Increased resistance against acute polymicrobial sepsis in mice challenged with immunostimulatory CpG oligodeoxynucleotides is related to an enhanced innate effector cell response

Recent reports support the concept that the major defect in polymicrobial sepsis is an impaired immunologic response to infection. Oligodeoxynucleotides containing CpG sequence motifs (CpG-ODN) were previously shown to induce immune protection in models of chronic infection with intracellular bacteria, parasites, and viruses due to their ability to augment IFN-gamma-dependent Th1 responses. Here, we demonstrate that challenging mice with CpG-ODN substantially increases the resistance against acute polymicrobial sepsis. Systemic levels of IL-12, IL-18, and IL-10 were not altered in CpG-ODN-treated mice as compared with controls. In contrast, administration of CpG-ODN resulted in a strongly enhanced accumulation of neutrophils at the primary site of infection. Neutrophils of CpG-ODN-treated mice exhibited an up-regulation of phagocytic receptors, an increased phagocytic activity, and an elevated production of reactive oxygen metabolites. These results suggest that the protective effects of CpG-ODNs in acute polymicrobial sepsis are related to an enhanced effector cell response of innate immunity. CpG-ODN may therefore represent potent agents for the treatment of sepsis-associated immunoparalysis.     

Activation of marginal zone B cells from lupus mice with type A(D) CpG-oligodeoxynucleotides

Several types of CpG-oligodeoxynucleotides (ODN) have been recently characterized. In mice, type A(D) CpG-ODNs primarily stimulate macrophages and dendritic cells, but fail to stimulate B cells. On the contrary, type B(K) CpG-ODNs are excellent B cell activators. Type C CpG-ODNs combine features of both types A(D) and B(K) CpG-ODNs. Despite cell type preferences, all CpG-ODNs require the presence of TLR9 for activation. In this study, we show that a subset of B cells from lupus mice responds to type A(D) CpG-ODN stimulation vigorously and directly with increased CD25 and CD86 expression and IL-10 secretion. Furthermore, these CpG-ODNs induce high surface IgM expression and promote 50- to 100-fold higher IgM and IgG3 secretion in lupus B cells than in controls. This response is similar to that seen with bacterial DNA stimulation of B cells. Type A(D)-responsive cells are enriched within lupus B cells with the marginal zone (MZ) phenotype. These cells are at least twice more numerous in lupus mice than in controls. The ability of lupus B cells to respond to type A(D) CpG-ODN stimulation is not due to differential TLR9 expression. Therefore, type A(D) CpG-ODNs may contribute to the lupus pathogenesis by inducing MZ-B cell activation, costimulatory molecule expression, and polyclonal Ig secretion. Through increased IL-10 secretion, MZ-B cells may also modify the activity of other cell types, particularly dendritic cells and macrophages.     

NF-kappaB-dependent regulation of tumor necrosis factor-alpha gene expression by CpG-oligodeoxynucleotides

Immunostimulatory activities of synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have gained attention as potentially useful immunotherapeutics. However, CpG-ODNs induce harmful and lethal shock effects because they greatly enhance the sequence-dependent induction of tumor necrosis factor-alpha (TNF-alpha). We have shown that phosphorothioate-modified oligodeoxynucleotides (PS-ODNs) of the CpG-ODN 1826 stimulate TNF-alpha gene expression, TNF-alpha promoter activity, IkappaB degradation, and NF-kappaB activation at higher levels compared with its phosphodiester ODN (PO-ODN). In contrast to the effects of CpG-ODN 1826, PS-ODN of the CpG-ODN 2006 showed lower stimulatory activities than its PO-ODN. Using transient transfection, it was found that myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated factor 6 are commonly required for activation of the TNF-alpha promoter by various CpG-ODNs with different potencies. These results strongly suggest a possibility to optimally activate the innate immune responses by modulating the potency of CpG-ODNs via sequence rearrangement and phosphorothioate backbone modification.     

CpG-containing oligonucleotides are efficient adjuvants for induction of protective antiviral immune responses with T-cell peptide vaccines

Synthetic nonmethylated oligonucleotides containing CpG dinucleotides (CpG-ODNs) have been shown to exhibit immunostimulatory activity. CpG-ODNs have the capacity to directly activate B cells, macrophages, and dendritic cells, and we show here that this is reflected by cell surface binding of oligonucleotides to these cell subsets. However, T cells are not directly activated by CpG-ODNs, which correlates with the failure to bind to the T-cell surface. Efficient competition for CpG-induced B-cell activation by non-CpG-containing oligonucleotides suggests that oligonucleotides might bind to an as yet undefined sequence-nonspecific receptor prior to cellular activation. Induction of protective T-cell responses against challenge infection with lymphocytic choriomeningitis virus (LCMV) or with recombinant vaccinia virus expressing the LCMV glycoprotein was achieved by immunizing mice with the immunodominant major histocompatibility complex class I-binding LCMV glycoprotein-derived peptide gp33 together with CpG-ODNs. In these experiments, B cells, potentially serving as CpG-ODN-activated antigen-presenting cells (APCs), were not required for induction of protective immunity since CpG-ODN-gp33-immunized B-cell-deficient mice were equally protected against challenge infection with both viruses. This finding suggested that macrophages and/or dendritic cells were sufficiently activated in vivo by CpG-ODNs to serve as potent APCs for the induction of naive T cells. Furthermore, treatment with CpG-ODN alone induced protection against infection with Listeria monocytogenes via antigen-independent activation of macrophages. These data suggest that CpG activation of macrophages and dendritic cells may provide a critical step in CpG-ODN adjuvant activity.     

Cutting edge: CpG oligonucleotides induce splenic CD19+ dendritic cells to acquire potent indoleamine 2,3-dioxygenase-dependent T cell regulatory functions via IFN Type 1 signaling

CpG oligodeoxynucleotides (CpG-ODNs) stimulate innate and adaptive immunity by binding to TLR9 molecules. Paradoxically, expression of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) is induced following i.v. CpG-ODN administration to mice. CpG-ODNs induced selective IDO expression by a minor population of splenic CD19+ dendritic cells (DCs) that did not express the plasmacytoid DC marker 120G8. Following CpG-ODN treatment, CD19+ DCs acquired potent IDO-dependent T cell suppressive functions. Signaling through IFN type I receptors was essential for IDO up-regulation, and CpG-ODNs induced selective activation of STAT-1 in CD19+ DCs. Thus, CpG-ODNs delivered systemically at relatively high doses elicited potent T cell regulatory responses by acting on a discrete, minor population of splenic DCs. The ability of CpG-ODNs to induce both stimulatory and regulatory responses offers novel opportunities for using them as immunomodulatory reagents but may complicate therapeutic use of CpG-ODNs to stimulate antitumor immunity in cancer patients.     

Endosomal translocation of CpG-oligodeoxynucleotides inhibits DNA-PKcs-dependent IL-10 production in macrophages

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODNs) function as powerful immune adjuvants by activating macrophages, dendritic cells, and B cells. However, the molecular recognition mechanism that initiates signaling in response to CpG-ODN has not fully been identified. We show in this study that peritoneal macrophages from SCID mice having mutations in the catalytic subunit of DNA-protein kinase (DNA-PKcs) were almost completely defective in the production of IL-10 and in ERK activation when treated with CpG-ODN. In contrast, IL-12 p70 production significantly increased. Furthermore, small interfering RNA (siRNA)-mediated knockdown of DNA-PKcs expression in the mouse monocyte/macrophage cell line RAW264.7 led to reduced IL-10 production and ERK activation by CpG-ODN. IL-10 and IL-12 p70 production, but not ERK activation, are blocked by chloroquine, an inhibitor of endosomal acidification. Endosomal translocation of CpG-ODN in a complex with cationic liposomes consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (CpG-DOTAP-liposomes) decreased IL-10 production and ERK activation, whereas the endosomal escape of CpG-ODN in a complex with cationic liposomes consisting of DOTAP and dioleyl-phosphatidylethanolamine (DOPE) (CpG-DOTAP/DOPE-liposomes) increased. In contrast, IL-12 p70 production was increased by CpG-DOTAP-liposomes and decreased by CpG-DOTAP/DOPE-liposomes. IL-10 production induced by CpG-DOTAP/DOPE-liposomes was not observed in macrophages from SCID mice. Thus, our findings suggest that DNA-PKcs in the cytoplasm play an important role in CpG-ODN-induced production of IL-10 in macrophages. In addition, DNA-PKcs-mediated production of IL-10 and IL-12 p70 can be regulated by manipulating the intracellular trafficking of CpG-ODN in macrophages.     

A single intradermal administration of soluble leishmanial antigen and plasmid expressing interleukin-12 protects BALB/c mice from Leishmania major infection.

In murine leishmaniasis, the induction of the T-helper type 1 (Th1) response contributes to infection resistance, whereas the establishment of the Th2 response makes the mice susceptible to infection. Interleukin-12 (IL-12) plays a pivotal role in the diversification of immune responses to the Th1 type. In this study, we tested whether the co-administration of IL-12 expression plasmid which compose p35 and p40 subunits and soluble leishmanial antigen (SLA) will skew the susceptible BALB/c mice to Th1 response and protect from leishmaniasis. When the mice were intradermally injected with the combination of IL-12 plasmid and SLA 7 days prior to the challenge with 1x10(6) promastigotes of Leishmania major, the local lesions completely healed and the parasite burden in the local lymph nodes significantly decreased. The cured mice attained long-term immunity, and were resistant to any subsequent rechallenge of the lethal dose of the parasite. The protective effect was associated with the development of a Th1 response, as demonstrated by the enhanced level of antigen-specific interferon-gamma (IFN-gamma) and dominant production of IgG2a in the serum. In contrast, the administration of empty plasmid plus SLA or IL-12 plasmid alone failed to protect the disease and shape the Th1 response. Furthermore, the protective efficiency induced by the vaccination was clearly prevented by the injection of either neutralizing anti-IL-12 mAb or anti-IFN-gamma mAb. The IL-12 expression plasmid is thus an effective adjuvant for the elicitation of a protective Th1 response against leishmaniasis and is therefore, considered to be appropriate for vaccinations that require the induction of Th1 type immunity.     

Protection of mice from lethal endogenous Candida albicans infection by immunization with Candida membrane antigen

The protective effects of immunization with Candida membrane antigen (CMA) on a systemic infection originating from intestinally colonized Candida albicans were examined. The colonization of orally inoculated C. albicans in the intestinal tract was established in BALB/c mice that had been concomitantly treated with oral doses of antibacterial drugs. In these animals, a systemic dissemination of C. albicans with fatal outcome was induced by a repeated dosing of prednisolone. In this endogenous infection model, the effects of immunization by CMA on the infection were examined. CMA-immunized mice showed a longer lifespan than unimmunized mice. The protective effect of CMA immunization in immunosuppressed mice was also measured by a decrease in body weight loss after treatment with prednisolone and in the number of viable Candida cells in the target organs, the kidneys and livers. However, the CFU of C. albicans in the intestinal tract was not significantly lowered. These results suggest that CMA immunization inhibited the dissemination of systemic Candida infection from the intestinal tract induced by treatment with prednisolone.     

Biotherapeutic effects of Bifidobacterium spp. on orogastric and systemic candidiasis in immunodeficient mice

Two commercially available Bifidobacterium spp. (Bifidobacterium infantis and Bifidobacterium lactis) were compared for their capacities to protect immunodeficient bg/bg-nu/nuand bg/bg-nu/+mice from orogastric and lethal candidiasis. Both Bifidobacterium spp. prolonged the survival of Candida albicans-colonized adult and neonatal bg/bg-nu/numice. The bifidobacteria affected the production of antibodies to C. albicans, inhibited disseminated candidiasis, suppressed weight loss associated with C. albicans infection, inhibited the growth of C. albicans in the alimentary tract, inhibited systemic candidiasis of endogenous origin, and decreased the severity of gastric candidiasis in both mouse strains. B. infantis inhibited systemic candidiasis of endogenous origin better than B. lactis; however, B. lactis was significantly more effective at inhibiting C. albicans colonization of the alimentary tract, suppressing gastric candidiasis, and protecting bg/bg-nu/numice from lethal candidiasis than B. infantis. These results show that Bifidobacterium spp. can protect immunodeficient mice from candidiasis but different species manifest quantitative and qualitative differences in their probiotic and biotherapeutic effects.     

Lethality in LPS-induced endotoxemia in C3H/HeCr mice is associated with prevalence of proinflammatory cytokines: lack of protective action of lactoferrin

C3H/HeCr mice are more susceptible to infection compared with other strains. Lactoferrin (LF), a protein involved in innate defense, was shown to protect mice against lethal endotoxemia. In this investigation we attempt to explain the cause of increased susceptibility of C3H/HeCr mice to LPS and lack of protective LF action in these mice. We found that C3H/HeCr mice produced up to 5-fold more serum TNFalpha and 66% higher IFNgamma levels in response to i.v. LPS injection than the control, CBA strain. 24 h pretreatment of C3H/HeCr mice with LF did not cause inhibition of the LPS-induced TNFalpha serum levels, whereas in CBA mice LF significantly decreased TNFalpha level. IL-6 serum levels, in turn, were lowered in C3H/HeCr mice but elevated in CBA mice. That differential regulation of cytokine production by LF in C3H/HeCr mice paralleled a decreased survival after lethal LPS injection - 10% vs. 60% in control, PBS treated mice. In addition, determination of colony forming units (CFU) in livers and spleens after administration of 10(8) Escherichia coli revealed that pretreatment of CBA mice with LF caused a marked reduction of CFU in these organs, whereas in C3H/HeCr mice the changes were insignificant. These results indicate that the altered TNFalpha/IL-6 ratio in C3H/HeCr mice, as compared to control CBA mice, as well as the increased IFNgamma level, may be responsible for the increased susceptibility to endotoxemia in that substrain. We also suggest that an association exists between the LF protective effect against endotoxic sequelae and the insult-induced systemic immune response.     

Influence of aging on murine neutrophil and macrophage function against Candida albicans

Previous work by our group showed that aged C57BL/6 mice develop an altered innate and adaptive immune response to Candida albicans and are more susceptible to systemic primary candidiasis. In this work, we used young (2-3 months old) and aged (18-20 months old) C57BL/6 mice to study in vitro the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines by resident peritoneal macrophages in response to C. albicans, and (3) cell surface Toll-like receptor (TLR) 2 expression on resident peritoneal macrophages. Our results indicate that murine phagocytes have a fungicidal activity well preserved with aging. In vitro production of proinflammatory cytokines (IL-6, IL-1beta, and tumor necrosis factor-alpha and chemokines (MIP-2) by purified (CD11b(+)) peritoneal macrophages in response to yeasts and hyphae of C. albicans was significantly lower in aged mice as compared with young mice. However, the production of IL-10 by macrophages, in response to C. albicans, was similar in both young and aged animals. Moreover, baseline TLR2 surface expression level was lower on aged macrophages than on control macrophages. Taken together, these data indicate that the increased susceptibility to C. albicans disseminated infections in aged mice is correlated with defects in TLR2 expression and in cytokine production, but not with an impaired fungicidal activity.     

Antifungal type 1 responses are upregulated in IL-10-deficient mice

C57BL/6 mice are highly resistant to infections caused by Candida albicans and Aspergillus fumigatus. To elucidate the role of IL-10 produced by C57BL/6 mice during these infections, parameters of infection and immunity to it were evaluated in IL-10-deficient and wild-type mice with disseminated or gastrointestinal candidiasis or invasive pulmonary aspergillosis. Unlike parasitic protozoan infection, C. albicans or A. fumigatus infection did not induce significant acute toxicity in IL-10-deficient mice, who, instead, showed reduced fungal burden and fungal-associated inflammatory responses. The increased resistance to infections as compared to wild-type mice was associated with upregulation of innate and acquired antifungal Th1 responses, such as a dramatically higher production of IL-12, nitric oxide (NO) and TNF-alpha as well as IFN-gamma by CD4+ T cells. Pharmacological inhibition of NO production greatly reduced resistance to gastrointestinal candidiasis, thus pointing to the importance of IL-10-dependent NO regulation at mucosal sites in fungal infections. These results are reminiscent of those obtained in genetically susceptible mice, in which IL-10 administration increased, and IL-10 neutralization decreased, susceptibility to C. albicans and A. fumigatus infections. Collectively, these observations indicate that the absence of IL-10 augments innate and acquired antifungal immunity by upregulating type 1 cytokine responses. The resulting protective Th1 responses lead to a prompt reduction of fungal growth, thus preventing tissue destruction and lethal levels of proinflammatory cytokines.     

Protective effect of chlorpromazine against the lethality of interleukin 1 in adrenalectomized or actinomycin D-sensitized mice

Interleukin 1 (IL-1) and Tumor Necrosis Factor (TNF) are thought to play a key role in septic shock and inflammation. We have tested the effect of dexamethasone (DEX) and chlorpromazine (CPZ) on the lethal effect of IL-1, TNF and endotoxin. Two different experimental models were used to sensitize mice to the lethal effect of IL-1: adrenalectomy and pretreatment with actinomycin D. CPZ (4 mg/kg) was found to protect mice against IL-1 and endotoxin toxicity in all cases, while DEX had a protective effect only in adrenalectomized mice. In contrast to its protective effect against IL-1 and endotoxin, CPZ did not protect mice against TNF. These findings might be useful in the analysis of the differences in the actions of IL-1 and TNF in vivo, and in the development of new drugs preventing their toxicity.     

Characterisation of Candida albicans infections of haematogenous and mucosal origin in mice lacking the interferon gamma receptor protein

Mice harbouring a null deletion mutation in the IFNgamma receptor gene were used to study the role of IFNgamma responsiveness during experimental systemic candidiasis of mucosal or haematogenous origin. After intravenous (i.v.) or intranasal (i.n.) challenge with Candida albicans the progression of infection and concomitant cellular and antibody anti-C. albicans immune responses were analysed. During the week following i.v. challenge, the rate of C. albicans multiplication in kidneys, liver and spleen was faster in IFNgammaR (-/-) than IFNgammaR (+/+) mice. As a result, IFNgammaR (-/-) mice perished earlier than IFNgammaR (+/+) mice when challenged with equal numbers of live yeast cells. However, the overall susceptibility of the two mouse strains, in terms of survival against different C. albicans challenge doses over a 60-day period, was similar. No differences were found in the cellular anti-C. albicans response generated by i.v. challenge in both mouse strains. In contrast the kinetics and strength of the serum anti-C. albicans antibody responses were markedly different. Significantly stronger, predominantly IgG2a antibody responses accompanied the eventual control of C. albicans infection in IFNgammaR (-/-) mice. Following intranasal challenge, there was no difference in the rate of C. albicans clearance from the lungs of IFNgammaR (-/-) and IFNgammaR (+/+) mice. However, 48 h after challenge, large, conspicuous abscesses appeared in the lungs, liver, kidneys and spleen of IFNgammaR (-/-) mice. These abscesses were characterised by the presence of C. albicans and abundant neutrophilic infiltrates, but very few macrophages. No such abscesses developed in i.n. challenged IFNgammaR (+/+) mice. In both mouse strains, i.n. challenge induced strong systemic anti-C. albicans cellular responses, but relatively low titre systemic antibody responses. Mucosal anti-C. albicans antibody responses were detected in IFNgammaR (+/+), but not IFNgammaR (-/-) mice. Splenic adherent macrophages obtained from IFNgammaR (-/-) mice exhibited a significantly lower candidacidal activity than those of IFNgammaR (+/+) mice, and as expected, were not responsive to IFNgamma. In summary, these data suggest that IFNgamma has a role in limiting C. albicans multiplication during the early stages of infection, as well as in preventing the development of C. albicans-associated abscesses. Activation of macrophages by IFNgamma might be pivotal in mediating this role.     

Effect of N-acetylchitohexaose against Candida albicans infection of tumor-bearing mice

A water-soluble oligosaccharide, N-acetylchitohexaose (NACOS-6), was able to enhance the protective effect against Candida albicans infection in mice during the early phase of tumor-bearing. A significant decrease in the number of C. albicans cells in the kidneys of NACOS-6-treated tumor-bearing mice was observed 8 days after the fungal infection, or 15 days after the tumor transplantation. The candidacidal activity of polymorphonuclear leukocytes from NACOS-6-treated tumor-bearing mice did not differ from that of NACOS-6-untreated tumor-bearing mice. On the other hand, the candidacidal activities of both macrophages and T lymphocytes increased following administration of NACOS-6 in the early phase of tumor-bearing. The culture supernatant of T lymphocytes from NACOS-6-treated tumor-bearing mice also potentiated the candidacidal activity of casein-induced macrophages. An enhancement of natural killer cell activity of splenic lymphocytes obtained from NACOS-6-treated tumor-bearing mice was also observed.     

Heat-killed Lactobacillus gasseri TMC0356 protects mice against influenza virus infection by stimulating gut and respiratory immune responses

This study investigated whether heat-killed Lactobacillus protects host animal against influenza virus infection and stimulates their immunity. Heat-killed Lactobacillus gasseri TMC0356 was orally administered to BALB/c mice for 19 days; the mice were intranasally infected with Flu A/PR/8/34 (H1N1) on day 14, and clinical symptoms were monitored. After 6 days, the mice were sacrificed, and pulmonary virus titres were determined. Splenic activation of natural killer (NK) cells and the mRNA expression of cytokines and other immune molecules in the lung and Peyer's patch (PP) were analysed. Clinical symptom scores of mice orally fed TMC0356 ameliorated significantly (P < 0.01); their pulmonary virus titres decreased significantly compared with those of control mice (P < 0.05); their mRNA expression of interleukin (IL)-12, IL-15 and IL-21 in PP and the pulmonary mRNA expression of IFN-γ, TNF, IL-12a, IL-12rbl, IL-2rb and perforin 1 increased significantly (P < 0.05). Oral administration of heat-killed lactobacilli may protect against influenza virus infection by stimulating local and systemic immune responses. Cellular components of lactobacilli may be pivotal in protecting against viral infection by enhancing gut and respiratory immune responses.     

Local delivery of GM-CSF protects mice from lethal pneumococcal pneumonia

The growth factor GM-CSF has an important role in pulmonary surfactant metabolism and the regulation of antibacterial activities of lung sentinel cells. However, the potential of intra-alveolar GM-CSF to augment lung protective immunity against inhaled bacterial pathogens has not been defined in preclinical infection models. We hypothesized that transient overexpression of GM-CSF in the lungs of mice by adenoviral gene transfer (Ad-GM-CSF) would protect mice from subsequent lethal pneumococcal pneumonia. Our data show that intra-alveolar delivery of Ad-GM-CSF led to sustained increased pSTAT5 expression and PU.1 protein expression in alveolar macrophages during a 28-d observation period. Pulmonary Ad-GM-CSF delivery 2-4 wk prior to infection of mice with Streptococcus pneumoniae significantly reduced mortality rates relative to control vector-treated mice. This increased survival was accompanied by increased inducible NO synthase expression, antibacterial activity, and a significant reduction in caspase-3-dependent apoptosis and secondary necrosis of lung sentinel cells. Importantly, therapeutic treatment of mice with rGM-CSF improved lung protective immunity and accelerated bacterial clearance after pneumococcal challenge. We conclude that prophylactic delivery of GM-CSF triggers long-lasting immunostimulatory effects in the lung in vivo and rescues mice from lethal pneumococcal pneumonia by improving antibacterial immunity. These data support use of novel antibiotic-independent immunostimulatory therapies to protect patients against bacterial pneumonias.     

TLR3 is essential for the induction of protective immunity against Punta Toro Virus infection by the double-stranded RNA (dsRNA), poly(I:C12U), but not Poly(I:C): differential recognition of synthetic dsRNA molecules

In the wake of RNA virus infections, dsRNA intermediates are often generated. These viral pathogen-associated molecular patterns can be sensed by a growing number of host cell cytosolic proteins and TLR3, which contribute to the induction of antiviral defenses. Recent evidence indicates that melanoma differentiation-associated gene-5 is the prominent host component mediating IFN production after exposure to the dsRNA analog, poly(I:C). We have previously reported that Punta Toro virus (PTV) infection in mice is exquisitely sensitive to treatment with poly(I:C(12)U), a dsRNA analog that has a superior safety profile while maintaining the beneficial activity of the parental poly(I:C) in the induction of innate immune responses. The precise host factor(s) mediating protective immunity following its administration remain to be elucidated. To assess the role of TLR3 in this process, mice lacking the receptor were used to investigate the induction of protective immunity, type I IFNs, and IL-6 following treatment. Unlike wild-type mice, those lacking TLR3 were not protected against PTV infection following poly(I:C(12)U) therapy and failed to produce IFN-alpha, IFN-beta, and IL-6. In contrast, poly(I:C) treatment significantly protected TLR3(-/-) mice from lethal challenge despite some deficiencies in cytokine induction. There was no indication that the lack of protection was due to the fact that TLR3-deficient mice had a reduced capacity to fight infection because they were not found to be more susceptible to PTV. We conclude that TLR3 is essential to the induction of antiviral activity elicited by poly(I:C(12)U), which does not appear to be recognized by the cytosolic sensor of poly(I:C), melanoma differentiation-associated gene-5.     

The in vivo antiviral activity of interleukin-12 is mediated by gamma interferon.

The injection of 20 ng of mouse interleukin-12 (IL-12) protects mice from a lethal infection with encephalomyocarditis virus. In vitro, an anti-gamma interferon (anti-IFN-gamma) monoclonal antibody but not an anti-IL-12 monoclonal antibody neutralizes the antiviral activity present in the supernatants of splenocytes stimulated with IL-12. Finally, IL-12 fails to protect 129 Sv/Ev IFN-gamma R0/0 mice against encephalomyocarditis virus infection. These results demonstrate that IL-12 exerts its antiviral activity through the induction of endogenous IFN-gamma.