Monocyte mRNA Phenotype and Adverse Outcomes From Pediatric Multiple
Organ Dysfunction Syndrome
Mark W. Hall MD, Mikhail A. Gavrilin PhD, Nina L. Knatz, Michelle D. Duncan,
Soledad A. Fernandez PhD, Mark D. Wewers MD
Online Data Supplement
Supplementary Methods
Monocyte isolation and mRNA quantification
Blood was drawn in Vacutainer® tubes (Becton Dickinson, Franklin Lakes, NJ)
containing EDTA and transported to the laboratory where processing began within 30
minutes of collection.
Monocytes were isolated from whole blood by density gradient centrifugation
using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) followed by anti-CD14 magnetic
bead isolation (Miltenyi Biotec, Auburn, CA). This procedure yielded monocyte cell
populations that were > 98% pure by flow cytometry. Monocytes were lysed using
Trizol reagent (Invitrogen, Carlsbad, CA) and frozen at -80ºC for batch RNA analysis.
RNA was extracted from the monocyte lysates and 1-2 g of total RNA was
reverse transcribed to cDNA by ThermoScript RNase H- Reverse Transcriptase
(Invitrogen, Carlsbad, CA) using Oligo(dT) (Invitrogen). Quantitative PCR with SYBR
Green I PCR Master Mix was then performed in the ABI PRIZM 7700 (both, Applied
Biosystems, Warrington, UK). We used relative quantification to evaluate the expression
of 28 selected genes representing both pro- and anti-inflammatory cytokines, TLR/NFκB
signaling pathway members, and elements of the inflammasome complex (see Table 1).
All gene-specific primers were designed with similar criteria: to give an 80-200
nucleotide amplicon, GC range 30-70%, Tm 59-60C, free of secondary structure and
delta G not to exceed –7 kcal/mole. Once selected, primer pairs were validated by PCR
and high resolution gel electrophoresis to have a single band of desired size, free of
primer dimers. In the event of limitation in RNA quantity from a given sample, genes
from the panel were analyzed in chronological order of primer development until the
sample was exhausted.
Relative copy numbers (RCN) of selected genes were normalized to the
expression of a group of housekeeping genes including: GAPDH, CAP-1 (cyclic AMPaccessory protein), and 18S ribosomal RNA. RCN was calculated according to the
equation: RCN=E-Ct x 100, where E = efficiency of PCR, Ct = cycle threshold, and Ct
= Ct target – Ct reference (average of housekeeping genes). PCR efficiency was
calculated by the equation: E=10(-1/slope). Based on our preliminary determination for this
reaction that E(target) = E(reference)  2, we substituted 2 for E in the equation,
representing an efficiency of nearly 100% for all selected genes in the PCR reaction. An
RCN of 100, therefore, represents target gene expression which is the same as the
average expression for the housekeeping genes.
Supplementary Data
Inflammasome
Toll-like receptor / NFκB signaling
Cytokines
Table E1. Monocyte mRNA expression profiles from four control patients in the PICU
with single or no organ failure.
mRNA
product
TNF
Coding for
TNFα
Relative Copy Number
median (IQR)
0.9 (0.52 – 1.9)
IL1B
IL-1β
15 (6.2 – 57)
IL6
IL-6
0.002 (0.001 – 0.009)
IL8
IL-8
2.6 (1.7 – 4.2)
IL10
IL-10*
0.53 (0.4 – 1.3)
IL18
IL-18
0.83 (0.17 – 1.5)
IL1RN
IL-1ra*
0.56 (0.24 – 0.82)
TLR4
TLR4
6.0 (4.6 – 7.1)
TLR2
TLR2
22 (11 – 33)
CD14
CD14
125 (108 – 140)
IRAK1
IRAK1
5.9 (5.1 – 7.6)
IRAK2
IRAK2*
0.13 (0.10 – 0.55)
IRAK3
IRAK-M*
3.1 (2.7 – 3.2)
NFKBIA
IκBα*
29 (18 – 40)
NFKBIZ
MAIL
4.0 (2.8 – 5.3)
NFKB1
NFκBp50
2.4 (1.3 – 3.5)
RELA
NFκBp65
1.8 (1.1 – 2.0)
NALP1
NALP1
4.05
CIAS1
NALP3
3.9 (2.5 – 5.7)
NALP6
NALP6
0.02 (0.018 – 0.021)
NALP12
NALP12
4.3 (3.8 – 4.6)
CARD4
NOD1
0.09 (0.07 – 0.12)
CARD15
NOD2
1.4 (1.2 – 1.5)
CARD12
IPAF
8.8 (7.2 – 11)
CASP1
Caspase 1
27 (25 – 29)
CASP5
Caspase 5
19 (13 – 25)
PYCARD
ASC
15 (12 – 16)
MEFV
Pyrin*
9.9 (7.6 – 13)
n = 4 for all genes. *Known or suspected anti-inflammatory mediator
Table E2. Monocyte mRNA expression in the first week of MODS in nonsurvivors and
survivors.
Inflammasome
Toll-like receptor / NFκB signaling
Cytokines
mRNA
product
TNF
Coding for
Relative Copy Number – median (IQR)
n
Nonsurvivors
Survivors
n
p value
TNFα
0.13 (0.06 – 0.47)
7
0.3 (0.12 – 0.82)
18
0.45
IL1B
IL-1β
2.8 (0.490 – 2.9)
7
3.2 (2.1 – 8.0)
19
0.13
IL6
IL-6
0.005 (0.003 -0.006)
6
0.006 (0.001 – 0.018)
13
0.82
IL8
IL-8
3.1 (1.0 – 16)
6
3.9 (0.92 – 55)
9
0.86
IL10
IL-10*
2.2 (0.4 – 3.3)
7
0.2 (0.04 – 1.0)
18
0.02
IL18
IL-18
0.26 (0.20 – 0.39)
3
0.23 (0.02 – 0.37)
13
0.57
TLR4
TLR4
3.5 (1.7 – 15)
7
3.0 (1.2 – 8.2)
8
0.69
TLR2
TLR2
11.6 (3.0 – 26)
6
3.8 (2.1 – 11)
7
0.37
CD14
CD14
73 (55 – 166)
5
196 (100 – 334)
16
0.15
IRAK1
IRAK1
3.9 (2.1 – 7.1)
4
1.9 (0.93 – 2.6)
11
0.33
IRAK2
IRAK2*
0.15 (0.02 – 0.40)
6
0.13 (0.06 – 0.6)
19
0.75
IRAK3
IRAK-M*
5.4 (0.66 – 32)
6
0.67 (0.07 – 4.3)
19
0.13
NFKBIA
IκBα*
12 (9.4 – 46)
7
34 (19 – 66)
18
0.1
NFKBIZ
MAIL
5.0 (1.0 – 7.4)
5
4.3 (2.9 – 10)
16
0.77
NFKB1
NFκBp50
1.2 (0.92 – 1.7)
7
0.63 (0.14 – 1.3)
19
0.34
RELA
NFκBp65
0.43 (0.28 – 0.63)
7
0.37 (0.22 – 0.84)
15
0.91
NALP1
NALP1
16 (7.4 – 120)
3
3.0 (1.5 – 9.1)
16
0.05
CIAS1
NALP3
3.2 (0.68 – 19)
6
7.7 (2.7 – 11)
18
0.71
NALP6
NALP6
0.12 (0.01 – 0.97)
6
0.04 (0.004 – 0.1)
15
0.51
NALP12
NALP12
4.6 (1.3 – 21)
6
8.1 (3.0 – 25)
18
0.41
CARD15
NOD2
1.9 (1.5 – 4.9)
3
0.77 (0.40 – 1.9)
16
0.16
CARD12
IPAF
16 (7.6 – 16)
3
3.9 (2.2 – 12)
17
0.09
CASP1
Caspase 1
9.7 (4.4 – 27)
5
6.1 (2.4 – 30)
18
0.53
CASP5
Caspase 5
25 (25 – 27)
3
23 (12 – 36)
17
0.6
PYCARD
ASC
14 (2.5 – 31)
6
10 (4.6 – 23)
18
0.82
MEFV
Pyrin*
9.1 (8.6 – 12)
4
1.4 (0.2 – 2.9)**
15
0.008
Only gene products with at least three nonsurvivor values are shown. *Known or suspected antiinflammatory mediator; **p < 0.05 vs control.
Table E3. Monocyte mRNA expression in the second week of MODS in nonsurvivors
and survivors.
Inflammasome
Toll-like receptor / NFκB signaling
Cytokines
mRNA
product
Relative Copy Number – median (IQR)
n
n
Nonsurvivors
Survivors
TNF
Coding for
TNFα
0.38 (0.04 – 0.9)
5
0.57 (0.11 – 3.3)
10
p
value
0.44
IL1B
IL-1β
5.1 (2.9 – 33)
5
7.6 (1.4 – 52)
10
0.99
IL6
IL-6
0.008 (0.001 – 0.058)
5
0.033 (0.009 – 5.9)
6
0.33
IL8
IL-8
12 (6.4 – 23)
4
4.7 (0.34 – 138)
5
0.9
IL10
IL-10*
5.0 (1.6 – 6.8)
5
1.2 (0.16 – 2.3)
10
0.13
IL18
IL-18
0.75 (0.32 – 1.28)
4
0.34 (0.10 – 1.9)
8
0.78
IL1RN
IL-1ra*
1.3 (0.46 – 2.4)
3
1.2 (0.13 – 29)
5
0.99
TLR4
TLR4
7.4 (4.5 – 12)
4
7.4 (3.5 – 8.3)
3
0.86
TLR2
TLR2
13 (7.0 – 30)
4
20 (4.5 – 36)
4
0.89
CD14
CD14
113 (52 – 233)
6
153 (105 – 529)
10
0.51
IRAK1
IRAK1
6.3 (5.4 – 13)
3
5.6 (2.9 – 27)
5
0.99
IRAK2
IRAK2*
0.10 (0.05 – 0.31)
4
0.38 (0.06 – 1.9)
9
0.41
IRAK3
IRAK-M*
5.3 (3.2 – 7.4)
4
1.0 (0.28 – 9.4)
10
0.3
NFKBIA
IκBα*
26 (13 – 75)
5
28 (15 – 163)
10
0.77
NFKBIZ
MAIL
2.3 (1.5 – 9.9)
3
3.8 (2.0 – 7.9)
9
0.86
NFKB1
NFκBp50
1.4 (0.73 – 2.4)
5
0.26 (1.3 – 4.8)
10
0.95
RELA
NFκBp65
0.68 (0.61 – 0.79)
4
0.31 (0.15 – 2.9)
9
0.41
CIAS1
NALP3
3.0 (2.1 – 3.6)
4
12 (5.6 – 17)
10
0.08
NALP6
NALP6
0.09 (0.04 – 0.17)
4
0.05 (0.02 – 0.10)
9
0.41
NALP12
NALP12
3.8 (2.5 – 5.9)
4
11 (3.2 – 22)
10
0.14
CASP1
Caspase 1
16 (11 – 64)
3
7.3 (3.8 – 22)
9
0.21
PYCARD
ASC
2.9 (2.9 – 21)
3
13 (4.1 – 16)
9
0.6
MEFV
Pyrin*
15 (11 – 16)
4
3.6 (1.8 – 7.9)
9
0.03
Only gene products with at least three nonsurvivor values are shown. *Known or suspected antiinflammatory mediator
Table E4. Monocyte mRNA expression on Day 3 of MODS in nonsurvivors and
survivors.
Inflammasome
Toll-like receptor / NFκB signaling
Cytokines
Relative Copy Number – median (IQR)
mRNA
product
TNF
Coding for:
TNFα
Nonsurvivors
0.16 (0.1 – 1.1)
n
4
Survivors
0.27 (0.09 – 1.2)
n
14
p value
0.71
IL1B
IL-1β
1. 2 (0.88 – 2.01)
3
2.5 (1.1 – 7.7)
15
0.29
IL6
IL-6
0.004 (0.003 – 0.005)
3
0.002 (0.0009 – 0.022)
10
0.64
IL8
IL-8
5.2 (3.8 – 7.8)
3
4.2 (0.22 – 72.2)
6
0.90
IL10
IL-10*
2.4 (1.5 – 3.1)
4
0.08 (0.02 – 0.94)
14
0.005
IL18
IL-18
0.2 (0.2 – 0.27)
3
0.2 (0.006 – 0.4)
10
0.69
TLR4
TLR4
4.4 (2.6 – 10.5)
4
1.4 (0.5 – 3.8)
12
0.06
TLR2
TLR2
14.1 (13.5 – 19.1)
3
2.1 (1.3 – 5.9)
4
0.06
CD14
CD14
72.7 (71.3 – 121)
4
130 (53.8 – 314)
12
0.60
IRAK1
IRAK1
3.0 (2.7 – 4.1)
3
1.5 (1.0 – 2.1)
8
0.05
IRAK2
IRAK2*
0.05 (0.02 – 0.13)
4
0.09 (0.04 – 0.2)
15
0.26
IRAK3
IRAK-M*
3.5 (1.3 – 7.6)
4
0.7 (0.1 – 4.0)
15
0.30
NFKBIA
IκBα*
14.9 (12.2 – 35)
4
32.7 (15.9 – 70.5)
14
0.33
NFKBIZ
MAIL
5.2 (1.9 – 7.4)
4
5.3 (1.5 – 10.3)
12
0.86
NFKB1
NFκBp50
0.7 (0.5 – 1.3)
4
0.58 (0.17 – 1.0)
15
0.53
RELA
NFκBp65
0.5 (0.3 – 0.8)
4
0.38 (0.11 – 0.8)
10
0.78
NALP1
NALP1
16 (11.7 – 68)
3
2.3 (1.2 – 10.4)
13
0.06
CIAS1
NALP3
2.1 (0.77 – 8.8)
4
3.6 (1.8 – 8.2)
14
0.57
NALP6
NALP6
0.135 (0.02 – 0.99)
4
0.02 (0.003 – 0.04)
11
0.17
NALP12
NALP12
2.7 (2.0 – 14.9)
4
5.7 (2.3 – 21.1)
14
0.44
CARD15
NOD2
1.9 (1.7 – 3.4)
3
0.8 (0.4 – 2.3)
11
0.18
CARD12
IPAF
15.5 (11.6 – 20)
3
3.9 (1.9 – 12.8)
13
0.11
CASP1
Caspase 1
12.6 (6.1 – 16.7)
4
5.0 (3.3 – 21.1)
14
0.80
CASP5
Caspase 5
25.3 (25.3 – 26.5)
3
19.5 (9.7 – 33.3)
13
0.35
PYCARD
ASC
11.6 (7.5 – 17.5)
4
9.9 (4.8 – 33.3)
14
0.99
MEFV
Pyrin*
8.6 (5.9 – 12.4)
4
0.59 (0.15 – 2.4)
11
0.003
Only gene products with at least three nonsurvivor values are shown. *Known or suspected antiinflammatory mediator
Table E5. Monocyte mRNA expression in nonsurvivors and survivors of pediatric
MODS from samples taken ≤ 72 hours before death (nonsurvivors) or ICU discharge
(survivors).
Inflammasome
Toll-like receptor / NFκB signaling
Cytokines
Relative Copy Number – median (IQR)
mRNA
product
TNF
Coding for
TNFα
Nonsurvivors
0.16 (0.06 – 1.1)
n
8
Survivors
0.19 (0.11 – 1.2)
n
18
p value
0.61
IL1B
IL-1β
3.5 (1.9 – 7.3)
8
3.3 (1.3 – 10.1)
18
0.99
IL6
IL-6
0.004 (0.001 – 0.006)
7
0.003 (0.0005 – 0.02)
11
0.93
IL8
IL-8
29.9 (4.9 – 75.5)
6
3.6 (0.24 – 39.4)
8
0.28
IL10
IL-10*
1.2 (0.4 – 5.4)
8
0.2 (0.03 – 0.87)
18
0.02
IL18
IL-18
0.37 (0.27 – 0.9)
4
0.22 (0.02 – 0.47)
11
0.28
IL1RN
IL-1ra*
2.2 (0.25 – 2.4)
5
0.06 (0.02 – 28.3)
6
0.23
TLR4
TLR4
7.2 (0.6 – 8.8)
7
1.1 (0.5 – 3.3)
13
0.09
TLR2
TLR2
12.6 (2.5 – 30.6)
6
2.1 (1.4 – 25.4)
6
0.18
CD14
CD14
117 (80.1 – 216)
7
153 (85 – 474)
15
0.53
IRAK1
IRAK1
5.4 (3.9 – 6.8)
4
2.1 (1.3 – 4.8)
8
0.21
IRAK2
IRAK2*
0.19 (0.02 – 1.1)
7
0.1 (0.05 – 0.19)
16
0.92
IRAK3
IRAK-M*
6.1 (3.4 – 9.6)
7
0.34 (0.08 – 2.4)
16
0.001**
NFKBIA
IκBα*
35.9 (12.8 – 98.8)
8
28.7 (16.3 – 39.1)
18
0.76
NFKBIZ
MAIL
7.5 (1.6 – 12.1)
5
3.4 (1.2 – 6.9)
14
0.39
NFKB1
NFκBp50
1.6 (0.7 – 2.2)
8
0.62 (0.16 – 2.8)
18
0.34
RELA
NFκBp65
0.7 (0.5 – 1.3)
7
0.26 (0.16 – 0.7)
13
0.18
NALP1
NALP1
26.2 (8.1 – 64.6)
5
4.5 (1.4 – 22.4)
14
0.03
CIAS1
NALP3
3.4 (2.6 – 28.7)
7
10 (2.5 – 14.5)
16
0.99
NALP6
NALP6
0.12 (0.02 – 2.2)
6
0.02 (0.007 – 0.04)
13
0.06
NALP12
NALP12
3.41 (2.1 – 13.6)
7
9.8 (2.9 – 32.3)
16
0.45
CARD4
NOD1
0.03 (0.03 – 0.12)
4
0.06 (0.01 – 0.14)
7
0.92
CARD15
NOD2
1.9 (1.7 – 10.4)
5
1 (0.23 – 8.2)
14
0.19
CARD12
IPAF
21.3 (8.8 – 22.9)
5
3.9 (2.3 – 11.7)
15
0.05
CASP1
Caspase 1
11 (3.6 – 32.5)
5
6.1 (2.6 – 9.8)
16
0.45
CASP5
Caspase 5
44.8 (18.3 – 76.7)
6
29.2 (10.7 – 39.6)
14
0.13
PYCARD
ASC
19.1 (1.7 – 21.6)
5
9.7 (4.9 – 16.2)
16
0.90
MEFV
Pyrin*
15.3 (14.2 – 56.8)
6
1.9 (0.33 – 5.1)
14
0.0006**
Only gene products with at least three nonsurvivor values are shown. *Known or suspected anti-inflammatory
mediator; **withstands Bonferroni correction for multiple comparisons