IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-gamma production by NK cells

The aim of this study was to examine the contribution of IL-18 in host defense against infection caused by Cryptococcus neoformans in mice with defective IL-12 production. Experiments were conducted in mice with a targeted disruption of the gene for IL-12p40 subunit (IL-12p40-/- mice). In these mice, host resistance was impaired, as shown by increased number of organisms in both lungs and brains, compared with control mice. Serum IFN-gamma was still detected in these mice at a considerable level (20-30% of that in control mice). The host resistance was moderately impaired in IL-12p40-/- mice compared with IFN-gamma-/- mice. Neutralizing anti-IFN-gamma mAb further increased the lung burdens of organisms. In addition, treatment with neutralizing anti-IL-18 Ab almost completely abrogated the production of IFN-gamma and also impaired the host resistance. Host resistance in IL-12p40-/- IL-18-/- mice was more profoundly impaired than in IL-12p40-/- mice. Administration of IL-12 as well as IL-18 increased the serum levels of IFN-gamma and significantly restored the reduced host resistance. Spleen cells obtained from infected IL-12p40-/- mice did not produce any IFN-gamma upon restimulation with the same organisms, while those from infected and IL-12-treated mice produced IFN-gamma. In contrast, IL-18 did not show such effect. Finally, depletion of NK cells by anti-asialo GM1 Ab mostly abrogated the residual production of IFN-gamma in IL-12p40-/- mice. Our results indicate that IL-18 contributes to host resistance to cryptococcal infection through the induction of IFN-gamma production by NK cells, but not through the development of Th1 cells, under the condition in which IL-12 synthesis is deficient.     

IL-12p40-dependent agonistic effects on the development of protective innate and adaptive immunity against Salmonella enteritidis.

To study a potential IL-12p40-dependent but IL-12p75-independent agonistic activity regulating the immune response against Salmonella Enteritidis, the course of infection in IL-12p35-deficient mice (IL-12p35(-/-), capable of producing IL-12p40) was compared with that of IL-12p40(-/-) mice. Mice lacking IL-12p40 revealed a higher mortality rate and higher bacterial organ burden than mice capable of producing IL-12p40. This phenotype was found in both genetically susceptible (BALB/c, Ity(s)) and resistant mice (129Sv/Ev, Ity(r)) indicating Ity-independent mechanisms. The more effective control of bacteria in the IL-12p35(-/-) mice was associated with elevated serum IFN-gamma and TNF-alpha levels. In contrast, IL-12p40(-/-) mice showed reduced IFN-gamma production, which was associated with significantly elevated serum IgE levels. Early during infection (days 3 and 4 postinfection), as well as late (day 20 postinfection), the number of infected phagocytes was strongly increased in the absence of IL-12p40 indicating impaired bactericidal activity when IL-12p40 was missing. Liver histopathology revealed a decreased number of mononuclear granulomas in IL-12p40(-/-) mice. Depletion of CD4(+) or CD8(+) T lymphocytes in vivo suggested that both T cell subpopulations contribute to the IL-12p40-dependent protective functions. Analysis of IL-12p40 vs IL-23p19 mRNA expression revealed an up-regulation of only IL-12p40 mRNA during Salmonella infection. Together these data indicate that IL-12p40 can induce protective mechanisms during both the innate and the adaptive type 1 immune response in Salmonella infection. This novel activity of IL-12p40 complements the well described dominant and essential role of IL-12p75 in protective immunity to Salmonella infection.     

IL-18 has IL-12-independent effects in delayed-type hypersensitivity: studies in cell-mediated crescentic glomerulonephritis

IL-18 (formerly known as IFN-gamma-inducing factor) enhances Th1 responses via effects that are thought to be dependent on and synergistic with IL-12. The potential for IL-18 to exert IL-12-independent effects in delayed-type hypersensitivity (DTH) responses was studied in a model of Th1-directed, DTH-mediated crescentic glomerulonephritis induced by planting an Ag in glomeruli of sensitized mice as well as in cutaneous DTH. Sensitized genetically normal (IL-12(+/+)) mice developed proteinuria and crescentic glomerulonephritis with a glomerular influx of DTH effectors (CD4(+) T cells, macrophages, and fibrin deposition) in response to the planted glomerular Ag. IL-12p40-deficient (IL-12(-/-)) mice showed significant reductions in crescent formation, proteinuria, and glomerular DTH effectors. Administration of IL-18 to IL-12(-/-) mice restored the development of histological (including effectors of DTH) and functional glomerular injury in IL-12(-/-) mice to levels equivalent to those in IL-12(+/+) mice. IL-18 administration to IL-12(-/-) mice increased glomerular ICAM-1 protein expression, but did not restore Ag-stimulated splenocyte IFN-gamma, GM-CSF, IL-2, or TNF-alpha production. Sensitized IL-12(+/+) mice also developed cutaneous DTH following intradermal challenge with the nephritogenic Ag. Cutaneous DTH was inhibited in IL-12(-/-) mice, but was restored by administration of IL-18. IL-12(+/+) mice given IL-18 developed augmented injury, with enhanced glomerular and cutaneous DTH, demonstrating the synergistic effects of IL-18 and IL-12 in DTH responses. These studies demonstrate that even in the absence of IL-12, IL-18 can induce in vivo DTH responses and up-regulate ICAM-1 without inducing IFN-gamma, GM-CSF, or TNF-alpha production.     

IL-12 and type-I IFN synergize for IFN-gamma production by CD4 T cells, whereas neither are required for IFN-gamma production by CD8 T cells after Listeria monocytogenes infection

Differentiation of Ag-specific T cells into IFN-gamma producers is essential for protective immunity to intracellular pathogens. In addition to stimulation through the TCR and costimulatory molecules, IFN-gamma production is thought to require other inflammatory cytokines. Two such inflammatory cytokines are IL-12 and type I IFN (IFN-I); both can play a role in priming naive T cells to produce IFN-gamma in vitro. However, their role in priming Ag-specific T cells for IFN-gamma production during experimental infection in vivo is less clear. In this study, we examine the requirements for IL-12 and IFN-I, either individually or in combination, for priming Ag-specific T cell IFN-gamma production after Listeria monocytogenes (Lm) infection. Surprisingly, neither individual nor combined defects in IL-12 or IFN-I signaling altered IFN-gamma production by Ag-specific CD8 T cells after Lm infection. In contrast, individual defects in either IL-12 or IFN-I signaling conferred partial ( approximately 50%) reductions, whereas combined deficiency in both IL-12 and IFN-I signaling conferred more dramatic (75-95%) reductions in IFN-gamma production by Ag-specific CD4 T cells. The additive effects of IL-12 and IFN-I signaling on IFN-gamma production by CD4 T cells were further demonstrated by adoptive transfer of transgenic IFN-IR(+/+) and IFN-IR(-/-) CD4 T cells into normal and IL-12-deficient mice, and infection with rLm. These results demonstrate an important dichotomy between the signals required for priming IFN-gamma production by CD4 and CD8 T cells in response to bacterial infection.     

Contribution of IL-18 to Th1 response and host defense against infection by Mycobacterium tuberculosis: a comparative study with IL-12p40

The present study was conducted to critically determine the protective role of IL-18 in host response to Mycobacterium tuberculosis infection. IL-18-deficient (knockout (KO)) mice were slightly more prone to this infection than wild-type (WT) mice. Sensitivity of IL-12p40KO mice was lower than that of IL-12p40/IL-18 double KO mice. IFN-gamma production caused by the infection was significantly attenuated in IL-18KO mice compared with WT mice, as indicated by reduction in the levels of this cytokine in sera, spleen, lung, and liver, and its synthesis by spleen cells restimulated with purified protein derivatives. Serum IL-12p40 level postinfection and its production by peritoneal exudate cells stimulated with live bacilli were also significantly lower in IL-18KO mice than WT mice, suggesting that attenuated production of IFN-gamma was secondary to reduction of IL-12 synthesis. However, this was not likely the case, because administration of excess IL-12 did not restore the reduced IFN-gamma production in IL-18KO mice. In further studies, IL-18 transgenic mice were more resistant to the infection than control littermate mice, and serum IFN-gamma level and its production by restimulated spleen cells were increased in the former mice. Taken together, our results indicate that IL-18 plays an important role in Th1 response and host defense against M. tuberculosis infection although the contribution was not as profound as that of IL-12p40.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.     

Vaccine-induced immunity against Helicobacter pylori infection is impaired in IL-18-deficient mice

Protective immunity against Helicobacter pylori infection in mice has been associated with a strong Th1 response, involving IL-12 as well as IFN-gamma, but recent studies have also demonstrated prominent eosinophilic infiltration, possibly linked to local Th2 activity in the gastric mucosa. In this study we investigated the role of IL-18, because this cytokine has been found to be a coregulator of Th1 development as well as involved in Th2-type responses with local eotaxin production that could influence gastric eosinophilia and resistance to infection. We found that IL-18(-/-) mice failed to develop protection after oral immunization with H. pylori lysate and cholera toxin adjuvant, indicating an important role of IL-18 in protection. Well-protected C57BL/6 wild-type (WT) mice demonstrated substantial influx of CD4(+) T cells and eosinophilic cells in the gastric mucosa, whereas IL-18(-/-) mice had less gastritis, few CD4(+) T cells, and significantly reduced numbers of eosinophilic cells. T cells in well-protected WT mice produced increased levels of IFN-gamma and IL-18 to recall Ag. By contrast, unprotected IL-18(-/-) mice exhibited significantly reduced gastric IFN-gamma and specific IgG2a Ab levels. Despite differences in gastric eosinophilic cell infiltration, protected WT and unprotected IL-18(-/-) mice had comparable levels of local eotaxin, suggesting that IL-18 influences protection via Th1 development and IFN-gamma production rather than through promoting local production of eotaxin and eosinophilic cell infiltration.     

Role of IL-12-independent and IL-12-dependent pathways in regulating generation of the IFN-gamma component of T cell responses to Salmonella typhimurium

Clearance of facultative intracellular pathogens such as Salmonella requires IFN-gamma from CD4 T cells. Mechanisms linking intracellular pathogen recognition with induction of IFN-gamma-producing T cells are still poorly understood. We show in this study that IL-12 is not required for commitment to the IFN-gamma-producing T cell response in infection with Salmonella typhimurium, but is needed for its maintenance. The IL-12-independent signals required for commitment depend on events during the first hour of infection and are related to Ag presentation. Even transient attenuation of Ag presentation early during infection specifically abrogates the IFN-gamma component of the resulting CD4 T cell response. The IL-12 needed for maintenance is also better induced by live rather than dead bacteria in vivo, and this difference is due to specific suppression of IL-12 induction by dead bacteria. Presence of exogenous IL-4 down-modulates IL-12 production by macrophages activated in vitro. Furthermore, macrophages from IL-4-null mice secrete high levels of both IL-12 and IL-18 in response to stimulation in vivo even with dead bacteria, but this does not lead to induction of IFN-gamma-secreting T cells in response to immunization with dead S. typhimurium. Early IL-4 is contributed by triggering of CD4 NK T cells by dead, but not live, bacteria. Thus, Ag presentation-related IL-12-independent events and IL-4-sensitive IL-12-dependent events play crucial complementary roles in the generation of the IFN-gamma-committed CD4 T cell component of the immune response in Salmonella infection.     

Selective suppression of IL-12 production by chemoattractants

We investigated the ability of chemoattractants to affect IL-12 production by human monocytes and dendritic cells. We found that pretreatment of monocytes with macrophage chemoattractant proteins (MCP-1 to -4), or C5a, but not stromal-derived factor-1, macrophage inflammatory protein-1alpha, RANTES, or eotaxin, inhibited IL-12 p70 production in response to stimulation with Staphylococcus aureus, Cowan strain 1 (SAC), and IFN-gamma. The production of TNF-alpha and IL-10, however, was minimally affected by any of the chemoattractants. The degree of inhibition of IL-12 p70 production by MCP-1 to -4 was donor dependent and was affected by the autocrine inhibitory effects of IL-10. In contrast, C5a profoundly suppressed IL-12 production in an IL-10-independent fashion. Neither TGF-beta1 nor PGE2 was important for the suppression of IL-12 by any of the chemoattractants tested. The accumulation of mRNA for both IL-12 p35 and p40 genes was inhibited by chemokine pretreatment. Interestingly, MCP-1 to -4 and C5a did not suppress IL-12 production by monocyte-derived dendritic cells (DC) stimulated with CD40 ligand and IFN-gamma or by SAC and IFN-gamma, suggesting that these factors may act at the site of inflammation to suppress IL-12 and IFN-gamma production rather than in the lymph node to affect T cell priming. Despite the inability of C5a to inhibit IL-12 production by DCs, the receptor for C5a (CD88) was expressed by these cells, and recombinant C5a induced a Ca2+ flux. Taken together, these results define a range of chemoattractant molecules with the ability to suppress IL-12 production by human monocytes and have broad implications for the regulation of immune responses in vivo.     

IL-12p40 and IL-18 modulate inflammatory and immune responses to respiratory syncytial virus infection

Respiratory syncytial virus-induced bronchiolitis has been linked to the development of allergy and atopic asthma. IL-12 and possibly IL-18 are central mediators orchestrating Th1 and/or Th2 immune responses to infection. To determine a possible role for IL-12 in regulating the immune response to acute respiratory syncytial virus infection, IL-12p40 gene-targeted (IL-12p40-/-) and wild-type mice were intratracheally infected with respiratory syncytial virus, and lung inflammatory and immune responses were assessed. Lung inflammation and mucus production were increased in the airways of IL-12p40-/- mice as compared with those of wild-type mice, concurrent with increased levels of the Th2 effector cytokines IL-5 and IL-13. Respiratory syncytial virus clearance and levels of Th1 effector cytokine IFN-gamma were not altered. Interestingly, IL-18, another mediator of IFN-gamma production, was significantly increased in the lungs of IL-12p40-/- mice early during the course of infection. Abrogation of IL-18-mediated signaling in IL-12p40-/- mice further enhanced Th2 immune response and mucus production in the airways during respiratory syncytial virus infection but failed to modulate IFN-gamma production or viral clearance. These findings implicate a role for IL-12 and IL-18 in modulating respiratory syncytial virus-induced airway inflammation distinct from that of viral clearance.     

Role of IFN-gamma in Th1 differentiation: IFN-gamma regulates IL-18R alpha expression by preventing the negative effects of IL-4 and by inducing/maintaining IL-12 receptor beta 2 expression

Two key events occur during the differentiation of IFN-gamma-secreting Th1 cells: up-regulation of IL-12Rbeta2 and IL-12-driven up-regulation of IL-18Ralpha. We previously demonstrated that IL-12-driven up-regulation of IL-18Ralpha expression is severely impaired in IFN-gamma(-/-) mice. However, it was unclear from these studies how IFN-gamma influenced IL-18Ralpha since IFN-gamma alone had no direct effect on IL-18Ralpha expression. In the absence of IL-4, IL-12-dependent up-regulation of IL-18Ralpha/IL-12Rbeta2 was independent of IFN-gamma. However, in the presence of IL-4, IFN-gamma functions to limit the negative effects of IL-4 on both IL-18Ralpha and IL-12Rbeta2. Neutralization of IL-4 restored IL-12-driven up-regulation of IL-18Ralpha/IL-12Rbeta2 in an IFN-gamma-independent fashion. In the absence of both IL-12 and IL-4, IFN-gamma up-regulates IL-12beta2 expression and primes IFN-gamma-producing Th1 cells. When T cells were primed in the presence of IL-4, no correlation was found between the levels of expression of the IL-18Ralpha or the IL-12Rbeta2 and the capacity of these cells to produce IFN-gamma, suggesting that IL-4 may also negatively affect IL-12-mediated signal transduction and thus Th1 differentiation. These data clarify the role of IFN-gamma in regulation of IL-18Ralpha/IL-12Rbeta2 during both IL-12-dependent and IL-12-independent Th1 differentiation.     

Endotoxin fails to induce IFN-gamma in endotoxin-tolerant mice: deficiencies in both IL-12 heterodimer production and IL-12 responsiveness

Mice exposed to sublethal endotoxemia develop short-term endotoxin tolerance, a state characterized by decreased monokine production and enhanced protection against endotoxic lethality. We confirmed that TNF-alpha production is markedly impaired in endotoxin-tolerant mice and additionally found 2- to 6-fold decreases in serum IFN-gamma in these animals following endotoxin challenge. The IFN-gamma deficiency of endotoxin tolerance correlated with 8-fold decreases in the bioactive p40/p35 heterodimeric form of IL-12. In contrast, total circulating IL-12 p40 was reduced by only 30-50%. Endotoxin-tolerant mice were less responsive to IL-12 than control mice, as evidenced by 3-fold lower levels of IFN-gamma inducible in vivo when rIL-12 was administered at the time of endotoxin challenge. Similarly, spleen cell cultures of endotoxin-tolerant mice produced 3-fold less IFN-gamma in the presence of optimal concentrations of both IL-12 and IL-18. Finally, levels of IL-12R beta 2 subunit mRNA and the percent composition of NK lymphocytes in the spleen were both decreased in endotoxin-tolerant mice relative to controls. We conclude that endotoxin-tolerant mice are profoundly impaired in their ability to produce IFN-gamma in response to endotoxin and that this is associated with acquired defects in both the production of circulating IL-12 heterodimer response and the response to IL-12 by NK cells.     

IL-18 is a potent coinducer of IL-13 in NK and T cells: a new potential role for IL-18 in modulating the immune response

IL-13 and IL-4 have similar biological activities and are characteristic of cytokines expressed by Th2 cells. In contrast, IL-12 and IL-18 have been shown to be strong cofactors for Th1 cell development. In this study, we found strong induction of IL-13 mRNA and protein by IL-2 + IL-18 in NK and T cells. In contrast, IL-12 did not enhance the IL-13 production induced by IL-2 alone. Moreover, IL-13 mRNA and protein expression induced by IL-2 + IL-18 in purified NK and T cells obtained from IFN-gamma knockout (-/-) mice were greater than seen in purified cells from normal controls. In contrast, IL-10 production induced by IL-2 and/or IL-12 was not significantly different in IFN-gamma (-/-) mice and normal controls. These results suggest IL-13 expression induced by IL-2 + IL-18 may be regulated by IFN-gamma in vivo, while IL-10 expression may be IFN-gamma-independent. Thus, depending upon the cell type, IL-18 may act as a strong coinducer of Th1 or Th2 cytokines. Our findings suggest that IL-12 and IL-18 have different roles in the regulation of gene expression in NK and T cells.     

IL-23 provides a limited mechanism of resistance to acute toxoplasmosis in the absence of IL-12

IL-23 and IL-12 are heterodimeric cytokines which share the p40 subunit, but which have unique second subunits, IL-23p19 and IL-12p35. Since p40 is required for the development of the Th1 type response necessary for resistance to Toxoplasma gondii, studies were performed to assess the role of IL-23 in resistance to this pathogen. Increased levels of IL-23 were detected in mice infected with T. gondii and in vitro stimulation of dendritic cells with this pathogen resulted in increased levels of mRNA for this cytokine. To address the role of IL-23 in resistance to T. gondii, mice lacking the p40 subunit (common to IL-12 and IL-23) and mice that lack IL-12 p35 (specific for IL-12) were infected and their responses were compared. These studies revealed that p40(-/-) mice rapidly succumbed to toxoplasmosis, while p35(-/-) mice displayed enhanced resistance though they eventually succumbed to this infection. In addition, the administration of IL-23 to p40(-/-) mice infected with T. gondii resulted in a decreased parasite burden and enhanced resistance. However, the enhanced resistance of p35(-/-) mice or p40(-/-) mice treated with IL-23 was not associated with increased production of IFN-gamma. When IL-23p19(-/-) mice were infected with T. gondii these mice developed normal T cell responses and controlled parasite replication to the same extent as wild-type mice. Together, these studies indicate that IL-12, not IL-23, plays a dominant role in resistance to toxoplasmosis but, in the absence of IL-12, IL-23 can provide a limited mechanism of resistance to this infection.     

IL-23 compensates for the absence of IL-12p70 and is essential for the IL-17 response during tuberculosis but is dispensable for protection and antigen-specific IFN-gamma responses if IL-12p70 is available

IL-12p70 induced IFN-gamma is required to control Mycobacterium tuberculosis growth; however, in the absence of IL-12p70, an IL-12p40-dependent pathway mediates induction of IFN-gamma and initial bacteriostatic activity. IL-23 is an IL-12p40-dependent cytokine containing an IL-12p40 subunit covalently bound to a p19 subunit that is implicated in the induction of CD4 T cells associated with autoimmunity and inflammation. We show that in IL-23 p19-deficient mice, mycobacterial growth is controlled, and there is no diminution in either the number of IFN-gamma-producing Ag-specific CD4 T cells or local IFN-gamma mRNA expression. Conversely, there is an almost total loss of both IL-17-producing Ag-specific CD4 T cells and local production of IL-17 mRNA in these mice. The absence of IL-17 does not alter expression of the antimycobacterial genes, NO synthase 2 and LRG-47, and the absence of IL-23 or IL-17, both of which are implicated in mediating inflammation, fails to substantially affect the granulomatous response to M. tuberculosis infection of the lung. Despite this redundancy, IL-23 is required to provide a moderate level of protection in the absence of IL-12p70, and this protection correlates with a requirement for IL-23 in the IL-12p70-independent induction of Ag-specific, IFN-gamma-producing CD4 T cells. We also show that IL-23 is required for the induction of an IL-17-producing Ag-specific phenotype in naive CD4 T cells in vitro and that absence of IL-12p70 promotes an increase in the number of IL-17-producing Ag-specific CD4 T cells both in vitro and in vivo.     

Cutting edge: IL-18-transgenic mice: in vivo evidence of a broad role for IL-18 in modulating immune function

IL-18 has been shown to be a strong cofactor for Th1 T cell development. However, we previously demonstrated that when IL-18 was combined with IL-2, there was a synergistic induction of a Th2 cytokine, IL-13, in both T and NK cells. More recently, we and other groups have reported that IL-18 can potentially induce IgE, IgG1, and Th2 cytokine production in murine experimental models. Here, we report on the generation of IL-18-transgenic (Tg) mice in which mature mouse IL-18 cDNA was expressed. CD8+CD44high T cells and macrophages were increased, but B cells were decreased in these mice while serum IgE, IgG1, IL-4, and IFN-gamma levels were significantly increased. Splenic T cells in IL-18 Tg mice produced higher levels of IFN-gamma, IL-4, IL-5, and IL-13 than control wild-type mice. Thus, aberrant expression of IL-18 in vivo results in the increased production of both Th1 and Th2 cytokines.     

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.