C3h hen

C3H/HeN and C57BL/J6 (5-week-old male) mice were purchased from Charles River (Frederick, MD). Mice were housed in ventilated filter-top cages at the USDA BHNRC animal facility under 12-h light/dark cycle. One week of acclimation on chow diet was conducted prior to the dietary treatments. Mice were then, randomized into four experimental groups (n = 8 per group): (1) Uninfected mice on control diet, (2) infected mice on control diet, (3) uninfected mice on treatment diet, and (4) infected mice on treatment diet. Mice were treated for two weeks prior to Cr infection and remained on their respective diets until the end of the experiment. Body weights and food consumption were recorded weekly. All experiments were approved by the USDA-ARS Beltsville Institutional Animal Care and Use Committee (18-027).
Mice were fed an AIN-93M diet with or without 1 µmol I3C/g diet. I3C was purchased from Sigma Chemical Company (St. Louis, MO, USA). A dose of 1 µmol I3C/g diet (147 mg/kg) in mice is roughly equivalent to a dose of 11.4 mg/kg in average adult human. In a clinical study, consumption of I3C was tolerated in doses up to 1200 mg/day in male and female cancer patients. Our selected concentration of I3C for this study was in the range achievable through dietary consumption as well as in a low dose chemo-preventive range [27 (link),28 (link),29 (link)].

Female C3H/HeN or C3H/HeN-SCID mice were acquired from Charles River Laboratories (Charles River Laboratories International, Inc., Wilmington, MA) and used at 4 wks of age for long term intradermal inoculation studies, or 8 wks of age or older for short term intravenous (i.v) inoculation experiments. For intravital microscopy Cd1d^-/- mice in a BALB/c background (Jax #2962) were bred in-house at the Clara Christie Centre for Mouse Genomics at the University of Calgary. Mice of both genders between 6–8 weeks of age were used.

Female C57BL/6, C3H/HeN, and nude mice (Balb/c nu/nu) (6–7 weeks old) were purchased from Charles River Laboratories Japan (Yokohama, Japan). Beige mice (C57BL/6J-bg/bg) were purchased from RIKEN BRC (Tsukuba, Japan). The animals were kept in standard laboratory cages in groups of five per cage under a 12 h light/dark cycle in a temperature- and humidity-controlled environment with food and water ad libitum. Clinical symptoms including body weight, tumor bleeding, behavior, appearance and general health condition were monitored daily. All care and experimental procedures were performed in accordance with national and regional legislation on animal protection, and all animal procedures were consistent with the University of Tsukuba's Regulation of Animal Experiments and were approved by the Animal Experiment Committee, University of Tsukuba (reference numbers 17–434). For tissue histology, mice were sacrificed by cervical dislocation under isoflurane anesthesia (2%, 2 L/min).

All mice were female C3H/HeN, weighed 25 to 30 g on delivery (Charles River Laboratories Inc., UK) and had free access to a commercial pelleted diet (R&M no.3 SDS LTD., Whitham, UK) and tap water in groups of 10 for one week of acclimation. They were then housed singly (Macrolon 2 cages; North Kent Plastics, UK) for another week prior to enrolment. Sawdust and wood shavings were used as bedding and cages were supplemented with ‘Sizzle Nest’, an aspen chew-block and a cardboard tube (B & K Universal). Room temperature was maintained at 21±1°C with 15–20 air changes per hour under a 12-hour light cycle (lights off at 19∶00 h). All testing was conducted between 10 am and 3 pm.

Infection experiments were approved by the LSHTM Ethics Committee and performed under UK Home Office licence PPL70/8207. All methods and manipulations were performed in accordance with the requirements of this licence. Female BALB/c and C3H/HeN mice were obtained from Charles River (UK) and CB17 SCID mice were bred in-house. Mice were maintained as described previously23 (link) and were aged 8–12 weeks when infected with bioluminescent CL Brener23 (link) or JRcl4 strains29 . Typically, 1 × 10⁴ bloodstream trypomastigotes (BTs) in 0.2 ml PBS were used to infect SCID mice via intraperitoneal (i.p.) inoculation. Parasitaemic blood from these mice was obtained 2–3 weeks later and adjusted to 5 × 10³ BTs ml⁻¹ with PBS. Mice were then infected i.p. with 1 × 10³ BTs23 (link). On occasions (~10%), infection with the JR strain led to a fatal outcome, prior to the initiation of treatment. These mice were not included in the data set (Table 1).

BALB/c, C57BL/6 and C3H/HeN mice were purchased from Charles River (UK), and CB17 SCID mice were bred in‐house. Animals were maintained under specific pathogen‐free conditions in individually ventilated cages. They experienced a 12 h light/dark cycle and had access to food and water ad libitum. Female mice aged 8–12 weeks were used, except where otherwise stated. SCID mice were infected with 1 × 10⁴in vitro‐derived tissue culture trypomastigotes in 0.2 ml PBS via i.p. injection. Unless otherwise stated, all other mice were infected by i.p injection of 1 × 10³ BTs derived from parasitaemic SCID mouse blood. All infected SCID mice developed fulminant infections and were euthanized at or before humane end‐points. At experimental end‐points, mice were sacrificed by ex‐sanguination under terminal anaesthesia. In some experiments chronically infected mice (>150 days post infection) were immunosuppressed with cyclophosphamide (200 mg kg⁻¹ day⁻¹) by i.p. injection at 3–4 day intervals, for a maximum of three doses (Francisco et al., 2015).