Development of an in vitro media perfusion model of Leishmania major macrophage infection

O’Keeffe, A., Hyndman, L., McGinty, S. , Riezk, A., Murdan, S. and Croft, S. L. (2019) Development of an in vitro media perfusion model of Leishmania major macrophage infection. PLoS ONE, 14(7), e0219985. (doi: 10.1371/journal.pone.0219985)

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Background: In vitro assays are widely used in studies on pathogen infectivity, immune responses, drug and vaccine discovery. However, most in vitro assays display significant differences to the in vivo situation and limited predictive properties. We applied medium perfusion methods to mimic interstitial fluid flow to establish a novel infection model of Leishmania parasites. Methods: Leishmania major infection of mouse peritoneal macrophages was studied within the Quasi Vivo QV900 macro-perfusion system. Under a constant flow of culture media at a rate of 360μl/min, L. major infected macrophages were cultured either at the base of a perfusion chamber or raised on 9mm high inserts. Mathematical and computational modelling was conducted to estimate medium flow speed, shear stress and oxygen concentration. The effects of medium flow on infection rate, intracellular amastigote division, macrophage phagocytosis and macropinocytosis were measured. Results: Mean fluid speeds at the macrophage cell surface were estimated to be 1.45 x 10−9 m/s and 1.23 x 10−7 m/s for cells at the base of the chamber and cells on an insert, respectively. L. major macrophage infection was significantly reduced under both media perfusion conditions compared to cells maintained under static conditions; a 85±3% infection rate of macrophages at 72 hours in static cultures compared to 62±5% for cultures under slow medium flow and 55±3% under fast medium flow. Media perfusion also decreased amastigote replication and both macrophage phagocytosis (by 44±4% under slow flow and 57±5% under fast flow compared with the static condition) and macropinocytosis (by 40±4% under slow flow and 62±5% under fast flow compared with the static condition) as measured by uptake of latex beads and pHrodo Red dextran. Conclusions: Perfusion of culture medium in an in vitro L. major macrophage infection model (simulating in vivo lymphatic flow) reduced the infection rate of macrophages, the replication of the intracellular parasite, macrophage phagocytosis and macropinocytosis with greater reductions achieved under faster flow speeds.

Item Type:Articles
Additional Information:This work was supported by Biotechnology and Biological Sciences Research Council [grant number BB/M009513/1] to AOK, SC; Kirkstall Ltd., UK to SM, LH; Engineering and Physical Sciences Research Council (EP/M506539/1 and EPM508056/1) to SM, LH; Alan & Kathie Stross Summer Fellowship to LH; Dr Hadwen Trust to LH; Medical Research Council Confidence in Concept award (number IF MC-PC_13069) to SC, AOK.
Glasgow Author(s) Enlighten ID:Mcginty, Dr Sean and Hyndman, Ms Lauren
Creator Roles:
Mcginty, S.Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Software, Supervision, Writing – original draft
Hyndman, L.Data curation, Formal analysis, Investigation, Methodology, Writing – original draft
Authors: O’Keeffe, A., Hyndman, L., McGinty, S., Riezk, A., Murdan, S., and Croft, S. L.
College/School:College of Science and Engineering > School of Engineering > Biomedical Engineering
Journal Name:PLoS ONE
Publisher:Public Library of Science
ISSN (Online):1932-6203
Copyright Holders:Copyright © 2019 O’Keeffe et al.
First Published:First published in PLoS ONE 14(7):e0219985
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
678901EPSRC DTG 2014Mary Beth KneafseyEngineering and Physical Sciences Research Council (EPSRC)EP/M506539/1R&I - RESEARCH STRATEGY & INNOVATION
701101EPSRC 2015 DTPMary Beth KneafseyEngineering and Physical Sciences Research Council (EPSRC)EP/M508056/1R&I - RESEARCH STRATEGY & INNOVATION