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Aquaporins in Infection and Inflammation
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The ability of eukaryotic cells to change their shape and to migrate directionally is highly dependent on active volume regulation in cells building up tissues as well as in individual cells. Transmembrane fluxes of water via specialized water channels, called aquaporins (AQPs), facilitate the changes of volume and shape, which additionally require a complex interplay between the plasma membrane and the cytoskeleton. AQPs have been shown to be involved in the development of inflammatory processes and diseases. The aims of the studies underlying this thesis were to further elucidate the expression and function of AQPs in both bacterial and viral infections as well as in the inflammatory disease, microscopic colitis. For this, molecular techniques qPCR, immunoblotting and live, holographic, confocal and super-resolution imaging were used.

When cells of the innate immune system encounter pathogens they need to respond and prepare for migration and phagocytosis and do so through volume regulatory processes. The Gramnegative bacterium Pseudomonas aeruginosa utilizes a small molecule-based communication system, called quorum sensing (QS) to control the production of its virulence factors and biofilms. We found that P. aeruginosa with a complete QS system elicits a stronger phagocytic response in human blood-derived macrophages compared to its lasI-/rhlI- mutant lacking the production of the QS molecules N-butyryl-L-homoserine lactone (C4-HSL) and N-3-oxododecanoyl-L-homoserine lactone (3O-C12-HSL). Infection with P. aeruginosa further increases the expression of AQP9 and induces re-localisation of AQP9 to the front and trailing ends of macrophages. Moreover, the 3O-C12-HSL alone elevates the expression of AQP9, redistribute the water channel to the front and rear ends and increases the cell area and volume of macrophages. Both infection with the wild type P. aeruginosa and the treatment with 3OC12-HSL change the nano-structural architecture of the AQP9 distribution in macrophages.

Viruses use the intracellular machinery of the invaded cells to produce and assemble new viral bodies. Intracellular AQPs are localised in a membranes of cellular organelles to regulate their function and morphology. C3H10T1/2 fibroblasts transiently expressing green fluorescent protein (GFP)-AQP6 show a reduced expression of AQP6 after Hazara virus infection and an increased cell area. Overexpressing AQP6 in C3H10T1/2 cells reduces the infectivity of Hazara virus indicating that AQP6 expression has a protective role in virus infections.

Ion and water channels in the epithelial cell lining tightly regulate the water homeostasis. In microscopic colitis (MC), patients suffer from severe watery diarrhoeas. For the first time, we have shown that the expression of AQP1, 8 and 11 and the sodium/hydrogen exchanger NHE1 are reduced in colonic biopsies from MC patients compared to healthy control individuals. Following treatment with the glucocorticoid budesonide the patients experienced a rapid recovery and we observed a restored or increased expression of the AQPs and NHE1 during treatment, suggesting a role for AQPs in the diarrhoeal mechanisms in MC.

Taken together, this thesis provides new evidence on the importance of water homeostasis regulation through AQPs during infections and inflammation and opens up a door for further investigations of roles for AQPs in inflammatory processes.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. , 58 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1520
National Category
Public Health, Global Health, Social Medicine and Epidemiology Clinical Laboratory Medicine Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-127500DOI: 10.3384/diss.diva-127500ISBN: 978-91-7685-792-2 (print)OAI: oai:DiVA.org:liu-127500DiVA: diva2:924453
Public defence
2016-06-02, Hasselqvistsalen, Campus US, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2016-04-28 Created: 2016-04-28 Last updated: 2016-04-28Bibliographically approved
List of papers
1. Pseudomonas aeruginosa lasI/rhlI quorum sensing genes promote phagocytosis and aquaporin 9 redistribution to the leading and trailing regions in macrophages
Open this publication in new window or tab >>Pseudomonas aeruginosa lasI/rhlI quorum sensing genes promote phagocytosis and aquaporin 9 redistribution to the leading and trailing regions in macrophages
2015 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, no 915Article in journal (Refereed) Published
Abstract [en]

Pseudomonas aeruginosa controls production of its multiple virulence factors and biofilm development via the quorum sensing (QS) system. QS signals also interact with and affect the behavior of eukaryotic cells. Host water homeostasis and aquaporins (AQP) are essential during pathological conditions since they interfere with the cell cytoskeleton and signaling, and hereby affect cell morphology and functions. We investigated the contribution of F? aeruginosa QS genes lasl/rhIl to phagocytosis, cell morphology, AQP9 expression, and distribution in human macrophages, using immunoblotting, confocal, and nanoscale imaging. Wild type F? aeruginosa with a functional QS system was a more attractive prey for macrophages than the lasl/rhIl mutant lacking the production of QS molecules, 30-C-12-HSL, and C-4 -HSL, and associated virulence factors. The F? aeruginosa infections resulted in elevated AQP9 expression and relocalization to the leading and trailing regions in macrophages, increased cell area and length; bacteria with a functional QS system lasl/rhIl achieved stronger responses. We present evidence for a new role of water fluxes via AQP9 during bacteria macrophage interaction and for the QS system as an important stimulus in this process. These novel events in the interplay between F? aeruginosa and macrophages may influence on the outcome of infection, inflammation, and development of disease.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2015
Keyword
host-bacteria relationship; quorum sensing; N-acylhomoserine lactone; innate immunity; macrophage; water homeostasis; aquaporin
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-122056 (URN)10.3389/fmicb.2015.00915 (DOI)000360626200001 ()26388857 (PubMedID)
Note

Funding Agencies|Swedish Research Council [2010-3045]; European Science foundation (TraPPs Euromembrane project); Euro-BioImaging Proof-of Concept Studies; Magnus Bergvalls Foundation; Faculty of Health Sciences, Linkoping University

Available from: 2015-12-18 Created: 2015-10-19 Last updated: 2016-04-28
2. Pseudomonas aeruginosa N-3-oxo-dodecanoyl-homoserine Lactone Elicits Changes in Cell Volume, Morphology, and AQP9 Characteristics in Macrophages
Open this publication in new window or tab >>Pseudomonas aeruginosa N-3-oxo-dodecanoyl-homoserine Lactone Elicits Changes in Cell Volume, Morphology, and AQP9 Characteristics in Macrophages
2016 (English)In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, no 32Article in journal (Refereed) Published
Abstract [en]

Quorum sensing (QS) communication allows Pseudomonas aeruginosa to collectively control its population density and the production of biofilms and virulence factors. QS signal molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (30-C-12-HSL), can also affect the behavior of host cells, e.g., by modulating the chemotaxis, migration, and phagocytosis of human leukocytes. Moreover, host water homeostasis and water channels aquaporins (AQP) are critical for cell morphology and functions as AQP interact indirectly with the cell cytoskeleton and signaling cascades. Here, we investigated how P aeruginosa 30-C-12-HSL affects cell morphology, area, volume and AQP9 expression and distribution in human primary macrophages, using quantitative PCR, immunoblotting, two- and three-dimensional live imaging, confocal and nanoscale imaging. Thus, 30-C-12-HSL enhanced cell volume and area and induced cell shape and protrusion fluctuations in macrophages, processes tentatively driven by fluxes of water across cell membrane through AQP9, the predominant AQP in macrophages. Moreover, 30-C-12-HSL upregulated the expression of AQP9 at both the protein and mRNA levels. This was accompanied with enhanced whole cell AQP9 fluorescent intensity and redistribution of AQP9 to the leading and trailing regions, in parallel with increased cell area in the macrophages. Finally, nanoscopy imaging provided details on AQP9 dynamics and architecture within the lamellipodial area of 30-C-12-HSL-stimulated cells. We suggest that these novel events in the interaction between P aeruginosa and macrophage may have an impact on the effectiveness of innate immune cells to fight bacteria, and thereby resolve the early stages of infections and inflammations.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2016
Keyword
host-bacteria interactions; quorum sensing; N-acylhomoserinelactone; innate immunity; macrophage; water homeostasis; aquaporin
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-127262 (URN)10.3389/fcimb.2016.00032 (DOI)000372710500001 ()27047801 (PubMedID)
Note

Funding Agencies|Swedish Research Council [2010-3045]; European Science foundation (TraPPs Euromembrane project); Magnus Bergvall Foundation; Faculty of Medicine and Health Sciences, Linkoping University

Available from: 2016-04-20 Created: 2016-04-19 Last updated: 2016-05-10
3. Protective role of host aquaporin 6 against Hazara virus, a model for Crimean–Congo hemorrhagic fever virus infection
Open this publication in new window or tab >>Protective role of host aquaporin 6 against Hazara virus, a model for Crimean–Congo hemorrhagic fever virus infection
Show others...
2016 (English)In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 363, no 8, fnw058Article in journal (Refereed) Published
Abstract [en]

Crimean–Congo hemorrhagic fever virus (CCHFV) is an arthropod-borne pathogen that causes infectious disease with severe hemorrhagic manifestations in vascular system in humans. The proper function of the cells in the vascular system is critically regulated by aquaporins (AQP), water channels that facilitate fluxes of water and small solutes across membranes. With Hazara virus as a model for CCHFV, we investigated the effects of viruses on AQP6 and the impact of AQP6 on virus infectivity in host cells, using transiently expressed GFP-AQP6 cells, immunofluorescent assay for virus detection, epifluorescent imaging of living cells and confocal microscopy. In GFP-AQP6 expressing cells, Hazara virus reduced both the cellular and perinuclear AQP6 distribution and changed the cell area. Infection of human cell with CCHFV strain IbAR 10200 downregulated AQP6 expression at mRNA level. Interestingly, the overexpression of AQP6 in host cells decreased the infectivity of Hazara virus, speaking for a protective role of AQP6. We suggest the possibility for AQP6 being a novel player in the virus–host interactions, which may lead to less severe outcomes of an infection.

Place, publisher, year, edition, pages
Oxford University Press, 2016
Keyword
Host–virus interactions; Nairovirus; Crimean–Congo hemorrhagic fever virus; aquaporin; virus infectivity; water homeostasis
National Category
Cell and Molecular Biology Microbiology in the medical area
Identifiers
urn:nbn:se:liu:diva-127499 (URN)10.1093/femsle/fnw058 (DOI)000377970600013 ()26976854 (PubMedID)
Funder
Swedish Research Council, 2010-3045European Science Foundation (ESF)Magnus Bergvall FoundationSwedish Research Council, 214–7495Linköpings universitet
Note

Funding agencies: Swedish Research Council [2010-3045]; European Science foundation; Magnus Bergvall Foundation; Faculty of Medicine and Health Sciences, Linkoping University; Infect-ERA Second Call (Swedish Research Council) [214-7495]

Available from: 2016-04-28 Created: 2016-04-28 Last updated: 2016-07-13Bibliographically approved

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