JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Immunology and Infection

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

Published: August 4th, 2021

DOI:

10.3791/62461

1Laboratory of Host - Parasite Interaction and Epidemiology, Gonçalo Moniz Institute
* These authors contributed equally

Here we study implications of Leishmania-host interaction by exploring Leishmania-infected dendritic cells migration. The differentiation and infection of dendritic cells, migration analysis, and the evaluation of adhesion complexes and actin dynamics are described. This method can be applied to other host cell migration studies when infected with Leishmania or other intracellular parasite species.

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous lesions to a highly fatal visceral form of the disease. An estimated 12 million people worldwide are currently infected, and another 350 million face risk of infection. It is known that host cells infected by Leishmania parasites, such as macrophages or dendritic cells, can migrate to different host tissues, yet how migration contributes to parasite dissemination and homing remains poorly understood. Therefore, assessing these parasites' ability to modulate host cell response, adhesion, and migration will shed light on mechanisms involved in disease dissemination and visceralization. Cellular migration is a complex process in which cells undergo polarization and protrusion, allowing them to migrate. This process, regulated by actin and tubulin-based microtubule dynamics, involves different factors, including the modulation of cellular adhesion to the substrate. Cellular adhesion and migration processes have been investigated using several models. Here, we describe a method to characterize the migratory aspects of host cells during Leishmania infection. This detailed protocol presents the differentiation and infection of dendritic cells, the analysis of host cell motility and migration, and the formation of adhesion complexes and actin dynamics. This in vitro protocol aims to further elucidate mechanisms involved in Leishmania dissemination within vertebrate host tissues and can also be modified and applied to other cell migration studies.

Leishmaniasis, a neglected tropical disease caused by protozoan parasites belonging to the genus Leishmania, results in a wide-ranging spectrum of clinical manifestations, from self-healing localized cutaneous lesions to fatal visceral forms of the disease. It has been estimated that up to one million new leishmaniasis cases arise annually, with a reported 12 million people currently infected worldwide1. Visceral leishmaniasis (VL), which can be fatal in over 95% of cases when left untreated, causes more than 50,000 deaths annually, affecting millions in South America, East Africa, South Asia, and the Mediterranean region.css-f1q1l5{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;-webkit-align-items:flex-end;-webkit-box-align:flex-end;-ms-flex-align:flex-end;align-items:flex-end;background-image:linear-gradient(180deg, rgba(255, 255, 255, 0) 0%, rgba(255, 255, 255, 0.8) 40%, rgba(255, 255, 255, 1) 100%);width:100%;height:100%;position:absolute;bottom:0px;left:0px;font-size:var(--chakra-fontSizes-lg);color:#676B82;}

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The procedures described herein were approved by the Institutional Review Board of the Gonçalo Moniz Institute (IGM-FIOCRUZ, protocol no. 2.751.345). Blood samples were obtained from healthy volunteer donors. Animal experimental procedures were conducted in accordance with the Ethical Principles in Animal Research adopted by the Brazilian law 11.784/2008 and were approved and licensed by the Ethical Committee for Animal Research of the Gonçalo Moniz Institute (IGM-FIOCRUZ, protocol no. 014/2019).

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This protocol described herein enables the evaluation of cell migration and its associated mechanisms, such as actin dynamics and adhesion, thereby providing a tool to determine the migration of Leishmania-infected host cells within the vertebrate host. The results presented here demonstrate that this in vitro assay provides rapid and consistent indications of changes in cellular adhesion, migration, and actin dynamics prior to in vivo experimentation.

First, cells w.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The method described here for evaluating cell migration using the cell culture membrane inserts system allows researchers to study the migratory response of cells in a two-dimensional environment. In this technique, some steps are considered critical. Firstly, the differentiation of human DCs and infection with Leishmania are determinative since the infection rate is donor-dependent. Using more than one donor per experiment and healthy Leishmania cultures will allow for more consistent results. It is al.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was supported by Bahia Research Support Foundation (Fapesb), grant number 9092/2015. The authors acknowledge CNPq, Capes and Fapesb for financial support via scholarships. The authors would like to thank Andris K. Walter for critical analysis, English language revision and manuscript copyediting assistance.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
16 Gauge needle Descarpack 353101
24 well cell culture plate JET-BIOFIL J011024
25 Gauge needle Descarpack 353601
Albumin from bovine serum Sigma Aldrich A2153-100G
Ammonium chloride Sigma Aldrich A-0171
Anti-mouse IgG, Alexa Fluor 488 Invitrogen A32723
Anti-mouse IgG, Alexa Fluor 594 Invitrogen A11032
Anti-rabbit IgG, Alexa Fluor 568 Invitrogen A11011
CD14 MicroBeads MACS Myltenyi Biotec 130-050-201
Cell Culture Flask 25cm2 SPL 70125
Cellstripper Corning 25-056-CI
Confocal microscope Leica TCS SP8
Coverslip circles 13mm Perfecta 10210013CE
Dissecting Forceps VWR 82027-406
EDTA Sigma Aldrich E6758
Falcon tube KASVI K19-0051
Fetal Bovine Serum gibco 16000044
Fluorescence microscope Olympus  BX51
Glass slide  25,4x76,2mm Perfecta 200
Hemin bovine Sigma Aldrich H2250
Hemocytometer Perfecta 7302HD
Histopaque® 1077 Sigma Aldrich 10771
MACS buffer MACS Myltenyi Biotec 130-091-221
Minimum Essential Medium Gibco 41090093
Mouse anti-Rac1 BD 610650
Paraformaldehyde Sigma Aldrich 158127
Phalloidin Alexa Fluor 488 Invitrogen A12379
Phosphate Buffered Saline ThermoFisher AM9624
Polycarbonate Membrane Transwell Inserts - Pore size 5.0 µm Corning 3421
ProLong Gold DAPI kit Invitrogen P36931
Rabbit anti-Cdc42 Invitrogen PA1-092X
Rabbit anti-FAK (pTyr397) Invitrogen RC222574
Rabbit anti-paxilin (pTyr118) Invitrogen QF221230
Rabbit anti-RhoA Invitrogen OSR00266W
Recombinant Human CCL3 R&D Systems 270-LD-010
Recombinant Human GM-CSF PeproTech 300-03
Recombinant Human IL-4 PeproTech 200-04
Recombinant Human M-CSF PeproTech 300-25
RPMI 1640 Medium Gibco 21870076
Saponin Sigma Aldrich 47036 – 50G – F
Syringe 3 mL Descarpack 324201
Trypan Blue Gibco 15250061

  1. Burza, S., Croft, S. L., Boelaert, M. Leishmaniasis. The Lancet. 392 (10151), 951-970 (2018).
  2. Bi, K., Chen, Y., Zhao, S., Kuang, Y., John Wu, C. H. Current Visceral Leishmaniasis Research: A Research Review to Inspire Future Study. BioMed Research International. 2018, (2018).
  3. Serafim, T. D., Iniguez, E., Oliveira, F. Leishmania infantum. Trends in Parasitology. 36 (1), 80-81 (2020).
  4. Van Assche, T., Deschacht, M., da Luz, R. A. I., Maes, L., Cos, P. Leishmania-macrophage interactions: Insights into the redox biology. Free Radical Biology and Medicine. 51 (2), 337-351 (2011).
  5. Podinovskaia, M., Descoteaux, A. Leishmania and the macrophage: A multifaceted interaction. Future Microbiology. 10 (1), 111-129 (2015).
  6. Antoine, J. C., Prina, E., Jouanne, C., Bongrand, P. Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH. Infection and Immunity. 58 (3), (1990).
  7. Antoine, J. C., Prina, E., Lang, T., Courret, N. The biogenesis and properties of the parasitophorous vacuoles that harbour Leishmania in murine macrophages. Trends in Microbiology. 6 (10), 392-401 (1998).
  8. Friedl, P., Wolf, K. Plasticity of cell migration: A multiscale tuning model. Journal of Cell Biology. 188 (1), 11-19 (2010).
  9. Sheetz, M. P., Felsenfeld, D., Galbraith, C. G., Choquet, D. Cell migration as a five-step cycle. Biochemical Society symposium. 65, 233-243 (1999).
  10. Yano, H., et al. Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion. Journal of Cell Biology. 166 (2), 283-295 (2004).
  11. Mitra, S. K., Hanson, D. A., Schlaepfer, D. D. Focal adhesion kinase: In command and control of cell motility. Nature Reviews Molecular Cell Biology. 6 (1), 56-68 (2005).
  12. Deramaudt, T. B., et al. Altering FAK-Paxillin Interactions Reduces Adhesion, Migration and Invasion Processes. PLoS One. 9 (3), (2014).
  13. Turner, C. E. Paxillin and focal adhesion signalling. Nature Cell Biology. 2 (12), (2000).
  14. Linder, S., Aepfelbacher, M. Podosomes: Adhesion hot spots of invasive cells. Trends in Cell Biology. 13 (7), 376-385 (2003).
  15. Huveneers, S., Danen, E. H. J. Adhesion signaling - Crosstalk between integrins, Src and Rho. Journal of Cell Science. 122 (8), 1059-1069 (2009).
  16. Jones, G. E. Cellular signaling in macrophage migration and chemotaxis. Journal of Leukocyte Biology. 68 (5), 593-602 (2000).
  17. De Fougerolles, A. R., Koteliansky, V. E. Regulation of monocyte gene expression by the extracellular matrix and its functional implications. Immunological Reviews. 186 (1), 208-220 (2002).
  18. Cortesio, C. L., Boateng, L. R., Piazza, T. M., Bennin, D. a., Huttenlocher, A. Calpain-mediated proteolysis of paxillin negatively regulates focal adhesion dynamics and cell migration. The Journal of Biological Chemistry. 286 (12), 9998-10006 (2011).
  19. Verkhovsky, A. B., et al. Orientational order of the lamellipodial actin network as demonstrated in living motile cells. Molecular Biology of the Cell. 14 (11), 4667-4675 (2003).
  20. Ridley, A. J., et al. Cell Migration: Integrating signals from front to back. Science. 302 (5651), 1704-1709 (2003).
  21. Hall, A. Small GTP-binding proteins, and the regulation of the actin cytoskeleton. Annual Review of Cell Biology. 10, 31-54 (1994).
  22. Machesky, L. M., Hall, A. Rho: A connection between membrane receptor signalling and the cytoskeleton. Trends in Cell Biology. 6 (8), 304-310 (1996).
  23. Ridley, A. J., Hall, A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 70 (3), 389-399 (1992).
  24. DeMali, K. A., Burridge, K. Coupling membrane protrusion and cell adhesion. Journal of Cell Science. 116 (12), 2389-2397 (2003).
  25. López-Colomé, A. M., Lee-Rivera, I., Benavides-Hidalgo, R., López, E. Paxillin: A crossroad in pathological cell migration. Journal of Hematology and Oncology. 10 (1), (2017).
  26. Carvalhal, D. G. F., et al. The modelling of mononuclear phagocyte-connective tissue adhesion in vitro: Application to disclose a specific inhibitory effect of Leishmania infection. Experimental Parasitology. 107 (3-4), 189-199 (2004).
  27. Pinheiro, N. F., et al. Leishmania infection impairs β1-integrin function and chemokine receptor expression in mononuclear phagocytes. Infection and Immunity. 74 (7), 3912-3921 (2006).
  28. de Menezes, J. P. B., et al. Leishmania infection inhibits macrophage motility by altering F-actin dynamics and the expression of adhesion complex proteins. Cellular Microbiology. 19 (3), (2017).
  29. Hermida, M. D. R., Doria, P. G., Taguchi, A. M. P., Mengel, J. O., dos-Santos, W. L. C. Leishmania amazonensis infection impairs dendritic cell migration from the inflammatory site to the draining lymph node. BMC Infectious Diseases. 14 (1), 450 (2014).
  30. Ballet, R., et al. Blocking junctional adhesion molecule c enhances dendritic cell migration and boosts the immune responses against Leishmania major. PLoS Pathogens. 10 (12), (2014).
  31. Rocha, M. I., et al. Leishmania infantum enhances migration of macrophages via a phosphoinositide 3-Kinase î-dependent pathway. ACS Infectious Diseases. 6 (7), 1643-1649 (2020).
  32. Hiasa, M., et al. GM-CSF and IL-4 induce dendritic cell differentiation and disrupt osteoclastogenesis through M-CSF receptor shedding by up-regulation of TNF-α converting enzyme (TACE). Blood. 114 (20), 4517-4526 (2009).
  33. de Melo, C. V. B., et al. Phenotypical characterization of spleen remodeling in murine experimental visceral leishmaniasis. Frontiers in Immunology. 11, 653 (2020).
  34. Gibaldi, D., et al. CCL3/macrophage inflammatory protein-1α is dually involved in parasite persistence and induction of a TNF- and IFNγ-enriched inflammatory milieu in Trypanosoma cruzi-induced chronic cardiomyopathy. Frontiers in Immunology. 11, 306 (2020).
  35. Finger, P. T., Papp, C., Latkany, P., Kurli, M., Iacob, C. E. Anterior chamber paracentesis cytology (cytospin technique) for the diagnosis of intraocular lymphoma. British Journal of Ophthalmology. 90 (6), 690-692 (2006).
  36. Zhang, C., Barrios, M. P., Alani, R. M., Cabodi, M., Wong, J. Y. A microfluidic Transwell to study chemotaxis. Experimental Cell Research. 342 (2), 159-165 (2016).
  37. Glynn, S. A., O'Sullivan, D., Eustace, A. J., Clynes, M., O'Donovan, N. The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, simvastatin, lovastatin and mevastatin inhibit proliferation and invasion of melanoma cells. BMC Cancer. 8, (2008).
  38. van de Merbel, A. F., vander Horst, G., Buijs, J. T., vander Pluijm, G. Protocols for migration and invasion studies in prostate cancer. Methods in Molecular Biology. 1786, 67-79 (2018).
  39. Omar Zaki, S. S., Kanesan, L., Leong, M. Y. D., Vidyadaran, S. The influence of serum-supplemented culture media in a transwell migration assay. Cell Biology International. 43 (10), 1201-1204 (2019).
  40. Borovikov, I. S. Izuchenie strukturnykh izmeneniǐ sokratitel'nykh belkov myshechnogo volokna s pomoshch'iu poliarizatsionnoǐ ul'trafioletovoǐ fluorestsentnoǐ mikroskopii. VIII. Vliianie glutaral'degida i falloidina na konformatsiiu F-aktina. Tsitologiya. 26 (11), 1262-1266 (1984).
  41. Cooper, J. A. Effects of cytochalasin and phalloidin on actin. The Journal of Cell Biology. 105 (4), 1473-1478 (1987).
  42. Allen, W. E., Jones, G. E., Pollard, J. W., Ridley, A. J. Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages. Journal of Cell Science. 110, 707-720 (1997).
  43. Lichtman, J. W., Conchello, J. A. Fluorescence microscopy. Nature Methods. 2 (12), 910-919 (2005).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved