Type III IFNs: Beyond antiviral protection.

The unexpected discovery of a novel family of antiviral mediators, type III IFNs or IFN-λs, challenged the widely accepted primacy of type I IFNs in antiviral immunity, and it is now well recognized that the IFN-λ-based antiviral system plays a major role in antiviral protection of epithelial barriers. The recent characterization of previously unknown IFN-λ-mediated activities has prompted further reassessment of the role of type I IFNs in innate and adaptive immune and inflammatory responses. Since type I and type III IFNs are co-produced in response to a variety of stimuli, it is likely that many physiological processes are simultaneously and coordinately regulated by these cytokines in pathological conditions, and likely at steady state, as baseline expression of both IFN types is maintained by microbiota. In this review, we discuss emerging differences in the production and signaling of type I and type III IFNs, and summarize results of recent studies describing the involvement of type III IFNs in anti-bacterial and anti-fungal, as well as antiviral, defenses.

Respiratory syncytial virus infection enhances Pseudomonas aeruginosa biofilm growth through dysregulation of nutritional immunity.

Clinical observations link respiratory virus infection and Pseudomonas aeruginosa colonization in chronic lung disease, including cystic fibrosis (CF) and chronic obstructive pulmonary disease. The development of P. aeruginosa into highly antibiotic-resistant biofilm communities promotes airway colonization and accounts for disease progression in patients. Although clinical studies show a strong correlation between CF patients' acquisition of chronic P. aeruginosa infections and respiratory virus infection, little is known about the mechanism by which chronic P. aeruginosa infections are initiated in the host. Using a coculture model to study the formation of bacterial biofilm formation associated with the airway epithelium, we show that respiratory viral infections and the induction of antiviral interferons promote robust secondary P. aeruginosa biofilm formation. We report that the induction of antiviral IFN signaling in response to respiratory syncytial virus (RSV) infection induces bacterial biofilm formation through a mechanism of dysregulated iron homeostasis of the airway epithelium. Moreover, increased apical release of the host iron-binding protein transferrin during RSV infection promotes P. aeruginosa biofilm development in vitro and in vivo. Thus, nutritional immunity pathways that are disrupted during respiratory viral infection create an environment that favors secondary bacterial infection and may provide previously unidentified targets to combat bacterial biofilm formation.

Contribution of type III interferons to antiviral immunity: location, location, location.

Type I interferons (IFN-α/ß) and the more recently identified type III IFNs (IFN-λ) function as the first line of defense against virus infection and regulate the development of both innate and adaptive immune responses. Type III IFNs were originally identified as a novel ligand-receptor system acting in parallel with type I IFNs, but subsequent studies have provided increasing evidence for distinct roles for each IFN family. In addition to their compartmentalized antiviral actions, these two systems appear to have multiple levels of cross-regulation and act coordinately to achieve effective antimicrobial protection with minimal collateral damage to the host.

Correction: Distinct Roles of Type I and Type III Interferons in Intestinal Immunity to Homologous and Heterologous Rotavirus Infections.

[This corrects the article DOI: 10.1371/journal.ppat.1005600.].

Distinct Roles of Type I and Type III Interferons in Intestinal Immunity to Homologous and Heterologous Rotavirus Infections.

Type I (IFN-α/ß) and type III (IFN-λ) interferons (IFNs) exert shared antiviral activities through distinct receptors. However, their relative importance for antiviral protection of different organ systems against specific viruses remains to be fully explored. We used mouse strains deficient in type-specific IFN signaling, STAT1 and Rag2 to dissect distinct and overlapping contributions of type I and type III IFNs to protection against homologous murine (EW-RV strain) and heterologous (non-murine) simian (RRV strain) rotavirus infections in suckling mice. Experiments demonstrated that murine EW-RV is insensitive to the action of both types of IFNs, and that timely viral clearance depends upon adaptive immune responses. In contrast, both type I and type III IFNs can control replication of the heterologous simian RRV in the gastrointestinal (GI) tract, and they cooperate to limit extra-intestinal simian RRV replication. Surprisingly, intestinal epithelial cells were sensitive to both IFN types in neonatal mice, although their responsiveness to type I, but not type III IFNs, diminished in adult mice, revealing an unexpected age-dependent change in specific contribution of type I versus type III IFNs to antiviral defenses in the GI tract. Transcriptional analysis revealed that intestinal antiviral responses to RV are triggered through either type of IFN receptor, and are greatly diminished when receptors for both IFN types are lacking. These results also demonstrate a murine host-specific resistance to IFN-mediated antiviral effects by murine EW-RV, but the retention of host efficacy through the cooperative action by type I and type III IFNs in restricting heterologous simian RRV growth and systemic replication in suckling mice. Collectively, our findings revealed a well-orchestrated spatial and temporal tuning of innate antiviral responses in the intestinal tract where two types of IFNs through distinct patterns of their expression and distinct but overlapping sets of target cells coordinately regulate antiviral defenses against heterologous or homologous rotaviruses with substantially different effectiveness.

Interferon induction and function at the mucosal surface.

Interferons (IFNs) are produced in response to virus infection and induce an antiviral state in virtually all cell types. In addition to upregulating the transcription of genes that inhibit virus replication, type I (or -α/ß) IFNs also act to orchestrate the adaptive immune response to virus infection. Recently a new family of antiviral cytokines, the type III (or -λ) IFNs, has been identified that activate the same antiviral pathways via a distinct receptor. Although the identical transcription factor, IFN-stimulated gene factor 3 is activated by either IFN-α/ß or IFN-λ signaling, differences in the induction and action of these two cytokine families are beginning to be appreciated. In this article, we review this emerging body of literature on the differing roles these cytokines play in host defense of the mucosal surface. Although many viruses enter the body through the respiratory and gastrointestinal tracts, we have focused the discussion on influenza A virus, respiratory syncytial virus, and rotavirus, three ubiquitous human pathogens that target the epithelial lining and are associated with a major disease burden.

Inflammatory impact of IFN-γ in CD8+ T cell-mediated lung injury is mediated by both Stat1-dependent and -independent pathways.

Influenza infection results in considerable pulmonary pathology, a significant component of which is mediated by CD8(+) T cell effector functions. To isolate the specific contribution of CD8(+) T cells to lung immunopathology, we utilized a nonviral murine model in which alveolar epithelial cells express an influenza antigen and injury is initiated by adoptive transfer of influenza-specific CD8(+) T cells. We report that IFN-γ production by adoptively transferred influenza-specific CD8(+) T cells is a significant contributor to acute lung injury following influenza antigen recognition, in isolation from its impact on viral clearance. CD8(+) T cell production of IFN-γ enhanced lung epithelial cell expression of chemokines and the subsequent recruitment of inflammatory cells into the airways. Surprisingly, Stat1 deficiency in the adoptive-transfer recipients exacerbated the lung injury that was mediated by the transferred influenza-specific CD8(+) T cells but was still dependent on IFN-γ production by these cells. Loss of Stat1 resulted in sustained activation of Stat3 signaling, dysregulated chemokine expression, and increased infiltration of the airways by inflammatory cells. Taken together, these data identify important roles for IFN-γ signaling and Stat1-independent IFN-γ signaling in regulating CD8(+) T cell-mediated acute lung injury. This is the first study to demonstrate an anti-inflammatory effect of Stat1 on CD8(+) T cell-mediated lung immunopathology without the complication of differences in viral load.

Plasticity and virus specificity of the airway epithelial cell immune response during respiratory virus infection.

Airway epithelial cells (AECs) provide the first line of defense in the respiratory tract and are the main target of respiratory viruses. Here, using oligonucleotide and protein arrays, we analyze the infection of primary polarized human AEC cultures with influenza virus and respiratory syncytial virus (RSV), and we show that the immune response of AECs is quantitatively and qualitatively virus specific. Differentially expressed genes (DEGs) specifically induced by influenza virus and not by RSV included those encoding interferon B1 (IFN-B1), type III interferons (interleukin 28A [IL-28A], IL-28B, and IL-29), interleukins (IL-6, IL-1A, IL-1B, IL-23A, IL-17C, and IL-32), and chemokines (CCL2, CCL8, and CXCL5). Lack of type I interferon or STAT1 signaling decreased the expression and secretion of cytokines and chemokines by the airway epithelium. We also observed strong basolateral polarization of the secretion of cytokines and chemokines by human and murine AECs during infection. Importantly, the antiviral response of human AECs to influenza virus or to RSV correlated with the infection signature obtained from peripheral blood mononuclear cells (PBMCs) isolated from patients with acute influenza or RSV bronchiolitis, respectively. IFI27 (also known as ISG12) was identified as a biomarker of respiratory virus infection in both AECs and PBMCs. In addition, the extent of the transcriptional perturbation in PBMCs correlated with the clinical disease severity. Our results demonstrate that the human airway epithelium mounts virus-specific immune responses that are likely to determine the subsequent systemic immune responses and suggest that the absence of epithelial immune mediators after RSV infection may contribute to explaining the inadequacy of systemic immunity to the virus.

VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents.

Ideally, an oncolytic virus will replicate preferentially in malignant cells, have the ability to treat disseminated metastases, and ultimately be cleared by the patient. Here we present evidence that the attenuated vesicular stomatitis strains, AV1 and AV2, embody all of these traits. We uncover the mechanism by which these mutants are selectively attenuated in interferon-responsive cells while remaining highly lytic in 80% of human tumor cell lines tested. AV1 and AV2 were tested in a xenograft model of human ovarian cancer and in an immune competent mouse model of metastatic colon cancer. While highly attenuated for growth in normal mice, both AV1 and AV2 effected complete and durable cures in the majority of treated animals when delivered systemically.

An intranasal recombinant NDV-BRSV Fopt vaccine is safe and reduces lesion severity in a colostrum-deprived calf model of RSV infection.

Human respiratory syncytial virus (HRSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, yet no safe, effective vaccine is commercially available. Closely related bovine RSV (BRSV) causes respiratory disease in young calves, with many similar features to those seen in HRSV. We previously showed that a Newcastle disease virus (NDV)-vectored vaccine expressing the F glycoprotein of HRSV reduced viral loads in lungs of mice and cotton rats and protected from HRSV. However, clinical signs and pathogenesis of disease in laboratory animals following HRSV infection differs from that observed in human infants. Thus, we examined whether a similar vaccine would protect neonatal calves from BRSV infection. Codon-optimized rNDV vaccine (rNDV-BRSV Fopt) was constructed and administered to colostrum-deprived calves. The rNDV-BRSV Fopt vaccine was well-tolerated and there was no evidence of vaccine-enhanced disease in the upper airways or lungs of these calves compared to the non-vaccinated calves. We found two intranasal doses reduces severity of gross and microscopic lesions and decreases viral load in the lungs. Furthermore, serum neutralizing antibodies were generated in vaccinated calves. Finally, reduced lung CXC chemokine levels were observed in vaccinated calves after BRSV challenge. In summary, we have shown that rNDV-BRSV Fopt vaccine is safe in colostrum-deprived calves, and is effective in reducing lung lesions, and decreasing viral load in upper respiratory tract and lungs after challenge.

Lambda interferon is the predominant interferon induced by influenza A virus infection in vivo.

The type I alpha/beta interferons (IFN-α/ß) are known to play an important role in host defense against influenza A virus infection, but we have now discovered that the recently identified type III IFNs (IFN-λ) constitute the major response to intranasal infection with this virus. Type III IFNs were present at much higher levels than type I IFNs in the lungs of infected mice, and the enhanced susceptibility of STAT2-/- animals demonstrated that only signaling through the IFN-α/ß or IFN-λ pathways was sufficient to mediate protection. This finding offers a possible explanation for the similar levels of antiviral protection found in wild-type (WT) mice and in animals lacking a functional type I IFN receptor (IFNAR-/-) but also argues that our current understanding of type III IFN induction is incomplete. While murine IFN-λ production is thought to depend on signaling through the type I IFN receptor, we demonstrate that intranasal influenza A virus infection leads to the robust type III IFN induction in the lungs of both WT and IFNAR-/- mice. This is consistent with previous studies showing that IFNAR-mediated protection is redundant for mucosal influenza virus infection and with data showing that the type III IFN receptor is expressed primarily by epithelial cells. However, the overlapping effects of these two cytokine families are limited by their differential receptor expression, with a requirement for IFN-α/ß signaling in combating systemic disease.

STAT1 negatively regulates lung basophil IL-4 expression induced by respiratory syncytial virus infection.

IL-4 contributes to immunopathology induced in mice by primary respiratory syncytial virus (RSV) infection. However, the cellular source of IL-4 in RSV infection is unknown. We identified CD3(-)CD49b(+) cells as the predominant source of IL-4 in the lungs of RSV-infected BALB/c mice. We ruled out T cells, NK cells, NKT cells, mast cells, and eosinophils as IL-4 expressors in RSV infection by flow cytometry. Using IL4 GFP reporter mice (4get) mice, we identified the IL-4-expressing cells in RSV infection as basophils (CD3(-)CD49b(+)FcepsilonRI(+)c-kit(-)). Because STAT1(-/-) mice have an enhanced Th2-type response to RSV infection, we also sought to determine the cellular source and role of IL-4 in RSV-infected STAT1(-/-) mice. RSV infection resulted in significantly more IL-4-expressing CD3(-)CD49b(+) cells in the lungs of STAT1(-/-) mice than in BALB/c mice. CD49b(+)IL-4(+) cells sorted from the lungs of RSV-infected STAT1(-/-) mice and stained with Wright-Giemsa had basophil characteristics. As in wild-type BALB/c mice, IL-4 contributed to lung histopathology in RSV-infected STAT1(-/-) mice. Depletion of basophils in RSV-infected STAT1(-/-) mice reduced lung IL-4 expression. Thus, we show for the first time that a respiratory virus (RSV) induced basophil accumulation in vivo. Basophils were the primary source of IL-4 in the lung in RSV infection, and STAT1 was a negative regulator of virus-induced basophil IL-4 expression.

Critical role of interferons in gastrointestinal injury repair.

The etiology of ulcerative colitis is poorly understood and is likely to involve perturbation of the complex interactions between the mucosal immune system and the commensal bacteria of the gut, with cytokines acting as important cross-regulators. Here we use IFN receptor-deficient mice in a dextran sulfate sodium (DSS) model of acute intestinal injury to study the contributions of type I and III interferons (IFN) to the initiation, progression and resolution of acute colitis. We find that mice lacking both types of IFN receptors exhibit enhanced barrier destruction, extensive loss of goblet cells and diminished proliferation of epithelial cells in the colon following DSS-induced damage. Impaired mucosal healing in double IFN receptor-deficient mice is driven by decreased amphiregulin expression, which IFN signaling can up-regulate in either the epithelial or hematopoietic compartment. Together, these data underscore the pleiotropic functions of IFNs and demonstrate that these critical antiviral cytokines also support epithelial regeneration following acute colonic injury.

Signal transducer and activator of transcription 1 in T cells plays an indispensable role in immunity to Leishmania major by mediating Th1 cell homing to the site of infection.

The signal transducer and activator of transcription 1 (STAT1) signaling pathway mediates the biological functions of IFN-gamma. We have previously shown that the STAT1 pathway is indispensable for host resistance against Leishmania major infection. In this study, we examined the role of STAT1 in lymphocytes and specifically CD4(+) and CD8(+) T cells in mediating immunity against L. major by transferring T cells from wild-type (WT) and STAT1(-/-) C57BL/6 mice into Rag2(-/-) C57BL/6 mice. Rag2(-/-) mice reconstituted with unfractionated STAT1(-/-) splenocytes (B cells and T cells) failed to mount an efficient Th1 response after L. major infection, produced more IL-4, and developed large lesions full of parasites. In contrast, Rag2(-/-) mice reconstituted with WT (STAT1(+/+)) splenocytes mounted a Th1 response and developed self-resolving lesions. Studies using Rag2(-/-) recipients that received a combination of purified CD4(+) and CD8(+) T cells from WT or STAT1(-/-) mice revealed that STAT1 deficiency in CD4(+) T cells, but not in CD8(+) T cells, leads to development of chronic, nonhealing lesions and systemic dissemination of parasites into the spleen after L. major infection. Further studies using Rag2(-/-) recipients of WT Thy1.1(+) and STAT1(-/-) Thy1.2(+) T cells showed that STAT1 in CD4(+) T cells was not required for Th1 differentiation during L. major infection. However, it was critical for up-regulation of CXCR3 on CD4(+) T cells and their migration to the regional lymph node and the cutaneous site of infection. Together, these studies indicate that the STAT1 pathway in CD4(+) T cells plays a critical role in immunity against L. major by controlling the migration of Th1 cells to the site of infection rather than their generation. Further, they reveal an essential role for CD4(+) T cell STAT1 in preventing systemic dissemination of L. major infection.

COVID-19 and emerging viral infections: The case for interferon lambda.

With the first reports on coronavirus disease 2019 (COVID-19), which is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the scientific community working in the field of type III IFNs (IFN-λ) realized that this class of IFNs could play an important role in this and other emerging viral infections. In this Viewpoint, we present our opinion on the benefits and potential limitations of using IFN-λ to prevent, limit, and treat these dangerous viral infections.

Influenza A virus inhibits alveolar fluid clearance in BALB/c mice.

RATIONALE: Pulmonary infections can impair alveolar fluid clearance (AFC), contributing to formation of lung edema. Effects of influenza A virus (IAV) on AFC are unknown. OBJECTIVES: To determine effects of IAV infection on AFC, and to identify intercellular signaling mechanisms underlying influenza-mediated inhibition of AFC. METHODS: BALB/c mice were infected intranasally with influenza A/WSN/33 (10,000 or 2,500 focus-forming units per mouse). AFC was measured in anesthetized, ventilated mice by instilling 5% bovine serum albumin into the dependent lung. MEASUREMENTS AND MAIN RESULTS: Infection with high-dose IAV resulted in a steady decline in arterial oxygen saturation and increased lung water content. AFC was significantly inhibited starting 1 hour after infection, and remained suppressed through Day 6. AFC inhibition at early time points (1-4 h after infection) did not require viral replication, whereas AFC inhibition later in infection was replication-dependent. Low-dose IAV infection impaired AFC for 10 days, but induced only mild hypoxemia. High-dose IAV infection increased bronchoalveolar lavage fluid ATP and UTP levels. Impaired AFC at Day 2 resulted primarily from reduced amiloride-sensitive AFC, mediated by increased activation of the pyrimidine-P2Y purinergic receptor axis. However, an additional component of AFC impairment was due to activation of A(1) adenosine receptors and stimulation of increased cystic fibrosis transmembrane regulator-mediated anion secretion. Finally, IAV-mediated inhibition of AFC at Day 2 could be reversed by addition of beta-adrenergic agonists to the AFC instillate. CONCLUSIONS: AFC inhibition may be an important feature of early IAV infection. Its blockade may reduce the severity of pulmonary edema and hypoxemia associated with influenza pneumonia.

A viral-vectored RSV vaccine induces long-lived humoral immunity in cotton rats.

Human respiratory syncytial virus (RSV) is the leading cause of lower airway disease in infants worldwide and repeatedly infects immunocompetent individuals throughout life. Severe lower airway RSV infection during infancy can be life-threatening, but is also associated with important sequelae including development of asthma and recurrent wheezing in later childhood. The basis for the inadequate, short-lived adaptive immune response to RSV infection is poorly understood, but it is widely recognized that RSV actively antagonizes Type I interferon (IFN) production. In addition to the induction of the anti-viral state, IFN production during viral infection is critical for downstream development of robust, long-lived immunity. Based on the hypothesis that a vaccine that induced robust IFN production would be protective, we previously constructed a Newcastle disease virus-vectored vaccine that expresses the F glycoprotein of RSV (NDV-F) and demonstrated that vaccinated mice had reduced lung viral loads and an enhanced IFN-γ response after RSV challenge. Here we show that vaccination also protected cotton rats from RSV challenge and induced long-lived neutralizing antibody production, even in RSV immune animals. Finally, pulmonary eosinophilia induced by RSV infection of unvaccinated cotton rats was prevented by vaccination. Overall, these data demonstrate enhanced protective immunity to RSV F when this protein is presented in the context of an abortive NDV infection.

Type III interferon is a critical regulator of innate antifungal immunity.

Type III interferons (IFN-λs) are the most recently found members of the IFN cytokine family and engage IFNLR1 and IL10R2 receptor subunits to activate innate responses against viruses. We have identified IFN-λs as critical instructors of antifungal neutrophil responses. Using Aspergillus fumigatus (Af) as a model to study antifungal immune responses, we found that depletion of CCR2+ monocytes compromised the ability of neutrophils to control invasive fungal growth. Using an unbiased approach, we identified type I and III IFNs as critical regulators of the interplay between monocytes and neutrophils responding to Af We found that CCR2+ monocytes are an important early source of type I IFNs that prime optimal expression of IFN-λ. Type III IFNs act directly on neutrophils to activate their antifungal response, and mice with neutrophil-specific deletion of IFNLR1 succumb to invasive aspergillosis. Dysfunctional neutrophil responses in CCR2-depleted mice were rescued by adoptive transfer of pulmonary CCR2+ monocytes or by exogenous administration of IFN-α and IFN-λ. Thus, CCR2+ monocytes promote optimal activation of antifungal neutrophils by initiating a coordinated IFN response. We have identified type III IFNs as critical regulators of neutrophil activation and type I IFNs as early stimulators of IFN-λ expression.

Animal models of respiratory syncytial virus infection and disease.

The study of human respiratory syncytial virus pathogenesis and immunity has been hampered by its exquisite host specificity, and the difficulties encountered in adapting this virus to a murine host. The reasons for this obstacle are not well understood, but appear to reflect, at least in part, the inability of the virus to block the interferon response in any but the human host. This review addresses some of the issues encountered in mouse models of respiratory syncytial virus infection, and describes the advantages and disadvantages of alternative model systems.

Acute and Chronic Airway Disease After Human Respiratory Syncytial Virus Infection in Cotton Rats (Sigmodon hispidus).

Infection with respiratory syncytial virus (RSV) generally presents as a mild, upper airway disease in human patients but may cause severe lower airway disease in the very young and very old. Progress toward understanding the mechanisms of RSV pathogenesis has been hampered by a lack of relevant rodent models. Mice, the species most commonly used in RSV research, are resistant to upper respiratory infection and do not recapitulate the pattern of virus spread in the human host. To address the need for better rodent models of RSV infection, we have characterized the acute and chronic pathology of RSV infection of a relatively permissive host, cotton rats (Sigmodon hispidus). We demonstrate that virus delivered to the upper airway results in widespread RSV replication in the ciliated respiratory epithelial cells of the nasal cavity and, to a lesser extent, of the lung. Although acute inflammation is relatively mild and rapidly eliminated after viral clearance, chronic, eosinophilic lung pathology persists. These data support the use of cotton rats as a robust rodent model of human RSV disease, including the association between RSV pneumonia and subsequent development of allergic asthma.