Nmri mice

Animal experiments were approved by the Dutch Central Commission for Animal experimentation (Centrale Commissie voor Dierproeven). Mouse experiments were performed at the Central Animal Facility of the Leiden University Medical Center (LUMC). All animals were SPF as commended by the FELASA recommendation (20 (link)). Six-month-old female NMRI-mice (Charles River laboratories, l’Arbresle, France) were used for all experiments except for studies evaluating fluorescence retention in the SLN, where 6–10 week-old Foxn1 mice (Charles River laboratories, l’Arbresle, France) were used. Each test condition consisted of three to five mice.

Naval Medical Research Institute (NMRI) mice, C57BL/6 mice, and Sprague Dawley (SD) rats were purchased from Charles River Laboratories (Sulzfeld, Germany). Anopheles stephensi mosquitoes were raised under a 14 h light/ 10 h dark cycle, 75% humidity and at 28 °C (non-infected) or 20 °C (infected). The Huh7 hepatoma cell line was cultured in RPMI supplemented with 10% FCS, 100 U penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 10 mM HEPES and 1× non-essential amino acids (NEAA) (Gibco). The HepG2 hepatoma cells were cultivated in EMEM (Gibco) containing 10% fetal calf serum, 1% L-Glutamine, 1% penicillin/Streptomycin and 1% MEM Non-essential amino acids (PAA Laboratories GmbH, Pasching, Austria). Wild-type P. berghei were either the P. berghei ANKA strain, ANKA clone 507 [26 (link)], which expresses GFP, or P. berghei NK65 [38 (link)].

Schistosoma mansoni (S. mansoni; Belo Horizonte strain) was held in a life cycle using Biomphalaria glabrata (B. glabrata) fresh water snails (Brazilian strain) as intermediate hosts and female NMRI mice as definitive hosts. Six- to eight-weeks-old NMRI mice (Charles River Laboratories, Germany) were housed in an animal facility with a 12:12 h light/dark cycle with ad libitum water and free access to standard chow (SSNIFF, Soest, Netherland). S. mansoni cercariae were obtained by mass shedding of 5–10 infected B. glabrata after day light exposure. With our experimental design we aim to investigate three widely differing infection intensities and degrees of liver damage^{34 (link)}. Each mouse was exposed to parasites by sitting in a water bath enriched with 25 (n = 4, light infection), 50 (n = 5, moderate infection) and 100 (n = 5, high infection) S. mansoni cercariae for 90 min. At a time when hepatic fibrosis is already established and the different infection intensities are apparent all animals were imaged via MRI (at day 42) and via PET/CT with [¹⁸F]FDG as radiopharmaceutical at day 43 after infection. Additionally, a healthy control group (n = 3) was imaged at the same days.

Human CD14⁺ monocytes were purified from one-day-old buffy coats, derived from healthy donors (Belgian Red Cross, Mechelen, Belgium), through density gradient centrifugation and positive selection (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described [25 (link)]. Human neutrophils were isolated from fresh blood derived from healthy donors via density gradient centrifugation as previously described [26 (link)].
The murine macrophage cell line RAW264.7 [American Type Culture Collection (ATCC), Manassas, VA, USA] was grown in Dulbecco's modified Eagle's medium (DMEM; Lonza, Verviers, Belgium) supplemented with glucose (4.5 g/l), 1 mM sodium pyruvate and 10% fetal calf serum (FCS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The murine fibroblast cell line L929 (ATCC) was grown in minimum essential medium (MEM) Rega (Thermo Fisher Scientific) supplemented with 10% FCS. Murine LLC cells (ATCC) were grown in DMEM supplemented with glucose (4.5 g/l) and 10% FCS. Murine peritoneal cells were extracted from healthy female NMRI mice (Charles River, Wilmington, MA, USA) that were kept in a specific pathogen-free environment. After euthanizing the mice, peritoneal lavages were carried out with 5 ml of phosphate-buffered saline (PBS) supplemented with 20 U/ml of heparin (LEO Pharma, Lier, Belgium) and 2% FCS.

Experiments were performed on 12–13-week-old male C57BL/6J (RRID:IMSR_JAX:000664), C57BL/6N (RRID:IMSR_CRL:027), and NMRI mice (RRID:IMSR_CRL:605), all acquired from Charles River. The mice were fed a standard rodent diet Ssniff Rat/mouse – Maintenance (V1534-000) (Ssniff, Soest, Germany) with 9, 24, and 67% of kcal derived from fat, protein, and carbohydrates, respectively, and water ad libitum. They were housed in individually ventilated cages (Allentown LLC, USA) in groups of 1-4 animals per cage at 20-24°C, 45-65% relative humidity, and a 12-hour day-night lighting cycle. Mice were weighed before sacrifice. Following the sacrifice with CO₂ and cervical dislocation, the mouse was placed on its abdomen, and body length was measured from the nose tip to the base of the tail. Glucose was measured within 5 minutes upon sacrifice in the morning between 8 AM and 9 AM from the tail vein using the Accu-Check Aviva glucometer (Roche, Switzerland). During analysis, body mass index (BMI) was calculated as the ratio between body weight and body surface area (g/m²), where body surface area was calculated according to the following formula (52 (link)):

C57BL/6 and NMRI mice were purchased from Charles River Laboratories. For all experiments six to eight weeks old female mice were used unless stated otherwise. P. berghei ANKA parasites were maintained by passaging via intra-peritoneal injections in NMRI mice and Anopheles stephensi mosquitoes. All animal experiments were performed according to FELASA standard guidelines and were approved by German authorities (Regierungspräsidium Karlsruhe).