The unparalleled spread of highly pathogenic avian influenza A (HPAI) H5N1 viruses has resulted in devastating outbreaks in domestic poultry and sporadic human infections with a high fatality rate. To better understand the mechanism(s) of H5N1 virus pathogenesis and host responses in humans, we utilized a polarized human bronchial epithelial cell model that expresses both avian alpha-2,3- and human alpha-2,6-linked sialic acid receptors on the apical surface and supports productive replication of both H5N1 and H3N2 viruses. Using this model, we compared the abilities of selected 2004 HPAI H5N1 viruses isolated from humans and a recent human H3N2 virus to trigger the type I interferon (IFN) response. H5N1 viruses elicited significantly less IFN regulatory factor 3 (IRF3) nuclear translocation, as well as delayed and reduced production of IFN-beta compared with the H3N2 virus. Furthermore, phosphorylation of Stat2 and induction of IFN-stimulated genes (ISGs), such as MX1, ISG15, IRF7, and retinoic acid-inducible gene I, were substantially delayed and reduced in cells infected with H5N1 viruses. We also observed that the highly virulent H5N1 virus replicated more efficiently and induced a weaker IFN response than the H5N1 virus that exhibited low virulence in mammals in an earlier study. Our data suggest that the H5N1 viruses tested, especially the virus with the high-pathogenicity phenotype, possess greater capability to attenuate the type I IFN response than the human H3N2 virus. The attenuation of this critical host innate immune defense may contribute to the virulence of H5N1 viruses observed in humans.

BACKGROUND

Respiratory viruses, predominantly rhinoviruses are the major cause of asthma exacerbations. Impaired production of interferon-beta in rhinovirus infected bronchial epithelial cells (BECs) and of the newly discovered interferon-lambdas in both BECs and bronchoalveolar lavage cells, is implicated in asthma exacerbation pathogenesis. Thus replacement of deficient interferon is a candidate new therapy for asthma exacerbations. Rhinoviruses and other respiratory viruses infect both BECs and macrophages, but their relative capacities for alpha-, beta- and lambda-interferon production are unknown.

METHODS

To provide guidance regarding which interferon type is the best candidate for development for treatment/prevention of asthma exacerbations we investigated respiratory virus induction of alpha-, beta- and lambda-interferons in BECs and peripheral blood mononuclear cells (PBMCs) by reverse transferase-polymerase chain reaction and enzyme-linked immunosorbent assay.

RESULTS

Rhinovirus infection of BEAS-2B BECs induced interferon-alpha mRNA expression transiently at 8 h and interferon-beta later at 24 h while induction of interferon-lambda was strongly induced at both time points. At 24 h, interferon-alpha protein was not detected, interferon-beta was weakly induced while interferon-lambda was strongly induced. Similar patterns of mRNA induction were observed in primary BECs, in response to both rhinovirus and influenza A virus infection, though protein levels were below assay detection limits. In PBMCs interferon-alpha, interferon-beta and interferon-lambda mRNAs were all strongly induced by rhinovirus at both 8 and 24 h and proteins were induced: interferon-alpha)-beta)-lambda. Thus respiratory viruses induced expression of alpha-, beta- and lambda-interferons in BECs and PBMCs. In PBMCs interferon-alpha)-beta)-lambda while in BECs, interferon-lambda>-beta)-alpha.

CONCLUSIONS

We conclude that interferon-lambdas are likely the principal interferons produced during innate responses to respiratory viruses in BECs and interferon-alphas in PBMCs, while interferon-beta is produced by both cell types.

In previous reports it was demonstrated that the Nipah virus V and W proteins have interferon (IFN) antagonist activity due to their ability to block signaling from the IFN-alpha/beta receptor (J. J. Rodriguez, J. P. Parisien, and C. M. Horvath, J. Virol. 76:11476-11483, 2002; M. S. Park et al., J. Virol. 77:1501-1511, 2003). The V, W, and P proteins are all encoded by the same viral gene and share an identical 407-amino-acid N-terminal region but have distinct C-terminal sequences. We now show that the P protein also has anti-IFN function, confirming that the common N-terminal domain is responsible for the antagonist activity. Truncation of this N-terminal domain revealed that amino acids 50 to 150 retain the ability to block IFN and to bind STAT1, a key component of the IFN signaling pathway. Subcellular localization studies demonstrate that the V and P proteins are predominantly cytoplasmic whereas the W protein is localized to the nucleus. In all cases, STAT1 colocalizes with the corresponding Nipah virus protein. These interactions are sufficient to inhibit STAT1 activation, as demonstrated by the lack of STAT1 phosphorylation on tyrosine 701 in IFN-stimulated cells expressing P, V, or W. Therefore, despite their common STAT1-binding domain, the Nipah virus V and P proteins act by retaining STAT1 in the cytoplasm while the W protein sequesters STAT1 in the nucleus, creating both a cytoplasmic and a nuclear block for STAT1. We also show that the IFN antagonist activity of the P protein is not as strong as that of V or W, perhaps explaining why Nipah virus has evolved to express these two edited products.

T cell-independent type 2 (TI-2), in contrast to T-dependent, antigens stimulate the production of murine IgG3. To investigate a possible role for cytokines in mediating the induction of this IgG subclass, we established an in vitro polyclonal model system for studying TI-2 antigen-mediated B cell activation by using dextran-conjugated anti-IgD antibody (alpha delta-dex). We demonstrate that interferon gamma (IFN-gamma) stimulates, and interleukin 4 inhibits, the expression of IgG3 by alpha delta-dexactivated cells. The production of IFN-gamma by non-T cells in response to bacterial products, possibly capsular polysaccharides, may provide an explanation underlying the ability of TI antigens, which are unable to directly stimulate T cell-derived cytokines to induce Ig isotype switching.

The interferon (IFN) regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the IFN-alpha/beta gene promoters, as well as the IFN-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the IFN-stimulated gene 15 (ISG15) promoter. Several recent studies have focused attention on the unique molecular properties of IRF-3 and its role in the regulation of IFN gene expression. IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. Following virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, located in the carboxy-terminus of IRF-3. Phosphorylation causes the cytoplasmic to nuclear translocation of IRF-3, stimulation of DNA binding, and increased transcriptional activation, mediated through the association of IRF-3 with the CBP/p300 coactivator. The purpose of this review is to summarize recent investigations demonstrating the important role of IRF-3 in cytokine gene transcription. These studies provide the framework for a model in which virus-dependent phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN and IFN-responsive genes.

Class II molecules of the major histocompatibility complex (MHC) are heterodimeric glycoproteins expressed on the surface of antigen-presenting B lymphocytes and macrophages. The genes encoding the alpha- and beta-chains of the class II heterodimers, A alpha A beta and E alpha E beta, have recently been characterized at the molecular level, and certain cloned genes were shown to be functionally expressed after introduction into cells by DNA-mediated gene transfer. One study found that a transfected Eb beta gene was expressed in a macrophage cell only after treatment of cells with gamma-interferon. DNA sequences associated with transfected Class II MHC genes may therefore have a regulatory role in their cell type-specific expression. We report here the identification of a cell type-specific transcriptional enhancer element associated with the mouse Ed beta gene.

Intercellular adhesion molecule 1 (ICAM-1) is a glycoprotein expressed on the surface of both hemopoietic and nonhemopoietic cells that mediates, in part, the emigration of leukocytes out of the vasculature. Expression of ICAM-1 on the surface of human umbilical vein endothelial cells and a human lung carcinoma cell line (A549) was increased by interleukin-1 beta, tumor necrosis factor alpha, and interferon gamma in a concentration-dependent manner. Phosphorothioate antisense oligonucleotides designed to hybridize to 10 target sites on the human ICAM-1 mRNA were tested for inhibition of ICAM-1 expression in both cell lines by an ICAM-1 enzyme-linked immunosorbent assay. Based upon potency and unique mRNA target sites, two oligonucleotides were studied in greater detail: ISIS 1570, which targeted the AUG translation initiation codon, and ISIS 1939, which targeted specific sequences in the 3'-untranslated region of the mRNA. Both oligonucleotides specifically inhibit expression of ICAM-1 as analyzed by immunoprecipitation of 35S-labeled proteins. Treatment of cells with ISIS 1939 promoted a reduction in ICAM-1 mRNA, whereas ISIS 1570 did not change the level of ICAM-1 mRNA, suggesting that the two oligonucleotides may be inhibiting ICAM-1 expression by two different mechanisms. The activity of both oligonucleotides was blocked by hybridization of the oligonucleotide to its complementary sense strand prior to addition to the cells. Neither ISIS 1570 nor ISIS 1939 changed the transcriptional rate of the ICAM-1 gene, demonstrating that both oligonucleotides were working through a post-transcriptional mechanism. 2'-O-Methyl phosphorothioate analogs, which do not support RNase H-mediated cleavage of target mRNA, were used to determine if the active antisense oligonucleotides inhibited ICAM-1 expression by an RNase H-dependent mechanism. The 2'-O-methyl phosphorothioate analog of ISIS 1939 did not significantly reduce interleukin-1 beta-induced ICAM-1 expression, whereas the 2'-O-methyl phosphorothioate analog of ISIS 1570 did inhibit ICAM-1 expression, suggesting that the reduction of ICAM-1 mRNA following treatment with ISIS 1939 was due, in part, to RNase H-mediated hydrolysis. Adherence of HL-60 cells to human umbilical vein cell monolayers was inhibited by ISIS 1570 and ISIS 1939, demonstrating that the reduced levels of ICAM-1 impact on ICAM-1-associated function.

ISGF2 was initially identified, purified and cloned as an interferon-alpha (IFN alpha) induced transcription factor that binds to the IFN-stimulated response element (ISRE) of IFN alpha/beta-stimulated genes (ISGs). It was reported to be transcriptionally regulated by several cytokines including IFN alpha and IFN gamma. IFN alpha and IFN gamma inducibility is mediated by a single element: a high affinity, nearly palindromic version of the IFN gamma activation site (GAS). The ISGF2 GAS is bound specifically by p91, which was previously identified as a subunit of the ISG activator ISGF3, and shown to mediate IFN gamma induction of the GBP gene via a GAS. Tyrosine phosphorylation and DNA binding activity of p91 parallel transcription of ISGF2 in response to IFN alpha and/or IFN gamma, consistent with induction mediated by only a GAS. Transcription of the genes that encode p91 and p113, another subunit of ISGF3, is activated only by IFN alpha. This result suggests induction mediated by an ISRE, and implies autoregulation, requiring the products of both genes. Specificity of the ISRE is the basis for the previous conclusion. In contrast, it appears likely that the ISGF2 GAS, and p91 or related factors, also mediate induction of ISGF2 by IL-6 and prolactin. Convergence of signalling pathways from at least four cytokines on this single site would thus be a key aspect of a general role for ISGF2 in cellular growth control.

We recently showed that human thyroid epithelial cells, which are normally negative for HLA-DR molecules, express HLA-DR in thyroid autoimmunity. Furthermore, induction of HLA-DR on normal thyroid cells can be achieved by culture with plant lectins. We have now found that recombinant human interferon-gamma (IFN-gamma) induces expression of HLA-DR molecules on cultured human thyroid cells, whereas Namalva IFN-alpha, recombinant IFN-beta or recombinant interleukin-2 (IL-2) do not. All three IFN, but not IL-2, enhanced thyroid cell HLA-A,B,C expression. The results strongly implicate T cells (which are the source of IFN-gamma) in the aberrant induction of DR on thyroid epithelial cells which is proposed to be a central feature of the immunopathological processes leading to autoimmunity.

The pathogenesis of rheumatoid arthritis (RA) may be mediated by Th1-type T cells. Since chemokine receptors CXCR3 and CCR5 are preferentially expressed on Th1 cells, we tested the expression and regulation of several chemokines, including those that signal through CXCR3 (interferon-gamma-inducible protein of 10 kDa, IP-10, CXCL10; and monokine induced by interferon-gamma, Mig, CXCL9) and CCR5 (macrophage inflammatory protein (Mip)-1 alpha, CCL3; and Mip-1 beta, CCL4) in RA synovial fluids, synovial tissues, and blood. Synovial fluid (SF) protein levels of IP-10 (32.1 +/- 10.5 ng/ml), Mig (15.0 +/- 6.4 ng/ml), Mip-1 beta (0.7 +/- 0.3 ng/ml), and Mip-1 alpha (0.8 +/- 0.1 ng/ml) were 100-, 50-, 25-, and 2-fold elevated in RASF compared to control SF (P < 0.001, P < 0.001, P < 0. 001, and P < 0.02, respectively). Tissue levels of IP-10, Mig, and Mip-1 beta were significantly higher in RA than in OA (P < 0.01). Serum levels of IP-10 (3.1 +/- 1.2 ng/ml) were higher in patients with seropositive RA compared to controls (1.2 +/- 0.2 ng/ml) (P < 0.02). There was a gradient of IP-10, Mig, Mip-1 alpha, and Mip-1 beta from the blood into the synovial fluid in RA. Infiltrating T cells around high endothelial venules in RA synovium and 90 +/- 3% of SF CD3(+)CD4(+) T cells expressed CXCR3, and 85 +/- 2% of SF CD3(+)CD4(+) T cells expressed CCR5. Chemokines, including IP-10, Mig, Mip-1 alpha, and Mip-1 beta, may participate in the selective recruitment of CCR5(+)CXCR3(+) T cells to the inflamed synovium.

Interferon (IFN) mediates its antiviral effects by inducing a number of responsive genes, including the double-stranded RNA (dsRNA)-dependent protein kinase, PKR. Here we report that inducible overexpression of functional PKR in murine fibroblasts sensitized cells to apoptosis induced by influenza virus, while in contrast, cells expressing a dominant-negative variant of PKR were completely resistant. We determined that the mechanism of influenza virus-induced apoptosis involved death signaling through FADD/caspase-8 activation, while other viruses such as vesicular stomatitis virus (VSV) and Sindbis virus (SNV) did not significantly provoke PKR-mediated apoptosis but did induce cytolysis of fibroblasts via activation of caspase-9. Significantly, treatment with IFN-alpha/beta greatly sensitized the fibroblasts to FADD-dependent apoptosis in response to dsRNA treatment or influenza virus infection but completely protected the cells against VSV and SNV replication in the absence of any cellular destruction. The mechanism by which IFN increases the cells' susceptibility to lysis by dsRNA or certain virus infection is by priming cells to FADD-dependent apoptosis, possibly by regulating the activity of the death-induced signaling complex (DISC). Conversely, IFN is also able to prevent the replication of viruses such as VSV that avoid triggering FADD-mediated DISC activity, by noncytopathic mechanisms, thus preventing destruction of the cell.

Expression of either the CD4 or CD8 glycoproteins discriminates two functionally distinct lineages of T lymphocytes. A null mutation in the gene encoding CD4 impairs the development of the helper cell lineage that is normally defined by CD4 expression. Infection of CD4-null mice with Leishmania has revealed a population of functional helper T cells that develops despite the absence of CD4. These CD8- alpha beta T cell receptor+ T cells are major histocompatibility complex class II-restricted and produce interferon-gamma when challenged with parasite antigens. These results indicate that T lymphocyte lineage commitment and peripheral function need not depend on the function of CD4.

Mounting an immune response to a viral pathogen involves the initial recognition of viral antigens through Toll-like receptor-dependent and -independent pathways and the subsequent triggering of signal transduction cascades. Among the many cellular kinases stimulated in response to virus infection, the noncanonical IKK-related kinases TBK1 and IKKepsilon have been shown to phosphorylate and activate interferon regulatory factor 3 (IRF-3) and IRF-7, leading to the production of alpha/beta interferons and the development of a cellular antiviral state. In the present study, we examine the activation of TBK1 and IKKepsilon kinases by vesicular stomatitis virus (VSV) infection in human lung epithelial A549 cells. We demonstrate that replication-competent VSV is required to induce activation of the IKK-related kinases and provide evidence that ribonucleoprotein (RNP) complex of VSV generated intracellularly during virus replication can activate TBK1 and IKKepsilon activity. In TBK1-deficient cells, IRF-3 and IRF-7 activation is significantly reduced, although transcriptional upregulation of IKKepsilon following treatment with VSV, double-stranded RNA, or RNP partially compensates for the loss of TBK1. Biochemical analyses with purified TBK1 and IKKepsilon kinases in vitro demonstrate that the two kinases exhibit similar specificities with respect to IRF-3 and IRF-7 substrates and both kinases target serine residues that are important for full transcriptional activation of IRF-3 and IRF-7. These data suggest that intracellular RNP formation contributes to the early recognition of VSV infection, activates the catalytic activity of TBK1, and induces transcriptional upregulation of IKKepsilon in epithelial cells. Induction of IKKepsilon potentially functions as a component of the amplification mechanism involved in the establishment of the antiviral state.

Adult immunocompetent mice inoculated with Ebola (EBO) or Marburg (MBG) virus do not become ill. A suckling-mouse-passaged variant of EBO Zaire '76 ('mouse-adapted EBO-Z') causes rapidly lethal infection in adult mice after intraperitoneal (i.p.) inoculation, but does not cause apparent disease when inoculated subcutaneously (s.c.). A series of experiments showed that both forms of resistance to infection are mediated by the Type I interferon response. Mice lacking the cell-surface IFN-alpha/beta receptor died within a week after inoculation of EBO-Z '76, EBO Sudan, MBG Musoke or MBG Ravn, or after s.c. challenge with mouse-adapted EBO-Z. EBO Reston and EBO Ivory Coast did not cause illness, but immunized the mice against subsequent challenge with mouse-adapted EBO-Z. Normal adult mice treated with antibodies against murine IFN-alpha/beta could also be lethally infected with i.p.-inoculated EBO-Z '76 or EBO Sudan and with s.c.-inoculated mouse-adapted EBO-Z. Severe combined immunodeficient (SCID) mice became ill 3-4 weeks after inoculation with EBO-Z '76, EBO Sudan or MBG Ravn, but not the other viruses. Treatment with anti-IFN-alpha/beta antibodies markedly accelerated the course of EBO-Z '76 infection. Antibody treatment blocked the effect of a potent antiviral drug, 3-deazaneplanocin A, indicating that successful filovirus therapy may require the active participation of the Type I IFN response. Mice lacking an IFN-alpha/beta response resemble primates in their susceptibility to rapidly progressive, overwhelming filovirus infection. The outcome of filovirus transfer between animal species appears to be determined by interactions between the virus and the innate immune response.

Severe respiratory syncytial virus (RSV)-induced bronchiolitis has been associated with a mixed "Th1" and "Th2" cytokine storm. We hypothesized that differentiation of "alternatively activated" macrophages (AA-M phi) would mediate the resolution of RSV-induced lung injury. RSV induced interleukin (IL)-4 and IL-13 by murine lung and peritoneal macrophages, IL-4R alpha/STAT6-dependent AA-M phi differentiation, and significantly enhanced inflammation in the lungs of IL-4R alpha(-/-) mice. Adoptive transfer of wildtype macrophages to IL-4R alpha(-/-) mice restored RSV-inducible AA-M phi phenotype and diminished lung pathology. RSV-infected Toll-like receptor (TLR)4(-/-) and interferon (IFN)-beta(-/-) macrophages and mice also failed to express AA-M phi markers, but exhibited sustained proinflammatory cytokine production (e.g., IL-12) in vitro and in vivo and epithelial damage in vivo. TLR4 signaling is required for peroxisome proliferator-activated receptor gamma expression, a DNA-binding protein that induces AA-M phi genes, whereas IFN-beta regulates IL-4, IL-13, IL-4R alpha, and IL-10 expression in response to RSV. RSV-infected cotton rats treated with a cyclooxygenase-2 inhibitor increased expression of lung AA-M phi. These data suggest new treatment strategies for RSV that promote AA-M phi differentiation.

The Toll-like receptor 3 (TLR3) and TLR4-signaling pathway that involves the adaptor protein TRIF activates type I interferon (IFN) and proinflammatory cytokine expression. Little is known about how TRIF pathway-dependent gene expression is regulated. SH2-containing protein tyrosine phosphatase 2 (SHP-2) is a widely expressed cytoplasmic tyrosine phosphatase. Here we demonstrate that SHP-2 negatively regulated TLR4- and TLR3-activated IFN-beta production. SHP-2 inhibited TLR3-activated but not TLR2-, TLR7-, and TLR9-activated proinflammatory cytokine IL-6 and TNF-alpha production. SHP-2 inhibited poly(I:C)-induced cytokine production by a phosphatase activity-independent mechanism. C-terminal domain of SHP-2 directly bound TANK binding kinase (TBK1) by interacting with the kinase domain of TBK1. SHP-2 deficiency increased TBK1-activated IFN-beta and TNF-alpha expression. TBK1 knockdown inhibited poly(I:C)-induced IL-6 production in SHP-2-deficient cells. SHP-2 also inhibited poly(I:C)-induced activation of MAP kinase pathways. These results demonstrate that SHP-2 specifically negatively regulate TRIF-mediated gene expression in TLR signaling, partially through inhibiting TBK1-activated signal transduction.

Secretion of interferon (IFN) by virus-infected cells is essential for activating autocrine and paracrine pathways that promote cellular transition to an antiviral state. In most mammalian cells, IFN production is initiated by the activation of constitutively expressed IFN regulatory factor 3, IRF3, which in turn leads to the induction of IRF7, the "master regulator" of IFN type I synthesis (alpha/beta IFN). Previous studies established that rotavirus NSP1 antagonizes IFN signaling by inducing IRF3 degradation. In the present study, we have determined that, in comparison to wild-type rotaviruses, rotaviruses encoding defective NSP1 grow to lower titers in some cell lines and that this poor growth phenotype is due to their failure to suppress IFN expression. Furthermore, we provide evidence that rotaviruses encoding wild-type NSP1 subvert IFN signaling by inducing the degradation of not only IRF3, but also IRF7, with both events occurring through proteasome-dependent processes that proceed with similar efficiencies. The capacity of NSP1 to induce IRF7 degradation may allow rotavirus to move across the gut barrier by enabling the virus to replicate in specialized trafficking cells (dendritic cells and macrophages) that constitutively express IRF7. Along with IRF3 and IRF7, NSP1 was found to induce the degradation of IRF5, a factor that upregulates IFN expression and that is involved in triggering apoptosis during viral infection. Our analysis suggests that NSP1 mediates the degradation of IRF3, IRF5, and IRF7 by recognizing a common element of IRF proteins, thereby allowing NSP1 to act as a broad-spectrum antagonist of IRF function.

Hepatitis B virus (HBV) infection is thought to be controlled by virus-specific cytotoxic T lymphocytes (CTL). We have recently shown that HBV-specific CTL can abolish HBV replication noncytopathically in the liver of transgenic mice by secreting tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) after antigen recognition. We now demonstrate that hepatocellular HBV replication is also abolished noncytopathically during lymphocytic choriomeningitis virus (LCMV) infection, and we show that this process is mediated by TNF-alpha and IFN-alpha/beta produced by LCMV-infected hepatic macrophages. These results confirm the ability of these inflammatory cytokines to abolish HBV replication; they elucidate the mechanism likely to be responsible for clearance of HBV in chronically infected patients who become superinfected by other hepatotropic viruses; they suggest that pharmacological activation of intrahepatic macrophages may have therapeutic value in chronic HBV infection; and they raise the possibility that conceptually similar events may be operative in other viral infections as well.

Among the molecules that determine the developmental fate of sympathetic neurons from noradrenergic to cholinergic function are two apparently unrelated proteins, cholinergic differentiation factor and ciliary neurotrophic factor (CDF and CNTF, respectively). The present work suggests a structural basis for their functional overlap: sequence pattern-matching and predictive structure analysis contends that CDF and CNTF are homologous and share a common helical framework. An integrated CDF/CNTF profile also reveals similar sequence/structure motifs in a group of hematopoietic cytokines composed of granulocyte colony-stimulating factor, interleukin-6, and a novel factor called oncostatin M; a more distant relationship is indicated with interleukin-3 and interferons-alpha/beta. Evolutionary ties between neuropoietic and hematopoietic cytokines predict a distinctive tertiary architecture for the uncharacterized CDF and CNTF receptors. The intertwined cytokine/receptor networks signal a closer relationship between the molecular mechanisms underlying neuro- and hematopoiesis.

Asthma is a highly prevalent chronic respiratory disease affecting 300 million people world-wide. A significant fraction of the cost and morbidity of asthma derives from acute care for asthma exacerbations. In the United States alone, there are approximately 15 million outpatient visits, 2 million emergency room visits, and 500,000 hospitalizations each year for management of acute asthma. Common respiratory viruses, especially rhinoviruses, cause the majority of exacerbations in children and adults. Infection of airway epithelial cells with rhinovirus causes the release of pro-inflammatory cytokines and chemokines, as well as recruitment of inflammatory cells, particularly neutrophils, lymphocytes, and eosinophils. The host response to viral infection is likely to influence susceptibility to asthma exacerbation. Having had at least one exacerbation is an important risk factor for recurrent exacerbations suggesting an 'exacerbation-prone' subset of asthmatics. Factors underlying the 'exacerbation-prone' phenotype are incompletely understood but include extrinsic factors: cigarette smoking, medication non-compliance, psychosocial factors, and co-morbidities such as gastroesophageal reflux disease, rhinosinusitis, obesity, and intolerance to non-steroidal anti-inflammatory medications; as well as intrinsic factors such as deficient epithelial cell production of the anti-viral type I interferons (IFN-alpha and IFN-beta). A better understanding of the biologic mechanisms of host susceptibility to recurrent exacerbations will be important for developing more effective preventions and treatments aimed at reducing the significant cost and morbidity associated with this important global health problem.

OBJECTIVE

Cytokines play a key role in the regulation of the immune response. The maximal capacity of cytokine production varies among individuals and correlates with the polymorphism in the cytokine gene promoters. The aim of this study was to characterize gene polymorphism in patients with chronic hepatitis B virus (HBV) infection and to determine the different patterns in patient subgroups.

METHODS

The study population consisted of 77 patients with chronic HBV infection (23 low-level HBV replicative carriers, 23 compensated high-level HBV replicative carriers, 21 decompensated liver transplant candidates, and 10 patients with documented hepatocellular carcinoma). The genetic profile of five cytokines was analyzed by polymerase chain reaction-sequence-specific primer (SSP), and subjects were genotyped as high or low producers of tumor necrosis factor-alpha and interleukin (IL)-6, and as high, intermediate, or low producers of transforming growth factor-beta(1), interferon (IFN)-gamma, and IL-10 based on single nucleotide substitutions. The control group included 10 healthy individuals who recovered from HBV infection and 48 healthy controls.

RESULTS

A highly statistically significant difference in the distribution of the IFN-gamma gene polymorphism (at position +879) was observed between patients with chronic HBV infection and controls. The majority of the patients (65.2%) exhibited the potential to produce low levels of IFN-gamma (A/A genotype) compared with 37.5% of the control group (p = 0.003). Healthy individuals who recovered from HBV infection had a similar distribution of IFN-gamma gene polymorphism as the healthy controls. No statistically significant difference in IFN-gamma production was found between patients with low- and high-level HBV replication and between compensated and decompensated patients. There was also no statistically significant difference in the genetic ability to produce tumor necrosis factor-alpha (at position -308), IL-6 (at position -174), IL-10 (at position -1082, -819, and -592), and transforming growth factor-beta(1) (at position +10 and +25).

CONCLUSIONS

These findings suggest an association between the genetic ability to produce low levels of IFN-gamma and the susceptibility to develop chronic HBV infection.

The induction of alpha/beta interferon (IFN-alpha/beta) is a powerful host defense mechanism against viral infection, and many viruses have evolved strategies to overcome the antiviral effects of IFN. In this study, we found that IFN-alpha had only some degree of antiviral activity against Japanese encephalitis virus (JEV) infection, in contrast to another flavivirus, dengue virus serotype 2, which was highly sensitive to IFN-alpha in the cultured cell system. JEV infection appeared to render cells resistant to IFN-alpha since the IFN-alpha-induced luciferase reporter activity driven by the IFN-stimulated response element (ISRE) was gradually reduced as the JEV infection progressed. Since the biological activities of IFNs are triggered by the Janus kinase (Jak) signal transducer and activation of transcription (Stat) signaling cascade, we then studied the activation of Jak-Stat pathway in the virus-infected cells. The IFN-alpha-stimulated tyrosine phosphorylation of Stat1, Stat2, and Stat3 was suppressed by JEV in a virus replication and de novo protein synthesis-dependent manner. Furthermore, JEV infection blocked the tyrosine phosphorylation of IFN receptor-associated Jak kinase, Tyk2, without affecting the expression of IFN-alpha/beta receptor on the cell surface. Consequently, expression of several IFN-stimulated genes in response to IFN-alpha stimulation was also reduced in the JEV-infected cells. Overall, our findings suggest that JEV counteracts the effect of IFN-alpha/beta by blocking Tyk2 activation, thereby resulting in inhibition of Jak-Stat signaling pathway.

Natural killer (NK) cells play an important role in early defense against viral infection. The cytotoxic activity of NK cells is increased by interferon-alpha/beta (IFN-alpha/beta), produced en masse in virally infected cells. However, the mechanism(s) by which IFN-alpha/beta contribute to the NK-cell-mediated antiviral response is not well understood. Here we provide evidence that the cytotoxicity of NK cells is enhanced by IFN-alpha/beta through induction of TNF-related apoptosis-inducing ligand (TRAIL). Isolation and analysis of the murine TRAIL promoter revealed the presence of an IFN-stimulated response element (ISRE), which binds to the transcription factor ISGF3 (interferon stimulated gene factor-3). This promoter is indeed activated by IFN-beta in ISGF3-dependent manner. We also show that virally infected cells, but not uninfected cells, are susceptible to TRAIL-mediated cytotoxicity in vitro, and that the TRAIL expressed in NK cells is indeed crucial in limiting virus replication in vivo. Thus, our study reveals a new molecular link between IFN-alpha/beta signaling and activation of NK cells in antiviral response of the host.

Humans and other mammals coexist with a diverse array of microbes colonizing the intestine, termed the microflora. The relationship is symbiotic, with the microbes benefiting from a stable environment and nutrient supply, and the host gaining competitive exclusion of pathogens and continuously maintenance of the gut immune homeostasis. Here we report novel crosstalk mechanisms between the human enterocyte cell line, Caco2, and underlying human monocyte-derived DC in a transwell model where Gram-positive (G+) commensals prevent Toll-like receptor-4 (TLR4)-dependent Escherichia coli-induced semimaturation in a TLR2-dependent fashion. These findings add to our understanding of the hypo-responsiveness of the gut epithelium towards the microflora. Gut DC posses a more tolerogenic phenotype than conventional DC. Here we show that Caco2 spent medium (SM) induces tolerogenic DC with lower expression of maturation markers, interleukin (IL)-12p70, and tumour necrosis factor-alpha when matured with G+ and Gram-negative (G-) commensals, while IL-10 production is enhanced in DC upon encountering G+ commensals and reduced upon encountering G- bacteria. The Caco2 SM-induced tolerogenic phenotype is also seen in DC priming of naive T cells with elevated levels of transforming growth factor-beta (TGF-beta) and markedly reduced levels of bacteria-induced interferon-gamma production. Caco2 cell production of IL-8, thymic stromal lymphopoietin (TSLP) and TGF-beta increases upon microbial stimulation in a strain dependent manner. TSLP and TGF-beta co-operate in inducing the tolerogenic DC phenotype but other mediators might be involved.