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1.  MyD88-Dependent Immunity to a Natural Model of Vaccinia Virus Infection Does Not Involve Toll-Like Receptor 2 
Journal of Virology  2014;88(6):3557-3567.
Although the pattern recognition receptor Toll-like receptor 2 (TLR2) is typically thought to recognize bacterial components, it has been described to alter the induction of both innate and adaptive immunity to a number of viruses, including vaccinia virus (VACV). However, many pathogens that reportedly encode TLR2 agonists may actually be artifactually contaminated during preparation, possibly with cellular debris or merely with molecules that sensitize cells to be activated by authentic TLR2 agonists. In both humans and mice, the most relevant natural route of infection with VACV is through intradermal infection of the skin. Therefore, we examined the requirement for TLR2 and its signaling adaptor MyD88 in protective immunity to VACV after intradermal infection. We find that although TLR2 may recognize virus preparations in vitro and have a minor role in preventing dissemination of VACV following systemic infection with large doses of virus, it is wholly disposable in both control of virus replication and induction of adaptive immunity following intradermal infection. In contrast, MyD88 is required for efficient induction of CD4 T cell and B cell responses and for local control of virus replication following intradermal infection. However, even MyD88 is not required to induce local inflammation, inflammatory cytokine production, or recruitment of cells that restrict virus from spreading systemically after peripheral infection. Thus, an effective antiviral response does require MyD88, but TLR2 is not required for control of a peripheral VACV infection. These findings emphasize the importance of studying relevant routes of infection when examining innate sensing mechanisms.
IMPORTANCE Vaccinia virus (VACV) provides the backbone for some of the most widely used and successful viral vaccine vectors and is also related to the human pathogens Cantagalo virus and molluscum contagiosum virus that infect the skin of patients. Therefore, it is vital to understand the mechanisms that induce a strong innate immune response to the virus following dermal infection. Here, we compare the ability of the innate sensing molecule Toll-like receptor 2 (TLR2) and the signaling molecule MyD88 to influence the innate and adaptive immune response to VACV following systemic or dermal infection.
PMCID: PMC3957935  PMID: 24403581
2.  Cross-talk between the Type 1 interferon and Nuclear Factor Kappa B pathways confers resistance to a lethal virus infection 
Cell host & microbe  2013;13(6):701-710.
Nuclear factor kappaB (NF-κB) and Type 1 interferon (T1-IFN) signaling are innate immune mechanisms activated upon viral infection. However, the role of NF-κB and its interplay with TI-IFN in antiviral immunity is poorly understood. We show that NF-κB is essential for resistance to ectromelia virus (ECTV), a mouse orthopoxvirus related to the virus causing human smallpox. Additionally, an ECTV mutant lacking an NF-κB inhibitor activates NF-κB more effectively in vivo, resulting in increased pro-inflammatory molecule transcription in uninfected cells and organs and decreased viral replication. Unexpectedly, NF-κB activation compensates for genetic defects in the T1-IFN pathway, such as a deficiency in the IRF7 transcription factor, resulting in virus control. Thus, overlap between the T1-IFN and NF-κB pathways allows the host to overcome genetic or pathogen-induced deficiencies in T1-IFN and survive an otherwise lethal poxvirus infection. These findings may also explain why some pathogens target both pathways to cause disease.
PMCID: PMC3688842  PMID: 23768494
3.  Stress-Induced Glucocorticoids at the Earliest Stages of Herpes Simplex Virus-1 Infection Suppress Subsequent Antiviral Immunity, Implicating Impaired Dendritic Cell Function 
The systemic elevation of psychological stress-induced glucocorticoids strongly suppresses CD8+ T cell immune responses resulting in diminished antiviral immunity. However, the specific cellular targets of stress/glucocorticoids, the timing of exposure, the chronology of immunological events, and the underlying mechanisms of this impairment are incompletely understood. In this study, we address each of these questions in the context of a murine cutaneous HSV infection. We show that exposure to stress or corticosterone in only the earliest stages of an HSV-1 infection is sufficient to suppress, in a glucocorticoid receptor-dependent manner, the subsequent antiviral immune response after stress/corticosterone has been terminated. This suppression resulted in early onset and delayed resolution of herpetic lesions, reduced viral clearance at the site of infection and draining popliteal lymph nodes (PLNs), and impaired functions of HSV-specific CD8+ T cells in PLNs, including granzyme B and IFN-γ production and the ability to degranulate. In knockout mice lacking glucocorticoid receptors only in T cells, we show that these impaired CD8+ T cell functions are not due to direct effects of stress/corticosterone on the T cells, but the ability of PLN-derivcd dendritic cells to prime HSV-1–specific CD8+ T cells is functionally impaired. These findings highlight the susceptibility of critical early events in the generation of an antiviral immune response to neuroendocrine modulation and implicate dendritic cells as targets of stress/glucocorticoids in vivo. These findings also provide insight into the mechanisms by which the clinical use of glucocorticoids contributes to altered immune responses in patients with viral infections or tumors.
PMCID: PMC3701455  PMID: 20089700
4.  A Marked Reduction in Priming of Cytotoxic CD8+ T Cells Mediated by Stress-Induced Glucocorticoids Involves Multiple Deficiencies in Cross-Presentation by Dendritic Cells 
Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8+ T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8+ T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA257–264-specific T cells. Using a murine model of psychological stress and OVA-loaded β2-microglobulin knockout “donor” cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide-MHC I complexes. In addition, the lymphoid tissue-resident CD11b−CD24+CD8α+ DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b−CD24+CD8α− DC precursors were increased, suggesting a block in development of CD8α+ DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice.
PMCID: PMC3701459  PMID: 21098225
5.  Local Activation of Dendritic Cells Alters the Pathogenesis of Autoimmune Disease In the Retina1 
Interest in the identities, properties, functions and origins of local antigen presenting cells (APC) in CNS tissues is growing. We recently reported that dendritic cells (DC) distinct from microglia were present in quiescent retina, and rapidly responded to injured neurons. In this study, the disease-promoting and regulatory contributions of these APC in experimental autoimmune uveoretinitis (EAU) were examined. Local delivery of purified, exogenous DC or monocytes from bone marrow substantially increased the incidence and severity of EAU induced by adoptive transfer of activated, autoreactive CD4 or CD8 T cells that was limited to the manipulated eye. In vitro assays of antigen presenting cell activity of DC from quiescent retina showed that they promoted generation of Foxp3+ T cells, and inhibited activation of naive T cells by splenic DC and antigen. Conversely, in vitro assays of DC purified from injured retina revealed an enhanced ability to activate T cells, and reduced induction of Foxp3+ T cells. These findings were supported by the observation that in situ activation of DC prior to adoptive transfer of β-galactosidase-specific T cells dramatically increased severity and incidence of EAU. Recruitment of T cells into retina by local delivery of antigen in vivo showed that quiescent retina promoted development of parenchymal Foxp3+ T cells, but assays of pre-injured retina did not. Together, these results demonstrated that local conditions in the retina determined APC function, and affected the pathogenesis of EAU by both CD4 and CD8 T cells.
PMCID: PMC3266064  PMID: 22219322
6.  Antibody Inhibition of a Viral Type 1 Interferon Decoy Receptor Cures a Viral Disease by Restoring Interferon Signaling in the Liver 
PLoS Pathogens  2012;8(1):e1002475.
Type 1 interferons (T1-IFNs) play a major role in antiviral defense, but when or how they protect during infections that spread through the lympho-hematogenous route is not known. Orthopoxviruses, including those that produce smallpox and mousepox, spread lympho-hematogenously. They also encode a decoy receptor for T1-IFN, the T1-IFN binding protein (T1-IFNbp), which is essential for virulence. We demonstrate that during mousepox, T1-IFNs protect the liver locally rather than systemically, and that the T1-IFNbp attaches to uninfected cells surrounding infected foci in the liver and the spleen to impair their ability to receive T1-IFN signaling, thus facilitating virus spread. Remarkably, this process can be reversed and mousepox cured late in infection by treating with antibodies that block the biological function of the T1-IFNbp. Thus, our findings provide insights on how T1-IFNs function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy.
Author Summary
Type 1 interferons are molecules important in the defense against viruses. Orthopoxviruses encode a Type 1 interferon binding protein that acts as a decoy for the Type 1 interferon receptor. Here we show that during infection with the Orthopoxvirus ectromelia virus, the agent of mousepox, Type 1 interferons protect the liver locally rather than systemically. We also show that the Type 1 interferon binding protein of ectromelia virus attaches to uninfected cells surrounding infected foci in the liver to impair their ability to receive Type 1 interferon signaling and facilitate virus spread and disease progression. We also show that this process can be reversed and mousepox cured late in infection by treating mice with antibodies that block the biological function of the Type 1 interferon binding protein. Because the Type 1 interferon binding proteins of different Orthopoxviruses are very well conserved, the antibodies also block the biological function of the Type 1 interferon binding proteins from variola virus (the virus of smallpox) and monkeypoxvirus. Thus, our findings provide insights on how Type 1 interferons function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy.
PMCID: PMC3252373  PMID: 22241999
7.  CD11b+, Ly6G+ Cells Produce Type I Interferon and Exhibit Tissue Protective Properties Following Peripheral Virus Infection 
PLoS Pathogens  2011;7(11):e1002374.
The goal of the innate immune system is containment of a pathogen at the site of infection prior to the initiation of an effective adaptive immune response. However, effector mechanisms must be kept in check to combat the pathogen while simultaneously limiting undesirable destruction of tissue resulting from these actions. Here we demonstrate that innate immune effector cells contain a peripheral poxvirus infection, preventing systemic spread of the virus. These innate immune effector cells are comprised primarily of CD11b+Ly6C+Ly6G- monocytes that accumulate initially at the site of infection, and are then supplemented and eventually replaced by CD11b+Ly6C+Ly6G+ cells. The phenotype of the CD11b+Ly6C+Ly6G+ cells resembles neutrophils, but the infiltration of neutrophils typically occurs prior to, rather than following, accumulation of monocytes. Indeed, it appears that the CD11b+Ly6C+Ly6G+ cells that infiltrated the site of VACV infection in the ear are phenotypically distinct from the classical description of both neutrophils and monocyte/macrophages. We found that CD11b+Ly6C+Ly6G+ cells produce Type I interferons and large quantities of reactive oxygen species. We also observed that depletion of Ly6G+ cells results in a dramatic increase in tissue damage at the site of infection. Tissue damage is also increased in the absence of reactive oxygen species, although reactive oxygen species are typically thought to be damaging to tissue rather than protective. These data indicate the existence of a specialized population of CD11b+Ly6C+Ly6G+ cells that infiltrates a site of virus infection late and protects the infected tissue from immune-mediated damage via production of reactive oxygen species. Regulation of the action of this population of cells may provide an intervention to prevent innate immune-mediated tissue destruction.
Author Summary
During a natural virus infection, small doses of infectious virus are deposited at a peripheral infection site, and then a “race” ensues, in which the replicating virus attempts to “outpace” the responding immune system of the host. In the early phases of infection, the innate immune system must contain the infection prior to the development of an effective adaptive response. Here we have characterized the cells of the innate immune system that move to a site of peripheral virus infection, and we find that a subset of these cells display atypical expression of cell surface molecules, timing of infiltration, and function. These cells protect the infected tissue from damage by producing reactive oxygen molecules, which are widely accepted to increase tissue damage. Therefore our findings indicate that during a peripheral virus infection, the typical rules governing the function of the innate immune system are altered to prevent tissue damage.
PMCID: PMC3213107  PMID: 22102816
8.  Interleukin-10 Prevents Diet-Induced Insulin Resistance by Attenuating Macrophage and Cytokine Response in Skeletal Muscle 
Diabetes  2009;58(11):2525-2535.
Insulin resistance is a major characteristic of type 2 diabetes and is causally associated with obesity. Inflammation plays an important role in obesity-associated insulin resistance, but the underlying mechanism remains unclear. Interleukin (IL)-10 is an anti-inflammatory cytokine with lower circulating levels in obese subjects, and acute treatment with IL-10 prevents lipid-induced insulin resistance. We examined the role of IL-10 in glucose homeostasis using transgenic mice with muscle-specific overexpression of IL-10 (MCK-IL10).
MCK-IL10 and wild-type mice were fed a high-fat diet (HFD) for 3 weeks, and insulin sensitivity was determined using hyperinsulinemic-euglycemic clamps in conscious mice. Biochemical and molecular analyses were performed in muscle to assess glucose metabolism, insulin signaling, and inflammatory responses.
MCK-IL10 mice developed with no obvious anomaly and showed increased whole-body insulin sensitivity. After 3 weeks of HFD, MCK-IL10 mice developed comparable obesity to wild-type littermates but remained insulin sensitive in skeletal muscle. This was mostly due to significant increases in glucose metabolism, insulin receptor substrate-1, and Akt activity in muscle. HFD increased macrophage-specific CD68 and F4/80 levels in wild-type muscle that was associated with marked increases in tumor necrosis factor-α, IL-6, and C-C motif chemokine receptor-2 levels. In contrast, MCK-IL10 mice were protected from diet-induced inflammatory response in muscle.
These results demonstrate that IL-10 increases insulin sensitivity and protects skeletal muscle from obesity-associated macrophage infiltration, increases in inflammatory cytokines, and their deleterious effects on insulin signaling and glucose metabolism. Our findings provide novel insights into the role of anti-inflammatory cytokine in the treatment of type 2 diabetes.
PMCID: PMC2768157  PMID: 19690064
9.  Dendritic Cell Migration Limits the Duration of CD8+ T-Cell Priming to Peripheral Viral Antigen▿  
Journal of Virology  2010;84(7):3586-3594.
CD8+ T cells (TCD8+) play a crucial role in immunity to viruses. Antiviral TCD8+ are initially activated by recognition of major histocompatibility complex (MHC) class I-peptide complexes on the surface of professional antigen-presenting cells (pAPC). Migration of pAPC from the site of infection to secondary lymphoid organs is likely required during a natural infection. Migrating pAPC can be directly infected with virus or may internalize antigen derived from virus-infected cells. The use of experimental virus infections to assess the requirement for pAPC migration in initiation of TCD8+ responses has proven difficult to interpret because injected virus can readily drain to secondary lymphoid organs without the need for cell-mediated transport. To overcome this ambiguity, we examined the generation of antigen-specific TCD8+ after immunization with recombinant adenoviruses that express antigen driven by skin-specific or ubiquitous promoters. We show that the induction of TCD8+ in response to tissue-targeted antigen is less efficient than the response to ubiquitously expressed antigen and that the resulting TCD8+ fail to clear all target cells pulsed with the antigenic peptide. This failure to prime a fully functional TCD8+ response results from a reduced period of priming to peripherally expressed antigen versus ubiquitously expressed antigen and correlated with a brief burst of pAPC migration from the skin, a requirement for induction of the response to peripheral antigen. These results indicate that a reduced duration of pAPC migration after virus infection likely reduces the amplitude of the TCD8+ response, allowing persistence of the peripheral virus.
PMCID: PMC2838146  PMID: 20089641
10.  Viral Sequestration of Antigen Subverts Cross Presentation to CD8+ T Cells 
PLoS Pathogens  2009;5(5):e1000457.
Virus-specific CD8+ T cells (TCD8+) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector TCD8+. Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated TCD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the TCD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into account during the rational design of antiviral vaccines.
Author Summary
Understanding the pathways by which protective immunity is mediated against viral pathogens is essential to allow the design of effective vaccines. No effective vaccine has been designed to activate killer cells of the immune system expressing CD8, although CD8+ T cells are the most effective cells at modulating anti-viral immunity. We have studied the process that activates the CD8+ T cell to better understand how the cells are triggered so future vaccines might readily activate these cells. CD8+ T cells are activated following recognition of small peptides derived from a virus that binds to a cell surface MHC molecule. Many viruses have evolved to prevent the presentation of these peptide-MHC complexes to CD8+ T cells. However, the immune system avoids these viral “evasion” mechanisms by allowing virus-derived peptides to be generated from viral proteins that are taken up by uninfected cells, a process termed “cross presentation”. We have shown that a poxvirus can specifically prevent the presentation of its proteins by uninfected cells, the first demonstration of evasion of cross presentation. This knowledge is vital in the use of certain viral vectors during vaccine design and adds to the numerous ways in which viruses can evade the immune system.
PMCID: PMC2680035  PMID: 19478869
11.  Deubiquitinating enzyme CYLD negatively regulates the ubiquitin-dependent kinase Tak1 and prevents abnormal T cell responses 
The Journal of Experimental Medicine  2007;204(6):1475-1485.
The deubiquitinating enzyme CYLD has recently been implicated in the regulation of signal transduction, but its physiological function and mechanism of action are still elusive. In this study, we show that CYLD plays a pivotal role in regulating T cell activation and homeostasis. T cells derived from Cyld knockout mice display a hyperresponsive phenotype and mediate the spontaneous development of intestinal inflammation. Interestingly, CYLD targets a ubiquitin-dependent kinase, transforming growth factor–β-activated kinase 1 (Tak1), and inhibits its ubiquitination and autoactivation. Cyld-deficient T cells exhibit constitutively active Tak1 and its downstream kinases c-Jun N-terminal kinase and IκB kinase β. These results emphasize a critical role for CYLD in preventing spontaneous activation of the Tak1 axis of T cell signaling and, thereby, maintaining normal T cell function.
PMCID: PMC2118606  PMID: 17548520
12.  Immunoproteasomes Shape Immunodominance Hierarchies of Antiviral Cd8+ T Cells at the Levels of T Cell Repertoire and Presentation of Viral Antigens 
The Journal of Experimental Medicine  2001;193(11):1319-1326.
Vertebrates express three cytokine-inducible proteasome subunits that are incorporated in the place of their constitutively synthesized counterparts. There is increasing evidence that the set of peptides generated by proteasomes containing these subunits (immunoproteasomes) differs from that produced by standard proteasomes. In this study, we use mice lacking one of the immunoproteasome subunits (LMP2) to show that immunoproteasomes play an important role in establishing the immunodominance hierarchy of CD8+ T cells (TCD8+) responding to seven defined determinants in influenza virus. In LMP2−/− mice, responses to the two most dominant determinants drop precipitously, whereas responses to two subdominant determinants are greatly enhanced. Adoptive transfer experiments with naive normal and transgenic TCD8+ reveal that the reduced immunogenicity of one determinant (PA224–233) can be attributed to decreased generation by antigen presenting cells (APCs), whereas the other determinant (NP366–374) is less immunogenic due to alterations in the TCD8+ repertoire, and not, as reported previously, to the decreased capacity of LMP2−/− APCs to generate the determinant. The enhanced response to one of the subdominant determinants (PB1F262–70) correlates with increased generation by LMP2−/− virus–infected cells. These findings indicate that in addition to their effects on the presentation of foreign antigens, immunoproteasomes influence TCD8+ responses by modifying the repertoire of responding TCD8+.
PMCID: PMC2193381  PMID: 11390439
antigen processing; immunodominance; T cells; proteasome; repertoire
13.  Lymph-Borne Chemokines and Other Low Molecular Weight Molecules Reach High Endothelial Venules via Specialized Conduits While a Functional Barrier Limits Access to the Lymphocyte Microenvironments in Lymph Node Cortex 
The Journal of Experimental Medicine  2000;192(10):1425-1440.
Lymph-borne, soluble factors (e.g., chemokines and others) influence lymphocyte recirculation and endothelial phenotype at high endothelial venules (HEVs) in lymph node cortex. Yet the route lymph-borne soluble molecules travel from the subcapsular sinus to the HEVs is unclear. Therefore, we injected subcutaneously into mice and rats a wide variety of fluorophore-labeled, soluble molecules and examined their distribution in the draining lymph nodes. Rather than percolating throughout the draining lymph node, all molecules, including microbial lipopolysaccharide, were very visible in the subcapsular and medullary sinuses but were largely excluded from the cortical lymphocyte microenvironments. Exclusion prevailed even during the acute lymph node enlargement accompanying viral infection. However, low molecular mass (MW) molecules, including chemokines, did gain entry into the cortex, but in a very defined manner. Low MW, fluorophore-labeled molecules highlighted the subcapsular sinus, the reticular fibers, and the abluminal and luminal surfaces of the associated HEVs. These low MW molecules were in the fibers of the reticular network, a meshwork of collagen fibers ensheathed by fibroblastic reticular cells that connects the subcapsular sinus floor and the HEVs by intertwining with their basement membranes. Thus, low MW, lymph-borne molecules, including chemokines, traveled rapidly from the subcapsular sinus to the HEVs using the reticular network as a conduit.
PMCID: PMC2193184  PMID: 11085745
reticular network; mouse; rat; lymphocyte recirculation; antigen

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