In this project we have combined proteomics, bioinformatics and immunological studies to characterize virus-induced changes in human primary macrophages and HaCaT keratinocytes.
We show that influenza A virus infection triggers significant changes in intracellular proteomes of human primary macrophages. The production of several antiviral proteins is initiated as a result of infection. In addition, several inflammatory pathways, especially NLRP3 inflammasome activation, are activated in influenza A virus infected and polyI:C transfected human primary macrophages. Inflammasome activation in human macrophages was regulated by cathepsins, src tyrosine kinase and P2X7 receptor activities and was followed by the initiation of apoptotic events only few hours post stimulation. Caspase-3-dependent apoptosis was detected in polyI:C transfected HaCaT keratinocytes as well as in influenza A virus infected and polyI:C transfected human primary macrophages. Caspase activation resulted in, for example, cytokeratin-18 cleavage and changes in 14-3-3 protein expression in HaCaT keratinocytes. Finally, we show that influenza A virus infection and polyI:C transfection trigger extensive secretion of various different proteins such as inflammatory, vesicle-related and danger signal proteins from human primary macrophages which might have an important role in cell-to-cell signaling after viral infection.
In conclusion, our studies show that quantitative subcellular proteomics and secretome analysis is a powerful tool for system-wide studies of cellular antiviral defense responses. Cellular signaling events are often regulated by protein posttranslational modifications such as phosphorylation and ubiquitination and especially protein ubiquitination has been recently associated with various innate immune signaling events. Therefore, large-scale studies of protein phosphorylation and ubiquitination could give deeper insights into regulation of host cell innate immune responses. Finally, to be able to utilize the full potential of large-scale proteomic studies, close collaboration between proteomics, bioinformatics and biology experts is required.
ACKNOWLEDGEMENTS
This project was carried out at the Institute of Biotechnology, University of Helsinki in close collaboration with the Unit of Excellence in Immunotoxicology, Finnish Institute of Occupational Health and the Department of Information Technology, University of Turku. The Institute of Biotechnology is acknowledged for providing excellent facilities for the research.
The work was funded by the Academy of Finland and the Helsinki Graduate Program in Biotechnology and Molecular Biology (GPBM). GPBM is also thanked for providing a variety of interesting courses for PhD studies.
I am extremely grateful to my supervisors Docent Tuula Nyman and Docent Sampsa Matikainen for all the guidance and help during these years. It has been a pleasure to work under your supervision. Your research enthusiasm and expertise have been important for the progress of this project.
I would like to thank the reviewers of this thesis, Docent Jaana Vesterinen and Docent Sampsa Hautaniemi, for their valuable comments. Docent Jaana Vesterinen and Professor Juho Rousu are thanked for the advises and discussions in the thesis committee meetings.
I am grateful to Docent Nisse Kalkkinen, the former head of the Protein Chemisry Research Group, for the opportunity to work in his well-equipped laboratory.
I express my gratitude to all the coauthors of the publications. I would especially like to thank Tiina Öhman: I couldn´n have hoped for a better teacher and lab partner. I am also thankful to Johanna Kerminen for all the hard work in immunology as well as all the encouraging words.
Olli Nevalainen and Jussi Salmi are thanked for the fruitful collaboration and for sharing their expertise in bioinformatics with us. Tero Aittokallio is thanked for the valuable help in the analysis of complex proteomic data.
I would also like to thank all the former and current colleagues in the Protein Chemistry Group and at FIOH for creating such a pleasant working environment. I would especially like to thank the other occupants of “Lastenhuone”, Pia, Juho and Wojciech, for all the humour and refreshing discussions.
Finally, I would like to thank my family, friends and Kalle for their love, patience and support.
Helsinki, April 2012
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