Electronic supplementary material
INCREASED MERTK EXPRESSION ON CIRCULATING INNATE IMMUNE CELLS IN SEPTIC
SHOCK PATIENTS
Caroline Guignant PharmD PhD*, Fabienne Venet PharmD PhD*, Séverine Planel PharmD
PhD, Julie Demaret MSc, Aurélie Gouel-Chéron MD, Christophe Nougier PharmD, Arnaud
Friggeri MD PhD, Bernard Allaouchiche MD PhD, Alain Lepape MD, Guillaume Monneret
PharmD PhD.
* These 2 authors contributed equally to this work.
Patients, Materials and Methods
Messenger RNA measurements
Messenger RNA (mRNA) extraction was performed on 300 µl of EDTA-whole blood sample
after lysis of red blood cells with Versalyse solution (Beckman-Coulter). After PBS washing,
leukocyte pellet was incubated with beta-mercaptoethanol and RLT buffer (from RNeasy
Mini kit Qiagen, Hilden, Germany) and put at -20°C prior to mRNA extraction. Then, total
RNA was extracted using the RNeasy Mini kit (Qiagen) and the residual genomic DNA was
digested using the RNase- Free DNase set (Qiagen).
Total RNA was reverse transcribed into complementary DNA using the SuperScript Vilo cDNA
synthesis kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. mRNA
expression was quantified using quantitative real-time PCR. Briefly, PCR reactions were
performed using a LightCycler 480 instrument with the LightCycler 480 Probes Master kit
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(Roche Molecular Biochemicals, Basel, Switzerland) according to the manufacturer’s
instructions. Thermocycling was performed in a final volume of 20 µL containing 0.9 µM of
each of the required primers and 0.25 µM of FAM dye-labeled MGB probe. PCR was
performed with an initial denaturation step of 10 min at 95°C followed by 45 cycles of a
touch-down PCR protocol (10 sec at 95°C, 29 sec annealing at 68–58°C, 1 sec extension at
72°C, and 10 sec cooling at 40°C). mRNA expression of MERTK was investigated using specific
cDNA standard and ready-to-use primers-probe mix (reference : Hs00179024, from Applied
Biosystem). The cDNA standard for MERTK was prepared from purified PCR amplicons
obtained from a pool of cDNA (LC Search).
The LightCycler 480 was used to determine the crossing point for individual samples.
Relative standard curves describing the PCR efficiency of the target gene and the
housekeeping gene peptidylpropyl isomerase B (PPIB, encoding for cyclophilin B) were
created and used to perform efficiency-corrected quantification with the LightCycler
software version 4. The results were expressed as a number-of-copy-concentration ratio of
target mRNA/PPIB mRNA.
Plasma protein concentration measurements
Plasma concentrations of total protein S were measured using an ELISA test kit (Asserachrom
protein S kit, Stago, Asnieres, France). In brief, for the measurement of total protein S
concentration, a 200 µL aliquot of citrated sample plasma diluted 1: 100 was added to antiprotein S antibody-precoated wells. The second monoclonal anti-protein S antibody that is
coupled with peroxidase was added at the same time as the plasma and incubated 1h at
room temperature. Following the washing peroxidase activity was measured at 450 nm. The
concentrations of total protein S were read from the standard curve and expressed as
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percentage against standard plasma for total protein S, which was regarded as 100% in
concentration.
Plasmatic concentrations of IL-6, IL-10 and TNFR2 were measured by multiplex ELISA
(Luminex technology) using commercial kit in accordance with manufacturer’s
recommendations (Life Technologies, Paisley, UK).
Supplementary Table 1. Clinical characteristics of survivors and non-survivors among septic
shock patients sampled both at D1-D2 and D3-D4.
Values are presented as median and interquartile ranges [IQR] in brackets for continuous
variables or as number of cases and percentages for categorical data.
SAPS II: Simplified Acute Physiology Score II, SOFA: Sepsis-related organ failure assessment
Total Population (n = 37)
Survivors (n = 22)
Non-survivors (n = 15)
Age at admission (years)
68 [60 – 77]
67 [58 – 75]
69 [65 – 78]
Gender - Male, n (%)
22 (59 %)
14 (64 %)
8 (53 %)
SAPS II at diagnosis of
67 [54 – 72]
64 [52 -72]
68 [63 – 73]
Medical, n (%)
22 (59 %)
12 (55 %)
10 (67 %)
Surgery + trauma, n (%)
15 (41%)
10 (45 %)
5 (33 %)
0, n (%)
8 (22 %)
6 (27 %)
2 (13 %)
≥ 1, n (%)
29 (78 %)
16 (73 %)
13 (87 %)
Charlson score
2 [1 - 3]
2 [0 – 3]
2 [2 – 4]
SOFA score at diagnostic
11 [9 – 12]
10 [9 – 12]
11 [9 – 14]
Bacilli gram -
13 (35 %)
8 (36 %)
5 (33 %)
Cocci gram +
12 (32 %)
8 (36 %)
4 (27 %)
Anaerobic bacteria
1 (3 %)
0 (0 %)
1 (67 %)
Fungi
7 (19 %)
4 (18 %)
3 (20 %)
shock
Main admission category
Number of comorbidities
of shock
Microbiologically
documented infections
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Supplementary Figures
Supplementary Figure 1. Flow cytometry gating strategy and representative examples in
one healthy donor, one trauma and one septic shock patient.
After red blood cell lysis, total lymphocytes (pink gate), monocytes (red gate) or neutrophils
(green gate) were specifically identified among circulating leukocytes based on respective
forward and side scatters characteristics (upper left dot-plot) as well as CD14 expression
(positive on monocytes, negative on lymphocytes and neutrophils – upper right dot-plot).
MerTK expression (mean of fluorescence intensity - MFI) was then measured on each cell
population. Lower left overlay represents MerTK expression on monocytes from one isotype
control staining, one healthy donor, one trauma patient and one septic shock patient. Lower
right overlay represents MerTK expression on neutrophils from one isotype control staining,
one healthy donor, one trauma patient and one septic shock patient.
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Supplementary Figure 2: AXL expression on circulating immune cells in septic shock
patients, trauma and healthy volunteers.
AXL expression was measured on circulating neutrophils, lymphocytes and monocytes in
whole blood from septic shock patients at day 1-2 after the onset of shock (Se, n = 47, dark
grey boxes), from severe trauma at D1-2 after admission (Tr, n= 51, light grey boxes) and
from healthy volunteers (HV, n = 23, open boxes). After red blood cell lysis, total
lymphocytes, monocytes or neutrophils were specifically identified among circulating
leukocytes based on respective forward and site scatters characteristics as well as CD14
expression (positive on monocytes, negative on lymphocytes and neutrophils) by flow
cytometry. Results are presented as box-plots of mean of fluorescence intensity (MFI).
Kruskal-Wallis test was performed with subsequent Dunn’s multiple comparison tests if
appropriate. * p<0.05, ** p<0.01, *** p<0.001.
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Supplementary Figure 3: TYRO-3 expression on circulating immune cells in septic shock
patients, trauma and healthy volunteers.
TYRO-3 expression was measured on circulating neutrophils, lymphocytes and monocytes in
whole blood from septic shock patients at day 1-2 after the onset of shock (Se, n = 47, dark
grey boxes), from severe trauma at D1-2 after admission (Tr, n= 51, light grey boxes) and
from healthy volunteers (HV, n = 23, open boxes). After red blood cell lysis, total
lymphocytes, monocytes or neutrophils were specifically identified among circulating
leukocytes based on respective forward and site scatters characteristics as well as CD14
expression (positive on monocytes, negative on lymphocytes and neutrophils) by flow
cytometry. Results are presented as box-plots of mean of fluorescence intensity (MFI).
Kruskal-Wallis test was performed with subsequent Dunn’s multiple comparison tests if
appropriate. * p<0.05, ** p<0.01, *** p<0.001.
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Supplementary Figure 4: Overtime evolution of MerTK on circulating neutrophils from
septic shock patients and relation with deleterious outcome.
MerTK expression was measured on circulating neutrophils from septic shock patients
sampled at D1-2 and D3-4 (n=37) after admission. After red blood cell lysis, neutrophils were
selected among leukocytes based on their side scatter characteristics and lack of CD14
expression by flow cytometry. Overtime evolution was analyzed in survivors (n = 22) vs nonsurvivors (n = 15) and in patients that developed (n = 9) or not (n = 28) a nosocomial episode.
Wilcoxon paired test and Mann-Whitney U test were performed to respectively assess the
overtime evolution within a group or the difference between groups at a given time-point.
Supplementary Figure 5: Protein S, pro and anti-inflammatory mediators’ concentrations
in plasma.
Concentrations of IL-6, IL-10, TNFR2 as well as protein S (antigen) were evaluated in the
plasma from 10 healthy volunteers (HV), 10 trauma patients (trauma) and 10 septic shock
patients (septic shock) that were used in the ex vivo experiments. Individual values are
presented as well as concentration measured in each respective pooled aliquot (red signs).
Values are presented as concentrations in pg/ml for cytokines and as percentage of normal
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values for protein S. Non parametric Mann Whitney test was used to compare differences
between groups.
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