Healthy, uninjured caspase-1(-\-) mice did not differ from wild-type mice in their ability to clear a Pseudomonas challenge. However, unlike wild-type mice, caspase-1(-/-) mice subjected to CLP had no impairment of bacterial clearance of the Pseudomonas challenge, suggesting that caspase-1 induction after CLP played a role in impairment of bacterial clearance. This was further substantiated by the use of a specific caspase-1 inhibitor, Ac-YVAD-CMK. Wild-type mice treated with Ac-YVAD-CMK (10 mg/kg s.c. twice daily, initiated at time of CLP) did not have impaired clearance of a Pseudomonas challenge compared with that of sham mice and had significantly improved bacterial clearance compared with that of untreated CLP mice. Increased caspase-1 expression and activity after CLP injury appears to contribute to diminished innate immune function.     

Cytokine profiling by multiplex immunoassay as an effective approach to assess immunomodulatory activity of bacterial product CANTASTIM

The bacterial product CANTASTIM (CS) is a purified extract of Pseudomonas aeruginosa that induces non-specific protection against bacterial infection, enhances macrophage effector functions and modulates production of cytokines. Most likely, it interacts with components of the innate immune response. Cytokine production can be used to assess the bioactivity of this product but these biomolecules operate in vivo in a complex regulatory network with reciprocal influences so there is a need for profiling an array of cytokines rather than an individual analysis. Current technology development of multiplex immunoassay for simultaneous measurement of multiple analytes in a single assay has greatly improved the throughput and cost effectiveness of cytokine profiling and proved to be an effective approach to evaluate the immunomodulatory activity of the bacterial product CS.     

The effects of recombinant interleukin-1 and recombinant tumor necrosis factor on non-specific resistance to infection

Natural and synthetic immunomodulators that increase non-specific resistance to infection induce the production of interleukin-1 (IL-1) and tumor necrosis factor (TNF). Therefore, we investigated the effect of IL-1 and of TNF on the survival of lethally-infected mice. Mice were injected with 1 x 10(6) Klebsiella pneumoniae in the thigh muscle. When recombinant human IL-1 beta was given as a single i.p. injection 24 h before the infection, survival was increased. Using 80 ng IL-1 beta per mouse, survival compared to control animals was 80% versus 20% 48 h after the infection (p less than 0.001). No effect of IL-1 was observed when it was given 1/2 h before or 6 h after the infection. IL-1 alpha proved to be at least as potent as IL-1 beta. Numbers of bacteria cultured from the blood, thigh muscle, liver, spleen, and kidney were similar in IL-1-treated and control animals. Protection against death by IL-1 was also investigated in granulocytopenic mice with a Pseudomonas aeruginosa infection. Administration of the cyclooxygenase-inhibitor, ibuprofen, did not affect the beneficial effect of IL-1. In this model human recombinant TNF was at least tenfold less active than IL-1 beta. Pretreatment with IL-1 also had a significant effect on survival of mice that received a high dose of bacterial lipopolysaccharide.     

Intratracheal immunization with pili protein protects against mortality associated with Pseudomonas aeruginosa pneumonia in mice

We examined the protective effect of intratracheal immunization with Pseudomonas aeruginosa pili protein against respiratory infection caused by P. aeruginosa. Mice were immunized intratracheally or subcutaneously with purified pili protein or bovine serum albumin as a control. Intratracheally but not subcutaneously pili protein-immunized mice showed significant improvement of survival after intratracheal challenge with the PAO1 strain. Furthermore, bacterial cell counts in pili protein-immunized murine lungs were significantly decreased compared to controls at 18 h after the challenge. Antipili protein antibody titers in bronchoalveolar lavage fluid of intratracheally pili protein-immunized mice were higher than in bovine serum albumin immunized mice. However, antipili antibody titers were not increased in bronchoalveolar lavage fluid of subcutaneously pili protein-immunized mice, despite the high serum antipili antibody titers. Inoculation of P. aeruginosa induced immediate increases in interleukin-12 and interferon-gamma in bronchoalveolar lavage fluid of pili protein-immunized mice, reflecting an adequate and rapid immune response against P. aeruginosa respiratory tract infection. Our findings suggest that intratracheal pili protein immunization is effective against respiratory tract infection caused by P. aeruginosa in mice.     

Protection of mice against bacterial infection by oral administration of bacterial lipopolysaccharide

The effect of orally administered bacterial lipopolysaccharide (LPS) on host resistance against bacterial infections was studied. LPS orally given for 5 consecutive days prior to infection caused no apparent toxic effect and protected mice against Pseudomonas aeruginosa and Listeria monocytogenes infections.     

Distinct modulatory effects of LPS and CpG on IL-18-dependent IFN-gamma synthesis

Innate cellular production of IFN-gamma is suppressed after repeated exposure to LPS, whereas CpG-containing DNA potentiates IFN-gamma production. We compared the modulatory effects of LPS and CpG on specific cellular and cytokine responses necessary for NK-cell dependent IFN-gamma synthesis. C3H/HeN mice pretreated with LPS for 2 days generated 5-fold less circulating IL-12 p70 and IFN-gamma in response to subsequent LPS challenge than did challenged control mice. In contrast, CpG-pretreated mice produced 10-fold more circulating IFN-gamma without similar changes in IL-12 p70 levels, but with 10-fold increases in serum IL-18 relative to LPS-challenged control or endotoxin-tolerant mice. The role of IL-18 in CpG-induced immune potentiation was studied in splenocyte cultures from control, LPS-conditioned, or CpG-conditioned mice. These cultures produced similar amounts of IFN-gamma in response to rIL-12 and rIL-18. However, only CpG-conditioned cells produced IFN-gamma when cultured with LPS or CpG, and production was ablated in the presence of anti-IL-18R Ab. Anti-IL-18R Ab also reduced in vivo IFN-gamma production by >2-fold in CpG-pretreated mice. Finally, combined pretreatment of mice with LPS and CpG suppressed the production of circulating IFN-gamma, IL-12 p70, and IL-18 after subsequent LPS challenge. We conclude that CpG potentiates innate IFN-gamma production from NK cells by increasing IL-18 availability, but that the suppressive effects of LPS on innate cellular immunity dominate during combined LPS and CpG pretreatment. Multiple Toll-like receptor engagement in vivo during infection can result in functional polarization of innate immunity dominated by a specific Toll-like receptor response.