The Journal of Immunology
A Single Nucleotide Polymorphism in NF-kB Inducing Kinase
Is Associated with Mortality in Septic Shock
Simone A. Thair,* Keith R. Walley,* Taka-aki Nakada,* Melissa K. McConechy,†
John H. Boyd,* Hugh Wellman,‡and James A. Russell*
We tested the hypothesis that single nucleotide polymorphisms (SNPs) within genes of the NF-kB pathway are associated with
altered clinical outcome of septic shock patients. We genotyped 59 SNPs in the NF-kB pathway in a discovery cohort of septic
shock patients (St. Paul’s Hospital [SPH], N = 589), which identified the C allele of rs7222094 T/C within MAP3K14 (NF-kB
inducing kinase; NIK) associated with increased 28-d mortality (uncorrected p = 0.00024, Bonferroni corrected p = 0.014). This
result was replicated in a second cohort of septic shock patients (Vasopressin and Septic Shock Trial [VASST; N = 616]) in which
the CC genotype of rs7222094 was associated with increased 28-d mortality (Cox regression: SPH cohort hazard ratio [HR], 1.35;
95% confidence interval [CI], 1.12–1.64; p = 0.002 Caucasian only; and VASST cohort HR, 1.24; 95% CI, 1.00–1.52; p = 0.048
Caucasian only). Patients having the CC genotype of rs7222094 in SPH experienced more renal and hematological dysfunction
(p = 0.003 and p = 0.011), while patients of the VASST cohort with the rs7222094 CC genotype showed the same trend toward
more renal dysfunction. In lymphoblastoid cell lines, we found the rs7222094 genotype most strongly associated with mRNA
expression of CXCL10, a chemokine regulated by NF-kB. Accordingly, we measured CXCL10 protein levels and found that the
CC genotype of rs7222094 was associated with significantly lower levels than those of the TT genotype in lymphoblastoid cell lines
(p , 0.05) and in septic shock patients (p = 0.017). This suggests that the CC genotype of NIK rs7222094 is associated with
increased mortality and organ dysfunction in septic shock patients, perhaps due to altered regulation of NF-kB pathway genes,
including CXCL10.The Journal of Immunology, 2011, 186: 2321–2328.
pathways involved in the pathogenesis of this pathological state
(1). Septic shock is defined as sepsis accompanied by organ failure
including cardiovascular failure (2). In response to infection,
NF-kB signaling (among other pathways) leads to the transcription
of a wealth of inflammatory mediators that contribute to the de-
velopment of cardiovascular failure, including cytokines, chemo-
kines, adhesion molecules, and reactive oxygen and nitrogen
species, to name a few (3, 4). The induction of this response is
necessary for the resolution of infection; however, this response,
when extreme, leads to organ damage and mortality in many cases.
eptic shock is an extreme manifestation of the host in-
flammatory response to severe infection, and the NF-kB
signaling pathway is one of the most important signaling
A better understanding of the key pathways and critical molecules
involved in the pathogenesis of septic shock is imperative.
NF-kB signaling is activated by a canonical (or classical)
pathway and by a noncanonical (or alternative) pathway involving
multiple genes (Supplemental Fig. 1) (5). Briefly, the canonical
NF-kB pathway requires the IkB kinase (IKK) complex composed
of IKKa/b/g (3). Activation of the IKK complex in response
to inflammatory stimuli results in phosphorylation and ubiquitin-
dependent degradation of IkBa or IkBb and translocation of p50-
related dimers into the nucleus (3). In response to inflammation
in the noncanonical pathway, NF-kB inducing kinase (NIK), a
docking molecule, recruits IKKa to p100 and then activates IKKa
(3, 6). IKKa phosphorylates p100, which is subjected to phos-
phorylation, ubiquitination, and proteosomal degradation, result-
ing in the release and nuclear translocation of p52 containing RelB
heterodimers (3, 6).
Genetic variation in key inflammatory genes contributes to
outcome in sepsis (7–12). Because NF-kB is a centrally important
signaling pathway, we tested the hypothesis that genetic variation
in genes involved in the NF-kB pathways would be associated
with mortality in septic shock. To accomplish this, we genotyped
59 single nucleotide polymorphisms (SNPs) in 19 genes in a sin-
gle center derivation cohort of septic shock patients. We next
tested for replication of an arising SNP association in a second
multicenter cohort of septic shock patients. We tested for similar
association with organ dysfunction to understand the involved
clinical phenotype in more detail. To test for biological plausi-
bility of the observed SNP association, we next measured gene
expression in genotyped lymphoblastoid cell lines and identified
a candidate gene whose expression was associated with the can-
didate SNP. We then tested for association of this gene product
with the candidate SNP both in vitro and in vivo.
*University of British Columbia Critical Care Research Laboratories, Heart and
Lung Institute, St. Paul’s Hospital, Vancouver, British Columbia V6Z 1Y6, Canada;
†Department of Pathology and Laboratory Medicine, University of British Columbia,
British Columbia Cancer Agency, Vancouver, British Columbia V5Z 4E6, Canada;
and‡Sirius Genomics Inc., Vancouver, British Columbia V6Z 2K8, Canada
Received for publication September 2, 2010. Accepted for publication December 7,
S.A.T. is the recipient of a Mathematics of Information Technology and Complex
Systems fellowship. J.H.B. is the recipient of a Providence Health Care Research
scholarship. T.-a.N. is the recipient of a Integrated and Mentored Pulmonary and
Cardiovascular Training fellowship. K.R.W. is a Michael Smith Foundation for
Health Research Distinguished Scholar. The Vasopressin and Septic Shock Trial is
supported by Canadian Institutes of Health Research Grant MCT 44152.
Address correspondence and reprint requests to James A. Russell, Heart and Lung
Institute, St. Paul’s Hospital, #166-1081 Burrard Street, Vancouver, British Colum-
bia, Canada, V6Z 1Y6. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this article: CI, confidence interval; HR, hazard ratio; IKK, IkB
kinase; NIK, NF-kB inducing kinase; SNP, single nucleotide polymorphism; SPH, St.
Paul’s Hospital; VASST, Vasopressin and Septic Shock Trial.
Materials and Methods
St. Paul’s Hospital cohort (discovery cohort). All patients admitted to the
St. Paul’s Hospital (SPH) Intensive Care Unit (Vancouver, British Colum-
bia, Canada) between July 2000 and January 2004 were screened. Using the
current consensus definition ($ 2 SIRS criteria, known or suspected in-
fection, hypotension unresponsive to fluid resuscitation alone) 601 patients
had septic shock on admission and had DNA available (12, 13). Twelve
patients in this cohort had also been enrolled in the Vasopressin and Septic
Shock clinical trial (14) and were therefore excluded. Thus 589 patients in
total were included in this analysis. This study was approved by the In-
stitutional Review Board at SPH and the University of British Columbia.
VASST cohort (replication cohort). The Vasopressin and Septic Shock Trial
(VASST) was a multicenter, randomized, double-blind, controlled trial
evaluating the efficacy of vasopressinversus norepinephrine in 779 patients
who were diagnosed with septic shock according to the current consensus
definition (15). Clinical phenotyping has been described elsewhere (14).
All patients were enrolled within 24 h of meeting the definition of septic
shock, and DNA was available from 616 patients. The research ethics
boards of all participating institutions approved this trial, and written in-
formed consent was obtained from all patients or their authorized repre-
sentatives. The research ethics board at the coordinating center (University
of British Columbia) approved the genetic analysis.
SNP selection and genotyping of patient cohorts
cytosolic genes in the NF-kB canonical and noncanonical pathway that also
had dense resequencing data publically available (Supplemental Fig. 1)
edu/; Cardiogenomics, http://cardiogenomics.med.harvard.edu/home; Innate
Immunity, http://www.pharmgat.org/IIPGA2/index_html, formerly http://
innateimmunity.net/; Berkeley University PGA, http://pga.jgi-psf.org/; and
SouthWestern PGA, http://pga.swmed.edu/). TagSNPs were identified for
genotyping in patient cohorts using a linkage disequilibrium-based tag SNP
selection method (16) and using an r2threshold of 0.65 for SNPs with
a minor-allele frequency .5% yielding 59 SNPs in 19 cytosolic genes; re-
ceptor and their ligands as well as downstream gene targets of NF-kB sig-
naling were excluded (Table I). DNA was extracted from peripheral blood
samples using a QIAamp DNA Blood Midi Kit (Qiagen, Mississauga,
Ontario, Canada) and genotyped using the Illumina Golden Gate Assay at
the UBC Centre for Molecular Medicine and Therapeutics genotyping core
probe sets are detailed in Supplemental Table IV.
The primary outcome was 28-d mortality. Secondary outcomes were days
aliveandfreeoforgandysfunctionduringthe first 28dcalculatedaccording
to the Brussels criteria (15).
Biological plausibility experiments
Microarray mRNA expression analysis in vitro. Lymphoblastoid DNA from
the Coriell Institute was genotyped for rs7222094 in 85 CEPH population
samples using Sanger sequencing of the region. Sequencing was performed
at the McGill University and Ge ´nome Que ´bec Innovation Centre (Montre ´al,
Que ´bec, Canada). Primers for sequencing the region surrounding rs7222094
are as follows: forward, 59-GGGTTCCCTATGGAGGAGAG-39; reverse,
59-CTGTCCAGCTCTCCAGGTTC-39. These 85 CEPH population lym-
phoblastoid cell lines of known genotype for NIK rs7222094 were cultured
in RPMI 1640 and subsequently stimulated in triplicate by the addition of
Cytomix (9, 17–19) (2.5 ng/ml of each TNF-a, IL-1b, IFN-g [R&D
Systems, Minneapolis, MN] and 12.5 mM CpG [Sigma-Aldrich, Oakville,
Ontario, Canada]) for 6 h. RNA was extracted from the three stimulated
samples as well as two control biological duplicates using the Qiagen
RNeasy kit (Qiagen), and mRNA expression was measured using the
Human WG-6 v.3 expression chip (Illumina, San Diego, CA; Ge ´nome
Que ´bec Innovation Centre, Montre ´al, Que ´bec, Canada). Microarray raw
data were normalized, and fold change was calculated using the Flexarray
(version 1.4.1) package available from Ge ´nome Que ´bec (http://genome-
quebec.mcgill.ca/FlexArray/license.php). MIAME compliant microarray
data are publically available at GEO (GSE25543; http://www.ncbi.nlm.nih.
ELISA protein expression analysis in vitro. Twenty-six CEPH population
lymphoblastoid cell lines of known genotype for NIK rs7222094 were
cultured in RPMI 1640 and subsequently stimulated by the addition of
Cytomix (9, 17–19) (2.5 ng/ml of each TNF-a, IL-1b, IFN-g [R&D
Systems] and 12.5 mM CpG [Sigma-Aldrich]) for 24 h. Of the 85 cell lines
used for the microarray experiment, 13 carry the minor allele, thus all 13
were interrogated, matched by a random selection of 13 major allele cell
lines. Supernatant was collected, and biological duplicate CXCL10 con-
centrations were measured by ELISA (R&D Systems).
genes of patients in the SPH cohort
Armitage trend test on mortality and SNPs of NF-kB pathway
2322 VARIANT IN NIK IS ASSOCIATED WITH SEPTIC SHOCK MORTALITY
ELISA protein expression analysis in vivo. Briefly, whole-blood samples
were drawn into chilled 7-ml EDTA Vacutainer tubes (BD, Mississauga,
Ontario, Canada), put on ice immediately, then spun at 3000 rpm for 15 min
at which point plasma was collected and stored at 270˚C until further use
(14). Baseline plasma samples from 60 patients of known genotype for
rs7222094 (30 TT and 30 CC) were randomly selected and assayed for
CXCL10 concentrations in duplicate by ELISA (R&D Systems).
We assessed baseline characteristics using a x2test for categorical data and
a Kruskal–Wallis test for continuous data and then reported the median and
interquartile ranges. We then tested for association between SNP genotype
and 28-d mortality in the SPH discovery cohort using an Armitage trend
test, as is commonly used for initial discovery surveys in genome-wide
association studies (Table I). One SNP emerged as statistically significant
after a Bonferroni correction for multiple comparisons. We then tested for
replication of this finding in the VASST cohort of septic shock patients. To
correct for potentially confounding variables, including age, gender, an-
cestry, and surgical versus medical diagnostic category, we used Cox re-
gression. We then tested for association between secondary outcome
measures of days alive and free of organ failure using Kruskal–Wallis tests
in both SPH and VASST cohorts. Vasopressin treatment by genotype (NIK
rs7222094 CT) interaction was assessed using logistic regression analysis
(interaction statistics): P(Death) ? vasopressin + genotype + vasopressin 3
A Student t test was performed to test for differences between genetic
groups (rs7222094 CC versus TT) of CXCL10 ELISA concentrations.
Analyses used SPSS (version 16; SPSS, Chicago, IL), R statistical soft-
ware package, and GraphPad Prism (version 5.02; GraphPad, La Jolla,
Twenty-eight–day mortality in septic shock patients
Of the 59 tagSNPs in 19 genes in canonical and noncanonical NF-
kB pathways, one SNP, rs7222094 in NIK, was significantly as-
sociated with 28-d mortality in the SPH discovery cohort (un-
corrected p = 0.00024, Bonferroni corrected p = 0.014) (Sup-
plemental Fig. 1, Table I).
Hardy–Weinberg equilibrium and minor allele frequencies of
all SNPs genotyped are presented along with literature based
minor allele frequencies in Supplemental Table I. Allele fre-
quencies of rs7222094 differed between ethnic groups within the
SPH and VASST cohorts (Supplemental Table II). Therefore, our
primary analysis was limited to Caucasians only (SPH, n = 453,
VASST, n = 517), and our secondary analysis of all patients in-
cluded ethnicity as a covariate (SPH, N = 589; VASST, N = 616).
To consider and correct for potential confounding variables due
to differences at baseline in septic shock patients, we used Cox
regression to test an additive model in the SPH septic shock cohort
and then used the same analysis in the VASST septic shock cohort.
Patients in the SPH cohort who had the CC genotype of NIK
rs7222094 had a significantly increased hazard of 28-d mortality
compared with that of patients having the CT or TT genotypes of
rs7222094 (hazard ratio [HR], 1.35; 95% confidence interval [CI],
1.12–1.64; p = 0.002 Caucasian only) (Table II). This finding was
replicated in the VASST cohort (HR, 1.24; 95% CI, 1.00–1.52; p =
0.048 Caucasian only) (Table II). The results were similar for all
patients with ethnicity included as a covariate in a Cox regression
model (Fig. 1, Table III).
Similarly, in an unadjusted univariate analysis, the C allele of
rs7222094 TC was associated with mortality in SPH (mortality,
Caucasian only: TT, 33.8%; CT, 42.4%; CC, 53.7%; p = 0.005;
mortality, all ethnicities: TT, 33.8%; CT, 43.2%; CC, 53.0%; p =
0.001), and a similar trend was observed in VASST (mortality,
Caucasian only: TT, 26.3%; CT, 34.7%; CC, 38.4%; p = 0.09;
mortality, all ethnicities: TT, 27.5%; CT, 33.2%; CC, 40.8%; p =
0.03). Allele frequencies of survivors versus nonsurvivors in both
Caucasian and all ethnicities are reported in Table IV.
In the SPH Caucasian cohort, patients having the CC genotype
of rs7222094 had greater baseline creatinine concentrations
(p = 0.04) and significantly higher PaO2/FIO2at baseline (p =
0.002) than patients having the CT or TT genotypes of rs7222094
(Table V). The only difference at baseline among VASST Cau-
casian patients was that patients having the CC genotype of
rs7222094 had significantly lower platelet counts than those of
patients having the CT or TT genotypes of rs7222094 (p = 0.02)
(Table V). Because the VASST cohort was a clinical trial com-
paring efficacy of vasopressin versus norepinephrine in septic
shock, in a secondary analysis we tested for an interaction by
logistic regression between NIK rs7222094 and vasopressin
treatment in Caucasian patients. We found no significant in-
teraction (interaction statistic p = 0.462).
Organ failure in septic shock patients
Patients homozygous for the C allele of rs7222094 in both the SPH
and VASST Caucasian cohorts had more organ dysfunction as
defined by Brussels criteria compared with that in patients having
the CT or TT genotypes of rs7222094 (15). SPH patients with
rs7222094 CC genotype had significantly fewer days alive and
free of renal dysfunction (p = 0.003) and fewer days alive and free
of acute renal replacement therapy (p = 0.008) as well as fewer
days alive and free of hematological dysfunction (p = 0.011)
Table II. Hazard ratios of 28-d mortality in Caucasian patients with septic shock (Cox regression)
SPH Cohort (n = 453)
HR (95% CI)p Value
VASST Cohort (n = 517)
HR (95% CI)p Value
Age, per year
NIK rs7222094 C allele
Table III.Hazard ratios of 28-d mortality in patients with septic shock (Cox regression)
SPH Cohort (N = 589)
HR (95% CI)p Value
VASST Cohort (N = 616)
HR (95% CI)p Value
Age, per year
NIK rs7222094 C allele
9.0 3 1024
8.0 3 1024
2.0 3 1024
The Journal of Immunology 2323
during the 28-d study period than those of patients having the
CT or TT genotypes of rs7222094 (Table VI). Patients of the
VASST cohort with the rs7222094 CC genotype show the same
trend toward more renal dysfunction (Table VI). The number of
patients affected by each type of organ dysfunction by genotypic
group is outlined in Table VII.
In the SPH cohort, patients with the rs7222094 CC genotype
also had significantly fewer days alive and free of cardiovascular
dysfunction (p = 0.008), with correspondingly fewer days alive
and free of vasopressor support (p = 0.01), which was also seen as
a trend in the VASST cohort (Table VI). SPH cohort patients also
experienced more hepatic and neurologic dysfunction (p = 0.007
and p = 0.006, respectively) (Table VI).
CXCL10 mRNA production by lymphoblastoid cell lines
The gene with the greatest difference (D) in fold change between
major (TT) and minor (CC) genotypes was CXCL10 (D fold
change, 0.67; uncorrected Student t test between groups, p = 0.055)
suggesting lower mRNA expression of CXCL10 for the CC geno-
type compared with that of TTor TC (Supplemental Table III).
CXCL10 protein production by lymphoblastoid cell lines
Protein levels of CXCL10 were measured in 26 cell lines of known
genotype for rs7222094. Cell lines homozygous for the C allele of
rs7222094 produced less CXCL10 at both baseline and after in-
flammatory stimulation than that of cell lines homozygous for the
T allele (p = 0.032 and p = 0.050, respectively) (Fig. 2).
CXCL10 protein production in VASST plasma samples
Baseline plasma specimens of a random sample of patients with
septic shock from the VASST cohort were assayed in duplicate for
CXCL10 by ELISA, and as was found in both the control and
stimulated lymphoblastoid cell lines, patients of the CC genotype
had significantly lower CXCL10 than that of the TT genotype
(p = 0.017) (Fig. 3).
We found that patients of the CC genotype of NIK rs7222094 had
significantly increased mortality compared with that of patients
having the CT or TT genotypes of rs7222094 in two cohorts of
patients who had septic shock. Specifically, Caucasian patients in
the SPH cohort who had the CC genotype of NIK rs7222094
experienced a significant increase in the hazard of death over the
28 d (HR, 1.35; 95% CI, 1.12–1.64; p = 0.002). This effect was
survivors and nonsurvivors
Allele frequencies in both SPH and VASST cohorts of
0.57 0.43 0.0050.52 0.48 0.09
0.450.55 0.45 0.55
0.52 0.480.0010.49 0.51 0.03
and VASST cohorts by NIK genotype of rs7222094. Patients who were
homozygous minor (CC) (gray dashed line) experienced increased mor-
tality in both the SPH and VASST cohorts.
Survival curves for patients with septic shock in the SPH
lines. Twenty-six lymphoblastoid cell lines of known genotype (13 TT and
13 CC) for rs7222094 were cultured and stimulated with Cytomix (2.5 ng/
ml of each TNF-a, IL-1b, IFN-g and 12.5 mM CpG) for 24 h. Supernatant
was collected, and CXCL10 concentrations were measured by ELISA. Cell
lines homozygous for the C allele produce significantly less CXCL10 at
both baseline (*p = 0.032) and under stimulation conditions (*p = 0.050).
CXCL10 protein concentrations in lymphoblastoid cellFIGURE 3.
the VASST replication cohort. Baseline plasma samples from 60 VASST
septic shock patients of known genotype (30 TTand 30 CC) for rs7222094
were randomly selected and assayed for CXCL10 concentrations by
ELISA. Patients homozygous for the C allele had significantly lower
concentrations of CXCL10 than those of patients who were homozygous
for the T allele. *p = 0.017.
CXCL10 protein concentrations in septic shock patients of
2324 VARIANT IN NIK IS ASSOCIATED WITH SEPTIC SHOCK MORTALITY
also found in the VASST Caucasian cohort (HR, 1.24; 95% CI,
1.00–1.52; p = 0.048). The results were similar for all patients
with ethnicity included as a covariate in a Cox regression model.
Also, patients of the CC genotype of rs7222094 of SPH experi-
enced more renal and hematological dysfunction compared with
that of patients having the CT or TT genotypes of rs7222094
(p = 0.003 and p = 0.011, respectively). Patients of the VASST
cohort with the rs7222094 CC genotype showed the same trend
toward more renal dysfunction as found in the SPH cohort.
NIK was first discovered in human B cells, and hence we chose
lymphoblastoid cell lines for this phase of our study (20). CXCL10
is a chemokine transcribed in response to NF-kB during in-
flammation and is generally thought to signal in response to ca-
nonical pathway stimuli (21, 22). In a recent study by Zarnegar
et al. (23), CXCL10 levels were dependent on NIK suggesting
noncanonical signaling. CXCL10 is transcribed in response to NF-
kB activation and has been shown to be downregulated upon in-
hibition of NIK (23). The CC genotype of NIK rs7222094 was
associated with significantly decreased levels of CXCL10 in
supernatant of lymphoblastoid cell lines at baseline and after
Cytomix stimulation (p = 0.032 and p = 0.050). Similarly,
CXCL10 levels were significantly lower in septic shock patients
of the CC genotype (p = 0.017), suggesting a biologically plau-
sible explanation for our observations.
CC genotype experienced increased mortality while supernatant
from cell lines of the same genotype had decreased levels of
CXCL10. This effect was replicated when CXCL10 concentration
CXCL10 is a proinflammatory chemokine released during in-
flammatory states, such as allograph rejection and infection (24).
It is plausible that proinflammatory molecules are necessary
to mount an effective immune response during septic shock.
CXCL10 is a chemokine that instigates chemotaxis of activated
T cells and NK cells (24). We speculate that without enough
CXCL10 to drive recruitment of inflammatory cells, it is possible
that these patients (who had the CC genotype of NIK rs7222094)
have an immunological disadvantage and so have increased mor-
tality of septic shock.
The original characterization of NIK indicated that NIK was
a powerful effector of canonical NF-kB signaling under TNF-a
and IL-1b stimulation (20). Later studies were unable to confirm
this mechanism, and instead the discovery of the noncanonical
pathway emerged as subsequent research appeared to distill the
two pathways into distinct and separate processes (6, 25–27).
Evidence is building suggesting that the IKK–NIK axis is a pivot
point for control of both noncanonical and canonical signaling
(23, 28, 29). It is possible that the timing of experimental proce-
dures is critical to our understanding of NIK, as many studies that
separated the two pathways and excluded NIK from canonical
signaling focused on early events (from minutes to less than 2 h)
(25, 27). In contrast, many studies find that NIK plays a pivotal
role in canonical signaling when evaluating effects at time points
of several hours to days (20, 23, 28). The current understanding of
the regulation of NIK is that NIK is constitutively transcribed,
translated, and degraded via its interaction with TRAF3. However,
after degradation of TRAF3 after appropriate stimuli, NIK recruits
IKKa to p100, activating IKKa thereby initiating proteosomal
degradation of p100 to p52 and consequently translocation of
heterodimers to the nucleus (6, 30). It is conceivable that accu-
mulation of NIK over time is a key facet to its mechanism.
Because NIK has been implicated in the host response to in-
fection, we speculate that NIK could modulate the immune re-
sponse during septic shock. NIK is important in the host response
Baseline characteristics by NIK genotype rs7222094 for Caucasian patients with septic shock in both SPH and VASST cohorts
TT (n = 139)
CT (n = 191)
CC (n = 123)
TT (n = 133)
CT (n = 251)
CC (n = 133)
Gender, percentage male
Preexisting conditions, %
Chronic heart failure
Chronic pulmonary disease
Chronic liver disease
Chronic renal failure
Chronic corticosteroid use
Cardiovascular variables day 1
Heart rate, bpm
Mean arterial pressure, mm Hg
Central venous pressure, mm Hg
Laboratory variables day 1
WBC count, 103/mm3
Platelet count, 103/mm3
aValues shown are median and interquartile range.
The Journal of Immunology 2325
to numerous infections including respiratory syncytial virus [with
evidence to suggest activation of both noncanonical and canonical
signaling (31)], HIV (32), hepatitis B (33), Escherichia coli (34),
as well as the response to LPS (35).
Several lines of evidence suggest why polymorphisms of NIK
may be predictors of outcome in septic shock (1). First, NIK stim-
ulates inflammation by upregulating the noncanonical (and possi-
bly the canonical) pathway of NF-kB activation. Second, NIK is
required for optimal IgG production by lymphocytes (36). Third,
of action of the calcium channel blocker nifedipine (37), and angio-
tensin II induces inflammation through NIK activation of the non-
canonical NF-kB pathway (38). However, polymorphisms of NIK
have not been widely studied. In a survey of 181 SNPs of 17 genes
in the NF-kB pathway, polymorphisms of NIK were not associated
with rheumatoid arthritis susceptibility (39). Notably, rs4792847 of
NIK was found to be significantly associated with response to anti-
TNF treatment in rheumatoid arthritis. Patients with GG genotype
had the greatest improvement at the 6-mo mark in a discovery co-
hort; however, this effect was not found in a replication cohort (40).
The GG genotype of rs4792847 (patients who may have had a more
favorable response to anti-TNF) is in high linkage disequilibrium
with the TT genotype of rs7222094 (r2= 1.0). This is consistent
with our observation of a protective effect of the TT genotype
(i.e., lower mortality and higher CXCL10 production) seen in our
study of patients who had septic shock and replicated in our invitro
To our knowledge, NIK has not been implicated in septic shock
to date. However, NIK is a drug target for other diseases (5).
Inhibitors of NIK have been synthesized for diseases such as
multiple myeloma and other cancers, as anti-inflammatory agents
for inflammatory diseases (41), and as a vaccine adjuvant (42).
Our data suggest that rs7222094 may be of interest in randomized
controlled trials of therapies for septic shock by defining risk
categories of patients and perhaps defining response to anti-
This study has several limitations. The analysis of the SPH and
VASST cohorts was performed retrospectively. The association
of rs7222094 with mortality, organ dysfunction, and CXCL10
as a marker for differences in NIK-induced NF-kB signaling. We
do not currently know of, nor did we test for, the influence of
CXCL10 itself on organ dysfunction or mortality. In view of the
large number of genes connected in some way to NF-kB signaling,
we chose to limit our analysis to genes of the cytosolic members
of the NF-kB pathway, excluding receptors and downstream tar-
gets. Therefore, this analysis does not include all potentially
functional variants, in particular those recently published after the
design of this study (43–45).
In conclusion, we found that NIK rs7222094 was consistently
and significantly associated with mortality in two independent
cohorts of patients who had septic shock. Patients homozygous
for the CC genotype of rs7222094 had increased mortality and
also experienced more renal and hematological failure in the
SPH cohort of Caucasian patients with a similar trend in VASST
Caucasian patients. Furthermore, we found that lymphoblastoid
cell lines homozygous for CC of rs7222094 produced less CXCL10
in vitro at baseline and after Cytomix stimulation than that of cell
Table VII. Caucasian septic shock patients affected by organ dysfunction according to NIK rs7222094 genotype
SPH Cohort VASST Cohort
TT (n = 139)
CT (n = 191)
CC (n = 123)
N (%)p Value
TT (n = 133)
CT (n = 251)
CC (n = 133)
N (%)p Value
Artificial organ support
Renal replacement therapy
NA, The p value was not calculated because all patients in each genotypic group are affected.
Table VI. Days alive and free of organ dysfunction in Caucasian septic shock patients according to NIK rs7222094 genotype
TT (n = 139) CT (n = 191)CC (n = 123)p Value TT (n = 133)CT (n = 251)CC (n = 133)p Value
Artificial organ support
Renal replacement therapy
aValues shown are median and interquartile range.
2326VARIANT IN NIK IS ASSOCIATED WITH SEPTIC SHOCK MORTALITY
lines having the TT genotype of NIK rs7222094 (p = 0.032 and p =
0.050, respectively). As well, patients who had septic shock in the
VASST cohort who were NIK rs7222094 CC genotype had sig-
nificantly lower plasma levels of CXCL10 than those of the TT
genotype (p = 0.017). We speculate that polymorphisms of NIK
could be used to predict risk of death from septic shock and to
predict response to anti-inflammatory treatment, such as inhibitors
S.A.T. has received grant support from MITACs made possible by an in-
dustry partnership with Sirius Genomics. J.A.R. holds stock in Sirius
Genomics Inc., which has submitted patents owned by the University of
British Columbia and licensed to Sirius Genomics that are related to the
genetics of vasopressin, NIK, and protein C. The University of British Co-
lumbia has also submitted a patent related to the use of vasopressin in septic
shock. J.A.R. is an inventor on these patents. J.A.R. has received consulting
fees from Ferring, which manufactures vasopressin; from Astra Zeneca,
which manufactures anti-TNF; and from Sirius Genomics Inc. J.A.R. has
received grant support from Sirius Genomics, Novartis, Ferring, and Eli
Lilly. J.A.R. has received speaking honoraria from Pfizer and Eli Lilly.
K.R.W. holds stock in Sirius Genomics Inc., which has submitted patents
owned by the University of British Columbia and licensed to Sirius
Genomics that are related to the genetics of vasopressin, NIK, and protein
C. The University of British Columbia has also submitted a patent related to
the use of vasopressin in septic shock. K.R.W. is an inventor on these pat-
serving as director of research and holding shares at Sirius Genomics Inc.,
which has submitted a patent owned by the University of British Columbia
and licensed to Sirius Genomics that is related to the genetics of LNPEP.
1. Liu, S. F., and A. B. Malik. 2006. NF-kappa B activation as a pathological
mechanism of septic shock and inflammation. Am. J. Physiol. Lung Cell. Mol.
Physiol. 290: L622–L645.
2. Levy, M. M., M. P. Fink, J. C. Marshall, E. Abraham, D. Angus, D. Cook,
J. Cohen, S. M. Opal, J. L. Vincent, G. Ramsay; SCCM/ESICM/ACCP/ATS/SIS.
2003. 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions
Conference. Crit. Care Med. 31: 1250–1256.
3. Bonizzi, G., and M. Karin. 2004. The two NF-kappaB activation pathways and
their role in innate and adaptive immunity. Trends Immunol. 25: 280–288.
4. Fischer, C., S. Page, M. Weber, T. Eisele, D. Neumeier, and K. Brand. 1999.
Differential effects of lipopolysaccharide and tumor necrosis factor on mono-
cytic IkappaB kinase signalsome activation and IkappaB proteolysis. J. Biol.
Chem. 274: 24625–24632.
5. Dejardin, E. 2006. The alternative NF-kappaB pathway from biochemistry to
biology: pitfalls and promises for future drug development. Biochem. Pharma-
col. 72: 1161–1179.
6. Xiao, G., A. Fong, and S. C. Sun. 2004. Induction of p100 processing by NF-
kappaB-inducing kinase involves docking IkappaB kinase alpha (IKKalpha) to
7. Majetschak, M., S. Flohe ´, U. Obertacke, J. Schro ¨der, K. Staubach, D. Nast-Kolb,
F. U. Schade, and F. Stu ¨ber. 1999. Relation of a TNF gene polymorphism to
severe sepsis in trauma patients. Ann. Surg. 230: 207–214.
8. Mira, J. P., A. Cariou, F. Grall, C. Delclaux, M. R. Losser, F. Heshmati,
C. Cheval, M. Monchi, J. L. Teboul, F. Riche ´, et al. 1999. Association of TNF2,
a TNF-alpha promoter polymorphism, with septic shock susceptibility and
mortality: a multicenter study. JAMA 282: 561–568.
9. Nakada, T. A., J. A. Russell, J. H. Boyd, R. Aguirre-Hernandez, K. R. Thain,
S. A. Thair, E. Nakada, M. McConechy, and K. R. Walley. 2010. beta2-
Adrenergic receptor gene polymorphism is associated with mortality in septic
shock. Am. J. Respir. Crit. Care Med. 181: 143–149.
10. Read, R. C., N. J. Camp, F. S. di Giovine, R. Borrow, E. B. Kaczmarski,
A. G. Chaudhary, A. J. Fox, and G. W. Duff. 2000. An interleukin-1 genotype is
associated with fatal outcome of meningococcal disease. J. Infect. Dis. 182:
11. Russell, J. A., H. Wellman, and K. R. Walley. 2008. Protein C rs2069912 C allele
is associated with increased mortality from severe sepsis in North Americans of
East Asian ancestry. Hum. Genet. 123: 661–663.
12. Walley, K. R., and J. A. Russell. 2007. Protein C -1641 AA is associated with
decreased survival and more organ dysfunction in severe sepsis. Crit. Care Med.
and septic shock: role in prognosis and potential for therapy. Chest 124: 1103–1115.
14. Russell, J. A., K. R. Walley, J. Singer, A. C. Gordon, P. C. He ´bert, D. J. Cooper,
C. L. Holmes, S. Mehta, J. T. Granton, M. M. Storms, et al; VASST Inves-
tigators. 2008. Vasopressin versus norepinephrine infusion in patients with septic
shock. N. Engl. J. Med. 358: 877–887.
15. Sibbald, W. J., and J. L. Vincent. 1995. Roundtable Conference on Clinical
Trials for the Treatment of Sepsis. Brussels, March 12-14, 1994. Chest 107:
16. Carlson, C. S., M. A. Eberle, M. J. Rieder, Q. Yi, L. Kruglyak, and
D. A. Nickerson. 2004. Selecting a maximally informative set of single-
nucleotide polymorphisms for association analyses using linkage disequilib-
rium. Am. J. Hum. Genet. 74: 106–120.
17. Cheung, V. G., L. K. Conlin, T. M. Weber, M. Arcaro, K. Y. Jen, M. Morley, and
R. S. Spielman. 2003. Natural variation in human gene expression assessed in
lymphoblastoid cells. Nat. Genet. 33: 422–425.
18. Farley, K. S., L. Wang, and S. Mehta. 2009. Septic pulmonary microvascular
endothelial cell injury: role of alveolar macrophage NADPH oxidase. Am. J.
Physiol. Lung Cell. Mol. Physiol. 296: L480–L488.
19. Liu, S., D. B. Stolz, P. L. Sappington, C. A. Macias, M. E. Killeen, J. J. Tenhunen,
R. L. Delude, and M. P. Fink. 2006. HMGB1 is secreted by immunostimulated
enterocytes and contributes to cytomix-induced hyperpermeability of Caco-2
monolayers. Am. J. Physiol. Cell Physiol. 290: C990–C999.
20. Malinin, N. L., M. P. Boldin, A. V. Kovalenko, and D. Wallach. 1997. MAP3K-
related kinase involved in NF-kappaB induction by TNF, CD95 and IL-1. Nature
21. Hoffmann, A., T. H. Leung, and D. Baltimore. 2003. Genetic analysis of NF-
kappaB/Rel transcription factors defines functional specificities. EMBO J. 22:
22. Neville, L. F., G. Mathiak, and O. Bagasra. 1997. The immunobiology of
interferon-gamma inducible protein 10 kD (IP-10): a novel, pleiotropic
member of the C-X-C chemokine superfamily. Cytokine Growth Factor Rev.
23. Zarnegar, B., S. Yamazaki, J. Q. He, and G. Cheng. 2008. Control of canonical
NF-kappaB activation through the NIK-IKK complex pathway. Proc. Natl. Acad.
Sci. USA 105: 3503–3508.
24. Charo, I. F., and R. M. Ransohoff. 2006. The many roles of chemokines and
chemokine receptors in inflammation. N. Engl. J. Med. 354: 610–621.
25. Senftleben, U., Y. Cao, G. Xiao, F. R. Greten, G. Kra ¨hn, G. Bonizzi, Y. Chen,
Y. Hu, A. Fong, S. C. Sun, and M. Karin. 2001. Activation by IKKalpha of
a second, evolutionary conserved, NF-kappa B signaling pathway. Science 293:
26. Xiao, G., E. W. Harhaj, and S. C. Sun. 2001. NF-kappaB-inducing kinase reg-
ulates the processing of NF-kappaB2 p100. Mol. Cell 7: 401–409.
27. Yin, L., L. Wu, H. Wesche, C. D. Arthur, J. M. White, D. V. Goeddel, and
R. D. Schreiber. 2001. Defective lymphotoxin-beta receptor-induced NF-
kappaB transcriptional activity in NIK-deficient mice. Science 291: 2162–
28. O’Mahony, A., X. Lin, R. Geleziunas, and W. C. Greene. 2000. Activation of the
heterodimeric IkappaB kinase alpha (IKKalpha)-IKKbeta complex is di-
rectional: IKKalpha regulates IKKbeta under both basal and stimulated con-
ditions. Mol. Cell. Biol. 20: 1170–1178.
29. Ramakrishnan, P., W. Wang, and D. Wallach. 2004. Receptor-specific signaling
for both the alternative and the canonical NF-kappaB activation pathways by
NF-kappaB-inducing kinase. Immunity 21: 477–489.
30. Qing, G., Z. Qu, and G. Xiao. 2005. Stabilization of basally translated NF-
kappaB-inducing kinase (NIK) protein functions as a molecular switch of pro-
cessing of NF-kappaB2 p100. J. Biol. Chem. 280: 40578–40582.
31. Choudhary, S., S. Boldogh, R. Garofalo, M. Jamaluddin, and A. R. Brasier. 2005.
Respiratory syncytial virus influences NF-kappaB-dependent gene expression
through a novel pathway involving MAP3K14/NIK expression and nuclear
complex formation with NF-kappaB2. J. Virol. 79: 8948–8959.
32. Leghmari, K., Y. Bennasser, and E. Bahraoui. 2008. HIV-1 Tat protein induces
IL-10 production in monocytes by classical and alternative NF-kappaB path-
ways. Eur. J. Cell Biol. 87: 947–962.
33. Park, S. G., H. M. Ryu, S. O. Lim, Y. I. Kim, S. B. Hwang, and G. Jung. 2005.
Interferon-gamma inhibits hepatitis B virus-induced NF-kappaB activation
through nuclear localization of NF-kappaB-inducing kinase. Gastroenterology
34. Kim, J. M., Y. K. Oh, J. H. Lee, D. Y. Im, Y. J. Kim, J. Youn, C. H. Lee, H. Son,
Y. S. Lee, J. Y. Park, and I. H. Choi. 2005. Induction of proinflammatory
mediators requires activation of the TRAF, NIK, IKK and NF-kappaB signal
transduction pathway in astrocytes infected with Escherichia coli. Clin. Exp.
Immunol. 140: 450–460.
35. Luo, S. F., W. N. Lin, C. M. Yang, C. W. Lee, C. H. Liao, Y. L. Leu, and
L. D. Hsiao. 2006. Induction of cytosolic phospholipase A2 by lipopolysac-
charide in canine tracheal smooth muscle cells: involvement of MAPKs and NF-
kappaB pathways. Cell. Signal. 18: 1201–1211.
36. Mills, D. M., G. Bonizzi, M. Karin, and R. C. Rickert. 2007. Regulation of late
B cell differentiation by intrinsic IKKalpha-dependent signals. Proc. Natl. Acad.
Sci. USA 104: 6359–6364.
37. Wu, L., M. Iwai, Z. Li, J. M. Li, M. Mogi, and M. Horiuchi. 2006. Nifedipine
inhibited angiotensin II-induced monocyte chemoattractant protein 1 expression:
involvement of inhibitor of nuclear factor kappa B kinase and nuclear factor
kappa B-inducing kinase. J. Hypertens. 24: 123–130.
38. Choudhary, S., M. Lu, R. Cui, and A. R. Brasier. 2007. Involvement of a novel
Rac/RhoA guanosine triphosphatase-nuclear factor-kappaB inducing kinase
signaling pathway mediating angiotensin II-induced RelA transactivation. Mol.
Endocrinol. 21: 2203–2217.
39. Dieguez-Gonzalez, R., S. Akar, M. Calaza, E. Perez-Pampin, J. Costas,
M. Torres, J. L. Vicario, M. L. Velloso, F. Navarro, J. Narvaez, et al. 2009.
The Journal of Immunology 2327
Genetic variation in the nuclear factor kappaB pathway in relation to suscepti- Download full-text
bility to rheumatoid arthritis. Ann. Rheum. Dis. 68: 579–583.
40. Bowes, J. D., C. Potter, L. J. Gibbons, K. Hyrich, D. Plant, A. W. Morgan,
A. G. Wilson, J. D. Isaacs, J. Worthington, A. Barton; BRAGGSS. 2009. In-
vestigation of genetic variants within candidate genes of the TNFRSF1B sig-
nalling pathway on the response to anti-TNF agents in a UK cohort of
rheumatoid arthritis patients. Pharmacogenet. Genomics 19: 319–323.
Y. G. Kwon, and Y. M. Kim. 2008. CT20126, a novel immunosuppressant, prevents
collagen-induced arthritis through the down-regulation of inflammatory gene ex-
pression by inhibiting NF-kappaB activation. Biochem. Pharmacol. 76: 79–90.
42. Andreakos, E., R. O. Williams, J. Wales, B. M. Foxwell, and M. Feldmann.
2006. Activation of NF-kappaB by the intracellular expression of NF-kappaB-
inducing kinase acts as a powerful vaccine adjuvant. Proc. Natl. Acad. Sci. USA
43. Toubiana, J., E. Courtine, F. Pe `ne, V. Viallon, P. Asfar, C. Daubin, C. Rousseau,
C. Chenot, F. Ouaaz, D. Grimaldi, et al. 2010. IRAK1 functional genetic variant
affects severity of septic shock. Crit. Care Med. 38: 2287–2294.
44. Vasl, J., P. Prohinar, T. L. Gioannini, J. P. Weiss, and R. Jerala. 2008. Functional
activity of MD-2 polymorphic variant is significantly different in soluble and
TLR4-bound forms: decreased endotoxin binding by G56R MD-2 and its rescue
by TLR4 ectodomain. J. Immunol. 180: 6107–6115.
45. Wurfel, M. M., A. C. Gordon, T. D. Holden, F. Radella, J. Strout, O. Kajikawa,
J. T. Ruzinski, G. Rona, R. A. Black, S. Stratton, et al. 2008. Toll-like receptor 1
polymorphisms affect innate immune responses and outcomes in sepsis. Am. J.
Respir. Crit. Care Med. 178: 710–720.
2328 VARIANT IN NIK IS ASSOCIATED WITH SEPTIC SHOCK MORTALITY