Secretome analysis of human macrophages activated by microbial stimuli

Show full item record

Permalink

http://urn.fi/URN:ISBN:978-951-51-3522-3
Title: Secretome analysis of human macrophages activated by microbial stimuli
Author: Lorey, Martina
Contributor: University of Helsinki, Faculty of Medicine, Clinicum, Department of Medicine, Faculty of Medicine
Thesis level: Doctoral dissertation (article-based)
Belongs to series: Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis
Abstract: The innate immune system is the first line of defence against microbial infections. Macrophages are the key cells of the innate immune system. Activated macrophages release inflammatory mediators through conventional and extracellular vesicle (EV)-mediated protein secretion. These protein secretion pathways are major components of intercellular communication in the immune system. Protein secretion is activated by inflammasomes in response to microbial components and endogenous danger signals. Inflammasomes are molecular platforms of the innate immune system that are critical to both local and systemic inflammation: they are involved in the pathogenesis of immune-mediated inflammatory diseases. Inflammasomes can be activated by both microbial stimuli as well as by endogenous danger signal molecules. The secretome of the cell is the pattern of all secreted molecules at a given time; it can provide important insights about the cell’s status as well as information about intercellular communication. The secretome can be studied by mass spectrometry (MS)-based proteomics; this technique provides a system-level overview of the current state of the cell. While traditional assays like ELISA or Luminex can quantify selected proteins in low pg/mL levels, they can only analyse a limited number of proteins. Proteomics offers the same sensitivity but supplemented with the capability of multiplex quantitative analysis of thousands of proteins in the same sample. The main goal of this work was to unravel the innate immune response of human macrophages activated by microbial stimuli with a focus on protein secretion using proteomics combined with bioinformatics and functional studies. The microbial stimuli used included Influenza A viruses (IAVs) and lipopolysaccharide (LPS), the cell wall components of Gram-negative bacteria. Both IAVs and Gram-negative bacteria are important human pathogens associated with aggressive infections. Influenza A virus and LPS are known to activate canonical NLRP3 inflammasome and non-canonical caspase-4/5 inflammasome, respectively. Inflammasome activation is followed by protein secretion which was studied in detail, with a special emphasis on unconventional protein secretion. In both studies, mediators of inflammation were released through EVs in response to influenza A virus infection and intracellular LPS stimulation. These included many danger signal proteins, cytokines and components of translational machinery. Our system-level characterization using modern mass spectrometry-based proteomics approach provides novel information how macrophages communicate to other cells through protein secretion. The results suggest that unconventional protein secretion is an essential part of the innate immune response to different activation stimuli.Luontainen immuunivaste on elimistön ensimmäinen puolustusreaktio mikrobi-infektiota vastaan. Makrofagit ovat luontaisen immuunijärjestelmän tärkeimpiä soluja, jotka aktivoituessaan erittävät tulehdusvälittäjäaineita ja muita proteiineja eri mekanismeilla, esimerkiksi membraanivesikkelien välityksellä. Proteiinien eritys muodostaa olennaisen osan solujen välisestä kommunikaatiota. Inflammasomit ovat luontaisen immuunivasteen proteiinikomplekseja, jotka käynnistävät proteiinierityksen mikrobien tai endogeenisten vaarasignaalien vaikutuksesta. Inflammasomeilla on keskeinen merkitys paikallisen ja systeemisen tulehduksen laukaisijoina. Niiden liiallinen aktivoituminen voi johtaa immuunivälitteisten tulehdussairauksien syntyyn. Tässä väitöskirjatyössä on tutkittu kokonaisvaltaisesti makrofagien erittämiä proteiineja luontaisen immuunivasteen aktivoitumisen aikana. Aktivaatiosignaaleina käytettiin influenssa A virus infektiota ja Gram-negatiivisten bakteerien soluseinärakennetta, lipolysakkaridia (LPS). Tutkimuksessa hyödynnettiin uusia systeemibiologian menetelmiä, proteomiikkaa ja bioinformatiikkaa. Näiden menetelmien avulla on mahdollista tutkia lähes kaikkia makrofagien erittämiä proteiineja samanaikaisesti. Sekä influenssa A virus infektio että LPS aktivoivat makrofageissa inflammasomi-kompleksin. Väitöskirjatutkimuksissa keskityttiin inflammasomiaktivaation jälkeisen proteiinierityksen tutkimiseen, erityisesti selvitettiin miten membraanivesikkelien proteiinikoostumus muuttuu aktivaation seurauksena. Tutkimuksessa löydettiin lukuisia uusia signalointiproteiineja, joita makrofagit erittivät aktivaation seurauksena. Tutkimustulokset osoittavat että vesikkelivälitteinen proteiinieritys on keskeinen osa luontaista immuunipuolustusjärjestelmää.
URI: URN:ISBN:978-951-51-3522-3
http://hdl.handle.net/10138/189282
Date: 2017-06-29
Subject:
Rights: This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.


Files in this item

Total number of downloads: Loading...

Files Size Format View
secretom.pdf 10.18Mb PDF View/Open

This item appears in the following Collection(s)

Show full item record