Development of an in vitro media perfusion model of Leishmania major macrophage infection.
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2019
2019
Historique:
received:
08
01
2019
accepted:
04
07
2019
entrez:
25
7
2019
pubmed:
25
7
2019
medline:
26
2
2020
Statut:
epublish
Résumé
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. 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. 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. 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.
Sections du résumé
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.
Identifiants
pubmed: 31339931
doi: 10.1371/journal.pone.0219985
pii: PONE-D-19-00661
pmc: PMC6656416
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0219985Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/M009513/1
Pays : United Kingdom
Organisme : Medical Research Council
ID : IF MC-PC_13069
Pays : United Kingdom
Déclaration de conflit d'intérêts
SM and LH gratefully acknowledge a financial donation from Kirkstall Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Références
Appl Environ Microbiol. 2006 Apr;72(4):2885-95
pubmed: 16597995
Am J Physiol. 1984 Jun;246(6 Pt 2):F835-44
pubmed: 6742132
Tissue Eng. 2006 Apr;12(4):905-15
pubmed: 16674302
Pharm Res. 1993 Jul;10(7):1093-5
pubmed: 8378254
Antimicrob Agents Chemother. 2016 May 23;60(6):3524-32
pubmed: 27021313
Int J Antimicrob Agents. 2011 Oct;38(4):341-7
pubmed: 21783345
Microvasc Res. 2004 Nov;68(3):258-64
pubmed: 15501245
J Biomech. 1984;17(5):377-94
pubmed: 6376512
Annu Rev Biomed Eng. 2000;2:691-713
pubmed: 11701528
Nat Rev Microbiol. 2011 Jul 11;9(8):604-15
pubmed: 21747391
J Biol Chem. 2012 Nov 16;287(47):39349-60
pubmed: 23035117
Exp Cell Res. 2006 Feb 1;312(3):289-98
pubmed: 16337626
Am J Physiol. 1993 Jan;264(1 Pt 2):H150-6
pubmed: 8381608
Arterioscler Thromb Vasc Biol. 2000 Oct;20(10):2220-5
pubmed: 11031207
J Control Release. 2012 Sep 10;162(2):259-66
pubmed: 22824784
Curr Protoc Immunol. 2008 Nov;Chapter 14:Unit 14.1
pubmed: 19016445
Cell Immunol. 2000 Jul 10;203(1):39-46
pubmed: 10915560
Biotechnol Bioeng. 2010 May 1;106(1):127-37
pubmed: 20091740
Exp Parasitol. 1996 Dec;84(3):400-9
pubmed: 8948329
Cancer Res. 2002 Nov 15;62(22):6731-9
pubmed: 12438274
Q J Exp Physiol. 1987 Oct;72(4):409-37
pubmed: 3321140
Integr Biol (Camb). 2012 Apr;4(4):401-9
pubmed: 22143066
Lancet Infect Dis. 2007 Sep;7(9):581-96
pubmed: 17714672
J Cell Sci. 1995 Apr;108 ( Pt 4):1497-508
pubmed: 7615670
Infect Immun. 1985 Aug;49(2):305-11
pubmed: 3160661
Ann Biomed Eng. 2006 Mar;34(3):446-54
pubmed: 16482410
Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):15779-84
pubmed: 16249343
Infect Immun. 1993 Apr;61(4):1553-8
pubmed: 8454363
Antimicrob Agents Chemother. 2014;58(4):2059-66
pubmed: 24449768
J Biomech Eng. 1985 Nov;107(4):341-7
pubmed: 4079361
PLoS Negl Trop Dis. 2015 Mar 31;9(3):e0003666
pubmed: 25826250
J Control Release. 2017 Jan 10;245:170-176
pubmed: 27916535
J Biomed Mater Res. 2002 Apr;60(1):148-58
pubmed: 11835170
Nat Rev Drug Discov. 2016 Nov;15(11):751-769
pubmed: 27616293
Biomicrofluidics. 2010 Sep 24;4(3):
pubmed: 20957065
Annu Rev Biomed Eng. 2007;9:229-56
pubmed: 17459001
Biologicals. 2005 Dec;33(4):257-60
pubmed: 16168668
Environ Microbiol. 2014 Jan;16(1):291-303
pubmed: 24148021
Nature. 1968 Sep 21;219(5160):1260-1
pubmed: 5677422
J Biomech Eng. 1981 Aug;103(3):177-85
pubmed: 7278196
Mol Pharm. 2018 Jun 4;15(6):2372-2383
pubmed: 29719153
Pharm Res. 2004 Feb;21(2):344-53
pubmed: 15032318