RIG-I Recognition of RNA Targets: The Influence of Terminal Base Pair Sequence and Overhangs on Affinity and Signaling.

Within the complex environment of the human cell, the RIG-I innate immune receptor must detect the presence of double-stranded viral RNA molecules and differentiate them from a diversity of host RNA molecules. In an ongoing effort to understand the molecular basis for RIG-I target specificity, here, we evaluate the ability of this sensor to respond to triphosphorylated, double-stranded RNA molecules that contain all possible terminal base pairs and common mismatches. In addition, we test the response to duplexes with various types of 5' and 3' overhangs. We conducted quantitative measurements of RNA ligand affinity, then tested RNA variants for their ability to stimulate the RIG-I-dependent interferon response in cells and in whole animals. The resulting data provide insights into the design of RNA therapeutics that prevent RIG-I activation, and they provide valuable insights into the mechanisms of evasion by deadly pathogens such as the Ebola and Marburg viruses.

RIG-I Selectively Discriminates against 5'-Monophosphate RNA.

The innate immune sensor RIG-I must sensitively detect and respond to viral RNAs that enter the cytoplasm, while remaining unresponsive to the abundance of structurally similar RNAs that are the products of host metabolism. In the case of RIG-I, these viral and host targets differ by only a few atoms, and a molecular mechanism for such selective differentiation has remained elusive. Using a combination of quantitative biophysical and immunological studies, we show that RIG-I, which is normally activated by duplex RNAs containing a 5'-tri- or diphosphate (5'-ppp or 5'-pp RNAs), is actively antagonized by RNAs containing 5'-monophosphates (5'-p RNAs). This is accomplished by a gating mechanism in which an alternative RIG-I conformation blocks the C-terminal domain (CTD) upon 5'-p RNA binding, thereby short circuiting the activation of signaling.

A minimal RNA ligand for potent RIG-I activation in living mice.

We have developed highly potent synthetic activators of the vertebrate immune system that specifically target the RIG-I receptor. When introduced into mice, a family of short, triphosphorylated stem-loop RNAs (SLRs) induces a potent interferon response and the activation of specific genes essential for antiviral defense. Using RNA sequencing, we provide the first in vivo genome-wide view of the expression networks that are initiated upon RIG-I activation. We observe that SLRs specifically induce type I interferons, subsets of interferon-stimulated genes (ISGs), and cellular remodeling factors. By contrast, polyinosinic:polycytidylic acid [poly(I:C)], which binds and activates multiple RNA sensors, induces type III interferons and several unique ISGs. The short length (10 to 14 base pairs) and robust function of SLRs in mice demonstrate that RIG-I forms active signaling complexes without oligomerizing on RNA. These findings demonstrate that SLRs are potent therapeutic and investigative tools for targeted modulation of the innate immune system.

Bifurcation of Toll-like receptor 9 signaling by adaptor protein 3.

Endosomal Toll-like receptors (TLRs) 7 and 9 recognize viral pathogens and induce signals leading to the activation of nuclear factor κB (NF-κB)-dependent proinflammatory cytokines and interferon regulatory factor 7 (IRF7)-dependent type I interferons (IFNs). Recognition of viral nucleic acids by TLR9 requires its cleavage in the endolysosomal compartment. Here, we show that TLR9 signals leading to the activation of type I IFN, but not proinflammatory cytokine genes, require TLR9 trafficking from endosomes to a specialized lysosome-related organelle. Furthermore, we identify adapter protein-3 as the protein complex responsible for the trafficking of TLR9 to this subcellular compartment. Our results reveal an intracellular mechanism for bifurcation of TLR9 signals by selective receptor trafficking within the endosomal system.

Differential roles of migratory and resident DCs in T cell priming after mucosal or skin HSV-1 infection.

Although mucosal surfaces represent the main portal of entry for pathogens, the mechanism of antigen presentation by dendritic cells (DCs) that patrol various mucosal tissues remains unclear. Instead, much effort has focused on the understanding of initiation of immune responses generated against antigens delivered by injection. We examined the contributions of migratory versus lymph node-resident DC populations in antigen presentation to CD4 and CD8 T cells after needle injection, epicutaneous infection, or vaginal mucosal herpes simplex virus (HSV) 1 infection. We show that upon needle injection, HSV-1 became lymph-borne and was rapidly presented by lymph node-resident DCs to CD4 and CD8 T cells. In contrast, after vaginal HSV-1 infection, antigens were largely presented by tissue-derived migrant DCs with delayed kinetics. In addition, migrant DCs made more frequent contact with HSV-specific T cells after vaginal infection compared with epicutaneous infection. Thus, both migrant and resident DCs play an important role in priming CD8 and CD4 T cell responses, and their relative importance depends on the mode of infection in vivo.

Dendritic cells and B cells maximize mucosal Th1 memory response to herpes simplex virus.

Although the importance of cytotoxic T lymphocytes and neutralizing antibodies for antiviral defense is well known, the antiviral mechanism of Th1 remains unclear. We show that Th1 cells mediate noncytolytic antiviral protection independent of direct lysis through local secretion of IFN-gamma after herpes simplex virus (HSV) 2 infection. IFN-gamma acted on stromal cells, but not on hematopoietic cells, to prevent further viral replication and spread throughout the vaginal mucosa. Importantly, unlike other known Th1 defense mechanisms, this effector function did not require recognition of virally infected cells via MHC class II. Instead, recall Th1 response was elicited by MHC class II(+) antigen-presenting cells at the site of infection. Dendritic cells (DCs) were not required and only partially sufficient to induce a recall response from memory Th1 cells. Importantly, DCs and B cells together contributed to restimulating memory CD4 T cells to secrete IFN-gamma. In the absence of both DCs and B cells, immunized mice rapidly succumbed to HSV-2 infection and death. Thus, these results revealed a distinct mechanism by which memory Th1 cells mediate noncytolytic IFN-gamma-dependent antiviral protection after recognition of processed viral antigens by local DCs and B cells.

Vaginal epithelial dendritic cells renew from bone marrow precursors.

Dendritic cells (DCs) represent key professional antigen-presenting cells capable of initiating primary immune responses. A specialized subset of DCs, the Langerhans cells (LCs), are located in the stratified squamous epithelial layer of the skin and within the mucosal epithelial lining of the vaginal and oral cavities. The vaginal mucosa undergoes cyclic changes under the control of sex hormones, and the renewal characteristics of the vaginal epithelial DCs (VEDCs) remain unknown. Here, we examined the origin of VEDCs. In contrast to the skin epidermal LCs, the DCs in the epithelium of the vagina were found to be repopulated mainly by nonmonocyte bone-marrow-derived precursors, with a half-life of 13 days under steady-state conditions. Upon infection with HSV-2, the Gr-1(hi) monocytes were found to give rise to VEDCs. Furthermore, flow cytometric analysis of the VEDCs revealed the presence of at least three distinct populations, namely, CD11b(+)F4/80(hi), CD11b(+)F4/80(int), and CD11b(-)F4/80(-). Importantly, these VEDC populations expressed CD207 at low levels and had a constitutively more activated phenotype compared with the skin LCs. Collectively, our results revealed mucosa-specific features of the VEDCs with respect to their phenotype, activation status, and homeostatic renewal potential.

Cutting Edge: Plasmacytoid dendritic cells provide innate immune protection against mucosal viral infection in situ.

Dendritic cells (DCs) are powerful APCs capable of activating naive lymphocytes. Of the DC subfamilies, plasmacytoid DCs (pDCs) are unique in that they secrete high levels of type I IFNs in response to viruses but their role in inducing adaptive immunity remains divisive. In this study, we examined the importance of pDCs and their ability to recognize a virus through TLR9 in immunity against genital HSV-2 infection. We show that a low number of pDCs survey the vaginal mucosa at steady state. Upon infection, pDCs are recruited to the vagina and produce large amounts of type I IFNs in a TLR9-dependent manner and suppress local viral replication. Although pDCs are critical in innate defense against genital herpes challenge, adaptive Th1 immunity developed normally in the absence of pDCs. Thus, by way of migrating directly into the peripheral mucosa, pDCs act strictly as innate antiviral effector cells against mucosal viral infection in situ.

Dual recognition of herpes simplex viruses by TLR2 and TLR9 in dendritic cells.

Dendritic cells (DCs) express multiple Toll-like receptors (TLR) in distinct cellular locations. Herpes simplex viruses (HSV) have been reported to engage both the surface TLR2 and intracellular TLR9 in conventional DCs. However, the contributions of these TLRs in recognition of HSV and the induction of proinflammatory cytokines in DCs remain unclear. Here, we demonstrate that a rare population of HSV, both in laboratory strains and in primary clinical isolates from humans, has the capacity to activate TLR2. This virus population is recognized through both TLR2 and TLR9 for the induction of IL-6 and IL-12 secretion from bone marrow-derived DCs. Further, we describe a previously uncharacterized pathway of viral recognition in which TLR2 and TLR9 are engaged in sequence within the same DC. Live viral infection results in two additional agonists of TLR2 and TLR9. These results indicate that in cells that express multiple TLRs, pathogens that contain multiple pathogen-associated molecular patterns can be detected in an orchestrated sequence and suggest that the innate immune system in DCs is optimized to linking uptake and degradation of pathogens to microbial recognition.

MAdCAM-1 expressing sacral lymph node in the lymphotoxin beta-deficient mouse provides a site for immune generation following vaginal herpes simplex virus-2 infection.

The members of the lymphotoxin (LT) family of molecules play a critical role in lymphoid organogenesis. Whereas LT alpha-deficient mice lack all lymph nodes and Peyer's patches, mice deficient in LT beta retain mesenteric lymph nodes and cervical lymph nodes, suggesting that an LT beta-independent pathway exists for the generation of mucosal lymph nodes. In this study, we describe the presence of a lymph node in LT beta-deficient mice responsible for draining the genital mucosa. In the majority of LT beta-deficient mice, a lymph node was found near the iliac artery, slightly misplaced from the site of the sacral lymph node in wild-type mice. The sacral lymph node of the LT beta-deficient mice, as well as that of the wild-type mice, expressed the mucosal addressin cell adhesion molecule-1 similar to the mesenteric lymph node. Following intravaginal infection with HSV type 2, activated dendritic cells capable of stimulating a Th1 response were found in this sacral lymph node. Furthermore, normal HSV-2-specific IgG responses were generated in the LT beta-deficient mice following intravaginal HSV-2 infection even in the absence of the spleen. Therefore, an LT beta-independent pathway exists for the development of a lymph node associated with the genital mucosa, and such a lymph node serves to generate potent immune responses against viral challenge.

In vivo role of nectin-1 in entry of herpes simplex virus type 1 (HSV-1) and HSV-2 through the vaginal mucosa.

Herpes simplex virus type 2 (HSV-2) is transmitted through the genital mucosa during sexual encounters. In recent years, HSV-1 has also become commonly associated with primary genital herpes. The mechanism of viral entry of HSV-1 and HSV-2 in the female genital tract is unknown. In order to understand the molecular interactions required for HSV entry into the vaginal epithelium, we examined the expression of herpesvirus entry mediator nectin-1 in the vagina of human and mouse at different stages of their hormonal cycle. Nectin-1 was highly expressed in the epithelium of human vagina throughout the menstrual cycle, whereas the mouse vaginal epithelium expressed nectin-1 only during the stages of the estrous cycle in which mice are susceptible to vaginal HSV infection. Furthermore, the ability of nectin-1 to mediate viral entry following intravaginal inoculation was examined in a mouse model of genital herpes. Vaginal infection with either HSV-1 or HSV-2 was blocked by preincubation of the virus with soluble recombinant nectin-1. Viral entry through the vaginal mucosa was also inhibited by preincubation of HSV-2 with antibody against gD. Together, these results suggest the importance of nectin-1 in mediating viral entry for both HSV-1 and HSV-2 in the genital mucosa in female hosts.