Leishmania amazonensis promastigotes in 3D Collagen I culture: an in vitro physiological environment for the study of extracellular matrix and host cell interactions

Ethics statement

The use of animal models was approved by the Ethics Committee for Animal Experimentation of the Health Sciences Centre, Federal University of Rio de Janeiro (Protocols n. IBCCF 096/097), according to the Brazilian federal law. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences, USA.

Leishmania amazonensis promastigotes culture

The MHOM/BR/75/Josefa strain of L. amazonensis used in this study is an anonymized strain that was isolated in 1975 from a patient with diffuse cutaneous leishmaniasis by Dr. Cesar A. Cuba-Cuba (Brasilia University, Brazil) and kindly provided by the Leishmania Collection of the Instituto Oswaldo Cruz (Code IOCL 0071–FIOCRUZ). It has been maintained by BALB/c footpad inoculation. Amastigote forms were obtained from these mice, and transformed into promastigotes that were axenically cultured in Warren’s medium (brain and heart infusion with 20 µg/ml hemin and 10 µg/ml folic acid) supplemented with 10% fetal bovine serum at 25 °C. Stationary-phase promastigotes were obtained from 5- to 6-day-old cultures and used throughout.

L. amazonensis promastigote cultivation on 3D COL I matrices

Rat tail extracted COL I solution was diluted with 5-times concentrated DMEM media and completed to the desired density (1.5 or 3.0 mg/ml) with DMEM. 0.1 M NaOH was used to neutralize the solution pH. 10⁷ promastigotes were mixed with 1 ml of diluted and neutralized COL I solution and incubated at 37 °C for a 1 h polymerization. After complete polymerization, serum-free RPMI medium was added to the top of the matrices to feed the cells and hydrate the COL I matrix. The culture was then left at 27 °C for no more than 72 h. Viability was measured using a fluorescent Live/Dead assay (Invitrogen, L3224) and only samples with viability scores higher than 90% were used.

Scanning electron microscopy

COL I matrix samples cultivated with L. amazonensis promastigotes were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 h and then washed overnight with PBS. After fixation, promastigotes in the COL I matrix were postfixed for 30 min in a solution containing 1% OsO₄, 1.25% potassium ferrocyanide and 5 mM CaCl₂ in 0.1 cacodylate buffer, washed in the same buffer and then dehydrated in an ethanol series from 30% to 100%. Finally, samples were critical point dried, coated with a thin gold layer in a Balzers gold sputtering system, and observed in a FEI-Quanta scanning electron microscope.

Protease inhibitors

Metalloprotease inhibitors included 200 nM Marimastat or 5 mM o-phenantroline, cysteine protease inhibitors included 20 ng/ml Cystatin or 100 µM of trans-epoxysuccinyl-L-leucylamido-(4-Guanidino)butane (E-64) and the serine protease inhibitor used was 1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF), (all protease inhibitors were purchased from Sigma-Aldrich). Protease inhibitors (PI) mix was used in some experiments. The PI mix used was composed of AEBSF, E-64, Cystatin and Marimastat.

Migration assay

The L. amazonensis promastigote migration was characterized for all experimental conditions. Samples were placed in a culture chamber, with controlled temperature (27 °C), adapted to an inverted microscope Nikon Eclipse TE 300 (Nikon, Melville, NY). Brightfield images were captured for 5 min with a Hamamatsu C2400 CCD camera (Hamamatsu, Japan) and digitalized by a SCION FG7 frame grabber (Scion Corporation, Torrance, CA) using a frame rate of 2 frames/s. The process was repeated every 24 h for a total period of 72 h. For each movie 15 different cells were marked with black dots. The distance covered by the black dot trajectories were then obtained by image analysis using the ImageJ software (National Institutes of Health, USA) and the indirect promastigote migration rates were determined by dividing the distance covered (µm) by the time in seconds (sec). Three different movies were made and analyzed for each condition.

Transmatrix migration (invasion) assay

For the transmatrix migration assay, the COL I matrix was previously prepared without L. amazonensis in a 96 well plate. Then, 100 µl serum free RPMI media containing 10⁶ promastigotes was added to the top of a 150 µl polymerized matrix. After 48 h, the RPMI media was washed out and the samples were fixed and processed for histology. The quantity of promastigotes inside the matrix (complete transmatrix process) and the distance migrated from the top to the bottom of the COL I matrix (transmatrix invasion distance) was used to determine the invasion ability of L. amazonensis. The transmatrix invasion distance was normalized considering 100% as the total distance between the top and the bottom of each COL I matrix histological cut. Similar results were obtained when the data were analyzed without normalization.

COL I matrix degradation assay

L. amazonensis promastigotes were cultivated inside of 3D COL I matrices prepared with a solution containing 5% FITC-labeled type I Collagen (Invitrogen, Molecular Probes). The degradation assay was as modified method from Sugiyama et al. (1980). After 0 h, 24 h, 48 h and 72 h, solid-phase collagens were pelleted (5 min, 16,000 g) and the supernatant containing released FITC-labeled type I Collagen fragments was measured with a spectrofluorimeter (485 nm excitation, 515 nm emission). The background signal and the total degradation were obtained from a cell-free matrix and from a matrix made with the presence of 25 U/ml type II collagenase (Gibco) respectively.

Zymography

Promastigotes cultivated for 48 h inside COL I matrix were placed in an eppendorf tube and centrifuged for 7 min at 1600 g to separate the COL I matrix and cells from the supernatant. After centrifugation, supernatant was filtered to remove any remaining cells. Proteins from the supernatant were then separated by SDS–PAGE gel electrophoresis using gels containing 1.5 mg/ml COL I and 10% acrylamide and bis-acrylamide. After electrophoresis, gels were washed with 50 mM Tris 2.5% Triton-X100 buffer (pH 6.8) for 30 min and then incubated with 50 mM Tris, 10 mM CaCl₂, 1 mM DTT buffer (pH 6.8) for 48 h. When indicated, the protease inhibitors o-phenantroline 5 mM or E-64 20 µM were added to the reaction buffer.

3D COL matrix model for promastigote–macrophage interaction

The murine macrophages RAW 264.7 cell line (3 × 10⁵) were mixed with 1 ml of diluted and neutralized COL I solution and incubated at 37 °C for a 1 h polymerization. After complete polymerization, RPMI medium complemented with 5% bovine serum was added to the top of the matrices to feed the cells and hydrate the COL I matrix. The culture was then left at 37 °C for 24 h. Macrophage 3D culture viability was analyzed using a fluorescent live dye fluorescein diacetate (Sigma). After 24 h of incubation, the medium was removed and Leishmania promastigotes were added to the 3D culture in two ways: 10⁷ promastigotes in 200 µl of RPMI were injected inside the matrix containing macrophages (injection mode) or promastigotes were added to the top of the matrix (invasion mode).

Confocal microscopy

Live promastigotes were labeled with 5 mM of cell tracker green (Invitrogen-C7025) for 15 min. Macrophages cultured in a 3D COL I matrix for 24 h were than labeled and put to interact with labeled L. amazonensis promastigotes for 4 h. After interaction the samples were fixed with 4% paraformaldehyde and than permeabilized with TritonX100 0.05% and incubated with rhodamine phalloidin (Invitrogen) overnight at 4 °C. The samples were visualized by the confocal microscopy LEICA TCS SP5 and the images were processed using the software LAS AF.

L. amazonensis promastigotes extensively interact with and remodel 3D COL I matrix fibers

Scanning electron microscopy (SEM) of promastigotes cultivated in a 3D COL I matrix showed a huge number of promastigotes extensively adhered to COL I fibers (Fig. 1). The presence of promastigotes altered the organization of the COL I fibers network (Figs. 1B–1F). This matrix remodeling transformed the original homogeneous COL I matrix (Fig. 1A) into a meshwork divided into areas of high fiber density and large fiber-free channels (Figs. 1E and 1F). Promastigote cell bodies and/or flagella were observed in contact with the COL I fibers (Fig. 1C). SEM revealed promastigotes completely trapped in the high density COL I fiber areas (Fig. 1D) and promastigotes in the fiber-free channels areas. Promastigotes had their cell body elongated over time during the 3D cultures on COL I matrices (Supplemental Information 1), a feature normally associated with standard liquid cultures.

3D COL I matrix degradation by L. amazonensis promastigotes

Promastigotes were cultivated inside 3D FITC-COL I matrices to measure their ability to degrade this network. L. amazonensis promastigotes degraded COL I fibers in a time-dependent manner and density-independent way (Figs. 2A and 2B). After 72 h, around 23% of the COL I total matrix was degraded. This degradation was significantly reduced in the presence of the cysteine protease inhibitors cystatin or E-64 (Fig. 3). The presence of the metalloprotease inhibitor marimastat also reduced COL I degradation, but no effect was observed with the serine protease inhibitor AEBSF (Fig. 3). Incubation with anti-gp63 caused a small but significant reduction of COL I degradation, suggesting that this metalloprotease is also capable of COL I cleavage. COL degradation was not completely abolished in the presence of protease inhibitor (PI) mix (Fig. 3). Although the concentration of inhibitors used could be insufficient for complete inhibition, we chose not to use higher concentrations of this inhibitor mix because they affected cell viability (Table S1). Zymography of culture supernatant from L. amazonensis grown in a COL I matrix detected two collagenase protease bands (Supplemental Information 2). The collagenase bands completely disappeared in the presence of the metalloprotease inhibitor O-phenanthroline, demonstrating the secretion of COL I-degrading metalloproteases by L. amazonensis promastigotes (Supplemental Information 2). Other classes of proteases could not be detected by COL zymography in the conditions tested. However, reaction buffers of pH lower than 6.8 destroy the COL gels, restricting the use of this assay for Leishmania cysteine proteases.

L. amazonensis migration and transmatrix migration in a 3D COL I matrix

L. amazonensis promastigote migration inside of a 3D COL I matrix was observed by video microscopy (Video S1). These flagellated protozoa displayed an intermittent migration mode with displacement periods (run phase) intercalated with non-displacement periods (freeze phase). The freeze and run migration mode contrasts with the continuous swimming migration mode found in conventional in vitro liquid culture. The migration speed in 3D cultures significantly increased over time (Fig. 4), reaching the highest mean rate (6 µm/s) after 72 h. Even at the first time point, just after COL I matrix polymerization, promastigotes had a slow but non-zero migration rate (Fig. 4). Over time, the displacement periods were more frequent and lasted longer, reflecting a higher migration speed (Fig. 4). Thus, there was a correlation between time, increased speed, and COL I matrix remodeling by promastigotes.

In addition to migration within a 3D COL I matrix, we tested the ability of promastigotes to adhere to and cross the matrix (transmatrix migration). Indeed, when promastigotes were added to the top of a polymerized 3D COL I matrix they were able to adhere to and penetrate through the COL I fiber meshwork (Fig. 5A). This matrix invasion ability (transmatrix migration) demonstrates that promastigotes are not only able to migrate inside of a 3D COL matrix (Fig. 4) but are also able to cross from a liquid environment (RPMI media) to a 3D meshwork (COL I matrix). Here it is important to note that promastigote transmatrix migration was observed without the addition of an external chemo-attractive factor, demonstrating a natural COL I matrix invasive ability for this intracellular parasite. The distance reached by promastigotes from the top of the matrix (transmatrix migration distance) was significantly reduced at higher COL I densities (3.0 mg/ml) compared to lower densities (1.5 mg/ml) (Fig. 5B).

Extracellular protease activity promotes L. amazonensis 3D COL I migration and invasion

The correlation between protease activity and promastigote migration in 3D was analyzed by videomicroscopy in the presence or absence of a protease inhibitor (PI) cocktail. The promastigote migration rates were analyzed using the software ImageJ. In the presence of a PI mix composed of E-64, Marimastat and Cystatin, the promastigote migration rates inside the 3D COL I matrix were significantly reduced, demonstrating that protease activity increases migration rates (Fig. 6). The fraction of promastigotes with migration rates exceeding 5 µm/s was also significantly decreased by the PI mix (Fisher’s exact test, Fig. 6). The reduction of migration rates could also be observed when only the run phase was considered in the analysis, indicating an inhibition of both frequency and maximum speed of migration.

In addition, the role of protease activities in promastigote transmatrix migration was evaluated after 72 h, in the presence or absence of PI mix (Fig. 7). Transmatrix migration is dependent on two processes: the ability of cells to penetrate into the matrix and, once inside, the ability to migrate. The PI mix presence reduced by almost 40% the number of promastigotes inside of the COL I matrix (Fig. 7A) and also significantly reduced the maximal transmatrix migration distance (in depth) achieved by the promastigotes (Fig. 7B).

Together the data demonstrate that extracellular protease activity is important for L. amazonensis promastigote transmatrix and migration in 3D cultures (Figs. 6 and 7), due to COL I matrix degradation/remodeling.

Interaction of macrophages and promastigotes in 3D COL I matrix

For an even better mimic of what would happen during Leishmania infection, we cultivated macrophages inside the COL I matrix for 24 h, after which L. amazonensis promastigotes were added to the top of the matrix (Fig. 8). After 6 h of interaction most of the parasites were found inside the matrix (Fig. 8), a shorter time than in the macrophage-free matrix. This suggests an influence of macrophages on the invasion ability of Leishmania promastigotes. 3D visualization by confocal microscopy of macrophage-promastigote interaction (Figs. 9A and 9B) showed macrophages possessing a round morphology with many actin-membrane filament projections (Fig. 9C). In some cases these thin actin-membrane projections were touching a promastigote’s surface (Fig. 9D). SEM microscopy observations showed the extensive contact of macrophages and promastigotes with COL I fibers in the 3D culture model (Fig. 10). The fibrotic characteristic of the ECM 3D environment seems to create a effective barrier for physical contact between Leishmania and macrophages, in contrast to 2D culture models in which macrophages become infected much earlier.

The injection of promastigotes directly into the matrix containing macrophages allowed a good distribution of promastigotes inside the COL meshwork and enabled the observation of macrophage-promastigote interaction in the first minutes of interaction (Video S2). That experiment demonstrated the promastigotes migrating further and faster than macrophages in the 3D culture. Additionally, the promastigotes exhibited the same freeze and run migration mode observed in the macrophage-free 3D matrix.