The proinflammatory nature of Ly6Chi monocytes is shaped differentially by parasitic and bacterial liver infection
The proinflammatory nature of Ly6Chi monocytes is shaped differentially by parasitic and bacterial liver infection
Monocytes from the livers of E. histolytica- or L. monocytogenes-infected C57BL/6J mice were sorted on the indicated days p.i., and surface staining was performed using CD11b (APC-Cy7; Ly6C (FITC) and Ly6G (APC). Cells were then sorted into collection tubes containing 2 mL of RNAprotect cell reagent (Qiagen). C57BL/6 (WT) and Ccr2-/- mice  were bred and kept in individually ventilated cages under specific pathogen-free conditions at the animal facility at the Bernhard Nocht Institute, Cd38-/- mice were provided by Prof. Mittrücker, University Medical Center Hamburg-Eppendorf, Hamburg. The mice were kept with a day/night cycle of 12 h, humidity of 50–60%, and a temperature of 21°C. Mice were euthanized with CO2 with a replacement rate of 20- 30% of the cage volume per minute, followed by cervical dislocation and cardiac puncture. Male C57BL/6 mice (aged 8–12 weeks) were used for infections. Briefly, 2 × 105 trophozoites of the highly pathogenic clone B2, generated from cell line B (HM-1:IMSS), were suspended in 20 µl of incomplete TYI-S-33 medium and injected into the left liver lobe, as described previously (47). Abscess size was calculated as % abscessed liver tissue. Mice were infected with 2 × 104 L. monocytogenes strain EGD or 1 × 107 L. monocytogenes actA in 200µl PBS via the lateral tail vein. Bacterial inoculi were controlled by plating serial dilutions on tryptic soy broth agar plates at 37°C. RNA was isolated using the RNeasy Plus Micro Kit (Qiagen) and RNA integrity was analyzed using an Agilent 6000 Pico Kit and an Agilent 2100 Bioanalyzer (Agilent). Samples used for transcriptome sequencing fulfilled the following criteria: total RNA ≥ 200 ng (≥ 20 ng/µL); RNA integrity number (RIN) ≥ 7.0, 28S/18S ≥1.0. RNA library preparation and sequencing were performed by BGI Genomics, China.