Effects of the hepatitis C virus core protein on innate cellular defense pathways.

The hepatitis C virus (HCV) core protein is thought to contribute to HCV pathogenesis through its interaction with various signal transduction pathways. In this study, we explored the interaction of the core protein with innate defense pathways (interferon [IFN] regulatory factor [IRF], Jak-Stat, and inducible nitric oxide synthase [iNOS]) in HeLa and Huh7 human cell lines. Expression of a patient-derived genotype 1b core protein activated human IRF-1 and guanylate-binding protein-2 (GBP-2) promoters, induced IRF-1 mRNA, but failed to induce IRF-3 phosphorylation. HCV core protein caused dose-dependent induction of the IFN-beta promoter and IFN-beta mRNA but not the IFN-alpha1 and IFN-alpha4 promoters. In the presence of IFN-alpha, core expression was associated with increased IFN-stimulated gene factor 3 (ISGF3) binding to the IFN-stimulated response element (ISRE) and tyrosine phosphorylation of Stat1. Core expression resulted in dose-dependent activation of the ISRE and gamma activated sequence (GAS) promoters, in both the absence and the presence of either IFN-alpha or IFN-gamma. Core stimulated the human iNOS promoter and induced iNOS protein. The data indicate that HCV core can modulate IRF, Jak-Stat, and iNOS pathways and suggest mechanisms by which core could affect HCV persistence and pathogenesis.     

Interferon-beta mediates opposing effects on interferon-gamma-dependent Interleukin-12 p70 secretion by human monocyte-derived dendritic cells

Interferon-beta (IFN-beta) exposure during tumour necrosis factor-alpha (TNF-alpha)-induced human monocyte-derived dendritic cell (DC) maturation augments the capacity of DC to promote the generation of T helper 1 (Th1) cells, while IFN-beta exposure during naive Th cell stimulation inhibits Th1 cell generation (Nagai et al., J Immunol, 2003 171:5233-43). Investigating these contradictory outcomes of IFN-beta exposure, we find that isolated DC matured with both TNF-alpha and IFN-beta secrete more IL-12 p70 upon CD40L stimulation than DC matured with TNF-alpha alone. mAb blocking studies indicate that the basis for this enhanced IL-12 p70 production is augmentation of two successive CD40-dependent autocrine pathways in the DC: (1) a pathway in which low levels of IL-12 p70, IL-27, IL-18 and, possibly, IL-23 act to mediate autocrine induction of DC IFN-gamma secretion; and (2) an IFN-gamma-initiated autocrine pathway promoting optimal DC IL-12 p70 secretion. In contrast to the IL-12 p70 promoting effects of IFN-beta during DC maturation, IFN-beta pre-treatment before CD40L stimulation was found to inhibit IFN-gamma-mediated enhancement of DC IL-12 p70 secretion. Thus, IFN-beta exposure during TNF-alpha-mediated DC maturation may promote Th1 polarization by increasing DC IL-12 p70 secretion, through enhancement of autocrine-acting IFN-gamma production by the DC. Moreover, IFN-beta exposure during naive Th cell stimulation may inhibit Th1 cell generation by blocking the IFN-gamma-induced signals required for optimal CD40L-induced DC IL-12 p70 secretion. IFN-beta pre-treatment was also observed to inhibit CD40L-induced DC IL-23 secretion. Our findings may account for some of the beneficial effects of IFN-beta therapy in patients with relapsing remitting multiple sclerosis.     

Type I interferon regulates respiratory virus infected dendritic cell maturation and cytokine production

Activation of dendritic cells (DCs) by viruses is critical for both innate and adaptive immune responses. In this report, we investigated the role of type I interferon (IFN) in the activation of DCs by respiratory syncytial virus (RSV). Using DCs from type I IFNR-/- mice, these studies indicate that maturation, including upregulation of co-stimulatory molecules and optimal cytokine production, by RSV infection was dependent on type I IFN receptor signaling. Subsequently, studies using DCs from wild type mice demonstrate that continued production of type I IFN during later stages of DC maturation could alter their activation profiles. IFN-alpha and IFN-beta were upregulated in DCs grown from bone marrow of wild type mice after infection with RSV. In order to determine their function in competent DCs, blocking antibodies were used to specifically inhibit IFN-alpha/beta . The data demonstrate that production of IFN-beta, but not IFN-alpha, in RSV-infected wild type DCs promotes chemokine production and toll-like receptor (TLR) expression, while limiting IL-12 production. The inhibition of IL-12p70 by IFN-beta correlated with suppressed IL-12p40 expression levels. Furthermore, the addition of recombinant IFN-beta potently inhibited IL-12p40 expression in mature DC subsets during RSV infection, while only the highest dose of IFN-alpha had any inhibitory effect. Together, our studies provide insight into the complex regulation of DC maturation and IL-12 production co-ordinated by type I interferons in RSV-infected dendritic cells, and demonstrate that type I IFN has specific roles depending upon the stage of DC maturation.     

IFN-beta modulates the response to TLR stimulation in human DC: involvement of IFN regulatory factor-1 (IRF-1) in IL-27 gene expression

Type I IFN are cytokines which play a central role in host resistance to viral or microbial infections and are important components linking innate and adaptive immunity. We and others have previously demonstrated that the production of IFN-beta by DC following bacterial infections or TLR triggering influences, in an autocrine manner, their maturation. In this study, we investigated whether IFN-beta release modulates the phenotype of the immature DC and their response to a subsequent TLR stimulation. The induction of CD86, HLA-DR, CD38 and B7H1 and the absence of CCR7 and CD83 expression upon IFN-beta treatment suggest that IFN-beta-primed DC remain at the site of infection acquiring an activated phenotype. These results prompted us to investigate the response of IFN-beta-primed DC to TLR stimulation. While IFN-beta pretreatment increases slightly the expression of maturation markers in TLR2- or TLR4-stimulated DC, it is able to modulate selectively the secretion of inflammatory and immuno-regulating cytokines. Interestingly, IL-27p28 subunit was induced by IFN-beta alone or during LPS-induced maturation of DC in a type I IFN-dependent manner through IFN regulatory factor-1 (IRF-1) activation. Taken together, our results shed light on the capacity of IFN-beta to finely tune DC response to invading pathogens.     

The neutralization of type I IFN biologic actions by anti-IFNAR-2 monoclonal antibodies is not entirely due to inhibition of Jak-Stat tyrosine phosphorylation.

A panel of monoclonal antibodies (mAb) derived against human interferon-alpha/beta receptor-2 (IFNAR-2) was evaluated for their ability to antagonize the biologic effects of type 1 interferons (IFN-alpha1, IFN-alpha2a, and IFN-beta). Anti-IFNAR-2 mAb 117.7, 35.9, 53.2, and 51.44 neutralized type I IFN-mediated antiviral, antiproliferative, and major histocompatibility complex (MHC) class I upregulation functions. However, only mAb 51.44 neutralized IFN-alpha2a and IFN-beta-mediated natural killer (NK) cell cytotoxicity. In BIAcore and cell binding studies, only mAb 51.44 and 234.28 inhibited IFN-alpha2a and IFN-beta binding to its receptor. The receptor blockade by mAb 51.44 and 234.28 resulted in the inhibition of IFN-alpha2a and IFN-beta-induced tyrosine phosphorylation of Jak1, Tyk2, Stat1/2/3, and IFNAR-1/2 and inhibition of IFN-stimulated gene factor 3 (ISGF3) formation. mAb 117.7, 35.9, and 53.2, although antagonists of IFN's biologic activities, did not block the binding of IFN-alpha/beta to its receptor. The 117.7 mAb, representative of this class of receptor nonblocking mAb, induced hyper-tyrosine phosphorylation of IFNAR-2 in the presence of IFN-alpha/beta but did not inhibit IFN-alpha/beta-induced Jak-Stat tyrosine phosphorylation and ISGF3 complex formation. These results show that the neutralization of type I IFN biologic actions by anti-IFNAR-2 mAb cannot be entirely explained by inhibition of Jak-Stat tyrosine phosphorylation.     

The sesquiterpene lactone parthenolide inhibits LPS- but not TNF-alpha-induced maturation of human monocyte-derived dendritic cells by inhibition of the p38 mitogen-activated protein kinase pathway

BACKGROUND: Dendritic cells (DCs) are the most potent antigen-presenting cells, and the manipulation of DC maturation provides a strategy for the treatment of allergic and inflammatory diseases. OBJECTIVE: In this study we examined the effect of the anti-inflammatory sesquiterpene lactone parthenolide (PTL) on DC maturation induced by LPS or TNF-alpha. METHODS: Human monocyte-derived DCs generated by means of culture with GM-CSF and IL-4 were pretreated with PTL and subsequently stimulated with LPS or TNF-alpha. RESULTS: PTL inhibited the upregulation of CD80, CD83, CD86, CD40, and MHC class II; the allostimulatory function; the production of TNF-alpha and IL-12; and the downregulation of FITC-labeled dextran uptake in human monocyte-derived DCs stimulated with LPS but not with TNF-alpha. The inhibitory effect of PTL on DC maturation was preceded by inhibition of the phosphorylation of p38 mitogen-activated protein kinase but not the nuclear translocation of NF-kappaB. CONCLUSION: These results might offer PTL not only as a promising compound for the treatment of LPS-induced disorders, including sepsis or septic shock, by inhibition of excessive DC maturation but also as a tool to further dissect the signaling pathways involved in DC maturation.     

Role of early- or late-phase activation of p38 mitogen-activated protein kinase induced by tumour necrosis factor-alpha or 2,4-dinitrochlorobenzene during maturation of murine dendritic cells

Dendritic cells (DCs) are maturated by a variety of stimuli. However, the precise mechanisms underlying the maturation of DCs are not fully understood. In the present study, we analysed the effects of tumour necrosis factor-alpha (TNF-alpha) and 2,4-dinitrochlorobenzene (DNCB) on phenotypic maturation and p38 mitogen activated protein kinase (MAPK) activity, using a murine DC line. TNF-alpha markedly increased the surface expression of major histocompatibility complex (MHC) and costimulatory molecules, CD86 and CD80, on DCs. DNCB more markedly enhanced the surface expression of costimulatory molecules, but showed less stimulatory capability on MHC molecules, compared with TNF-alpha. Simultaneous treatment of DCs with TNF-alpha and DNCB showed additive enhancement of costimulatory molecule expression. TNF-alpha activated p38 MAPK in DCs only at an early time-point (15 min). In contrast, DNCB activated p38 MAPK at later time-points (3-6 hr). SB203580, a specific inhibitor of p38 MAPK, partially or markedly inhibited the phenotypic changes of DCs induced by TNF-alpha or DNCB, respectively. In addition, N-acetyl-l-cysteine, a reducing supplier, completely inhibited the DNCB-induced expression of MHC and costimulatory molecules, but not those induced by TNF-alpha. These findings demonstrate that TNF-alpha and DNCB activate the p38 MAPK pathway at an early and a late phase, respectively, and thereby induce DC maturation through different signal pathways.     

Engagement of human monocyte-derived dendritic cells into interleukin (IL)-12 producers by IL-1beta + interferon (IFN)-gamma

Dendritic cells (DCs) are potent antigen-presenting cells and can induce tumour- or pathogen-specific T cell responses. For adoptive immunotherapy purposes, immature DCs can be generated from adherent monocytes using granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4, and further maturation is usually achieved by incubation with tumour necrosis factor (TNF)-alpha. However, TNF-alpha-stimulated DCs produce low levels of IL-12. In this study, we compared the effects of TNF-alpha, interferon (IFN)-gamma, IL-1beta or IFN-gamma + IL-1beta on the phenotypic and functional maturation of DCs. Our results show that IFN-gamma, but not IL-1beta, augmented the surface expression of CD80, CD83 and CD86 molecules without inducing IL-12 production from DCs. However, IL-1beta, but not IFN-gamma, induced IL-12 p40 production by DCs without enhancing phenotypic maturation. When combined, IFN-gamma + IL-1beta treatment profoundly up-regulated the expression of CD80, CD83, CD86 and major histocompatibility complex (MHC) class II antigens. Furthermore, IFN-gamma + IL-1beta-treated DCs produced larger amounts of IL-12 and induced stronger T cell proliferation and IFN-gamma secretion in primary allogeneic mixed lymphocyte reaction (MLR) than did TNF-alpha-treated DCs. Our results show that IFN-gamma + IL-1beta induced human monocyte-derived DCs to differentiate into Th1-prone mature DCs.     

Counter-regulation mechanism of IL-4 and IFN-α signal transduction through cytosolic retention of the pY-STAT6:pY-STAT2:p48 complex

IFN‐α and IL‐4 induce Th1 and Th2 responses, respectively, and often display antagonistic actions against each other. To elucidate the molecular mechanism of counter‐regulation, we have investigated the signal interception by IFN‐α and IL‐4, employing a human B‐cell line Ramos, sensitive to both cytokines. In these cells, IFN‐α effectively inhibited IL‐4‐induced Fc epsilon receptor II (CD23) expression, whereas IL‐4 suppressed IFN‐α‐mediated IRF7 expression. The counter‐regulatory action by IL‐4 and IFN‐α proceeded with a delayed kinetics requiring 4 h. Notably, IFN‐α did not affect the IL‐4‐induced tyrosine phosphorylation of STAT6, but induced a time‐dependent cytoplasmic accumulation of phosphotyrosine(pY)‐STAT6 and a corresponding decrease in nuclear pY‐STAT6. By confocal analysis and co‐immunoprecipitation assays, we demonstrated the colocalization and molecular interaction of IL‐4‐induced pY‐STAT6 with IFN‐α‐induced pY‐STAT2:p48 in the cytosol. In addition, the over‐expression of STAT2 or STAT6 induced the concomitant cytosolic accumulation of pY‐STAT6 or pY‐STAT2, leading to the suppression of IL‐4‐induced CD23 or IFN‐α‐induced IRF7 gene expression, respectively. Our data suggest that the signals ensued by IFN‐α and IL‐4 induce cytoplasmic sequestration of IL‐4‐activated STAT6 and IFN‐α‐activated STAT2:p48 in B cells through the formation of pY‐STAT6:pY‐STAT2:p48 complex, which provides a novel mechanism by which IFN‐α and IL‐4 cross‐regulate their signaling into the nucleus.     

IFN regulatory factor (IRF) 3/7-dependent and -independent gene induction by mammalian DNA that escapes degradation

DNase II in macrophages cleaves the DNA of engulfed apoptotic cells and of nuclei expelled from erythroid precursor cells. Macrophages in DNase II-deficient mice accumulate undigested DNA and constitutively produce IFN-beta as well as TNF-alpha. The IFN-beta causes severe anemia in the DNase II(-/-) embryos, which die prenatally. On the other hand, when the DNase II gene is inactivated postnatally, mice develop polyarthritis owing to the TNF-alpha produced by macrophages. Here, we showed that the IFN-beta gene activation in DNase II(-/-) mice is dependent on IFN regulatory factor (IRF) 3 and 7. Accordingly, DNase II(-/-)IRF3(-/-)IRF7(-/-) mice do not suffer from anemia, but they still produce TNF-alpha, and age-dependently develop chronic polyarthritis. A microarray analysis of the gene expression in the fetal liver revealed a set of genes that is induced in DNase II(-/-) mice in an IRF3/IRF7-dependent manner, and another set that is induced independent of these factors. These results indicate that the mammalian chromosomal DNA that accumulates in macrophages due to inefficient degradation activates genes in both IRF3/IRF7-dependent and -independent manners.     

Synergism of nitric oxide and maturation signals on human dendritic cells occurs through a cyclic GMP-dependent pathway

Nitric oxide (NO), generated by phagocytes at inflammation sites, contributes to regulate immune responses through autocrine and paracrine actions on bystander cells. Among the latter are dendritic cells (DCs). Little is known about regulation of DC function by NO, especially in the human system. We exposed human monocyte-derived DCs to the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2 diolate (DETA-NO) during their maturation process induced by treatment with tumor necrosis factor alpha or lipopolysaccharide or by CD40 activation. We report here that after exposure to DETA-NO, DCs exhibit a significantly increased ability to activate T lymphocytes stimulated by mycobacterial antigens, Staphylococcus aureus Cowen strain B, allo-antigens, or cross-linking of the CD3-T cell receptor complex. This effect persists after removal of DETA-NO, depends on the generation of cyclic guanosine 5'-monophosphate, and is a result of enhanced release by DCs of soluble factors, in particular interleukin (IL)-12. This modulation of DC function is a result of a synergism between NO and the various maturation stimuli, as neither enhanced T cell activation nor IL-12 release was observed after DC exposure to DETA-NO only. These results provide the first evidence that NO acts as a cosignaling molecule regulating human DC response to maturation stimuli.