Parvalbumin-containing Interneurons in Rat Hippocampus Have an AMPA Receptor Profile Suggestive of Vulnerability to Excitotoxicity

Journal of Chemical Neuroanatomy. May, 2002  |  Pubmed ID: 12048108

alpha-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors mediate excitatory neurotransmission in the central nervous system, and contain combinations of four subunits (GluR1-4). We developed a GluR3-specific monoclonal antibody and quantified the cellular distribution of GluR3 in rat hippocampus. GluR3 immunoreactivity was detected in all pyramidal neurons and most interneurons. In addition, we found a subset of parvalbumin (PV)-containing interneurons in the hippocampus and neocortex that was notable for its intense GluR3 immunoreactivity and lack of GluR2 immunoreactivity. Such an expression pattern of AMPA receptor subunits is likely to make these interneurons selectively vulnerable to excitotoxicity.

Immune Responses to Viruses, Bacteria, Parasites and Fungi in Humans

International Reviews of Immunology. Jul-Oct, 2002  |  Pubmed ID: 12486815

Antiviral Immunity and the Role of Dendritic Cells

International Reviews of Immunology. Jul-Oct, 2002  |  Pubmed ID: 12486818

Using our experimental model we demonstrate the need for Th1 immune responses for recovery from influenza virus infection. Inoculation of IL-4 concurrent with infection significantly delays virus clearance and converts the immune response to a Th2 response. Immunization using live virus in the presence of IL-4 leads to generation of Th2 memory cells that fail to facilitate recovery upon subsequent virus infection. Inactivated virus expands Th2 cells, leading to responses similar to those observed following IL-4 infusion. Immunization using cultured dendritic cells incubated with live or inactivated virus mimics the results observed with direct virus injection. We demonstrate that in contrast to live virus-infected dendritic cells, inactivated virus fails to elicit Th1 immunity. This failure correlates with the inactivated viruses' inability to induce dendritic cell maturation. Thus, our data suggest that the polarity of the immune response is dictated by the nature of the antigen, and the trigger for influenza virus-induced DC maturation leading to Th1 immunity is dependent on virus replication.

Autocrine Type I IFN and Contact with Endothelium Promote the Presentation of Influenza A Virus by Monocyte-derived APC

Journal of Immunology (Baltimore, Md. : 1950). Jan, 2003  |  Pubmed ID: 12517968

Purified monocytes infected with influenza A virus do not become mature dendritic cells (DCs) and they present viral peptides poorly to autologous memory T cells. In this study, we investigated whether influenza A-infected monocytes matured to DCs with a high capacity to stimulate T cells when they were infected with influenza A virus in a model tissue setting wherein they were cocultured with endothelium grown on a type I collagen matrix. Intercellular interactions with endothelium strongly promoted the Ag-presenting capacity of monocyte-derived cells infected with influenza A virus, and the heterologous coculture system also enhanced production of IFN-alpha by monocytes in the absence of plasmacytoid cells. Production of IFN-alpha in the presence of endothelium correlated with monocyte differentiation to mature DCs and their ability to stimulate proliferation and IFN-gamma production by autologous T cells. Monocyte-derived cells that developed into migratory DCs promoted proliferation of influenza A virus-specific CD4(+) and CD8(+) cells, whereas those that developed into macrophages promoted proliferation of CD8(+) T cells only. This onset of APC activity could be partially blocked with Ab to the IFN-alphabeta receptor when monocytes were infected with UV-treated virus, but neutralizing this pathway was inconsequential when monocytes were infected with live virus. Thus, type I IFN and direct contact with endothelium promote development of influenza A virus-presenting activity in monocyte-derived cells in a setting in which this differentiation does not depend on plasmacytoid cells. However, when infected with live influenza virus, the role of type I IFN in mediating differentiation and Ag-presenting capacity is expendable, apparently due to other mechanisms of virus-mediated activation.

Type I Interferon Induction Pathway, but Not Released Interferon, Participates in the Maturation of Dendritic Cells Induced by Negative-strand RNA Viruses

The Journal of Infectious Diseases. Apr, 2003  |  Pubmed ID: 12660927

Signaling through toll-like receptors (TLRs) is essential for dendritic cell (DC) maturation induced by bacteria and other pathogens. The mechanism for virus-induced DC maturation is not known. By use of pairs of live viruses with different abilities to induce the interferon (IFN) pathway, a strong correlation between DC maturation and the ability of the virus to induce type I IFN synthesis was demonstrated. The secreted IFN was not necessary, nor was it sufficient to induce full DC maturation. Intracellular viral replication is necessary for this process, and the transcription factor nuclear factor-kappaB was crucial for cytokine induction. The double-stranded RNA-dependent protein kinase was not essential for DC maturation. Similar to TLR-induced DC maturation, after virus infection, separate pathways for the induction of cytokine secretion and the up-regulation of major histocompatibility complex and costimulatory molecules were activated. It was demonstrated that these pathways have different sensitivities to the presence of viral stimulus.

Glutamate Receptor Subunit 3 (GluR3) Immunoreactivity Delineates a Subpopulation of Parvalbumin-containing Interneurons in the Rat Hippocampus

The Journal of Comparative Neurology. Jul, 2003  |  Pubmed ID: 12761821

Rasmussen's encephalitis is a childhood disease resulting in intractable seizures associated with hippocampal and neocortical inflammation. An autoantibody against the GluR3 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors is implicated in the pathophysiology of Rasmussen's encephalitis. AMPA receptors mediate excitatory neurotransmission in the brain and contain combinations of four subunits (GluR1-4). Although the distributions of GluR1, GluR2, and GluR4 are known in some detail, the cellular distribution of GluR3 in the mammalian brain remains to be described. We developed and characterized a GluR3-specific monoclonal antibody and quantified the cellular distribution of GluR3 in CA1 of the rat hippocampus. GluR3 immunoreactivity was detected in all pyramidal neurons and astrocytes and in most interneurons. We quantified the intensity of GluR3 immunoreactivity in interneuron subtypes defined by their calcium-binding protein content. GluR3 immunofluorescence, but not GluR1 or GluR2 immunofluorescence, was significantly elevated in somata of parvalbumin-containing interneurons compared to pyramidal somata. Strikingly, increased GluR3 immunofluorescence was not observed in calbindin- and calretinin-containing interneurons. Furthermore, 24% of parvalbumin-containing interneurons could be distinguished from surrounding neurons based on their intense GluR3 immunoreactivity. This subpopulation had significantly elevated GluR3 immunoreactivity compared to the rest of parvalbumin-containing interneurons. Electron microscopy revealed enriched GluR3 immunoreactivity in parvalbumin-containing perikarya at cytoplasmic and postsynaptic sites. Parvalbumin-containing interneurons, potent inhibitors of cortical pyramidal neurons, are vulnerable in the brains of epileptic patients. Our findings suggest that the somata of these interneurons are enriched in GluR3, which may render them vulnerable to pathological states such as epilepsy and Rasmussen's encephalitis.

Influenza Virus-induced Dendritic Cell Maturation is Associated with the Induction of Strong T Cell Immunity to a Coadministered, Normally Nonimmunogenic Protein

The Journal of Experimental Medicine. Jul, 2003  |  Pubmed ID: 12847140

We evaluated the proposal that during microbial infection, dendritic cells (DCs) undergo maturation and present a mixture of peptides derived from the microbe as well as harmless environmental antigens. Mice were exposed to an aerosol of endotoxin free ovalbumin (OVA) in the absence or presence of influenza virus. In its absence, OVA failed to induce B and T cell responses and even tolerized, but with influenza, OVA-specific antibodies and CD8+ cytolytic T lymphocytes developed. With or without infection, OVA was presented selectively in the draining mediastinal lymph nodes, as assessed by the comparable proliferation of infused, CD8+ and CD4+, TCR transgenic T cells. In the absence of influenza, these OVA-specific T cells produced little IL-2, IL-4, IL-10, and IFN-gamma, but with infection, both CD4+ and CD8+ T cells made high levels of IL-2 and IFN-gamma. The OVA plus influenza-treated mice also showed accelerated recovery to a challenge with recombinant vaccinia OVA virus. CD11c+ DCs from the mediastinal lymph nodes of infected mice selectively stimulated both OVA- and influenza-specific T cells and underwent maturation, with higher levels of MHC class II, CD80, and CD86 molecules. The relatively slow (2-3 d) kinetics of maturation correlated closely to the time at which OVA inhalation elicited specific antibodies. Therefore respiratory infection can induce DC maturation and simultaneously B and T cell immunity to an innocuous antigen inhaled concurrently.

The C-type Lectin SIGN-R1 Mediates Uptake of the Capsular Polysaccharide of Streptococcus Pneumoniae in the Marginal Zone of Mouse Spleen

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2004  |  Pubmed ID: 14694198

SIGN-R1, a recently discovered C-type lectin expressed at high levels on macrophages within the marginal zone of the spleen, mediates the uptake of dextran polysaccharides by these phagocytes. We now find that encapsulated Streptococcus pneumoniae are rapidly cleared by these macrophages from the bloodstream, and that capture also takes place when different cell lines express SIGN-R1 after transfection. To assess the role of the capsular polysaccharide of S. pneumoniae (CPS) in the interaction of SIGN-R1 with pneumococci, we first studied binding and uptake of serotype 14 CPS in transfected cells. Binding was observed and was of a much higher avidity (3000-fold) for CPS 14 than dextran. The CPSs from four different serotypes were also cleared by marginal zone macrophages in vivo. To establish a role for SIGN-R1 in this uptake, we selectively down-regulated expression of the lectin by pretreatment of the mice with SIGN-R1 antibodies, including a newly generated hamster monoclonal called 22D1. For several days after this transient knockout, the marginal zone macrophages were unable to take up either CPSs or dextrans. Therefore, marginal zone macrophages in mice have a receptor that interacts with capsular pneumococcal polysaccharides, setting the stage for further studies of the functional consequences of this interaction.

In Vivo Targeting of Antigens to Maturing Dendritic Cells Via the DEC-205 Receptor Improves T Cell Vaccination

The Journal of Experimental Medicine. Mar, 2004  |  Pubmed ID: 15024047

The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic alpha-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the alphaDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell-mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models.

TLR-independent Induction of Dendritic Cell Maturation and Adaptive Immunity by Negative-strand RNA Viruses

Journal of Immunology (Baltimore, Md. : 1950). Dec, 2004  |  Pubmed ID: 15557183

TLR signaling leads to dendritic cell (DC) maturation and immunity to diverse pathogens. The stimulation of TLRs by conserved viral structures is the only described mechanism leading to DC maturation after a virus infection. In this report, we demonstrate that mouse myeloid DCs mature normally after in vivo and in vitro infection with Sendai virus (SeV) in the absence of TLR3, 7, 8, or 9 signaling. DC maturation by SeV requires virus replication not necessary for TLR-mediated triggering. Moreover, DCs deficient in TLR signaling efficiently prime for Th1 immunity after infection with influenza or SeV, generating IFN-gamma-producing T cells, CTLs and antiviral Abs. We have previously demonstrated that SeV induces DC maturation independently of the presence of type I IFN, which has been reported to mature DCs in a TLR-independent manner. The data presented here provide evidence for the existence of a novel intracellular pathway independent of TLR-mediated signaling responsible for live virus triggering of DC maturation and demonstrate its critical role in the onset of antiviral immunity. The revelation of this pathway should stimulate invigorating research into the mechanism for virus-induced DC maturation and immunity.

Reepithelialized Orthotopic Tracheal Allografts Expand Memory Cytotoxic T Lymphocytes but Show No Evidence of Chronic Rejection

Transplantation. Apr, 2005  |  Pubmed ID: 15849536

Acute rejection of mouse tracheal allografts is characterized by infiltration of the lamina propria with CD4+/CD8+ T cells that leads to the destruction of the epithelium and luminal obliteration. The donor epithelium is progressively replaced by recipient-derived epithelium. Once allograft reepithelialization has occurred, immunosuppression can be withdrawn without inciting acute rejection. We hypothesize that reepithelialization will also prevent chronic rejection of the trachea after withdrawal of immunosuppression.

Induction of Mucosal Tolerance in Peyer's Patch-deficient, Ligated Small Bowel Loops

The Journal of Clinical Investigation. Aug, 2005  |  Pubmed ID: 16041410

To explore the requirement for M cells and the Peyer's patch (PP) in induction of oral tolerance and address the potential in vivo role of intestinal epithelial cells as nonprofessional APCs, we have attempted to induce tolerance in mice with ligated small bowel loops without M cells and Peyer's patches. A 2-centimeter section of vascularized small bowel was spliced away from the gut without disruption of the mesenteric attachments. We introduced OVA directly into the lumen of the loop prior to footpad immunization. By excising segments of bowel that contain PPs in some mice and segments without patches in others, we could study the necessity of the M cell and the underlying patch versus epithelial cells in induction of mucosal tolerance. We show that OVA-specific T cell proliferation and serum antibody responses are reduced in mice that have previously been given OVA both in PP-containing loops and in loops without patches. Furthermore, both high- and low-dose tolerance could be induced in the absence of PPs. Low-dose tolerance is associated with bystander suppression and requires IL-10, which indicates active suppression and the induction of regulatory cells. These data suggest that there is a critical role for components of the mucosal immune system other than PPs in antigen sampling and induction of oral tolerance.

Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Nonintegrin/CD209 is Abundant on Macrophages in the Normal Human Lymph Node and is Not Required for Dendritic Cell Stimulation of the Mixed Leukocyte Reaction

Journal of Immunology (Baltimore, Md. : 1950). Oct, 2005  |  Pubmed ID: 16177066

The C-type lectin dendritic cell-specific ICAM 3-grabbing nonintegrin (DC-SIGN)/CD209 efficiently binds several pathogens, including HIV-1. DC-SIGN is expressed on monocyte-derived DCs in culture, and importantly, it is able to sequester HIV-1 within cells and facilitate transmission of virus to CD4+ T cells. To investigate DC-SIGN function, we have generated new mAbs. We report in this study that these and prior anti-DC-SIGN mAbs primarily label macrophages in the medullary sinuses of noninflamed human lymph node. In contrast, expression is not detected on most DCs in the T cell area, except for occasional cells. We also noted that IL-4 alone can induce expression of DC-SIGN in CD14+ monocytes and circulating blood DCs. However, blockade of DC-SIGN with Abs and DC-SIGN small interfering RNA did not result in a major reduction in the capacity of these DCs to transfer HIV to T cells, confirming significant DC-SIGN-independent mechanisms. The blocking approaches did reduce HIV-1 transmission by DC-SIGN-transfected cells by >90%. DC-SIGN blockade also did not reduce the ability of DCs to stimulate T cell proliferation in the MLR. These results indicate that DC-SIGN has the potential to contribute to macrophage function in normal human lymph node, and that DCs do not require DC-SIGN to transmit HIV or to initiate T cell responses.

Attenuation and Immunogenicity in Mice of Temperature-sensitive Influenza Viruses Expressing Truncated NS1 Proteins

The Journal of General Virology. Oct, 2005  |  Pubmed ID: 16186237

It was previously shown that two mutant influenza A viruses expressing C-terminally truncated forms of the NS1 protein (NS1-81 and NS1-110) were temperature sensitive in vitro. These viruses contain HA, NA and M genes derived from influenza A/WSN/33 H1N1 virus (mouse-adapted), and the remaining five genes from human influenza A/Victoria/3/75 virus. Mice intranasally infected with the NS1 mutant viruses showed undetectable levels of virus in lungs at day 3, whereas those infected with the NS1 wild-type control virus still had detectable levels of virus at this time. Nevertheless, the temperature-sensitive mutant viruses induced specific cellular and humoral immune responses similar to those induced by the wild-type virus. Mice immunized with the NS1 mutant viruses were protected against a lethal challenge with influenza A/WSN/33 virus. These results indicate that truncations in the NS1 protein resulting in temperature-sensitive phenotypes in vitro correlate with attenuation in vivo without compromising viral immunogenicity, an ideal characteristic for live attenuated viral vaccines.

Intensified and Protective CD4+ T Cell Immunity in Mice with Anti-dendritic Cell HIV Gag Fusion Antibody Vaccine

The Journal of Experimental Medicine. Mar, 2006  |  Pubmed ID: 16505141

Current human immunodeficiency virus (HIV) vaccine approaches emphasize prime boost strategies comprising multiple doses of DNA vaccine and recombinant viral vectors. We are developing a protein-based approach that directly harnesses principles for generating T cell immunity. Vaccine is delivered to maturing dendritic cells in lymphoid tissue by engineering protein antigen into an antibody to DEC-205, a receptor for antigen presentation. Here we characterize the CD4+ T cell immune response to HIV gag and compare efficacy with other vaccine strategies in a single dose. DEC-205-targeted HIV gag p24 or p41 induces stronger CD4+ T cell immunity relative to high doses of gag protein, HIV gag plasmid DNA, or recombinant adenovirus-gag. High frequencies of interferon (IFN)-gamma- and interleukin 2-producing CD4+ T cells are elicited, including double cytokine-producing cells. In addition, the response is broad because the primed mice respond to an array of peptides in different major histocompatibility complex haplotypes. Long-lived T cell memory is observed. After subcutaneous vaccination, CD4+ and IFN-gamma-dependent protection develops to a challenge with recombinant vaccinia-gag virus at a mucosal surface, the airway. We suggest that a DEC-targeted vaccine, in part because of an unusually strong and protective CD4+ T cell response, will improve vaccine efficacy as a stand-alone approach or with other modalities.

Toll-like Receptor-independent Triggering of Dendritic Cell Maturation by Viruses

Journal of Virology. Apr, 2006  |  Pubmed ID: 16537581

Sendai Virus Infection Induces Efficient Adaptive Immunity Independently of Type I Interferons

Journal of Virology. May, 2006  |  Pubmed ID: 16611914

Adaptive immunity in response to virus infection involves the generation of Th1 cells, cytotoxic T cells, and antibodies. This type of immune response is crucial for the clearance of virus infection and for long-term protection against reinfection. Type I interferons (IFNs), the primary innate cytokines that control virus growth and spreading, can influence various aspects of adaptive immunity. The development of antiviral immunity depends on many viral and cellular factors, and the extent to which type I IFNs contribute to the generation of adaptive immunity in response to a viral infection is controversial. Using two strains (Cantell and 52) of the murine respiratory Sendai virus (SeV) with differential abilities to induce type I IFN production from infected cells, together with type I IFN receptor-deficient mice, we examined the role of type I IFNs in the generation of adaptive immunity. Our results show that type I IFNs facilitate virus clearance and enhance the migration and maturation of dendritic cells after SeV infection in vivo; however, soon after infection, mice clear the virus from their lungs and efficiently generate cytotoxic T cells independently of type I IFN signaling. Furthermore, animals that are unresponsive to type I IFN develop long-term anti-SeV immunity, including CD8+ T cells and antibodies. Significantly, this memory response is able to protect mice against challenge with a lethal dose of virus. In conclusion, our results show that primary and secondary anti-SeV adaptive immunities are developed normally in the absence of type I IFN responsiveness.

Influenza Virus Evades Innate and Adaptive Immunity Via the NS1 Protein

Journal of Virology. Jul, 2006  |  Pubmed ID: 16775317

Both antibodies and T cells contribute to immunity against influenza virus infection. However, the generation of strong Th1 immunity is crucial for viral clearance. Interestingly, we found that human dendritic cells (DCs) infected with influenza A virus have lower allospecific Th1-cell stimulatory abilities than DCs activated by other stimuli, such as lipopolysaccharide and Newcastle disease virus infection. This weak stimulatory activity correlates with a suboptimal maturation of the DCs following infection with influenza A virus. We next investigated whether the influenza A virus NS1 protein could be responsible for the low levels of DC maturation after influenza virus infection. The NS1 protein is an important virulence factor associated with the suppression of innate immunity via the inhibition of type I interferon (IFN) production in infected cells. Using recombinant influenza and Newcastle disease viruses, with or without the NS1 gene from influenza virus, we found that the induction of a genetic program underlying DC maturation, migration, and T-cell stimulatory activity is specifically suppressed by the expression of the NS1 protein. Among the genes affected by NS1 are those coding for macrophage inflammatory protein 1beta, interleukin-12 p35 (IL-12 p35), IL-23 p19, RANTES, IL-8, IFN-alpha/beta, and CCR7. These results indicate that the influenza A virus NS1 protein is a bifunctional viral immunosuppressor which inhibits innate immunity by preventing type I IFN release and inhibits adaptive immunity by attenuating human DC maturation and the capacity of DCs to induce T-cell responses. Our observations also support the potential use of NS1 mutant influenza viruses as live attenuated influenza virus vaccines.

A Novel Role for Viral-defective Interfering Particles in Enhancing Dendritic Cell Maturation

Journal of Immunology (Baltimore, Md. : 1950). Oct, 2006  |  Pubmed ID: 16982887

Dendritic cell (DC) maturation is a crucial event in the development of adaptive immune responses that confer long-lasting protection against reinfection with the same virus. Sendai virus strain Cantell has a particularly strong ability to mature DCs independently of type I IFNs and TLR signaling, currently the best-described pathways for the induction of DC maturation. In this study, we demonstrate that defective-interfering (DI) particles present in Sendai virus-Cantell stocks are required for its robust DC maturation ability. DI particles contain incomplete genomes that are unable to replicate unless the viral polymerase is supplied by coinfection with complete virus. Accordingly, the improvement in the virus-induced maturation of DCs provided by DI particles requires standard virus coinfection and likely results from increased production of dsRNA replication intermediaries. This unique ability of DI particles to stimulate DC maturation cannot be mimicked by simply increasing the dose of standard virus. Furthermore, viruses with weak DC maturation abilities can be converted into potent DC stimulators with the addition of DI particles, supporting a potential application for DI particles as a novel natural adjuvant for viral immunizations.

A Novel Model of Demyelinating Encephalomyelitis Induced by Monocytes and Dendritic Cells

Journal of Immunology (Baltimore, Md. : 1950). Nov, 2006  |  Pubmed ID: 17082601

Local inflammation may be a precipitating event in autoimmune processes. In this study, we demonstrate that regulated influx of monocytes and dendritic cells (DC) into the CNS causes an acute neurological syndrome that results in a demyelinating encephalomyelitis. Expansion of monocytes and DC by conditional expression of Flt3 ligand in animals expressing CCL2 in the CNS promoted parenchymal cell infiltration and ascending paralysis in 100% of the mice within 9 days of Flt3 ligand induction. Depletion of circulating monocytes and DC reduced disease incidence and severity. Unlike the classical models of experimental autoimmune encephalomyelitis, depletion of CD4+ and CD8+ T cells did not affect disease induction. T cells and demyelinating lesions were observed in the CNS at a later stage as a result of organ-specific inflammation. We propose that alterations in the numbers or function of monocytes and DC coupled to dysregulated expression of chemokines in the neural tissues, favors development of CNS autoimmune disease.

Cytokine-independent Upregulation of MDA5 in Viral Infection

Journal of Virology. Jul, 2007  |  Pubmed ID: 17475649

The RNA helicases RIG-I and MDA5 detect virus infection of dendritic cells (DCs) leading to cytokine induction. Maximal sensitivity for virus detection by these helicases is obtained after their upregulation, which is thought to occur primarily through type I interferon (IFN) signaling. Here we demonstrate that in response to paramyxovirus infection, RIG-I upregulation requires type I IFN whereas MDA5 expression is increased by Sendai virus infection independently of signaling mediated by type I IFN, STAT1, tumor necrosis factor alpha, or NF-kappaB. This MDA5 upregulation is largely lost in IRF3 knockout DCs and is achieved in type I IFN-deficient cells expressing constitutively active IRF3.

Chromosome-specific and Noisy IFNB1 Transcription in Individual Virus-infected Human Primary Dendritic Cells

Nucleic Acids Research. 2007  |  Pubmed ID: 17675303

The induction of interferon beta (IFNB1) is a key event in the antiviral immune response. We studied the role of transcriptional noise in the regulation of the IFNB1 locus in primary cultures of human dendritic cells (DCs), which are important 'first responders' to viral infection. In single cell assays, IFNB1 mRNA expression in virus-infected DCs showed much greater cell-to-cell variation than that of a housekeeping gene, another induced transcript and viral RNA. We determined the contribution of intrinsic noise by measuring the allelic origin of transcripts in each cell and found that intrinsic noise is a very significant part of total noise. We developed a stochastic model to investigate the underlying mechanisms. We propose that the surprisingly high levels of IFNB1 transcript noise originate from the complexity of IFNB1 enhanceosome formation, which leads to a range up to many minutes in the differences within each cell in the time of activation of each allele.

A Prospective, Comparative Study of the Immune Response to Inactivated Influenza Vaccine in Pediatric Liver Transplant Recipients and Their Healthy Siblings

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. Mar, 2008  |  Pubmed ID: 18230041

Annual influenza vaccination is routinely recommended for pediatric solid organ transplant recipients. However, there are limited data defining the immune response to the inactivated vaccine in this population.

MDA5 Participates in the Detection of Paramyxovirus Infection and is Essential for the Early Activation of Dendritic Cells in Response to Sendai Virus Defective Interfering Particles

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2008  |  Pubmed ID: 18354215

Defective interfering (DI) particles are byproducts of virus replication that potently enhance dendritic cell (DC) maturation by virus infection. DI particles have been reported for many different viruses and are strong inducers of type I IFNs. The cellular mechanisms involved in the response to DI particles are not known. In this study, we show that 1) DI particles are recognized by more than one viral sensor independently of TLRs and type I IFN signaling; 2) The helicase MDA5 participates in the detection of DI genomes as MDA5-deficient DCs respond inefficiently to Sendai virus stocks containing DI particles; 3) DI particles stimulate the expression of IRF3-responsive genes by a uniquely potent mechanism when compared with other prototypic viral stimulus; and 4) the efficient detection of DI particles overcomes virus immune antagonism. These data highlight the outstanding adjuvant capacity of DI particles in stimulating mouse and human DCs. They also offer biological relevance to the previously reported inhibition of MDA5 by different paramyxovirus V proteins. The unique mechanism by which DI particles trigger the maturation of DCs represents a novel strategy that could be further exploited for the development of potent adjuvant molecules.

The Tumour Suppressor CYLD is a Negative Regulator of RIG-I-mediated Antiviral Response

EMBO Reports. Sep, 2008  |  Pubmed ID: 18636086

On detecting viral RNAs, the RNA helicase retinoic acid-inducible gene I (RIG-I) activates the interferon regulatory factor 3 (IRF3) signalling pathway to induce type I interferon (IFN) gene transcription. How this antiviral signalling pathway might be negatively regulated is poorly understood. Microarray and bioinformatic analysis indicated that the expression of RIG-I and that of the tumour suppressor CYLD (cylindromatosis), a deubiquitinating enzyme that removes Lys 63-linked polyubiquitin chains, are closely correlated, suggesting a functional association between the two molecules. Ectopic expression of CYLD inhibits the IRF3 signalling pathway and IFN production triggered by RIG-I; conversely, CYLD knockdown enhances the response. CYLD removes polyubiquitin chains from RIG-I as well as from TANK binding kinase 1 (TBK1), the kinase that phosphorylates IRF3, coincident with an inhibition of the IRF3 signalling pathway. Furthermore, CYLD protein level is reduced in the presence of tumour necrosis factor and viral infection, concomitant with enhanced IFN production. These findings show that CYLD is a negative regulator of RIG-I-mediated innate antiviral response.

A Comprehensive Evaluation of Human Plasmacytoid Dendritic Cells Using Small Volumes of Human Blood

Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research. Aug, 2008  |  Pubmed ID: 18729740

Elucidation of the role played by cells of the innate immune system, particularly the dendritic cell (DC) populations, has led to a precipitous growth in our understanding of immunity to pathogens and foreign antigens. Much of this information has been derived from studies using mouse model systems. However, mice and human DCs differ drastically in the relative distribution of the toll-like receptors (TLRs) critical for immune activation. This is particularly true for the plasmacytoid DCs (pDCs), which are activated almost exclusively through TLR signaling. Variation in this DC subpopulation has been implicated in a number of pathological syndromes; therefore, a thorough understanding of their steady state and activation profiles in human patients is essential. A number of factors, including the relatively low numbers of these cells in blood, have precluded careful analysis in clinical trials. To overcome these limitations, we have developed a technique for studying the steady state and activation profile of pDCs collected from small amounts of human blood. This technique can be performed with 10,000 cells to obtain the immune transcriptome of the pDCs analyzed by quantitative PCR using amplified RNA. In addition, we have used multiplex enzyme-linked immunosorbent assays to measure secreted proteins. We demonstrate the validity of this technique and document its potential for use with blood from human study populations.

Estrogen Inhibits Dendritic Cell Maturation to RNA Viruses

Blood. Dec, 2008  |  Pubmed ID: 18802009

Dendritic cells (DCs) play a central role in initiating and polarizing the immune response. Therefore, DC maturation represents a key control point in the shift from innate to adaptive immunity. It is suspected that during pregnancy, hormones are critical factors that modulate changes reported to occur in maternal immunity. Here we examined the effect of 17-beta-estradiol (E2) on the maturational response triggered by virus in human DCs and its influence on their ability to activate naive T cells. We developed an in vitro system to measure the response of DCs to virus infection with Newcastle disease virus (NDV) after a 24-hour E2 treatment. Using this system, we demonstrated that E2 pretreatment down-regulated the antiviral response to RNA viruses in DCs by profoundly suppressing type I interferon (IFN) synthesis and other important inflammatory products. In addition, the DCs capacity to stimulate naive CD4 T cells was also reduced. These results suggest an important role for E2 in suppressing the antiviral response and provide a mechanism for the reduced immunity to virus infection observed during pregnancy.

Interferon-beta Pretreatment of Conventional and Plasmacytoid Human Dendritic Cells Enhances Their Activation by Influenza Virus

PLoS Pathogens. Oct, 2008  |  Pubmed ID: 18974865

Influenza virus produces a protein, NS1, that inhibits infected cells from releasing type I interferon (IFN) and blocks maturation of conventional dendritic cells (DCs). As a result, influenza virus is a poor activator of both mouse and human DCs in vitro. However, in vivo a strong immune response to virus infection is generated in both species, suggesting that other factors may contribute to the maturation of DCs in vivo. It is likely that the environment in which a DC encounters a virus would contain multiple pro-inflammatory molecules, including type I IFN. Type I IFN is a critical component of the viral immune response that initiates an antiviral state in cells, primarily by triggering a broad transcriptional program that interferes with the ability of virus to establish infection in the cell. In this study, we have examined the activation profiles of both conventional and plasmacytoid dendritic cells (cDCs and pDCs) in response to an influenza virus infection in the context of a type I IFN-containing environment. We found that both cDCs and pDCs demonstrate a greater activation response to influenza virus when pre-exposed to IFN-beta (IFN priming); although, the priming kinetics are different in these two cell types. This strongly suggests that type I IFN functions not only to reduce viral replication in these immune cells, but also to promote greater DC activation during influenza virus infections.

Antiviral-activated Dendritic Cells: a Paracrine-induced Response State

Journal of Immunology (Baltimore, Md. : 1950). Nov, 2008  |  Pubmed ID: 18981106

Infection of immature dendritic cells (DCs) by virus stimulates their maturation into APC. Infected DCs can also expose uninfected DCs to a panoply of cytokines/chemokines via paracrine signaling. Mathematical modeling suggests that a high rate of paracrine signaling is likely to occur among DCs located in three-dimensional space. Relatively little is known about how secreted factors modify the early response to virus infection. We used a transwell experimental system that allows passage of secreted factors, but not direct contact, between virus-infected DCs and uninfected DCs to investigate paracrine signaling responses. Paracrine signaling from infected DCs induced an antiviral-primed DC state distinct from that of mature virus-infected DCs that we refer to as antiviral-activated DCs (AVDCs). AVDCs had increased surface MHC class II and CD86 levels, but in contrast to virus-infected DCs, their MHC class I levels were unchanged. Imaging flow cytometry showed that AVDCs had an increased rate of phagocytosis compared with naive DCs. Experiments with IFN-beta cytokine indicated that it may be responsible for CD86, but not MHC class II regulation in AVDCs. Both IFN-inducible and IFN-independent genes are up-regulated in AVDCs. Notably, AVDCs are relatively resistant to virus infection in comparison to naive DCs and achieve accelerated and augmented levels of costimulatory molecule expression with virus infection. AVDCs show a distinct antiviral-primed state of DC maturation mediated by DC paracrine signaling. Although further in vivo study is needed, the characteristics of the AVDC suggest that it is well suited to play a role in the early innate-adaptive transition of the immune system.

Impact of Alpha-defensins1-3 on the Maturation and Differentiation of Human Monocyte-derived DCs. Concentration-dependent Opposite Dual Effects

Clinical Immunology (Orlando, Fla.). Jun, 2009  |  Pubmed ID: 19237318

alpha-defensins1-3 are potent antimicrobial molecules that also link innate and adaptive immunity, depending on the concentration range. However, their effects on the biology of human DCs remain largely unknown. We analyzed the impact of different concentrations of alpha-defensins1-3 on the maturation and differentiation of monocyte-derived DCs (MDDCs). Low doses of alpha-defensins1-3 up-regulated CD83, CD86 and HLA-DR expression, increased TNF-alpha, IL-1beta, IL-12p40, IL-10 and IL-8 secretion, and slightly augmented allostimulatory capacity. By contrast, high doses down-regulated CD86 and HLA-DR expression, TNF-alpha, IL-1beta, IL-12p40 and IL-10 secretion and allostimulatory capacity, whereas strongly up-regulated IL-8. Furthermore, during the MDDC differentiation process, high doses of alpha-defensins1-3 affected CD14, CD11c and CD86 expression and strongly up-regulated IL-8. Results suggest that alpha-defensins1-3 might modulate the maturation and differentiation of MDDCs in vivo and therefore could be of special interest in the field of vaccine development.

Alpha-C-galactosylceramide As an Adjuvant for a Live Attenuated Influenza Virus Vaccine

Vaccine. Jun, 2009  |  Pubmed ID: 19464560

There is a substantial need to develop better influenza virus vaccines that can protect populations that are not adequately protected by the currently licensed vaccines. While live attenuated influenza virus vaccines induce superior immune responses compared to inactivated vaccines, the manufacturing process of both types of influenza virus vaccines is time consuming and may not be adequate during a pandemic. Adjuvants would be particularly useful if they could enhance the immune response to live attenuated influenza virus vaccines so that the amount of vaccine needed for a protective dose could be reduced. The glycolipid, alpha-galactosylceramide (alpha-GalCer), has recently been shown to have adjuvant activity for both inactivated and replicating recombinant vaccines. The goal of these experiments was to determine whether a derivative of alpha-GalCer, alpha-C-galactosylceramide (alpha-C-GalCer) can enhance the immune response elicited by a live attenuated influenza virus vaccine containing an NS1 protein truncation and reduce the amount of vaccine required to provide protection after challenge. Our results indicated that the adjuvant reduced both morbidity and mortality in BALB/c mice after challenge with wild type influenza virus. The adjuvant also increased the amount of influenza virus specific total IgG, IgG1, and IgG2a antibodies as well as IFN-gamma secreting CD8(+) T cells. By using knockout mice that are not able to generate NKT cells, we were able to demonstrate that the mechanism of adjuvant activity is dependent on NKT cells. Thus, our data indicate that stimulators of NKT cells represent a new avenue of adjuvants to pursue for live attenuated virus vaccines.

Cutting Edge: Stealth Influenza Virus Replication Precedes the Initiation of Adaptive Immunity

Journal of Immunology (Baltimore, Md. : 1950). Sep, 2009  |  Pubmed ID: 19717515

A timely immune response is crucial for the effective control of virus infection. The influenza virus NS1 protein interferes with the expression of key proinflammatory cytokines from infected cells in vitro. To investigate the effect of NS1 during the onset of immunity in vivo, we systematically studied the early events that occur in the lungs and draining lymph nodes upon infection with influenza virus. Strikingly, no sign of innate immunity was detected in the lungs for almost 2 days after infection until a sudden inflammatory burst, including IFNs, cytokines, and chemokines, occurred. This burst preceded the robust dendritic cell migration and T cell activation in the lymph nodes. An NS1-deficient virus triggered rapid inflammation in the lungs whereas a wild-type virus did not. Thus, we demonstrate that, in vivo, influenza virus uses the NS1 protein to replicate for almost 2 days after infection before detection by the immune system.

The Functional Impairment of Natural Killer Cells During Influenza Virus Infection

Immunology and Cell Biology. Nov-Dec, 2009  |  Pubmed ID: 19721456

Natural killer (NK) cells have a critical role in clearing influenza virus, which primarily infects the lung epithelial cells. However, the ability of influenza virus to infect and manipulate NK cells has not been studied. In this context, we hypothesized that influenza virus can target NK cells leading to a functional impairment in their ability to mediate cytotoxicity and cytokine/chemokine generations. Here, we show influenza virus, PR8, can enter and infect NK cells. This infection did not alter the expression levels of activating, inhibitory or developmental receptors of NK cells. However, infection of NK cells by PR8 reduced the cytotoxicity to tumor cells that represent 'induced-self' and 'missing-self'. PR8-infection also significantly downregulated the NCR1, NKG2D, Nkpr1c, Ly49D and CD244 receptors-mediated generation of pro-inflammatory cytokines and chemokines. Mutations in the non-structural protein 1 (NS1) of influenza virus further augmented the functional impairment of NK cells. Our observations show the presence of a new, but yet to be explored, mechanism by which the influenza virus can evade immune detection.

IL-11 Regulates Autoimmune Demyelination

Journal of Immunology (Baltimore, Md. : 1950). Oct, 2009  |  Pubmed ID: 19734214

Current therapies for the autoimmune demyelinating disease multiple sclerosis (MS) target inflammation, but do not directly address neuroprotection or lesion repair. Cytokines of the gp130 family regulate survival and differentiation of both neural and immune cells, and we recently identified expression of the family member IL-11 in active MS plaques. In this study, we show that IL-11 regulates the clinical course and neuropathology of experimental autoimmune encephalomyelitis, a demyelinating model that mimics many of the clinical and pathologic features of MS. Importantly, the effects of IL-11 are achieved via a combination of immunoregulation and direct neuroprotection. IL-11R-alpha-null (IL-11Ralpha(-/-)) mice displayed a significant increase in clinical severity and neuropathology of experimental autoimmune encephalomyelitis compared with wild-type littermates. Inflammation, demyelination, and oligodendrocyte and neuronal loss were all exacerbated in IL-11Ra(-/-) animals. Conversely, wild-type mice treated with IL-11 displayed milder clinical signs and neuropathology than vehicle-treated controls. In cocultures of murine myelin oligodendrocyte glycoprotein(35-55)-specific CD4+ T lymphocytes and CD11c+ APCs, IL-11 treatment resulted in a significant decrease in T cell-derived effector cytokine production. This effect was generated via modulation of CD11c+ APC-mediated lymphocyte activation, and was associated with a decrease in the size of the CD11c+ cell population. Conversely, IL-11 strongly reduced apoptosis and potentiated mitosis in primary cultures of mouse oligodendrocyte progenitors. Collectively, these data reveal that IL-11 regulates inflammatory demyelination via a unique combination of immunoregulation and neuroprotection. IL-11 signaling may represent a therapeutic avenue to restrict CNS inflammation and potentiate oligodendrocyte survival in autoimmune demyelinating disease.

Hemagglutinin-pseudotyped Green Fluorescent Protein-expressing Influenza Viruses for the Detection of Influenza Virus Neutralizing Antibodies

Journal of Virology. Feb, 2010  |  Pubmed ID: 19939917

Influenza virus is a highly contagious virus that causes yearly epidemics and occasional pandemics of great consequence. Influenza virus neutralizing antibodies (NAbs) are promising prophylactic and therapeutic reagents. Detection of NAbs in serum samples is critical to evaluate the prevalence and spread of new virus strains. Here we describe the development of a simple, sensitive, specific, and safe screening assay for the rapid detection of NAbs against highly pathogenic influenza viruses under biosafety level 2 (BSL-2) conditions. This assay is based on the use of influenza viruses in which the hemagglutinin (HA) gene is replaced by a gene expressing green fluorescent protein (GFP). These GFP-expressing influenza viruses replicate to high titers in HA-expressing cell lines, but in non-HA-expressing cells, their replication is restricted to a single cycle.

Immune Response Modeling of Interferon Beta-pretreated Influenza Virus-infected Human Dendritic Cells

Biophysical Journal. Feb, 2010  |  Pubmed ID: 20159146

The pretreatment of human dendritic cells with interferon-beta enhances their immune response to influenza virus infection. We measured the expression levels of several key players in that response over a period of 13 h both during pretreatment and after viral infection. Their activation profiles reflect the presence of both negative and positive feedback loops in interferon induction and interferon signaling pathway. Based on these measurements, we have developed a comprehensive computational model of cellular immune response that elucidates its mechanism and its dynamics in interferon-pretreated dendritic cells, and provides insights into the effects of duration and strength of pretreatment.

The Virion Host Shut-off (vhs) Protein Blocks a TLR-independent Pathway of Herpes Simplex Virus Type 1 Recognition in Human and Mouse Dendritic Cells

PloS One. 2010  |  Pubmed ID: 20174621

Molecular pathways underlying the activation of dendritic cells (DCs) in response to Herpes Simplex Virus type 1 (HSV-1) are poorly understood. Removal of the HSV virion host shut-off (vhs) protein relieves a block to DC activation observed during wild-type infection. In this study, we utilized a potent DC stimulatory HSV-1 recombinant virus lacking vhs as a tool to investigate the mechanisms involved in the activation of DCs by HSV-1. We report that the release of pro-inflammatory cytokines by conventional DC (cDC) during HSV-1 infection is triggered by both virus replication-dependent and replication-independent pathways. Interestingly, while vhs is capable of inhibiting the release of cytokines during infection of human and mouse cDCs, the secretion of cytokines by plasmacytoid DC (pDC) is not affected by vhs. These data prompted us to postulate that infection of cDCs by HSV triggers a TLR independent pathway for cDC activation that is susceptible to blockage by the vhs protein. Using cDCs isolated from mice deficient in both the TLR adaptor protein MyD88 and TLR3, we show that HSV-1 and the vhs-deleted virus can activate cDCs independently of TLR signaling. In addition, virion-associated vhs fails to block cDC activation in response to treatment with TLR agonists, but it efficiently blocked cDC activation triggered by the paramyxoviruses Sendai Virus (SeV) and Newcastle Disease Virus (NDV). This block to SeV- and NDV-induced activation of cDC resulted in elevated SeV and NDV viral gene expression indicating that infection with HSV-1 enhances the cell's susceptibility to other pathogens through the action of vhs. Our results demonstrate for the first time that a viral protein contained in the tegument of HSV-1 can block the induction of DC activation by TLR-independent pathways of viral recognition.

Broadly Protective Monoclonal Antibodies Against H3 Influenza Viruses Following Sequential Immunization with Different Hemagglutinins

PLoS Pathogens. Feb, 2010  |  Pubmed ID: 20195520

As targets of adaptive immunity, influenza viruses are characterized by the fluidity with which they respond to the selective pressure applied by neutralizing antibodies. This mutability of structural determinants of protective immunity is the obstacle in developing universal influenza vaccines. Towards the development of such vaccines and other immune therapies, our studies are designed to identify regions of influenza viruses that are conserved and that mediate virus neutralization. We have specifically focused on viruses of the H3N2 subtype, which have persisted as a principal source of influenza-related morbidity and mortality in humans since the pandemic of 1968. Three monoclonal antibodies have been identified that are broadly-neutralizing against H3 influenza viruses spanning 40 years. The antibodies react with the hemagglutinin glycoprotein and appear to bind in regions that are refractory to the structural variation required for viral escape from neutralization. The antibodies demonstrate therapeutic efficacy in mice against H3N2 virus infection and have potential for use in the treatment of human influenza disease. By mapping the binding region of one antibody, 12D1, we have identified a continuous region of the hemagglutinin that may act as an immunogen to elicit broadly protective immunity to H3 viruses. The anti-H3 monoclonal antibodies were identified after immunization of mice with the hemagglutinin of four different viruses (A/Hong Kong/1/1968, A/Alabama/1/1981, A/Beijing/47/1992, A/Wyoming/3/2003). This immunization schedule was designed to boost B cells specific for conserved regions of the hemagglutinin from distinct antigenic clusters. Importantly, our antibodies are of naturally occurring specificity rather than selected from cloned libraries, demonstrating that broad-spectrum humoral immunity to influenza viruses can be elicited in vivo.

Antiviral Instruction of Bone Marrow Leukocytes During Respiratory Viral Infections

Cell Host & Microbe. May, 2010  |  Pubmed ID: 20478536

Respiratory viral infections trigger a robust inflammatory response in the lung, producing cytokines, chemokines, and growth factors that promote infiltration of effector leukocytes. Whereas the role of chemokines and infiltrating leukocytes in antiviral immunity is well studied, the effect that lung cytokines have on leukocytes in distal hematopoietic and lymphoid tissues and their role in antiviral immunity is unknown. We show that, during infection with influenza or Sendai virus, the lung communicates with the sterile bone marrow, the primary site of hematopoiesis, through type I interferons. While in the bone marrow, leukocytes exposed to type I interferons activate an antiviral transcriptional program and become resistant to infection with different viruses. The protected bone marrow leukocytes are capable of migrating to the infected lung and contribute to virus clearance. These findings show that appropriate instruction of cells during their development in the bone marrow is needed for effective control of infection.

Palmitoylome Profiling Reveals S-palmitoylation-dependent Antiviral Activity of IFITM3

Nature Chemical Biology. Aug, 2010  |  Pubmed ID: 20601941

Identification of immune effectors and the post-translational modifications that control their activity is essential for dissecting mechanisms of immunity. Here we demonstrate that the antiviral activity of interferon-induced transmembrane protein 3 (IFITM3) is post-translationally regulated by S-palmitoylation. Large-scale profiling of palmitoylated proteins in a dendritic cell line using a chemical reporter strategy revealed over 150 lipid-modified proteins with diverse cellular functions, including innate immunity. We discovered that S-palmitoylation of IFITM3 on membrane-proximal cysteines controls its clustering in membrane compartments and its antiviral activity against influenza virus. The sites of S-palmitoylation are highly conserved among the IFITM family of proteins in vertebrates, which suggests that S-palmitoylation of these immune effectors may be an ancient post-translational modification that is crucial for host resistance to viral infections. The S-palmitoylation and clustering of IFITM3 will be important for elucidating its mechanism of action and for the design of antiviral therapeutics.

Peripheral Blood Cytokine Profiling During Pregnancy and Post-partum Periods

American Journal of Reproductive Immunology (New York, N.Y. : 1989). Dec, 2010  |  Pubmed ID: 20712812

Pregnancy requires that the maternal immune system adapt to prevent rejection of the fetal semi-allograft. This immunologic adaptation may contribute to pregnancy-related alterations in disease susceptibility and severity of infections from viral pathogens such as influenza virus.

Vaccination with a Synthetic Peptide from the Influenza Virus Hemagglutinin Provides Protection Against Distinct Viral Subtypes

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2010  |  Pubmed ID: 20956293

Current influenza virus vaccines protect mostly against homologous virus strains; thus, regular immunization with updated vaccine formulations is necessary to guard against the virus' hallmark remodeling of regions that mediate neutralization. Development of a broadly protective influenza vaccine would mark a significant advance in human infectious diseases research. Antibodies with broad neutralizing activity (nAbs) against multiple influenza virus strains or subtypes have been reported to bind the stalk of the viral hemagglutinin, suggesting that a vaccine based on this region could elicit a broadly protective immune response. Here we describe a hemagglutinin subunit 2 protein (HA2)-based synthetic peptide vaccine that provides protection in mice against influenza viruses of the structurally divergent subtypes H3N2, H1N1, and H5N1. The immunogen is based on the binding site of the recently described nAb 12D1, which neutralizes H3 subtype viruses, demonstrates protective activity in vivo, and, in contrast to a majority of described nAbs, appears to bind to residues within a single α-helical portion of the HA2 protein. Our data further demonstrate that the specific design of our immunogen is integral in the induction of broadly active anti-hemagglutinin antibodies. These results provide proof of concept for an HA2-based influenza vaccine that could diminish the threat of pandemic influenza disease and generally reduce the significance of influenza viruses as human pathogens.

Buying Time-the Immune System Determinants of the Incubation Period to Respiratory Viruses

Viruses. Nov, 2010  |  Pubmed ID: 21994630

Respiratory viruses cause disease in humans characterized by an abrupt onset of symptoms. Studies in humans and animal models have shown that symptoms are not immediate and appear days or even weeks after infection. Since the initial symptoms are a manifestation of virus recognition by elements of the innate immune response, early virus replication must go largely undetected. The interval between infection and the emergence of symptoms is called the incubation period and is widely used as a clinical score. While incubation periods have been described for many virus infections the underlying mechanism for this asymptomatic phase has not been comprehensively documented. Here we review studies of the interaction between human pathogenic respiratory RNA viruses and the host with a particular emphasis on the mechanisms used by viruses to inhibit immunity. We discuss the concept of the "stealth phase", defined as the time between infection and the earliest detectable inflammatory response. We propose that the "stealth phase" phenomenon is primarily responsible for the suppression of symptoms during the incubation period and results from viral antagonism that inhibits major pathways of the innate immune system allowing an extended time of unhindered virus replication.

Unique Type I Interferon Responses Determine the Functional Fate of Migratory Lung Dendritic Cells During Influenza Virus Infection

PLoS Pathogens. Nov, 2011  |  Pubmed ID: 22072965

Migratory lung dendritic cells (DCs) transport viral antigen from the lungs to the draining mediastinal lymph nodes (MLNs) during influenza virus infection to initiate the adaptive immune response. Two major migratory DC subsets, CD103(+) DCs and CD11b(high) DCs participate in this function and it is not clear if these antigen presenting cell (APC) populations become directly infected and if so whether their activity is influenced by the infection. In these experiments we show that both subpopulations can become infected and migrate to the draining MLN but a difference in their response to type I interferon (I-IFN) signaling dictates the capacity of the virus to replicate. CD103(+) DCs allow the virus to replicate to significantly higher levels than do the CD11b(high) DCs, and they release infectious virus in the MLNs and when cultured ex-vivo. Virus replication in CD11b(high) DCs is inhibited by I-IFNs, since ablation of the I-IFN receptor (IFNAR) signaling permits virus to replicate vigorously and productively in this subset. Interestingly, CD103(+) DCs are less sensitive to I-IFNs upregulating interferon-induced genes to a lesser extent than CD11b(high) DCs. The attenuated IFNAR signaling by CD103(+) DCs correlates with their described superior antigen presentation capacity for naïve CD8(+) T cells when compared to CD11b(high) DCs. Indeed ablation of IFNAR signaling equalizes the competency of the antigen presenting function for the two subpopulations. Thus, antigen presentation by lung DCs is proportional to virus replication and this is tightly constrained by I-IFN. The "interferon-resistant" CD103(+) DCs may have evolved to ensure the presentation of viral antigens to T cells in I-IFN rich environments. Conversely, this trait may be exploitable by viral pathogens as a mechanism for systemic dissemination.

P53 Serves As a Host Antiviral Factor That Enhances Innate and Adaptive Immune Responses to Influenza A Virus

Journal of Immunology (Baltimore, Md. : 1950). Dec, 2011  |  Pubmed ID: 22105999

Several direct target genes of the p53 tumor suppressor have been identified within pathways involved in viral sensing, cytokine production, and inflammation, suggesting a potential role of p53 in antiviral immunity. The increasing need to identify immune factors to devise host-targeted therapies against pandemic influenza A virus (IAV) led us to investigate the role of endogenous wild-type p53 on the immune response to IAV. We observed that the absence of p53 resulted in delayed cytokine and antiviral gene responses in lung and bone marrow, decreased dendritic cell activation, and reduced IAV-specific CD8(+) T cell immunity. Consequently, p53(-/-) mice showed a more severe IAV-induced disease compared with their wild-type counterparts. These findings establish that p53 influences the antiviral response to IAV, affecting both innate and adaptive immunity. Thus, in addition to its established functions as a tumor suppressor gene, p53 serves as an IAV host antiviral factor that might be modulated to improve anti-IAV therapy and vaccines.

Characterizing the Pregnancy Immune Phenotype: Results of the Viral Immunity and Pregnancy (VIP) Study

Journal of Clinical Immunology. Dec, 2011  |  Pubmed ID: 22198680

PURPOSE: The increased risk of morbidity and mortality from certain microbial infections and the demonstrated improvements in the clinical course of some autoimmune diseases support the existence of pregnancy-related alterations in immune status. Elucidating the changes in innate and adaptive immunity during gestation may improve pregnancy outcomes and facilitate the development of targeted therapies for autoimmune diseases. METHOD: The Viral Immunity and Pregnancy (VIP) study evaluated over 50 subjects longitudinally at three time points during pregnancy and at two time points post-delivery. Leukocyte enumeration was performed; functional responses of NK cells and CD4 T cells were analyzed, and soluble factors such as cytokines, defensins, and steroid hormones were measured in maternal blood. RESULTS: In comparison to the post-partum period, the latter part of pregnancy was characterized by significant increases in blood phagocytes and pDCs and decreases in the number and activity of NK and T cells. Alterations were found in antimicrobial proteins and serum cytokines. CONCLUSIONS: These data show that pregnancy is not a period of immunosuppression but an alteration in immune priorities characterized by a strengthening of innate immune barriers and a concomitant reduction in adaptive/inflammatory immunity in the later stages of pregnancy.

Alpha-defensins 1-3 Release by Dendritic Cells is Reduced by Estrogen

Reproductive Biology and Endocrinology : RB&E. 2011  |  Pubmed ID: 21861873

During pregnancy the immune system of the mother must protect any activation that may negatively affect the fetus. Changes in susceptibility to infection as well as resolution of some autoimmune disorders represent empirical evidence for pregnancy related alterations in immunity. Sex hormones reach extremely high levels during pregnancy and have been shown to have direct effects on many immune functions including the antiviral response of dendritic cells. Among the immunologically active proteins secreted by monocyte derived DCs (MDDC) are the alpha-defensins 1-3. This family of cationic antimicrobial peptides has a broad spectrum of microbicidal activity and has also been shown to link innate to adaptive immunity by attracting T cells and immature DCs, which are essential for initiating and polarizing the immune response.

The Virion Host Shutoff Protein of Herpes Simplex Virus 1 Blocks the Replication-independent Activation of NF-κB in Dendritic Cells in the Absence of Type I Interferon Signaling

Journal of Virology. Dec, 2011  |  Pubmed ID: 21937652

Immune evasion is a defining feature of the virus-host relationship. During infection, herpes simplex virus type 1 (HSV-1) utilizes multiple proteins to manipulate the host immune response. In the present study, we investigated the mechanism by which the virion host shutoff (vhs) protein blocks the activation of dendritic cells (DCs). Previously, we found that coinfection of wild-type HSV-1 with a panel of RNA viruses resulted in a block to DC activation that was attributable to vhs. These observations led us to hypothesize that the vhs-mediated inhibition was dependent on signaling through the RIG-I-like receptor (RLR) signaling pathway. By examining DCs generated from MAVS (IPS-1) knockout (KO) mice, we determined that RLR/MAVS signaling is not essential for the DC response to HSV-1. We also evaluated the requirement for the type I interferon (IFN) signaling pathway in DC activation following infection with HSV-1 and found that stimulation of DCs with wild-type HSV-1 required intact type I IFN signaling for the production of cytokines, whereas the vhs deletion (vhs(-)) mutant virus activated DCs without the need for exogenous IFN signaling. Comparisons of transcription factor activation in DCs infected with wild-type HSV and the vhs(-) mutant virus revealed that NF-κB activation was inhibited by vhs in the early phase of the infection. In contrast, IRF3 activation was not influenced by vhs. In these studies, measurement of proinflammatory cytokines and type I IFN release from the infected DCs reflected the activation status of these transcription factors. Taken together, the work presented here (i) describes a novel role for the vhs protein as an inhibitor of the early activation of NF-κB during HSV-1 infection of DCs and (ii) offers a mechanistic explanation of how this protein interferes with DC activation.