key: cord-0971672-6njxf06i
authors: Major, Jack; Crotta, Stefania; Llorian, Miriam; McCabe, Teresa M.; Gad, Hans Henrik; Priestnall, Simon L.; Hartmann, Rune; Wack, Andreas
title: Type I and III interferons disrupt lung epithelial repair during recovery from viral infection
date: 2020-06-11
journal: Science
DOI: 10.1126/science.abc2061
sha: fbf04430329a5db9ac814c22c9475020150e8a99
doc_id: 971672
cord_uid: 6njxf06i

Excessive cytokine signaling frequently exacerbates lung tissue damage during respiratory viral infection. Type I (IFN-α/β) and III (IFN-λ) interferons are host-produced antiviral cytokines. Prolonged IFN-α/β responses can lead to harmful proinflammatory effects, whereas IFN-λ mainly signals in epithelia, inducing localized antiviral immunity. Here we show that IFN signaling interferes with lung repair during influenza recovery, with IFN-λ driving these effects most potently. IFN-induced p53 directly reduces epithelial proliferation and differentiation, increasing disease severity, and susceptibility to bacterial superinfections. Thus, excessive or prolonged IFN-production aggravates viral infection by impairing lung epithelial regeneration. Therefore, timing and duration are critical parameters of endogenous IFN action and should be considered carefully for IFN therapeutic strategies against viral infections like influenza and coronavirus disease 2019 (COVID-19).

During infection with respiratory viruses, disease severity is linked to lung epithelial destruction, due to both cytopathic viral effects and immune-mediated damage. Epithelial loss contributes to acute respiratory distress syndrome, pneumonia, and increased susceptibility to bacterial superinfections. Restoration of damaged epithelial tissues is therefore paramount in order to maintain lung function and barrier protection.

Interferons (IFNs) are key to the antiviral host defense. IFN-α/β and IFN-λ are induced upon viral recognition and trigger transcription of interferon-stimulated genes with antiviral function in infected and bystander cells. Due to widespread expression of the type I IFN receptor (IFNAR) in immune cells, IFN-α/β responses can result in immunopathology during viral infections, including influenza virus and severe acute respiratory syndrome coronavirus (SARS-CoV-1) (1) (2) (3) (4) . The IFN-λ receptor (IFNLR) is mainly expressed at epithelial barriers, and responses are therefore often characterized by their ability to confer localized antiviral protection at the site of infection, without driving damaging proinflammatory responses associated with IFN-α/β. In addition to antiviral and proinflammatory activity, IFNs exert antiproliferative and proapoptotic functions (5) . Despite a growing understanding of immunopathology in respiratory viral infection, it is unknown how IFN responses affect lung epithelial repair.

Influenza virus infection in C57BL/6 (B6) wild-type (WT) mice resulted in weight loss, accompanied by significant immune cell infiltration and lung damage (fig. S1, A to D). Recovery from infection coincided with the onset of epithelial regeneration (fig. S1, C and D). To further investigate the dynamics of lung repair following influenza virus infection, epithelial cell proliferation was analyzed by flow cytometry, using the proliferation marker Ki67 (gating strategy in fig.  S2 ). During steady-state conditions, type II alveolar epithelial cells (AT2; EpCam + MHCII + CD49fl lo ) (6) (7) (8) showed a low rate of turnover (Fig. 1A) . However, following influenza virus-induced lung damage, AT2 cells underwent rapid proliferation starting at days 5-7 post infection, correlating with mouse recovery and weight gain ( Fig. 1A and fig. S1B ).

To compare the dynamics of epithelial recovery with IFN production, we analyzed IFN subtypes (IFN-α, IFN-β, and IFN-λ) in bronchoalveolar lavage fluid (BALF) throughout infection. IFNs were produced rapidly, peaking 2 days post infection (Fig. 1B) . The magnitude of IFN-λ production was significantly greater than that of IFN-α/β, both in duration and in length of peak production. Importantly, only IFN-λ was detected 7-8 days post infection, coinciding with the onset of epithelial recovery (Fig. 1, A and B) . Thus, following influenza virus infection, signaling triggered by IFNs, in particular by IFN-λ, overlaps with the onset of lung repair.

To compare the effects of equipotent amounts of IFN-α, IFN-β, and IFN-λ on lung repair, mice were treated during recovery from influenza virus infection (7 to 10 days post Type I and III interferons disrupt lung epithelial repair during recovery from viral infection Excessive cytokine signaling frequently exacerbates lung tissue damage during respiratory viral infection. Type I (IFN-α/β) and III (IFN-λ) interferons are host-produced antiviral cytokines. Prolonged IFN-α/β responses can lead to harmful proinflammatory effects, whereas IFN-λ mainly signals in epithelia, inducing localized antiviral immunity. Here we show that IFN signaling interferes with lung repair during influenza recovery, with IFN-λ driving these effects most potently. IFN-induced p53 directly reduces epithelial proliferation and differentiation, increasing disease severity, and susceptibility to bacterial superinfections. Thus, excessive or prolonged IFN-production aggravates viral infection by impairing lung epithelial regeneration. Therefore, timing and duration are critical parameters of endogenous IFN action and should be considered carefully for IFN therapeutic strategies against viral infections like influenza and coronavirus disease 2019 (COVID-19). In chimeric mice, both IFN-α and IFN-β treatments significantly reduced the proliferation of AT2 cells on day 11 post influenza virus infection (Fig. 1C) . Similarly, IFN-λ treatment reduced AT2 cell proliferation in WT mice (Fig. 1D ). Reductions in proliferation were independent from changes in viral burden ( fig. S3 , B and C). The IFN-λ-mediated reduction in AT2 cell proliferation did not require IFN-λ signaling in neutrophils (9) (10) (11) , as neutrophil depletion in WT mice using an anti-Ly6G monoclonal antibody had no effect ( fig. S3 , D and E). A caveat when using inbred mouse strains for influenza virus infection is the lack of a functional Mx1 protein, a crucial IFN-inducible influenza virus restriction factor in both mice and humans (12) . We therefore infected mice expressing functional Mx1 alleles (B6-Mx1) with the influenza virus strain hvPR8-ΔNS1 for a more clinically relevant influenza model. IFN-λ treatment significantly reduced epithelial proliferation in the presence of functional Mx1 as well (Fig. 1E) .

We next used Ifnar1 −/− and Ifnlr1 −/− mice to determine the role of endogenous IFNs during lung repair. AT2 cells were analyzed on day 8 post influenza virus infection, the time when IFN signaling and epithelial cell proliferation overlapped (Fig. 1, A and B ). Both Ifnar1 −/− and Ifnlr1 −/− mice had improved AT2 cell proliferation, compared to WT controls ( Fig. 1, F and G) . This was dependent on IFN signaling specifically through the epithelium, as receptor deficiency in the stromal compartment alone was sufficient to increase lung epithelial cell proliferation (Fig. 1H ). Improved proliferation was independent of major changes in viral burden ( fig. S5A ). Viral control in individual IFN receptor-knockout mice was likely unaffected due to redundancy between type I and III IFN antiviral responses in epithelial cells (13, 14) . Despite type I and III IFN redundancy in viral control ( fig. S5A ), the lack of redundancy in antiproliferative IFN responses, with both Ifnar1 −/− and Ifnlr1 −/− mice displaying enhanced epithelial proliferation (Fig. 1 , F to H), led us to further interrogate the phenotype. IFNAR signaling has previously been shown to be important for the production of IFN-λ during influenza virus infection (15, 16) . Consistently, we observed a significant reduction in IFN-λ (and indeed IFN-α/β) production in If-nar1 −/− mice compared to WT, yet we saw little change in IFNα/β levels in Ifnlr1 −/− mice ( fig. S5B ). Thus, the improved epithelial proliferation in Ifnar1 −/− mice may result from reduced IFN-λ. IFN production defects in Ifnar1 −/− mice are linked to reduced steady-state priming in the absence of tonic IFNAR activation in immune cells (17) . To circumvent this, we administered an anti-IFNAR monoclonal antibody (MAR1-5A3) only from the onset of influenza virus infection. Anti-IFNAR treatment maintained steady-state priming required for IFNλ production ( fig. S5C ), despite blocking IFN-α/β signaling through IFNAR ( fig. S5D ). Importantly, anti-IFNAR treatment from day 0 or day 3 post infection had no effect on lung epithelial cell proliferation ( fig. S5E ). Thus, in murine influenza virus infection, endogenous IFN-λ responses are most effective in disrupting epithelial regeneration during influenza recovery, through direct effects on epithelial cells.

To understand mechanistically how IFNs exert the observed antiproliferative effects, we set up primary murine airway epithelial cell (AEC) cultures. AECs undergo rapid proliferation and differentiation upon exposure to an air-liquid interface (ALI), recapitulating lung repair processes observed in vivo (18, 19) . IFNs used for in vitro assays were titrated on AEC cultures, to compare IFN subtypes at equivalent biological potency ( fig. S6 ). All three IFN subtypes significantly impaired the growth of AEC cultures, with IFN-β and IFN-λ having the most significant effects ( However, the growth inhibitory effects of IFNs were only observed in actively dividing cultures ( fig. S7 , E to G). Thus, the increase in apoptosis observed may occur as a result of failed progression through the cell cycle following IFN treatment, as seen previously (20) .

We next examined the effects of IFNs on AEC differentiation. Following acute damage, populations of basal cells and Scgb1a1 + secretory cells give rise to secretory and multiciliated cell subtypes (21) . To study effects of IFNs on AEC differentiation, we initiated IFN treatment late during the course of AEC growth, during air exposure, when AEC differentiation is induced ( fig. S8A ). IFN-β and IFN-λ treatment significantly reduced the expression of genes pertaining to multiciliated (Mcidas and Ccno) and secretory cell (Muc5AC and Scb1a1) differentiation (Fig. 2F) . Expression of the basal cell marker Krt5 remained unchanged, or was increased by IFN-λ treatment, suggesting maintenance of stemness ( fig.  S8B ). We also found reduced numbers of multiciliated cells in AEC cultures (acetylated α-tubulin + ) following IFN-λ treatment, but not with IFN-α or IFN-β ( Fig. 2G and fig. S8C ). In vivo, Ifnlr1 −/− mice displayed increased multiciliated cells in repairing conducting airways on day 10 post influenza virus infection (Fig. 2H ). Using flow cytometry, we quantified this increase in the frequency of differentiated AECs (EpCamhi CD49f hi CD24 + ), composed of multiciliated, goblet, and club cells ( Fig. 2I and fig. S2 ) (22) . Thus, IFN-λ signaling reduces the capacity for basal cell differentiation during recovery from influenza virus infection. To understand how IFNs mediate antiproliferative effects, we performed RNA-sequencing on IFN-treated AEC cultures (Fig. 3A) . Principal component analysis (PCA) clustered 4hour IFN treated samples together regardless of subtype (Fig.  3B) , confirming equal subtype dosage based on previous titrations ( fig. S9A ). Five-days of IFN-β or IFN-λ treatment clustered AECs together separate from untreated controls on both PC1 and PC2 (Fig. 3B ). Gene ontology analysis confirmed genes contributing to this variance are involved in IFNsignaling and epithelial cell development (supplementary text and fig. S9B ). Ingenuity Pathway Analysis revealed induction of pathways regulating cell cycle and cell death following prolonged IFN treatment, most significantly induced by IFN-λ across all timepoints (Fig. 3C) . Predicted upstream transcriptional regulators identified typical regulators of IFN function, including STAT and IRF proteins, in addition to cell cycle regulators (Fig. 3D) . We identified the tumor suppressor protein p53 as a top candidate regulating IFN-inducible antiproliferative effects. p53 has previously been shown to directly regulate IFN-α/β antitumor responses (23) . Gene set enrichment analysis (GSEA) identified IFN-mediated induction of the p53 pathway ( fig. S9C ), and we identified induction of p53-regulated downstream targets in expression data ( fig. S9D ). To confirm the role of p53, we utilized Tp53 −/− AEC cultures. IFN-mediated reduction in AEC growth, differentiation, and induction of antiproliferative downstream p53 target genes Gadd45 g and Dusp5 (24, 25) was p53-dependent, with no changes observed in Tp53 −/− AECs (Fig. 3, E to G, and  fig. S9E ). We next examined whether IFNs regulate p53 activity in epithelial cells during lung repair in vivo. To study IFN effects specifically in the lung epithelium, we once again generated Ifnar1 −/− → WT BM chimeric mice for IFN-α or IFN-β treatment, or depleted neutrophils in WT mice with anti-Ly6G for IFN-λ treatment ( fig. S3A ). IFN-β and IFN-λ, but not IFN-α, significantly up-regulated p53 expression in repairing lung epithelial cells (Fig. 3, H and I) . Thus, IFN-β and IFN-λ mediate antiproliferative effects in AECs via the induction of p53.

Our data supports a key role for IFN signaling, particularly IFN-λ, in the reduction of epithelial proliferation and differentiation during lung repair. We therefore tested whether IFNs alter the state or barrier function of lung epithelia. RNA-sequencing of sorted lung epithelial cells (Ep-Cam + CD31 − CD45 − ) from influenza virus-infected WT or Ifnlr1 −/− mice confirmed an up-regulation of pathways pertaining to proliferation and multiciliogenesis in Ifnlr1 −/− mice (Fig. 4A) . Improved repair correlated with reduced lung damage, with a reduction in both the total, and red blood cell number in the BALF of Ifnlr1 −/− mice day 8 post infection (Fig.  4, B and C, and fig. S10A ). Additionally, Ifnlr1 −/− mice had fewer immune cells infiltrating lung tissue (Fig. 4D) . In humans, influenza virus-induced epithelial damage increases susceptibility to infection by opportunistic bacterial pathogens, including S. pneumoniae (26) . To measure the effects of IFN-λ on lung barrier function, we challenged influenza virus-infected mice with S. pneumoniae. Both full IFNLR knockout mice, and mice lacking IFNLR in the stromal compartment (WT → Ifnlr1 −/− ), had improved survival following bacterial superinfection (Fig. 4E and fig. S10B ). Thus, IFN-λ signaling reduces the capacity for epithelial repair, resulting in prolonged lung damage, compromised barrier function, and increased susceptibility to bacterial superinfection.

Here we describe a mechanism by which type I and III IFN signaling aggravates lung pathology during respiratory viral infection. Although all three IFN subtypes reduced lung proliferation following treatment during influenza recovery, only endogenous IFN-λ compromised repair. This is likely due to increased IFN-λ production during infection combined with greater induction of antiproliferative pathways, compared to IFN-α/β. A recent study has shown that IFN-λ produced by dendritic cells inhibits lung epithelial repair following viral recognition (27) . Influenza virus-infected macaques revealed an elevated IFN signature late during infection bronchial tissue (28) . Additionally, COVID-19 patients displayed strong induction of IFN and p53 signaling in collected BALF (29) . Analysis of lung tissue and BALF from respiratory virus infected patients experiencing severe disease will provide insight into the mechanisms regulating disease pathogenesis. IFN-λ treatment early during influenza virus infection is protective in mice, offering antiviral protection without the proinflammatory responses associated with IFN-α/β (30, 31) . By studying specific effects in the respiratory epithelium, we identify a mechanism by which IFN exacerbates respiratory virus disease, independent of immunomodulation. Our data indicate the need for effective regulation of host IFN responses, and the importance of timing and duration when considering IFNs as therapeutic strategies to treat respiratory virus infections. Optimal protection would be achieved by strong induction of IFN-stimulated genes early during infection to curb viral replication, followed by timely down-regulation of IFN responses, enabling efficient lung epithelial repair. license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.

science.sciencemag.org/cgi/content/full/science.abc2061/DC1 Materials and Methods Supplementary Text Figs. S1 to S10 

Persistent LCMV infection is controlled by blockade of type I interferon signaling

Pathogenic potential of interferon αβ in acute influenza infection

Dysregulated type I interferon and inflammatory monocytemacrophage responses cause lethal pneumonia in SARS-CoV-infected mice

Blockade of chronic type I interferon signaling to control persistent LCMV infection

Antitumour actions of interferons: Implications for cancer therapy

Fraction of MHCII and EpCAM expression characterizes distal lung epithelial cells for alveolar type 2 cell isolation

Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells

Regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor

IFN-λ resolves inflammation via suppression of neutrophil infiltration and IL-1β production

IFN-λ suppresses intestinal inflammation by non-translational regulation of neutrophil function

Type III interferon is a critical regulator of innate antifungal immunity

Mx GTPases: Dynamin-like antiviral machines of innate immunity

Interferon-λ contributes to innate immunity of mice against influenza A virus but not against hepatotropic viruses

Wack, Type I and type III interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia

Gene expression and antiviral activity of alpha/beta interferons and interleukin-29 in virus-infected human myeloid dendritic cells

Constitutive type I interferon modulates homeostatic balance through tonic signaling

Growth and differentiation of mouse tracheal epithelial cells: Selection of a proliferative population

Cellular crosstalk in the development and regeneration of the respiratory system

Type I interferon induction of the Cdk-inhibitor p21WAF1 is accompanied by ordered G1 arrest, differentiation and apoptosis of the Daudi B-cell line

Repair and regeneration of the respiratory system: Complexity, plasticity, and mechanisms of lung stem cell function

Influenza virus infects epithelial stem/progenitor cells of the distal lung: Impact on Fgfr2b-driven epithelial repair

Integration of interferon-α/β signalling to p53 responses in tumour suppression and antiviral defence

Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen

Dual-specificity phosphatase 5 (DUSP5) as a direct transcriptional target of tumor suppressor p53

The co-pathogenesis of influenza viruses with bacteria in the lung

Type III interferons disrupt the lung epithelial barrier upon viral recognition

Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus

Heightened innate immune responses in the respiratory tract of COVID-19 patients

IFNλ is a potent anti-influenza therapeutic without the inflammatory side effects of IFNα treatment

Interferon-λ mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness

Polyclonal and monoclonal antibodies to the interferon-inducible protein Mx of influenza virus-resistant mice

Strong interferon-inducing capacity of a highly virulent variant of influenza A virus strain PR8 with deletions in the NS1 gene

Human interferon-λ3 is a potent member of the type III interferon family

Rho-associated protein kinase inhibition enhances airway epithelial Basal-cell proliferation and lentivirus transduction

Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement

USP18-based negative feedback control is induced by type I and type III interferons and