Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response
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 combination of factors associated with host immune response, which both define clinical outcomes3,4. Leishmania braziliensis is the main species that causes CL in Brazil, with cases reported throughout all states of the country5. The immune response against L. braziliensis is considered protective, since it restricts the parasite to the inoculation site, and involves several immune cell types, such as macrophages, neutrophils e lymphocytes4,6,7.
Macrophages are multifunctional cells essential for the immune system, since they are specialized in the detection and phagocytosis of microorganisms, and can present antigens and activate other cell types. Macrophages are able to regulate processes from inflammation to tissue repair and the maintenance of homeostasis8,9. These cells play an essential role in the early immune response against intracellular parasites, such as Leishmania, being important for their elimination10,11,12.
During L. braziliensis infection, macrophages can respond through different mechanisms to eliminate the parasite, such as the production of reactive oxygen species (ROS) and inflammatory mediators13,14. Immune responses can be guided by the production of proinflammatory or anti-inflammatory cytokines, which contribute to an exacerbated inflammatory state or tissue repair processes6,15,16. The plasticity of macrophages is fundamental to the immunopathogenesis of CL, as well as to parasite-host interaction, and these cells are considered crucial to the elucidation of disease mechanisms and to the development of new therapeutic approaches.
As CL is a complex disease, investigations require researchers to explore cell types that mimic those found in humans. The immune responses observed in different experimental models can vary and produce results that do not reflect the immune response observed in naturally infected humans. Thus, the protocol presented herein was designed to enable the study of human macrophages and their immune responses during CL caused by L. braziliensis.
Protocol
Representative Results
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 have nothing to disclose.
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
| AlamarBlue Cell Viability Reagent | Invitrogen | DAL1100 | |
| Cell Culture Flask 25 cm² | SPL | 70125 | |
| CellROX Green Reagent | Invitrogen | C10444 | |
| Coverslip circles 13 mm | Perfecta | 10210013CE | |
| DAPI (4',6-diamidino-2-phenylindole) | ThermoFisher | D1306 | |
| Disposable support for blood collection | BD Vacutainer | 364815 | |
| Eclipse blood collection needle 21 g x 1.25 in | BD Vacutainer | 368607 | |
| Entellan | Sigma Aldrich | 107961 | |
| Falcon Conical Tubes, 15 mL | Sigma Aldrich | CLS430791-500EA | |
| Falcon Conical Tubes, 50 mL | StemCell Technologies | 100-0090 | |
| Fetal Bovine Serum | Gibco | A4766801 | |
| Formaldehyde 3.7% | Merck | 252549 | |
| Glass slide 25,4×76,2mm | Perfecta | 0200 | |
| Histopaque | Sigma Aldrich | 10771 | |
| Human IL-6 ELISA Kit | RD | DY206 | |
| Human M-CSF Recombinant Protein | PeproTech | 300-25 | |
| Human TNF-a ELISA Kit | RD | DY210 | |
| Leukotriene B4 ELISA Kit | Cayman | 520111 | |
| Methanol | Merck | MX0482 | |
| Penilicin-Sreptomycin-Glutamine (100x) | ThermoFisher | 10378-016 | |
| Phosphate Buffered Saline pH 7.2 (10x) | Gibco | 70013032 | |
| Plasma tube, 158 USP units of sodium heparin (spray coated) | BD Vacutainer | 367874 | |
| Quick H&E Staining Kit (Hematoxylin and Eosin) | abcam | ab245880 | |
| RPMI 1640 Medium | Gibco | 11875093 | |
| Schneider's Insect Medium | Sigma Aldrich | S0146 | |
| Tissue Culture 24-wells Plate | TPP | Z707791-126EA | |
| Trypan Blue | Gibco | 15250061 |
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