In order to mimic in vivo systemic inflammation associated with endotoxemia, we used a model in which healthy volunteers are exposed to lipopolysaccharide (LPS), the endotoxin derived from gram negative bacteria 7 . Informed consent was obtained from healthy Caucasian male volunteers for the study, which was approved by the Institutional Review Board of the Academic Medical Center Amsterdam. Inclusion criteria included: no history of sepsis or CVD; not having previously received endotoxin intravenously; non-smokers, no use of any medication and free from any febrile illness in the month preceding the study. In all subjects, a medical history, physical examination, routine laboratory examination, electrocardiogram and chest X-ray were performed. All experiments were performed after an overnight fast.

A bolus of Escherichia coli endotoxin (LPS; catalog number 1235503, lot G2B274; Pharmacopeial Convention, Inc, Rockville, USA; 1 ng/kg) was infused intravenously in healthy volunteers. For controls, an equal amount of endotoxin-free 0.9% NaCl (saline) was infused. Vital signs, including temperature of the study subjects, were monitored at the Intensive Care Unit by a medical doctor who was present throughout the experiments. The incidence, time and severity of clinical symptoms associated with endotoxemia, were recorded. Blood was regularly sampled for clinical chemistry and hematological parameters. IL-6 levels were determined using the Cytometric Bead Array technique (R&D systems, Minneapolis, MN, USA). Whole blood samples in 4% trisodium citrate were obtained at baseline (T = 0), one hour (T = 1) and four hours (T = 4) after LPS infusion.

Whole blood was centrifuged 12 minutes at 300 g and plasma was replaced with an equal volume of PBS buffer containing 1.25% BSA and 2 mM EDTA (PBS/BSA/EDTA). The samples were then layered onto 0.5 volume Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 400 g for 20 minutes. Mononuclear cells were removed and washed twice with PBS/BSA/EDTA buffer. An aliquot of 1 million peripheral mononuclear blood cells were removed for flow cytometric analysis. The remaining cells were used for monocyte isolation using magnetic CD14+ microbeads (Miltenyi Biotech GMBH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Briefly, 20 μl microbeads and 80 μl PBS/BSA/EDTA containing 10⁶ cells were mixed and incubated on ice for 15 minutes. Cells were washed with PBS/BSA/EDTA before being run on an MS column on a varioMACS (Miltenyi Biotech, Cologne, GMBH). Cell purity was assessed on an aliquot of cells by flow cytometry using anti-CD14 (CLB-mon/1, 8G3, Mouse IgG2a, Sanquin Reagents, Amsterdam, The Netherlands). The purified cells were then lysed in RNA Bee (TelTest Inc., Friendswood, Texas, USA) and RNA extracted according to manufacturer's protocol. The concentration of purified RNA was quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA) and quality assessed using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). RNA purity was assessed by Real-Time PCR (RT-PCR) using Taqman transcripts specific for B-cells (CD19/Hs00174333_m1), T-cells (CD3/Hs00167894_m1), erythroid cells (CD235a/Hs00266777_m1), platelets (CD41/HS00166246_m1), neutrophils (CD66a/Hs00174351_m1), monocytes (CD14/Hs00169122_g1) and GAPDH (Hs99999905_m1) as control. A total of 12 ng of RNA was reverse transcribed and amplified using Taqman Transcription Reagents according to the manufacturer's protocol (Applied Biosystems, Foster City, CA, USA). Assays were run on an Mx4000 Multiplex Quantitative PCR System (Stratagene Inc, La Jolla, California, USA). Only those samples with a FACS purity > 80% were processed to ensure reliability of results.

Template Switching Polymerase Chain Reaction (TS-PCR) was used to prepare amplified double stranded (ds) copy DNA (cDNA) following manufacturer's instructions (BD Biosciences Clontech, Palo Alto, CA, USA). Amplifications were performed with either 10 ng (23 cycles) or 100 ng (17 cycles) of starting material, as described previously 8 . Amplified cDNA was subsequently purified using the QIAquick PCR Purification Kit (QIAGEN Inc., Chatsworth, UK) according to manufacturer's protocol with two amendments. Firstly, samples were washed three times in 0.7 mL PE buffer. Secondly, for elution, 50 μL of sterile water was added, the column was left to stand for 2 minutes and then centrifuged. Purified cDNA was quantified using a NanoDrop and fragment length assessed on 1.0% agarose gel. Performance of the amplification was assessed using RT-PCR as above.

Out of the 11 LPS infusion experiments the 5 samples with the highest purity based on FACS and RT-PCR and sufficient yield were chosen for arraying at T = 0 vs. T = 1 and T = 0 vs. T = 4. Similarly, 4 out of the 5 were chosen for the control experiments for the T = 0 vs. T = 1 and 2 of the 5 samples for the T = 0 vs. T = 1. A total of 250 ng of amplified cDNA was labelled by incorporation of Cy3 or Cy5 dCTP using the Bioprime Labelling Kit (Invitrogen Ltd, Paisley, UK). Labelled products were purified on Autoseq G-50 columns (GE Healthcare, Buckinghamshire, United Kingdom) before differential labelling. Biologically paired pre- and post-infusion samples were pooled and hybridized to Hver 2.1.1 cDNA micro-arrays (Wellcome Trust Sanger Institute; WTSI). Hybridization and washing of the arrays were performed according to WTSI protocols. Processed slides were promptly scanned at 10- μm resolution on an Agilent Micro-array Scanner (G2505B; Agilent Technologies, Stockport, United Kingdom). Images were exported into GenePix version 4.1 (Molecular Devices, Sunnyvale, CA, USA) for spotfinding and feature extraction. In accordance with MIAME (Minimum Information About a Micro-array Experiments) regulations, all data were deposited into ArrayExpress database at http://www.ebi.ac.uk 9 10 .

Data was analyzed using R-project version 2.2.0. Features were included if all of the following criteria were met: (i) Manually unflagged in Genepix 6.0 (Axon Instruments, Foster City, CA, USA); (ii) (Δ median-mean)/median intensity difference < 0.1;

(iii) Saturation < 20%; (iv) Mean/standard deviation (SD) > 2; (v) Intensities of background (BG) + 1SD > 90% and G + 2 SD > 80%. In addition, the feature had to be either up or down regulated in at least 50% of the arrays, in order to pass the inclusion threshold. LPS and control samples were analysed separately in a linear mixed effects model with corrections for false discovery rates (FDR) As cut-off we selected a FDR < 0.005, a posterior probability of differential expression > 0.95. The foldchanges had to be greater than two and present in at least 70% of the arrays.

RT-PCR was used to confirm the observations from the comparative microarray study in the 6 LPS samples not used for Microarray for the following transcripts chosen at random: SLA1 (Hs00153504_m1); BATF (Hs00232390_m1); C3aR1 (Hs00377780_m1); AKIP (Hs00610917_g1); ILRN (Hs00174099_m1); TIMP1 (Hs00171558_m1); VCAN (Hs00171642_m1).

RNA (100 ng) was reverse transcribed using the TaqMan RT kit (Applied Biosystems), from which 0.5 ng cDNA was then used as template for RT-PCR following the manufacturer's instructions (Applied Biosystems). Reactions were incubated at 50°C for 2 minutes then 95°C for 10 minutes, and RT-PCR reactions were performed over 40 cycles (95°C for 15 seconds, 60°C for 1 minute) on an MX-4000 (Stratagene, La Jolla, CA). Threshold values (Ct) were normalized to GAPDH to allow comparison between samples.

In total, 16 healthy men with an average age of 23 ± 1 years (mean ± SD) meeting the inclusion criteria participated; 11 for LPS infusion and five as controls. Medical history, physical examination, routine laboratory examination, electrocardiogram and chest X-ray were normal. All subjects receiving LPS experienced a clinical response consisting of nausea, malaise, chills, muscle ache and fever. Plasma/Serum IL-6 increased significantly (Figure 1). No such effects were observed in the control group.

The monocyte count differed significantly between LPS and control experiments at the first hour (p < 0.05) (Figure 2). In the LPS infusion experiments, monocyte count dropped 55% in the first hour followed by a slow increase towards normal at four hours. In the control experiments, monocyte count varied slightly but did not decrease as observed after LPS infusion (Figure 2). Summary data for all samples and those included in the study is shown in additional file 2 and 3; supplementary table 1 and 2. RT-PCR performed pre- and post-amplification demonstrated minimal contamination of selected RNA samples by other cell types and non-biased amplification (Data not shown).

For the T = 0 vs. T = 1 experiments, 10 arrays were hybridised with LPS-stimulated samples and 8 with control samples. On average 19% of spots passed the stringent quality control criteria. One hour after LPS infusion (T = 1), 11 genes with a known function were differentially expressed, of which, 1 was down regulated and 10 unregulated (Table 1 and Figure 3) with fold-changes ranging from 2.0 to 5.8.

For the T = 0 vs. T = 4 experiments, 10 arrays were hybridised with LPS-stimulated samples and 4 arrays with control samples. On average 26% of spots passed the stringent quality control criteria. In total 28 genes were identified as being differentially expressed in monocytes following LPS stimulation, of which 3 were down regulated and 25 up regulated (see Table 2 and Figure 3). The fold changes ranged from 2.0 to 5.7. Interestingly, AKIP was found to be differentially expressed at both one and four hours after LPS infusion.

We did not identify any genes that were significantly differentially expressed in the control experiments at either T = 1 or T = 4 confirming that the differential expression observed, whilst limited, is associated with LPS infusion.

Using quantitative RT-PCR, we were able to confirm the differential expression of 7 of 8 genes tested (Figure 3). Validation was performed on RNA from individuals who had not been part of the subset analysed by microarray, thus highlighting that the changes observed are universal. Absolute fold changes varied between the micro-array and Taqman results, however in all cases, except for AKIP (both at T = 1 and T = 4), the direction of differential expression was conserved.