Effects of type I interferons on Friend retrovirus infection

The type I interferon (IFN) response plays an important role in the control of many viral infections. However, since there is no rodent animal model for human immunodeficiency virus, the antiviral effect of IFN-alpha and IFN-beta in retroviral infections is not well characterized. In the current study we have used the Friend virus (FV) model to determine the activity of type I interferons against a murine retrovirus. After FV infection of mice, IFN-alpha and IFN-beta could be measured between 12 and 48 h in the serum. The important role of type I IFN in the early immune defense against FV became evident when mice deficient in IFN type I receptor (IFNAR(-/-)) or IFN-beta (IFN-beta(-/-)) were infected. The levels of FV infection in plasma and in spleen were higher in both strains of knockout mice than in C57BL/6 wild-type mice. This difference was induced by an antiviral effect of IFN-alpha and IFN-beta and was most likely mediated by antiviral enzymes as well as by an effect of these IFNs on T-cell responses. Interestingly, the lack of IFNAR and IFN-beta enhanced viral loads during acute and chronic FV infection. Exogenous IFN-alpha could be used therapeutically to reduce FV replication during acute but not chronic infection. These findings indicate that type I IFN plays an important role in the immediate antiviral defense against Friend retrovirus infection.     

Activation of Toll-like receptor 3 impairs the dengue virus serotype 2 replication through induction of IFN-β in cultured hepatoma cells

Toll-like receptors (TLRs) play an important role in innate immunity against invading pathogens. Although TLR signaling has been indicated to protect cells from infection of several viruses, the role of TLRs in Dengue virus (DENV) replication is still unclear. In the present study, we examined the replication of DENV serotype 2 (DENV2) by challenging hepatoma cells HepG2 with different TLR ligands. Activation of TLR3 showed an antiviral effect, while pretreatment of other TLR ligands (including TLR1/2, TLR2/6, TLR4, TLR5 or TLR7/8) did not show a significant effect. TLR3 ligand poly(I:C) treatment prior to viral infection or simultaneously, but not post-treatment, significantly down-regulated virus replication. Pretreatment with poly(I:C) reduced viral mRNA expression and viral staining positive cells, accompanying an induction of the type I interferon (IFN-β) and type III IFN (IL-28A/B). Intriguingly, neutralization of IFN-β alone successfully restored the poly(I:C)-inhibited replication of DENV2. The poly(I:C)-mediated effects, including IFN induction and DENV2 suppression, were significantly reversed by IKK inhibitor, further suggesting that IFN-β is the dominant factor involved in the poly(I:C) mediated antiviral effect. Our study presented the first evidence to show that activation of TLR3 is effective in blocking DENV2 replication via IFN-β, providing an experimental clue that poly(I:C) may be a promising immunomodulatory agent against DENV infection and might be applicable for clinical prevention.     

Toll-Like Receptor 7 Inhibits Early Acute Retroviral Infection through Rapid Lymphocyte Responses

Early events during retroviral infection play a critical role in determining the course of infection and pathogenesis, but the mechanisms that regulate this phase of infection are poorly understood. Toll-like receptor 7 (TLR7) is required for promoting germinal center reactions and virus-specific neutralizing antibodies at later time points postinfection, but TLR7''s role in early acute infection has not been determined. By infecting TLR7-deficient mice with a retroviral pathogen, Friend virus (FV), I determined that TLR7 potently inhibits retroviral replication during the first 5 days of infection and is required for rapid secretion of virus-specific IgM and interleukin-10 (IL-10) in response to infection. Although the IgM response was nonneutralizing, plasmas from wild-type mice but not TLR7-deficient mice inhibited FV replication when passively transferred to infected mice, suggesting an indirect mechanism of antibody function. Interestingly, IL-10 was secreted primarily by CD4 T cells, and IL-10-deficient mice also exhibited accelerated early virus spread, demonstrating that this response inhibits acute infection. Surprisingly, TLR7-deficient mice exhibited normal or elevated secretion of proinflammatory cytokines during acute infection, revealing the existence of a TLR7-independent retrovirus-sensing pathway that drives inflammatory cytokine secretion. Together, these results establish a previously unappreciated role for lymphocytes in mediating rapid TLR7-dependent inhibition of early retroviral infection through nonneutralizing IgM and IL-10.     

Toll-like receptor 3 activation induces antiviral immune responses in mouse sertoli cells

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and elicit antimicrobial immune responses. In the testis, viruses can induce pathological conditions, such as orchitis, and may participate in the etiology of testicular cancer; however, the molecular mechanisms involved remain under investigation. It has been suggested that because they constitutively express interferon (IFN)-inducible antiviral proteins, Sertoli cells participate in the testicular antiviral defense system. Previously, we demonstrated a key function of mouse Sertoli cells in the bactericidal testicular defense mechanism mediated by a panel of TLRs. To better characterize the potential role of Sertoli cells in the response against testicular viral infections, we investigated the TLR3 expression and function in these cells. Sertoli cells express TLR3, and under stimulation with the synthetic double-stranded RNA analogue poly (I:C), they produce the proinflammatory molecule ICAM1 and secrete functionally active CCL2 chemokine. Using both pharmacological and genetic approaches, we found that these effects are TLR3-dependent. Moreover, using ELISA, we found that IFNA is constitutively produced and not further inducible, whereas IFNB1 is absent and dramatically induced only by transfected poly (I:C), indicating different control mechanisms underlying IFNA and IFNB1 production. To conclude, poly (I:C) elicits both inflammatory and antiviral responses in Sertoli cells.     

Polyinosinic-polycytidylic acid limits tumor outgrowth in a mouse model of metastatic lung cancer

Polyinosinic-polycytidylic acid (poly I:C), a TLR3 ligand, is currently being tested in human clinical trials as an adjuvant to anti-cancer vaccines and in combination with other therapies. However, little is known about its activity in established pulmonary metastasis. The aim of our study was to elucidate the effect of poly I:C (1, 10, or 100 μg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth was arrested after a single administration of poly I:C. This was associated with higher influx of mature dendritic cells (DCs), which drove toward a Th1-like, Th17-like, and cytotoxic immune environment. The interference with IFN type I receptor signaling by means of a specific mAb reversed poly I:C-mediated tumor regression due to lower presence of myeloid DCs, cytotoxic DCs (CD11c(+)CD8(+)), NKT cells, CD8(+) T cells, and Th1-like cytokines. Moreover, the adoptive transfer of poly I:C-activated bone marrow-derived DCs into tumor-bearing mice resulted in activities similar to those of the systemic administration of poly I:C on lung tumor burden. In conclusion, our data prove that poly I:C has potential anti-tumor activity in a mouse model of established pulmonary metastasis. The activation of DCs and the production of IFN type I are responsible for an effective T cytotoxic immune response against metastatic lung cancer progression after poly I:C treatment.     

Interferon-alpha Subtype 11 Activates NK Cells and Enables Control of Retroviral Infection

The innate immune response mediated by cells such as natural killer (NK) cells is critical for the rapid containment of virus replication and spread during acute infection. Here, we show that subtype 11 of the type I interferon (IFN) family greatly potentiates the antiviral activity of NK cells during retroviral infection. Treatment of mice with IFN-α11 during Friend retrovirus infection (FV) significantly reduced viral loads and resulted in long-term protection from virus-induced leukemia. The effect of IFN-α11 on NK cells was direct and signaled through the type I IFN receptor. Furthermore, IFN-α11-mediated activation of NK cells enabled cytolytic killing of FV-infected target cells via the exocytosis pathway. Depletion and adoptive transfer experiments illustrated that NK cells played a major role in successful IFN-α11 therapy. Additional experiments with Mouse Cytomegalovirus infections demonstrated that the therapeutic effect of IFN-α11 is not restricted to retroviruses. The type I IFN subtypes 2 and 5, which bind the same receptor as IFN-α11, did not elicit similar antiviral effects. These results demonstrate a unique and subtype-specific activation of NK cells by IFN-α11.     

Poly I:C enhances susceptibility to secondary pulmonary infections by gram-positive bacteria

Secondary bacterial pneumonias are a frequent complication of influenza and other respiratory viral infections, but the mechanisms underlying viral-induced susceptibility to bacterial infections are poorly understood. In particular, it is unclear whether the host's response against the viral infection, independent of the injury caused by the virus, results in impairment of antibacterial host defense. Here, we sought to determine whether the induction of an "antiviral" immune state using various viral recognition receptor ligands was sufficient to result in decreased ability to combat common bacterial pathogens of the lung. Using a mouse model, animals were administered polyinosine-polycytidylic acid (poly I:C) or Toll-like 7 ligand (imiquimod or gardiquimod) intranasally, followed by intratracheal challenge with Streptococcus pneumoniae. We found that animals pre-exposed to poly I:C displayed impaired bacterial clearance and increased mortality. Poly I:C-exposed animals also had decreased ability to clear methicillin-resistant Staphylococcus aureus. Furthermore, we showed that activation of Toll-like receptor (TLR)3 and Retinoic acid inducible gene (RIG-I)/Cardif pathways, which recognize viral nucleic acids in the form of dsRNA, both contribute to poly I:C mediated impairment of bacterial clearance. Finally, we determined that poly I:C administration resulted in significant induction of type I interferons (IFNs), whereas the elimination of type I IFN signaling improved clearance and survival following secondary bacterial pneumonia. Collectively, these results indicate that in the lung, poly I:C administration is sufficient to impair pulmonary host defense against clinically important gram-positive bacterial pathogens, which appears to be mediated by type I IFNs.     

Innate immunity in the human female reproductive tract: antiviral response of uterine epithelial cells to the TLR3 agonist poly(I:C)

The objective of this study was to examine the expression of TLR by human primary uterine epithelial cells (UEC) and to determine whether exposure to the TLR agonist poly(I:C) would induce an antiviral response. The secretion of several cytokines and chemokines was examined as well as the mRNA expression of human beta-defensin-1 and -2 (HBD1 and HBD2), IFN-beta, and the IFN-beta-stimulated genes myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase. The expression of TLR1-9 by UEC was demonstrated by RT-PCR, with only TLR10 not expressed. Stimulation of UEC with the TLR3 agonist poly(I:C) induced the expression of the proinflammatory cytokines TNF-alpha, IL-6, GM-CSF, and G-CSF, as well as the chemokines CXCL8/IL-8, CCL2/MCP-1, and CCL4/MIP-1beta. In addition, poly(I:C) exposure induced the mRNA expression of HBD1 and HBD2 by 6- and 4-fold, respectively. Furthermore, upon exposure to poly(I:C) UEC initiated a potent antiviral response resulting in the induction of IFN-beta mRNA expression 70-fold and myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase mRNA expression (107- and 96-fold), respectively. These results suggest that epithelial cells that line the uterine cavity are sensitive to viral infection and/or exposure to viral dsRNA released from killed epithelial cells. Not only do UEC release proinflammatory cytokines and chemokines that mediate the initiation of an inflammatory response and recruitment of immune cells to the site of infection, but they also express beta-defensins, IFN-beta, and IFN-beta-stimulated genes that can have a direct inhibiting effect on viral replication.     

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.     

A temporal role of type I interferon signaling in CD8+ T cell maturation during acute West Nile virus infection

A genetic absence of the common IFN-α/β signaling receptor (IFNAR) in mice is associated with enhanced viral replication and altered adaptive immune responses. However, analysis of IFNAR(-/-) mice is limited for studying the functions of type I IFN at discrete stages of viral infection. To define the temporal functions of type I IFN signaling in the context of infection by West Nile virus (WNV), we treated mice with MAR1-5A3, a neutralizing, non cell-depleting anti-IFNAR antibody. Inhibition of type I IFN signaling at or before day 2 after infection was associated with markedly enhanced viral burden, whereas treatment at day 4 had substantially less effect on WNV dissemination. While antibody treatment prior to infection resulted in massive expansion of virus-specific CD8(+) T cells, blockade of type I IFN signaling starting at day 4 induced dysfunctional CD8(+) T cells with depressed cytokine responses and expression of phenotypic markers suggesting exhaustion. Thus, only the later maturation phase of anti-WNV CD8(+) T cell development requires type I IFN signaling. WNV infection experiments in BATF3(-/-) mice, which lack CD8-α dendritic cells and have impaired priming due to inefficient antigen cross-presentation, revealed a similar effect of blocking IFN signaling on CD8(+) T cell maturation. Collectively, our results suggest that cell non-autonomous type I IFN signaling shapes maturation of antiviral CD8(+) T cell response at a stage distinct from the initial priming event.     

Uterine epithelial cells specifically induce interferon-stimulated genes in response to polyinosinic-polycytidylic acid independently of estradiol

Interferon β (IFNβ) is an antiviral cytokine secreted in response to pathogenic exposure that creates a restrictive intracellular environment through the action of downstream interferon-stimulated genes (ISG). The objective of this study was to examine the expression of IFNβ and ISG in both human uterine epithelial cells (UEC) and the ECC-1 uterine epithelial cell line and determine if expression changes with TLR stimulation and hormone exposure. Stimulation of primary uterine epithelial cells and ECC-1 cells with the TLR3 agonist poly (I:C) induced the mRNA expression of IFNβ, MxA, OAS2 and PKR. Other TLR agonists including imiquimod and CpG had no effect on either IFNβ or ISG expression. In contrast to ECC-1 cell responses which were slower, maximal IFNβ upregulation in UEC occurred 3 hours post-stimulation and preceded the ISG response which peaked approximately 12 hours after poly (I:C) exposure. Unexpectedly, estradiol, either alone or prior to treatment with poly (I:C), had no effect on IFNβ or ISG expression. Blockade of the IFN receptor abrogated the upregulation of MxA, OAS2 and PKR. Furthermore, neutralizing antibodies against IFNβ partially inhibited the upregulation of all three ISG. Estradiol, directly and in the presence of poly (I:C) had no effect on IFNβ and ISG expression. These results indicate that uterine epithelial cells are important sentinels of the innate immune system and demonstrate that uterine epithelial cells are capable of mounting a rapid IFN-mediated antiviral response that is independent of estradiol and is therefore potentially sustained throughout the menstrual cycle to aid in the defense of the uterus against potential pathogens.     

Effective postexposure treatment of retrovirus-induced disease with immunostimulatory DNA containing CpG motifs

Therapeutic strategies for the treatment of acute retroviral infections have relied mainly on antiviral drugs. In this study we used the Friend virus model system to demonstrate that enhancement of the immune system can also have dramatic therapeutic effects. Since resistance to Friend virus-induced leukemia in mice is associated with T helper cell type 1 (Th1) immune responses, we enhanced these responses in susceptible mice by treatment with synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN). Treatments begun at 4 days postinfection increased recovery from 6% in the control group to 74% in the CpG-treated group. CpG-mediated recovery was associated with a significant reduction of viral loads in the blood and spleens of treated mice compared to those of control animals. The treatment promoted Th1-type cytokine production by splenocytes of Friend virus-infected mice and augmented Friend virus-specific cytotoxic T-cell responses, but no influence on the virus-specific neutralizing antibody response was observed. Friend virus-specific CD8(+) T cells were critical for effective treatment with CpG-ODN, since in vivo depletion of these cells from treated mice prevented their recovery. Our results demonstrate that CpG-ODN therapy can significantly enhance virus-specific cellular immune responses and prevent retrovirus-induced disease. These findings may have implications for antiviral therapy in general.     

Mechanism of up-regulation of human Toll-like receptor 3 secondary to infection of measles virus-attenuated strains

PolyI:C, a synthetic double-stranded (ds)RNA, and viruses act on cells to induce IFN-beta which is a key molecule for anti-viral response. Although dsRNA is a virus-specific signature and a ligand for human Toll-like receptor 3 (TLR3), largely uncharacterized multiple pathways associate virus-mediated IFN-beta induction. Here, we demonstrated that laboratory-adapted but not wild-type strains of measles virus (MV) up-regulated TLR3 expression both in dendritic cells and epithelial cell line A549. The kinetics experiments with the laboratory MV strain revealed that TLR3 was induced late compared to IFN-beta and required new protein synthesis. Furthermore, neutralizing antibodies against IFN-beta or IFNAR (Interferon-alpha/beta receptor) suppressed MV-induced TLR3 induction, indicating that type I IFN, IFN-alpha/beta, is critical for MV-mediated TLR3 induction. Yet, a recently identified virus-inducible IFN, the IFN-lambda, did not contribute to TLR3 expression. A virus-responsive element that up-regulates TLR3 was identified in the TLR3-promoter region by reporter gene experiments. The ISRE, a recently reported site for IFN-beta induction, but not STAT binding site, located around -30bp of TLR3 promoter responded to MV to induce TLR3 expression. This further indicates the importance of type I IFN for TLR3 up-regulation in the case of viral infection. In HeLa and MRC5 cells, augmented production of IFN-beta was observed in response to dsRNA when TLR3 had been induced beforehand. Thus, the MV-induced expression of TLR3 may reflect amplified IFN production that plays a part in host defense to viral infection.     

Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-α/β) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-α/β production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.     

TLR activation of Langerhans cell-like dendritic cells triggers an antiviral immune response

Langerhans cells (LC) are a unique subset of dendritic cells (DC), present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses, we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1-10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types, stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-gamma) and CXCL11 (IFN-gamma-inducible T cell alpha chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally, CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together, our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system.     

Attrition of bystander CD8 T cells during virus-induced T-cell and interferon responses

Experiments designed to distinguish virus-specific from non-virus-specific T cells showed that bystander T cells underwent apoptosis and substantial attrition in the wake of a strong T-cell response. Memory CD8 T cells (CD8(+) CD44(hi)) were most affected. During acute viral infection, transgenic T cells that were clearly defined as non-virus specific decreased in number and showed an increase in apoptosis. Also, use of lymphocytic choriomeningitis virus (LCMV) carrier mice, which lack LCMV-specific T cells, showed a significant decline in non-virus-specific memory CD8 T cells that correlated to an increase in apoptosis in response to the proliferation of adoptively transferred virus-specific T cells. Attrition of T cells early during infection correlated with the alpha/beta interferon (IFN-alpha/beta) peak, and the IFN inducer poly(I:C) caused apoptosis and attrition of CD8(+) CD44(hi) T cells in normal mice but not in IFN-alpha/beta receptor-deficient mice. Apoptotic attrition of bystander T cells may make room for the antigen-specific expansion of T cells during infection and may, in part, account for the loss of T-cell memory that occurs when the host undergoes subsequent infections.     

Toll-like receptors 2 and 4 regulate the frequency of IFNγ-producing CD4+ T-cells during pulmonary infection with Chlamydia pneumoniae

TLR2 and TLR4 are crucial for recognition of Chlamydia pneumoniae in vivo, since infected TLR2/4 double-deficient mice are unable to control the infection as evidenced by severe loss of body weight and progressive lethal pneumonia. Unexpectedly, these mice display higher pulmonary levels of the protective cytokine IFNγ than wild type mice. We show here, that antigen-specific CD4(+) T-cells are responsible for the observed IFNγ-secretion in vivo and their frequency is higher in TLR2/4 double-deficient than in wild type mice. The capacity of TLR2/4 double-deficient dendritic cells to re-stimulate CD4(+) T-cells did not differ from wild type dendritic cells. However, the frequency of CD4(+)CD25(+)Foxp3(+) T-cells was considerably higher in wild type compared to TLR2/4 double-deficient mice and was inversely related to the number of IFNγ-secreting CD4(+) effector T-cells. Despite increased IFNγ-levels, at least one IFNγ-mediated response, protective NO-secretion, could not be induced in the absence of TLR2 and 4. In summary, CD4(+)CD25(+)Foxp3(+) regulatory T-cells fail to expand in the absence of TLR2 and TLR4 during pulmonary infection with C. pneumoniae, which in turn enhances the frequency of CD4(+)IFNγ(+) effector T-cells. Failure of IFNγ to induce NO in TLR2/4 double-deficient cells represents one possible mechanism why TLR2/4 double-deficient mice are unable to control pneumonia caused by C. pneumoniae and succumb to the infection.     

Bovine plasmacytoid dendritic cells are the major source of type I interferon in response to foot-and-mouth disease virus in vitro and in vivo

Type I interferons (alpha/beta interferons [IFN-α/β]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3(-) CD14(-) CD21(-) CD11c(-) NK(-) TCRδ(-) CD4(+) MHC II(+) CD45RB(+) CD172a(+) CD32(+)). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.