Innate Immune Response to Influenza Virus at Single-Cell Resolution in Human Epithelial Cells Revealed Paracrine Induction of Interferon Lambda 1.

Irene Ramos,Frederique Ruf-Zamojski,Miguel Fribourg,Veronica A Dejesus,Randy A Albrecht,Carles Martínez-Romero,Elena Zaslavsky,Stuart C Sealfon,Michel Zamojski,Edwin A Carbajal,Venugopalan D Nair,Gene S Tan,Paula L Monteagudo,Tinaye Mutetwa,Gregory R Smith ,C Jayaprakash ,Nada Marjanović ,Boris Hartmann ,Adolfo Garcı́a-Sastre ,Ana Fernández-Sesma

doi:10.1128/jvi.00559-19

Abstract

Early interactions of influenza A virus (IAV) with respiratory epithelium might determine the outcome of infection. The study of global cellular innate immune responses often masks multiple aspects of the mechanisms by which populations of cells work as organized and heterogeneous systems to defeat virus infection, and how the virus counteracts these systems. In this study, we experimentally dissected the dynamics of IAV and human epithelial respiratory cell interaction during early infection at the single-cell level. We found that the number of viruses infecting a cell (multiplicity of infection [MOI]) influences the magnitude of virus antagonism of the host innate antiviral response. Infections performed at high MOIs resulted in increased viral gene expression per cell and stronger antagonist effect than infections at low MOIs. In addition, single-cell patterns of expression of interferons (IFN) and IFN-stimulated genes (ISGs) provided important insights into the contributions of the infected and bystander cells to the innate immune responses during infection. Specifically, the expression of multiple ISGs was lower in infected than in bystander cells. In contrast with other IFNs, IFN lambda 1 (IFNL1) showed a widespread pattern of expression, suggesting a different cell-to-cell propagation mechanism more reliant on paracrine signaling. Finally, we measured the dynamics of the antiviral response in primary human epithelial cells, which highlighted the importance of early innate immune responses at inhibiting virus spread.IMPORTANCE Influenza A virus (IAV) is a respiratory pathogen of high importance to public health. Annual epidemics of seasonal IAV infections in humans are a significant public health and economic burden. IAV also causes sporadic pandemics, which can have devastating effects. The main target cells for IAV replication are epithelial cells in the respiratory epithelium. The cellular innate immune responses induced in these cells upon infection are critical for defense against the virus, and therefore, it is important to understand the complex interactions between the virus and the host cells. In this study, we investigated the innate immune response to IAV in the respiratory epithelium at the single-cell level, providing a better understanding on how a population of epithelial cells functions as a complex system to orchestrate the response to virus infection and how the virus counteracts this system.

Highlights

Interactions of influenza A virus (IAV) with respiratory epithelium might determine the outcome of infection
Signaling events downstream of IFNAR1-IFNAR2 and IFNLR1-IL10R2 are highly similar, despite the sequence and structure of these receptors being extremely different [11]. These events include activation of Janus kinase (JAK) family kinases, phosphorylation and dimerization of the signal transducer and activator of transcription 1 (STAT1) and STAT2, association between STAT complexes and interferon responsive factor 9 (IRF9) to form interferon-stimulated gene factor 3 (ISGF3), and translocation of ISGF3 to the nucleus, where it binds to specific DNA elements to induce IFN-stimulated genes (ISGs) expression [12]
When we measured the median fluorescence intensity per cell in the infected cells (GFPϩ cells), we found that a higher multiplicity of infection (MOI) was associated with higher levels of nonstructural protein 1 (NS1) expression per cell at every time point (Fig. 1C)

Summary

Introduction

Interactions of influenza A virus (IAV) with respiratory epithelium might determine the outcome of infection. The cellular innate immune responses induced in these cells upon infection are critical for defense against the virus, and it is important to understand the complex interactions between the virus and the host cells. We investigated the innate immune response to IAV in the respiratory epithelium at the single-cell level, providing a better understanding on how a population of epithelial cells functions as a complex system to orchestrate the response to virus infection and how the virus counteracts this system. Signaling events downstream of IFNAR1-IFNAR2 and IFNLR1-IL10R2 are highly similar, despite the sequence and structure of these receptors being extremely different [11] These events include activation of Janus kinase (JAK) family kinases, phosphorylation and dimerization of the signal transducer and activator of transcription 1 (STAT1) and STAT2, association between STAT complexes and interferon responsive factor 9 (IRF9) to form interferon-stimulated gene factor 3 (ISGF3), and translocation of ISGF3 to the nucleus, where it binds to specific DNA elements to induce ISG expression [12]. The PB1-F2 and PB2 viral proteins have been shown to prevent mitochondrial antiviral-signaling protein (MAVS) activation and IFN induction [37, 38], and the PA-X protein has been reported to degrade cellular mRNA [46]

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Results

Conclusion