Lung cell fates during influenza

Abstract

Roughly 1 billion people are infected by Influenza A viruses (IAVs) worldwide each year, resulting in approximately half a million deaths. Particularly concerning is the threat of IAV spillover from avian and other animal reservoirs. The recent outbreak of highly pathogenic avian influenza H5N1 in US dairy cows highlights this concern. While viruses that enter human populations from such zoonotic transmission typically lack the ability to transmit effectively between humans, they may be only a few mutations from acquiring this capacity. These newly adapted viruses have the potential to be significantly more virulent than seasonal strains. A major contributor to influenza pathology is the over-exuberant immune response to the virus, particularly when the infection is present in distal pulmonary tissues. Maladaptive immune pathway over-activation can drive tissue damage and pathology, often independently of effective viral control. Anti-inflammatories targeting host-initiated pathological processes hold promise, but these avenues require a thorough understanding of virus-triggered lung inflammation before they can be fully exploited. In this review, we will discuss recent advances in our understanding of the cell types that are targeted by IAV, the consequences of IAV infection on the biology of these cells, and their contribution to lung pathology in influenza. We will also discuss how virus-induced hyper-inflammatory responses present new entry-points for therapeutic intervention, showcasing Z-form nucleic acid-binding protein 1 (ZBP1)-initiated necroptosis as an example of one such pathway.

Fig. 1. Features of IAV infection in lung cell types of the airway and alveolar epithelia.

The airway epithelium (top) is composed of ciliated, goblet, club, basal, tuft cells and PNECs, which together enable MCC and maintain airway barrier integrity during homeostasis. IAV-infected epithelial cells undergo widespread lytic and programmed cell death. Even in the absence of cell death, infection can disrupt key cellular functions, such as ciliary activity. Protective host responses include increased secretion of mucin MUC5AC, and basal cell-driven epithelial regeneration. ISGs and cytokines, expressed by both infected and bystander epithelial cells, amplify antiviral responses and promote the recruitment of immune cells, such as DCs, to sites of infection. The alveolar epithelium (bottom) is the parenchymal tissue of the lung and is primarily composed of type I and type II AECs. Other significant cell types making up alveoli include endothelial cells, fibroblasts, and AMs. During infection, endothelial cells are significant producers of cytokines promoting immune cell recruitment, including that of cytotoxic T and NK cells, which can restrict infection, but also cause epithelial injury depending on disease severity. AM depletion hampers antiviral defense while excessive neutrophil recruitment and NET formation cause tissue damage. Aberrant ECM remodeling and hyaluronan production by fibroblasts, as well as tuft cell dysplasia, can further contribute to the disruption of alveolar function. Type II AEC-driven regeneration is a critical reparative response to infection-induced damage but can be overwhelmed in severe illness where there is extensive AEC cell death. IAV infection has also been found to compromise alveolar epithelium integrity in ways besides cell death, such as the direct disruption of cellular junctions in infected AECs. Loss of alveolar integrity and ensuing fluid leakage into alveolar luminal space are key features of viral pneumonia and ARDS. Overall, these changes to the lung epithelia highlight the intricate cascade of events and host–pathogen interactions that occur during IAV infection, particularly in cases of severe illness. Created with BioRender.com.

Fig. 2. scRNAseq analysis of lung cells isolated from IAV-infected mice.

a–d Adult (8–12-week-old) C57BL/6 mice were infected intranasally with a 2500 EID₅₀ dose of mouse-adapted influenza A/Puerto Rico/8/1934 virus, and lung cells isolated from infected mice were profiled by scRNAseq analysis. Violin plots of percent total cellular transcripts mapped to IAV genes in lung structural (a) and immune compartment (c) cell types, plotted on log scale, range 0–10% (dotted line represents estimated infection threshold). Proportion of cells infected within individual lung structural (b) and immune compartment (d) cell types based on estimated infection threshold. Data are from combined 3 and 6 dpi datasets. Cells were isolated from the lungs of 4–5 mice for each time point. The data have been published previously (NCBI BioProject PRJNA613670).

Fig. 3. Programmed cell death pathways activated by IAV infection.

IAV infection results in the production of Z-RNA, which is sensed by ZBP1. Once activated by binding Z-RNA, ZBP1 interacts with RIPK3 via RHIMs found in both proteins. RIPK3 can then induce either apoptosis via RIPK1-Fas-associated protein with death domain (FADD)-caspase-8 signaling, or necroptosis via mixed lineage kinase domain-like protein (MLKL). ZBP1-RIPK3 signaling can also activate the NLRP3 inflammasome to induce pyroptosis, via caspase-1 cleavage and activation of GSDMD. IAV infection can also induce intrinsic apoptosis via BAX/BAK/caspase-9 activation. Both PB1-F2 and NS1 proteins have been implicated in mediating mitochondrial cytochrome c release to trigger this pathway. Additionally, IAV infection can result in the initiation of extrinsic apoptosis via Fas ligand (FasL) and TRAIL-driven death receptor activation in lung cells. Created with BioRender.com.