RIG-I-Like Receptor Regulation of Immune Cell Function and Therapeutic Implications

Abstract

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are cytosolic RNA sensors critical for initiation of antiviral immunity. Activation of RLRs following RNA recognition leads to production of antiviral genes and interferons (IFNs) for induction of broad antiviral immunity. While the RLRs are ubiquitously expressed, much of our understanding of these molecules comes from their study in epithelial cells and fibroblasts. However, RLR activation is critical for induction of immune function and long-term protective immunity. Recent work has focused on the roles of RLRs in immune cells and their contribution to programming of effective immune responses. This new understanding of RLR function in immune cells and immune programming has led to the development of vaccines and therapeutics targeting the RLRs. This review covers recent advances in our understanding of RLR contribution to immune cell function during infection and the emerging RLR-targeting strategies for induction of immunity against cancer and viral infection.

Introduction

The recognition of pathogen-associated molecular patterns (PAMPs) by membrane-bound and cytosolic pattern recognition receptors (PRRs) results in the induction of type I and III interferon (IFN) responses. IFNs play a critical role in providing antiviral protection through paracrine and autocrine induction of hundreds of effector antiviral genes and are critical to the initiation of adaptive immune responses (1–3). IFN induction is critical not only for antiviral immunity, but also induction of anti-tumor immunity and protection against bacteria, fungal, and parasitic infection (4–6). Further, aberrant expression of IFN promotes autoimmune diseases such as systemic lupus erythematosus (SLE), inflammatory myositis, Aicardi-Goutières syndrome (AGS), rheumatoid arthritis and other rheumatic diseases (7–9). Multiple therapeutics targeted at limiting IFN responses have been clinically developed and are in clinical trials or approved for treatment of specific autoimmune disorders (7, 8). As such, recombinant IFN has been developed as an antiviral therapeutic. However, IFN therapies are frequently accompanied by severe side effects, limiting clinical application (10). Given the dual role of IFN in promoting antiviral immunity while exacerbating autoimmunity, understanding the functions of RLRs beyond production of IFN is important for harnessing this pathway for therapeutic benefit. Particularly unravelling the cell types and mechanisms required for RLR promotion of adaptive immune responses have important implications for human health and disease (11, 12).

Nucleic Acid Sensing Pathways

IFNs are induced by multiple pathogen sensing pathways including the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) (the focus of this review), toll-like receptors (TLRs), nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), and the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) DNA sensing pathways. These are localized in distinct cellular compartments and recognize unique ligands to allow for effective immunity against a variety of pathogens. These pathways have been described in detail in other recent reviews (13–16). TLRs are type I transmembrane proteins expressed at the plasma membrane or in endosomes/lysosomes that broadly recognize membrane components and nucleic acids derived from pathogens leading to activation of transcription factors, such as nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1), to induce pro-inflammatory and IFN responses (13, 17–19). NLRs generally detect components of peptidoglycan and nucleic acid PAMPs in the cytosol, leading to type I IFN as well as interleukin 1 (IL-1) release (20–24). The cGAS/STING pathway recognizes host and foreign DNA for activation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), inducing a robust type I IFN response (16, 25, 26).

RLR Pathway

The RLRs are cytoplasmic RNA sensors that can initiate IFN, antiviral, and pro-inflammatory responses (27, 28). The family of RLRs consist of RIG-I, melanoma differentiation-associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), encoded by DDX58, IFIH1, and DHX58, respectively (29). All of the RLRs contain a structurally conserved DExD/H box RNA helicase and carboxy-terminal domain (CTD) (30). In addition, both RIG-I and MDA5 (but not LGP2) contain two amino-terminal caspase activation and recruitment domains (CARDs)(27) (Figure 1a). Interaction of cognate RNA ligands with the DExD/H helicase causes a confirmational change to displace the CARDs, allowing for their oligomerization and interaction with mitochondrial antiviral signaling proteins (MAVS) CARD domains (27, 29–31). MAVS is embedded in the mitochondrial-associated membranes (MAMs) and in oxidative organelles, peroxisomes (27). Canonically, MAVS forms a scaffold at MAM with tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), TNF receptor-associated factor 5 (TRAF5), and TRAF6 to activate inhibitor of nuclear kappa-B kinase (IKK) family and TBK1 (32). IKK family kinases and TBK1 phosphorylate NF-κB, IRF-3 and IRF-7, which allows for their translocation to the nucleus and transcription of IFN, antiviral genes, pro-apoptotic genes, and immune cell programming genes (27). LGP2 plays a dual role in propagating MDA5 signaling during the acute stage of infection, but limits signaling downstream of MAVS during resolution of infection (33–35) (Figure 1b).

Figure 1. RLR domains, polymorphisms, and signaling.

A) The RLRs RIG-I, MDA5, and LGP2 all contain a C-terminal repressor domain (CTD) and a helicase domain. RIG-I and MDA5 contain two caspase activation and recruitment domains (CARDs). The numbered dashes represent relative locations of single nucleotide polymorphisms (SNPs) present in coding regions of the RLRs with numbers corresponding to Table I. B) Upon virus infection, RIG-I and MDA5 sense cognate RNAs and undergo confirmational change to allow for interaction with and activation of MAVS at mitochondrial-associated membranes (MAM) for downstream activation of NF-kB and IRF3 leading to antiviral gene programming, IFN production, immune cell programming genes, and pro-apoptotic genes. The role of LGP2 is still under investigation, but during acute infection it stabilizes and propagates MDA5 signaling. However, during resolution of infection LGP2 dampens RLR signaling.

As RLRs recognize RNA, many studies have focused on delineating features that allow for RIG-I, MDA5, and LGP2 discrimination of self vs non-self RNA. RIG-I preferentially recognizes short RNAs containing 5’ triphosphate or diphosphate groups (36–41). MDA5 recognizes long RNA duplexes in high molecular weight RNAs (41, 42). LGP2 binds RNA with high affinity regardless of their length or the modifications in the 5’ end, but its direct role in independent signal activation vs regulation of other RLRs remains unclear (43). In addition, these helicases are under control of regulatory enzymes and interacting proteins that can further enhance RLR activation (44–46). Despite these conserved features, RIG-I and MDA5 have been described to recognize self RNA and contribute to antiviral immunity as well as autoimmunity (47–51), and several recent reviews have comprehensively covered known RNA ligands of the RLRs (27, 52, 53). Overall, these studies have led to an understanding that each RLR plays unique and distinct roles in antiviral control and potentially immune programming (27, 53, 54).

Virus recognition and induction of immunity in infected target cells (epithelial, fibroblasts, and others) leads to antiviral programming and cytokine/chemokine production that promotes antigen presenting cell (APC) maturation and migration for adaptive immune programming (55). However, RLRs are expressed within nearly all cell types, suggesting both extrinsic (e.g. other immune cell-, epithelial cell-, or fibroblast-mediated) and intrinsic roles for RLRs in immune cell function (27). While relatively few studies have focused specifically on RLR functions in specific immune cell subsets, there has been a renewed interest in delineating immune cell roles of these pathways with a focus on developing therapeutics and vaccine adjuvants that target host immune pathways normally activated following virus infection.

RLR Polymorphisms and Disease

Single nucleotide polymorphisms (SNPs) in RIG-I (DExD-Box Helicase 58, DDX58), MDA5 (Interferon Induced With Helicase C Domain 1, IFIH1), and LGP2 (DExH-Box Helicase 58, DHX58) have been identified with linkage to disease susceptibility (Figure 1a and Table I). For example, minor alleles of DDX58 (rs10813831 and rs3824456) and DHX58 (rs2074160) are associated with spontaneous clearance of hepatitis C virus (HCV) and hepatitis B virus (HBV) (56, 57). Conversely, minor alleles of DDX58 (rs3739674) and IFIH1 (rs1990760) are associated with severe enterovirus 71(EV71) infection or more severe HCV following solid organ transplant (58, 59). Despite their canonical role in antiviral immunity, SNPs in DDX58, IFIH1, and DHX58 are linked to enhanced risk of developing autoimmune diseases such as atypical singleton-merton syndrome, systemic lupus erythematosus (SLE), and type 1 diabetes (T1D) (60–65). However, several of these SNPs are protective against psoriasis/psoriatic arthritis and T1D, highlighting the critical role for appropriate RLR function in regulating inflammation and immunity (59, 66–68).

Table I.

RLR Polymorphisms and Disease Implications¹

#	SNP	Genotype	Mutation	Disease Implications	Source
RIG-I (DDX58)
1	rs3739674	G>C	Upstream mutation; reduced expression	Increased EV71 severity	(58)
2	rs10813831	G>A	Coding mutation; missense variant	Spontaneous HCV Clearance; Conversely, Severe Recurrent HCV following Liver Transplant	(56, 59)
3	rs3824456	C>G	Intron mutation	Spontaneous HBV Clearance	(57)
4	rs55789327	G>A	Coding mutation; Missense variant	Rotavirus vaccine IgA seroconversion	(69)
5	rs786204848	G>T	Coding mutation; missense variant	Atypical Singleton-Merten Syndrome; Congenital Glaucoma	(60)
6	rs786204847	A>C	Coding mutation; missense variant	Atypical Singleton-Merten Syndrome	(60)
MDA5 (IFIH1)
7	rs10930046	T>C	Coding mutation; Missense variant	Decreased Psoriasis Risk; Increased SLE Risk	(59, 63, 66)
8	rs35744605	C>A	Coding mutation; loss of function	Reduced T1D Risk	(67)
9	rs3747517	T>C	Coding mutation; Missense variant	Increased SLE Risk	(61, 62)
10	rs35667974	T>C	Coding mutation; Loss of function	Decreased Psoriasis and Psoriatic Arthritis Risk; Reduced T1D Risk	(66–68)
11	rs1990760	C>T	Coding mutation; decreased expression, increased methylation	Increased T1D and SLE Risk; Increased EV71 Severity	(58, 61–64)
12	rs2111485	A>G	Intergenic mutation; unknown, no expression change	Increased T1D Risk; Spontaneous HBV Clearance	(57, 64)
LGP2 (DHX58)
13	rs12600570	C>T	Intronic; unknown	Elevated Fasting Plasma Glucose	(65)
14	rs2074160	G>A	Coding mutation; unknown	Spontaneous HBV Clearance	(57)

¹Table 1 shows SNPs in RIG-I, MDA5, and LGP2 as identified by NCBI dbSNP designation, genotype (major allele>minor allele), associated mutation, disease association, and references. SNPs with mutations in coding regions are depicted in Figure 1a using the # designation from the first column.

While many associations with autoimmunity and chronic viral infection have been described, how RLR SNPs regulate susceptibility to these illnesses is not well understood. However, the emergence of novel mouse models containing SNPs equivalent to those found in humans will shed further light on the importance of these RLR SNPs in autoimmune and infectious disease and allow for greater understanding of their mechanism. For example, the IFIH1 rs1990760 minor allele is associated with multiple autoimmune diseases, but the mechanisms underlying this enhanced risk due to this polymorphism had not been elucidated. A murine model expressing risk (rs1990760 minor allele) and non-risk (rs1990760 major allele) alleles of IFIH1 was developed to determine the role of these haplotypes in IFN regulation, antiviral immunity, and susceptibility to autoimmune disease (62). Mice expressing the risk allele had higher basal IFNβ expression, enhanced protection against encephalomyocarditis virus (EMCV) infection, as well as increased autoantibody production and incidence of diabetes. Similar models and mechanistic studies will broaden our understanding of the implications of RLR SNPs in pathogen resistance and autoimmunity and allow for delineation of how these variants impact immune cell function.

For example, epistatic interaction between the rs55789327 minor allele of DDX58 and a polymorphism in TLR3 is associated with enhanced IgA seroconversion following rotavirus vaccination (69). However, the specific contribution of RIG-I to this process and in which cell types it is important remains unknown. Based on the relative number of peer-reviewed publications, much of our understanding of RLR regulation derives from in vitro studies on epithelial cells and fibroblasts. Thus, it is less well understood how cell identity further contributes to the functions and regulation of RLRs. As immune cells are critical effectors of both viral protection and drivers of autoimmunity, understanding the specific roles of RLRs in immune cells is critical for understanding the mechanisms of protective vs deleterious RLR responses and allow for design of targeted therapeutics promoting health while minimizing deleterious tissue damage. Recently, multiple studies have begun to unravel the roles of the RLRs and their functions in programming immune cell-specific responses, described below (Figure 2).

Figure 2. RLR functions in immune cell subsets.

The roles of RIG-I, MDA5 and LGP2 in dendritic cells, macrophages, natural killer cells, B lymphocytes, T lymphocytes, and granulocytes during infection are summarized. References for each function are color-coded: RIG-I (orange), MDA5 (teal), LGP2 (magenta).

RLR Regulation of Immune Cell Function and Antiviral Responses

Macrophages

Macrophages play a vital role in controlling viral infection through the induction of downstream ISGs and pro-inflammatory cytokines. However, infected macrophages are disabled in their cytopathic function and require RLR sensing to control viral load (70). Murine bone marrow macrophages (BMMs) require MAVS downstream of RIG-I and MDA5 to induce type I IFN and pro-inflammatory cytokine expression in response to Vesicular Stomatitis Virus (VSV)(71). There is also evidence that both RIG-I and MDA5 contribute to production of MIP1α, RANTES, CXCL10 and IFNγ in human monocyte derived macrophages (MDM) during Middle East respiratory syndrome coronavirus (MERS-CoV) infection (72). Porcine alveolar macrophages depend upon both RIG-I and MDA5 in a redundant fashion to induce IFNβ and interferon stimulated gene 15 (ISG15) in response to African Swine Fever Virus (ASFV), VSV, Sendai Virus (SeV), EMCV, and Influenza A Virus (IAV), which suggest overlapping function between RIG-I and MDA5 in the context of these infections (73, 74). Further, in vivo murine model of Ebola virus infection found the RLR signaling adapter MAVS is required for secretion of IFNβ and protection against lethality (75). While the specific role of LGP2 in regulating RLR signaling is unclear, studies suggest LGP2 synergizes with RIG-I and MDA5 to promote proinflammatory cytokine and IFN induction in murine and porcine macrophages (76, 77) (Figure 1b).

Despite the canonical role for the RLRs in regulating antiviral immunity in macrophages, there is evidence that the RLRs play unique roles in regulating virus infection and macrophage function. RIG-I, but not MDA5, is required in human monocyte-derived macrophages for type I and type III IFN induction following H5N1 IAV infection (78). Murine BMM lacking MDA5 have diminished IFNβ production compared to wild-type and RIG-I deficient BMM during Murine Hepatitis Virus (MHV) infection (79). These differential requirements are due to the specific RNAs produced by these viruses during infection. Importantly, utilization of whole body murine genetic double knockouts of MDA5 and RIG-I demonstrate that both RLRs have non-redundant roles in mediating protection against West Nile Virus (WNV) as measured by replicating virus and diminished IFNβ production (80, 81). During WNV infection, the RLRs reinforce pro-inflammatory M1 macrophage programming, while suppressing wound healing M2 macrophages in both murine and human cells (81). Overall, these studies show that the RLRs regulate not only IFN and antiviral gene programming but play a direct role in regulating macrophage polarization.

Dendritic Cells

Among APCs, dendritic cells (DCs) play a critical role in presenting foreign antigen for the activation of adaptive immune responses important for pathogen clearance and long-term memory protection. While extrinsic inflammation induced by RLR signaling in epithelial and other cells impacts DC activation, there is evidence that DC activation, maturation, and antigen presentation capability are dependent upon intrinsic RLR signaling. Human monocyte derived DCs (moDCs) infected with IAV or Measles Virus rely on MDA5 and RIG-I for type I IFN induction, similar to murine bone marrow-derived DCs (BMDCs) during rabies virus and WNV infection (80, 82–84). MDA5 signaling in BMDCs is required for cytokine production during norovirus and EMCV infection (85, 86). LGP2 deficient DCs infected with reovirus, EMCV, SeV, and VSV have diminished production of type I IFN, thereby suggesting LGP2 regulates MDA5 and/or RIG-I signaling (77) (Figure 1b).

While human moDCs and murine BMDCs serve as tractable models, there is evidence that different DC subsets present in vivo rely differentially upon PRR pathways. Infection of murine embryonic fibroblasts (MEF) and conventional DCs (cDC) with Newcastle disease virus (NDV) led to a RIG-I-dependent induction of type I IFN. By contrast, the absence of RIG-I did not affect the production of IFN in plasmacytoid DCs (pDC). In pDC, NDV-induced IFN expression required the expression of the RNA sensors TLR7 and TLR9 (87). However, treatment of human pDC with a RIG-I activating HCV-derived PAMP led to production to type III IFN (88). Mass spectrometry analysis of splenic DC subsets revealed that RIG-I and MDA5 was readily detected in CD4⁺ and double negative (CD4⁻CD8α⁻) cDC subsets, but low or undetectable in CD8α⁺ cDC, suggesting that RLR expression unique to specific DC subsets may regulate their functions during acute viral infection (89). In addition, differences in the expression and utilization of signal transduction molecules downstream of PRRs across dendritic cell subsets could further dictate the cell type specific regulation of dsRNA detection (90).

Temporal changes in RLR expression may also allow for varying roles of RIG-I, MDA5 and/or LGP2 in specific cell subsets over the course of infection. During in vivo lymphocytic choriomeningitis virus (LCMV) infection, induction of type I IFN (day 1 post infection) is RIG-I-dependent, while the later IFN response (day 2 post infection) is MAVS-MDA5-dependent (91). Human DCs upregulate RIG-I, MDA5, and LGP2 following Zika Virus, suggesting these RLRs may contribute differentially to DC function and immune responses as infection progresses (92). Together, this supports that abundance of nucleic acid sensors, co-regulatory molecules, and downstream effectors all play a critical role in the spatial and temporal antiviral functions of RLRs. As we continue to uncover additional functions for RLRs in coordinating innate and adaptive responses beyond antiviral control, focusing on understanding their unique roles in immune cells will allow for the development of targeted therapeutics and adjuvants to fine-tune DC function for optimal activation of adaptive immunity.

In support of this, the RLRs play a role in regulation of DC function for downstream activation of adaptive immunity. RIG-I and MAVS signaling in DCs are required for generation of cytolytic CD8⁺ T cells function following IAV infection in vitro and in vivo (84, 93). Particularly, RIG-I signaling in murine BMDCs is necessary for presenting antigen to both CD4⁺ and CD8⁺ T cells during IAV infection (93). RLR signaling in dendritic cells also regulates CD4⁺ T cell immunity. Upon infection with dengue Virus, human DCs become activated to secrete IL-27, leading to polarization of T_FH and production of IgM and IgG in a RIG-I and MDA5-dependent manner as shown by siRNA knockdown of MAVS (94, 95). Full MAVS knockout or deletion of MAVS in CD11c⁺ cells, but not B cells, led to increased virus titers and enhanced WNV-specific IgG and neutralizing antibody responses (96). Overall, RLR signaling in DCs is required not only for antiviral activity, but also support DC activation and generation of robust adaptive immune responses.

T cells

APCs, primarily DCs, play a critical role in regulating T cell activation. As such, both cell intrinsic and extrinsic dependencies on RLR expression have been described for T cell activation and function across various infectious models. RLRs can play non-redundant roles in CD4⁺ T cell activation. RIG-I is required for priming of CD4⁺ T cells during IAV infection in mice (93). During H5N1 IAV infection RIG-I is required for priming of CD4⁺ T cells and Th1 polarization (97). During dengue virus infection, both RIG-I and MDA5 are required for Th1 polarization (94). Interestingly, the absence of RLR expression in DCs appeared to be required for Th1 polarization during dengue virus. Further, both RIG-I and MDA5 in DCs are required for CD4⁺ T_FH responses during dengue virus infection (95). T_FH play an important role in germinal centers to support B cell and antibody-mediated responses. Therefore, it is likely this role for RIG-I and MDA5 in regulating T_FH also impacts the development of B cell and antibody responses during infection.

While the RLRs play a role in CD4⁺ T cell function, much more is known about RLR regulation of CD8⁺ T cell responses. During IAV infection, RIG-I is important for CD8⁺ T cell priming, and mice lacking MAVS and RIG-I have reduced CD8⁺ T cell production of IFNγ, TNF, and Granzyme B (93). Mixed bone-marrow chimeras (lethal irradiation of wild type mice and reconstitution with RIG-I deficient bone marrow) and in vitro studies showed that RIG-I in both DCs and CD8⁺ T cells is required for CD8⁺ T cell priming and function during IAV infection (93). Mice lacking MDA5 have dysfunctional effector CD8⁺ T cell responses during WNV and MHV infection (98). During WNV infection, T cell extrinsic MDA5 signaling is required for CD8⁺ T cell priming to control CNS infection (99). Additionally, virus specific CD8⁺ T cell responses were significantly reduced in MDA5 deficient mice following chronic LCMV infection (100). This study showed MDA5-dependent induction of type I IFN extrinsic of T cells was required to prevent CD8⁺ T cell exhaustion (100). MDA5 also contributes to memory generation as MDA5 expression in non-hematopoietic cells enhanced survival and memory of CD8⁺ T cells during herpesvirus infection (101). LGP2 also contributes to regulation of T cell immunity. During WNV infection, WNV-specific CD8⁺ T cell populations and cell survival are reduced in mice lacking LGP2, but whether LGP2 functions intrinsically in CD8⁺ T cells or extrinsically in another cell type, such as an APC, to regulated WNV-specific CD8⁺ T cell immunity is currently unknown (102).

It is important to note that the individual contributions of RIG-I, MDA5, and LGP2 cannot be directly compared in vivo as commonly used mice deficient in RIG-I are embryonic lethal in a full C57BL/6 background and have to be maintained on a mixed Sv129 × C57BL/6 background or on an Sv129 background (87, 103, 104). As MDA5 and LGP2 deficient mice are on the C57BL/6 background, currently any comparison of these mice compared to those lacking RIG-I include the caveat that differences in experimental models may be due to background strain differences. Additionally, a lack of floxed RLR mice has prevented the investigation of the RLRs in individual cell subsets to date. However, regardless of whether the contribution is intrinsic to T cells or extrinsically regulated by RLR signaling in cells known to regulate T cell immunity, RLRs clearly contribute to T cell immunity. Given this, the RLRs have been targeted, particularly in the context of cancer, to prime cytotoxic anti-tumor T cell immunity (11, 12, 105, 106).

B cells

Our understanding of the mechanisms by which RLRs support B cell responses is starting to emerge, and multiple studies have demonstrated that adjuvant use of RLR agonists can promote B cell and antibody responses. Administration of RIG-I agonist RNA as an adjuvant administered with H5N1 virus-like particles to mice led to a decrease in viral titers and an increase in IgG2a responses (97). Inclusion of a RIG-I agonist in an influenza virus DNA vaccine vector led to activation of IFNβ in cell culture and increased serum antibody levels (107). The use of in vitro transcribed defective interfering RNA from SeV as an adjuvant for H1N1 IAV vaccination led to increased IgG subtypes, IgA in the airways, and enhancement of survival following lethal challenge (108). In addition to enhancement of antibody titers, one study utilizing administration of a RIG-I targeting adjuvant in combination with H1N1 IAV vaccination to mice found it led to a higher IgG antibody titer, enhanced germinal center formation, and earlier accumulation of virus-specific plasma cells in the bone marrow compared to vaccination alone (109). These results suggest targeting of RIG-I as an adjuvant may not only increase antibody titers, but also may enhance the durability of these responses. In humans, one polymorphism of RIG-I is correlated with enhanced IgA seroconversion following vaccination against rotavirus infection (69). Together, these studies indicate an important role for the RLRs in regulating B cell immunity that can be harnessed to boost protective immunity, but to date these studies have not delineated whether RIG-I signaling in DCs and/or B cells is required for this adjuvant effect. While it is likely RLR regulation of DC function contributes to this effect, B cells isolated from peripheral blood mononuclear cells of HCV positive patients displayed upregulation of both protein and mRNA transcripts for RIG-I and IRF-2, suggesting there may be an intrinsic role for RIG-I in regulating B cell responses during HCV infection (110).

Natural Killer Cells

There are few studies addressing the role of RLRs in natural killer (NK) cells during virus infection. However, the effects of synthetic agonists on viral sensors, such as RLRs, in NK cells has been investigated. Human and murine NK cells co-cultured with DCs and treated with poly(I:C), a synthetic dsRNA analogue known to activate MDA5, promotes IFNγ production by NK cells (111). This IFNγ production by NK cells is significantly reduced with the loss of MAVS and is dependent upon DCs (111). As poly(I:C) also activates the membrane-bound dsRNA sensor, TLR3, defining the target of this ligand that modulates NK function is necessary. Injection of mice with poly(I:C) led to IFNγ production in NK cells that was dependent upon both MDA5 and TLR3 (112). Mixed bone marrow chimera mice identified a requirement for MDA5 in the stromal compartment for NK cell type I IFN production in response to poly(I:C) in vivo (112). As observed in murine models, RLR agonists can also activate human NK cells (111). Human NK cells infected with HBV and treated with poly(I:C) have diminished levels of perforin, Granzyme B and IFNγ, compared to control (113). HBV infection reduced RIG-I expression and dampened NF-kB and mitogen activated protein kinase (MAPK) signaling (113). Targeting RIG-I activation by 5’triphosphate ssRNA treatment of NK cells led to increased surface expression of TNF-related apoptosis inducing ligand (TRAIL), suggesting there is also an NK cell intrinsic RLR role (114). Together these studies demonstrate a requirement for both RIG-I and MDA5 in regulating NK cell function.

Granulocytes

Mast cells primarily reside in the skin and mucosal tissues, and act as immune sentinels for various types of viruses. The expression of RLRs in mast cells is vital for initiating a state of inflammation to combat replicating virus. Silencing of MDA5 and RIG-I using small interfering RNA (siRNA) in human mast cells led to an increase in VSV titer during in vitro infection (115). Further, both RIG-I and MDA5 are required for murine mast cell production of IFNα, IFNβ, CXCL10, and IL-6 production during VSV infection (116). Additionally, knockdown of MDA5 and RIG-I sensors in KU812 mast cell line diminished CXCL10, CCL4, and CCL5 in response to antibody-enhanced dengue Virus (117). Primary human cord blood-derived mast cells infected with dengue virus in the presence of dengue immune sera led to an increase in mRNA expression of MDA5, RIG-I, as well as type I IFNs (117). Together, these studies support an important and potentially inducible role for RIG-I and MDA5 in regulating anti-viral responses and chemokine/cytokine production in mast cells.

Eosinophils contribute to fungal and allergic disease where they secrete chemokines/cytokines, degranulate, and form extracellular traps (118). However, whether eosinophils can respond to challenge for control of viral infection is an emerging area of research (119). Murine models have shown that RLR mRNA expression is lower than that observed in DCs (119). Expression of both Ddx58 and Ifih1 was induced by in vitro infection with IAV H1N1. Similarly, human eosinophils have low expression of RIG-I and MDA5 compared to expression of other PRRs known to promote eosinophil activation, such as NOD1 and NOD2 (120, 121). However, upon IAV challenge RIG-I mRNA expression can be rapidly induced, suggesting there may be a temporal role for RIG-I in eosinophils following upregulation of the receptor (122).

Neutrophils rapidly infiltrate infected tissue to phagocytose pathogens, debris, and cells and form extracellular traps to aid in clearance of viral infection. Transfection of human neutrophils with poly(I:C) to activate MDA5 results in upregulation of type I IFN, TNF, IL-12, multiple chemokines/cytokines, and ISGs, demonstrating neutrophils sense and respond to virus infection in a canonical fashion (123). IFNβ and TNF production by murine neutrophils during EMCV infection is dependent upon MDA5 (123). Human neutrophils treated with single-stranded RNA (ssRNA40) derived from Herpes Immunodeficiency Virus-1 (HIV-1) led to upregulation RIG-I and MDA5, similar to elevated levels in neutrophils isolated from HIV-1 positive patients (124, 125). Intriguingly, RIG-I and MDA5 have been observed in secretory vesicles of neutrophils and presence of RIG-I bound to 5’triphosphate ssRNA at the plasma membrane of neutrophils (126). While the relevance of this non-canonical distribution of the RLRs in neutrophils is unknown, they suggest RLRs may function in a unique fashion in neutrophils that can be exploited to modulate their function during infection or inflammation.

Targeting RLRs as Therapeutics

Given the role of RLRs in priming immune responses, there has been much interest in development of RLR agonists as therapeutics and vaccine adjuvants. A number of studies in the area of cancer immunity have shown that activation of RIG-I or MDA5 leads to apoptosis of tumor cells, releasing DAMPS, and activating DCs to present tumor antigen for activation of anti-tumor cytotoxic CD8⁺ T cell responses (11, 12, 105, 106) As such, multiple groups have developed and investigated synthetic RNAs and small molecule compounds to harness RIG-I activation as a therapeutic. The treatment of tumor cell lines with 5’triphosphate RNA or poly(I:C) leads to cell death, upregulation of proinflammatory cytokines and IFN, and antigen presentation by DCs for activation of tumor-specific T cells (127). A double-stranded, triphosphorylated, 14 base pair stem-loop RNA (SL14) that specifically activates RIG-I in a MAVS-dependent manner to rapidly upregulate ISGs and IFN following intravenous injection (128). A double-stranded, triphosphorylated stem-loop RNA of 20 base pairs (SLR20) induced RIG-I signaling and slowed tumor progression in an in vivo murine model via induction of apoptosis and pyroptosis for enhanced activation and tumor infiltration of CD4⁺ and CD8⁺ T cells (129). Intratumoral administration of SL14 in subcutaneous melanoma, colon cancer, and breast cancer tumor lines significantly slowed tumor progression and metastasis in mice (130). This stem-loop RNA was taken up by CD11b⁺ cells that migrated to draining lymph nodes and led to an increase in cytotoxic T cells in the tumor (130). Depletion studies showed that both CD4⁺ and CD8⁺ T cells were partially required to mediate RIG-I induced anti-tumor immunity (130).

There is also growing evidence that specific targeting of the RLRs can function as effective antivirals and/or vaccine adjuvants independent of the induction of apoptosis. The same stem-loop RNA RIG-I agonist investigated in pre-clinical models as a cancer therapeutic provided mice with protection against SARS-CoV-2 infection with multiple variants when administered prophylactically or as a therapeutic (131). RIG-I agonist derived from the 3’UTR of HCV blocked Zika virus (ZIKV) infection of human moDCs more efficiently than type I IFN (92). A 5’triphosphate-containing RNA agonist, known as M8, activates and differentiates human monocyte derived DCs (97). Administration of M8 as an adjuvant with an H5N1 virus-like particle-based vaccine to mice significantly increased survival, reduced virus titer, and pulmonary pathology upon challenge (97). Additionally, M8 enhanced IgG2a, IFNγ production by CD8⁺ T cells, and IL-2, TNF, and IFNγ production by CD4⁺ T cells compared to vaccination alone(97). A small molecule compound that activates IRF3 downstream of RIG-I adjuvants 2009 pandemic H1N1 split subunit vaccine (pH1N1 SV) to enhance survival and reduced pulmonary viral burden upon subsequent lethal challenge in mice compared to vaccination alone (132). Administration of this compound with pH1N1 SV led to enhanced neutralizing antibody responses and production of IL-4 and IL-10 by CD4⁺ T cells compared to vaccination alone (132). Together, these studies show targeting of the RLR pathways, primarily RIG-I, as a promising therapeutic avenue for enhancement of immunity in multiple vaccination, infection, and cancer. In sum, RLR activation induces gene programs beyond IFN for promotion of adaptive immune responses, but work is still needed to understand the cell type and context-specific programs regulated by RIG-I, MDA5, and LGP2 in modulating innate and adaptive immune responses.

Conclusions

While unique roles for RIG-I, MDA5, and LGP2 in regulating immune-cell specific responses have been identified, the cell intrinsic and extrinsic nature of this requirements and their mechanisms of immune cell programming downstream of the RLRs largely remains to be elucidated. The studies described above reveal differences in RLR substrates, receptor abundance, basal expression levels, and varying temporal usage during infection across cell types all contribute to differences in RLR regulation of immune cell function. However, our ability to understand these intricacies of RLR function has been hampered by the current unavailability of conditional floxed animal models. The emergence of new tools such as genome editing technologies and advances in generation of transgenic murine models will aid in these efforts. Studies investigating the roles of these RLR agonists utilizing RIG-I, MDA5 and LGP2 knockout mice, potentially in specific cell subtypes, will aid in unravelling the complex functions of RLR signaling in regulating anti-tumor and antiviral adaptive immune responses. Further understanding of the requirement for each RLR in specific subtypes will aid in development and refinement of specific RLR agonists for optimal context-specific immune activation.