MATERIALS AND METHODS Mice, parasites and cells. All animal care and procedures were performed in accordance with institutional guidelines and European regulations. Experiments were conducted using AQP9-/- and their litternate control aged from 7 to 10 weeks. P. yoelii (265BY) P. berghei (ANKA strain) and P. falciparum (NF54 strain) sporozoites were obtained by dissection of infected Anopheles stephensi mosquito salivary glands.
Human hepatocarcinoma cell lines HepG2-A16-hCD81EGFP (Yalaoui et al 2008 plos pathogens), HepG2-CD81 (Bartosh et al 2003 JBC) and Huh-7 were cultured in DMEM (Invitrogen) supplemented with 10% FCS, 2 mM glutamine and antibiotics (Invitrogen, Gibco). Primary mouse hepatocytes were isolated as previously described (Renia et al. 1990), seeded at a density of 4 x 104 cells per well in 96-well microplates and cultured at 37 °C in 5% CO2 in William’s E medium (Invitrogen, Gibco) supplemented with 2% penicillin-streptomycin, 1% sodium pyruvate, 1% L-glutamine, 1% insulin-transferrin-selenium, 1% non essentials amino acids and 10% fetal bovine serum (Invitrogen, Gibco). Human liver fragments were collected following informed consent provided from patients undergoing a partial hepatectomy. The collection and use of these tissues were undertaken in ccordance with French government ethical regulations. Primary human hepatocytes were isolated from healthy parts of the liver biopsies
Small interfering RNA and lentiviral transduction. We used small double stranded RNA oligonucleotides targeting human CD81 (5’- GCA CCA AGT GCA TCA AGT A -3’), human AQP3 (Qiagen HP validated siRNA AQP3_5), human AQP9 (5’- CTG CTG ATC GTG GGA GAA A-3’ and 4 Qiagen HP GenomeWide siRNA). A siRNA oligonucleotide targeting human CD92 (5’- AAG GCA AGA ACT GAA AAC T -3’) was used as a control siRNA throughout the study. Primary hepatocytes were transfected with 30nM of siRNA with Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s recommendations.
Following siRNA transfection, cells were cultured for 72 hours before western blot, flow cytometry analysis or sporozoite infection. The TRIP? 3-CMV-AQP9 plasmid was constructed by inserting the human AQP9 cDNA sequence in the TRIP? 3-CMV vector (Ref). Vector particles were produced by transient calcium phosphate cotransfection of 293T cells by the TRIP? 3-CMV-AQP9 plasmid, an encapsidation plasmid and a vesicular stomatitis virus envelope expression plasmid, as described. Cells were transduced by incubating 1/1000 of concentrated viral preparation 3h before washing and viral particles expressing GFP were used as a control. Note personnelle Patrick; J-3+Si => J-2lavage+DMSO (cad elimination des Si => J0 spz => J3->5 schizontes => => il n’y a pas de prolif avec HH => pas ou peu de synthese de proteines; cependant, il est possible qu’il y ait une neosynthese de prot => probleme pour interpreter le role des proteines sur developpement/maturation. Real-time PCR quantification of liver-stage parasite burden. Wild-type (n = 4) and AQP91-/- (n = 4) male mice were infected by retro-orbital injection with 4 x 104 P. erghei ANKA-GFP sporozoites and sacrificed 40h post inoculation. Livers were harvested, homogenized using a Polytron homogenizer and total RNA was isolated using the Micro-to-midi Total RNA purification system (Invitrogen). The detection and quantification of the liver stage parasite load was performed on the corresponding total cDNAs, as detailed previously (Rodrigues et al. , 2008). Amplified P. berghei 18S sequences were normalized to mouse Hypoxanthine Guanine Phosphoribosyltransferase (HPRT). All test samples were performed in triplicate.
Parasite infection and quantification by immunofluorescence analysis. For infection assays in 96-well plates, the plates were centrifuged after inoculation of 3 x 104 (P. falciparum) or 2 x 104 (P. berghei and P. yoelii) Plasmodium sporozoites to allow the parasite to come immediately into contact with the target cells. After sporozoite penetration into hepatocytes for 3 hours at 37°C, cultures were washed and further incubated in fresh medium for 2 to 4 days (for P. yoelii and P. berghei or P. falciparum, respectively) until EEF quantification.
After fixation of the cultures with cold methanol, EEFs were stained using the anti-HSP70 serum {Renia, 1990 #128} and revealed with FITC-conjugated (Sigma) or Alexa Fluor 680-conjugated (Invitrogen, Molecular Probes) goat anti-mouse immunoglobulin and counted under a fluorescence microscope or using the Odyssey Infrared Imaging System (LI-COR Biosciences) as described previously {Gego, 2006 #14}. Nuclei were stained with 1 ? g/ml diamidino-phenyl-indole (DAPI; Sigma). All assays were performed in triplicate.
Quantification of extracellular and intracellular parasites was carried out using double immunostaining as described before {Renia, 1988 #54}{Silvie, 2003 #4}. AQP9 activity inhibition and cell viability assays Phloretin (Sigma) was diluted at 0. 5M in DMSO as a stock. Phloretin was added to primary human hepatocyte 24h before infection with Plasmodium sporozoites and maintained during infection. Infected cells were washed and culture in standard medium without phloretin for 3 days. For cell viability assays, phloretin was added to cells 24h before a MTT test (Mosmann et al, 1983). Western Blot and cell immunofluorescence.
Primary human hepatocytes or cell lines cultured for 24h were lysed at 4°C for 30 minutes in 30 mM Tris, pH 7. 4, 150 mM NaCl, 0. 02% NaN3, protease inhibitors (PMSF, INH) and 1% Triton X-100. AQP9 was analyzed by western-blotting with a mouse anti-AQP9 mAb (G-3) (Santa Cruz) (1/100) followed by a goat anti-mouse Alexa Fluor 680 (Invitrogen) (1:15000). Monoclonal mouse anti-tubulin (Abcam) (1:5000) was used as a standard. Data were acquired and quantified with an Odyssey Infrared Imaging System. Statistical analysis. All data were analyzed for statistical significance using the OneWay ANOVA followed by the Tukey multiple comparison test.
