Master sensors of pathogenic RNA – RIG-I like receptors

Initiating the immune response to invading pathogens, the innate immune system is constituted of immune receptors (pattern recognition receptors, PRR) that sense microbe-associated molecular patterns (MAMPs). Detection of pathogens triggers intracellular defense mechanisms, such as the secretion of cytokines or chemokines to alarm neighboring cells and attract or activate immune cells. The innate immune response to viruses is mostly based on PRRs that detect the unusual structure, modification or location of viral nucleic acids. Most of the highly pathogenic and emerging viruses are RNA genome-based viruses, which can give rise to zoonotic and epidemic diseases or cause viral hemorrhagic fever. As viral RNA is located in the same compartment as host RNA, PRRs in the cytosol have to discriminate between viral and endogenous RNA by virtue of their structure or modification. This challenging task is taken on by the homologous cytosolic DExD/H-box family helicases RIG-I and MDA5, which control the innate immune response to most RNA viruses. This review focuses on the molecular basis for RIG-I like receptor (RLR) activation by synthetic and natural ligands and will discuss controversial ligand definitions.     

Toll-like receptors 7, 8, and 9: linking innate immunity to autoimmunity

Summary: Toll-like receptors (TLRs) detect infections by highly conserved components of pathogens that are either not present in our own cells or are normally sequestered in cellular compartments that are inaccessible to the TLRs. Most TLRs are expressed on the cell surface, where they have been shown to detect pathogen-expressed molecules such as lipopolysaccharides and lipopeptides. A subset of TLRs, including TLR3, TLR7, TLR8, and TLR9, are expressed intracellularly within one or more endosomal compartments and detect nucleic acids. Because pathogen and host nucleic acids have very similar structures, these endosomal TLRs may face an extra challenge to induce anti-pathogen immune responses while avoiding the induction of autoimmune diseases. With the rapid growth in understanding of the biology of the TLRs has come an increasing awareness of their effects on autoimmunity, several aspects of which are the focus of this review. First, recent studies have revealed an inappropriate activation of TLR7, TLR8, and TLR9 in systemic lupus erythematosus and several other autoimmune diseases. Secondly, the potential for therapeutic development of TLR antagonists is considered. Finally, with the rapid progress in the development of therapeutic agonists for the TLRs, there is accompanying attention to the theoretical possibility that such therapy may induce autoimmunity or autoimmune diseases.     

Recognition of nucleic acids by pattern-recognition receptors and its relevance in autoimmunity

Host cells trigger signals for innate immune responses upon recognition of conserved structures in microbial pathogens. Nucleic acids, which are critical components for inheriting genetic information in all species including pathogens, are key structures sensed by the innate immune system. The corresponding receptors for foreign nucleic acids include members of Toll-like receptors, RIG-I-like receptors, and intracellular DNA sensors. While nucleic acid recognition by these receptors is required for host defense against the pathogen, there is a potential risk to the host of self-nucleic acids recognition, thus precipitating autoimmune and autoinflammatory diseases. In this review, we discuss the roles of nucleic acid-sensing receptors in guarding against pathogen invasion, discriminating between self and non-self, and contributing to autoimmunity and autoinflammatory diseases.     

The potential role of retroviruses in autoimmunity

In the last 20 years research in Immunology underwent fundamental changes. Most importantly, the identification of the key role of innate immune pattern recognition receptors (PRRs) that recognize evolutionarily conserved molecular patterns on infectious pathogens. This results in priming of innate immune cells, which in turn activate and direct the adaptive immune response. Progress in innate immune recognition instigated the current working hypothesis, that recognition of endogenous ligands by PRRs results in innate immune cell activation (autoinflammation) or activation of adaptive cells, with self-reactive antigen receptors (autoimmunity). In particular, nucleic acid-sensing innate immune receptors seem to be prime candidates for a mechanistic understanding of autoreactive activation of the immune system. However, it remains uncertain what the actual source of nucleic acid ligands is and what other signals are needed to drive activation of autoreactive innate immune cells and break self-tolerance of the adaptive immune system. Here, I will review our present understanding about whether the infection with exogenous retroviruses or the reactivation of endogenous retroviruses might play an etiological role in certain autoimmune conditions of humans and murine experimental models.     

Innate signaling and regulation of Dendritic cell immunity

Dendritic cells are crucial in pathogen recognition and induction of specific immune responses to eliminate pathogens from the infected host. Host recognition of invading microorganisms relies on evolutionarily conserved, germline-encoded pattern-recognition receptors (PRRs) that are expressed by DCs. The best-characterized PRR family comprises the Toll-like receptors (TLRs) that recognize bacteria or viruses. In addition to TLRs, intracellular Nod-like receptors and the membrane-associated C-type lectins (CLRs) function as PRRs. Many of these innate receptors also have an important function in natural host homeostatic responses, such as the maintenance of gut homeostasis. Clearly, more indications are hinting at a fine-tuning of immune responses by a concerted action of these PRRs on the recognition of pathogen components and the consequent signalling events that are created. It is becoming increasingly clear that these PRRs can initiate specific signalling events that modulate the production of inflammatory cytokines, phagocytosis, intracellular routing of antigen, release of oxidative species and DC maturation and the subsequent development of adaptive immunity. Notably, members within one family of PRRs can trigger opposite signalling features, indicating that the ultimate outcome of pathogen-induced immune responses depends on the pathogen signature and the collective PRRs involved.     

Distinct Strategies Employed by Dendritic Cells and Macrophages in Restricting Mycobacterium tuberculosis Infection: Different Philosophies but Same Desire

Dendritic cells (DCs) and macrophages (M?s) are professional antigen-presenting cells (APCs) that can efficiently phagocytose Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). It is quite interesting to mention here that DCs and M?s use distinct strategies to combat and eliminate Mtb. Similarly, Mtb employs different mechanisms to counteract the action of DCs and M?s. M?s are evolved with specialized, innate, defensive machinery to restrict growth of Mtb at the initial phase of infection. However, DCs are more endowed toward initiating adaptive immunity by activating naïve T cells. During encounter with Mtb, DCs and M?s deliver discrete functions via triggering through different pattern recognition receptors (PRRs) expressed by these APCs. Mtb-infected DCs and M?s show differential expression of genes encoding cytokines, chemokines, costimulatory molecules, and adhesion molecules. Interestingly, Mtb impairs the immune defensive machinery by exploiting various PRRs. Remarkably, selective signaling through PRRs by Mtb abrogates the bactericidal activity of M?s, but subverts differentiation of monocytes to DCs. In this article, we highlight the role of PRRs in inducing distinct immune response by DCs and M?s against Mtb. Concurrently, we also discuss smart strategies exploited by Mtb to impair the function of host DCs and M?s.     

Cytosolic nucleic acid sensors and innate immune regulation

During viral and bacterial infections, pathogen-derived cytosolic nucleic acids are recognized by the intracellular RNA sensors retinoic acid-inducible gene I and melanoma-differentiated gene 5 and intracellular DNA sensors, including cyclic-di-GMP-AMP synthase, absent in melanoma 2, interferon (IFN)–gamma inducible protein 16, polymerase III, and so on. Binding of intracellular nucleic acids to these sensors activates downstream signaling cascades, resulting in the production of type I IFNs and pro-inflammatory cytokines to induce appropriate systematic immune responses. While these sensors also recognize endogenous nucleic acids and activate immune responses, they can discriminate between self- and non-self-nucleic acids. However, dysfunction of these sensors or failure of regulatory mechanisms causes aberrant activation of immune response and autoimmune disorders. In this review, we focus on how intracellular immune sensors recognize exogenous nucleic acids and activate the innate immune system, and furthermore, how autoimmune diseases result from dysfunction of these sensors.     

Type I interferon in systemic lupus erythematosus and other autoimmune diseases

Different genetic alterations may lead to type I interferon (IFN) overproduction in human systemic lupus erythematosus (SLE). The increased bioavailability of type I IFN contributes to peripheral tolerance breakdown through the activation of immature myeloid dendritic cells (mDCs). IFN-matured mDCs activate autoreactive T cells. These cells, together with plasmacytoid DCs, help expand autoreactive B cells. IFN-matured DCs also activate cytotoxic CD8+ T cells, possibly increasing apoptotic cell availability. The capture of apoptotic cells by mDCs and of nucleic acid-containing immune complexes by plasmacytoid DCs and B cells amplifies the autoimmune reaction leading to disease manifestations. Genetic alterations in lineages other than B cells might explain other autoimmune syndromes where type I IFNs appear to be involved.     

Interferon-�� and interleukin-12 are induced, respectively, by double-stranded DNA and single-stranded RNA in human myeloid dendritic cells

Dendritic cells (DCs) are initiators of innate immunity and acquired immunity as cells linking these two bio‐defence systems through the production of cytokines such as interferon‐α (IFN‐α) and interleukin‐12 (IL‐12). Nucleic acids such as DNA from damaged cells or pathogens are important activators not only for anti‐microbial innate immune responses but also in the pathogenesis of IFN‐related autoimmune diseases. Plasmacytoid DCs are regarded as the main effectors for the DNA‐mediated innate immunity by possessing DNA‐sensing toll‐like receptor 9 (TLR9). We here found that double‐stranded DNA (dsDNA) complexed with lipotransfectants triggered activation of human monocyte‐derived DCs (moDCs), leading to the preferential production of IFN‐α but not IL‐12. This indicates that myeloid DCs also function as supportive effectors against the invasion of pathogenic microbes through the DNA‐mediated activation in innate immunity. The dsDNA with lipotransfectants can be taken up by moDCs without co‐localization of endosomal LAMP1 staining, and the dsDNA‐mediated IFN‐α production was not impaired by chloroquine. These findings indicate that moDC activation by dsDNA does not involve the endosomal TLR pathway. In contrast, single‐stranded RNA (ssRNA) stimulated moDCs to secrete IL‐12 but not IFN‐α. This process was inhibited by chloroquine, suggesting an involvement of the TLR pathway in ssRNA‐mediated moDC activation. As might be inferred from our findings, myeloid DCs may function as a traffic control between innate immunity via IFN‐α production and acquired immunity via IL‐12 production, depending on the type of nucleic acids. Our results provide a new insight into the biological action of myeloid DCs underlying the DNA‐mediated activation of protective or pathogenic immunity.     

Recognition of viral nucleic acids in innate immunity

Viral infections are detected by sensor molecules, which initiate innate antiviral responses, including the activation of type I interferons (IFNs) and proinflammatory cytokines. These cytokines are responsible for not only inhibiting viral replication in infected cells but also regulating the induction of adaptive immunity, leading to the swift eradication of viruses. Recent advances in the identification of pathogen receptors in the innate immune system have revealed that distinct types of sensors play a role in the detection of viral nucleic acids in different ways; Toll‐like receptors (TLRs), which detect viral DNA or RNA in endosomal compartments in immune cells, retinoic acid inducible gene‐I (RIG‐I)‐like receptors (RLRs), which recognise viral RNA in the cytoplasm, and DNA sensors, which detect cytoplasmic viral DNA. Since these sensors have to exclusively recognise viral infections, it is intriguing to understand how they distinguish self nucleic acids from foreign viral ones. Here, we review the current knowledge of the recognition of viral nucleic acids by these sensor molecules and the signal transduction machinery. Copyright © 2009 John Wiley & Sons, Ltd.     

Reading the viral signature by Toll-like receptors and other pattern recognition receptors

Successful host defense against viral infections relies on early production of type I interferon (IFN) and subsequent activation of a cellular cytotoxic response. The acute IFN and inflammatory response against virus infections is mediated by cellular pattern-recognition receptors (PRRs) that recognize specific molecular structures on viral particles or products of viral replication. Toll-like receptors (TLRs) constitute a class of membrane-bound PRRs capable of detecting microbial infections. While TLR2 and TLR4, which were first identified to recognize Gram-positive and Gram-negative bacteria, respectively, sense specific viral proteins on the cell surface, TLRs 3, 7, 8, and 9 serve as receptors for viral nucleic acids in endosomic compartments. In addition to TLRs, cells express cytoplasmic PRRs such as the RNA helicase retinoic acid inducible gene I and the kinase double-stranded RNA-activated protein kinase R, both of which sense dsRNA, a characteristic signature of viral replication, and initiate a protective cellular response. Here we review the recent progress in our understanding of PRRs and viral infections and discuss the molecular and cellular responses evoked by virus-activated PRRs. Finally, we look into what is currently known about the role of PRRs in viral infections in vivo.     

Dendritic cells and the immunopathogenesis of systemic lupus erythematosus

Over the last decade, the role of dendritic cells (DCs) in the immunopathogenesis of systemic lupus erythematosus (SLE) has become apparent. As unique mediators of both tolerance and immunity, aberrant myeloid and plasmacytoid DC function can promote autoimmune responses via a number of mechanisms and proinflammatory pathways. This review provides an overview of DC function, the potential role of DCs in promoting autoimmune responses in SLE, and how other abnormalities in lupus can lead to an enhanced engagement of DCs in immune responses. How medications used to treat SLE and other autoimmune conditions may exert effects on DCs is also explored.     

Evaluation of Microfluidic Biosensor Development Using Microscopic Analysis of Molecular Beacon Hybridization Kinetics

Molecular beacons, oligonucleotide probes that fluoresce upon hybridization to a target nucleic acid, can be used in microfluidic devices to detect and quantify nucleic acids in solution as well as inside bacterial cells. Three essential steps towards the development of such devices as integrated microfluidic biosensors using molecular beacons were investigated in the present study. First, experiments using real-time confocal microscopy indicated that diffusion of DNA molecular beacons across a 100-m diameter microfluidic channel took less than one minute after the flow of reagents was stopped. Second, experiments to evaluate hybridization kinetics of DNA molecular beacons with target nucleic acids in solution showed that DNA molecular beacons can be used to characterize hybridization kinetics in real time in microfluidic channels and that hybridization signals approached their maximum in approximately three minutes. Finally, it was demonstrated that peptide nucleic acid molecular beacons can be used to detect bacterial cells in microfluidic devices. These results suggest that the use of microfluidic devices to detect nucleic acids in solution and in bacterial cells is promising and that further development of an integrated microfluidic biosensor for bacterial detection based on this concept is warranted.     

Adoptive transfer of autoimmune splenic dendritic cells to lupus-prone mice triggers a B lymphocyte humoral response

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by increased autoantibody production that leads to multiple tissue injuries. Dendritic cells (DCs) are important orchestrators of immune responses and key components in fine-tuning the balance between tolerance and immunity. However, their role in autoimmune disorders such as SLE remains uncertain. We analyzed the contribution of DCs in triggering SLE by adoptively transferring splenic DCs from aged autoimmune [NZB×NZW]F1 (BWF1) mice to young healthy BWF1 mice. We observed that the transfer of DCs from autoimmune mice to pre-autoimmune mice induced high autoantibody titers in the serum of recipient mice. Moreover, autoimmune DCs from aged BWF1 mice were crucial for the expansion and differentiation of plasmablasts and CD5⁺ B cells or B1-like cells in the peripheral blood, and spleen of recipient BWF1 mice, a phenomenon that is observed in autoimmune BWF1 mice. On the other hand, DCs from aged BWF1 mice participated in the expansion and differentiation of DCs and IFN-γ-producing T cells. These results reveal that DCs from autoimmune BWF1 mice exhibit functional and phenotypic characteristics that allow them to trigger B cell hyperactivation, as well as DC and T cell expansion and differentiation, thereby promoting an exacerbated humoral response in lupus-prone mice.     

Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins

Pattern-recognition receptors (PRRs) detect molecular signatures of microbes and initiate immune responses to infection. Prototypical PRRs such as Toll-like receptors (TLRs) signal via a conserved pathway to induce innate response genes. In contrast, the signaling pathways engaged by other classes of putative PRRs remain ill defined. Here, we demonstrate that the beta-glucan receptor Dectin-1, a yeast binding C type lectin known to synergize with TLR2 to induce TNF alpha and IL-12, can also promote synthesis of IL-2 and IL-10 through phosphorylation of the membrane proximal tyrosine in the cytoplasmic domain and recruitment of Syk kinase. syk-/- dendritic cells (DCs) do not make IL-10 or IL-2 upon yeast stimulation but produce IL-12, indicating that the Dectin-1/Syk and Dectin-1/TLR2 pathways can operate independently. These results identify a novel signaling pathway involved in pattern recognition by C type lectins and suggest a potential role for Syk kinase in regulation of innate immunity.     

Inflammatory signals in dendritic cell activation and the induction of adaptive immunity

Summary:  Pathogen invasion induces a rapid inflammatory response initiated through the recognition of pathogen-derived molecules by pattern recognition receptors (PRRs) expressed on both immune and non-immune cells. The initial wave of pro-inflammatory cytokines and chemokines limits pathogen spread and recruits and activates immune cells to eradicate the invaders. Dendritic cells (DCs) are responsible for initiating a subsequent phase of immunity, dominated by the action of pathogen-specific T and B cells. As for the early pro-inflammatory response, DC activation is triggered by PRR signals. These signals convert resting DCs into potent antigen-presenting cells capable of promoting the expansion and effector differentiation of naive pathogen-specific T cells. However, it has been argued that signals from PRRs are not a prerequisite for DC activation and that pro-inflammatory cytokines have the same effect. Although this may appear like an efficient way to expand the number of DCs that initiate adaptive immunity, evidence is accumulating that DCs activated indirectly by inflammatory cytokines are unable to induce functional T-cell responses. Here, we review the differences between PRR-triggered and cytokine-induced DC activation and speculate on a potential role for DCs activated by inflammatory signals in tolerance induction rather than immunity.     

The response of human dendritic cells to co-ligation of pattern-recognition receptors

Dendritic cells (DCs) are key antigen-presenting cells that express a wide variety of pattern-recognition receptors (PRRs). Triggering of a single PRR, especially Toll-like receptors (TLRs) and C-type lectins, induces maturation of DCs, but cooperativity between multiple PRRs is needed in order to achieve an effective immune response. In this review, we summarize the published data related to the effect of individual and joint PRR agonists on DCs and Langerhans-like cells derived from monocytes (MoDCs and MoLCs, respectively). Our results demonstrate that MoDCs co-stimulated with TLR3/TLR7 and TLR3/Dectin-1 ligands induced superior T helper (Th)1 and Th17 immune responses, compared to effects of single agonists. The opposite outcome was observed after co-ligation of TLR3 and Langerin on MoLCs. These findings may be relevant to improve strategy for tumor immunotherapy.     

Antimicrobial peptides and self-DNA in autoimmune skin inflammation

Toll-like receptor (TLR)-mediated detection of viral nucleic acids and production of type I interferons (IFNs) by plasmacytoid dendritic cells (pDCs) are key elements of antiviral defense. By contrast, inappropriate recognition of self-nucleic acids with induction of IFN responses in pDCs can lead to autoimmunity. In this review we describe how pDC responses to self-DNA are normally avoided and focus on our recent finding that in psoriasis, a common autoimmune disease of the skin, these barriers can be breached by the cationic antimicrobial peptide LL37. LL37 binds extracellular self-DNA fragments into aggregated particles that enter pDCs and trigger robust IFN responses by activating endosomal TLR9 as if they were viruses. We also describe the mechanisms that normally control production and activity of LL37 in human skin and propose that the persistent overexpression of LL37 in psoriasis leads to uncontrolled IFN responses that drive autoimmune skin inflammation.     

Nucleic acid-associated autoantigens: Pathogenic involvement and therapeutic potential

Autoimmunity to ubiquitously expressed macromolecular nucleic acid–protein complexes such as the nucleosome or the spliceosome is a characteristic feature of systemic autoimmune diseases. Disease-specificity and/or association with clinical features of some of these autoimmune responses suggest pathogenic involvement which, however, has been proven in only a few cases so far. Although the mechanisms leading to autoimmunity against nucleic acid-containing complexes are still far from being fully understood, there is increasing experimental evidence that the nucleic acid component may act as a co-stimulator or adjuvans via activation of nucleic acid-binding receptor systems such as Toll-like receptors in antigen-presenting cells. Dysregulated apoptosis and inappropriate stimulation of nucleic acid-sensing receptors may lead to loss of tolerance against the protein components of such complexes, activation of autoreactive T cells and formation of autoantibodies. This has been demonstrated to occur in systemic lupus erythematosus and seems to represent a general mechanism that may be crucial for the development of systemic autoimmune diseases. This review provides a comprehensive overview of the most thoroughly-characterized nucleic acid-associated autoantigens, describing their structure and biological function, as well as the nature and pathogenic importance of the reactivities directed against them. Furthermore, recent advances in immunotherapy such as antigen-specific approaches targeted at nucleic acid-binding antigens are discussed.     

Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1

The mechanisms by which tumor microenvironments modulate nucleic acid-mediated innate immunity remain unknown. Here we identify the receptor TIM-3 as key in circumventing the stimulatory effects of nucleic acids in tumor immunity. Tumor-associated dendritic cells (DCs) in mouse tumors and patients with cancer had high expression of TIM-3. DC-derived TIM-3 suppressed innate immune responses through the recognition of nucleic acids by Toll-like receptors and cytosolic sensors via a galectin-9-independent mechanism. In contrast, TIM-3 interacted with the alarmin HMGB1 to interfere with the recruitment of nucleic acids into DC endosomes and attenuated the therapeutic efficacy of DNA vaccination and chemotherapy by diminishing the immunogenicity of nucleic acids released from dying tumor cells. Our findings define a mechanism whereby tumor microenvironments suppress antitumor immunity mediated by nucleic acids.