Leishmania amazonensis impairs phagosome acidification in B-1 phagocytes as an unrecognized parasite infection and proliferation mechanism

Categories: Disease & Virus

September 30, 2025

Experimental animals

Female mice of 8 to 12-week-old C57BL/6 strain were used. The animals were kept with no water or dietary restriction, in light/dark cycles under a temperature of 22–25 °C, in the experimental animal facility at the Department of Parasitology, University of São Paulo. All experiments were carried out in accordance with international ARRIVE guidelines and in full agreement with institutional and local regulations [Brazilian Federal Law #11,794, Decree #6,899 and Normative Resolutions published by the National Council for the Control of Animal Experimentation (CONCEA)] and approved by the Institutional Animal Care and Use Committee (IACUC) under protocols #104/2013 and #95/2013 at the Institute of Biomedical Sciences, University of São Paulo, Brazil.

Bone marrow-derived macrophages isolation

Bone marrow-derived macrophages (BMM) were prepared as described¹⁰. Briefly, BMM from C57BL/6 mice were obtained after 7 days of bone marrow differentiation in RPMI medium (Vitrocell) supplemented with supplemented with 25 mM HEPES, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 20% (v/v) heat-inactivated fetal bovine serum (iFBS, from Vitrocell) and 20% (v/v) L-929 cell conditioned medium, pH 7.2, at 37 °C in a 5% CO₂ standard humidified incubator.

Peritoneal macrophages and B-1P cells isolation

B-1P cells were obtained from C57BL/6 mice by using an adapted two-step differentiation protocol²⁰. Step One: Peritoneal cavity cells were collected by washes with RPMI-1640 medium supplemented with 25 mM HEPES, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, without iFBS, pH 7.2. The cells were incubated for 1 h at 37 °C in a 5% CO₂ standard humidified incubator. Non-adherent cells were removed, and adherent monolayers were incubated for 5 days in RPMI medium supplemented with 10% (v/v) iFBS. Step Two: After the 5 days of incubation, the adherent cells were used as peritoneal macrophages. For B-1P cells differentiation, the cells present in the supernatant were then incubated in RPMI medium containing 10% (v/v) iFBS and 50% (v/v) L-929 cells conditioned medium for at least 24 h. The number of adhered B-1P cells (37.14% average from the total) was determined by subtracting the total peritoneal cells initially plated minus the number of non-adherent cells counted in the supernatant.

RAW cell line culture

RAW 264.7 macrophage-like cells (American Tissue Type Collection, ATCC) were maintained in RPMI medium supplemented with supplemented with 25 mM HEPES, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 10% (v/v) iFBS, pH 7.2, at 37 °C in a 5% CO₂ standard humidified incubator. Subcultures were prepared by washing the cells twice with PBS, followed by the addition of trypsin for 5 min, then dilution in complete medium and thoroughly re-suspension.

Leishmania amazonensis parasites

Leishmania amazonensis (IFLA/BR/67/PH8) parasites were obtained from lesions in C57BL/6 mice and then propagated as promastigotes in M199 medium (Vitrocell) supplemented with 40 mM HEPES, 2.5 µg/mL hemin, 10 mM adenine, 2 mM L-glutamine, 2 µg/mL D-biotin, 100 U/mL penicillin, 100 µg/mL streptomycin and 20% (v/v) iFBS, pH 7.2, at 26 °C in a bio-oxygen demand incubator, as described^27,28. Subcultures were made weekly at an initial density of 5 × 10⁵ promastigotes/mL up to six passages. Parasites were washed three times in phosphate-buffered saline (PBS) before use in experiments.

In vitro infection

The cells were seeded at least 24 h prior to infection. For immunofluorescence assays, the cells were seeded on top of glass coverslips in 24-well plates. L. amazonensis promastigotes were added at a multiplicity of infection (MOI) of 2 in RPMI supplemented with 5% (v/v) iFBS and 2% (v/v) L-929 cell supernatant for 2 h at 34 °C in a 5% CO₂ standard humidified incubator. The cells were then washed three times with PBS to remove non-internalized parasites, followed by the addition of RPMI supplemented with 10% (v/v) iFBS and 2% (v/v) L-929 cell supernatant and incubated at 34 °C in a 5% CO₂ humidified incubator for the indicated periods.

In vivo infection and tissue processing

Wild-type and TLR9^−/− mice were injected with 10⁶ L. amazonensis stationary phase promastigotes in the left hind footpad. Lesion size progression was followed by weekly measurements with a caliper by an observer blinded to the experimental group assignments. The parasite load in the lesions was quantified by a limiting dilution assay. Footpad tissue homogenates were dissociated by incubation with collagenase (2 mg/mL, Sigma-Aldrich; St. Louis, MO) in Tyrode buffer (140 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 10 mM HEPES, 2 mM MgCl₂, pH 7.2) for 2 h at 37 °C under mild agitation. The homogenate was filtered through a 70-mm pore-size cell strainer (Falcon^®; Corning, NY), centrifuged at 20 x g for 5 min, and washed twice with PBS (230 x g for 10 min). The cellular suspension was diluted 100-fold in complete M199 media, followed by another 10-fold serial dilution in 96-well plates performed in triplicates. After 10 days of incubation at 26 °C, the presence or absence of viable parasites in each well was determined by direct observation under an inverted light microscope. Results are presented in logarithm scale in base 10 (Log₁₀) of the highest dilution in which viable promastigotes were observed. In some experiments, 2.5 × 10⁷ amastigotes were injected in the peritoneal cavity and cells were collected by peritoneal washes with RPMI-1640 medium after 48 h of infection.

In some experiments, footpads of animals were removed and fixed in freshly made 10% formaldehyde (v/v) in PBS, followed by dehydration and paraffin embedding. Serial 5-µm sections were stained by hematoxylin and eosin (H&E, Sigma Aldrich). The histological sections were evaluated and captured under a light microscope (AxioImager M2, Zeiss) coupled with a high-resolution camera (AxioCam HRc, Zeiss).

Phenotypic characterization of B-1 and B-1P cells by flow cytometry

Cells isolated from footpad lesions, peritoneal cells, or the non-adherent cells fraction of five days of culture (B-1 cells) were counted, suspended in PBS containing 2% FBS and incubated with an anti-mouse CD16/CD32 antibodies (BD Pharmingen) to block Fc receptors, followed by staining with fluorochrome-conjugated mAb for surface markers, namely: phycoerythrin (PE)-labeled anti-mouse CD19 (Caltag Medsystems), Pacific Blue (PB)-labeled anti-mouse F4/80 (Thermo Scientific), and fluorescein isothiocyanate (FITC)-labeled anti-mouse CD23 (BD-Pharmingen). Fluorescence-minus-one (FMO) controls were used to determine the cut-off point between background fluorescence and positive populations in multicolor flow cytometry experiments. All the surface markers were incubated for 30 min at 4 °C in the dark. After washes, stained cells were fixed with 4% paraformaldehyde solution for 15 min at room temperature (RT). Cells were then washed, and incubated in PBS containing 2% SFB and then the samples were acquired by using an LSRFortessa™ flow cytometer (BD Bioscience) or an Attune Acoustic Focusing Flow Cytometer (Applied Biosystems) acquiring at least 50,000 events. Data were analyzed by using the FlowJo software, version 10.0.7 (Tree Star Inc. Ashland, OR, USA) as shown (Fig. S1).

Drug treatment in vitro

To evaluate the potential association between the susceptibility to infection with the size of PV, B-1P cells and BMM were pretreated with 1 µM of vacuolin-1 (compound 5114069, Cayman Chemical) for 1 h. The cells were then washed with fresh medium and infected. The phagocytosis capacities of B-1P cells and BMM were evaluated by pretreating the cells with 5 mM latrunculin A (Sigma-Aldrich) for 30 min at 37 °C. After the treatment, the cells were washed with fresh medium and infected on ice for 30 min to allow parasite attachment, followed by incubation of the cells at 34 °C for an additional 30 min.

Vacuole pH detection with acridine orange

Infected cells were incubated for 15 min at 34 °C with RPMI medium containing acridine orange (AO) [2 µg/mL 3,6-bis-(dimethylamino) acridine; Sigma-Aldrich] in 5% CO₂. After washes with PBS to remove excess AO, the cells were incubated in RPMI with 5% FBS medium supplemented with 2% L-929 conditioned media, and allowed to settle at 37 °C in a 5% CO₂ humidified incubator for 30 min before imaging.

Immunofluorescence and image analyses

For the immunofluorescence assays, the samples were processed and stained using two strategies. For staining and counting, coverslips containing the infected cells were washed and fixed with 100% ice-cold methanol for 5 min. After fixation, the samples were treated with blocking and permeabilization solution [1% BSA, 0.1% saponin, and 0.1% sodium azide in 25 mM Tris-buffered saline (TBS)] for 30 min. The coverslips were incubated with anti-LAMP-1 polyclonal antibody (Developmental Studies Hybridoma Bank) followed by anti-rat IgG antibody Alexa Fluor 568-conjugated (Thermo Scientific); anti-Leishmania serum followed by anti-rabbit IgG antibody Alexa Fluor 488-conjugated (Thermo Scientific); PE-labeled anti-mouse CD19 (BD Bioscience); FITC-labeled anti-mouse IgM (BD Bioscience); anti-EEA-1 (BD Bioscience) polyclonal antibody followed by anti-mouse IgG antibody Alexa Fluor 488 (Thermo Scientific); and anti-V-ATPase polyclonal antibody (Anti-V-ATP6V0D2, Sigma Aldrich) followed by anti-rabbit IgG antibody Alexa Fluor 488 (Thermo Scientific). Cells were incubated with 10 µg/mL DAPI (Sigma-Aldrich) to detect the nuclei of parasites and host cells. For the assessment of phagocytosis, the samples were processed without permeabilization (staining only OUT parasites) and incubated with anti-Leishmania polyclonal antibody followed by anti-rabbit IgG antibody Alexa Fluor 488 and 10 µg/mL propidium iodide (PI; Sigma-Aldrich). The images were randomly acquired (an observer blinded to the experimental group assignments) in a fluorescence microscope (Leica DMI6000B/AF6000) coupled to a digital camera system (DFC365FX) moving through visual fields in parallel rows across each coverslip, and analyzed by using ImageJ. An appropriate filter set was used depending on the sample fluorescence labeling. The regions of interest (ROIs) were delineated manually to measure diameter (Fig. 3) or fluorescence intensity. The number of events analyzed is annotated in corresponding figure legends.

Additionally, vacuole pH detection with AO images was acquired in the InCell Analyzer High Content Imaging System (GE), version 2200, by using a 20x objective. The images from the Cell Analyzer High Content Imaging System were analyzed by CellProfiler software, providing the following outputs per image. Total number of cells, total number of infected cells, number of parasites per infected cell, the ratio of infected cells to the total number of cells, and intensity of Cy3 fluorescence channel per vacuole (Fig. S5).

Infection quantification

The infection quantification was performed by examining stained slides on a fluorescence microscope with an objective lens under immersion oil (Leica DMI6000B/AF6000; 100x lens). Quantification was performed on a regular basis by an investigator blinded to the group assignments. Moving through visual fields in parallel rows across each coverslip, the number of macrophages and the number of intracellular parasites was quantified with a manual click-counter. At least 300 cells per coverslip were counted. Parasite load was determined by counting the number of intracellular parasites in at least 100 infected cells. The infection index was obtained by multiplying the percentage of infection per the average number of intracellular parasites per cell. In the phagocytic capacity assay, the attached parasites (OUT) were visualized by microscopy in green and red (anti-Leishmania and Nuclei PI-staining, respectively), while internalized (IN) parasites were visualized only in red (Propidium iodide, PI).

Cytokine quantification

Cell culture supernatants were used for cytokines and/or nitrite quantification. IL-10 and TNF-α were assayed by using the Cytometric Bead Array (CBA) kit for Mouse inflammation (BD Biosciences). The sample acquisitions were carried out on FACSCanto II (BD Biosciences) and the data analysis was performed with the FACSDiva™ software.

Quantitative real-time PCR

The RNA extraction and purification were performed by using the Quick-RNA™ MiniPrep kit (Zymo Research). Quantification was carried out in a NanoDrop instrument (Thermo Scientific) and cDNA synthesis was performed by using a kit (SuperScript III Reverse Transcriptase, Invitrogen). Quantitative PCR reaction was performed on the equipment StepOnePlus™ Real-Time PCR System (Applied Biosystems) by using the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific). Oligonucleotide primers, forward: 5’-AGC AGA AGG TGA TAG ACC AGA A-3’ and reverse: 5’-CCC ACA CTT GGA TCA TCA ATG C-3’ were used to amplify a portion of LYST/Beige cDNA; and forward: 5’-TCA GTC AAC GGG GGA CAT AAA-3’ and reverse: 5’-GGG GTC GTA CTG CTT AAC CAG-3’ to amplify the housekeeping gene (Hprt1). The reaction was incubated for 10 min at 95 °C and then for 40 cycles of 15 s at 95 °C, followed by 30 s at 55 °C and 30 s at 72 °C. Fluorescence was detected at each annealing step. Technical triplicates were performed for each reaction and negative controls were included. The data were presented as relative quantification normalized by Hprt1 expression levels calculated through the 2-ΔΔCt methodology²⁹.

Western blot

A total of 20 µg of protein/well were separated under reducing conditions on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel and blotted onto nitrocellulose membranes with a transfer system (BioRad Laboratories, Hercules; CA). The membranes were probed with an anti-LAMP-1 polyclonal antibody followed by peroxidase-conjugated anti-rat IgG mAb (Imuny-VBP Biotecnologia) or anti-V-ATPase polyclonal antibody (Anti-V-ATP6V0D2, Sigma Aldrich) followed by peroxidase-conjugated anti-rabbit IgG mAb (Imuny-VBP Biotecnologia). Immunoblots were developed by using the Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific) and detected with a ChemiDoc Imaging System (BioRad) on the ImageLab (BioRad) software.

Statistical analysis

Data were analyzed with Prism 6.0 (GraphPad Software, San Diego, CA). Statistical significance was determined by the Student’s t-test (two-tailed), two-way analysis of variance (ANOVA), or ANOVA one-way for data with Gaussian distribution and similar variation between groups. After the test for normal and log non-normal distribution, non-Gaussian data were analyzed by Mann-Whitney or Kruskal-Wallis, or Wilcoxon test, as indicated. Statistically significant differences were defined as * when p-values were p p

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