Additional Methods
Neutrophil and PBMC isolation
Freshly obtained EDTA anti-coagulated blood was layered on Ficoll-paque (GE Healthcare Life Sciences, Little Chalfont, United Kingdom) and centrifuged for 10 minutes at 1000 G. The PBMC fraction was aspirated while neutrophils were recovered by performing erythrocyte lysis of the neutrophil+erythrocyte fraction.
Neutrophil and PBMC cell fractions were taken up in Qiagen RNAprotect Cell Reagent (Qiagen, Venlo, The Netherlands) prior to RNA isolation. RNA was isolated using a Qiagen AllPrep kit (Qiagen) according to manufacturer's instructions.
Quantitative PCR
RNA obtained from neutrophils and PBMCs was reverse transcribed using oligo (dT) primer and MLV reverse transcriptase (Promega, Madison, WI) according to the manufacturer’s recommendations. Quantitative PCR (qPCR) was performed using Sensifast (Bioline, Luckenwalde, Germany) on a LightCycler 480 (Roche Applied Science, Penzberg, Germany). Primers are shown in Table S1. qPCR data were analyzed with LinRegPCR (17) and normalized with hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT) and set relative to the median of the healthy controls.
Assessment of TNFα release in whole blood
Heparinized blood was stimulated for 3 hours with LPS 100 ng/ml (Escherichia coli 0111:B4, Invivogen) or medium. TNFα was measured in supernatants by cytometric bead array (BD Biosciences) using a FACSCalibur flow cytometer (BD Biosciences).
Table S1: Primer sequences
Target gene Primer sequence
p38 MAPK Forward GCTGACATAATTCACAGGGACCT
A
Reverse GCCACGTAGCCTGTCATTTC
P65 Forward TTTCTCCTCAATCCGGTGAC
Reverse CTCCTGTGCGTGTCTCCAT
MKP-1 Forward CAACCACAAGGCAGACATCA
Reverse CTTCGCCTCTGCTTCACAA
A20 Forward TCCAGAACACCATTCCGTG
Reverse TGAGGTGCTTTGTGTGGTTC
HPRT Forward GGATTTGAAATTCCAGACAAGTTT
Reverse GCGATGTCAATAGGACTCCAG P38 MAPK: P38 mitogen activated protein kinase
P65: nuclear factor kappa-light-chain-enhancer of activated B cells subunit p65 MKP-1: MAP kinase phosphatase 1
A20: Tumor necrosis factor, alpha-induced protein 3 HPRT: hypoxanthine phosphoribosyltransferase 1
Figure S1: Sepsis patient whole blood produces less TNFα in response to ex vivo stimulation than healthy controls
Blood of 12 sepsis patients (Sepsis) and 5 healthy controls (HC) was treated ex vivo with LPS or vehicle (medium) for 3 hours and TNFα levels were measured in supernatants. * P <0.05
Figure S2: Nuclear location of phosphorylated NFκB in response ex vivo blood stimulation
Healthy controls blood was treated ex vivo with vehicle (A) (medium) or PMA / Ionomycin (B) for 10 minutes and stained for phosphorylated NFkB (p65). Nuclei were visualized by DAPI staining.
Figure S3: gMFI data
Blood of 19 healthy controls (HC, circles) and 19 sepsis patients (Sepsis, squares) was drawn within 24 hours after ICU admission. Blood was stimulated for 10 minutes with 100 ng/ml LPS (black) or 1 μg/ml PMA / 100 ng/ml Ionomycin (grey). Medium controls are show in open symbols. Phosphorylated p38 MAPK (A, depicted as p-p38) and NFκB (B, p-NFκB) levels were determined in CD4 T-cells, CD8 T-cells, B- cells, monocytes and neutrophils. Data are presented as gMFIs. Horizontal lines represent medians.