A systems genomics approach uncovers molecular associates of RSV severity.

Respiratory syncytial virus (RSV) infection results in millions of hospitalizations and thousands of deaths each year. Variations in the adaptive and innate immune response appear to be associated with RSV severity. To investigate the host response to RSV infection in infants, we performed a systems-level study of RSV pathophysiology, incorporating high-throughput measurements of the peripheral innate and adaptive immune systems and the airway epithelium and microbiota. We implemented a novel multi-omic data integration method based on multilayered principal component analysis, penalized regression, and feature weight back-propagation, which enabled us to identify cellular pathways associated with RSV severity. In both airway and immune cells, we found an association between RSV severity and activation of pathways controlling Th17 and acute phase response signaling, as well as inhibition of B cell receptor signaling. Dysregulation of both the humoral and mucosal response to RSV may play a critical role in determining illness severity.

CX3CR1 Engagement by Respiratory Syncytial Virus Leads to Induction of Nucleolin and Dysregulation of Cilia-related Genes.

Respiratory syncytial virus (RSV) contains a conserved CX3C motif on the ectodomain of the G-protein. The motif has been indicated as facilitating attachment of the virus to the host initiating infection via the human CX3CR1 receptor. The natural CX3CR1 ligand, CX3CL1, has been shown to induce signaling pathways resulting in transcriptional changes in the host cells. We hypothesize that binding of RSV to CX3CR1 via CX3C leads to transcriptional changes in host epithelial cells. Using transcriptomic analysis, the effect of CX3CR1 engagement by RSV was investigated. Normal human bronchial epithelial (NHBE) cells were infected with RSV virus containing either wildtype G-protein, or a mutant virus containing a CX4C mutation in the G-protein. RNA sequencing was performed on mock and 4-days-post-infected cultures. NHBE cultures were also treated with purified recombinant wild-type A2 G-protein. Here we report that RSV infection resulted in significant changes in the levels 766 transcripts. Many nuclear associated proteins were upregulated in the WT group, including nucleolin. Alternatively, cilia-associated genes, including CC2D2A and CFAP221 (PCDP1), were downregulated. The addition of recombinant G-protein to the culture lead to the suppression of cilia-related genes while also inducing nucleolin. Mutation of the CX3C motif (CX4C) reversed these effects on transcription decreasing nucleolin induction and lessening the suppression of cilia-related transcripts in culture. Furthermore, immunohistochemical staining demonstrated decreases in in ciliated cells and altered morphology. Therefore, it appears that engagement of CX3CR1 leads to induction of genes necessary for RSV entry as well as dysregulation of genes associated with cilia function.ImportanceRespiratory Syncytial Virus (RSV) has an enormous impact on infants and the elderly including increased fatality rates and potential for causing lifelong lung problems. Humans become infected with RSV through the inhalation of viral particles exhaled from an infected individual. These virus particles contain specific proteins that the virus uses to attach to human ciliated lung epithelial cells, initiating infection. Two viral proteins, G-protein and F-protein, have been shown to bind to human CX3CR1and nucleolin, respectively. Here we show that the G-protein induces nucleolin and suppresses gene transcripts specific to ciliated cells. Furthermore, we show that mutation of the CX3C-motif on the G-protein, CX4C, reverses these transcriptional changes.

Airway gene-expression classifiers for respiratory syncytial virus (RSV) disease severity in infants.

BACKGROUND: A substantial number of infants infected with RSV develop severe symptoms requiring hospitalization. We currently lack accurate biomarkers that are associated with severe illness. METHOD: We defined airway gene expression profiles based on RNA sequencing from nasal brush samples from 106 full-tem previously healthy RSV infected subjects during acute infection (day 1-10 of illness) and convalescence stage (day 28 of illness). All subjects were assigned a clinical illness severity score (GRSS). Using AIC-based model selection, we built a sparse linear correlate of GRSS based on 41 genes (NGSS1). We also built an alternate model based upon 13 genes associated with severe infection acutely but displaying stable expression over time (NGSS2). RESULTS: NGSS1 is strongly correlated with the disease severity, demonstrating a naïve correlation (ρ) of ρ = 0.935 and cross-validated correlation of 0.813. As a binary classifier (mild versus severe), NGSS1 correctly classifies disease severity in 89.6% of the subjects following cross-validation. NGSS2 has slightly less, but comparable, accuracy with a cross-validated correlation of 0.741 and classification accuracy of 84.0%. CONCLUSION: Airway gene expression patterns, obtained following a minimally-invasive procedure, have potential utility for development of clinically useful biomarkers that correlate with disease severity in primary RSV infection.

Airway Gene Expression Correlates of Respiratory Syncytial Virus Disease Severity and Microbiome Composition in Infants.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants. The causes and correlates of severe illness in the majority of infants are poorly defined. METHODS: We recruited a cohort of RSV-infected infants and simultaneously assayed the molecular status of their airways and the presence of airway microbiota. We used rigorous statistical approaches to identify gene expression patterns associated with disease severity and microbiota composition, separately and in combination. RESULTS: We measured comprehensive airway gene expression patterns in 106 infants with primary RSV infection. We identified an airway gene expression signature of severe illness dominated by excessive chemokine expression. We also found an association between Haemophilus influenzae, disease severity, and airway lymphocyte accumulation. Exploring the time of onset of clinical symptoms revealed acute activation of interferon signaling following RSV infection in infants with mild or moderate illness, which was absent in subjects with severe illness. CONCLUSIONS: Our data reveal that airway gene expression patterns distinguish mild/moderate from severe illness. Furthermore, our data identify biomarkers that may be therapeutic targets or useful for measuring efficacy of intervention responses.

Unbiased analysis of peripheral blood mononuclear cells reveals CD4 T cell response to RSV matrix protein.

Respiratory syncytial virus (RSV) is the most important cause of respiratory tract illness especially in young infants that develop severe disease requiring hospitalization, and accounting for 74,000-126,000 admissions in the United States (Rezaee et al., 2017; Resch, 2017). Observations of neonatal and infant T cells suggest that they may express different immune markers compared to T-cells from older children. Flow cytometry analysis of cellular responses using "conventional" anti-viral markers (IL2, IFN-γ, TNF, IL10 and IL4) upon RSV-peptide stimulation detected an overall low RSV response in peripheral blood. Therefore we sought an unbiased approach to identify RSV-specific immune markers using RNA-sequencing upon stimulation of infant PBMCs with overlapping peptides representing RSV antigens. To understand the cellular response using transcriptional signatures, transcription factors and cell-type specific signatures were used to investigate breadth of response across peptides. Unexpected from the ICS data, M peptide induced a response equivalent to the F-peptide and was characterized by activation of GATA2, 3, STAT3 and IRF1. This along with upregulation of several unconventional T cell signatures was only observed upon M-peptide stimulation. Moreover, signatures of natural RSV infections were identified from the data available in the public domain to investigate similarities between transcriptional signatures from PBMCs and upon peptide stimulation. This analysis also suggested activation of T cell response upon M-peptide stimulation. Hence, based on transcriptional response, markers were chosen to validate the role of M-peptide in activation of T cells. Indeed, CD4+CXCL9+ cells were identified upon M-peptide stimulation by flow cytometry. Future work using additional markers identified in this study could reveal additional unconventional T cells responding to RSV infections in infants. In conclusion, T cell responses to RSV in infants may not follow the canonical Th1/Th2 patterns of effector responses but include additional functions that may be unique to the neonatal period and correlate with clinical outcomes.

Flow-based sorting of neonatal lymphocyte populations for transcriptomics analysis.

RATIONALE: Emerging data suggest an important role for T lymphocytes in the pathogenesis of chronic lung disease in preterm infants. Comprehensive assessment of the lymphocyte transcriptome may identify biomarkers and mechanisms of disease. METHODS: Small volume peripheral blood samples were collected from premature infants enrolled with consent in the Prematurity and Respiratory Outcomes Program (PROP), at the time of discharge from the hospital. Blood samples were collected at two sites and shipped to a central laboratory for processing. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation and separated into individual lymphocyte cell types by fluorescence-activated cell sorting. Gating strategies were optimized to ensure reproducible recovery of highly purified lymphocyte populations over a multi-year recruitment period. RNA was isolated from sorted cells and characterized by high-throughput sequencing (RNASeq). RESULTS: Blood volumes averaged 2.5ml, and sufficient PBMCs were collected from 165 of the 246 samples obtained (67%) from the 277 recruited subjects to complete sorting and RNASeq analysis on the resulting sorted cells. The number of total lymphocytes per ml of blood in the neonatal subjects was approximately 4 million/ml. Total lymphocyte frequencies recovered following sort varied widely among subjects, as did the frequency of individual lymphocyte and NK cell sub-populations. RNA yield from sorted cells varied according to cell type, but RNA of sufficient quantity and quality was recovered to enable RNASeq. SUMMARY: Our results describe a validated procedure for the generation of genome-wide expression data from isolated lymphocyte sub-populations obtained from newborn blood.