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 outcomes^3,4. 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 lymphocytes^4,6,7.

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 homeostasis^8,9. These cells play an essential role in the early immune response against intracellular parasites, such as Leishmania, being important for their elimination^10,11,12.

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 mediators^13,14. Immune responses can be guided by the production of proinflammatory or anti-inflammatory cytokines, which contribute to an exacerbated inflammatory state or tissue repair processes^6,15,16. 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

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 density gradient to 50 mL tubes.
4. Carefully overlay up to 40 mL of the diluted blood sample on top of density gradient. Separate the blood and density gradient layers.
5. First centrifugation: centrifuge tubes containing blood and density gradient layers at 400 x g for 30 min at 24 ºC.
   NOTE: Switch-off the brake before starting the centrifuge.
6. Remove the plasma above the PBMC ring with a pipette (the buffy coat layer is located between the plasma and density gradient layers; below that are red blood cells/granulocytes pellet).
7. Transfer the cloud-like PBMC layer (buffy coat layer) with a pipette to a 15 mL tube and fill with cold saline (kept on ice or at 4 °C).
8. Second centrifugation: centrifuge tubes at 300 x g for 10 min at 4 °C with the brake switched-on.
9. Discard the supernatant and resuspend the pellet by filling the tube with cold saline.
10. Third centrifugation: centrifuge tubes at 250 x g for 10 min at 4 °C with the brake switched-on.
11. Discard the supernatant and resuspend the pellet, filling the tube with cold saline.
12. Fourth centrifugation: centrifuge the tube at 200 x g for 10 min at 4 °C with the brake switched-on.
13. Discard the supernatant and resuspend the pellet with 1 mL of cold RPMI medium.
14. Count cells to determine the number of cells obtained.

2. Differentiation into human macrophages

NOTE: For a 24-well plate, calculate the amount of total cells needed to plate 2 x 10⁶ cells per well, which will yield 2 x 10⁵ macrophages. This yield is based on an average of 10% monocytes in human blood. Alternatively, monocytes can also be released by non-enzymatic methods and then counted for plating.

1. On a 24-well plate, place 13 mm round glass coverslips at the bottom of each well. Plate the equivalent of 2×10⁶ cells in 1 mL of RPMI incomplete per well.
   NOTE: Limit the number of cells in each well, since overestimated amounts can hinder cell adhesion and compromise culturing. In addition, all coverslips must be clean and sterile.
2. Incubate plates for 30 min at 37 ºC under 5% CO₂ for cell adhesion.
3. Remove the supernatants and wash once with 0.9% saline at room temperature to remove any non-adherent cells.
4. After washing, remove the saline and add 250 µL of supplemented RPMI medium at room temperature (10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 50 ng/mL M-CSF) to each well.
5. Incubate the cells for 7 days at 37 ºC under 5% CO₂.
   1. Add 125 µL of supplemented RPMI medium to each well every two days. At the end of cell differentiation, the final volume will be 500 µL per well.
   2. To analyze cell viability, perform another culture in parallel on a 96-well plate (2 x 10⁵ per well).
   3. After differentiation into macrophages (7 days), add 20 µL of AlamarBlue reagent.
   4. After 7 hours of incubation, read plates on a spectrophotometerat wavelengths of 570 nm and 600 nm.

3. Leishmania culture and infection

NOTE: L. braziliensis promastigotes from two different strains (MHOM/BR/01/BA788 and MHOM/BR88/BA-3456) were used in this assay.

1. Count Lb parasites and calculate the volume to obtain 5 x 10⁵/mL parasites.
2. Prepare supplemented Schneider's Insect medium (10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin).
3. Incubate parasites in a total volume of 5 mL in a 24 ºC incubator until the stationary phase is reached (4 to 6 days).
   NOTE: Count parasites every day to assess growth.
4. After reaching the stationary phase, centrifuge Leishmania cultures at 100 x g for 10 min at 4 ºC to remove any dead parasites (precipitated at the bottom of the tube).
5. Transfer the supernatant to a new tube and centrifuge at 1,800 x g for 10 min at 4 ºC to recover viable parasites. Discard the supernatant.
6. Resuspend the pellet with 1 mL of supplemented RPMI medium to count parasites.
7. Calculate the volume of parasites to obtain a 10:1 parasite:cell ratio. Transfer parasites to culture plates containing macrophages previously cultured in supplemented RPMI medium (~300 µL/well) at room temperature.
8. Remove the supernatant from each well containing differentiated macrophages.
   NOTE: As soon as possible, replace the medium in each well to avoid cells spending extended periods without medium. We suggest removing and replacing medium in three wells at a time.
9. Transfer the calculated amount of Lb to each well containing differentiated macrophages.
10. Infect cells for 4 or 24 hours at 37 ºC under 5% CO₂.
    1. After 4 hours, wash macrophages 3 times with saline at room temperature to remove any non-internalized parasites.
    2. For the 24-hour infection period, add 300 µL of supplemented RPMI medium to each well after washing, and then reincubate for another 20 hours at 37 °C under 5% CO₂.
11. Remove the supernatant to measure inflammatory mediators using an enzyme-linked immunosorbent assay (ELISA) following the manufacturer's instructions.
    1. Centrifuge the collected supernatant at 1,800 x g for 10 min at room temperature. Transfer the supernatant to a new tube. This procedure is performed to remove any non-internalized parasites. Supernatants can be frozen and kept at -80^oC until the time of future analysis.
       NOTE: Cells may be used to assess infection rate, parasite viability or ROS production.

4. Evaluation of infection

1. Quantification of infection rate
   1. Add 300 µL of methanol to each well after removing the supernatant. Allow 15 min to fix cells adhered to coverslips.
   2. Remove the coverslips from the wells and place on a support to soak in the cell staining solution (e.g., Quick Panoptic 2) for 1 min.
   3. Submerge coverslips twice in cell staining solution (e.g., Quick Panoptic 3) and wait until dried.
   4. Place 15 µL of mounting medium (e.g., Entellan) on slides, and then place coverslips over the medium.
      ​NOTE: All macrophages attached to coverslips should be in contact with the mounting medium.
   5. Count 100 cells at random under an optical microscope using an 100x objective to quantify infection rate and number of internalized amastigotes.
2. Parasite viability
   1. After 4 hours of infection, wash cells with saline at room temperature 3 times.
   2. Add 300 µL of supplemented Schneider's Insect medium.
   3. Incubate in a 24 ºC incubator.
   4. Quantify parasite growth after 48, 72, 96 and 120 hours.

5. Evaluation of ROS production by fluorescence microscopy

1. After the infection period, remove the supernatant and wash cells with 500 µL of saline.
2. Add 300 µL of the fluorogenic probe reagent (e.g., CellROX Green Reagent at 5 µM) to each well.
3. Incubate cells for 30 min at 37 ºC and 5% CO₂.
4. Wash cells 3x with 500 µL of phosphate buffered saline (PBS).
5. Fix cells with 300 µL of 3.7% formaldehyde and let sit for 15 min.
   ​NOTE: Measure the florescence signal within 24 hours.
6. Add 5 µL of DAPI staining agent (e.g., DAPI ProLong Gold antifade) for cell staining. Place the coverslip with the surface containing macrophages in direct contact with the DAPI staining agent.
7. Analyze the florescence signal by fluorescence microscopy at an excitation wavelength of 485/520 nm.
8. Calculate the Corrected Total Cell Fluorescence (CTCF) from 30 cells for each coverslip using ImageJ.
   CTCF = Integrated Density (Area of a selected cell x Mean fluorescence of background readings)

6. Statistical analysis

1. Use the Mann-Whitney test to compare two groups with unpaired samples. Perform the statistical analyses using GraphPad Prism 7.0. Consider differences statistically significant when p < 0.05.

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...

Materials

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