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How viruses use the immune system to promote infection of polarized cells

Nicola F Fletcher* ABSTRACT: To establish infection and access bodily compartments including the gut, lung, liver and brain, viruses must traverse polarized epithelial and endothelial cell sheets. Many viruses use components of the immune system to successfully infect epithelial cells and gain access to underlying tissue. Recently, several reports have highlighted new and surprising ways by which viruses can hijack the immune system to invade polarized cells. This review will summarize recent advances in our understanding of how viruses interact with the immune system, and with polarized cells, for successful infection. These studies raise important questions about the design and screening of therapeutics and vaccines that activate the immune system, which may need to consider the role of immune cells and the inflammatory microenvironment. Many viruses gain entry to the body, and to individual organs, by traversing polarized epithelial and endothelial cell sheets. These specialized cells restrict the passage of substances, including pathogens, both into and out of organs due to the presence of apical and basolateral membrane domains that express distinct receptors on the apical and basolateral surfaces. Furthermore, the cells are connected via tight junctions, which regulate the paracellular transport of immune cells, macromolecules and pathogens. Tight junctions are the most apical element of the junctional complex and are formed of the transmembrane proteins occludin, claudins and junctional adhesion molecule (JAM) proteins that are connected to scaffolding proteins such as zonula occludens (ZO)-1 and -2. These scaffolding proteins organize the transmembrane proteins, link them to the actin cytoskeleton and facilitate signaling between the junctional complex and the interior of the cells [1] . Below the tight junctions lie adherens junctions, which are formed of the transmembrane proteins cadherin and nectins. Tight and gap junctions, together with desmosomes, form the ‘epithelial junctional complex’ [2] . Polarized epithelial and endothelial cell sheets are present in various bodily sites, including the gut, lung, liver and brain. Gut and lung epithelia form barriers between the gut lumen and airway, and the underlying bloodstream, respectively (Figure 1) . Hepatocytes of the liver form complex polarity, with the basolateral face of the cells in contact with fenestrated endothelium and with many cells sharing an apical surface to form bile canaliculi. The brain is an immune-privileged site that is protected by the blood–brain barrier (BBB) and blood–cerebrospinal fluid (CSF) barriers, which are formed of polarized endothelial and epithelial cells, respectively. Viruses have evolved many mechanisms to overcome these barriers and gain access to underlying tissue. Recent studies highlight several novel mechanisms by which diverse viruses both invade and infect polarized epithelial and endothelial cells. This review will highlight some recent examples of ways that viruses use cellular components and hijack elements of the adaptive immune response to gain access to, and subsequently promote, infection of target cells.

KEYWORDS

• chemokine • cytokine • innate immunity • occludin • tight junction

*Veterinary Sciences Centre, University College Dublin, Ireland; and Centre for Human Virology, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK; Tel.: +44 121 4146845; Fax: +44 121 4143599; [email protected]

10.2217/FVL.14.46 © 2014 Future Medicine Ltd

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Apical (bloodstream) BBB endothelial cells TJ AJ GJ Basolateral (brain parenchyma)

Apical (lumen) TJ AJ GJ

Epithelial cells (e.g., gut and lung)

Basolateral (bloodstream)

Basolateral (sinusoid)

GJ AJ TJ Hepatocytes Apical (bile canaliculus) Basolateral

Figure 1. Morphology or polarized cells. (A) Polarized endothelial cell, (C) simple columnar epithelial cell and (C) hepatocyte morphology. AJ: Apical junction; BBB: Blood–brain barrier; GJ: Gap junction; TJ: Tight junction.

Viruses use novel mechanisms to infect polarized epithelia Epithelial tight junctions are important barriers to prevent the dissemination of viral infections through the body, and ultimately, spread to other hosts. While many viruses are effectively excluded from sites such as the brain, due to the presence of the BBB, other viruses have evolved diverse mechanisms to traverse epithelial barriers (Figure 1) . Direct infection of epithelial cells, which may or may not result in tight junction perturbation, is the mechanism by which some viruses overcome epithelial barriers. Infection occurs through viral binding to receptors located on the apical or basolateral face of the epithelium, and some viruses use tight junction proteins to infect epithelia. Coxsackie B virus (CBV) and

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adenoviruses use the coxackie and adenovirus receptor (CAR) for viral attachment, which is located at the tight junction in polarized cells [3] . In addition, the tight junction protein, occludin, is required for CBV and hepatitis C virus (HCV) entry [4,5] . CBV does not directly interact with occludin during viral entry, and the HCV envelope protein, E2, does not directly interact with occludin during viral entry [6] . It is surprising that viruses have evolved to use tight junctions as viral receptors, given that these proteins are relatively inaccessible from either the apical or basolateral side of epithelial cells [7] and, moreover, in vitro studies reveal that polarization restricts viral entry to epithelial cells [8,9] . Recent studies investigating the kinetics of HCV infection of primary human hepatocytes (PHHs) revealed

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How viruses use the immune system to promote infection of polarized cells that bile canaliculi development increased over time and, moreover, occludin localized to bile canaliculi together with multidrug resistance transporter-2 (MRP-2), indicating functional tight junction development in these cells [10] . Interestingly, the development of tight junctions was associated with a reduction in HCV entry to PHH, which is in agreement with previous studies demonstrating that polarization reduces HCV entry to HepG2 hepatoma cells [8] . Moreover, polarization also restricts HCV entry to BBB endothelial cells [11] , indicating that viral access to one or more HCV entry factors is restricted in both polarized hepatocytes and endothelial cells. CBV initially engages an apically expressed receptor, decay accelerating factor (DAF), and this triggers intracellular signals that lead to actin rearrangement and movement of virus to the tight junction where it binds to CAR [3] . However, as CBV particles internalize into the cell, they are closely associated with occludin and the small GTPases Rab34 and Rab5, highlighting that CBV entry occurs by a process that combines aspects of caveolar endocytosis with features characteristic of macropinocytosis [4] . These data highlight a mechanism by which CBV directly facilitates viral engagement with the tight junction-expressed receptor CAR, followed by virus internalization in association with occludin, by binding to the apically expressed receptor, DAF. Similarly, HCV entry to permissive cells is dependent on the second extracellular loop (ECL2) of occludin, although occludin ECL2 did not associate with the E2 protein of HCV but with dynamin II, an important GTPase required for endocytosis, indicating that HCV and EBV may interact in similar ways with occludin during viral entry [6] . HCV requires four essential entry factors for viral entry: the tetraspanin CD81, scavenger receptor BI (SR-BI) and the tight junction proteins, claudin-1 and occludin [5,12,13] . HCV glycoproteins initially engage SR-BI and CD81 on the cellular surface [14] and, subsequently, HCV–CD81–claudin-1 complexes internalize via a clathrin- and dynamin-dependent process [15,16] . Importantly, claudin-1 associates with CD81 at the basolateral membrane of polarized hepatocytes, whereas tight junction-associated pools of claudin-1 demonstrate a minimal association with CD81 [16] . However, the role of occludin in HCV entry is poorly understood. Unlike claudin-1, occludin is localized exclusively at the tight junction in polarized


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hepatocytes, raising questions on how HCV accesses occludin and whether there is any direct interaction between HCV envelope glycoproteins and occludin. It has been proposed that occludin plays a role in the late stages of HCV entry [17] , and recent studies have confirmed and extended this observation by demonstrating that an anti-occludin antibody inhibits HCV postbinding entry events [18] . However, the precise nature of the role of occludin in HCV entry, and how this virus accesses occludin at the tight junction, remain to be elucidated. Measles virus (MeV) infects peripheral immune cells, including T and B cells, and activated monocytes and dendritic cells, together with epithelial cells and cells of the central nervous system (CNS). The primary receptor for wild-type measles strains, CD150/SLAM, facilitates MeV infection of airway-resident macrophages and DCs. These cells then carry the virus across the airway epithelium where MeV disseminates to secondary lymphoid organs and viral replication takes place. Viral exit from the airway, and spread to other hosts, takes place via infection of the airway epithelium via an epithelial cell receptor, nectin-4 [19,20] . Nectin-4 is an adherens junction protein that is also expressed on the basolateral surface of airway epithelia. As MeV initially encounters airway epithelium via the apical (or air) side, where nectin-4 is not expressed, epithelial cell infection is not thought to take place during the initial stages of infection, but epithelial infection occurs during the late stages of infection when virus can access the basolateral cell surface. This highlights a novel mechanism by which MeV initially crosses the epithelium within trafficking, activated immune cells, and subsequently uses a basolaterally expressed receptor to directly infect the epithelium. Proinflammatory cytokines promote viral infection For successful infection, all viruses must evade the host immune system. Viruses have evolved diverse mechanisms of immune evasion, including inhibition of interferon responses, IL-10 mimics and manipulation of the host ubiquitin pathway [21–23] . Recently, several studies have shown that some viruses use proinflammatory cytokines to promote infection of polarized epithelial cells. Adenovirus-infected macrophages secrete IL-8 (CXCL8) that promotes adenovirus infection of polarized epithelium [24] . The

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Review Fletcher secretion of IL-8 was triggered by adenovirus infection, which in turn caused a re-localization of the adenovirus receptor, ανβ3 integrin, to the apical side of polarized human bronchial epithelial cells. This occurred in the absence of epithelial cell tight junction disruption; rather, CXCL8 activated a Src-family tyrosine kinase via the apical CXCR1 and CXCR2 receptors, which relocated ανβ3 integrin to the apical surface, and enabled apical binding and infection with adenovirus that was dependent on the primary adenovirus receptor, CAR. Recently, IL-1β and TNF-α were demonstrated to promote infection of polarized hepatocytes with HCV [9] . Peripheral bloodderived macrophages activated with bacterial lipopolysaccharide (LPS), flagellin or ssRNA40, which stimulate TLR4, -5 and -9, respectively, secreted high levels of TNF-α that promoted infection with HCV together with MeV, Lassa and vesicular stomatitis virus pseudoviruses. The increased levels of infection were associated with tight junction disruption and a re-localization of occludin to the basolateral membrane of the hepatocytes. Since hepatocyte tight junctions are located at the bile canaliculi, and it is thought that viruses first entering the liver, via the sinusoidal blood, encounter the basolateral membrane of hepatocytes, this likely facilitates viral binding and subsequent entry via enhanced receptor binding at the basolateral surface of the cells (Figure 1) . Viral entry, rather than translation or replication, was specifically enhanced following IL-1β and TNF-α treatment. This observation was not restricted to polarized hepatocytes; TNF-α promotes viral entry to polarized gut and lung epithelial cells and BBB endothelial cells in addition to hepatocytes [11] [Fletcher NF, Unpublished Data] . These examples highlight that viruses not only overcome the innate immune response for successful infection; they can also hijack elements of the innate immune system that are classically considered to be antiviral to promote their own infection. Immune cells act as ‘Trojan horses’ to facilitate viral invasion of tissues As part of normal immune surveillance, immune cells, including lymphocytes and monocytes, traverse epithelial cell sheets at various sites in the body. This occurs in both an apical-tobasolateral direction and vice versa, and occurs without significant tight junction disruption [25] . During inflammatory states, immune cells

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become activated and secrete proinflammatory cytokines such as IL-1β, TNF-α and IL-6 that disrupt tight junctions and facilitate immune cell invasion and pathogen clearance. Some viruses, such as HIV and its feline counterpart, feline immunodeficiency virus (FIV), gain entry to sites such as the brain within infected, trafficking lymphocytes, in a process known as the ‘Trojan horse’ mechanism [26,27] (Figure 1) . HIVinfected lymphocytes traverse the BBB, which then leads to infection of underlying astrocytes, microglia and perivascular macrophages (reviewed in [27]). Similarly, MeV first traverses polarized airway epithelia within activated, trafficking DCs and other immune cells, where it can then disseminate throughout the body and access the basolateral side of the epithelium for direct infection via the epithelial cell receptor, nectin-4 [19,20] . Recent studies have demonstrated that, in addition to immune cells trafficking virus across polarized epithelial cell sheets, lymphocytes can also transfer virus to permissive epithelial cells in a process termed ‘transfer infection’ (Figure 1) . Epstein–Barr virus (EBV) is a human herpesvirus that exhibits a distinct tropism for B cells and epithelial cells. EBV, present in salivary secretions, initially infects B cells through binding to the complement receptor, CD21, on the B-cell surface [28,29] , and fusion takes place through binding of the envelope glycoprotein, gp42, with HLA class II [30] . Virus can readily be detected in epithelial cells of oral hairy leukoplakia, as well as nasopharyngeal and gastric carcinoma [31] . However, epithelial cells do not express CD21 on their cellular surface, which raises questions on how epithelial cell infection takes place. EBV is normally transmitted to a new host via salivary secretions to the oropharynx, and so B cells normally first encounter epithelial cells via their basolateral surface [32] . Recent studies have highlighted that B cells can transfer virus to the basolateral surface of polarized epithelial cells [33] . This occurs via EBV-mediated firm adhesion of B cells to the epithelial cell surface, followed by ‘capping’ of the virus to one pole of the B cell. Viral transfer from the B cell to the epithelium takes place via a process that requires CD11b on the B-cell surface interacting with heparan sulphate moieties of CD44v3 and LEEP-CAM on epithelial cells. Consequently, transfer infection was mediated via CD11b-positive memory B cells but not by CD11b-negative naive B cells [32] . Importantly, transfer infection only occurs


How viruses use the immune system to promote infection of polarized cells when virus is delivered to the basolateral side of epithelial cells. Interestingly, cell-free infection of polarized epithelium, although inefficient, can occur in the absence of viral receptor expression on the epithelium, and the process by which this occurs has yet to be elucidated. HCV also infects hepatocytes via transfer of infection from activated B cells [34] . Interestingly, this occurs in the absence of viral infection of the B cell, in contrast to EBV, which replicates in B cells. Rather, viral particles bind to SR-BI, DC-SIGN and L-SIGN on the B-cell surface and are then temporarily internalized by B cells. HCV promotes the adhesion of B cells to the surface of hepatoma cells and transfer infection takes place. While hepatoma infection could be inhibited using antibodies directed against SR-BI and DC-SIGN/L-SIGN, B-cellassociated virus was resistant to HCV-specific neutralizing antibodies. This study indicates that B cells may provide a vehicle for HCV to evade humoral immune responses and transmit to the liver. Recently, elegant in vitro and in vivo studies have demonstrated, using GFP-expressing MeV, that immune cell-to-epithelial cell transfer of MeV occurs in the respiratory tract epithelium and the nasal epithelium of macaques [35] . Viral transfer is dependent on the expression of nectin-4 within the epithelium, and high levels of infection were observed in the ciliated pseudostratified columnar epithelial cells in the nasal cavity of macaques at the peak of infection, indicating a key role of these cells in MeV transmission. Furthermore, using a novel in vitro co-culture model of B cells with primary differentiated normal human bronchial epithelial cells grown as an air–liquid interface (ALI), transfer of infection was observed with wild-type virus but not with a nectin-4 ‘blind’ recombinant wild-type MeV, confirming the requirement for nectin-4 in transfer of infection to epithelial cells. These studies highlight a novel role for immune cells to transfer diverse viruses to polarized epithelium, which is an efficient mechanism for viruses to infect polarized cells, which capitalizes on the interactions between epithelium and trafficking immune cells, and which also protects viruses from neutralizing antibodies. Tight junction disruption facilitates viral transmigration across polarized cells Disruption of epithelial and endothelial tight junctions, by a variety of mechanisms, allows


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viruses and other pathogens to traverse polarized cells via a paracellular route (Figure 2) . While some viruses, such as HIV-1, are thought to invade bodily compartments such as the BBB following immune-mediated tight junction disruption (reviewed in [27]), other viruses directly drive tight junction disruption by a variety of mechanisms. West-Nile virus (WNV) is an arthropod-borne flavivirus that replicates through a dsRNA intermediate (reviewed in [36] ). This dsRNA binds to TLR3 on peripheral lymphoid cells, which stimulates TNF-α production. TNF-α and other proinflammatory cytokines such as IL-1β activate a canonical NF-κB pathway in polarized cells that leads to myosin light-chain kinase activation, a reorganization of perijunctional F-actin and relocalization of tight junction proteins away from the tight junction, resulting in tight junction disruption [37,38] . This transient depolarization of BBB endothelial cells allows WNV to cross the BBB via a paracellular route, without infecting the BBB, and enter the CNS, followed by viral replication in neuronal cells [39] . More recently, matrix metalloproteinases (MMPs), which can disrupt BBB endothelial cells, have been shown to be elevated in WNV infection, and this study also confirmed that astrocytes were a source of MMP [40] . WNV-induced MMP also reduces expression of the junctional proteins ZO-1, claudin-1, occludin and JAM-A at the BBB [41] . Since astrocytes play a critical role in the maintenance of BBB integrity, and immune cell infiltration to the brain takes place during WNV infection, it is possible that initial infiltration of virus across the BBB during early infection results in exacerbated BBB disruption and further infiltration of virus and immune mediators to the brain. Rhinovirus can directly induce airway epithelial cell disruption that is reactive oxygen species (ROS) dependent [42] . Rhinovirus dsRNA binds to a novel receptor, Nod-like receptor X-1 (NLRX-1), that is expressed on polarized airway epithelium. This stimulates mitochondrial ROS, which drives tight junction disruption and a relocalization of junctional proteins away from the tight junction, thus facilitating rhinovirus invasion of the lung. These studies demonstrate that, in addition to viruses taking advantage of a pre-existing inflammatory microenvironment and barrier disruption for paracellular transport across polarized epithelium and the BBB, some viruses can directly orchestrate barrier disruption to promote invasion of bodily compartments.


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Infection using apical, basolateral or tight junction protein (HCV, EBV, MeV, adenovirus)

Tight junction disruption by activated monocytes/macrophages, facilitating infection via cytokines (HCV) or paracellular transcytosis (WNV)

Viral infection of trafficking leucocyte (HIV) B-cell transfer infection (EBV, HCV)

TNF-α IL-1β

Tight junction proteins

Transcytosis (EBV)

Figure 2. Mechanisms of viral infection and transport across polarized epithelial and endothelial cells. Viruses must traverse polarized epithelial cells at various sites in the body in order to gain access to underlying tissues. (A) Some viruses directly infect polarized cells via receptors expressed on the apical or basolateral side of the cells, or via tight junction proteins. (B) Inflammatory cytokines, such as IL-1β and TNF-α, which are secreted by activated immune cells, disrupt tight junction integrity. This facilitates direct infection of epithelial cells by HCV, or paracellular transport across polarized cells and access to underlying tissue, which is exploited by WNV. (C) Viral transport across polarized epithelium and endothelium can occur via transcellular transport. (D) Viruses that infect immune cells can cross polarized barriers within activated, trafficking leukocytes. (E) Direct transfer from immune cells to epithelial and endothelial cells. EBV: Epstein–Barr virus; HCV: Hepatitis C virus; MeV: Measles virus; WNV: West Nile virus.

Clinical implications Vaccine strategies frequently make use of replication-deficient viral vectors derived from viruses such as adenovirus, poxvirus and vaccinia virus [43] . Viral vectors have been used to develop vaccines against diverse pathogens including Japanese encephalitis virus (Imojev, Sanofi-Pasteur) [44] and efforts to develop a vaccine against HIV [45] and, more recently, to deliver zinc-finger nucleases that knock out CCR5 to patient T cells as a potential HIV therapy [46] , have used viral vectors. Viral vectors potently activate the innate immune system [47] and, given that viruses such as adenovirus, which are commonly used as viral vectors, can subvert the innate immune response to promote their own infection [24] , this should be considered when evaluating host responses to these vaccine design approaches. Anti-TNF monoclonal antibody therapy has been used to treat HCV-infected patients with

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chronic inflammatory conditions such as rheumatoid arthritis and Crohn’s disease [48] . One Phase II trial reported that the anti-TNF-α antibody, etanercept, in combination with interferon and ribavirin significantly reduced HCV replication, with undetectable levels of HCV RNA in 63% of etanercept-treated patients after 24 weeks, compared with 32% of patients receiving interferon and ribavirin therapy [49] . The finding that TNF-α can promote HCV infection provides a possible mechanism for these findings, and suggests that restoration of hepatocyte junctional integrity may restrict HCV infection in vivo. Future perspective Recent studies have highlighted many novel ways that diverse viruses can hijack the innate and adaptive immune responses to promote infection of polarized epithelial and endothelial cells. Epithelial cell sheets exist to restrict the


How viruses use the immune system to promote infection of polarized cells passage of substances, including pathogens, into and out of various bodily compartments, including the gut and airway, which protect these sites from constant exposure to pathogens. In addition, organs including the brain are regarded as ‘immune-privileged sites’, and the passage of substances into the CNS are strictly controlled by the BBB and blood–CSF barriers. Many host cellular responses are exploited by viruses to gain access to these tissues. These studies highlight the importance of including immune components when modeling viral infections both in vitro and in vivo. These studies have implications for our understanding of pathogen interactions with the host immune system, and highlight that viral infection models should consider the host immune response. Many in vitro studies use single cell types to study viral infection, and small animal models, in many

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cases, use immunodeficient animals. Recent studies raise important questions about the design and screening of future antiviral therapeutics and vaccines such as the use of replicationincompetent adenoviral vectors that activate the immune system. These therapeutic approaches may need to consider the role of immune cells and the inflammatory microenvironment. Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

EXECUTIVE SUMMARY Viruses use novel mechanisms to infect polarized epithelia ●●

Viruses have evolved diverse mechanisms to traverse epithelial barriers.

●●

Infection occurs through viral binding to receptors located on the apical or basolateral face of the epithelium, and some viruses use tight junction proteins to infect epithelia.

●●

Recent studies highlight that measles virus initially traverses lung epithelia via trafficking immune cells, but

subsequently encounters epithelia via the basolateral face, where it uses the basolaterally expressed receptor, nectin-4, for epithelial infection and dissemination. Proinflammatory cytokines promote viral infection ●●

Recently, several studies have shown that some viruses use proinflammatory cytokines to promote infection of polarized epithelial cells.

●●

Adenovirus-infected macrophages secrete IL-8 (CXCL8), which re-localizes the adenovirus receptor, ανβ3 integrin, to the apical side of polarized human bronchial epithelial cells, thereby promoting infection.

●●

IL-1β and TNF-α promote hepatitis C virus infection by disrupting hepatocyte tight junctions and promoting occludin re-localization to the basolateral membrane of the cells, thus promoting infection.

Immune cells act as ‘Trojan horses’ to facilitate viral invasion of tissues ●●

Some viruses gain entry to sites such as the brain within infected, trafficking lymphocytes, in a process known as the ‘Trojan horse’ mechanism.

●●

Recent studies have demonstrated that lymphocytes can also transfer viruses to permissive epithelial cells in a process termed ‘transfer infection’.

Tight junction disruption facilitates viral transmigration across polarized cells ●●

Recent studies have demonstrated that West Nile virus-induced TNF-α and matrix metalloproteinases promote blood–brain barrier tight junction disruption that facilitates West Nile virus invasion of the brain.

Clinical implications ●●

Viral vectors potently activate the innate immune system and, given that viruses used to generate viral vectors can

subvert the innate immune response to promote their own infection, this should be considered when evaluating host responses to these vaccine design approaches.



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