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Interferon Lambda: Modulating Immunity in Infectious Diseases

Mohammedyaseen Syedbasha and Adrian Egli

 Interferon lambdas (IFN-λs; IFNL1-4) modulate immunity in the context of infections and autoimmune diseases, through a network of induced genes. IFN-λs act by binding to the heterodimeric IFN-λ receptor (IFNLR), activating a STAT phosphorylation-dependent signaling cascade. 
Thereby hundreds of IFN-stimulated genes are induced, which modulate various immune functions via complex forward and feedback loops.

 When compared to the well-characterized IFN-α signaling cascade, three important differences have been discovered. 

• First, the IFNLR is not ubiquitously expressed: in particular, immune cells show significant variation in the expression levels of and susceptibilities to IFN-λs. 
•  Second, the binding affinities of individual IFN-λs to the IFNLR varies greatly and are generally lower compared to the binding affinities of IFN-α to its receptor.
•  Finally, genetic variation in the form of a series of single-nucleotide polymorphisms (SNPs) linked to genes involved in the IFN-λ signaling cascade has been described and associated with the clinical course and treatment outcomes of hepatitis B and C virus infection.

 The clinical impact of IFN-λ signaling and the SNP variations may, however, reach far beyond viral hepatitis. Recent publications show important roles for IFN-λs in a broad range of viral infections such as human T-cell leukemia type-1 virus, rotaviruses, and influenza virus. IFN-λ also potentially modulates the course of bacterial colonization and infections as shown for Staphylococcus aureus and Mycobacterium tuberculosis. 

 Although the immunological processes involved in controlling viral and bacterial infections are distinct, IFN-λs may interfere at various levels: as an innate immune cytokine with direct antiviral effects; or as a modulator of IFN-α-induced signaling via the suppressor of cytokine signaling 1 and the ubiquitin-specific peptidase 18 inhibitory feedback loops.

 In addition, the modulation of adaptive immune functions via macrophage and dendritic cell polarization, and subsequent priming, activation, and proliferation of pathogen-specific T- and B-cells may also be important elements associated with infectious disease outcomes. 

This review summarizes the emerging details of the IFN-λ immunobiology in the context of the host immune response and viral and bacterial infections.

Type I and III Interferon in the Gut: Tight Balance between Host Protection and Immunopathology

Johanna Pott and Silvia Stockinger

 The intestinal mucosa forms an active interface to the outside word, facilitating nutrient and water uptake and at the same time acts as a barrier toward the highly colonized intestinal lumen. A tight balance of the mucosal immune system is essential to tolerate harmless antigens derived from food or commensals and to effectively defend against potentially dangerous pathogens. Interferons (IFN) provide a first line of host defense when cells detect an invading organism. Whereas type I IFN were discovered almost 60 years ago, type III IFN were only identified in the early 2000s. 

It was initially thought that type I IFN and type III IFN performed largely redundant functions. However, it is becoming increasingly clear that type III IFN exert distinct and non-redundant functions compared to type I IFN, especially in mucosal tissues. Here, we review recent progress made in unraveling the role of type I/III IFN in intestinal mucosal tissue in the steady state, in response to mucosal pathogens and during inflammation.

Introduction

The intestinal tract is a major entry site for viruses and bacteria. Mucosal innate and adaptive immune cells are equipped to respond to and fight invading pathogens. At the same time, the intestinal lumen is densely colonized by commensal microflora, which at steady state does not provoke an exacerbated inflammatory response. 

The intestinal lumen is separated from the underlying sterile lamina propria harboring the body’s largest immune cell compartment by a single layer of polarized intestinal epithelial cells (IECs). This epithelial cell layer undergoes rapid and perpetual self-renewal without disrupting the functional integrity of cell–cell junctions. In addition, IECs not only form a passive physical barrier but also participate actively in the immune response against major enteric pathogens and cross talk with the commensal flora (1, 2). However, pathogenic viruses, bacteria, and parasites exploit opportunities for breaching the epithelial barrier.

Upon infection, host cells communicate by means of production and secretion of signaling molecules. Interferons (IFN) are a large family of cytokines with diverse functions during a successful host defense. The family of type I IFN comprises more than 20 members with multiple IFN-α and one IFN-β being the most important. Classically, the most prominent function of type I IFN is to induce antiviral immunity, whereas IFN-γ, the only type II IFN, promotes the response to intracellular bacteria. However, a vast amount of studies has found that type I IFN are also produced during bacterial infection. In contrast to their action in viral infections, their activity against bacteria can be either favorable or detrimental for the host (3–6).

Recently, a novel family of IFN, the type III IFN or IFN-λ family, was described (7, 8). This family consists of IFN-λ1, IFN-λ2, IFN-λ3 (also called IL-29, IL-28A, and IL-28B), and IFN-λ4 in humans, whereas mice have only two functional genes encoding IFN-λ (Ifnl2 and Ifnl3) and two Ifnl1 pseudogenes (9). Similar to type I IFN, type III IFN are induced by viral infection and show antiviral activity. However, they are structurally distinct from type I IFN and interact with a heterodimeric class II cytokine receptor consisting of the IFN-λR1 (also called IL-28Rα) chain in complex with the IL-10R2 chain, opposed to the type I IFN receptor (IFNAR).

A number of studies have addressed the functional importance of type III IFN compared to type I IFN in the context of viral infections (10–15). Less is known about the role of type I IFN and almost nothing on the role of type III IFN in the host defense against bacterial enteropathogens, intestinal homeostasis, and colitis. Therefore, we review recent progress made on the importance of type I and III IFN during enteric viral infections and focus on the role of type I IFN in the intestinal mucosal tissue during steady state, in response to bacterial infections and during inflammation.

 Induction of Type I and III IFN

The induction of type I and III IFN has been recently reviewed elsewhere (16), therefore we will only briefly summarize the major mechanism leading to IFN expression. Virtually all cells are equipped with the machinery to recognize viral infection and express type I and III IFN in response. Similar stimuli and pathways lead to the expression of type I and III IFN; however, differences between cell types as well as in magnitude and kinetics have been described (14, 16, 17). Comparable expression patterns of type I and III IFN result from a similar requirement of transcription factors for the expression of their encoding genes, such as IFN regulatory factors (IRFs) and NF-κB. There are however some differences in the promoter region, with IFN-β expression relying on the binding of the constitutively expressed IRF-3 to its promoter, which allows rapid induction. By contrast, IFN-α requires IRF-7 binding, which is an interferon-stimulated gene (ISG) itself and needs to be upregulated in most cell types following infection (3). Type III IFN are more dependent on the activation of NF-κB (18) and require the combined action of IRFs and NF-κB for full induction (19–21).

During systemic viral infections hematopoietic cells are the major source of type I IFN. Plasmacytoid dendritic cells (pDCs), which are designated as being the “professional” type I IFN-producing cells, produce large amounts in response to a wide range of viruses, parasites, and bacteria and are particularly important in the early phase of type I IFN production (22–24). However, depending on the infectious agent, myeloid cells are also involved in systemic type I IFN production. During systemic Listeria infection, the vast amount of systemic IFN-β production is independent of pDCs but seems to be produced by LysM-Cre-expressing macrophage/monocyte-like cells including TipDCs but not neutrophils (25–27).

 In the intestinal lamina propria, dendritic cells (DCs) as well as mononuclear phagocytes produce IFN-β and IFN-α5 in the steady state (14, 23, 28).

Epithelial cells are thought to be the major producer of type III IFN at steady state and during enteric viral infection, while lamina propria leukocytes (LPLs) also produce type III IFN under certain conditions (14, 29). 

Intraepithelial lymphocytes (IEL) produce IFN-α and IFN-λ upon TCR activation, which contributes to protection during norovirus infection (30). 

Moreover, Th17 cells are the main source of IFN-λ in psoriatic lesions of the skin (31).

Bacteria trigger similar intracellular signaling cascades to viral infections and many bacterial infections lead to the production of type I IFN [reviewed in Ref. (32, 33)].

 Induction of type III IFN has been demonstrated only for a limited number of bacterial species. A human epithelial colon cancer cell line expresses type III IFN upon infection with Gram-positive bacteria such as Listeria monocytogenes (34, 35), Staphylococcus aureus, and Enterococcus faecalis but fails to produce considerable amounts of type III IFN when infected with Gram-negative bacteria such as Salmonella enterica ssp. Typhimurium, Shigella flexneri, and Chlamydia trachomatis (35). Induction seems to be cell type, species, and gene specific (36, 37).

Signaling in Response to IFN

Binding of IFN to their corresponding receptors triggers the stimulation of a Janus kinase (JAK)–signal transducer of transcription (STAT) pathway. The type I IFN receptor (IFNAR) consists of two subunits, IFNAR1 and IFNAR2. Engagement of IFNAR with its ligand ultimately results in the activation of the transcription factor complex ISGF3 comprised of STAT1/STAT2 heterodimers in conjunction with IRF-9 and subsequently the induction of ISGs (3, 38).

 

The type III IFN receptor consists of the unique IFN-λR1 chain and the IL-10R2 chain, which is shared with the IL-10 receptor. Engagement of this receptor complex results in the activation of a signal transduction cascade in a manner highly similar to that caused by type I IFN signaling. Interestingly, signaling by type III IFN is additionally regulated at the level of receptor expression. Whereas IFNAR is ubiquitously present, the IFN-λR1 chain of the type III IFN receptor is only expressed in a limited number of cell types, preferentially located at mucosal surfaces. Epithelial cells in mucosal tissues are a major target of type III IFN (39, 40). Additional responsiveness to type III IFN has recently been suggested for a restricted panel of immune cells (9). Type III IFN was proposed to have a role in the direct regulation of NK cell effector function (41). A suppressive function of type III IFN in autoimmune and inflammatory diseases was also proposed recently. In a model of collagen-induced arthritis, treatment with type III IFN inhibits the recruitment of IL-1β-expressing neutrophils, which have been shown to express high levels of IFN-λR1 and respond directly to type III IFN (42). In addition, there are controversial data on the responsiveness of T cells, DCs, and monocytes to type III IFN (9). In human cells, expression of the type III IFN receptor seems to be less restricted than in mouse cells and a wider panel of immune cells, including B cells, is responsive to type III IFN (43).

Signaling by IFNs induces the transcription of hundreds of ISGs. These include pattern-recognition receptors, antiviral effectors such as myxovirus resistance (Mx) gene 1 and 2, pro-apoptotic genes, MHC class I genes, inducible nitric oxide synthase, and genes encoding members of the GTPase superfamily which alter the maturation of phagosomes to counteract pathogen strategies based on survival in intracellular compartments.

 Moreover genes involved in the desensitization to IFNs are also induced, allowing cells to recover from the IFN response (38, 44).

 The importance of IFNs in the immediate defense against pathogens has been shown by the generation of gene-targeted mice. Mice deficient in components of the type I IFN signal transduction pathway are highly susceptible to a variety of viruses (5, 45). The role of type I IFN in bacterial infections is more complex. Whereas type I IFN protect mice against systemic infection with most extracellular bacteria tested, they exacerbate disease during infection of mice with L. monocytogenes or Mycobacterium tuberculosis (3–6).

Enteric Viral Infections and IFN

Studies investigating the functional importance of type I IFN versus type III IFN in the context of systemic viral infections found a dominant phenotype for type I IFN and only a small contribution of type III IFN in the absence of type I IFN. The first indication for a tissue-specific role of type III IFN arose from studies with organ-tropic viral infections suggesting that type III IFN are important in enforcing and strengthening the antiviral response at mucosal sites (Table 1) (10–12, 46–48). The gastrointestinal tract, lung, vagina, and salivary glands respond strongly to systemic IFN-λ expression (40). 

In the lung and gastrointestinal tract, epithelial cells were identified to express high levels of the type III IFN receptor and represent the major target of type III IFN (11). These findings explain why mice deficient for both IFN systems are more susceptible to lung-tropic viruses, such as influenza A and B virus, respiratory syncytial virus, and severe acute respiratory syndrome coronavirus than single type I IFN receptor-deficient mice (11). The remaining part of this section focuses on the role of type III IFN in enteric viral infections.

TABLE 1

Table 1. Role of type I interferons (IFN) and type III IFN during enteric viral infections.

Rotavirus

Rotavirus belongs to the family of reoviridae and infection of humans leads to severe diarrhea in children younger than 5 years. The susceptibility of infants can be recapitulated in a mouse model, where suckling mice are highly susceptible to infection compared to adult mice. The strict host cell tropism of rotavirus for IECs makes it a clean model to study epithelial-specific effects of IFN.

Mice can be infected with a homologous strain of murine rotavirus or with a heterologous strain such as rhesus or simian rotavirus. Homologous strains are better equipped to evade the host immune response, which generally leads to higher viral titers and a more severe pathology at a lower infectious dose (49).

A protective role of type I IFN and IFN-γ has been questioned, since mice impaired in type I IFN or IFN-γ signaling infected with a murine rotavirus strain do not show differences in viral load, and treatment with either type I IFN or IFN-γ did not result in a clinical benefit (53). However, simian and rhesus rotavirus show enhanced systemic replication in mice deficient for type I IFN and IFN-γ receptor or STAT1.

By contrast, type III IFN were protective in a homologous infection model of suckling and adult mice (12). Of note, a very distinct cell tropism for type III IFN responsiveness in the intestine was reported: IECs were solely activated by type III IFN and are not responsive to type I IFN, whereas cells in the lamina propria respond to type I IFN induced during viral infection (12). Supporting these findings it was shown that IL-22 augments the antiviral effects of type III IFN signaling and contributes to the protective effect during homologous rotavirus infection (29). However, this model has been questioned by another study reporting type I IFN- and type III IFN-mediated protection only for heterologous but not for homologous rotavirus infection of suckling mice (50). Experimental discrepancies between those studies are not apparent suggesting that flora differences between mouse facilities or genetic strategy of the knock-out mouse lines might impact on the efficacy of IFN signaling. Of note, Lin et al. reported age-dependent responsiveness of IECs toward IFNs with neonatal IECs being responsive to both type I IFN and type III IFN, whereas adult IECs were responsive to type III IFN only (50).

Norovirus

Norovirus is the cause of the majority of non-bacterial gastroenteritis in adults. In contrast to rotavirus, the host cell tropism of norovirus is broad and not fully characterized. Ex vivo and most in vivo studies could not show productive virus replication in IECs (54). Phagocytes allow productive virus replication and during in vivo infection, virus was detected in LPLs (54, 55). Although the virus does not replicate in IECs, it has been suggested that it translocates across the epithelium or enters the host via M cells (56).

Type I IFN and IFN-γ restrict murine norovirus replication in macrophages and DCs in vitro (57, 58). In vivo, the antiviral activity of type I IFN mediates some protection from systemic replication of an acute strain (51) and after high-dose oral infection (59). However, local replication in the colon and fecal shedding of a persistent norovirus strain is controlled by type III IFN. Treatment with type III IFN resolves persistent infection, independent of adaptive immune responses, by acting on non-hematopoietic cells (15). By contrast, type I IFN controls the systemic spread and persistency of the acute norovirus strain CW3 by activation of the host DCs (48). These findings demonstrate the distinct cell-type specificities of type I IFN and type III IFN during infection: local protection in the colon through type III IFN stimulation of epithelial cells and prevention of systemic spread and persistency by type I IFN in myeloid cells.

The commensal bacterial flora was reported to promote norovirus persistency in the intestine and antibiotic treatment of mice prevented persistent infection with norovirus. The protective effect was only observed in the presence of functional type III IFN signaling (13). The antibiotic treatment did not alter type III IFN signaling and therefore the authors concluded that the microflora might render the virus susceptible to the antiviral action of type III IFN. Alternatively, the absence of type III IFN signaling might increase the host’s vulnerability to persistent viral infection so dramatically that minor changes by antibiotic treatment do not impact on the overall susceptibility under those conditions.

Reovirus

Reovirus has a broad host cell tropism and replicates in epithelial cells and immune cells of the intestinal mucosa. After oral infection, it enters the host via M cells into Peyer’s patches and can spread further during infection. Type I IFN produced by hematopoietic cells is essential to limit systemic spread of the virus and to prevent lethality (60). In a study using type I IFN or type III IFN signaling-deficient mice, it was demonstrated that type III IFN signaling specifically prevents replication of the virus in IECs, whereas type I IFN signaling limits replication in lamina propria cells and systemic spread of the virus (14). This study confirms the compartmentalized action of IFN in the intestinal mucosa and provides an explanation by showing that IECs only express low levels of IFNAR (Figure 1). Furthermore, it was shown that the production of IFN is cell type specific in that IECs produce higher levels of type III IFN and LPLs predominantly produce type I IFN.

FIGURE 1

 

Figure 1. Cell-type-specific responsiveness to type I interferons (IFN) and type III IFN at the intestinal mucosa. (A) C57BL/6 mice were injected with IFN-β (1,000 U) (middle panel) or IFN-λ (1 μg) (right panel) and 3 h later, the small intestine was processed for histological assessment. Staining was performed for the interferon-stimulated gene IFIT3 (red) as a marker for IFN response and for the epithelial cell marker E-cadherin (green). (B) Schematic of the IFN production and responsiveness at the intestinal mucosa. When type I IFN levels are high, lamina propria cells readily respond with a strong IFN response whereas IECs are rather unresponsive but might respond under certain conditions [(A) middle panel; (B) left panel]. In contrast, IECs are the most responsive cells to type III IFN (A,B) right panel. Most virus-infected cells express type I IFN. Hematopoietic cells, such as plasmacytoid dendritic cells and macrophages produce the highest amounts of type I IFN whereas IECs seem to express preferentially type III IFN. T, T cell; DC, dendritic cell; MΦ, macrophage; IEC, intestinal epithelial cell.

Taken together, the studies of enteric viral models with rotavirus, reovirus, and norovirus show a strong and specific responsiveness of IECs to type III IFN (12, 15, 29). Therefore, type III IFN might specifically enforce the intestinal barrier against enteric viruses and also against viral entry via the intestinal route. Additionally, a strong IFN response by type III IFN signaling within the epithelial lining prevents viral spreading (12, 14, 15). Studies showing that type III IFN treatment protects against oral EMCV (52) infection but not from systemic infection (47) support the conclusion that type III IFN protects the host not only from enteric viruses but also from viral entry via the oral route. By contrast, the contribution of type I IFN to the epithelial antiviral response in the intestine is less clear and conflicting results suggest it to be context dependent (12, 29, 50).

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