Type III interferons disrupt the lung epithelial barrier upon viral recognition

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

T he ability to resolve viral infections of the lung is dependent on the actions of interferons (IFNs) and inflammatory cytokines, yet their relative contributions to host defense and return to homeostasis remain undefined. In particular, type III IFNs (IFN-l) have attracted much attention, as they operate primarily at mucosal surfaces (1). Recent work established that, unlike other IFNs, IFN-l signaling induces antiviral activities while simultaneously limiting the tissuedamaging functions of neutrophils (2)(3)(4). When considered in the context of respiratory viral infections in which inflammation appears to be the primary driver of life-threatening symptoms, including the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (5), the ability of IFN-l to limit immunopathology but maintain antiviral activity is noteworthy. Discussions on the possible use of IFN-l against SARS-CoV-2 have begun (6), and clinical trials have been initiated. However, despite this interest in the use of IFNl to treat viral infections, the long-term effects of IFN-l on lung physiology remain largely overlooked. For example, during viral infections of the lung, immunopathology may predispose the host to opportunistic bacterial infections, and IFN-l impairs bacterial control during super-infections (7,8). It remains unresolved whether this is due to the anti-inflammatory activity of IFN-l, which reduces host resistance, or to the capacity of IFN-l to alter lung physiology upon a viral encounter. Indeed, superinfections represent the first cause of lethality upon influenza virus infection (9) and correlate with severity in coronavirus disease 2019 (COVID-19) patients (10).
Mouse models of SARS, Middle East respiratory syndrome (MERS) (11,12), and influenza (1,13) are characterized by a robust induction of type I and III IFNs. However, the involvement of these cytokines in COVID-19 is controversial (14,15). To directly evaluate the capacity of SARS-CoV-2 to induce IFNs, we tested nasooropharyngeal swabs of COVID-19 patients and healthy controls, as well as the bronchoalveolar lavage fluid (BALF) of SARS-CoV-2positive patients with severe COVID-19. Levels of IFN mRNAs in the upper airways of COVID-19 patients were not significantly different from levels in healthy controls. By contrast, BALF of patients with severe disease presented elevated levels of both inflammatory cytokines as well as type I and III IFNs (Fig. 1, A to E).
To evaluate the contribution of IFN-l to the immunopathology driven by RNA respiratory viruses uncoupled from its effect on viral replication, we devised an experimental system in which pattern recognition receptors (PRRs) involved in viral sensing were stimulated with their cognate ligands. RNA viruses are sensed via either endosomal Toll-like receptor (TLR) 3 and TLR7 or cytoplasmic retinoic acid-inducible gene I (RIG-I) and melanoma differentiationassociated protein 5 (MDA5) (16). We intratracheally instilled the TLR7 ligand, R848, or the synthetic analog of double-stranded RNA, polyinosine:polycytidylic acid [poly (I:C)], that stimulates both TLR3 and the RIG-I-MDA5 pathway in vivo (17). PRRs were stimulated over the course of 6 days to elicit prolonged innate immune activation in the lung. Both ligands induced hypothermia (Fig. 1F) and weight loss ( fig. S1A), but only poly (I:C) compromised barrier function ( Fig. 1G and fig. S1B). IFN mRNAs were strongly up-regulated by poly (I:C) but not R848 (Fig. 1, H and I). By contrast, R848 treatment induced the up-regulation of proinflammatory cytokines (i.e., Il1b), but this did not correlate with barrier function decrease ( Fig. 1, G to J, and fig. S1B).
Alterations in the epithelial barrier predispose mice to lethal bacterial superinfections (18). We therefore infected mice treated with either R848 or poly (I:C) with Staphylococcus aureus. Mice treated with poly (I:C) died upon S. aureus infection (Fig. 1K) and had higher bacterial burdens (Fig. 1L In contrast to wild-type (WT) mice, mice deficient in IFN-l receptor 1 (Ifnlr1) expression were protected from poly (I:C)-induced morbidity and barrier damage ( efficiently after poly (I:C) administration, in the presence or absence of S. aureus (Fig. 3, D to G). The most down-regulated gene in Ifnlr1 −/− epithelial cells compared with WT cells was the E3 ubiquitin-protein ligase makorin-1 (Mkrn1) (Fig. 3A and data S1). The protein encoded by this gene induces p21 degradation and favors apoptosis via p53 under oxidative stress conditions and after DNA damage (hallmarks of severe viral infections) (19). Indeed, Ifnlr1 −/− epithelial cells showed elevated levels of p21 (Fig. 3, H and I). Thus, the ability of IFN-l to reduce tissue tolerance stems from its capacity to inhibit tissue repair by directly influencing epithelial cell proliferation and viability. We next investigated the cellular source and molecular pathways that drive IFN-l production. Upon poly (I:C) administration, lungresident dendritic cells (DCs) expressed the highest levels of IFN-l transcript, during both the early and late phases after poly (I:C) administration ( Fig. 4A and fig. S8A). By contrast, epithelial cells, alveolar macrophages, and monocytes expressed type I IFNs and proinflammatory cytokines but no IFN-l transcripts ( fig. S8, A to C). Depletion of CD11c + DCs was sufficient to abolish the production of IFN-l but not type I IFNs (Fig. 4, B and C, and fig.  S8, D and E). Alveolar macrophages were not depleted upon diphtheria toxin administration ( fig. S8F) and did not produce IFN-l in response to poly (I:C) (Fig. 4A). By using in vitro-generated DCs, we found that IFN-l was induced only when the TLR3 pathway was activated (Fig. 4D and fig. S9, A and B). Consistent with in vivo data, TLR7 stimulation in vitro induced only the up-regulation of proinflammatory cytokines (Fig. 4D and fig.  S9, A and B). Ex vivo analysis showed that conventional DC1 (cDC1) are the major pro-ducer of IFN-l ( fig. S10). Activation of RIG-I and MDA5 via intracellular delivery of poly (I:C) (Fig. 4D and fig. S9, A and B) and of triphosphate hairpin RNA (3p-hpRNA; fig. S11, A to E) induced high levels of type I IFNs, but not type III IFNs, in a mitochondrial antiviral signaling protein (MAVS)-dependent manner. Blockade of endosomal acidification via chloroquine treatment confirmed the importance of TLR3 for IFN-l induction ( fig. S12, A and B). WT mice or mice that do not respond to TLR3 stimulation [Toll-like receptor adaptor molecule 1 deficient (Ticam1 −/− )] were treated in vivo with poly (I:C). Only DCs sorted from Ticam1 −/− mice did not express IFN-l mRNA, although they still expressed type I IFN mRNA (Fig. 4, E and F). Furthermore, Ticam1 −/− mice were protected against S. aureus superinfections (Fig. 4G). Ticam1 −/− mice also showed lower levels of IFN-l mRNA (but not type I IFN Broggi    mRNA) than WT mice (Fig. 4, H and I). Similar results were obtained when only hematopoietic cells were deficient in Ticam1 (Fig. 4, J to L).
The immune system evolved to protect against pathogens, but doing so often threatens host fitness and can cause immunopathologies (20). In COVID-19, SARS, MERS, and flu, severe symptoms and death occur late, and after the peak in viral load, indicating a central role for the immune system in driving the pathology (21-24). In our system, we isolated the effect of immune activation from resistance to lung viral infections and demonstrated that sustained IFN-l is produced by DCs via TLR3. TLR3 detects replication intermediates from dying cells (25) and thus is insensitive to viral immune evasion. Correspondingly, IFN-l acts on lung epithelial cells and compromises lung barrier function, predisposing the host to lethal secondary bacterial infections.
Previous findings suggested that IFN-l protects against viral infections (26) and increases the barrier functions of gut epithelial cells and endothelial cells (27)(28)(29). These discrepancies may have arisen because, in those studies, the particular cell types targeted by IFN-l were different. Furthermore, our data support the hypothesis that the detrimental activities of IFN-l occur only upon chronic exposure and in the presence of tissue damage. Early administration of IFN-l in a mouse model of COVID-19 could instead confer protection (30). Our data enjoin clinicians to carefully analyze the duration of IFN-l administration and to consider the severity of disease when IFN-l is used as a therapeutic agent against lung viral infections.