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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes the process for obtaining human macrophages from monocytes for infection with Leishmania braziliensis. It also allows researchers to evaluate infection rate and parasite viability, ROS production by fluorescence microscopy, and the production of inflammatory mediators in culture supernatants to investigate macrophage response to infection.

Abstract

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106 cells per well on 24-well plates in order to obtain 2 x 105 macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

Introduction

The intracellular protozoan parasite of the genus Leishmania is the causative agent of a neglected disease complex known as leishmaniasis1. These tropical diseases have a wide range of clinical manifestations that can range from skin lesions to complications arising from the visceral form of the disease, which can be fatal if not treated. Cutaneous leishmaniasis (CL) is the most frequent form of leishmaniasis and is characterized by a single or few ulcerated skin lesions with exacerbated chronic inflammation2. The development of disease is dependent on the Leishmania species, in addition to a combinatio....

Protocol

The Institutional Review Board for Ethics in Human Research at the Gonçalo Moniz Institute (Oswaldo Cruz Foundation-IGM-FIOCRUZ, Salvador, Bahia-Brazil), approved this study (protocol number: CAAE 95996618.8.0000.0040).

1. Isolation of human PBMCs

  1. Ensure that the blood samples, 1.077 g/mL density gradient (e.g., Ficoll-Histopaque), and saline solution are at room temperature.
  2. Dilute blood samples with saline solution at 1:1 ratio.
  3. Transfer 10-12 mL of dens.......

Representative Results

The comprehension of parasites and host cells interaction is crucial to elucidate mechanisms involved in the pathogenesis of several diseases. Although cultured human cells are less used due to limitations of cell culture compared to cell lineages, the protocol presented herein shows a robust and reproducible differentiation of human macrophages. This protocol enables the analysis of several aspects of the immune response and cell biology, from the production of inflammatory mediators up to the susceptibility of an infec.......

Discussion

The protocol presented herein for human monocytes differentiation into macrophages followed by the infection with two strains of L. braziliensis allows the evaluation of several aspects of parasite-cell interaction. These tools can be crucial to elucidate unanswered questions about CL. With the establishment of this protocol, our group was able to uncover some aspects of the immune response of macrophages obtained from individuals with diabetes and CL14.

The di.......

Disclosures

The authors declare they have no competing financial interests.

Acknowledgements

This work was supported by Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB) under Grant number PET0009/2016 and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) under Finance Code 001.

....

Materials

NameCompanyCatalog NumberComments
AlamarBlue Cell Viability ReagentInvitrogenDAL1100
Cell Culture Flask 25 cm²SPL70125
CellROX Green ReagentInvitrogenC10444
Coverslip circles 13 mmPerfecta10210013CE
DAPI (4',6-diamidino-2-phenylindole)ThermoFisherD1306
Disposable support for blood collectionBD Vacutainer364815
Eclipse blood collection needle 21 g x 1.25 inBD Vacutainer368607
EntellanSigma Aldrich107961
Falcon Conical Tubes, 15 mLSigma AldrichCLS430791-500EA
Falcon Conical Tubes, 50 mLStemCell Technologies100-0090
Fetal Bovine SerumGibcoA4766801
Formaldehyde 3.7%Merck252549
Glass slide  25,4x76,2mmPerfecta0200
HistopaqueSigma Aldrich10771
Human IL-6 ELISA KitRDDY206
Human M-CSF Recombinant ProteinPeproTech300-25
Human TNF-a ELISA KitRDDY210
Leukotriene B4 ELISA KitCayman520111
MethanolMerckMX0482
Penilicin-Sreptomycin-Glutamine (100x)ThermoFisher10378-016
Phosphate Buffered Saline pH 7.2 (10x)Gibco70013032
Plasma tube, 158 USP units of sodium heparin (spray coated)BD Vacutainer367874
Quick H&E Staining Kit (Hematoxylin and Eosin)abcamab245880
RPMI 1640 MediumGibco11875093
Schneider's Insect MediumSigma AldrichS0146
Tissue Culture 24-wells PlateTPPZ707791-126EA
Trypan BlueGibco15250061

References

  1. Desjeux, P. Leishmaniasis: current situation and new perspectives. Comparative Immunology, Microbiology and Infectious Diseases. 27 (5), 305-318 (2004).
  2. Burza, S., Croft, S. L., Boelaert, M. Leishmaniasis. The Lancet. 392 (10151), 951-970....

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MacrophagesHuman MonocytesLeishmania BraziliensisInfectionImmune ResponsePBMCFicoll HypaqueM CSFDifferentiationMicroscopyParasite Load

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