Type I and III interferons disrupt lung epithelial repair during recovery from viral infection

Interferons interfere with lung repair Interferons (IFNs) are central to antiviral immunity. Viral recognition elicits IFN production, which in turn triggers the transcription of IFN-stimulated genes (ISGs), which engage in various antiviral functions. Type I IFNs (IFN-α and IFN-β) are widely expressed and can result in immunopathology during viral infections. By contrast, type III IFN (IFN-λ) responses are primarily restricted to mucosal surfaces and are thought to confer antiviral protection without driving damaging proinflammatory responses. Accordingly, IFN-λ has been proposed as a therapeutic in coronavirus disease 2019 (COVID-19) and other such viral respiratory diseases (see the Perspective by Grajales-Reyes and Colonna). Broggi et al. report that COVID-19 patient morbidity correlates with the high expression of type I and III IFNs in the lung. Furthermore, IFN-λ secreted by dendritic cells in the lungs of mice exposed to synthetic viral RNA causes damage to the lung epithelium, which increases susceptibility to lethal bacterial superinfections. Similarly, using a mouse model of influenza infection, Major et al. found that IFN signaling (especially IFN-λ) hampers lung repair by inducing p53 and inhibiting epithelial proliferation and differentiation. Complicating this picture, Hadjadj et al. observed that peripheral blood immune cells from severe and critical COVID-19 patients have diminished type I IFN and enhanced proinflammatory interleukin-6– and tumor necrosis factor-α–fueled responses. This suggests that in contrast to local production, systemic production of IFNs may be beneficial. The results of this trio of studies suggest that the location, timing, and duration of IFN exposure are critical parameters underlying the success or failure of therapeutics for viral respiratory infections. Science, this issue p. 706, p. 712, p. 718; see also p. 626

D uring infection with respiratory viruses, disease severity is linked to lung epithelial destruction, owing to both cytopathic viral effects and immune-mediated damage. Epithelial loss contributes to acute respiratory distress syndrome, pneumonia, and increased susceptibility to bacterial superinfections. Thus, the restoration of damaged epithelial tissues is paramount to maintain lung function and barrier protection.
Interferons (IFNs) are key to the antiviral host defense. IFN-a and IFN-b (IFN-a/b) and IFN-l are induced upon viral recognition, and they trigger transcription of IFN-stimulated genes with antiviral functions in infected and bystander cells. Because of widespread expression of the type I IFN receptor (IFNAR) in immune cells, IFN-a/b responses can result in immunopathology during viral infections, including influenza virus and severe acute respiratory syndrome-coronavirus 1 (SARS-CoV-1) (1)(2)(3)(4). The IFN-l receptor (IFNLR) is mainly expressed at epithelial barriers, and IFN-l responses are therefore often characterized by their ability to confer localized antiviral protection at the site of infection without driving damaging proinflammatory responses like those associated with IFN-a/b. In addition to antiviral and proinflammatory activity, IFNs exert antiproliferative and proapoptotic functions (5). Despite a growing understanding of immunopathology in respiratory viral infec-tion, 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 substantial immune cell infiltration and lung damage (fig. S1, A to D). Recovery from infection coincided with the onset of epithelial regeneration ( fig. S1, B to D). To further investigate the dynamics of lung repair after influenza virus infection, epithelial cell proliferation was analyzed by flow cytometry using the proliferation marker Ki67 (see gating strategy in fig. S2). During steady-state conditions, type II alveolar epithelial (AT2) cells (EpCam + MHCII + CD49f lo ) (6)(7)(8) showed a low rate of turnover (Fig. 1A). However, after influenza virus-induced lung damage, AT2 cells underwent rapid proliferation starting at days 5 to 7 after infection, which correlated 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-a, IFN-b, and IFN-l) in bronchoalveolar lavage fluid (BALF) throughout infection. IFNs were produced rapidly, peaking 2 days after infection (Fig. 1B). The magnitude of IFN-l production was significantly greater than that of IFN-a/b, both in duration and in length of peak production. Notably, only IFN-l was detected 7 to 8 days after infection, coinciding with the onset of epithelial recovery (Fig. 1, A and B). Thus, after influenza virus infection, signaling triggered by IFNsparticularly by IFN-l-overlaps with the onset of lung repair.
To compare the effects of equipotent amounts of IFN-a, IFN-b, and IFN-l on lung repair, mice were treated during recovery from influenza virus infection (7 to 10 days after infection; figs. S3A and S4). To study the effects of IFN treatment specifically on epithelial cells, we generated irradiation bone marrow (BM) chimeras in which WT recipients were given Ifnar1 −/− BM cells (Ifnar1 −/− → WT BM chimeric), thereby restricting IFNAR expression to the stromal compartment.
In chimeric mice, both IFN-a and IFN-b treatments significantly reduced the proliferation of AT2 cells on day 11 after influenza virus infection (Fig. 1C). Similarly, IFN-l treatment reduced AT2 cell proliferation in WT mice (Fig. 1D). Reductions in proliferation were independent of changes in viral burden ( fig. S3, B and C). The IFN-l-mediated reduction in AT2 cell proliferation did not require IFN-l 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 to bear in mind when using inbred mouse strains for influenza virus infection is their 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-DNS1 for a moreclinically relevant influenza model. IFN-l 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 after 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 with WT controls (Fig. 1, F and G). This was dependent on IFN signaling specifically through the epithelium, because 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 owing 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 been previously shown to be important for the production of IFN-l during influenza virus infection (15,16). Consistent with these findings, we observed a significant reduction in IFN-l (and in IFN-a/b) production in Ifnar1 −/− mice compared with WT; yet, we saw little change in IFN-a/b levels in Ifnlr1 −/− mice ( fig. S5B). Thus, the improved epithelial proliferation in Ifnar1 −/− mice may result from reduced IFN-l. IFN production defects in Ifnar1 −/− mice are linked to reduced steadystate 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 IFN-l production ( fig.  S5C), despite blocking IFN-a/b signaling through IFNAR ( fig. S5D). Notably, anti-IFNAR treatment from day 0 or day 3 after infection had no effect on lung epithelial cell proliferation ( fig. S5E). Thus, in murine influenza virus infection, endogenous IFN-l 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), which recapitulates 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 potencies ( fig. S6). All three IFN subtypes significantly impaired the growth of AEC cultures, with IFN-b and IFN-l having the most significant effects (Fig. 2 fig. S7, E to G). Thus, the increase in apoptosis observed may occur as a result of failed progression through the cell cycle after IFN treatment, as has been seen previously (20).
We next examined the effects of IFNs on AEC differentiation. After acute damage, populations of basal cells and Scgb1a1 + secretory cells give rise to secretory and multiciliated cell subtypes (21). To study the 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-b and IFN-l treatment significantly reduced the expression of genes pertaining to multiciliated (Mcidas and Ccno) and secretory (Muc5AC and Scb1a1) cell differentiation (Fig. 2F). Expression of the basal cell marker Krt5 remained unchanged or was increased by IFN-l treatment, which suggests maintenance of stemness ( fig. S8B). We also found reduced numbers of multiciliated cells in AEC cultures (acetylated a-tubulin + ) after IFN-l treatment, but we did not observe this with IFN-a or IFN-b treatment ( Fig. 2G and  fig. S8C). In vivo, Ifnlr1 −/− mice displayed increased multiciliated cells in repairing conducting airways on day 10 after influenza virus infection (Fig. 2H). Using flow cytometry, we quantified this increase in the frequency of differentiated AECs (EpCam hi CD49f hi CD24 + ) composed of multiciliated, goblet, and club cells ( Fig. 2I and fig. S2) (22). Thus, IFN-l 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 components analysis (PCA) clustered 4-hour IFN-treated samples together regardless of subtype (Fig. 3B), which confirmed equal sub-type dosage on the basis of previous titrations ( fig. S9A). Five days of IFN-b or IFN-l treatment clustered AECs together separately from untreated controls on both PC1 and PC2 (Fig.  3B). Gene ontology analysis confirmed that genes contributing to this variance are involved in IFN signaling and epithelial cell development (supplementary text and fig.  S9B). Ingenuity pathway analysis revealed induction of pathways regulating cell cycle and cell death after prolonged IFN treatment, most significantly induced by IFN-l across all time points (Fig. 3C). Predicted upstream transcriptional regulators identified typical regulators of IFN function-including STAT (signal transducer and activator of transcription) and IRF (IFN regulatory factor) 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-a/b 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 Gadd45g 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-a or IFN-b treatment, and we depleted neutrophils in WT mice with anti-Ly6G for IFN-l treatment ( fig. S3A). IFN-b and IFN-l, but not IFN-a, significantly up-regulated p53 expression in repairing lung epithelial cells (Fig. 3, H and I). Thus, IFN-b and IFN-l mediate antiproliferative effects in AECs via the induction of p53.
Our data support a key role for IFN signaling, particularly IFN-l, 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 (EpCam + 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 number of cells and the number of red blood cells in the BALF of Ifnlr1 −/− mice at day 8 after 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 Streptococcus pneumoniae (26).
To measure the effects of IFN-l 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 after bacterial superinfection (Fig. 4E and fig. S10B). Thus, IFN-l signaling reduces the capacity for epithelial repair, which results in prolonged lung damage, compromised barrier function, and increased susceptibility to bacterial superinfection.
In this work, 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 after treatment during influenza recovery, only endogenous IFN-l compromised repair. This is likely because of increased IFN-l production during infection combined with greater induction of antiproliferative pathways. A recent study has shown that IFN-l produced by dendritic cells inhibits lung epithelial repair after viral recognition (27). Influenza virus-infected macaques have been found to have an elevated IFN signature late during infection in bronchial tissue (28). Additionally, COVID-19 patients have displayed strong induction of IFN and p53 signaling in collected BALF samples (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-l treatment early during influenza virus infection is protective in mice, offering antiviral protection without the proinflammatory responses associated with IFN-a/b (30,31). By studying specific effects in the respiratory epithelium, we identified 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 could be achieved by strong induction of IFN-stimulated genes early during infection to curb viral replication followed by timely down-regulation of IFN responses, thereby enabling efficient lung epithelial repair.