Impact of class A, B and C CpG-oligodeoxynucleotides on in vitro activation of innate immune cells in human immunodeficiency virus-1 infected individuals

Oligodeoxynucleotides (ODN) with unmethylated deoxycytidyl-deoxyguanosine dinucleotides (CpG-ODNs) stimulate Toll-like receptor 9 (TLR9) in plasmacytoid dendritic cells (pDC) and B cells and activate innate and adaptive immunity. Three classes of synthetic CpG-ODNs, class A, B and C, activate cells through TLR9; our goal was to evaluate their effect on cells from human immunodeficiency virus (HIV)-1(+) individuals. We compared the frequencies and the unstimulated activation status of immune effector cells in HIV-1(+) and HIV-1(-) individuals. Fewer pDC, myeloid dendritic cells (mDC), B cells, natural killer (NK) cells and invariant natural killer T cells (iNKT) were present in HIV-1(+) peripheral blood mononuclear cells (PBMC) and their baseline activation status was higher than HIV-1(-) PBMC. Exposure of HIV-1(+) PBMC to all classes of CpG-ODNs led to activation and maturation of pDC based on CD86, CD80, and CD83 expression similar to that of cells from HIV-1(-) individuals. The percentage of CpG-ODN stimulated pDC that express CD40 was dramatically higher when cells were obtained from HIV-1(+) than from HIV-1(-) individuals. B-lymphocytes were activated similarly in HIV-1(+) and HIV-1(-) individuals. mDC, NK and iNKT cell, which lack TLR9, were indirectly activated. Interferon-alpha (IFN-alpha) and interferon inducible protein 10 (IP-10) secretion was induced by class A or C but not class B CpG-ODN, but the concentrations were less than those produced by HIV-1(-) PBMC. HIV-1 infected individuals have fewer innate effector cells that are chronically activated, but these cells can be further activated by CpG-ODN, which suggests that synthetic CpG-ODNs could be used to enhance the immune system in HIV-1 infected individuals.     

Granulin is a soluble cofactor for toll-like receptor 9 signaling

Toll-like receptor (TLR) signaling plays a critical role in innate and adaptive immune responses and must be tightly controlled. TLR4 uses LPS binding protein, MD-2, and CD14 as accessories to respond to LPS. We therefore investigated the presence of an analagous soluble cofactor that might assist in the recruitment of CpG oligonucleotides (CpG-ODNs) to TLR9. We report the identification of granulin as?an essential secreted cofactor that potentiates TLR9-driven responses to CpG-ODNs. Granulin, an unusual cysteine-rich protein, bound to CpG-ODNs and interacted with TLR9. Macrophages from granulin-deficient mice showed not only impaired delivery of CpG-ODNs to endolysosomal compartments, but?also decreased interaction of TLR9 with CpG-ODNs. As a consequence, granulin-deficient macrophages showed reduced responses to stimulation with CpG-ODNs, a trait corrected by provision of exogenous granulin. Thus, we propose that granulin contributes to innate immunity as a critical soluble cofactor for TLR9 signaling.     

Role of scavenger receptor MARCO in macrophage responses to CpG oligodeoxynucleotides

The macrophage Class A scavenger receptor MARCO (macrophage receptor with a collagenous structure) functions as a pattern-recognition receptor for bacterial components, but its role in responses to CpG oligonucleotide sequences (CpG-ODN) in microbial DNA has not been characterized. Phosphorothioate (PS)-linked CpG-ODN stimulated IL-12 and NO production in wild-type but not in MARCO-deficient, thioglycollate-elicited peritoneal macrophages. MARCO and the related class A receptor SR-A belong to a redundant system of receptors for PS ODNs. The ability of MARCO to bind CpG-ODNs and conversely, to costimulate IL-12 and NO production upon specific ligation with immobilized mAb is consistent with MARCO being a signaling receptor for CpG-ODNs, costimulating TLR9-mediated NO and IL-12 production in macrophages. In contrast to MARCO, SR-A is likely to mediate negative regulation of macrophage responses to CpG-ODNs. In particular, increased affinity toward SR-A may contribute to decreased potency of oligo G-modified CpG-ODNs in stimulating IL-12 production. The results suggest that differential involvement of activating and inhibitory membrane receptors, such as SR-A and MARCO, may underlie profound differences observed in biological activities of different ODN sequences.     

CD40-dependent and -independent activation of human tonsil B cells by CpG oligodeoxynucleotides

Immunostimulatory CpG oligodeoxynucleotide (CpG-ODN) sequences are known to directly activate B cells. We investigated the expression of the CpG receptor, Toll-like receptor 9 (TLR9), in human tonsil B cells, and determined functional responses following stimulation by a well-characterized stimulatory CpG-containing ODN sequence in the human immune system, ODN 2006. Tonsil B cells were found to express high amounts of TLR9 mRNA and protein, and exposure of B cells to CpG-ODN but not to an inactive control ODN induced a concentration- and time-dependent up-regulation of the activation markers CD23, CD25, CD40, CD54, CD80, CD86 and HLA-DR. However, significant induction of proliferation and the release of IL-6, IL-10, IgG and IgM were only noted when B cells were co-incubated with irradiated CD40L-expressing CHO cells. Endogenous IL-10 was identified as a critical mediator of Ig production, whereas all activating effects were independent of IL-6. Further, CpG-ODN counteracted IgE production induced by IL-4. Collectively, these findings suggest a synergistic role of the TLR9/CD40 system and a critical role for the immunomodulatory cytokine IL-10 in the orchestration of CpG-ODN-induced responses in B lymphocytes.     

Characterization of suppressive oligodeoxynucleotides that inhibit Toll-like receptor-9-mediated activation of innate immunity

Synthetic oligodeoxynucleotides containing unmethylated CpG sequences (CpG-ODNs) stimulate Toll-like receptor-9 (TLR-9), thereby activating innate immunity. Stimulatory CpG-ODNs have been shown to be valuable in modifying immune responses in allergy, infection and cancer. Recently, it has been reported that the stimulation of TLR-9 by endogenous DNA might contribute to the pathogenesis of autoimmune diseases. We here report the identification of a suppressive, guanosine-rich ODN (G-ODN) that inhibited the activation of TLR-9 by stimulatory CpG-ODNs. The G-ODN was suppressive in murine macrophages and dendritic cells as well as in human plasmacytoid dendritic cells in vitro. G-ODN blocked the secretion of tumour necrosis factor-alpha (TNF-alpha) and interleukin-12p40 and interfered with the up-regulation of major histocompatibility complex (MHC) class II and costimulatory molecules. G-ODN was inhibitory even at a molar ratio of 1:10 (G-ODN:CpG-ODN) and when administered up to 7 hr after stimulation with CpG. G-ODN specifically inhibited TLR-9 but not other TLRs. Inhibition was dependent on a string of five guanosines. G-ODN was also inhibitory in an in vivo model of CpG/galactosamin (GalN) lethal shock. G-ODN interfered with upstream TLR-9 signalling. However, by extensive analysis we can exclude that G-ODN acts at the stage of cellular uptake. G-ODN therefore represents a class of suppressive ODNs that could be of therapeutic use in situations with pathologic TLR-9 activation, as has been proposed for certain autoimmune diseases.     

Human monocytes represent a competitive source of interferon-alpha in peripheral blood

Interferon-alpha (IFN-alpha) has a critical role in antiviral immunity and plasmacytoid dendritic cells (pDCs) have been demonstrated as the principal IFN-alpha source after Toll-like receptor (TLR) 7 and 9 stimulation. Little is known about the contribution of pDC-independent IFN-alpha sources to total IFN-alpha production capacity of human peripheral blood. Using an array of pathogen associated molecular patterns (PAMPs), Poly(I:C)/Dotap represented the second strongest IFN-alpha stimulus in total PBMC. Poly(I:C)/Dotap induced three times more IFN-alpha, when compared to TLR7-stimulation (R848) and four times less, when compared to TLR9-stimulation. Dotap (mediator of cellular uptake) dramatically increased Poly(I:C)-induced IFN-alpha production. Sorting experiments and ELISpot assays revealed that monocytes and not myeloid DCs are the main IFN-alpha source after Poly(I:C)/Dotap stimulation. ELISpot analyses demonstrated the highest IFN-alpha spot numbers after Poly(I:C)/Dotap stimulation. Although pDCs produced highest IFN-alpha levels per cell, monocytes represent a competing IFN-alpha source in total PBMC due to their high frequency.     

Toll-like receptor 9 mediates CpG-DNA signaling

Among the bacterial products known to activate the innate immune '1system is bacterial DNA. This activity resides within the nonmethylated CpG motifs of the DNA and is recapitulated using appropriate synthetic CpG containing oligodeoxynucleotides (CpG-ODN). TLR9-deficient mice were shown to exhibit a nonresponsive phenotype-to-bacterial DNA and CpG-ODN. Here, we describe a model system to further characterize CpG-ODN and TLR9 interactions using ectopically expressed TLR9 in HEK293 cells. Expression of TLR9 confers cellular responsiveness to CpG-ODN but not to the other bacterial products. Previous studies identified species-specific CpG-containing sequences; here, we show that expression of murine TLR9 favors responses to CpG-ODN motifs specific to mouse cells, and expression of human TLR9 favors CpG-ODN known to preferentially activate human cells. Response patterns to various CpG-ODN motifs were parallel when cells containing an ectopically expressed TLR9 and endogenous receptor were compared. Here, we also show that TLR9 acts at the cell surface and engages an intracellular signaling pathway that includes MyD88, IRAK, and TRAF6.     

Degranulation of paneth cells via toll-like receptor 9

The release of antimicrobial peptides and growth factors by Paneth cells is thought to play an important role in protecting the small intestine, but the mechanisms involved have remained obscure. Immunohistochemistry and immunofluorescence showed that Paneth cells express Toll-like receptor 9 (TLR9) in the granules. Injection of mice with oligonucleotides containing CpG sequence (CpG-ODNs) led to a down-modulation of TLR9 and a striking decrease in the number of large secretory granules, consistent with degranulation. Moreover CpG-ODN treatment increased resistance to oral challenge with virulent Salmonella typhimurium. Moreover, our findings demonstrate a sentinel role for Paneth cells through TLR9.     

Co-stimulation with TLR3 and TLR21 ligands synergistically up-regulates Th1-cytokine IFN-γ and regulatory cytokine IL-10 expression in chicken monocytes

Toll-like receptors (TLRs) are pattern recognition receptors of the innate immune system for various conserved pathogen-associated molecular motifs. Chicken TLR3 and TLR21 (avian equivalent to mammalian TLR9) recognize poly I:C (double-stranded RNA) and CpG-ODN (a CpG-motif containing oligodeoxydinucleotide), respectively. Interaction between TLR3 and TLR21 agonists poly I:C and CpG-ODN has been reported to synergize in expression of proinflammatory cytokines and chemokines and the production of nitric oxide in chicken monocytes. However, the interaction between poly I:C and CpG-ODN on the expression of interferons (IFNs) and Th1/Th2 cytokines remains unknown. The objective of the present study was to investigate the effect of the interaction between poly I:C and CpG-ODN on the mRNA expression levels of IFN-α and IFN-β, Th1 cytokines IFN-γ and IL-12, Th2 cytokine IL-4, and regulatory IL-10 in chicken monocytes. When stimulated with either agonist alone, CpG-ODN significantly up-regulated the expression of INF-γ, IL-10, and IL-12p40, but not IFN-α and IFN-β; whereas poly I:C induced the expression of INF-γ, IFN-α, IFN-β, and IL-10; but not IL-12p40. However, stimulation with a combinatory CpG-ODN and poly I:C further synergistically increased the expression of IFN-γ and IL-10 mRNA. Our results provide strong evidence supporting the critical role of TLR3 and TLR21 in avian innate immunity against both viral and bacterial infections; and the synergistic interaction between the TLR3 and TLR21 pathways produces a stronger Th1-biased immune response in chicken monocytes. Our result also suggest a potential use of poly I:C and CpG-ODN together as a more efficient adjuvant for poultry vaccine development.     

Human monocyte-derived dendritic cells express TLR9 and react directly to the CpG-A oligonucleotide D19

Oligodeoxynucleotides (ODNs) containing unmethylated CpG exhibit their immunostimulatory activities by binding to TLR. Here, we show that human monocyte-derived dendritic cells (moDC) contain TLR9 protein, surprisingly, in amounts comparable with plasmacytoid DC (pDC). Immature moDC but not mature moDC nor monocytes captured CpG-ODNs. moDC stimulation with the CpG-A ODN D19 up-regulated CD83, CD86, and HLA-DR. Without CD40 ligand costimulation, full maturation was not achieved. D19-stimulated moDC primed allogeneic CD4(+)-T cells for proliferation and differentiation into IFN-gamma-secreting Th1 cells. Neither IL-12 nor IL-6 or TNF-alpha was involved. Microarray analysis pointed to a participation of Type I IFNs. In fact, D19-stimulated moDC secreted considerable amounts of IFN-alpha. This indicates that moDC themselves sense viral and bacterial DNA and do not need help from pDC.     

CG sequence- and phosphorothioate backbone modification-dependent activation of the NF-kappaB-responsive gene expression by CpG-oligodeoxynucleotides in human RPMI 8226 B cells

Oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have gained attention because of their stimulatory effects on innate immune responses. CpG-ODN 1826 containing two GACGTT motifs is well known to activate the mouse immune cells while CpG-ODN 2006 containing three GTCGTT motifs is optimal for human cells. We have shown that stimulation of the human B cell line RPMI 8226 with CpG-ODN 1826 or 2006 results in the activation of IL-8 promoter and nuclear localization of NF-kappaB in the CG sequence- and phosphorothioate backbone modification-dependent manner. It was also demonstrated that myeloid differentiation protein and tumor necrosis factor receptor-associated factor 6 are involved in the signal transduction pathway triggered by the CpG-ODNs. Furthermore, phosphorothioate-modified CpG-ODN 1826 led to induce the NF-kappaB-responsive inflammatory cytokine gene expression in the cells. Experimental results indicated that the phosphorothioate derivative of CpG-ODN 1826 not only activates the mouse immune cells, but also stimulates NF-kappaB responsive gene expression in the human B cell line.     

Association of polymorphisms of toll-like receptor 4 with a reduced prevalence of hay fever and atopy.

BACKGROUND: The response to innate immune stimuli seems to be critical to conditioning adaptive immunity. Early exposure to endotoxin initiates immune responses that have been shown to alter the risk of asthma and allergic diseases. The toll-like receptor 4 (TLR4) gene encodes the principal innate immunity receptor in humans for bacterial endotoxin. Polymorphisms in the TLR4 gene may regulate the effects of endotoxin exposure and could play a role in the development of asthma and atopy-related phenotypes. OBJECTIVE: To investigate the association between TLR4 polymorphisms and allergic phenotypes in nonsmokers. METHODS: The data from 915 nonsmoking students were available for the study. The TLR4 299 and 399 polymorphisms were genotyped using mouthwash samples. The TLR4 299 and 399 polymorphisms were grouped together to define the TLR4 polymorphic group. Skin prick tests were conducted in a subgroup of healthy participants. A brief questionnaire was administered to determine demographic characteristics and chronic health conditions. RESULTS: The prevalence of hay fever was 0% in the TLR4 polymorphic group and 7.5% in the wild-type group (P = .01). After controlling for age group and sex using logistic regression, the odds of having hay fever were reduced by 88% (P = .009) in the TLR4 polymorphic group compared with the wild-type group. In a subgroup analysis, the association between TLR4 polymorphisms and atopy was only observed among females. CONCLUSIONS: To our knowledge, this study is the first to report an association between TLR4 polymorphisms and atopy-related phenotypes in a nonsmoking population. Further investigation of the role of TLR4 polymorphisms in asthma and atopy-related phenotypes is warranted.     

CpG-ODN对肺腺癌细胞增殖的抑制作用及其与Runx3关系的初步研究

目的探讨CpG-ODN对人肺腺癌A549细胞的增殖及其抑癌基因Runx相关转录因子3(Runx3)表达的影响,探讨TLR9与肺腺癌发生发展的关系,为基于TLR9的肿瘤治疗寻找理论依据。方法不同浓度CpG-ODN作用于人肺腺癌A549细胞系,用RT-PCR和Western blot分别检测Runx3在mRNA和蛋白水平的表达情况。针对Runx3基因的特定靶位,采用化学合成法合成Runx3 siRNA,并瞬时转染A549细胞,用MTT法检测CpG-ODN对转染前后的细胞生长作用的影响。结果 CpG-ODN能明显抑制A549细胞的增殖;不同浓度CpG-ODN刺激A549后,细胞中Runx3基因在mRNA和蛋白表达水平均增加;Runx3 siRNA的瞬时转入对CpG-ODN刺激的A549增殖抑制作用明显减弱。结论 TLR9的配体CpG-ODN可抑制A549细胞的增殖,同时正向调控Runx3的表达。由此推测,CpG-ODN可能通过上调Runx3抑制A549细胞的增殖。     

CpG-ODN enhances ingestion of apoptotic neutrophils by macrophages

The clearance of apoptotic neutrophils by macrophages plays an important role in the process of inflammatory response. In the present study, we examined the ability of macrophages to ingest apoptotic neutrophils after activated by synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODN) in vitro. The results showed that, while CpG-ODN at the experimental concentration had no cytotoxic effect on the viability of macrophages, the percentage of macrophages with ingested apoptotic neutrophils was increased from 23.6 to 42.30% by CpG-ODN stimulation. This effect was silenced when macrophages were treated with the mutation of CpG-ODN motifs. Both the total and cell surface protein of Toll-like receptor 9 (TLR9) expression in macrophages was up-regulated after CpG-ODN stimulation. While chloroquine (CHQ) had no effect on TLR9 expression in macrophages, it abolished the enhanced uptake of apoptotic neutrophils by macrophages. Although CpG-ODN had no significant effect on the IL-6 production, it was able to induce the increase of TNF-α protein expression and this effect was inhibited by CHQ pretreatment. Increased TNF-α production from macrophages induced by CpG-ODN stimulation was down-regulated after phagocytosis of apoptotic neutrophils. In conclusion, CpG-ODN could enhance the ingestion of apoptotic neutrophils by macrophages via TLR9 accompanied with an increasing in the level of TNF-α. After phagocytosis of apoptotic neutrophils, the increased TNF-α production from macrophages induced by CpG-ODN stimulation was down-regulated which the implications in the immune response remains for the further study.     

Cross-talk between toll-like receptors 5 and 9 on activation of human immune responses

The recognition of pathogen-associated molecular patterns by TLRs triggers the activation of innate and adaptive immune responses. Flagellin, the agonist of TLR5, is expressed by prokaryotes and eukaryotes, and DNA sequences containing unmethylated CpG dinucleotides, agonists of TLR9, are present essentially in prokaryotes. To test the potential modulating effects of simultaneous activation of different TLRs on the immune response, we compared the outcomes in different immune cell compartments induced by triggering TLR5 and TLR9 individually and in combination. PBMCs, monocytes, and monocyte-derived DC (MoDC) secreted high levels of IL-10 in response to flagellin, whereas oligodeoxynucleotides (ODN) containing the CpG sequence (CpG-ODN), synthetic ligands of TLR9, did not induce IL-10 secretion in any of the three cell types but synergized with flagellin in this induction. In contrast, PBMC production of IFN-alpha induced by CpG-ODN was strongly inhibited by flagellin. Conversely, CpG-ODN did not enhance the up-regulation of activation markers in MoDC induced to mature in the presence of flagellin. Flagellin-matured, but not CpG-ODN-matured, MoDC stimulated the expansion of allogeneic CD4+CD25+ T cells, and the extent of expansion induced by MoDC, matured in the presence of flagellin and CpG-ODN, was similar to that induced by flagellin-matured MoDC. Moreover, flagellin and CpG-ODN differentially affected NK-mediated cytotoxicity, and flagellin completely abrogated the NK-mediated immune response induced by CpG-ODN stimulation. Together, these results suggest that flagellin inhibits the TLR9-induced cell activation and cytokine production, which favor Th1-type immune responses, possibly because the signals evoked by flagellin to indicate the presence of extracellular pathogens must favor a Th2-polarized response. Thus, TLR5 and TLR9, alerted by the presence of microorganisms, influence each other to mount the more efficient and appropriate immune response to contain the infection of a specific pathogen.     

Monocyte-derived dendritic cells from highly atopic individuals are not impaired in their pro-inflammatory response to toll-like receptor ligands

BACKGROUND: Toll-like receptor (TLR) agonists are widely used as adjuvants in specific immune therapy protocols for patients with atopic disposition. Monocyte-derived dendritic cells (mDCs) are thought to be important target cells for these compounds. OBJECTIVES: To compare surface markers, TLR expression, TLR functionality after ligand stimulation, and genetic polymorphisms in the TLR 2-, 3-, and 4-genes in mDCs from atopic vs. non-atopic patients. METHODS: mDCs from highly atopic individuals (total serum IgE >1000 IU/mL) and healthy control persons (total serum IgE <75 IU/mL) were screened for TLR 1-10 expression by real-time PCR. Receptor function was analysed by IL-12 and TNF-alpha production after incubation with the respective ligands peptidoglycan (PGN) (TLR 2), polyriboinosinic-polyribocytidylic acid (poly IC) (TLR 3), lipopolysaccharide (LPS) (TLR 4), flagellin (TLR 5), and CpG-DNA/non-CpG-DNA (TLR 9). Haplotype-tagging single-nucleotide polymorphisms of the TLR 2-, 3-, and 4- genes were analysed for genetic associations. RESULTS: mDC from atopic patients showed a very similar pattern of TLR expression as controls with strong expression of TLR 2, 4, 5, 6, and 8, moderate expression of TLR 1 and 3, and no or very low expression of TLR 7, 9, and 10. After stimulation with TLR ligands, mDCs from atopic patients acquired a mature phenotype with a tendency towards a higher up-regulation of the co-stimulatory molecules CD80, CD83, and CD86 than control mDCs. IL-12 and TNF-alpha were produced at a similar level in both groups of DCs. Among the different TLR agonists, poly IC showed the strongest activation of DCs, followed by LPS, PGN, and flagellin. This was paralleled by a strong functional expression of protein kinase R and retinoid-inducible gene-I (RIG-I), two additional poly IC-sensing receptors in both groups. Genetic analysis of single-nucleotide polymorphisms in the TLR 2-, 3-, and 4-genes in both groups revealed no major allele or genotype differences. CONCLUSIONS: mDC from atopic patients are not restricted in their response to TLR-ligands. TLR agonists seem to be suitable to induce pro-inflammatory immune responses and maturation in mDCs from highly atopic individuals and represent reasonable adjuvants for specific immunotherapy reagents.     

Interleukin receptor-associated kinase-4 deficiency impairs Toll-like receptor-dependent innate antiviral immune responses

BACKGROUND: Engagement of all known Toll-like receptors (TLRs) causes the production of inflammatory cytokines, including TNF-alpha, whereas in humans, engagement of TLRs 3, 7, 8, and 9 also induces type I IFNs. IRAK-4 is a critical effector in signaling by TLRs and the IL-1 receptor, which share homology in their intracellular domain and recruit IRAK-4 via the adaptor myeloid differentiation factor 88 (MyD88). Patients with IRAK-4 deficiency are susceptible to invasive bacterial infections but have so far not been reported to be susceptible to viral infection. Blood cells from these patients are impaired in their ability to make TNF-alpha in response to activation by TLRs. A recent report has described concomitant impairment of type I IFN production after activation of TLRs 7, 8, and 9, but not TLR3. OBJECTIVES: We sought to evaluate the role of IRAK-4 in TLR-induced production of the type I IFN, IFN-alpha, in humans. METHODS: We examined TLR-induced production of TNF-alpha and IFN-alpha in PBMCs from an IRAK-4-deficient patient, his heterozygous carrier parents, and normal controls. RESULTS: TNF-alpha production in response to TLR agonists was severely impaired in the patient. IFN-alpha production induced by TLR7, TLR8, and TLR9, as well as TLR3 agonists, was low or absent. CONCLUSIONS: IRAK-4 plays an important role in the production of type I IFN, as well as TNF-alpha, induced by all TLRs, including TLR3. CLINICAL IMPLICATIONS: IRAK-4 may play a broader role in human innate antiviral immunity than previously appreciated.     

IFNs activate toll-like receptor gene expression in viral infections

Toll-like receptors (TLRs) mediate innate immune responses to microbes. TLR2, TLR5, TLR6, and TLR9 have been implicated in responses to bacterial components, and TLR4 is the receptor for Gram-negative bacteria. Recently, TLR4 was described to function in respiratory syncytial virus-induced NF-kappaB activation. Here we have analyzed TLR1-9 mRNA expression in human primary macrophages infected with influenza A and Sendai viruses. TLR1, TLR2, TLR4, TLR6, and TLR8 mRNAs were expressed at basal levels in macrophages. Viral infection enhanced TLR1, TLR2, TLR3, and TLR7 mRNA expression, and neutralizing anti-IFN-alpha/beta antibodies downregulated gene expression of these TLRs. Exogenously added IFN-alpha upregulated TLR1, TLR2, TLR3, and TLR7 mRNA expression in macrophages, as well as TLR3 mRNA expression in epithelial and endothelial cell lines. IFN-gamma enhanced the expression of TLR1 and TLR2 mRNA in macrophages, and TLR3 in epithelial and endothelial cells. The data suggests a novel role for IFNs in the activation of innate immunity.