Mice and ethics statement:
Design and realization of animal experiments follow the ARRIVE guidelines.
Female BALB/c mice were purchased from Charles River (France).
Mice were maintained and handled according to the regulations of the European Union, the French Ministry of Agriculture and to FELASA (Federation of Laboratory Animal Science Associations) recommendations.
Experiments were approved by the ethics committee of the Nice School of Medicine, France (Protocol number: 2017-56).
Leishmania culture:
L. infantum MON-1 (MHOM/FR/94/LPN101), was isolated from a patient with Mediterranean visceral leishmaniasis contracted in the Nice area (South of France). We used this isolate to generate a recombinant L. infantum - expressing the Green Fluorescent Protein reporter (GFP-L. infantum) and L. infantum - expressing the Luciferase reporter (LUC-L. infantum)9. L. infantum promastigotes were routinely grown at 26°C in Schneider’s Insect Medium (Sigma®) supplemented with NaHCO3 0.4g/L (Janssen chimica®), CaCl 0.6g/L (Fluka Chemika®), Fetal Bovine Serum 10% (Gibco®), 10 mL urine pool for 500 mL of medium, Phenol Red 0,1%, Hepes 1M pH 7.3, penicillin/streptomycin 1% (Gibco®), and L-Glutamine 1% (Gibco®).
Parasite preparation and inoculation in mice:
Briefly the promastigote forms were washed three times in PBS, and 2.108 parasites were injected by intravenous route in 200µL of PBS. Control mice were injected with 200µL of PBS.
Minced BAT tissue from infected BALB/c:
BAT from infected BALB/c mice was sampled and freshly minced using a potter. The minced infected BAT was injected by intraperitoneal route in naive BALB/c mice.
Separation of an adipocyte-enriched fraction and a stromal vascular fraction (SVF):
Briefly BAT and WAT were minced and then digested for 45 min at 37°C in collagenase type 2. The tissue digest was passed through 250 µm nylon sheets. Floating adipocytes were separated from the SVF after decantation. The floating fraction corresponding to the adipocyte-enriched fraction was carefully removed.
Quantification of parasites by quantitative PCR:
Each sample of Adipose tissue, liver and spleen (25 mg) was put in a sterile tube of Lysing Kits (Precellys®), and then homogenized by Precellys® (2 x 30 sec, with a break of 15 sec) in lysis buffer of the Qiagen Kit QIAmp DNA Mini Kit®. DNA extraction was conducted according to the recommendations of Qiagen®. The extracts were kept at -20°C for conservation. Quantitative PCR was implemented for detection and quantification of L. infantum targeting minicircle kinetoplast DNA (kDNA). Primers and probes previously described by Mary et al.5 containing 20 pmol of each forward (5’-CTTTTCTGGTCCTCCGGGTAGG-3’) and reverse (5’-CCACCCGGCCCTATTTTACACCAA-3’) primer and 3.33 pmol TaqMan probe (FAM-TTTTCGCAGAACGCCCCTACCCGC-TAMRA) were used for Leishmania screening and quantification 5. The assays were performed with a final volume of 10 µL, including the 2.5µL DNA sample. A standard curve was obtained from the primary DNA extraction source of 2.5x107 parasites and diluted serially at a 1/10 rate, which corresponded to 50 000 to 0.05 parasites in 2.5 µL. The PCR program was implemented with two temperature steps of 95°C and 60°C for 40 cycles. The standard curve and a pair of negative controls were used for each assay.
Histology and microscopic observation:
Organs were fixed in 4% PFA (ParaFormAldehyde). Samples were embedded in paraffin automatically with the spin tissue processor STP120 (ThermoFisher®). The tissue processor STP120 uses alcohol to remove water from tissues and replace it with a medium that allows sectioning of tissue. Thin sections (2.5 µm) were cut with a Microtome Microme HM340E (Leica BIOSYSTEMS®). Sections were deparaffinized by immersing 3x in xylene, rehydrated by successive immersion in ethanol solutions of different percentages (100%, 95%, 70%, 50%) and in water. Unmasking was conducted boiling in 10 mM Sodium Citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by a solution of 3.0% hydrogen peroxide. The immuno-histochemistry labeling consisted of a primary human antibody directed against Leishmania. This antibody was recognized by an anti-human goat antibody, which was recognized by biotinylated anti-goat, the signal was amplified and revealed by Streptavidin-HRP. The brightfield microscope was an Eclipse Ci upright stand (Nikon, Japan), using objectives 20X dry NA 0.40. Acquisitions were done with a DS – Ri 1 camera (Nikon, Japan).
Mouse primary pre-adipocyte purification and differentiation:
The method for generating white, brite and brown adipocytes from stromal vascular fraction (SVF) cells was adapted from a previous publication 23. Briefly, fat deposits were sampled, minced and then digested for 45 min at 37°C in DMEM (Lonza, BE12–707F) containing 2 mg/mL collagenase A (Roche Diagnostics, 11088793011) and 20 mg/mL BSA (Sigma-Aldrich Chemie Gmbh, A7030). The digestion was successively filtrated through 250, 100 and 27 µm nylon sheets, and finally centrifuged at 500 g for 5 min. The pellet containing the SVF was cleared from red blood cells using specific buffer (Sigma) before being plated and maintained in DMEM containing 10% (v/v) fetal calf serum (FCS) until confluence. Differentiation was induced by supplementation with 1 µM dexamethasone (Sigma-Aldrich Chemie Gmbh, D4902), 0.5 mM isobutylmethylxanthine (Sigma-Aldrich Chemie Gmbh, I5879) and 860 nM insulin (Invitrogen, 12585014) for 2 days. Cells were then maintained for 7–10 days in presence of 100 nM insulin for white adipogenesis or a mixture containing 100 nM insulin, 1 µM rosiglitazone (BertinPharma, 71740) and 0.2 nM triiodothyronine (Sigma-Aldrich Chemie Gmbh, T6397) for brown or brite adipogenesis.
Differentiation and generation of BMDM (Bone Marrow Derived Macrophage):
Femurs were removed and purified from the surrounding muscles and connective tissue. Under sterile conditions, the bone marrow was flushed by pressure after needle penetration in epiphyses with BMDM medium containing RPMI and decomplemented FBS 10% and gentamycin 0,001%. The cells were centrifuged (400 g, 5 min) and resuspended in BMDM medium supplemented with M-CSF 10 ng/mL. Cells were seeded at 5x105 cells /well.
Human adipocytes differentiation:
Human adipose tissue primary progenitor cells were from a previous study 19 and differentiated as follow. Cells were cultivated in DMEM containing 10% FCS until confluence. When the cells reached confluence, they were induced to differentiate for 3 days in DMEM/Ham's F12 (1:1) media supplemented with 10 µg/mL transferrin, 10 nM insulin, 0.2 nM triiodothyronine, 1 µM dexamethasone and 500 µM isobutyl-methylxanthine. The cells were next differentiated into white adipocytes using a media supplemented with 10 µg/mL transferrin, 10 nM insulin, 0.2 nM triiodothyronine or into brite adipocytes in the same media supplemented with 100 nM rosiglitazone.
Microscopy:
The BMDM and adipocytes of pre-adipocyte murine origin were infected after 7 days of differentiation with a ratio of 10:1 GFP-L.infantum per cell. Human adipocytes were infected after 14 days of differentiation with a ratio of 10:1 GFP-L.infantum per cell.
Epifluorescence:
For epifluorescence acquisition, we used the EVOS FL microscope (AMF-4302-EU; Labtech, France), using the 10X dry Ph and 20X dry FL objectives. Acquisitions were done with a Sony ICK285AL monochrome CCD, 2/3’’ 1360x1024, 1.4 Megapixel camera (Labtech, France).
Electron Microscopy:
For ultrastructural analysis, cells were fixed in 1.6% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C, rinsed in 0.1 mol/L cacodylate buffer, and fixed for 1 hour in 1% osmium tetroxide and 1% potassium ferrocyanide in 0.1 mol/L cacodylate buffer to enhance the staining of membranes. Cells were rinsed in cold distilled water, quickly dehydrated in cold ethanol, and lastly embedded in epoxy resin. Contrasted ultrathin sections (70 nm) were analyzed under a JEOL 1400 transmission electron microscope (EM) mounted with a Morada Olympus charge-coupled device camera.