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Protocol

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Immunology and Infection

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

Published: August 7th, 2021

DOI:

10.3791/62555

1Oswaldo Cruz Foundation, Gonçalo Moniz Institute, 2Federal University of Bahia, 3National Institute of Science and Technology (INCT), Research Institute in Immunology, 4Senai Institute for Innovation in Advanced Health Systems, National Service for Industrial Learning, Integrated Manufacturing and Technology Campus, SENAI - CIMATEC Salvador

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.

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.

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....

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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.......

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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.......

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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.......

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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.

....

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Name Company Catalog Number Comments
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  25,4x76,2mm 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

  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 (2018).
  3. Arenas, R., Torres-Guerrero, E., Quintanilla-Cedillo, M. R., Ruiz-Esmenjaud, J. Leishmaniasis: A review. F1000Research. 6, 1-15 (2017).
  4. Scott, P., Novais, F. O. Cutaneous leishmaniasis: Immune responses in protection and pathogenesis. Nature Reviews Immunology. 16 (9), 581-592 (2016).
  5. Bittencourt, A. L., Barral, A. Evaluation of the histopathological classifications of American cutaneous and mucocutaneous leishmaniasis. Memórias do Instituto Oswaldo Cruz. 86 (1), 51-56 (1991).
  6. Scorza, B. M., Carvalho, E. M., Wilson, M. E. Cutaneous manifestations of human and murine leishmaniasis. International Journal of Molecular Sciences. 18 (6), (2017).
  7. Carvalho, L. P., Passos, S., Schriefer, A., Carvalho, E. M. Protective and pathologic immune responses in human tegumentary leishmaniasis. Frontiers in Immunology. 3, 1-9 (2012).
  8. Wood, W., Martin, P. Macrophage Functions in Tissue Patterning and Disease: New Insights from the Fly. Developmental Cell. 40 (3), 221-233 (2017).
  9. Okabe, Y., Medzhitov, R. Tissue biology perspective on macrophages. Nature Immunology. 17 (1), 9-17 (2016).
  10. Ribeiro-Gomes, F. L., et al. Macrophage Interactions with Neutrophils Regulate Leishmania major Infection. The Journal of Immunology. 172 (7), 4454-4462 (2004).
  11. Liu, D., Uzonna, J. E. The early interaction of Leishmania with macrophages and dendritic cells and its influence on the host immune response. Frontiers in Cellular and Infection Microbiology. 2, 1-8 (2012).
  12. Giudice, A., et al. Macrophages participate in host protection and the disease pathology associated with Leishmania braziliensis infection. BMC Infectious Diseases. 12, (2012).
  13. Morato, C. I., et al. Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages. Microbes and Infection. , (2014).
  14. Bonyek-Silva, I., et al. Unbalanced Production of LTB 4/PGE 2 Driven by Diabetes Increases Susceptibility to Cutaneous Leishmaniasis. Emerging Microbes & Infections. , (2020).
  15. Tomiotto-Pellissier, F., et al. Macrophage Polarization in Leishmaniasis: Broadening Horizons. Frontiers in Immunology. 9, 2529 (2018).
  16. Anderson, C. F., Mosser, D. M. A novel phenotype for an activated macrophage: the type 2 activated macrophage. Journal of Leukocyte Biology. 72 (1), 101-106 (2002).
  17. Jin, X., Kruth, H. S. Culture of macrophage colony-stimulating factor differentiated human monocyte-derived macrophages. Journal of Visualized Experiments. 2016 (112), 6-11 (2016).
  18. Rios, F. J., Touyz, R. M., Montezano, A. C. Isolation and differentiation of human macrophages. Methods in Molecular Biology. 1527, 311-320 (2017).
  19. Hamilton, T. A., Zhao, C., Pavicic, P. G., Datta, S. Myeloid colony-stimulating factors as regulators of macrophage polarization. Frontiers in Immunology. 5, 1-6 (2014).
  20. Jaguin, M., Houlbert, N., Fardel, O., Lecureur, V. Polarization profiles of human M-CSF-generated macrophages and comparison of M1-markers in classically activated macrophages from GM-CSF and M-CSF origin. Cellular Immunology. 281 (1), 51-61 (2013).
  21. Wozencraft, A. O., Blackwell, J. M. Increased infectivity of stationary-phase promastigotes of Leishmania donovani: Correlation with enhanced C3 binding capacity and CR3-mediated attachment to host macrophages. Immunology. 60 (4), 559-563 (1987).
  22. Sinha, R., et al. Genome plasticity in cultured Leishmania donovani: Comparison of early and late passages. Frontiers in Microbiology. 9, 1-20 (2018).
  23. Rebello, K. M., et al. Leishmania (Viannia) braziliensis: Influence of successive in vitro cultivation on the expression of promastigote proteinases. Experimental Parasitology. 126 (4), 570-576 (2010).
  24. Maspi, N., Abdoli, A., Ghaffarifar, F. P. r. o. -. Pro- and anti-inflammatory cytokines in cutaneous leishmaniasis: a review. Pathogens and Global Health. 110 (6), 247-260 (2016).
  25. Nunes, S., et al. Integrated analysis reveals that miR-193b, miR-671, and TREM-1 correlate with a good response to treatment of human Localized cutaneous leishmaniasis caused by Leishmania braziliensis. Frontiers in Immunology. 9, 1-13 (2018).
  26. De Moura, T. R., et al. Toward a Novel Experimental Model of Infection To Study American Cutaneous Leishmaniasis Caused by Leishmania braziliensis. Infection and Immunity. 73 (9), 5827-5834 (2005).
  27. Lima, F. R., Ferreira, L. D. M., Bonyek-silva, I., Santos, R. L., Tavares, N. M. Metformin promotes susceptibility to experimental Leishmania braziliensis infection. Memórias do Instituto Oswaldo Cruz. 115, 1-8 (2020).

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