Role of interferon regulatory factor 7 in T cell responses during acute lymphocytic choriomeningitis virus infection.

Type I interferons (IFNs), predominantly IFN-α and -ß, play critical roles in both innate and adaptive immune responses against viral infections. Interferon regulatory factor 7 (IRF7), a key innate immune molecule in the type I IFN signaling pathway, is essential for the type I IFN response to many viruses, including lymphocytic choriomeningitis virus (LCMV). Here, we show that although IRF7 knockout (KO) mice failed to control the replication of LCMV in the early stages of infection, they were capable of clearing LCMV infection. Despite the lack of type I IFN production, IRF7 KO mice generated normal CD4(+) T cell responses, and the expansion of naïve CD8(+) T cells into primary CD8(+) T cells specific for LCMV GP(33-41) was relatively normal. In contrast, the expansion of the LCMV NP(396)-specific CD8(+) T cells was severely impaired in IRF7 KO mice. We demonstrated that this defective CD8(+) T cell response is due neither to an impaired antigen-presenting system nor to any intrinsic role of IRF7 in CD8(+) T cells. The lack of a type I IFN response in IRF7 KO mice did not affect the formation of memory CD8(+) T cells. Thus, the present study provides new insight into the impact of the innate immune system on viral pathogenesis and demonstrates the critical contribution of innate immunity in controlling virus replication in the early stages of infection, which may shape the quality of CD8(+) T cell responses.

Induction and inhibition of type I interferon responses by distinct components of lymphocytic choriomeningitis virus.

Type I interferons (IFNs) play a critical role in the host defense against viruses. Lymphocytic choriomeningitis virus (LCMV) infection induces robust type I IFN production in its natural host, the mouse. However, the mechanisms underlying the induction of type I IFNs in response to LCMV infection have not yet been clearly defined. In the present study, we demonstrate that IRF7 is required for both the early phase (day 1 postinfection) and the late phase (day 2 postinfection) of the type I IFN response to LCMV, and melanoma differentiation-associated gene 5 (MDA5)/mitochondrial antiviral signaling protein (MAVS) signaling is crucial for the late phase of the type I IFN response to LCMV. We further demonstrate that LCMV genomic RNA itself (without other LCMV components) is able to induce type I IFN responses in various cell types by activation of the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5. We also show that expression of the LCMV nucleoprotein (NP) inhibits the type I IFN response induced by LCMV RNA and other RIG-I/MDA5 ligands. These virus-host interactions may play important roles in the pathogeneses of LCMV and other human arenavirus diseases.

Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1.

Type 5 adenovirus (Ad5) is a human pathogen that has been widely developed for therapeutic uses, with only limited success to date. We report here the novel finding that human erythrocytes present Coxsackie virus-adenovirus receptor (CAR) providing an Ad5 sequestration mechanism that protects against systemic infection. Interestingly, erythrocytes from neither mice nor rhesus macaques present CAR. Excess Ad5 fiber protein or anti-CAR antibody inhibits the binding of Ad5 to human erythrocytes and cryo-electron microscopy shows attachment via the fiber protein of Ad5, leading to close juxtaposition with the erythrocyte membrane. Human, but not murine, erythrocytes also present complement receptor (CR1), which binds Ad5 in the presence of antibodies and complement. Transplantation of human erythrocytes into nonobese diabetic/severe combined immunodeficiency mice extends blood circulation of intravenous Ad5 but decreases its extravasation into human xenograft tumors. Ad5 also shows extended circulation in transgenic mice presenting CAR on their erythrocytes, although it clears rapidly in transgenic mice presenting erythrocyte CR1. Hepatic infection is inhibited in both transgenic models. Erythrocytes may therefore restrict Ad5 infection (natural and therapeutic) in humans, independent of antibody status, presenting a formidable challenge to Ad5 therapeutics. "Stealthing" of Ad5 using hydrophilic polymers may enable circumvention of these natural virus traps.

MyD88 intrinsically regulates CD4 T-cell responses.

Myeloid differentiation factor 88 (MyD88) is an essential adaptor protein in the Toll-like receptor-mediated innate signaling pathway, as well as in interleukin-1 receptor (IL-1R) and IL-18R signaling. The importance of MyD88 in the regulation of innate immunity to microbial pathogens has been well demonstrated. However, its role in regulating acquired immunity to viral pathogens and neuropathogenesis is not entirely clear. In the present study, we examine the role of MyD88 in the CD4(+) T-cell response following lymphocytic choriomeningitis virus (LCMV) infection. We demonstrate that wild-type (WT) mice developed a CD4(+) T-cell-mediated wasting disease after intracranial infection with LCMV. In contrast, MyD88 knockout (KO) mice did not develop wasting disease in response to the same infection. This effect was not the result of MyD88 regulation of IL-1 or IL-18 responses since IL-1R1 KO and IL-18R KO mice were not protected from weight loss. In the absence of MyD88, naïve CD4(+) T cells failed to differentiate to LCMV-specific CD4 T cells. We demonstrated that MyD88 KO antigen-presenting cells are capable of activating WT CD4(+) T cells. Importantly, when MyD88 KO CD4(+) T cells were reconstituted with an MyD88-expressing lentivirus, the rescued CD4(+) T cells were able to respond to LCMV infection and support IgG2a antibody production. Overall, these studies reveal a previously unknown role of MyD88-dependent signaling in CD4(+) T cells in the regulation of the virus-specific CD4(+) T-cell response and in viral infection-induced immunopathology in the central nervous system.

Respiratory syncytial virus activates innate immunity through Toll-like receptor 2.

Respiratory syncytial virus (RSV) is a common cause of infection that is associated with a range of respiratory illnesses, from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children <1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected to contribute to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs, including TLR2, TLR6, TLR3, TLR4, and TLR7, that can interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting tumor necrosis factor alpha, interleukin-6, CCL2 (monocyte chemoattractant protein 1), and CCL5 (RANTES). As previously noted, TLR4 also contributes to cytokine activation (L. M. Haynes, D. D. Moore, E. A. Kurt-Jones, R. W. Finberg, L. J. Anderson, and R. A. Tripp, J. Virol. 75:10730-10737, 2001, and E. A. Kurt-Jones, L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg, Nat. Immunol. 1:398-401, 2000). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation.

Lymphocytic choriomeningitis virus (LCMV) infection of CNS glial cells results in TLR2-MyD88/Mal-dependent inflammatory responses.

In response to invading pathogens, Toll-like receptors (TLR) play a critical role in the initiation of the innate immune response, which can be either beneficial or detrimental to the host. In the present study, we demonstrated that central nervous system (CNS) glial cells are activated by Lymphocytic Choriomeningitis Virus (LCMV) in a TLR2-MyD88/Mal-dependent manner. Specifically, in response to LCMV, both astrocytes and microglial cells isolated from wild-type (WT) mice produced chemokines, such as MCP-1, RANTES and TNF-alpha. Similar responses occurred in TLR3 KO and TLR4 KO glial cells. In striking contrast, both astrocytes and microglial cells isolated from mice deficient in TLR2, MyD88, and Mal did not produce any of these chemokines. In addition, LCMV infection of glial cells induced up-regulation of TLR2, MHC class-I and II, CD40, CD86 in a MyD88-dependent manner. These results define a functional role for TLR signaling in viral infection-induced activation of CNS glial cells as well as for the immunopathology in the CNS.

Methods for in vivo and in vitro analysis of innate immune responses to Helicobacter pylori infection.

It is estimated that half of the world's population is infected by Helicobacter pylori (H. pylori) (Polk and Peek, Nat Rev Cancer 10:403-414, 2010; Peek et al., Physiol Rev 90:831-858, 2010). Following infection, H. pylori induces a chronic innate immune response that is thought to contribute to gastric complications. Due to the widespread prevalence of H. pylori, it is important to study the innate immune responses that result from the infection. A variety of in vitro and in vivo techniques have been developed by our laboratory to study this immune response (Fox et al., Am J Pathol 171:1520-1528, 2007; Kurt-Jones et al., Infect Immun 75:471-480, 2007; Kurt-Jones et al., J Endotoxin Res 10:419-424, 2004). These methods are described here.

CD200R1 supports HSV-1 viral replication and licenses pro-inflammatory signaling functions of TLR2.

The CD200R1:CD200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. We generated CD200R1(-/-) mice and employed them to explore both the role of CD200R1 in regulating macrophage signaling via TLR2 as well as the host response to an in vivo, TLR2-dependent model, herpes simplex virus 1 (HSV-1) infection. CD200R1(-/-) peritoneal macrophages demonstrated a 70-75% decrease in the generation of IL-6 and CCL5 (Rantes) in response to the TLR2 agonist Pam(2)CSK(4) and to HSV-1. CD200R1(-/-) macrophages could neither up-regulate the expression of TLR2, nor assemble a functional inflammasome in response to HSV-1. CD200R1(-/-) mice were protected from HSV-1 infection and exhibited dysfunctional TLR2 signaling. Finally, both CD200R1(-/-) mice and CD200R1(-/-) fibroblasts and macrophages showed a markedly reduced ability to support HSV-1 replication. In summary, our data demonstrate an unanticipated and novel requirement for CD200R1 in "licensing" pro-inflammatory functions of TLR2 and in limiting viral replication that are supported by ex vivo and in vivo evidence.

Discovery of a novel TLR2 signaling inhibitor with anti-viral activity.

Blockade of Toll-like receptor (TLR)-mediated inflammatory responses represents a new approach in the development of anti-inflammation therapeutics. In the present study, we have screened for TLR2-mediated inflammation inhibitors from small molecule compound libraries using a sensitive cell line stably expressing TLR2, CD14, and an NF-kappaB-driven-luciferase reporter gene. Lymphocytic choriomeningitis virus (LCMV) was used as a virus model. This arenavirus activates a TLR2/CD14-dependent NF-kappaB signaling pathway. We have identified 10 potential anti-inflammatory compounds out of 101,306 compounds. We further evaluated 1 of these positive compounds, E567. We demonstrated that compound E567 efficiently inhibits both LCMV and Herpes simplex virus 1 (HSV-1) induced cytokine responses in both human and mouse cell cultures. We also demonstrated that E567 inhibits cytokine responses in the mouse. Remarkably, E567 is also capable of inhibiting LCMV replication in mice. This is a new model for developing drugs for use in treating viral illnesses.

Role of MyD88 in route-dependent susceptibility to vesicular stomatitis virus infection.

TLRs are important components of the innate immune response. The role of the TLR signaling pathway in host defense against a natural viral infection has been largely unexplored. We found that mice lacking MyD88, an essential adaptor protein in TLR signaling pathway, were extremely sensitive to intranasal infection with vesicular stomatitis virus, and this susceptibility was dose dependent. We demonstrated that this increased susceptibility correlates with the impaired production of IFN-alpha and defective induction and maintenance of neutralizing Ab. These studies outline the important role of the TLR signaling pathway in nasal mucosae-respiratory tracts-neuroepithelium environment in the protection against microbial pathogen infections. We believe that these results explain how the route of infection, probably by virtue of activating different cell populations, can lead to entirely different outcomes of infection based on the underlying genetics of the host.

The erythrocyte viral trap: transgenic expression of viral receptor on erythrocytes attenuates coxsackievirus B infection.

Viruses rely on attachment to specific cell surface receptors to infect host cells. Selective expression of viral receptors has the potential to attenuate infection of susceptible tissues by redirecting virus to cells that cannot support viral replication. We propose that erythrocytes are an ideal instrument for this strategy, because they are present in vast numbers, permeate every organ, and cannot serve as hosts for viral propagation. To test this hypothesis, we generated a transgenic mouse, termed globin transcription factor 1 (GATA1)-coxsackie and adenovirus receptor (CAR), that expressed the CAR on erythrocytes. Coxsackievirus group B (CVB) adhered to the surface of CAR-expressing erythrocytes and was rendered noninfectious. Upon infection with CVB, GATA1-CAR mice had diminished viremia and reduced viral replication in heart, brain, and liver. Furthermore, when faced with a CVB challenge that was lethal to WT littermates, the survival of GATA1-CAR mice was prolonged, and their ultimate mortality was reduced. The GATA1-CAR mouse model presented here demonstrates that erythrocyte expression of CAR limits CVB pathogenesis. Erythrocytes also may be coated with a variety of receptors by nontransgenic methods, making this a very flexible model for the treatment of infectious diseases in humans.

Coxsackievirus and adenovirus receptor is essential for cardiomyocyte development.

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein that is known to be a site of viral attachment and entry, but its physiologic functions are undefined. CAR expression is maximal in neonates and wanes rapidly after birth in organs such as heart, muscle, and brain, suggesting that CAR plays a role in the development of these tissues. Here, we show that CAR deficiency resulted in an embryonic lethal condition associated with cardiac defects. Specifically, commencing approximately 10.5 days postconception (dpc), CAR-/- cardiomyocytes exhibited regional apoptosis evidenced by both histopathologic features of cell death and positive staining for the apoptotic marker cleaved caspase 3. CAR-/- fetuses invariably suffered from degeneration of the myocardial wall and thoracic hemorrhaging, leading to death by 11.5 dpc. These findings are consistent with the view that CAR provides positive survival signals to cardiomyocytes that are essential for normal heart development.