Dissection of influenza infection in vivo by single-cell RNA-sequencing
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ABSTRACT: The influenza virus is a major cause of morbidity and mortality worldwide, yet, the impact of intracellular viral invasion and the cellular response diversity remain uncharacterized. By massively parallel single-cell RNA-seq we comprehensively mapped the host lung response to in-vivo influenza infection in wild-type and Irf7-knockout mice across nine immune and non-immune cell types. We found an unexpected high prevalence of infected cells in all cell types, showed that infection is a characteristic property of cell types that is independent of type-I interferon activity, and demonstrated that all cell types responded primarily with a robust generic transcriptional response. Analysis of the viral and host transcriptomes in the same single cell enabled us to resolve the heterogeneity of bystander (exposed but uninfected) as compared to viral-infected cells. Our results highlight novel markers specific for influenza-infected as opposed to bystander cells, opening new avenues for targeted therapy aimed exclusively at infected cells.
Project description:The host response to influenza A infections is strongly influenced by host genetic factors. Animal models of genetically diverse mouse strains are well suitable to identify host genes involved in severe pathology, viral replication and immune responses. Here, we have utilizing a dual RNAseq approach that allowed us to investigate both viral and host gene expression in the same individual from a single expression assay after H1N1 infection. We performed a comparative expression analysis to identify (i) correlations between host genes and the viral gene expression, (ii) host genes involved in viral replication, and (iii) genes showing differential expression between the two mouse strains after infection. These genes may be key players involved in regulating the differences in pathogenesis and host defense mechanisms after influenza A infections. Expression levels of influenza segments correlated well with the viral load and may thus be used as surrogates for conventional viral load measurements. Furthermore, we investigated the functional role of two genes, Reg3g and Irf7, in knock-out mice and found that deletion of the Irf7 gene renders the host highly susceptible to H1N1 infection. Female, 10-12 weeks old mice were anesthetized by intra-peritoneal injection with Ketamine/Xylazine (85% NaCl (0.9%), 10% Ketamine, 5% Xylazine) with doses adjusted to the individual body weight. Mice were then intra-nasally infected with 20µl virus solution (2x10³ FFU PR8M) or mock-treated with PBS.
Project description:Despite intense study, the complexity of interactions between viral and host cell mechanisms have ensured that the cellular innate immune response to influenza infection is still not well understood, especially at early time points following infection. Single cell RNA sequencing provides a new approach with which we can not only examine these early time points of cellular response, but also examine the heterogeneity of that response and more intensely examine patterns of expression that are hidden from bulk sample sequencing. As part of a larger study investigating the immune response of lung epithelial cells to influenza infection, we performed single-cell RNA expression profiling on A549 cells, a lung epithelial cell line, undergoing either a mock infection or infection by the influenza strain PR8-NS1-GFP at MOIs of 2.0 and 0.2. We measured single cell RNA expression at 4 hours and 12 hours post infection. We found a MOI-dependent negative correlation between expression of viral genes and cellular genes, suggesting an antagonist effect of the viral proteins in the innate immune response at the transcriptional level. Also, we found an unexpected early induction of some interferon inducible genes at higher levels in infected than bystander cells, consistent with autocrine activation. Finally, IFN lambda 1 showed broad expression among infected and bystander cells, indicating a possible paracrine component to induction.hile the cellular innate immune response to influenza infection has been studied intensely, there are still many unanswered questions, especially relating to the early
Project description:Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, where the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly-infected and un-infected bystander cells and heightened in Alpha-infection. Alpha highly-infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.
Project description:The host response to influenza A infections is strongly influenced by host genetic factors. Animal models of genetically diverse mouse strains are well suitable to identify host genes involved in severe pathology, viral replication and immune responses. Here, we have utilizing a dual RNAseq approach that allowed us to investigate both viral and host gene expression in the same individual from a single expression assay after H1N1 infection. We performed a comparative expression analysis to identify (i) correlations between host genes and the viral gene expression, (ii) host genes involved in viral replication, and (iii) genes showing differential expression between the two mouse strains after infection. These genes may be key players involved in regulating the differences in pathogenesis and host defense mechanisms after influenza A infections. Expression levels of influenza segments correlated well with the viral load and may thus be used as surrogates for conventional viral load measurements. Furthermore, we investigated the functional role of two genes, Reg3g and Irf7, in knock-out mice and found that deletion of the Irf7 gene renders the host highly susceptible to H1N1 infection.
Project description:Virus and host factors contribute to cell-to-cell variation in viral infection and determine the outcome of the overall infection. However, the extent of the variability at the single cell level and how it impacts virus-host interactions at a systems level are not well understood. To characterize the dynamics of viral transcription and host responses, we used single-cell RNA sequencing to quantify at multiple time points the host and viral transcriptomes of human A549 cells and primary bronchial epithelial cells infected with influenza A virus. We observed substantial variability of viral transcription between cells, including the accumulation of defective viral genomes (DVGs) that impact viral replication. We show a correlation between DVGs and viral-induced variation of the host transcriptional program and an association between differential induction of innate immune response genes and attenuated viral transcription in subpopulations of cells. These observations at the single cell level improve our understanding of the complex virus-host interplay during influenza infection.
Project description:IRF7 plays a critical role in the production and amplification of the antiviral type I and III interferon response. Autosomal recessive IRF7-deficiency resulted in life-threatening influenza disease in a 3-year-old child. We studied the impact of IRF7-deficiency in non-hematopoietic tissues (fibroblasts and lung epithelial cells) as well in hematopoietic cells (peripheral blood mononuclear cells (PBMCs)). Genome-wide gene expression analysis demonstrated a profound loss of type I and III IFNs in PBMCs infected with influenza virus. PBMCs were isolated from 4 healthy donors and patients with deficiencies for IRF7 and UNC93B. The cells were infected with influenza virus A/CA/4/2009 at a multiplicity of infection (MOI) of 2 for 8 and 16 hours. Uninfected cells were cultured for 16 hours.
Project description:The upper respiratory tract (nasopharynx or NP) is the first site of influenza replication, allowing the virus to disseminate to the lower respiratory tract or promoting community transmission. The host response in the NP regulates an intricate balance between viral control and tissue pathology. The hyper-inflammatory responses promote epithelial injury, allowing for increased viral dissemination and susceptibility to secondary bacterial infections. However, the pathologic contributors to influenza upper respiratory tissue pathology are incompletely understood. In this study, we investigated the role of IL-17RA as a modulator of influenza host response and inflammation in the upper respiratory tract. We used a combined experimental approach involving IL-17RA-/- mice and an air-liquid interface (ALI) epithelial culture model to investigate the role of IL-17 response in epithelial inflammation, barrier function, and tissue pathology. Our data show that IL-17RA-/- mice exhibited significantly reduced neutrophilia, epithelial injury, and viral load. The reduced NP inflammation and epithelial injury in IL-17RA-/- mice correlated with increased resistance against co-infection by Streptococcus pneumoniae (Spn). IL-17A treatment, while potentiating the apoptosis of IAV-infected epithelial cells, caused bystander cell death and disrupted the barrier function in ALI epithelial model, supporting the in vivo findings.
Project description:Viral infection outcomes are governed by the complex and dynamic interplay between the infecting virus population and the host response. It is increasingly clear that both viral and host cell populations are highly heterogeneous, but little is known about how this heterogeneity influences infection dynamics or viral pathogenicity. To dissect the interactions between influenza A virus (IAV) and host cell heterogeneity, we examined the combined host and viral transcriptomes of thousands of individual cells, each infected with a single IAV virion. We observed complex patterns of viral gene expression and the existence of multiple distinct host transcriptional responses to infection at the single cell level. We show that human H1N1 and H3N2 strains differ significantly in patterns of both viral and host anti-viral gene transcriptional heterogeneity at the single cell level. Our analyses also reveal that semi-infectious particles that fail to express the viral NS can play a dominant role in triggering the innate anti-viral response to infection. Altogether, these data reveal how patterns of viral population heterogeneity can serve as a major determinant of antiviral gene activation.
Project description:Antiviral responses must be regulated to rapidly defend against infection while minimizing inflammatory damage, but the mechanisms for establishing the magnitude of response within an infected cell are not well understood. miRNAs are small non-coding RNAs that negatively regulate protein levels by binding target sequences on their cognate mRNA. Here we identify miR-144 as a negative regulator of the host antiviral response. Ectopic expression of miR-144 resulted in increased replication of three RNA viruses, influenza, EMCV, and VSV, in primary mouse lung epithelial cells. To elucidate the mechanism whereby miR-144 increases influenza replication within lung epithelial cells, TC-1 cells stably over-expressing miR-144 were infected with influenza A for 24 hours and the transcriptional profile was compared with those of infected control cells. This systems biology approach identified the transcriptional network regulated by miR-144 and demonstrate that it controls the TRAF6/IRF7 antiviral response by post-transcriptionally suppressing TRAF6 levels. In vivo ablation of miR-144 reduced influenza replication within the lung. TC-1 lung epithelial cells stably expressing miR144+miR451 or control vector were unstimulated (n=1) or infected with Influenza A/PR/8/34 (MOI=5) for 24 hours (n=3).
Project description:A. Esteban Hernandez-Vargas & Michael Meyer-Hermann. Innate Immune System Dynamics to Influenza Virus. IFAC Proceedings Volumes 45, 18 (2012).
The understanding of how influenza virus infection activates the immune system is crucial to designing prophylactic and therapeutic strategies against the infection. Nevertheless, the immune response to influenza virus infection is complex and remains largely unknown. In this paper we focus in the innate immune response to influenza virus using a mathematical model, based on interferon-induced resistance to infection of respiratory epithelial cells and the clearance of infected cells by natural killers. Simulation results show the importance of IFN-I to prevent new infections in epithelial cells and to stop the viral explosion during the first two days after infection. Nevertheless, natural killers response might be the most relevant for the first depletion in viral load due to the elimination of infected cells. Based on the reproductive number, the innate immune response is important to control the infection, although it would not be enough to clear completely the virus. The effective coordination between innate and adaptive immune response is essential for the virus eradication.