Quantitative Subcellular Proteome and Secretome Profiling of Influenza A Virus-Infected Human Primary Macrophages

Abstract

Influenza A viruses are important pathogens that cause acute respiratory diseases and annual epidemics in humans. Macrophages recognize influenza A virus infection with their pattern recognition receptors, and are involved in the activation of proper innate immune response. Here, we have used high-throughput subcellular proteomics combined with bioinformatics to provide a global view of host cellular events that are activated in response to influenza A virus infection in human primary macrophages. We show that viral infection regulates the expression and/or subcellular localization of more than one thousand host proteins at early phases of infection. Our data reveals that there are dramatic changes in mitochondrial and nuclear proteomes in response to infection. We show that a rapid cytoplasmic leakage of lysosomal proteins, including cathepsins, followed by their secretion, contributes to inflammasome activation and apoptosis seen in the infected macrophages. Also, our results demonstrate that P2X7 receptor and src tyrosine kinase activity are essential for inflammasome activation during influenza A virus infection. Finally, we show that influenza A virus infection is associated with robust secretion of different danger-associated molecular patterns (DAMPs) suggesting an important role for DAMPs in host response to influenza A virus infection. In conclusion, our high-throughput quantitative proteomics study provides important new insight into host-response against influenza A virus infection in human primary macrophages.

Global Characterization of Protein Secretion from Human Macrophages Following Non-canonical Caspase-4/5 Inflammasome Activation

“…These cells are much more heterogeneous starting material than cell lines. We have seen this in our previous studies (19,29), and here these differences manifest in quantification fold differences obtained from different biological replicates.…”

Differential proteomic analysis of respiratory samples from patients suffering from influenza

“…Till date, however, only one scientific report [19] has been documented in literature studying nasal secretions from naturally influenza infected patients. Proteins identified in this study are different from those described in influenza-infected cell lines and primary macrophages [7,22]. Ten proteins were found to be differentially upregulated in the infected children including PLUNC, cystatin S, cystatin SA, S100A9, lipocalin 1 fragments (n52), truncated lactotransferrin, two immunoglobulin (Ig) kappa fragments and one immunoglobulin (Ig) lambda fragment.…”

Quantitative Changes in Gimap3 and Gimap5 Expression Modify Mitochondrial DNA Segregation in Mice

“…After labeling, the samples were pooled and dried, and the peptides were fractionated by strong cation exchange chromatography using an Ettan HPLC system (Amersham Biosciences) connected to a Polysulfoethyl A column. Each selected SCX fraction containing labeled peptides was then processed with C18 Spin Columns (Pierce) according to the manufacturer instructions before being analyzed twice with nano-LC-ESI-MS/ MS using Ultimate 3000 nano-LC (Dionex) and QStar Elite mass spectrometer (Applied Biosystems) as previously described (Lietzen et al 2011). MS data were acquired automatically using Analyst QS 2.0 software.…”

“…To identify such factors we used a quantitative proteomic approach with iTRAQ (isobaric tags for relative and absolute quantification) labeling followed by LC-MS/MS analysis of postnuclear membrane enrichments generated from spleens of wild type and Gimap5 tm1Wlr heterozygote littermates (File S1 and File S2) (Lietzen et al 2011). Three independent biological replicates for each genotype were used for the iTRAQ analysis, which led to the identification of .1100 distinct proteins (File S1).…”