Receptors of the Innate Immune System Detect Conserved Determinants of Viral Origin

The innate immune response is the first line of defense against infectious disease. The principal challenge for the host is to detect the pathogen and mount a rapid defensive response. Receptors of the innate immune system detect conserved determinants of viral origin. Activation of these receptors initiates signaling events that culminate in an effective immune response. The first line of cellular defense is not the specialized immune cells but is the non-immune cells. Non-immune cells have traditionally been viewed as a physical barrier. However, the discovery that many of the functions traditionally attributed to immune cells are also performed by non-immune cells has caused a shift to a multidirectional hypothesis in which non-immune cells play active roles.

The surprising abilities of non-immune cells are now becoming clearer when non-immune cells also recognize viral nucleic acid and initiate an innate immune response. These new observations highlight the importance of non-immune cells in innate immunity against viral infection, which is the focus of this review paper.

Introduction

Viral infection constitutes a significant portion of human morbidity and mortality, which has led to extensive investigation into the countermeasures employed by the host to combat such infection (1). Our innate immune system has evolved a range of receptors to detect viral pathogens. However, viruses have adapted strategies to evade or even inhibit key elements of host immune responses and make innate immune system carrying a substantial burden of the defense against viral pathogens (2). The smart host also has evolved means of activating a broad range of defense mechanisms to ensure effective protection. In the midst of this power struggle, a remarkable picture is emerging of the importance of non-immune cells for antiviral defense. The non-immune cells express a variety of pattern recognition receptors (PRRs) to elicit the expression of the type Ⅰ and Ⅲ interferons (IFNs), cytokines, and chemokines protected itself and innate immune cells from virus infection. In this paper, we review the recent development in no-immune cells-mediated innate immunity and the significance of these new observations.

Innate immunity and IFN responses

The innate immune system has evolved over millennia to nonspecifically control and eliminate potentially infectious threats. Unlike the adaptive immune system, which is composed of highly specialized receptors to recognize foreign antigens, the innate immune system uses a series of PRRs to detect patterns associated with bacteria, viruses, and/or parasites. The intracellular innate antiviral response in human cells is an essential component of immunity against virus infection. As obligate intracellular parasites, all viruses must evade the actions of the host cells innate immune response in order to replicate and persist. Innate immunity is induced when PRRs of the host cell sense viral products including nucleic acid as “non-self”. Five classes of PRRs have been identified to date, including the Toll-like receptors (TLRs), RIG-I like receptors (RLRs), NOD-like receptors (NLRs), the AIM2-like receptors (ALRs) and C-type lectin receptors (CLRs) (Table 1) (3).  An important part of a PRR-induced innate immune response is transcriptional, which leads to the production of inflammatory cytokines and IFN; these chemical messages are critical for initiating innate and adaptive immune responses. PRRs activation also initiates non-transcriptional responses such as the induction of phagocytosis, autophagy, cell death, and cytokine processing (4, 5). The coordination of these immune responses, which contain the spread of an initial infection and direct the appropriate adaptive response (6).

Table 1 Pattern-recognition receptor families

IFN are secreted cytokines that impact numerous host processes and are well known for their antiviral properties. Among the three main IFN families, Type I (IFN-α/β) and III (IFN-λ) IFNs are considered the primary antiviral IFNs. IFNs bind their cognate receptors, and initiate signaling through the JAK/STAT pathway. The result is the transcriptional induction of interferon-stimulated genes (ISGs), which encode direct antiviral effectors or molecules with the potential to positively and negatively regulate IFN signaling and other host responses. For the ISGs, their mechanisms generally target conserved aspects of viral infection. Examples include ISGs that inhibit virus entry (Myxovirus resistance (Mx), IFITM proteins, and TRIM proteins), virus translation and replication (IFN-induced protein with tetratricopeptide repeats (IFIT) family, the OAS-RNase L pathway, and PKR), viral egress (Tetherin and Viperin) (7).

Function of Innate Immune Cells and Evasion by Viruses

The first line of the host defense is provided by the innate immune system and comprises cell types such as circulating dendritic cells (DCs), natural killer (NK) cells, monocytes, along with tissue-resident mast cells and macrophages (8). These cells that express PRRs control opportunistic invasion by a wide range of viral pathogens,  in part by releasing cytokines and chemokines to recruit and activate adaptive immune cells including T and B cells and thereby to orchestrate immune responses (9).  Our previous studies showed that macrophages expressed functional TLR3 and RIG-I, activation of which resulted in induction of type Ⅰ and Ⅲ IFNs, ISGs and CC chemokines significantly and inhibited  HIV infection and replication (10, 11). NK cell-mediated anti-HCV activity showed that NK cells culture supernatants-treated hepatocytes expressed higher levels of IFN-α/β (12). Several studies showed that plasmacytoid DCs (pDCs) recognize dengue virus (DENV) via TLR7 in endocytic vesicles (13), become activated, produce high amounts of IFN-α (14) and may thus limit DENV replication. Further, pDCs sense DENV-infected cells by direct cell-to-cell contact and immature DENV particles were found to trigger higher IFN responses in pDCs (14).

However, to secure persistence or latency, many viruses dedicate a substantial part of their genomes to down-modulating the IFN pathways. A general mechanism used by several viruses is inhibition of cellular gene expression by inhibiting transcription, RNA processing, and/or translation (2). Virus-induced shutoff of cellular protein expression prevents the synthesis of IFN and of ISG products. Most viruses also encode viral products that specifically target pathways involved in the response to IFN. For example, HIV blocks IFN Induction in DCs and macrophages by dysregulation of TANK-binding kinase 1(TBK1) via two different viral accessory proteins (vprandvif) (15). HIV also downregulate the transcription of known anti-viral ISGs (IFI44, ISG15, and OAS1) in primary macrophages (16). In addition, inhibition of type I IFN protein translation was observed during Zika virus (ZIKV) infection of DCs. ZIKV not only antagonized type I IFN-mediated phosphorylation of STAT1 and STAT2 but also subverted DCs immunogenicity during infection (17). Intriguingly, V proteins of several paramyxoviruses, and the NS1 and NS2 proteins of the respiratory syncytial virus, have the remarkable property of inhibiting both IRF3 and STAT activation. Antagonism of type 1 IFN-inducible genes and their products (18).

Non-Immune Cells Have Functional PRRs and Produce IFNs and ISGs

Most viruses need to establish a solid infection and to provide an outcrop of progeny virus for host-to-host transmission, they have learned to cope with the IFN system.  How can non-immune cells deal with this situation in the presence of a damaged innate immune defense? The answer is that non-immune cells have functional PRRs and produce IFNs and ISGs. Non-immune cells are the most abundant and widely distributed cell type in the body, such as epithelial cells, endothelial cells, and fibroblasts, among others, also contribute to immunity development (19). Thus, the outcome of the immune response in a target tissue depends not exclusively on the immune cells but also on the intricate network and signals given by immune and non-immune cells. Of the PRRs characterized to date, several have been linked to antiviral immunity. Among these, TLR3, TLR7, TLR8, TLR9 and RIG-I/MDA5 detect distinct forms of viral nucleic acids and are critical in the recognition of viral genetic materials in endolysosomal compartments and initiate antiviral responses. For example, TLR3 has a relatively wide tissue distribution, with its transcripts detected in many human tissues such as the placenta, lung, liver, heart, lymph node, spleen, and brain (20). TLR3 protein is expressed by key sentinel cells of the innate immune system in non-immune cells including epithelial cells, fibroblasts, astrocytes, hepatocytes, and endothelial cells (21-23).  In addition, type I IFNs receptor complex (IFNAR1 and IFNAR2) have a broad tissue distribution, as a result, nearly all cells are capable of responding to type I IFNs (24). Type Ⅲ IFNs receptor complex (IFNLR1 and IL-10Rβ) also have widely distributed across cell type. Although IFNLR1 and IL10Rβ are expressed broadly on many cell types and tissues, IFNLR1 is expressed preferentially on epithelial cells (25). Consistent with this pattern, the antiviral effects of Type Ⅲ IFNs are most evident against pathogens targeting epithelial tissues (26).  Recent studies provide evidence that the liver has specific immunological properties and contains a large number of resident and nonresident cells that participate in immune responses (27). Although Kupffer cells are considered the primary cells to respond to PAMPs, supporting our results, multiple populations of nonhematopoietic liver cells including hepatic stellate cells (28) and hepatocytes (29) express functional TLR3 and lead to the induction of IFNs and ISGs. In addition, TLR3 and RIG-I on brain microvascular endothelial cells and neuronal cells in the central nervous system (CNS) have been demonstrated to be involved in innate immunity against viral infection (30-32). Furthermore, intestinal epithelial cells (IECs) express both RIG-I and MDA5, which is required for the activation of IFN-β production by rotavirus infected IECs and has a functionally important role in determining the magnitude of rotavirus replication in the intestinal epithelium (33).

Self- and joint- defense against viral infection of non-immune cells

As we’ve described, multiple offensive/defensive mechanisms have evolved to result in a balance for the coexistence of hosts and viruses, and type Ⅰ /Ⅲ IFNs have emerged as pivotal in this conflict. Since non-immune cells have functional PRRs and produce IFNs and ISGs, it will be of interest to see whether the non-immune cells directly or indirectly involved in the antiviral activity (Table 2).

Table 2 Self- and joint- defense against viral infection of non-immune cells:

On the one hand, as viral target cells, non-immune cells showed strong self-defense mechanism against viral infection.  In the gastrointestinal (GI) system, type I IFN produced by IECs in response to RIG-I and MDA5 sensing of rotavirus infection and signaling through MAVS, has a pivotal functional role in determining the amount of virus produced by the intestinal epithelium (34). Moreover, In EV71-infected IECs, TLR/TRIF signaling was essential to IFNs induction; viral replication increased and the induction of IFN-α, -β, -ω, -κ, and -ε decreased markedly in TRIF-silenced IECs (35). It was reported that dsRNA receptor TLR3 acts to induce both RIG-I and MDA5 gene and protein expression in human bronchial epithelial cells in a model of RV infection. Both RIG-I and MDA5 were required for maximal IFN and pro-inflammatory cytokine induction, and control of RV replication indicating that they have non-redundant roles in RV infection (36). Lebre et al. have demonstrated that activation of TLR3/9 by human keratinocytes leads to a predominant T-helper type Ⅰ immune response and to the production of type I IFN, which have both been implicated in eliciting cell-mediated immunity against HPV (37). During HSV infection, we have reported, human neuronal cells expressed TLR family members 1-10 and IFN-α/β. The activation of TLR3 or TLR8 by poly I: C or single-stranded RNA (ssRNA) induced IFN-α/β expression. In addition, HSV-1 infection of human neuronal cells induced IFN-α expression. Investigation of the mechanisms showed that poly I: C or ssRNA treatment enhanced the expression of several IFN regulatory factors. Importantly, the activation of TLR3 or TLR8 by poly I: C or ssRNA prior to HSV-1 infection reduced the susceptibility of the neuronal cells to infection.  We also showed that human cervical epithelial cells expressed functional TLR3 and RIG-I, which could be activated by poly I: C and 5’-ppp dsRNA, resulting in the induction of endogenous IFN-λ. The induced IFN-λ contributed to TLR3/RIG-I-mediated inhibition of HSV-2 replication in human cervical epithelial cells. Further studies showed that TLR3/RIG-I signaling in the cervical epithelial cells by dsRNA induced the expression of the ISGs: ISG56, OAS-1and MxA, the key antiviral elements in the IFN signaling pathway. In addition, we observed that the topical treatment of genital mucosa with poly I: C could protect mice from genital HSV-2 infection (41). TLR3 activation results in the production of type I/Ⅲ IFNs in different cell types. Wieland et al. showed that TLR3 ligand poly I: C induces intrahepatic IFN-β production and inhibits HBV replication by non-cytolytic mechanisms that either destabilize pregenomic (pg) RNA-containing capsids or prevent their assembly (53). A great number of ISGs are activated by IFNs and may inhibit the different steps of the HBV life cycle (54). A recent publication suggests that HBV covalently closed circular (ccc) DNA could be degraded by the action of APOBEC3A/B cytidine deaminases (55). Our recent study showed that poly I: C could activate TLR3 and induce IFNs expression in Huh7.5.1 cells, and inhibit HCV replication by more than 90% (46). In the respiratory system, signaling through RIG-I or TLR3 is important for IFN induction by IAV and RSV in human lung alveolar epithelial cell and tracheobronchial epithelial cell.

On the other hand, to circumvent this inhibition, infected cells can transfer viral dsRNA extracellularly to uninfected bystander cells, thereby promoting IFN-I/Ⅲ production and limiting viral replication in vitro (56, 57). Our accumulative evidence demonstrates that, as a bystander cells, human brain microvascular endothelial cell (30), intestinal epithelial cell and cervical epithelial cell (unpublish data) possess functional TLR3, the activation of which resulted in induction of key IFN regulatory factors (IRF3 and IRF7), IFN-β, IFN-λ and CC chemokines (MIP-1α, MIP-1β, RANTES), the ligands of HIV entry co-receptor CCR5. TLR3-activated IECs release exosomes that contained the anti-HIV factors, including IFN stimulated genes (ISGs: ISG15, ISG56, MxB, OAS-1, GBP5, and Viperin) and HIV restriction miRNAs (miRNA-17, miRNA-20, miRNA-28, miRNA-29 family members and miRNA-125b). Importantly, treatment of macrophages with supernatant (SN) from the activated cell cultures inhibited HIV replication. Further studies showed that IEC SN could also induce the expression of antiviral ISGs and cellular HIV restriction factors (Tetherin and APOBEC3G/3F) in HIV-infected macrophages. Recent studies provide evidence that IFN-α induced the transfer of resistance to HBV from nonpermissive liver nonparenchymal cells (LNPCs) to permissive hepatocytes via exosomes. Exosomes from IFN-α-treated LNPCs were rich in molecules with antiviral activity. Moreover, exosomes from LNPCs were internalized by hepatocytes, which mediated the intercellular transfer of antiviral molecules (50). We also showed that hepatic stellate cell (HSC) can produce antiviral factors that inhibit HCV replication in human hepatocytes. When HCV JFH-1-infected Huh7 cells were co-cultured with hepatic stellate cells activated with poly I: C or incubated in media conditioned with SN from poly I: C-activated HSC, HCV replication was significantly suppressed. This HSC SN action on HCV inhibition was mediated through IFN-λ. The role of IFN-λ in HSC-mediated anti-HCV activity is further supported by the observation that HSC SN treatment induced the expression of IRF-7 and ISGs: OAS-1 and MxA in HCV-infected Huh7 cells (51).

Conclusion

As innate immunity research becomes better characterized, non-immune cells are coming to light as respected contenders in the pathophysiology of viral infection. Intricate networks of immune and non-immune cell interactions concurrently initiate and sustain both innate and adaptive immune responses. The networks elucidated thus far probably represent the proverbial “tip of the iceberg”, and the non-immune cell is now important to engage the powerful “ordinary people” to control viral infection. Nonetheless, it has already become abundantly obvious that nonimmune cells are not merely self-defense against viral infection but are also joint-defense against viral infection in innate immune cells (Figure 1). Therefore, non-immune cell emerge as potentially crucial and alternative targets for therapeutic intervention.

Figure 1. Schema of the Self- and joint- defense against viral infection of non-immune cells. Virus-induced shutoff of cellular protein expression prevents the synthesis of IFN and of ISG products in innate immune cells. The sensors for detecting viral products include components in the cytoplasm that are particularly sensitive to viral blocks, initiating the transcription of type Ⅰand Ⅲ IFN, cytokines and chemokines, and releasing exosomes in the non-immune cells.  IFNs bind their cognate receptors, and initiate signaling through the JAK/STAT pathway. The result is the transcriptional induction of interferon-stimulated genes (ISGs), which encode direct antiviral effectors