Isotype control

Irinotecan hydrochloride (CPT-11, Camptosar®) and SN-38 (7-Ethyl-10-hydroxycamptothecin) were purchased from Sigma-Aldrich. Recombinant murine IL-33 (rmIL-33) was obtained from Biolegend. Mouse IL-33 MAb (Clone 396118) and Isotype control were purchased from R&D Systems. Purified Rat anti-mouse Ly6G (clone 1A8) and Isotype control were purchased from BD Biosciences. Recombinant murine sST2 was generated and purified from HEK293T cells at VIB Protein Service Facility, VIB Inflammation Research Center, Ghent, Belgium. DF-2156A was obtained from Dompé S.p.A., L’Aquila, Italy.

Male C57Bl/6J mice (Jackson Laboratory) aged 10–18 weeks were used for the CNI treatment studies as well as controls in all experiments. Male FK12Tie2 KO mice were generated as described previously and were used between the ages of 10–18 weeks.^{8 (link)} All mice were maintained on a 12:12 light/dark cycle and had access to standard chow ad libitum. Tail-cuff systolic blood pressures (IITC, Inc.) were measured at baseline and on day 7 of daily treatment with CSA (50 mg/kg/day, i.p.; Alamone, Isreal), TAC (10 mg/kg/day, i.p.; LC Laboratories) or diluent (saline and DMSO, 0.2% final concentration) as described previously.^{8 (link), 20 (link), 21 (link)} Mice were trained for this procedure for 3 days prior to baseline measurements. Some mice were given daily i.p. injections of RA (300 ug/mouse/day; Sigma). Other control, CSA-treated, TAC-treated, and FK12Tie2 KO mice were given i.p. injections of either an IL-17 neutralizing antibody (100 ug/mouse; R&D Systems) or the isotype control (100 ug/mouse; R&D Systems) on days 1, 4, and 7. Animals were anesthetized on day 8 with isoflurane and euthanized by cervical dislocation. All procedures were approved by the Institutional Animal Care and Use Committees in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Male mice aged 8-15 weeks were infected by intravenous injection of viable P. chabaudi AS parasites (WT) or PccASluc (luciferase-expressing; [45 (link)]). To ensure viability of the parasites, a frozen aliquot was thawed and injected intraperitoneally into a transfer mouse. The number of asexual parasites intravenously injected into each mouse was adjusted according to body weight so that every animal received 1x10⁴ iRBCs per 20 grams. Parasitemia was monitored from day 5 post infection every 48 hours by Giemsa-stained thin blood smear. Anti-GCSF antibody (150 μl per mouse, R&D) or isotype control (150 μl per mouse, R&D) were injected intravenously on day 7 post infection.
Mice were bled by cardiac puncture under non-recovery deep anesthesia. Blood was kept from coagulating by addition of 50 μM final concentration of EDTA (Sigma). Plasma was generated by centrifugation at 10,000 x g at 4°C for 10 minutes. Plasma was aliquoted, snap frozen in liquid nitrogen and stored at -80°C until further use. Plasma was always thawed on ice.
Organs were harvested without additional perfusion (except in parasite sequestration experiments) as blood was removed by terminal bleeding of the animals. The organs were fixed for 20h at room temperature in 2% PFA.