Search

Fas-induced apoptosis is a critical process for normal immune system development and function. Although many molecular components in the Fas signaling pathway have been identified, a systematic understanding of how they work together to determine network dynamics and apoptosis itself has remained elusive. To address this, we generated a computational model for interpreting and predicting effects of pathway component properties. The model integrates current information concerning the signaling network downstream of Fas activation, through both type I and type II pathways, until activation of caspase-3. Unknown parameter values in the model were estimated using experimental data obtained from human Jurkat T cells. To elucidate critical signaling network properties, we examined the effects of altering the level of Bcl-2 on the kinetics of caspase-3 activation, using both overexpression and knockdown in the model and experimentally. Overexpression was used to distinguish among alternative hypotheses for inhibitory binding interactions of Bcl-2 with various components in the mitochondrial pathway. In comparing model simulations with experimental results, we find the best agreement when Bcl-2 blocks the release of cytochrome c by binding to both Bax and truncated Bid instead of Bax, truncated Bid, or Bid alone. Moreover, although Bcl-2 overexpression strongly reduces caspase-3 activation, Bcl-2 knockdown has a negligible effect, demonstrating a general model finding that varying the expression levels of signal molecules frequently has asymmetric effects on the outcome. Finally, we demonstrate that the relative dominance of type I vs type II pathways can be switched by varying particular signaling component levels without changing network structure.

The generation of many HLA class I peptides entails a final trimming step in the endoplasmic reticulum that, in humans, is accomplished by two 'candidate' aminopeptidases. We show here that one of these, ERAP1, was unable to remove several N-terminal amino acids that were trimmed efficiently by the second enzyme, ERAP2. This trimming of a longer peptide required the concerted action of both ERAP1 and ERAP2, both for in vitro digestion and in vivo for cellular antigen presentation. ERAP1 and ERAP2 localized together in vivo and associated physically in complexes that were most likely heterodimeric. Thus, the human endoplasmic reticulum is equipped with a pair of trimming aminopeptidases that have complementary functions in HLA class I peptide presentation.

RNA interference (RNAi) is an ancient and evolutionarily conserved process. In some eukaryotes, RNAi silences parasitic genetic elements. In plants, RNAi serves as an immune system against RNA viruses and transgenes and in worms, RNAi silences transposons. In mammals, RNAi has yet unknown functions. However, emerging roles for short RNAs and the factors that interact with them in other eukaryotes include chromatin modification, DNA deletion and DNA methylation, which may provide clues to the roles for short RNA function in mammals. For example, antigen receptor expression in lymphocytes is a highly regulated process and although much is known about chromatin modification and DNA deletion in the immune system, several molecular details of chromatin regulation remain elusive. This review compares emerging roles for short RNA function to processes required for antigen receptor expression in mammalian lymphocytes and predicts that short RNAs direct events required for successful lymphocyte-restricted gene expression.

The amenability of the nematode Caenorhabditis elegans for genetic analysis and other experimentation provides a powerful tool for studying host-pathogen interactions. Our current understanding of how C. elegans responds to pathogen challenges is in its infancy, but the discovery that the worm has inducible defence responses, which to some extent parallel those of other organisms, demonstrates the potential of this model organism for the study of innate immunity. Most progress in dissecting the C. elegans antimicrobial response has focused around signal transduction pathways and the expression of genes activated by the worm in response to microbial infections.

Nucleic acid-induced gene silencing, such as RNA interference (RNAi), induces a multitude of responses in addition to the knockdown of a gene. This is best understood in the context of the antiviral immune response, from which the processes of RNAi are thought to be derived. Viral challenge of a vertebrate host leads to an intricate series of responses that orchestrate antiviral immunity. The success of this multifaceted system in overcoming viral encounters hinges on complex pathogen-host interactions. One aspect of these interactions, the nucleic acid-based immune response, is key to the successful resolution of a viral challenge. In particular, dsRNA, a nucleic acid associated with viral replication, is involved in numerous interactions contributing to induction, activation and regulation of antiviral mechanisms. Specifically, dsRNA is responsible for stimulating important protective responses, such as the activation of dicer-related antiviral pathways, induction of type 1 IFN, and stimulation of dsRNA-activated protein kinase and oligoadenylate synthetase. Furthermore, the modulation and shaping of this overall immune response is facilitated through nucleic acid interactions with pattern recognition receptors such as toll-like receptor 3. These diverse dsRNA-induced antiviral responses have implications for biotechnologies that use dsRNA to harness one arm of the host antiviral machinery for silencing a specific target gene. The interlinked nature of these response elements means that it may be difficult to completely isolate one element from the other arms of the antiviral response program of an organism. Thus, it is beneficial to understand all aspects of the immune response to dsRNA in order to manipulate these systems and minimize unwanted non-specific effects.

Sequence-specific gene silencing by double-strand RNA has been observed in many eukaryotes. Accumulating data suggest that it is the major antiviral defense mechanism in plants and invertebrates. The discovery that this cellular mechanism is also highly conserved though somewhat impaired in mammals has stimulated debate about the evolution of antiviral systems. Here we suggest that the existence of the interferon response as an evolutionary intermediate could account for both the relative decline of RNA silencing and the development of protein-based immune systems in vertebrates. In addition, we emphasize the opportunities presented by RNA silencing and the deeper understanding of vertebrate antiviral systems that is needed.

RNA interference (RNAi) is triggered in eukaryotic organisms by double-stranded RNA (dsRNA), and it destroys any mRNA that has sequence identity with the dsRNA trigger. The RNAi pathway in Anopheles gambiae can be silenced by transfecting cells with dsRNA derived from exon sequence of the A. gambiae Argonaute2 (AgAgo2) gene. We hypothesized that RNAi may also act as an antagonist to alphavirus replication in A. gambiae because RNA viruses form dsRNA during replication. Silencing AgAgo2 expression would make A. gambiae mosquitoes more permissive to virus infection. To determine whether RNAi conditions the vector competence of A. gambiae for O'nyong-nyong virus (ONNV), we engineered a genetically modified ONNV that expresses enhanced GFP (eGFP) as a marker. After intrathoracic injection, ONNV-eGFP slowly spread to other A. gambiae tissues over a 9-day incubation period. Mosquitoes were then coinjected with virus and either control beta-galactosidase dsRNA (dsbetagal; note that "ds" is used as a prefix to indicate the dsRNA derived from a given gene throughout) or ONNV dsnsP3. Treatment with dsnsP3 inhibited virus spread significantly, as determined by eGFP expression patterns. ONNV-eGFP titers from mosquitoes coinjected with dsnsP3 were significantly lower at 3 and 6 days after injection than in mosquitoes coinjected with dsbetagal. Mosquitoes were then coinjected with ONNV-eGFP and dsAgAgo2. Mosquitoes coinjected with virus and AgAgo2 dsRNA displayed widespread eGFP expression and virus titers 16-fold higher than dsbetagal controls after 3 or 6 days after injection. These observations provide direct evidence that RNAi is an antagonist of ONNV replication in A. gambiae, and they suggest that the innate immune response conditions vector competence.

Transforming growth factor (TGF)-beta is the key molecule implicated in impaired immune function in human patients with malignant gliomas. Here we report that patients with glioblastoma, the most common and lethal type of human glioma, show decreased expression of the activating immunoreceptor NKG2D in CD8(+) T and natural killer (NK) cells. TGF-beta is responsible for the down-regulation of NKG2D expression in CD8(+) T and NK cells mediated by serum and cerebrospinal fluid of glioma patients in vitro. Moreover, TGF-beta inhibits the transcription of the NKG2D ligand MICA. Interference with the synthesis of TGF-beta1 and TGF-beta2 by small interfering RNA technology prevents the down-regulation of NKG2D on immune cells mediated by LNT-229 glioma cell supernatant and strongly enhances MICA expression in the glioma cells and promotes their recognition and lysis by CD8(+) T and NK cells. Furthermore, TGF-beta silencing results in a less migratory and invasive glioma cell phenotype in vitro. LNT-229 glioma cells deficient in TGF-beta exhibit a loss of subcutaneous and orthotopic tumorigenicity in nude mice, and NK cells isolated from these mice show an activated phenotype. RNA interference targeting TGF-beta1,2 results in a glioma cell phenotype that is more sensitive to immune cell lysis and less motile in vitro and nontumorigenic in nude mice, strongly confirming TGF-beta antagonism as a major therapeutic strategy for the future treatment of malignant gliomas.

Dendritic cells (DC) are important regulators of immune function, transporting Ags from the periphery to draining lymph nodes (dLN) where they prime Ag-specific T lymphocytes. The magnitude of the immune response generated depends upon the longevity of the Ag-bearing DC in lymphoid tissues. We hypothesized that the control of DC survival is regulated by the antiapoptotic factor bcl-x(L). Gene gun immunization of dual-expression DNA vaccines into a bcl-x(fl/fl) mouse resulted in the delivery of Ag, as well as selective deletion of the bcl-x gene in directly transfected, skin-residing DC. bcl-x-deficient DC failed to mount effective immune responses, and this corresponded to their rapid disappearance from the dLN due to apoptosis. We confirmed these results using RNA interference to specifically silence the antiapoptotic bcl-x(L) isoform in targeted skin-residing DC of C57BL/6 mice. In addition, delivery of bcl-x(L) in trans complemented the bcl-x deficiency in DC of bcl-x(fl/fl) mice, resulting in the maintenance of normal levels of Ag-bearing DC in the dLN. Taken together, our work demonstrates that the bcl-x(L) isoform is critical for survival of skin-derived, Ag-bearing DC in vivo.

RNA interference technology has been used to modulate dendritic cell (DC) function by targeting the expression of genes such as IL-12 and NF-kappa B. In this paper, we demonstrate that transfection of DC with IL-10-specific double strands of small interference RNA (siRNA) resulted in potent suppression of IL-10 gene expression without inducing DC apoptosis or blocking DC maturation. Inhibition of IL-10 by siRNA was accompanied by increased CD40 expression and IL-12 production after maturation, which endowed DC with the ability to significantly enhance allogeneic T cell proliferation. IL-10 siRNA transfection did not affect MHC class II, CD86, CD83, or CD54 expression in mature DC. To further test the ability of IL-10 siRNA-treated DC to induce a T cell response, naive CD4 T cells were stimulated by autologous DC pulsed with KLH. The results indicated that IL-10 siRNA-transfected DC enhanced Th1 responses by increasing IFN-gamma and decreasing IL-4 production. These findings suggest the potential for a novel immunotherapeutic strategy of using IL-10 siRNA-transfected antigen-presenting cells as vaccine delivery agents to boost the Th1 response against pathogens and tumors that are controlled by Th1 immunity.

The respiratory syncytial virus (RSV) is a global health problem affecting infants and the elderly and claiming more lives than AIDS in many parts of the world. Only two antibody drugs are approved for its prevention, and ribavarin, a relatively nonspecific antiviral, is used for treatment. In the mid-1990s, a number of pharmaceutical and biotech companies initiated research programs against RSV. Together, the academic and the industrial R&D covered the whole spectrum of antibodies, vaccines, synthetic small molecule antiviral and antisense technology, and at one point, accounted for at least 25 active R&D programs. However, coincident to the marketing of the monoclonal antibody palivizumab (Synagis) in 1998, a sharp decline in such projects ensued. Many companies recently cancelled RSV projects during a prioritisation of their R&D portfolios although the continuing medical need, large market size and sales projections clearly indicate that a safe and effective RSV drug or vaccine is likely to attain blockbuster status. Today RSV receives an insignificant fraction of the R&D budget compared with AIDS, for example. This article reviews the present status of the anti-RSV regimen, covers drugs in the market and in development, and attempts to link basic research to industrial drug development, animal models of RSV, clinical trials, current clinical management, and present and future market projections. It is hoped that the unmet medical need of the victims of RSV will encourage continued involvement of the pharmaceutical and biotechnology industry in developing safe and effective prevention and treatments for RSV., (Copyright 2004 John Wiley & Sons, Ltd.)

Stimulation of murine macrophages with LPS results in the coordinated activation of a set of proinflammatory cytokines and costimulatory molecules, including TNF-alpha, IL-6, IL-1, IL-8, IL-12, and CD80. Macrophage LPS-induced synthesis of IL-12 is inhibited following FcgammaR ligation; TNF-alpha secretion is unchanged. We report that microtubule-associated serine/threonine kinase-205 kDa (MAST205) is required for LPS-induced IL-12 synthesis. RNA interference-mediated suppression of MAST205 results in the inhibition of LPS-stimulated IL-12 promoter activity and IL-12 secretion, from both J774 cells and bone marrow-derived macrophages. Similarly, dominant-negative MAST205 mutants inhibit LPS-stimulated IL-12 synthesis and NF-kappaB activation, but do not affect IL-1 or TNF-alpha signaling. Finally, macrophage FcgammaR ligation regulates MAST205 by inducing the rapid ubiquitination and proteasomal degradation of the protein.

The human gut microflora is important in regulating host inflammatory responses and in maintaining immune homeostasis. The cellular and molecular bases of these actions are unknown. Here we describe a unique anti-inflammatory mechanism, activated by nonpathogenic bacteria, that selectively antagonizes transcription factor NF-kappaB. Bacteroides thetaiotaomicron targets transcriptionally active NF-kappaB subunit RelA, enhancing its nuclear export through a mechanism independent of nuclear export receptor Crm-1. Peroxisome proliferator activated receptor-gamma (PPAR-gamma), in complex with nuclear RelA, also undergoes nucleocytoplasmic redistribution in response to B. thetaiotaomicron. A decrease in PPAR-gamma abolishes both the nuclear export of RelA and the anti-inflammatory activity of B. thetaiotaomicron. This PPAR-gamma-dependent anti-inflammatory mechanism defines new cellular targets for therapeutic drug design and interventions for the treatment of chronic inflammation.

Dendritic cells (DC) reside at the center of the immunological universe, possessing the ability both to stimulate and inhibit various types of responses. Tolerogenic/regulatory DC with therapeutic properties can be generated through various means of manipulations in vitro and in vivo. Here we describe several attractive strategies for manipulation of DC using the novel technique of RNA interference (RNAi). Additionally, we overview some of our data regarding yet undescribed characteristics of RNAi in DC such as specific transfection strategies, persistence of gene silencing, and multi-gene silencing. The advantages of using RNAi for DC genetic manipulation gives rise to the promise of generating tailor-made DC that can be used effectively to treat a variety of immunologically mediated diseases.

New opportunities in mammalian functional genomics are emerging through the combination of high throughput technology and methods that allow manipulation of gene expression in living cells. Here we describe the application of an RNAi-based forward genomics approach toward understanding the biology and mechanism of TRAIL-induced apoptosis. TRAIL is a TNF superfamily member that induces selective cytotoxicity of tumor cells when bound to its cognate receptors. In addition to detecting well-characterized genes in the apoptosis pathway, we uncover several modulators including DOBI, a gene required for progression of the apoptotic signal through the intrinsic mitochondrial cell death pathway, and MIRSA, a gene that acts to limit TRAIL-induced apoptosis. Moreover, our data suggest a role for MYC and the WNT pathway in maintaining susceptibility to TRAIL. Collectively, these observations offer several insights on how TRAIL mediates the selective killing of tumor cells and demonstrate the utility of large-scale RNAi screens in mammalian cells.

RNA interference (RNAi) is a powerful tool used to manipulate gene expression or determine gene function. One technique of expressing the short double-stranded (ds) RNA intermediates required for interference in mammalian systems is the introduction of short-interfering (si) RNAs. Although RNAi strategies are reliant on a high degree of specificity, little attention has been given to the potential non-specific effects that might be induced. Here, we found that transfection of siRNAs results in interferon (IFN)-mediated activation of the Jak-Stat pathway and global upregulation of IFN-stimulated genes. This effect is mediated by the dsRNA-dependent protein kinase, PKR, which is activated by 21-base-pair (bp) siRNAs and required for upregulation of IFN-beta in response to siRNAs. In addition, we show by using cell lines deficient in specific components mediating IFN action that the RNAi mechanism itself is independent of the interferon system. Thus, siRNAs have broad and complicating effects beyond the selective silencing of target genes when introduced into cells. This is of critical importance, as siRNAs are currently being explored for their potential therapeutic use.

Specific NF-kappaB/Rel proteins regulate murine dendritic cell (DC) survival, differentiation, and activation, but little is known of their role in human cells because of limited loss-of-function analyses. RNA interference (RNAi) is a mechanism to effectively silence gene expression via sequence-specific double-stranded small interfering RNAs (siRNAs). RNAi was used to assess the role of the p50 (NF-kappaB1) protein in the maturation and activation of cultured human monocyte-derived DC (MoDC). Transfection of cultured MoDC with siRNAs reduced p50 mRNA and protein levels in a specific, dose-dependent, and time-dependent manner. Basal or maturation-induced expression of HLA-DR and costimulatory molecules were not affected, whereas transcription of the IL-12 p40 gene and the secretion of IL-12alphabeta were reduced. Such MoDC induced less IFN-gamma production by T cells in MLR. This is the first report of RNAi-induced phenotype in human primary DC with a method that caused no measurable toxicity or type-I IFN response. siRNAs appear useful for the study of signaling pathways in immune cells, revealing a pivotal requirement for p50 in MoDC for IL-12 production and induction of optimal type-1 immune responses.

An autoantibody against SS-A/Ro52 (Ro52) is most frequently found in the sera of patients with Sjögren's syndrome, systemic lupus erythematosus, and congenital heart block from anti-Ro52 Ab-positive mother. However, the physiological function of the autoantigen SS-A/Ro52 has not yet been elucidated. In this study, we describe the role of Ro52 protein in T cell activation. Overexpression of SS-A/Ro52 in Jurkat T cell resulted in enhanced IL-2 production following CD28 stimulation. Furthermore, transfection of anti-Ro52-specific small RNA duplexes partially blocked the expression of native and overexpressed Ro52 in Jurkat T cell, resulting in decreased IL-2 production via CD28 pathway in these cells. Finally, intracellular localization of Ro52 dramatically changed following CD28 stimulation. Our data reveal a novel function of Ro52 in CD28-mediated pathway, which eventually contributes to cytokine production and expression of the T cell biological programs.

Many biotechnological applications require high-level expression of transgenes in plants. One strategy to achieve this goal was the production of potato virus X (PVX) "amplicon" lines: transgenic lines that encode a replicating RNA virus vector carrying a gene of interest. The idea was that transcription of the amplicon transgene would initiate viral RNA replication and gene expression, resulting in very high levels of the gene product of interest. This approach failed, however, because every amplicon transgene, in both tobacco and Arabidopsis thaliana, was subject to post-transcriptional gene silencing (PTGS). In PTGS, the transgene is transcribed but the transcripts fail to accumulate as a result of sequence-specific targeting and destruction. Even though the amplicon locus is silenced, the level of beta-glucuronidase (GUS) activity in a PVX/GUS line is similar to that in some transgenic lines expressing GUS from a conventional (not silenced) GUS locus. This result suggested that the very high levels of expression originally envisioned for amplicons could be achieved if PTGS could be overcome and if the resulting plants did not suffer from severe viral disease. Here we report that high-level transgene expression can be achieved by pairing the amplicon approach with the use of a viral suppressor of PTGS, tobacco etch virus (TEV) helper component proteinase (HC-Pro). Leaves of mature tobacco plants co-expressing HC-Pro and a PVX/GUS amplicon accumulate GUS to approximately 3% of total protein. Moreover, high-level expression occurs without viral symptoms and, when HC-Pro is expressed from a mutant transgene, without detrimental developmental phenotypes.