Immunological research using RNA interference technology

Abstract

RNA interference (RNAi) is a potent method of gene silencing that has developed rapidly over the past few years as a result of its extensive importance in the study of genetics, molecular biology and physiology. RNAi technology has also recently yielded significant insight into the innate and adaptive immune systems by helping to elucidate numerous mechanisms that regulate the development, activation and function of cells that mediate immunity. In addition, because of its ability to suppress gene expression effectively, this technique may be used to regulate the immune response for clinical purposes. Nonetheless, before RNAi can be successfully administered into human patients as a medical treatment, it is necessary to overcome several major limitations of this technology, such as inefficient in vivo delivery, incomplete silencing of target genes, non-specific immune responses, and off-target effects. As novel developments and discoveries in molecular biology swiftly continue to unfold, it is likely that RNAi may soon translate into a potent form of in vivo gene silencing with profound applications to vaccination and immunotherapy. In the present review, we examine the current progress of immunological studies employing RNAi and discuss the prospects for the implementation of this technique in the clinical arena.

Introduction

The phenomenon of RNA interference (RNAi) was first observed in the late 1980s and has since evolved into a powerful laboratory technique for potent and specific gene silencing.^1–3 The first event in RNAi involves the cleavage of cytoplasmic double-stranded RNA into 21–28 nucleotide duplexes [termed small interfering RNA (siRNA)] by the RNase-III enzyme Dicer. A single strand of the siRNA duplex is then incorporated into the multiprotein RNA-inducing silencing complex (RISC) and serves as a template for the recognition of complementary target messenger RNA (mRNA), which is then degraded by the RISC enzyme Slicer (Argonaute-2). RNAi exhibits relatively long-term efficacy because during Slicer activity, the siRNA is protected within the RISC and is preserved to facilitate mRNA degradation in subsequent endonucleolytic reactions. Figure 1 summarizes the steps involved in RNAi. Multiple types of small artificial double-stranded RNA have been inserted into a variety of cells and organisms and have demonstrated that RNAi can be widely employed as an effective method for specific gene knockdown. Thus, this technology may have vast potential as a tool for determining gene function and as a form of therapy for pathologies characterized by aberrant cellular protein expression, such as infectious diseases, genetic disorders, neoplasia and immunological dysfunction.

Figure 1.

The RNA interference process. dsRNA enters the cellular cytoplasm (1) where it is processed by the RNase-III enzyme Dicer into 21–28-nucleotide double-stranded siRNA duplexes (2). A single strand of the siRNA duplex is incorporated into the RISC (3) and serves as a template for the recognition of complementary target mRNA (4). The RISC-associated enzyme Slicer (Argonaute-2) degrades the target mRNA (5) and is preserved for further RNAi activity.

Immunology is an emerging field that has been extensively studied using RNAi technology. The pivotal role of the immune system in the prevention and treatment of diseases suggests that RNAi may also be employed to modulate the immune response in a clinical context. The immune system can be subdivided into two intricately related components: innate and adaptive immunity.⁴ Cells of the innate immune system − which comprise neutrophils, macrophages, and natural killer cells − mount an initial immune response against pathogens through non-antigen-specific recognition and eliminatory strategies including inflammation and phagocytosis. By contrast, cells of the adaptive immune system − which comprise B and T cells − produce neutralizing/opsonizing antibodies against pathogens or directly control infected cells through a cell-mediated process, which is highly antigen-specific. The innate and adaptive immune responses co-ordinate tightly to remove pathogens from the body,⁵ a process mediated by professional antigen-presenting cells (APCs) such as B cells, macrophages and dendritic cells (DCs). These cells phagocytose and present components of pathogens as small fragments to lymphocytes, which are subsequently primed to recognize and eliminate the pathogen, thus initiating the adaptive immune response. In turn, the adaptive immune system can bolster the innate immune response by activating macrophages and facilitating phagocytosis. Thus, innate and adaptive immunity represent two distinct but interdependent systems of physiological defence against pathogens.

RNA interference-mediated gene-silencing techniques have frequently been used to investigate both the innate immune system and the adaptive immune system, and these studies have focused on the functions of critical genes, important signal transduction pathways, immune cell development and differentiation, and initiator and effector mechanisms of immune responses. RNA interference has also contributed to the understanding of immune system disorders as well as mechanisms by which pathogens are able to evade the host immune response. The information acquired from these studies suggests that RNAi may furthermore be used to regulate the immune system in a clinical setting. Specifically, the potential medical applications of this technology include enhancement of immune responses against pathogens and cancers, treatment of autoimmune disease and allergies, and minimization of graft rejection.

In this review article, we examine the current progress of studies of the innate and adaptive immune systems employing RNAi and discuss the potential clinical modulation of immune system function.

Application of RNAi technology to innate immune system studies

The innate immune system was originally considered an evolutionarily primitive preliminary response to infection. However, as this system was further explored, it became clear that the innate immune system is composed of highly sophisticated defence mechanisms and that it is crucial in initiating the adaptive immune system. As a result, research of the innate immune system has revolved around characterizing the molecular pathways regulating innate immunity, the process of antigen presentation by professional APCs, and the consequent activation of the adaptive immune system. RNA interference technology is a particularly effective technique for studying the innate immune system, and in this section we summarize the application of RNAi to examine and modify neutrophils, natural killer (NK) cells, mast cells, macrophages and the complement system (see Fig. 2).

Figure 2.

Innate immune responses and potential molecular targets for RNAi intervention. RNAi can be used to target molecules that mediate innate immune responses. RNAi-mediated silencing of Btk²⁰ and RasGRP4²¹ inhibits the release of Btk and RasGRP4 from mast cells, respectively. RNAi-mediated silencing of Gal is a potential strategy for overcoming immune rejection during xenotransplantation^22,23 and the complement protein C3 may be suppressed to prevent ischaemia-reperfusion injury.²⁵ RNAi-mediated silencing of IL-12 in DCs inhibits NK-cell activation¹⁶ whereas silencing of GATA-3 induces NK-cell activation.¹⁴ RNAi-mediated silencing of IL-8^7,8 and syntaxin 4⁶ inhibits the release of inflammatory and chemotactic signals from endothelial cells. RNAi-mediated silencing of TRADD enhances macrophage activation.²⁶ RNAi-mediated silencing of ODC allows for H. pylori clearance by macrophages²⁸ and silencing of p21 inhibits HIV-1 replication in macrophages.³⁰ Molecules associated with innate immune cells that have been silenced using the RNAi technique are indicated with an asterisk. The immunological and clinical significance of down-regulating these molecular targets is discussed in the section on Application of RNAi Technology to Innate Immune System Studies.

RNAi for the study and modulation of neutrophils

The phagocytic family of neutrophils circulates in abundant quantity in the blood and represents an important cellular player in innate immunity once a site of infection has been established. Upon stimulation by inflammatory signals, neutrophils extravasate from capillaries and cluster into infected tissue in a process termed chemotaxis. The neutrophils can then either recognize, phagocytose, and kill pathogens or release factors that enhance the immune response against these pathogens.

RNA interference technology has been used to study neutrophil recruitment to sites of infection by endothelial cells. Upon infection or activation by proinflammatory molecules such as thrombin and histamine, endothelial cells exocytose Weibel–Palade bodies (WPBs), which strengthen the adhesion between neutrophils and the endothelium. Studies with siRNA have recently shown that the exocytosis of WPBs from endothelial cells is mediated by the soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor protein syntaxin 4,⁶ and that WPBs are able to attract neutrophils largely through the presence of the cytokine interleukin-8 (IL-8) in these vesicles.^7,8 After WPBs recruit neutrophils to the blood vessels surrounding a focus of infection, rearrangement of the endothelium is required for leucocyte extravasation. The introduction of siRNA into endothelial cells has shown that signalling through the mitogen-activated protein kinase kinases 3 and 6 pathways induces alterations in the endothelial cell cytoskeleton, thus facilitating neutrophil infiltration of infected tissue.⁹ Altogether, these studies suggest that RNAi may be used to regulate neutrophil recruitment to sites of inflammation in vivo and therefore represents a potential strategy for the prevention of tissue injury by inappropriate inflammation.

Indeed, RNAi has been applied in vivo to attenuate neutrophil-mediated inflammatory responses that often result in acute lung injury in preclinical models. For example, Lomas-Neira et al. have shown that silencing of the neutrophil chemokine macrophage inflammatory protein-2 (MIP-2) with siRNA in mouse lung tissue reduces neutrophil influx into the lung following traumatic shock or haemorrhage, which frequently leads to the pathogenesis of acute lung injury.¹⁰ Thus, this study demonstrates the influential role of MIP-2 in acute lung injury and also suggests a potential therapeutic intervention to protect trauma patients from this type of secondary lung disease. In addition, it has been shown that delivery of siRNA targeting Fas to the lungs can be used to treat acute lung injury because apoptosis of lung epithelial cells often contributes to the pathogenesis of acute lung injury by causing excessive neutrophil infiltration of lung tissue.¹¹ These studies have suggested several important genes that may be silenced with RNAi to study and prevent acute lung injury, thus highlighting the potential of this technology to serve as both a powerful research tool and a direction for future clinical translation.

RNAi for the study and modulation of NK cells

Natural killer cells of the innate immune system kill pathogens and infected cells through the secretion of cytotoxic granules and probably have an important role in antiviral and antitumour immunity.^12,13 Although the current understanding of NK cells is very limited, several RNAi-based studies have helped elucidate molecular pathways controlling NK-cell activation and function.

The activation of NK cells represents one area of immunological research that has recently been studied with RNAi technology. This process is largely regulated by the inhibitory receptor NKG2A, which is expressed on the surface of most NK cells.¹⁴ The siRNA-mediated silencing of the transcription factor GATA-binding protein 3 (GATA-3) has shown that GATA-3 regulates NKG2A gene expression.¹⁴ Furthermore, NK-cell activity can be influenced by DCs, which form synapses with NK cells and stimulate them to secrete antiviral and macrophage-activating cytokines such as interferon-γ (IFN-γ).¹⁵ Silencing of IL-12 in DCs using siRNA has demonstrated that this cytokine is crucial for the activation of NK cells by DCs.¹⁶ Interestingly, IL-12 also behaves as an activation signal for the adaptive immune system, and GATA-3 is an important factor in T-cell development (see section on RNAi for the study and modulation of lymphocytes).¹⁷ Thus, these studies suggest a strong link between the innate and adaptive immune systems.

RNA interference has also been employed to explore the function of NK cells. For example, the use of short hairpin RNA targeting perforin revealed that NK cells lyse the fungus Cryptococcus neoformans by secreting perforins to make the fungal cell membranes permeable.¹⁸ In addition, RNAi has been applied to investigate the survival mechanisms of NK cells. For instance, it is believed that NK cells are able to kill tumour cells through the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) death signal.¹² TRAIL has been shown to induce apoptosis in several cancer cell lines and may hold potential as an antitumour therapy. However, because NK cells also express the TRAIL receptor, the NK cells may themselves be susceptible to the TRAIL-induced death signal. In a recent study, RNAi was used to down-regulate expression of the cellular Fas-associated death domain protein-like IL-1-converting enzyme-inhibitory protein (c-FLIP) in NK cells, resulting in their apoptosis by the TRAIL pathway.¹⁹ This shows that c-FLIP confers resistance to TRAIL-mediated apoptosis on NK cells and suggests that c-FLIP may be used to protect NK cells in cancer treatments employing TRAIL. With a better understanding of the activity and function of NK cells, it may further be possible to employ siRNA for the enhancement of their function or survival for therapeutic purposes.

RNAi for the study and modulation of mast cells

Mast cells are responsible for the mediation of allergic responses, which frequently results in extremely unpleasant experiences and even fatality. Upon binding immunoglobulin E (IgE) cross-linked with allergen, these cells secrete potent factors that stimulate smooth muscle contraction as well as blood vessel dilatation, which lead to numerous allergic symptoms. These symptoms are mainly caused by histamine and prostaglandin, and therefore a reduction in the synthesis or release of these molecules in mast cells mediated by RNAi should diminish the allergic responses. For example, Bruton's tyrosine kinase (Btk) is a central downstream signal transduction molecule of the B-cell receptor in mast cells, and siRNA targeting Btk has been shown to disrupt the allergen–IgE signal and decrease the release of histamine from these cells by 25%.²⁰ Furthermore, the tyrosine protein kinase oncogene c-kit signalling pathway regulates the production of the enzyme cyclo-oxygenase, which is involved in the synthesis of prostaglandin D₂ in mast cells. This reaction cascade depends on the Ras guanine nucleotide-releasing protein 4 (RasGRP4), and siRNA knockdown experiments have demonstrated that silencing of RasGRP4 inhibits the c-kit pathway and reduces prostaglandin D₂ synthesis.²¹ These studies suggest that RNAi can effectively suppress the release of proinflammatory substances by mast cells. As the molecular biology of the immune response to allergens becomes more precisely understood, it may be possible to use RNAi technology for the clinical control of allergies by knocking down other proteins that are essential for the generation of histamine and prostaglandin.

RNAi for the study and modulation of the complement system

The complement system comprises a family of zymogens − cleavage-activated proteases – and represents an important component of innate immunity. Complement proteins bind to immunoglobulin or distinct molecular patterns on the pathogen surface and can promote inflammation, opsonize the pathogen, or form a complex to lyse the pathogen. The role of the complement system in hyperacute xenograft rejection has recently been a subject of much investigation because of the shortage of human organs available for transplantation. This issue may be resolved with improvements in organ xenotransplantation from pigs to humans and the prevention of xenorejection. The disaccharide antigen Gal α1,3 Gal (Gal), expressed by porcine cells, activates the complement system in humans and is considered to be a major obstacle in porcine-to-human xenotransplantation.²² Recently, two groups have developed xenograft strategies that use siRNA to down-regulate the expression of α1,3 galactosyltransferase − an enzyme involved in the synthesis of Gal − in a porcine vascular endothelial cell line. The treated cells expressed reduced amounts of Gal, induced a substantially weaker human complement reaction, and were consequently immune to killing by human serum.^22,23 Thus, RNAi may provide a method for overcoming human xenorejection of porcine organs and have an impact on xenotransplantation.

In addition to xenorejection, the complement system may also cause renal ischaemia–reperfusion injury.²⁴ DNA vector-based delivery of siRNA targeting C3, the central complement protein, has recently been used to suppress renal complement reaction in a murine ischaemia–reperfusion injury model. Administration of C3-specific siRNA correlated with decreased levels of C3 expression in the kidneys, reduced degrees of renal injury, and prolonged survival of mice.²⁵ This study implies that RNAi may be safely delivered to the kidneys in vivo and may represent a promising technique for the treatment of ischaemia–reperfusion injury.

RNAi for the study and modulation of macrophages

It is now well established that macrophages are the primary mediators of the innate immune response. These phagocytes exhibit potent antimicrobial activity, contribute to the inflammatory process, and can differentiate into professional APCs and initiate adaptive immunity. Numerous RNAi studies have been conducted to characterize the function of macrophages as well as to modify their behaviour for clinical purposes.

Small interfering RNA has been extensively used to investigate the signal transduction pathways regulating macrophage activation and macrophage-induced inflammation. IFN-γ secretion by NK or T cells is required for macrophage activation, and this process is highly regulated to prevent excessive macrophage damage to self-tissue. Wesemann et al. have discovered that the activation signal of IFN-γ is negatively controlled in the macrophage nucleus by the tumour necrosis factor receptor 1-associated death domain protein (TRADD).²⁶ This molecule forms a complex with signal transducer and activator of transcription 1-α − the major downstream messenger of IFN-γ − which in effect inhibits IFN-γ signalling. The siRNA-mediated silencing of TRADD resulted in IFN-γ-induced macrophage activation²⁶ and may provide an approach for strengthening the activation of macrophages.

Another important subject of research is the mechanism of phagocytosis, and RNAi has generated insight into pathogen ingestion by macrophages. For example, it is believed that membrane fusion between the endoplasmic reticulum (ER) of macrophages and phagosomes has an essential role in phagocytosis. Recently it has been shown that silencing of the ER-localized SNARE protein syntaxin 18 using siRNA reduces the efficiency and rate of phagocytosis.²⁷ Thus, this study indicates that syntaxin 18 is important in macrophage phagocytosis of pathogens, probably by mediating the fusion between the ER and phagosomes.

The siRNA has also been used to study the evasion by pathogens of macrophage-mediated immunity. Although the innate immune system effectively clears the vast majority of pathogens before a focus of infection can be established, certain pathogens occasionally escape macrophage detection and killing by inhibiting the innate immune response, and this phenomenon has become a strong focus of immunological research. For instance, it has been shown that upon encounter with the bacterium Helicobacter pylori, which is often associated with peptic ulcer disease and gastric cancer, macrophages are stimulated to produce spermine, a polyamine that inhibits the translation of inducible nitric oxide synthase (iNOS) and the production of antimicrobial nitric oxide (NO).²⁸ The siRNA-mediated silencing of ornithine decarboxylase (ODC), an enzyme that is critical for spermine synthesis, in macrophages resulted in elevated amounts of iNOS and NO, thereby enhancing H. pylori clearance.²⁸ This study identifies a pathway of immune evasion by H. pylori and suggests that the macrophage response to this pathogen may be restored through the targeting of ODC. In addition, when human immunodeficiency virus-1 (HIV-1) infects macrophages, it substantially up-regulates the expression of cyclin-dependent kinase inhibitor A1 (p21) by these cells, which stimulates the transcription coactivator p300 and facilitates viral replication.²⁹ Reduction of p21 expression in vitro using siRNA has been shown to inhibit HIV-1 replication and viral infection.³⁰ Thus, the use of RNAi targeting p21 or other proteins of similar function may represent a plausible strategy for inhibiting HIV-1 replication in the clinical arena. Continual employment of this technology to analyse pathogen-induced macrophage dysregulation is necessary because these pathogens frequently cause chronic diseases that are difficult to cure and can lead to mortality.

Several non-infectious diseases that are thought to disrupt the function of macrophages have been studied, in many cases with RNAi technology. One of these serious illnesses is the cardiovascular inflammatory disease atherosclerosis. It is believed that plasma high-density lipoprotein (HDL) levels are closely related to the pathogenesis of atherosclerosis. It has been shown that HDL promotes the removal of cholesterol from foam macrophages lining the arterial walls and lowers the risk of atherosclerotic disease.³¹ Targeting by siRNA of ATP-binding cassette transporters G1 and G4 decreased the expression of these proteins in liver-derived macrophages, which correlated with reduced cholesterol efflux from macrophages to HDL.³¹ Thus, from this siRNA study it was demonstrated that ATP-binding cassettes (particularly G1 and G4) may be vital in the removal of cholesterol from foam macrophages as well as in the prevention of blood vessel inflammation and atherosclerosis.

Other macrophage-related non-infectious diseases that have been investigated with RNAi are bone metastasis and osteoporosis. Osteoclasts, highly differentiated bone-reabsorbing macrophages that are essential for bone remodelling, have received much attention because of their pivotal role in these diseases. It is well established that the development of osteoclasts is regulated by a variety of extracellular signals, such as the cytokine receptor activator of nuclear factor-κB ligand (RANKL).³² It has been shown that RANKL activates the receptor-like molecule dendritic cell-specific transmembrane protein (DC-STAMP) on osteoclasts, and Kukita et al. have demonstrated that down-regulation of DC-STAMP with siRNA in precursor cells suppressed their differentiation into osteoclasts.³³ Therefore, this study revealed that DC-STAMP is essential in RANKL-induced osteoclastogenesis, and RNAi silencing of this gene may provide a potential method for blocking osteoclast differentiation and clinically modulating the bone reabsorption process. Although the majority of these studies with siRNA were performed in vitro, many of these molecular targets related to the innate immune system are very promising for therapeutic RNAi interventions and warrant further clinical translation.

Application of RNAi technology to adaptive immune system studies

For decades, immunological research has centred on the characterization of the adaptive immune system, and recently RNAi technology has led to numerous advances in the understanding of this system. In this section, we discuss the current progress of experiments using RNAi to study the two principle cellular components of adaptive immunity − DCs and lymphocytes (see Fig. 3). Clinical applications of RNAi that build on the knowledge established by this technique, such as the enhancement of the adaptive immune response or the control of immunological diseases, appear promising and may be expected in the future.

Figure 3.

Adaptive immune responses and potential molecular targets for RNAi intervention. RNAi can be used to target molecules that mediate adaptive immune responses. RNAi-mediated silencing of SOCS1 in DCs enhances T-ell activation by DCs.^1–43 RNAi-mediated silencing of IL-10 in DCs promotes the differentiation of CD4⁺ T cells into Th1 cells⁴⁴ whereas silencing of IL-12 promotes differentiation into Th2 cells.⁴⁵ Similarly, RNAi-mediated silencing of GATA-3 in CD4⁺ T cells promotes their differentiation into Th1 cells,⁴⁷ whereas silencing of T-bet promotes differentiation into Th2 cells.⁴⁸ RNAi-mediated silencing of TRPM4 in T cells increases the proliferative rate of these cells. RNAi-mediated silencing of Ape1⁵⁰ and Fas⁵³ in target cells renders them more susceptible to killing by CTLs. Molecules associated with adaptive immune system cells that have been silenced using the RNAi technique to enhance T-cell activation and function are indicated with an asterisk. The immunological and clinical significance of down-regulating these molecular targets is discussed in the section on Application of RNAi Technology to Adaptive Immune System Studies.

RNAi for the study and modulation of DCs

Dendritic cells provide an essential link between the innate and adaptive immune responses, as they are the most potent activators of the adaptive immune system. Immature DCs are present in normal tissue and function as phagocytes. Upon antigen encounter and subsequent activation, these cells differentiate into highly effective, professional APCs. Mature DCs migrate to the draining lymph nodes, where they prime naive T cells, thus triggering the adaptive immune response. A significant number of studies have targeted proteins expressed by DCs using RNAi and have revealed many critical biological properties of these cells.

RNA interference technology has been used to understand the process of antigen presentation by DCs. The proteasome has a critical role in DC processing of antigens for presentation via the major histocompatibility complex (MHC) class I pathway. These proteolytic complexes cleave the cytosolic proteins derived from intracellular pathogens, such as viruses, facilitating antigenic peptide binding to MHC. Studies with RNAi have indicated that, in addition to the proteasome, the endoproteolytic tripeptidyl peptidase II may be indispensable for the generation of particular epitopes, including the HIV epitope HIV-Nef(73–82).³⁴ Thus, this study illustrates that the nature of antigen processing and presentation is complex and not entirely regulated by the proteasome.

Studies with RNAi interventions have shown that molecules important for interactions between pathogens and DCs can be targeted to prevent pathogens from evading immune-system-mediated protection. In general, antigen presentation by DCs is remarkably successful in activating the adaptive immune system. However, several pathogens can escape detection and processing by DCs and may even proliferate by exploiting the migratory activity of these cells. Most notably, studies using siRNA have revealed that HIV can adhere to the DC surface lectin DC-SIGN and upon DC activation can be transported to the lymph nodes, where the virus can infect large amounts of susceptible CD4⁺ T cells.³⁵ Silencing of DC-SIGN has been shown to inhibit HIV transmission in in vitro assays.³⁶ This discovery may lead to the development of novel strategies for the control or prevention of HIV infection that involve the down-regulation of adhesion molecules that facilitate HIV-DC binding.

RNA interference has been employed to study the differentiation of DCs. For example, siRNA-mediated knockdown of the transcription factor Spi-B in CD34⁺ plasmacytoid DC precursors inhibited their development into plasmacytoid DCs both in vitro and in injected mice.³⁷ These results indicate that Spi-B is critical in the differentiation of CD34⁺ precursors into plasmacytoid DCs.

In addition to studying the biochemical and physiological properties of DCs, RNAi has been used to modulate their function to improve vaccine potency. For example, one important application of this technology is the prolongation of DC life through the silencing of proapoptotic genes such as Bax and Bak. Extended DC survival has been correlated with enhanced antigen-presenting ability, and vaccine potency has been significantly improved through the incorporation of strategies to prolong DC life. Kim et al. have used the gene gun to specifically deliver DNA vaccines into DCs in the skin.³⁸ Using this system, they have delivered a DNA vaccine encoding the human papillomavirus (HPV) type 16 oncoprotein E7 in conjunction with siRNA targeting Bax/Bak to generate E7-specific immune responses as well as antitumour effects against E7-expressing tumours in a preclinical model.³⁸ These results indicate that intradermal administration via gene gun may represent an efficient method for the delivery of siRNA in vivo.

Small interfering RNA has also been used to modify the properties of DCs to enhance adaptive immune responses when administered in vivo. For example, the antiapoptotic strategy mentioned above has been used to prepare DC-based vaccines. Peng et al. demonstrated that HPV E7-loaded DCs transfected with siRNA targeting Bax/Bak could become resistant to killing by T cells, leading to enhanced E7-specific CD8⁺ T-cell-mediated immune responses in E7-expressing tumour-challenged mice.³⁹ Similarly, Kang et al. have used a DC-based vaccine to enhance E7 antigen presentation to CD8⁺ and CD4⁺ T cells and to prolong DC life. DCs transfected with HPV E7 linked to the sorting signal of lysosome-associated membrane protein 1 were able to target E7 to the lysosomal/endosomal compartment and dramatically enhanced the activation of E7-specific CD4⁺ and CD8⁺ T cells in vaccinated mice.⁴⁰ Furthermore, the DC-based vaccination resulted in a significantly more potent antitumour effect against E7-expressing tumours.⁴⁰

Another strategy to modify the properties of DCs to enhance vaccine potency is to target immunosuppressive proteins expressed by DCs using RNAi. For example, it is now clear that the suppressor of cytokine signalling 1 (SOCS1) negatively regulates DC production of and signalling through IL-12.⁴¹ The siRNA-mediated silencing of SOCS1 in DCs has been shown to attenuate the immunosuppressive signals controlling these cells and allow them to break self-tolerance.⁴¹ Song et al. have silenced SOCS1 in DCs using siRNA and shown that these DCs are able to generate significantly enhanced numbers of HIV-1 envelope-specific CD8⁺ T cells and CD4⁺ T cells in mice.⁴² In the same study, delivery of siRNA targeting SOCS1 in conjunction with HIV DNA vaccines was shown to enhance vaccine potency in mice.⁴² Furthermore, treatment of mice with DCs knocked down for SOCS1 by siRNA has been reported to enhance tumour-specific immunity in tumour-challenged mice.⁴³ Therefore, inhibition of SOCS1 (and other suppressors of cytokine signalling) may be exploited for enhancement of tumour- and virus-specific adaptive immune responses.

The RNAi technology may also be used to modify DCs to select for a particular type of immune response to infection. Dendritic cells can induce two types of CD4⁺ T helper (Th) cell responses − the Th1 response stimulates macrophages to produce antimicrobial peptides, inflammatory cytokines and cytotoxic chemicals, and augments macrophage killing of phagocytosed pathogens, while the Th2 response promotes humoral immunity by stimulating B cells to produce neutralizing antibodies. Normally, one of the two Th responses becomes predominant, and suppressing or boosting either type of response will oppositely affect the other response. Although both Th1 and Th2 cells are vital components of adaptive immunity, a particular Th response may occasionally be inappropriate and may result in a variety of immune disorders. The Th1 response is associated with cell-mediated autoimmune disease, and a dominant Th2 response is often implicated in allergic reactions or in failure to clear intracellular pathogens.⁴⁴ In the event of these immunological dysfunctions, it may be desirable to redirect the Th response. Small interfering RNA has been employed for the knockdown of IL-10 expression in DCs, resulting in a magnified Th1 response and a suppressed Th2 response.⁴⁴ The RNAi-induced silencing of IL-12 in DCs elicits the opposite effect.⁴⁵ Therefore, clinical treatment of DCs with siRNA targeting IL-10 or IL-12 may represent an effective method for the minimization of allergic reaction or autoimmune responses, respectively.

RNAi for the study and modulation of lymphocytes

Lymphocytes (comprising T and B cells) are the primary cellular mediators of the adaptive immune response. These cells mount antigen-specific responses against pathogens. B cells can release neutralizing or opsonizing antibodies, and T cells can directly perform cell-mediated killing of micro-organisms. RNAi technology has been extensively used to investigate the developmental, activating and effector mechanisms of lymphocytes. These studies have greatly contributed to our knowledge of adaptive immune responses and have suggested several strategies for controlling or treating diseases related to lymphocyte dysfunction.

RNA interference technology has been applied to study and modify T-cell and B-cell differentiation. Lymphocytes develop from stem cells in the bone marrow, and unique genetic recombination in precursor cells provides T and B cells with remarkable plasticity. Towards the end of the maturation process, T cells are selected for either the CD4 or CD8 surface marker, which helps to determine the functional role of the cell. CD8⁺ CD4^– T cells become cytotoxic T lymphocytes (CTLs), and CD8^– CD4⁺ T cells become Th cells. The transcription factor GATA-3 promotes CD4⁺ T-cell differentiation, and down-regulation of GATA-3 using siRNA has been shown to inhibit this process.⁴⁶ Naive CD4⁺ T cells become armed effector Th cells upon priming by antigen-bearing APCs through the MHC class II pathway. As previously mentioned, Th cells can be classified as Th1 or Th2 depending on the type of leucocyte they activate. In addition to the modulation of cytokines secreted by DCs (e.g. IL-10, IL-12), direct modification of CD4⁺ T cells with siRNA can influence their development into Th1 or Th2 cells. It has been shown that siRNA-based silencing of GATA-3 in CD4⁺ T cells suppresses the Th2 response,⁴⁷ indicating that continual cellular expression of GATA-3 is necessary for differentiation into Th2 cells. By contrast, siRNA-based silencing of the transcription factor T-bet impaired the Th1 response.⁴⁸ Thus, T-bet may represent an ideal target for RNAi therapy in the treatment of autoimmune disorders, and GATA-3 may be silenced to boost the immune response to macrophage infection.

The signal transduction pathways that mediate T-cell clonal expansion upon activation by APCs have also been studied with RNAi. For example, one particularly important pathway is triggered by fluctuations in the T-cell cytosolic concentration of calcium. Accumulation of calcium in the cytoplasm results in the activation of protein kinase C, which in turn initiates a signalling cascade that results in the release of cytokine IL-2, a strong stimulator of T-cell proliferation.⁴⁹ The calcium-activated transient receptor potential channel protein TRPM4 was recently reported to be essential in the control of T-cell calcium concentration. Experiments using siRNA have shown that knockdown of TRPM4 elevates cytosolic calcium concentration and IL-2 production in vitro.⁴⁹ Thus, TRPM4 may be an endogenous regulator of T-cell activation.

The RNAi technology has also helped to identify several key molecules involved in lymphocyte effector mechanisms. For example, apurinic endonuclease-1 (Ape1) is important in the DNA base excision repair process, and Fan et al. have used siRNA to show that knockdown of Ape1 in cells sensitizes them to DNA damage and apoptosis.⁵⁰ Thus, Ape1 confers resistance to apoptosis, and this study has led to the theory that the CTL-secreted protease granzyme A may mediate the killing of target cells by cleaving their intracellular Ape1. In addition, lymphocytes possess natural mechanisms of defence against pathogens that are essential in preventing infection by certain viruses, including HIV. For example, RNAi-mediated silencing of the antiviral deoxycytidine deaminase human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) showed that APOBEC3G is a restriction factor that blocks HIV-1 reverse transcription and replication after entry into unstimulated CD4⁺ T cells.⁵¹ In another example, silencing of the copper toxicosis gene Murr1 in inactive CD4⁺ T cells showed that Murr1 is a genetic restriction factor that provides protection against HIV infection by inhibiting viral replication through the NF-κB signalling pathway.⁵² The results from these studies provide excellent examples of how RNAi can be used to study the functions of lymphocytes, facilitating the development of innovative strategies for the enhancement of CTL killing or for antiviral therapy.

Indeed, RNAi techniques have great potential for improving current treatments for disease. For instance, studies have shown that cancer cell lines expressing Fas ligand (a member of the tumour necrosis factor receptor superfamily) are capable of evading the adaptive immune response.⁵³ The Fas ligand binds to Fas on the surface of CTLs and stimulates T-cell death through the extrinsic apoptosis pathway. Down-regulation of Fas in CTLs with siRNA abolishes the apoptotic effect of Fas-expressing cancer cells.⁵³ Thus, clinical suppression of Fas in CTLs may represent a remedy for tumour evasion of host immunity and may enhance the efficacy of T-cell-mediated immunotherapy. In addition, manipulation of CD40 ligand (CD40L) expression on Th cells may be a promising approach for controlling inflammatory responses. The CD40 surface receptor is present on macrophages, B cells, and endothelial cells, and upon binding its ligand, signals the up-regulation of cellular adhesion molecules. Thus, CD40L–CD40 interaction is crucial for leucocyte adhesion to endothelium and for subsequent leucocyte extravasation. In clinical trials, anti-CD40L antibodies have been employed to inhibit T-cell binding to endothelial cells and suppress inflammation; however, these antibodies have been shown to also cause thrombotic events.⁵⁴ Small interfering RNA targeting CD40L has effectively blocked signalling through CD40 in endothelial cells and may prove to be a safer and more successful method for modulating the inflammatory response.⁵⁴ Also, siRNA-based silencing of the HIV receptors in T cells could help limit HIV transmission among these cells. Experiments that used siRNA to decrease expression of the receptors CXCR4 and CCR5 generated significant antiviral protection in vitro.^55,56 In addition, CCR5 signals through the G protein Gα_i to facilitate viral replication, and it has recently been shown that siRNA-mediated silencing of Gα_i expression in CCR5-transduced peripheral blood mononuclear cells markedly reduces their susceptibility to HIV-1 virion infection.⁵⁷ Therefore, CXCR4, CCR5 and Gα_i may represent promising molecular targets for HIV therapy using RNAi. Many more clinical applications for RNAi technology in lymphocytes and in the adaptive immune system should emerge in the near future.

Challenges to the application of RNAi technology in clinical immunology

Although the RNAi technique is gradually maturing as a powerful molecular biological tool, several obstacles remain to be overcome before this technology can be safely and efficiently used in medical practice. In this section, we analyse the three major limitations to the implementation of RNAi in vivo − siRNA delivery, non-specific gene suppression and off-target effects.

Delivery of siRNA to target immune cells in vivo

The efficacy of systemic delivery of siRNA into mammals is limited by the transient nature of gene silencing as well as the inherent instability of the RNA molecule. It has been shown that extracellular siRNA degradation usually occurs within 2 days after administration, and the potency of gene knockdown is unpredictable because of the variation in the efficiency of siRNA uptake by different cells.⁵⁸

To overcome these obstacles, several groups have developed chemically modified siRNAs that possess increased stability or are protected from degradation. The siRNA molecules can be encased in lipid complexes or cationic liposomes⁵⁹ altered to resist serum RNase activity⁶⁰ linked with fusogenic peptides⁶¹ or conjugated to antibodies that bind to membrane-associated receptors for cell-specific delivery.⁶² Other strategies to selectively silence genes in particular cell types include use of the highly cell-specific RNA polymerase II promoter⁶³ (as opposed to the ubiquitous RNA polymerase III) or stabilized nanoparticles that are preferentially taken up into target cells.⁶⁴ It is quite likely that significant progress in the delivery of siRNA into specific cells will occur in the near future.

The issue of in vivo siRNA administration persists as a significant challenge in transgenic experiments, and particularly in those involving adaptive immunity, as mature lymphocytes are difficult targets for efficient RNA interference.⁶⁵ Numerous methods have been developed for in vitro introduction of siRNA, such as viral vector-based delivery⁵³ and electroporation.⁶⁶ However, extensive developments are still needed to deliver siRNA effectively, specifically and safely into animals and humans.

In vivo siRNA delivery methods include intravascular injection, inhalation, gene gun administration and in vivo electroporation. Intravascular injection is commonly used for drug delivery but needs to be modified (for example, by using high-pressure injection) to efficiently carry nucleic acids to specific sites.⁶⁷ When targeting pulmonary tissue with siRNA, intratracheal instillation is a feasible delivery method and has been shown to be effective in a mouse model.¹⁰ In addition, co-immunization of mice with DNA vaccines and siRNA has been successfully tested using the gene gun method^38,39 and in vivo electroporation has achieved potent gene silencing in muscle tissue.⁶⁸ Nonetheless, specific in vivo targeting of siRNA remains particularly difficult. Ex vivo cellular engineering followed by adoptive cell transfer represents a plausible delivery strategy that may be a promising alternative.³⁷ Also, siRNA can be incorporated into viral vectors to achieve more potent gene silencing in target cells.⁶⁹ These viruses have the ability to integrate into the host genome and induce long-term gene knockdown. Numerous studies have explored RNAi delivery with lentiviral,^70–72 adenoviral,^71,73,74 retroviral^72,75,76 and adeno-associated viral vectors.⁷⁷ Nevertheless, these vectors generally have low cell specificity and raise concerns about toxicity associated with the viruses themselves. Currently a limited number of successful in vivo RNAi studies have been reported, and in vivo siRNA delivery is undeniably the greatest obstacle to RNAi application in humans.

Non-specific effects

Mammalian immune cells are capable of secreting interferons upon entry of double-stranded RNA into the cytoplasm. The interferon response provides a natural defence mechanism against viruses and results in non-specific RNA degradation and translational inhibition. Interferons also induce significant alterations in the physiology of the cell and in the behaviour of the immune system. Thus, it is of serious concern that siRNAs are also capable of activating an interferon response.⁷⁸ A related non-specific response is the potential initiation of the Toll-like receptor-3 or -7 signalling pathways by siRNA. This may result in sequence-independent gene suppression as well as immune activation, particularly in DCs.^79,80 It is especially important to prevent these undesirable effects when using RNAi technology in both immunological research and medical practice. The length³ and sequence⁸¹ of the siRNA strand are notable factors that probably influence the immune response to RNA.

The use of viral vectors to administer siRNA into tissues raises additional concerns of non-specific effects. For example, these vectors may alter the physiology of target cells (and immune cells in particular). Lentiviruses are popular gene transduction tools and are commonly used for the delivery of siRNA. However, studies have shown that lentiviruses can alter the immunological function of transfected DCs.⁷⁶

In summary, minimization of non-specific immune responses is necessary for accurate and consistent analysis of results from RNAi-based experiments. Strategies that enable sensitive detection and effective prevention of these responses are very important for the widespread future application of interference RNA technology in the clinical arena.

Off-target effects

Although RNAi is a highly specific process, it may still alter the expression of non-target genes as a result of sequence similarity.⁸² This unintended outcome is frequently referred to as the off-target effect. It is believed that occasionally non-target genes with up to three or four base mismatches can still be silenced, and GU wobble base pairing is likely to be tolerated by cellular RNAi-associated enzymes.⁸³ Nevertheless, the off-target effect is usually not as appreciable as non-specific responses, unless the siRNA alters the expression of a critical non-target gene and immediate results are produced.⁸⁴

The limitations of RNAi technology mean that drugs or vaccines that employ this technique should be very carefully designed to ensure safety of administration. The synthesis of siRNA using modified nucleic acids has been shown to be able to enhance specificity and reduce the probability of off-target effects.⁸⁵ In addition, computer algorithms have also been developed for the prediction of potential off-target effects and for the optimization of siRNA sequence design.^86,87 Despite these advances however, off-target effects should still be considered as a possible confounding factor when interpreting data collected from an RNAi-based experiment.

Conclusion

To date, RNAi technology has generated much insight into the development, activation and function of cells comprising the innate and adaptive immune systems. In addition, RNAi has contributed significantly to the understanding of immune responses to pathogenesis. The abundant amount of immunobiological information revealed by this technique suggests its usefulness in modulating the immune system to bolster immune responses, control inflammation, or treat autoimmune disorders. Current efforts to regulate the innate and adaptive immune systems with siRNA have primarily employed cell culture systems. In the future, it will be important to pursue further RNAi studies in animal models, to determine whether the in vitro efficacy of this technology corresponds to potent and specific gene silencing in vivo. The issues of delivery as well as non-specific and off-target effects represent considerable challenges to the testing of RNAi in animals. Nonetheless, as both the phenomenon of RNAi and the science of immunology become more thoroughly understood, it is likely that novel strategies will be developed to overcome these obstacles. Initial results derived from in vivo siRNA experimentation have in general achieved remarkable success, generating optimism that in the future this technique may be used as a highly effective form of immunotherapy.

Abbreviations

Ape1

apurinic endonuclease-1

APC

antigen-presenting cell

APOBEC3G

apolipoprotein B messenger RNA-editing enzyme-catalytic polypeptide-like 3G

Btk

Bruton's tyrosine kinase

c-FLIP

cellular Fas-associated death domain protein-like interleukin-1-converting enzyme-inhibitory protein

CD40L

CD40 ligand

CTL

cytotoxic T lymphocyte

DC

dendritic cell

DC-STAMP

dendritic cell-specific transmembrane protein

ER

endoplasmic reticulum

Gal

Gal α1,3 Gal

GATA-3

GATA-binding protein 3

HDL

high-density lipoprotein

HIV

human immunodeficiency virus

HPV

human papillomavirus

IFN-γ

interferon-γ

IgE

immunoglobulin E

IL

interleukin

iNOS

inducible nitric oxide synthase

MHC

major histocompatibility complex

MIP-2

macrophage inflammatory protein-2

NK cell

natural killer cell

NO

nitric oxide

ODC

ornithine decarboxylase

p21

cyclin-dependent kinase inhibitor A1

RANKL

receptor activator of nuclear factor-κB ligand

RasGRP4

Ras guanine nucleotide-releasing protein 4

RISC

RNA-induced silencing complex

RNAi

RNA interference

siRNA

small interfering RNA

SOCS1

suppressor of cytokine signalling 1

Th cell

T helper cell

TRADD

tumour necrosis factor receptor 1-associated death domain protein

TRAIL

tumour necrosis factor-related apoptosis-inducing ligand

WPBs

Weibel–Palade bodies