GENE PROFILING IN HUMAN BLOOD LEUCOCYTES DURING RECOVERY FROM SEPTIC SHOCK
Payen D(1), Lukaszewicz A(1), Belikova I(1), Faivre V(1), Gelin C(3), Russwurm S(4), Launay JM (2), Sevenet N(2).
Additional data
Hierachical clustering of gene expression according to outcome. List of gene of interest for predicting outcome.
In the hierarchical clustering representation of gene expression at D1 referred to D0, according to outcome (Figure 1suppl), two dead patients (number 1 and 2) were grouped. The 3rd patient who died (number 9) was not grouped with other dead patients because the first cluster profile was less clear. This gene profile contains thirty six genes of interest (p < 0.05) grouped in 3 different clusters (figure 1suppl and table 1suppl). The first cluster of genes (genes 1 to 7) showed an under-expression between D1 and D0 (green). These genes encode membrane proteins or chemokines implicated in innate immunity cell activation. The second cluster grouped 26 genes (genes 8 to 33), for which dead patients presented a significant upregulation (red). This group of genes encoded inflammatory processes, antigen presentation machinery, signal transduction pathway, and apoptosis inducers. In the 3rd cluster (genes 34 to 36), the 3 genes were over-expressed in all patients.
Detailed methods
Microarray procedure according to MIAME criteria:
Experiment Design:
Type of experiment:
Peripheral blood mononuclear cells of 17 patients (59±16 yrs) with severe sepsis or septic shock were studied. First, genomic dependence of early mortality was determined by comparing the transcriptomes at D1 relatively to D0 (respectively 24 hrs after meeting inclusion criteria, and the day after) for 14 survivors and 3 patients who died on the second day. Second, among the 14 patients who recovered, the variability of gene expression was investigated at day 1 (D1), day 7 (D7) and day 28 (D28) after ICU admission. Every time point was compared to D0 for each patient.
Experimental factors: Experiments done on samples of patients: no experimental factors studied.
The number of hybridizations performed in the experiment.
Thirty four hybridizations Dx/D0 were performed because some samples were missing or could not be used because of the RNA quality. All tables of hybridization have been registered in GEO data bank under the accession number GSE 5262 and GSE 5271.
The type of reference used for the hybridizations.
D0 time point cDNA was used as a reference for each hybridization.
Hybridization design: co-hybridizations for all survivals: D1/D0; D7/D0; D28/D0; + 3 hybridizations for the 3 patients died: D1/D0. The reference time D0 was always labeled with Cy3 fluorochrome; samples at time points (D1, D7, D28) were always labeled with Cy5 fluorochrome. Two series of hybridization were carried out one for outcome (GEO GSE 5262), and one for time evolution (GEO GSE 5271).
Quality control steps taken:
All control elements of the Lab-Arraytor human 500-1 were spotted/programmed in the same way on 24 diagonal positions of each of four blocks of the internal triplicates. Then, each block included a special positioning of a full set of reliable controls (e.g., negative controls and Arabidobsis thaliana control clones).
URL of any supplemental websites or database accession numbers
http://www.sirs-lab.com
http://www.ncbi.nlm.nih.gov/geo/
Samples used, extract preparation and labeling:
The origin of the biological sample
Peripheral blood mononuclear cells (PBMC)
Manipulation of biological samples and protocols used:
Peripheral EDTA-blood samples (15 ml) were collected from patients at time points D0, D1, D7 and D28. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation on Histopaque.
Protocol for preparing the hybridization extract:
Total RNA was extracted using the Qiagen Rneasy Mini kit (Qiagen). All RNA samples were treated with Rnase-free DNAse I during the extraction as a step of the extraction protocol. The quality was estimated on the bioanalyzer (Agilent) and quantity of total RNA was measured on Nanodrop spectrophotometer (Labtech International). Fifteen micrograns of total RNA were processed for microarray analysis. Double-strand cDNA was obtained by the reverse transcription using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions.
Labeling protocol(s).
Incorporation of a nucleotide containing an alkyl amino group (AA-dUTP) allowed post-reverse transcription conjugation of the fluorescence dyes. The reaction was blocked by hydroxylamine hydrochloride. Cy-3 and Cy-5 containing probes were combined and purified with QIAquick PCR Purification kit (Qiagen).
For each patient, the cDNA of the treatment probes (from time points D1, D7 and D28) were labeled using the fluorescence dye (Cy5) and the reference cDNA (from time point D0) using the fluorescent dye (Cy3).
External controls (spikes).
Spike RNAs were added in the different steps of the experimental procedure according to the manufacturer’s instructions.
Hybridization procedures and parameters:
The protocol and conditions used during hybridization, blocking and washes.
The same amount of a treated and reference cDNA was used for the experiment. Cy-3 and Cy-5 containing probes were denaturated in the presence of human Cot-1 DNA, yeast tRNA and polyA RNA for 2 min at 100oC. The hybridization was performed on the Lab-Arraytor® human 500-1 cDNA (SIRS-Lab GmbH, Jena, Germany) in 3.5xSSC and 0.3% SDS at 65oC for 20 hours. The slides were then washed in 2xSSC, 0.03% SDS, in 2xSSC, 0.01% SDS and 0.25xSSC for washes lasting 2 min each.
Measurement data and specifications:
The quantitations based on the images.
Signal intensities of a microarray were measured with the GMS418 Scanner (Affymetrix. The resulting images were analyzed using the AIDA Array Evaluation software (Raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany). After detecting the location of each spot the corresponding signal intensities were calculated. For each dye, the signal for any given spot was measured by the mean of the pixel intensities within the spot. The approach for the background calculation included taking the mean intensities of selected background spots (blanks) within a block of 12 by 12 spots. Block by block, the mean value of the background intensity was subtracted from the intensities of the spots in red and green channels. Intensity ratios were calculated, log10 transformed then divided by the corresponding mean intensity of Arabidobsis Th. spikes. Changes in expression levels were also given as ratios of signals in Cy5 versus Cy3 (ratios higher than 1 indicated an increase in transcript level, ratios smaller than 1 indicated a decrease).
The set of quantitations from several arrays upon which the authors base their conclusions:
Type of scanning hardware and software used: MWG GMS418 Scanner and Genetic microsystem Software (Affymetrix) were used to obtain the image data, according to the manufacturer’s instructions.
Type of image analysis software used: specifications should be stated in the materials and methods. AIDA Array Evaluation software (raytest Isotopenmessgeräte GmbH, Straubenhardt).
A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
Measure of fluorescence intensities over each colored spot by AIDA with subtraction of background noise intensity.
The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
Data selection and transformation procedures
Calculation of ratio Cy5/Cy3 for each spot
Log10 transformation of the Cy5/Cy3 ratio
Subtraction of the mean value of all Ara 1 spots of the considered slide.
Normalization with the lowess method.
Unpaires t-test comparison based on outcome, Anova analysis of comparisons based on trend over time.
Cluster analysis by hierarchical descendant classification of the most statistically significant Cy5/Cy3 ratios (p<0.05).
Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
Array Design:
General array design, including the platform type.
The experiment was performed on the Lab-Arraytor® human 500-1 cDNA from the SIRS-Lab GmbH, Jena. The array contained 340 probes for well-characterized human genes composed of blocks with 12 by 12 spots. Each block included a special positioning of a full set of reliable controls (e.g., negative controls and Arabidobsis thaliana control clones). The chip consisted of three identical subarrays of four blocks, thus obtaining three replicates for each probe.
For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given.
The microarray chip is described in GEO data bank under the accession number GPL1936.
For each reporter, its type (e.g., cDNA or oligonucleotide) should be given, along with information that characterizes the reporter molecule unambiguously, in the form of appropriate database reference(s) and sequence (if available).
For commercial arrays: a reference to the manufacturer should be provided, including a catalogue number and references to the manufacturer’s website if available.
Lab-Arraytor human 500-1 (SIRS Lab, Jena, www.sirs-lab.com).
Gene expression assays (Applied Biosystems)
The probes contain a 6-carboxy-fluorescein phosphoramidite (FAM dye) label at the 5' end of the gene and a minor groove binder and nonfluorescent quencher at the 3' end, and are designed to hybridize across exon junctions. The assays are supplied with primers and probe concentrations of 900 nM and 250 nM, respectively. For each gene, the assay with the highest amplification efficiency was selected for this study. All of the 4 assays selected were referenced as “inventoried” assays on the Celera database; the TaqMan probes and primer sequences are presented in supplementary table 1.
To control for possible variations among PCR runs performed on different days, for each plate of amplification and for each TaqMan Gene Expression Assay, 2 types of negative PCR control were done (PCR substrate resulting in an RT reaction without template = no template control (NTC); PCR substrate resulting in an RT reaction without polymerase = No amplification control (NAC)) and generated no flurorescence signal indicating that we did not have any contamination of our PCR mix. For each plate of amplification and for each TaqMan Gene Expression Assay, one type of positive PCR control was performed (Reference RNA (Clontech, BD Biosciences)) allowing the comparison of the efficiency of the PCR reactions over the time of experimentation. Eukaryotic 18S ribosomal RNA was used as the endogenous RNA controls (Assay ID: Hs99999901_s1; Applied Biosystems). For calibration, we used cDNA prepared from Universal Human Reference RNA (Clontech). Correlations in figure 2suppl.
Flow cytometry for HLA-DR expression measurement:
The whole blood was incubated with appropriate antibodies conjugated with fluorochrome (Fluorescein isothiocyanate (FiTC) or Phycoerythrin (PE)). Whole blood samples were labeled following these two conditions: (i) condition 1 : anti CD14-FiTC (clone RMO52, Beckman Coulter, Marseille, France) and PE conjugated irrelevant isotype control antibodies (Simultest control, BD Biosciences, San Jose, CA), (ii) condition 2 : anti CD14-FiTC and anti HLA-DR-PE (clone L243, BD Biosciences). The red –labeled cells were lyzed (FACS Lysing Solution; BD Biosciences). Resting cells were fixed in PBS by paraformaldehyde, and stored at 4°C in the dark until the flow cytometry study was performed within the next 24 hrs. Mean fluorescence intensities of each condition were converted into number of Antibodies Bound per Cell (AB/C) using QFCM (Quantitative Flow CytoMetry). Final level of monocyte HLA-DR expression was obtained by the subtracting expression in the two conditions.
S100A8 protein ELISA:
Briefly, 100 µl of diluent solution were added to each well of 96 –well polycarbonate plate coated with the primary antibody (Mo2B9; 0,166 mg/L; BMA, Basel, Switzerland), Plasma samples were diluted either in working Block-AceTM (Dainippon Pharmacology Co.LTD) solution, or in MRP8/14 complex calibrator solution and the plate was mixed for 15 s with a plate mixer. The plate was incubated for 1 hr at 37°C to allow the immunologic reaction to proceed. After this one-hour incubation at RT, followed by 5 washes, plates were incubated with the secondary antibody Mo3D2 (BMA) at RT. After 5 washes, plates were further incubated in 100 µl of streptavidin-HRP conjugate diluted 1:1500-in the working Block-Ace solution for 30 min. Finally, the HRP activity was classically determined by colorimetry. Within-run CVs were 3.9-5.6%, between-day CVs were 5.9-7.6% and mean recovery was 98% (range 85-103%).
Table 1suppl: Clinical characteristics, outcome, and type of infection of the septic patient population.
Age / Sex / OF / SAPSII / SOFA / outcome / Bacteria / Site / Postop1 / 60 / M / 4 / 81 / 12 / dead / G+ / Catheter / No
2 / 87 / M / 4 / 62 / 15 / dead / G- / Abdo+blood / Yes
3 / 50 / M / 4 / 32 / 7 / alive / ND / Soft tissues / Yes
4 / 61 / F / 3 / 55 / 10 / alive / ND / Lung / No
5 / 47 / M / 3 / 38 / 8 / alive / Poly / Abdo+blood / Yes
6 / 64 / F / 2 / 42 / 6 / alive / ND / Cervical / No
7 / 69 / M / 3 / 43 / 10 / alive / G+ / Hip joint / Yes
8 / 65 / M / 2 / 42 / 6 / alive / Poly / Abdomen / Yes
9 / 93 / M / 4 / 70 / 9 / dead / Poly / Abdomen / Yes
10 / 36 / M / 2 / 49 / 8 / alive / Poly / Lung / No
11 / 50 / M / 3 / 32 / 8 / alive / ND / Urinary tract / No
12 / 76 / M / 4 / 64 / 14 / alive / Poly / Abdo+blood / Yes
13 / 62 / M / 3 / 60 / 14 / alive / G+ / Abdomen / Yes
14 / 36 / M / 3 / 41 / 14 / alive / Poly / Soft tissues / Yes
15 / 44 / M / 3 / 56 / 10 / alive / Poly / Abdo+pelvis / Yes
16 / 52 / F / 3 / 33 / 7 / alive / G- / Abdomen / Yes
17 / 71 / M / 3 / 55 / 15 / alive / G- / Abdomen / Yes
mean±SD / 59±16 / 50±15 / 10±3
OF: number of organ failure; SAPSII: severity score; SOFA: organ failure scoring at day 0 of the protocol entry. ND: non determined. G+: gram positive, G-: gram negative, Poly: polymicrobial.
Table 2 suppl: Genes of interest whose expression exhibited a relation with outcome.
SIRS-lab / Gene ID / Description / Category of function
1 / AF144103 / CXCL14 / chemokine (C-X-C motif) ligand 14 / Monocyte, dendritic cell attraction
2 / XM_003933 / CD180 / CD180 antigen / TLR4 pathway inhibition
3 / XM_058179 / CD244 / CD244 natural killer cell receptor 2B4 / NK activation
4 / NM_031409 / CCR6 / chemokine (C-C motif) receptor 6 / Monocytic cell attraction
5 / XM_046798 / PIK3C2B / phosphoinositide-3-kinase, class 2, beta polypeptide / Signal transduction, cell polarisation
6 / XM_002335 / ITGA6 / integrin, alpha 6 / integrin
7 / NM_003874 / CD84 / CD84 antigen (leukocyte antigen) / Monocytic cell activation, costimilatory molecule AG presentation
8 / AF332468 / WHSC1L1 / Wolf-Hirschhorn syndrome candidate 1-like 1 / Transcription factor
9 / XM_034770 / PAFAH1B1 / platelet-activating factor acetylhydrolase, isoform Ib, alpha subunit / inactivation of proinflammatory PAF
10 / XM_012039 / FUT4 / fucosyltransferase 4 (alpha (1,3) fucosyltransferase, myeloid-specific) / Leucocyte tissu invasion
11 / XM_033972 / ATF6 / activating transcription factor 6 / Stress response element
12 / XM_038900 / SDFR1 / stromal cell derived factor receptor 1 / Leucocyte migration, platelet aggregation
13 / XM_042665 / MAP3K10 / mitogen-activated protein kinase kinase kinase 10 / Signal transduction, monocytic cells activation and differentiation
14 / XM_003594 / BST1 / bone marrow stromal cell antigen 1 / Membrane protein with inflammatory role
15 / XM_036154 / LAMP2 / lysosomal-associated membrane protein 2 / Leucocyte adhesion to endothelium
16 / XM_027216 / ATF2 / activating transcription factor 2 / Regulation CD1a
17 / XM_055849 / SRP54 / signal recognition particle 54kD / RNA molecule trafficking
18 / NM_002856 / PVRL2 / poliovirus receptor-related 2 (herpesvirus entry mediator B) / Cell adhesion
19 / XM_037854 / BMP6 / bone morphogenetic protein 6 / Growth factor
20 / NM_006017 / PROM1 / prominin 1 / Membrane protein
21 / XM_009219 / EDG6 / endothelial differentiation, G-protein-coupled receptor 6 / Cell activation
22 / XM_012608 / CD79B / CD79B antigen (immunoglobulin-associated beta) / B lymphocyte AG presentation
23 / NM_005037 / PPARG / peroxisome proliferative activated receptor, gamma / T cell apoptosis
24 / XM_043574 / DAP3 / death associated protein 3 / Apoptosis
25 / XM_010177 / DUSP9 / dual specificity phosphatase 9 / Signal transduction
26 / XM_059235 / HBXIP / hepatitis B virus x-interacting protein / Apoptosis inhibition
27 / NM_000880 / IL7 / Interleukin 7 / Growth factor
28 / XM_048792 / CD1A / CD1A antigen / Lipid presentation
29 / XM_030523 / MAP3K8 / mitogen-activated protein kinase kinase kinase 8 / Signal transduction
30 / XM_002762 / TNFAIP6 / tumor necrosis factor, alpha-induced protein 6 / Inhibition PMN migration
31 / NM_000418 / IL4R / Interleukin 4 Receptor / Inhibition cytokine response
32 / NM_003684 / MKNK1 / MAP kinase-interacting serine/threonine kinase 1 / Transcription factor and kinase
33 / XM_002682 / IL18R1 / interleukin 18 receptor 1 / Cell surface receptor
34 / NM_002211 / ITGB1 / integrin, beta 1 (fibronectin receptor, beta polypeptide) / integrin
35 / NM_017526 / LEPROT / leptin receptor overlapping transcript / Unknown
36 / NM_022845 / CBFB / core-binding factor, beta subunit / T cell development
Table 3 suppl: Fold Change D1/D0 according to outcome, expressed in mean and SD