Comment[ArrayExpressAccession]	E-GEOD-45170
MAGE-TAB Version	1.1							
Public Release Date	2013-03-15
Investigation Title	Different responses of murine and human macrophages to leptospiral infection revealed by comparative array analysis							
Comment[Submitted Name]	Different responses of murine and human macrophages to leptospiral infection revealed by comparative array analysis
Experiment Description	Leptospirosis is a re-emerging tropical infectious disease caused by the pathogenic Leptospira spp. The different host innate immune responses were partially related to the different severity of leptospirosis. In this study, we employed transcriptomics and cytokine array to comparatively calculate the responses of murine peritoneal macrophages (MPM) and human peripheral blood monocytes (HBM) to leptospiral infection, and uncover a series of different expression regulations of these two immune cells. The regulation percentages in several biological processes of MPM, such as the antigen process and presentation, the regulation of membrane potential, and the innate immune response, etc., were much greater than those of HBM (>2 folds). In HBM and MPM, the CASP8 and FADD-like apoptosis regulator genes were significantly up-regulated, which supported previous results that the caspase-8/3 pathway plays an important role in macrophage apoptosis during leptospiral infection. The key component of complement pathway, C3, was only up-regulated in MPM. Several cytokines (e.g. IL-10, TNF-alpha) were differently expressed at both mRNA and protein level in MPM and HBM. The differences in the transcriptomics and the cytokine expression revealed in this study were partially consistent with the different outcomes of the chronic and acute leptospirosis; thus, these findings facilitated further molecular study on the innate immune response to leptospiral infection. Murine peritoneal macrophages (MPM) and human peripheral blood monocytes (HBM) were infected by pathogenic Leptospira. The host cells were collected at 1h, 2h, and 4h after infection. The uninfected macrophage sample was designed as a negative control. Three biological replicates were designed for each sample type. There were totally 24 hybridizations (12 for MPM, and 12 for HBM) included in this submission.							
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl						
Person Last Name	Xue	Xue	Zhao					
Person First Name	Feng	Feng	Jinping					
Person Email	geo@ncbi.nlm.nih.gov							
Person Affiliation	Beijing Tropical Medicine Research Institute, Beijing Friendship Hospital, Capital Medical University							
Person Address	Department of Microbiology and Parasitology, Beijing Tropical Medicine Research Institute, Beijing Friendship Hospital, Capital Medical University, No. 95, Yong'an Road, Beijing, Beijing, China							
Person Roles	submitter							
Protocol Name	P-GSE45170-1	P-GSE45170-6	P-GSE45170-3	P-GSE45170-8	P-GSE45170-7	P-GSE45170-2	P-GSE45170-4	P-GSE45170-5
Protocol Description	ID_REF =  VALUE = quantile normalized	The labeled samples were hybridized to the OneArrayTM whole genome microarray with Phalanx hybridization buffer using cover slides. After overnight hybridization at 50M-0C, non-specific binding targets were washed away using 3 different washing steps.	Murine peritoneal macrophages (MPMs) were isolated from male BALB/c mice (6 to 8 weeks old) by washing the peritoneal cavities with cold RPMI 1640 medium. Macrophages were seeded in 75cm2 tissue culture flasks (Corning. Inc., Big Flats, NY) and the cell numbers were counted in a haemocytometer. The cells were cultured in RPMI 1640 medium (supplemented with 10% heat-inactivated fetal bovine serum, 100 M-NM-<g/ml of penicillin, and 100 M-NM-<g/ml of streptomycin) for 2 h at 37M-0C under a humidified atmosphere containing 5% CO2. After the pre-incubation, non-adherent cells were removed gently by washing with PBS (0.01M, pH 7.4), and the purity of the remaining macrophages was tested by WrightM-bM-^@M-^Ys staining. Human peripheral blood monocytes (HBMs) were isolated from 6 healthy donors using standard Ficoll-Hypaque gradient centrifugation as previously described. The monocytes were seeded and counted according to the above-mentioned protocol of MPM, then pre-incubated overnight at 37M-0C under a humidified atmosphere containing 5% CO2. After the pre-incubation, the monocytes was thoroughly washed with autocleaved PBS to remove non-adherent cells, and then continuously incubated for 5 days in a medium containing 500 U/ml of granulocyte-macrophage colony stimulating factor (GM-CSF; eBioscience, CA, USA) to differentiate the cells into macrophages.	Spots that passed the above-mentioned criteria were normalized using quantile normalization according to the manufacturerM-bM-^@M-^Ys recommendation and tested for differential expression. The differential expressed genes were further analyzed using Agilent GeneSpring GX software (version 10.0). Considering that primary cells were a mixture of several related cell types with limited purity, regulated genes with more than 3 folds (P<0.05) of up-regulation or down-regulation were defined as significantly regulated genes.	The slides were dried using centrifugation and scanned using a GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA, USA). The Cy5 fluorescent intensities of each spot were analyzed using GenePix Pro 6.0 software (Molecular Devices). The signal intensity of each spot was corrected by subtracting background signals in the immediate surroundings. Spots which flag <0 and SNR <3, and the control probes were filtered out.	The MPM and HBM cell cultures were washed 3 times with autocleaved PBS, renewed with new medium without antibiotics, and further cultured for 12 h before infection. Leptospiral cells were harvested by centrifugation at 8,000 g for 10 min at 20M-bM-^DM-^C, and then washed twice with PBS. The bacterial pellets were re-suspended in 37M-bM-^DM-^C RPMI 1640 medium to final concentration of 108 per ml. Then 10 ml of leptospiral suspension (10^9) were added into 10^7 cells (bacteria: cell = 100:1) and incubated at 37M-0C under a humidified atmosphere containing 5% CO2. Cell samples were collected at 3 intervals (1, 2 and 4 hours) respectively in the cell infection models, and 3 biological replicates were designed for each sample. Previous cytokine mRNA kinetic expression research showed that most of the cytokine transcripts can be detected as early as 1h after infection in animal infection model, which indicated that the rapid gene regulation of innate immune response can be detected within 4-hour window period in this study. The MPM and HBM cell cultures not infected by L. interrogans were used as negative controls.	At sample harvesting, MPM and HBM cell cultures were washed 3 times using autocleaved PBS to remove leptospiral cells. The total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA), then purified using RNeasy Mini Kit (QIAGEN, Hilden, Germany) with on-column DNase digestion (QIAGEN, Hilden, Germany) according to the RNeasy Mini handbook. RNA quantity and integrity was determined using RNA 6000 Nano Laboratory-on-a-Chip kit and the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).	For each sample, about 10 M-NM-<g of total RNA was mixed with 600 ng of random hexamer primers (TaKaRa, Otsu, Japan) and denaturalized at 65M-bM-^DM-^C for 5 min. Then the first strand cDNA was synthesized using 2 M-NM-<l (400 U) SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) according to the protocol recommended by the manufacturer. The double strand cDNA (ds cDNA) sample was then synthesized using 2nd Strand Synthesis section of M-MLV RTase cDNA Synthesis Kit (TaKaRa, Otsu, Japan) according to the manufacturersM-bM-^@M-^Y instructions. Following RNase H (Invitrogen, Carlsbad, CA) and RNase A (Ambion, Texas) digestion for 1 h, ds cDNA sample was purified using QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) according to QIAquick Spin handbook. The double-stranded cDNA templates were labeled using Amersham Mono-functional Cy5 CyDye.
Protocol Type	bioassay_data_transformation	hybridization	grow	feature_extraction	image_aquisition	specified_biomaterial_action	nucleic_acid_extraction	labeling
Experimental Factor Name	ORGANISM	TIME	CELL TYPE	INFECTED WITH				
Experimental Factor Type	Organism	time	cell type	infected with				
Comment[SecondaryAccession]	GSE45170							
Comment[GEOReleaseDate]	2013-03-15							
Comment[ArrayExpressSubmissionDate]	2013-03-14							
Comment[GEOLastUpdateDate]	2013-03-16							
Comment[AEExperimentType]	transcription profiling by array							
SDRF File	E-GEOD-45170.sdrf.txt