Here we study implications of Leishmania-host interaction by exploring Leishmania-infected dendritic cells migration. The differentiation and infection of dendritic cells, migration analysis, and the evaluation of adhesion complexes and actin dynamics are described. This method can be applied to other host cell migration studies when infected with Leishmania or other intracellular parasite species.
Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous lesions to a highly fatal visceral form of the disease. An estimated 12 million people worldwide are currently infected, and another 350 million face risk of infection. It is known that host cells infected by Leishmania parasites, such as macrophages or dendritic cells, can migrate to different host tissues, yet how migration contributes to parasite dissemination and homing remains poorly understood. Therefore, assessing these parasites’ ability to modulate host cell response, adhesion, and migration will shed light on mechanisms involved in disease dissemination and visceralization. Cellular migration is a complex process in which cells undergo polarization and protrusion, allowing them to migrate. This process, regulated by actin and tubulin-based microtubule dynamics, involves different factors, including the modulation of cellular adhesion to the substrate. Cellular adhesion and migration processes have been investigated using several models. Here, we describe a method to characterize the migratory aspects of host cells during Leishmania infection. This detailed protocol presents the differentiation and infection of dendritic cells, the analysis of host cell motility and migration, and the formation of adhesion complexes and actin dynamics. This in vitro protocol aims to further elucidate mechanisms involved in Leishmania dissemination within vertebrate host tissues and can also be modified and applied to other cell migration studies.
Leishmaniasis, a neglected tropical disease caused by protozoan parasites belonging to the genus Leishmania, results in a wide-ranging spectrum of clinical manifestations, from self-healing localized cutaneous lesions to fatal visceral forms of the disease. It has been estimated that up to one million new leishmaniasis cases arise annually, with a reported 12 million people currently infected worldwide1. Visceral leishmaniasis (VL), which can be fatal in over 95% of cases when left untreated, causes more than 50,000 deaths annually, affecting millions in South America, East Africa, South Asia, and the Mediterranean region2. The main etiological agent of VL in the new world, Leishmania infantum, is transmitted to humans by infected female sandflies during blood-feeding3. These parasites are recognized and internalized by phagocytes, e.g., macrophages and dendritic cells3,4,5. Inside these cells, parasites differentiate into their intracellular forms, known as amastigotes, which will then multiply and be transported via the lymphatic system and bloodstream to different host tissues6,7. However, the mechanisms by which Leishmania parasites are disseminated in the vertebrate host, as well as the role played by host cell migration in this process, remain unclear.
Cell migration is a complex process executed by all nucleated cells, including leukocytes8. According to the classic cycling model of cell migration, this process involves several integrated molecular events that can be divided into five steps: leading-edge protrusion; adhesion of the leading edge to matrix contacts; contraction of cellular cytoplasm; release of the rear edge of the cell from contact sites; and the recycling of membrane receptors from the rear to the front of the cell9.
For cell migration to occur, protrusions must be formed and then stabilized through attachment to the extracellular matrix. Among the different receptor types involved in the promotion of cell migration, integrins are notable. Integrin activation results in migration-related signaling; intracellular signaling then occurs via focal adhesion kinase (FAK) and Src family kinases, in addition to talin, vinculin, and paxillin molecues10,11,12. The phosphorylation of paxillin by activated kinases, including FAK, leads to the recruitment of effector molecules, which transduces external signals that prompt cell migration. It has been shown that paxillin is an intracellular molecule that is crucial to cell adhesion, actin polymerization, and cell migration processes13,14,15.
The actin cytoskeleton plays a central role in the polarization and migration of phagocytes16. During cell migration process, protrusions formed due to actin polymerization become stabilized through cell adhesion to the extracellular matrix. This process may be modulated by integrin receptors associated with the actin cytoskeleton17,18,19. Several actin-binding proteins regulate the rate and organization of actin polymerization in cellular protrusions20. Studies have shown that RhoA, Rac, and Cdc42 regulate actin reorganization after the stimulation of adherent cells by extracellular factors21,22. During migration, Rac1 and Cdc42 are located at the leading edge of the cell, controlling the extension of lamellipodia and filopodia, respectively, while RhoA, located at the rear of the cell, regulates the contraction of the actomyosin cytoskeleton15,23,24,25.
Studies have shown that Leishmania infection modulates host cell functions, such as adhesion to the cellular substrate and migration26,27,28,29,30,31. Immature DCs reside in peripheral tissues; upon interaction with PAMPS, these cells become activated and migrate to the draining lymph nodes, prompting antigen presentation to T cells. A previous study using a mouse model showed that L. amazonensis infection provokes a reduction in the migration of DCs to draining lymph nodes29. It was also demonstrated that the inhibition of the adhesion process reduced DC migration after infection with L. major30. Nonetheless, the impact of DC migration on parasite dissemination in the host, as well as the mechanisms involved in this process, remain poorly understood.
Here we present a compiled step-by-step protocol to perform an in vitro adhesion and migration assay involving human DCs infected by Leishmania. This method comprises not only the differentiation and infection of DCs, but also permits the analysis of host cell motility and migration, the formation of adhesion complexes, as well as actin dynamics. The presently described in vitro protocol allows researchers to further investigate the mechanisms involved in Leishmania dissemination within vertebrate host tissues and can also be manipulated and applied to other cell migration studies.
The procedures described herein were approved by the Institutional Review Board of the Gonçalo Moniz Institute (IGM-FIOCRUZ, protocol no. 2.751.345). Blood samples were obtained from healthy volunteer donors. Animal experimental procedures were conducted in accordance with the Ethical Principles in Animal Research adopted by the Brazilian law 11.784/2008 and were approved and licensed by the Ethical Committee for Animal Research of the Gonçalo Moniz Institute (IGM-FIOCRUZ, protocol no. 014/2019).
1. Isolation and differentiation of human dendritic cells
2. Leishmania infantum cultivation
NOTE: Leishmania infantum (MCAN/BR/89/BA262) parasites are used in this assay. Hamsters were intravenously infected with 20 µL of the solution containing 1 x 106 L. infantum promastigotes in sterile saline. After 1 to 2 months, animals were euthanized and the amastigote forms of Leishmania were recovered from their spleens and differentiated into promastigotes33.
3. Human dendritic cell infection
4. Migration assay using cell culture inserts
5. Adhesion assay and evaluation of actin polymerization by immunofluorescence
NOTE: For this assay, use 24-well plates with coverslips.
Primary solutions | Chemical compound | Diluent | |
Ammonium chloride solution | 0,134 g of NH4Cl | 50 ml of PBS 1X | |
Saponin 15% | 150 mg of saponin | 1 mL de PBS 1X | |
Albumin from bovine serum (ABS) 10% | 1 g of ABS | 10 mL of PBS 1X | |
Secondary solutions | Component 1 | Component 2 | Component 3 |
Saponin 0,15% | 1mL of saponin 15% | 100 mL of PBS 1X | – |
PBS 1X / ABS 3% / Saponin 0,15% | 13,8 mL of PBS 1X | 6 mL of ABS 10% | 200 µL of Saponin 15% |
PBS1X / ABS 0,3% / Saponin 0,15%: | 19,2 mL of PBS 1X | 0,6 mL of ABS 10% | 200 µL of Saponin 15% |
PBS 1X / ABS 1% / Saponin 0,15% | 17,8 mL of PBS 1X | 2 mL of ABS 10% | 200 µL of Saponin 15% |
Table 1: Buffer recipes.
6. Confocal microscopy, image acquisition, and quantification using FIJI
NOTE: To acquire/capture immunofluorescence images, use a confocal laser scanning microscope. Oil-immersion 63x objective lens is recommended for optimal resolution.
7. Statistical analysis
This protocol described herein enables the evaluation of cell migration and its associated mechanisms, such as actin dynamics and adhesion, thereby providing a tool to determine the migration of Leishmania-infected host cells within the vertebrate host. The results presented here demonstrate that this in vitro assay provides rapid and consistent indications of changes in cellular adhesion, migration, and actin dynamics prior to in vivo experimentation.
First, cells were successfully cultured following aseptic techniques and lab protocols. Data generated via migration analysis using cell culture membrane inserts allowed us to evaluate the migration of L. infantum-infected or uninfected human dendritic cells. DAPI staining permitted the facile visualization of migratory cells, enabling us to discriminate between infected and non-infected cells as staining procedures incorporate both dendritic cell and parasite nuclei. Infected cells can be identified by visualizing the large macrophage nuclei and the number of smaller amastigote nuclei clustered around each macrophage nucleus. Next, infected cells (L. infantum infected group) and uninfected cells (control group) were then counted for each field of vision using a manual counter. Finally, the number of migratory infected cells was compared to the number of uninfected cells (control group) that migrated. Our results indicate higher rates of cell migration following L. infantum infection when compared to non-infected controls (Figure 1).
The evaluation of actin dynamics and the formation of adhesion complexes, factors critical to cellular migration, allows for an enhanced understanding of how infection may modulate host cell migration. To assess these mechanisms, we performed immunostaining for molecules involved in actin dynamics (phalloidin, Rac1, Cdc42, and RhoA) and adhesion complex formation (FAK and paxillin). The expression of each protein was evaluated using confocal microscopy. The differences in protein expression were assessed by comparing the fluorescence intensity between infected and uninfected cells for each protein analyzed. Our results demonstrate actin polymerization in infected and non-infected cells and the formation and localization of adhesion complexes. DAPI staining enabled the identification of infected cells through the staining of parasite nuclei. Fluorescence analysis indicated increased FAK and paxillin expression in DCs following L. infantum infection (Figure 2). To evaluate the organization of actin filaments in DCs, actin was labeled with fluorescent phalloidin. The resulting images revealed more areas with actin polymerization, yet no differences in phalloidin staining comparing infected and non-infected cells (Figure 3A). However, considering that the structure of actin is highly dynamic, the evaluation of actin-associated molecules may provide additional insight into the organization of this structural protein. Thus, we evaluated the expression of Rho GTPase proteins. Although phalloidin staining yielded similar results in infected and non-infected cells, increases were noted in Rac1, Cdc42 and RhoA expression after L. infantum infection when compared to uninfected controls (Figure 3B,C,D). These results reinforce the need for further investigation of molecules involved in actin polymerization to gain a more comprehensive understanding of its associated dynamics.
Figure 1: Evaluation of dendritic cell migration in L. infantum infection. Dendritic cells were infected by L. infantum at a ratio of 20:1 for 4 h. At 6, 12, 24 or 48 h after infection, dendritic cells could migrate in the presence of CCL3 chemoattractant through the cell culture insert system for an additional 4 h. Migrating cells were washed, fixed, and stained with DAPI. Bars represent numbers of migratory cells after L. infantum infection from random counts in 10 fields using confocal microscopy. Each dot represents one cell. *p<0.05 (Student's t-test). Please click here to view a larger version of this figure.
Figure 2: Evaluation of adhesion complex formation in L. infantum-infected dendritic cells. Dendritic cells infected or not with L. infantum were stained with anti-pFAK or anti-paxillin antibodies. (A) Fluorescence intensity of FAK expression. (B) Fluorescence intensity of paxillin expression. For each group, 30 cells were analyzed using FIJI software. Red: anti-pFAK, or anti-paxillin; Blue: DAPI; Grayscale: differential interference contrast (DIC). Scale bar = 0.18 inches. *p<0.05 (Student's t-test). Please click here to view a larger version of this figure.
Figure 3: Evaluation of actin dynamics in L. infantum-infected dendritic cells. Dendritic cells infected or not with L. infantum were stained with anti-Rac1, anti-RhoA and anti-Cdc42 antibodies or fluorescent phalloidin. (A) Fluorescence intensity of phalloidin expression (green). (B) Fluorescence intensity of Rac1 expression (red). (C) Fluorescence intensity of Cdc42 expression (red) (D) Fluorescence intensity of RhoA expression (red). For each group, 30 cells were analyzed using FIJI software. Red: anti-Rac1 or anti-Cdc42; Green: anti-RhoA or phalloidin; Blue: DAPI; Grayscale: differential interference contrast (DIC). Scale bar = 0,18 inches. *, p<0,05 (Student's t-test). Please click here to view a larger version of this figure.
The method described here for evaluating cell migration using the cell culture membrane inserts system allows researchers to study the migratory response of cells in a two-dimensional environment. In this technique, some steps are considered critical. Firstly, the differentiation of human DCs and infection with Leishmania are determinative since the infection rate is donor-dependent. Using more than one donor per experiment and healthy Leishmania cultures will allow for more consistent results. It is also crucial that parasites be maintained in host animals, which favors the selection and maintenance of virulent strains and the ability to readily colonize host cells. Following DC differentiation, we recommend checking the expression of surface markers CD80 and CD11c to verify that the cells being used in experimentation are, in fact, dendritic cells. Despite variability in infection rates, increased DC migration following L. infantum infection was observed in all experiments.
The cell culture membrane inserts system assay, employed herein, entails cell migration through a porous polycarbonate membrane from the upper to the lower compartment of the cell culture membrane inserts system. A chemoattractant is placed in the bottom of each well to direct cell migration through the porous membrane during incubation36. It is important to use an appropriate pore size for the cell type of interest. For DCs, we used a cell culture membrane inserts system with a 5μm pore size. Of note, after the fixation step, the surfaces of the insert membranes were scraped with a swab to remove cells that had not migrated. This step was implemented to ensure that only those cells that successfully migrated through the membrane were evaluated. Another critical point that warrants consideration is the use of DAPI staining, a rapid procedure that allows investigators to not only identify host cells but also parasite nuclei, thus enabling the convenient identification of infected cells.
Although the cell culture membrane inserts system has been extensively used as an effective tool for assessing migration28,36,37,38,39, there are some limitations associated with this technique. During cell washing and fixation steps, less-adherent cells may be lost when analyzing the membrane, resulting in an underestimation of the number of cells that migrated. Another limitation is time related. After long incubation periods, gradient loss may occur due to diffusion through the porous membrane. Thus, this system should be considered more efficient for shorter incubation periods36. On the other hand, the use of the cell culture membrane inserts system is advantageous compared to other methods such as the scratch assay or random migration since it allows the study of directional migration in the presence of a chemoattractant.
Another essential component of this protocol is the use of immunofluorescence to investigate mechanisms involved in cellular migration, such as adhesion complex formation and actin dynamics. This technique allows investigators to visualize specific targets in tissues or cells using specific antibodies for proteins of interest. In this protocol, to assess cellular adhesion, we evaluated the expression of phosphorylated FAK and paxillin, both crucial proteins involved in the formation of adhesion complexes in different cell types, including leukocytes13, and consequently excellent tools for studying cell adhesion. Previous studies have shown that increased FAK signaling promotes cellular motility11. Also, the use of this technique to evaluate cell adhesion does not require the use of more labor-intensive techniques, such as the use of inflamed connective tissue26.
The phalloidin staining technique40,41 provides information about the expression of polymerized actin, F-actin, and the regions of cells with higher polymerization rates. To gain further insight into actin dynamics in L. infantum-infected cells, we also evaluated the expression of Rac1, Cdc42, RhoA, and Rho GTPase, which participate in the polymerization of actin filaments42. The expression of these molecules was also performed using immunofluorescence. This technique is not only an alternative to the use of transgenic mice expressing fluorescent actin28 but also provides further information about molecules modulated during the actin polymerization process.
Several factors can affect immunofluorescence quality and efficacy. Antibody dilution, for example, when not carefully determined, can impair the acquisition of images and lead to non-specific staining due to elevated levels of background signals. Samples must also always be protected from light to avoid any loss of cell fluorescence or staining43.
In summary, here we describe an in vitro protocol that allows for the evaluation of cell adhesion and migration processes in the context of Leishmania infection. Our primary focus was on DCs, which are known to play a significant role in the immunopathogenesis of leishmaniasis; however, how these cells participate in parasite dissemination in the vertebrate host remains poorly understood. This protocol can also be modified to investigate cellular migration in other types of host cells for the investigation of other species of intracellular parasites. Also, extracellular matrix, such as collagen I or Matrigel, can be added to the cell culture membrane inserts system to evaluate 3D migration and cellular invasion in different fields of study.
The authors have nothing to disclose.
This work was supported by Bahia Research Support Foundation (Fapesb), grant number 9092/2015. The authors acknowledge CNPq, Capes and Fapesb for financial support via scholarships. The authors would like to thank Andris K. Walter for critical analysis, English language revision and manuscript copyediting assistance.
16 Gauge needle | Descarpack | 353101 | |
24 well cell culture plate | JET-BIOFIL | J011024 | |
25 Gauge needle | Descarpack | 353601 | |
Albumin from bovine serum | Sigma Aldrich | A2153-100G | |
Ammonium chloride | Sigma Aldrich | A-0171 | |
Anti-mouse IgG, Alexa Fluor 488 | Invitrogen | A32723 | |
Anti-mouse IgG, Alexa Fluor 594 | Invitrogen | A11032 | |
Anti-rabbit IgG, Alexa Fluor 568 | Invitrogen | A11011 | |
CD14 MicroBeads | MACS Myltenyi Biotec | 130-050-201 | |
Cell Culture Flask 25cm2 | SPL | 70125 | |
Cellstripper | Corning | 25-056-CI | |
Confocal microscope | Leica | TCS SP8 | |
Coverslip circles 13mm | Perfecta | 10210013CE | |
Dissecting Forceps | VWR | 82027-406 | |
EDTA | Sigma Aldrich | E6758 | |
Falcon tube | KASVI | K19-0051 | |
Fetal Bovine Serum | gibco | 16000044 | |
Fluorescence microscope | Olympus | BX51 | |
Glass slide 25,4×76,2mm | Perfecta | 200 | |
Hemin bovine | Sigma Aldrich | H2250 | |
Hemocytometer | Perfecta | 7302HD | |
Histopaque® 1077 | Sigma Aldrich | 10771 | |
MACS buffer | MACS Myltenyi Biotec | 130-091-221 | |
Minimum Essential Medium | Gibco | 41090093 | |
Mouse anti-Rac1 | BD | 610650 | |
Paraformaldehyde | Sigma Aldrich | 158127 | |
Phalloidin Alexa Fluor 488 | Invitrogen | A12379 | |
Phosphate Buffered Saline | ThermoFisher | AM9624 | |
Polycarbonate Membrane Transwell Inserts – Pore size 5.0 µm | Corning | 3421 | |
ProLong Gold DAPI kit | Invitrogen | P36931 | |
Rabbit anti-Cdc42 | Invitrogen | PA1-092X | |
Rabbit anti-FAK (pTyr397) | Invitrogen | RC222574 | |
Rabbit anti-paxilin (pTyr118) | Invitrogen | QF221230 | |
Rabbit anti-RhoA | Invitrogen | OSR00266W | |
Recombinant Human CCL3 | R&D Systems | 270-LD-010 | |
Recombinant Human GM-CSF | PeproTech | 300-03 | |
Recombinant Human IL-4 | PeproTech | 200-04 | |
Recombinant Human M-CSF | PeproTech | 300-25 | |
RPMI 1640 Medium | Gibco | 21870076 | |
Saponin | Sigma Aldrich | 47036 – 50G – F | |
Syringe 3 mL | Descarpack | 324201 | |
Trypan Blue | Gibco | 15250061 |
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