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 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.
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
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.
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.
4. Evaluation of infection
5. Evaluation of ROS production by fluorescence microscopy
6. Statistical analysis
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 infectious agent in human macrophages.
The first evidence that cellular differentiation is taking place is macrophage morphology (Figure 1A). On the plating day, cells are rounded and small when compared to the morphology after seven days of culture. The cellular spreading is observed when cultures are treated with macrophage colony-stimulating factor (M-CSF). In the absence of M-CSF, cell differentiation takes more time and results in an heterogenous population of macrophage-like cells (data not shown). After 7 days of differentiation, macrophages were incubated with the Alamarblue reagent for 24 hours until reading. This method allows the quantification of the cellular capacity to reduce resazurin to resorufin, thus differentiating viable from dead cells. The "ctrl" group refers to macrophages cultured for 7 days in supplemented medium, while the "dead" group refers to macrophages submitted to osmotic lysis during differentiation, which serves as a control for the technique. Once the differentiation in complete (for seven days), the macrophages derived from human monocytes remain viable and prompt to further assays that can last up to 24 hours after differentiation (Figure 1B) or few days (data not shown).
The first moments of interaction between Leishmania and a phagocyte is marked by close contact that will culminate in phagocytosis and internalization of the parasite. To understand the process of infection will help to explain the mechanisms involved in parasite killing or susceptibility to a certain pathogen. Based on the results, the first four hours of infection present the highest infection rate of L. braziliensis (both BA788 and BA3456 tested strains). After 24 hours of infection, there is a reduction in the infection rate of both strains, but we found statistical significance only for BA788 (Figure 2A, D). Considering longer periods of infection, no internalized parasites were found inside the cells after 72 hours (data not shown), suggesting that human macrophages are able to control L. braziliensis infection in vitro. The infection rate is measured by the count of 100 cells and, among those, the infected ones. This estimates the percentage of infection, which can vary due to the immune response of the cell donor, the amount of Leishmania parasites in the stationary phase and also due to the experimenter bias. Figure 2H shows representative images of a low infection rate (left) and a higher rate (right) by optical microscope.
Another data that can be assessed in cultured human macrophages is the parasite load, which is important to indicate the ability of macrophages to control the infection. It is measured by the average of internalized parasites in each cell and, after different time points, it is possible to determine whether the number of parasites has increased or reduced (Figure 2B, E). Finally, results of parasite viability further assemble information about the infection control. It is measured by the count of viable parasites in the culture after replacing RPMI to Schneider medium. Regarding L. braziliensis infection, we have already tested different time points, such as 48 h, 72 h, 96 h and 120 h to quantify viable parasites. The results indicate that 72 hours is recommended to evaluate parasite viability in these conditions (Figure 2G).
Based on our protocol, it is also possible to measure the inflammatory response against L. braziliensis infection in culture supernatant as soon as 4 hours after the infection. We were able to detect IL-6, TNF-α and LTB4 (Figure 3A-C), but IL-10 and IL-1β production was below the detection level (data not shown).
Another important aspect of the immune response against L. braziliensis is the production of reactive oxygen-derived species (ROS) by macrophages. This is one of the main mechanisms for parasite killing. The protocol presented herein show that ROS production from macrophages is significantly increased after 4 hours of infection (Figure 4A). The quantification of ROS based on corrected total cell fluorescence (CTCF) allowed the detection of almost twice the ROS production between infected and uninfected macrophages (Figure 4B).
Together, the results show that macrophage derived from humanmonocytes allows the study of several aspects of the immune response against L. braziliensis infection in vitro. Thus, this protocol enables research groups to further explore the role of human macrophages in leishmaniasis, minimizing the bias of lineage or murine cell models.
Figure 1. Cell morphology during human macrophage differentiation in vitro. (A) Representative images of the morphology of adherent cells at first day of culture (left) and after seven days of differentiation. (B) Cell viability after culture for seven days for differentiation. Ctrl = macrophages in culture with supplemented medium; Dead = macrophages subjected to osmotic lysis; Objective 60x; n = 3. Please click here to view a larger version of this figure.
Figure 2. Several parameters of infection caused by two strains of Leishmania braziliensis can be assessed by optical microscopy. (A, D) Infection rate (B, E), parasite load (four and 24 hours of infection) and (C, F) representative images of macrophages infected by L. braziliensis from BA5456 (A-C) or BA788 (D-E) strains (four hours of infection). (G) Macrophage-derived viable L. braziliensis after four hours of infection with BA788 strain. (H) Representative images of human macrophages infected by L. braziliensis (BA788 strain) showing low (left) and high infection rate (right). Arrows = intracelular amastigotes; Scale bar 10 µm; *p < 0.05; (A,B) n = 3; (D,E) n = 6; (G) n = 6. Please click here to view a larger version of this figure.
Figure 3. L. braziliensis-induced production of inflammatory mediators by human macrophages in four hours after infection. (A) LTB4, (B) IL-6 and (C) TNF-α production in culture supernatant after four hours infection by L. braziliensis (BA788 strain) measured by ELISA. **p < 0.01; n = 5. Please click here to view a larger version of this figure.
Figure 4. ROS production by human macrophages after in vitro infection with L. braziliensis. (A) Representative images of ROS fluorescent labelling in cultured human macrophages after four hours of infection by L. braziliensis (BA788 strain). (B) Quantification of ROS production based on the corrected total cell fluorescence (CTCF) using Image J. Green = ROS; Blue = nucleus; Scale bar 10 µm; * p < 0.05; n =6. Please click here to view a larger version of this figure.
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 differentiation process of monocytes into human macrophages is complex and requires attention from the first day of culture. The researcher must monitor the differentiation, checking the development of the culture by cellular morphology daily. Usually, the culture of monocytes for seven days with M-CSF containing medium is sufficient for complete differentiation. It is important to mention that cell morphology depends on the donor, thus several stages of cellular differentiation can be observed between donors. This can be overcome with an increase in the number of donors, which will allow the identification of outliers. Moreover, the use of M-CSF is crucial for fully differentiation into macrophages17; otherwise, it will result in a highly heterogeneous population of dendritic cells-like, macrophage-like cells and monocytes. Other growth factors or a combination of these has been used as tolls to further polarize macrophages into M1 or M2 profile17,18. Several studies have shown that macrophages cultured with M-CSF develop an M2 profile, while cells treated with GM-CSF exhibit an M1 profile19. However, macrophages cultured with M-CSF can polarize to the M1 profile after stimulation20. Based on the results, we were unable to determine a macrophage profile prior to infection, but hypothesize that this probably trended toward an M2 profile. On the other hand, after 4 hours of infection, macrophages produced high levels of pro-inflammatory cytokines and ROS, which is characteristic of a classic macrophage profile. Another relevant aspect about the infection of macrophages with Leishmania is the dispersion observed in the percentage of infected cells (infection rate). This is a marked feature of assays with human macrophages, also due to the responsiveness of each donor. To minimize this effect, the stationary-phase of Leishmania cultures should be confirmed and methods to purify metacyclic promastigotes can be considered21,22,23. In addition, our findings show that increased infection periods result in a decrease in the infection rate, since human macrophages seem to be able to control L. braziliensis.
The interaction of macrophages and Leishmania involves the production of mediators that are a combination of a protective response of the host cell to kill the parasite and escape mechanisms developed by each Leishmania species. Thus, to define the profile of mediators produced during the infection is essential to understand the pathogenesis of CL24. Based on our protocol, it is possible to measure pro-inflammatory mediators after 4 hours of infection with L. braziliensis. With this method, we were also able to show this inflammatory response of macrophages from individuals with diabetes after infection in vitro with L. braziliensis14. The possibility to evaluate different inflammatory mediators is crucial to better understand CL as a chronic inflammatory skin disease24,25,26.
The production of mediators involved in parasite killing also play a significant role in the disease development and outcome. It has already been described that ROS production is one of the most efficient mechanism to control L. braziliensis infection13,14. The protocol presented herein allow the evaluating of ROS production within the macrophages infected by L. braziliensis using fluorescence microscope. ROS production seems to be key to define susceptibility to the infection14,25. Unlike other methods, the quantification of ROS through fluorescence microscopy has the advantage of locally identifying production inside the cell. Other methods only allow the indirect quantification of ROS production from an entire cell population, without considering that cells can respond differently to the same stimulus13,25.
In summary, the results show that the protocol described herein enables studies that aim to explore the interaction between macrophages and L. braziliensis, assessing aspects such as the infection rate, parasite killing and production of mediators. This allows to extrapolate the findings and to correlate it with naturally occurring mechanisms.
The authors have nothing to disclose.
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.
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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