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A Single Nucleotide Polymorphism in NF-κB Inducing Kinase Is Associated with Mortality in Septic Shock

This information is current as Simone A. Thair, Keith R. Walley, Taka-aki Nakada, of September 30, 2021. Melissa K. McConechy, John H. Boyd, Hugh Wellman and James A. Russell J Immunol 2011; 186:2321-2328; Prepublished online 21 January 2011;

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Supplementary http://www.jimmunol.org/content/suppl/2011/01/21/jimmunol.100286 Material 4.DC1 http://www.jimmunol.org/ References This article cites 45 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/186/4/2321.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 Downloaded from 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 http://www.jimmunol.org/ 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.

eptic shock is an extreme manifestation of the host in- A better understanding of the key pathways and critical molecules flammatory response to severe infection, and the NF-kB involved in the pathogenesis of septic shock is imperative. S signaling pathway is one of the most important signaling NF-kB signaling is activated by a canonical (or classical) pathways involved in the pathogenesis of this pathological state pathway and by a noncanonical (or alternative) pathway involving by guest on September 30, 2021 (1). Septic shock is defined as sepsis accompanied by organ failure multiple genes (Supplemental Fig. 1) (5). Briefly, the canonical including cardiovascular failure (2). In response to infection, NF-kB pathway requires the IkB kinase (IKK) complex composed NF-kB signaling (among other pathways) leads to the transcription of IKKa/b/g (3). Activation of the IKK complex in response of a wealth of inflammatory mediators that contribute to the de- to inflammatory stimuli results in phosphorylation and ubiquitin- velopment of cardiovascular failure, including cytokines, chemo- dependent degradation of IkBa or IkBb and translocation of p50- kines, adhesion molecules, and reactive oxygen and nitrogen related dimers into the nucleus (3). In response to inflammation species, to name a few (3, 4). The induction of this response is in the noncanonical pathway, NF-kB inducing kinase (NIK), a necessary for the resolution of infection; however, this response, docking molecule, recruits IKKa to p100 and then activates IKKa when extreme, leads to organ damage and mortality in many cases. (3, 6). IKKa phosphorylates p100, which is subjected to phosphorylation, ubiquitination, and proteosomal degradation, result- *University of British Columbia Critical Care Research Laboratories, Heart and ing in the release and nuclear translocation of p52 containing RelB Lung Institute, St. Paul’s Hospital, Vancouver, British Columbia V6Z 1Y6, Canada; heterodimers (3, 6). † Department of Pathology and Laboratory Medicine, University of British Columbia, Genetic variation in key inflammatory genes contributes to British Columbia Cancer Agency, Vancouver, British Columbia V5Z 4E6, Canada; and ‡Sirius Genomics Inc., Vancouver, British Columbia V6Z 2K8, Canada outcome in sepsis (7–12). Because NF-kB is a centrally important Received for publication September 2, 2010. Accepted for publication December 7, signaling pathway, we tested the hypothesis that genetic variation 2010. in genes involved in the NF-kB pathways would be associated S.A.T. is the recipient of a Mathematics of Information Technology and Complex with mortality in septic shock. To accomplish this, we genotyped 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 59 single nucleotide polymorphisms (SNPs) in 19 genes in a sin- Cardiovascular Training fellowship. K.R.W. is a Michael Smith Foundation for gle center derivation cohort of septic shock patients. We next Health Research Distinguished Scholar. The Vasopressin and Septic Shock Trial is tested for replication of an arising SNP association in a second supported by Canadian Institutes of Health Research Grant MCT 44152. multicenter cohort of septic shock patients. We tested for similar Address correspondence and reprint requests to James A. Russell, Heart and Lung Institute, St. Paul’s Hospital, #166-1081 Burrard Street, Vancouver, British Colum- association with organ dysfunction to understand the involved bia, Canada, V6Z 1Y6. E-mail address: [email protected] clinical phenotype in more detail. To test for biological plausi- The online version of this article contains supplemental material. bility of the observed SNP association, we next measured gene Abbreviations used in this article: CI, confidence interval; HR, hazard ratio; IKK, IkB expression in genotyped lymphoblastoid cell lines and identified kinase; NIK, NF-kB inducing kinase; SNP, single nucleotide polymorphism; SPH, St. a candidate gene whose expression was associated with the can- Paul’s Hospital; VASST, Vasopressin and Septic Shock Trial. didate SNP. We then tested for association of this gene product Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 with the candidate SNP both in vitro and in vivo. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002864 2322 VARIANT IN NIK IS ASSOCIATED WITH SEPTIC SHOCK MORTALITY

Materials and Methods Cytomix (9, 17–19) (2.5 ng/ml of each TNF-a, IL-1b, IFN-g [R&D Patient cohorts 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 St. Paul’s Hospital cohort (discovery cohort). All patients admitted to the were interrogated, matched by a random selection of 13 major allele cell St. Paul’s Hospital (SPH) Intensive Care Unit (Vancouver, British Colum- lines. Supernatant was collected, and biological duplicate CXCL10 con- bia, Canada) between July 2000 and January 2004 were screened. Using the centrations were measured by ELISA (R&D Systems). current consensus definition ($ 2 SIRS criteria, known or suspected infection, 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 Table I. Armitage trend test on mortality and SNPs of NF-kB pathway total were included in this analysis. This study was approved by the In- genes of patients in the SPH cohort stitutional Review Board at SPH and the University of British Columbia. VASST cohort (replication cohort). The Vasopressin and Septic Shock Trial Bonferroni (VASST) was a multicenter, randomized, double-blind, controlled trial Uncorrected Corrected evaluating the efficacy of vasopressin versus norepinephrine in 779 patients Pathway Genes SNP p Value p Value who were diagnosed with septic shock according to the current consensus definition (15). Clinical phenotyping has been described elsewhere (14). Canonical All patients were enrolled within 24 h of meeting the definition of septic MYD88 rs2239621 0.443 1.000 shock, and DNA was available from 616 patients. The research ethics rs6853 0.489 1.000 boards of all participating institutions approved this trial, and written in- rs7744 0.006 0.356 formed consent was obtained from all patients or their authorized repre- IRAK1 rs1059701 0.070 1.000 sentatives. The research ethics board at the coordinating center (University IRAK4 rs4251513 0.839 1.000 Downloaded from of British Columbia) approved the genetic analysis. rs4251520 0.969 1.000 TIRAP rs1786697 0.310 1.000 rs614700 0.342 1.000 SNP selection and genotyping of patient cohorts rs646005 0.223 1.000 We used Ingenuity IPA (version 8.6, build 93815, content 3003) to identify 19 MAP3K7 rs1145727 0.712 1.000 cytosolic genes in the NF-kB canonical and noncanonical pathway that also rs157688 0.946 1.000 rs205342 0.810 1.000

had dense resequencing data publically available (Supplemental Fig. 1) http://www.jimmunol.org/ (Seattle SNPs Program for Genomic Applications, http://pga.mbt.washington. rs967779 0.691 1.000 edu/; Cardiogenomics, http://cardiogenomics.med.harvard.edu/home; Innate MAP3K7IP1 rs5750824 0.117 1.000 Immunity, http://www.pharmgat.org/IIPGA2/index_html, formerly http:// rs6001585 0.039 1.000 innateimmunity.net/; Berkeley University PGA, http://pga.jgi-psf.org/; and rs739141 0.028 1.000 SouthWestern PGA, http://pga.swmed.edu/). TagSNPs were identified for rs7949 0.080 1.000 genotyping in patient cohorts using a linkage disequilibrium-based tag SNP IKBKB rs10958715 0.272 1.000 selection method (16) and using an r2 threshold of 0.65 for SNPs with rs3747811 0.759 1.000 a minor-allele frequency .5% yielding 59 SNPs in 19 cytosolic genes; re- rs5029748 0.508 1.000 ceptor and their ligands as well as downstream gene targets of NF-kB sig- RIPK1 rs11242823 0.693 1.000 naling were excluded (Table I). DNA was extracted from peripheral blood rs2326173 0.430 1.000 samples using a QIAamp DNA Blood Midi Kit (Qiagen, Mississauga, rs4959774 0.455 1.000 by guest on September 30, 2021 Ontario, Canada) and genotyped using the Illumina Golden Gate Assay at MAP2K3 rs7218577 0.297 1.000 the UBC Centre for Molecular Medicine and Therapeutics genotyping core rs8065233 0.260 1.000 facility (Luminex Molecular Diagnostics, Toronto, Ontario, Canada). Primer MAP2K6 rs12453226 0.066 1.000 probe sets are detailed in Supplemental Table IV. rs1548444 0.331 1.000 rs2586545 0.152 1.000 NFKBIA rs1957106 0.945 1.000 Clinical phenotype rs2233415 0.181 1.000 The primary outcome was 28-d mortality. Secondary outcomes were days rs3138055 0.338 1.000 alive and free of organ dysfunction during the first 28 d calculated according rs696 0.693 1.000 to the Brussels criteria (15). NFKBIB rs10775533 0.006 0.335 rs4803006 0.033 1.000 NFKBIE rs2282151 0.362 1.000 Biological plausibility experiments rs3799962 0.650 1.000 Microarray mRNA expression analysis in vitro. Lymphoblastoid DNA from rs730775 0.522 1.000 the Coriell Institute was genotyped for rs7222094 in 85 CEPH population RELA rs1049728 0.669 1.000 samples using Sanger sequencing of the region. Sequencing was performed rs11227247 0.722 1.000 at the McGill University and Geńome Que´bec Innovation Centre (Montreál, NFKB1 rs11722146 0.817 1.000 Que´bec, Canada). Primers for sequencing the region surrounding rs7222094 rs230521 0.557 1.000 are as follows: forward, 59-GGGTTCCCTATGGAGGAGAG-39; reverse, rs230542 0.627 1.000 59-CTGTCCAGCTCTCCAGGTTC-39. These 85 CEPH population lym- rs3774934 0.808 1.000 phoblastoid cell lines of known genotype for NIK rs7222094 were cultured Noncanonical in RPMI 1640 and subsequently stimulated in triplicate by the addition of TRAF2 rs10283820 0.349 1.000 Cytomix (9, 17–19) (2.5 ng/ml of each TNF-a, IL-1b, IFN-g [R&D rs10781520 0.661 1.000 Systems, Minneapolis, MN] and 12.5 mM CpG [Sigma-Aldrich, Oakville, rs2784069 0.402 1.000 Ontario, Canada]) for 6 h. RNA was extracted from the three stimulated TRAF6 rs2303439 0.152 1.000 samples as well as two control biological duplicates using the Qiagen rs5030411 0.772 1.000 RNeasy kit (Qiagen), and mRNA expression was measured using the rs540386 0.683 1.000 Human WG-6 v.3 expression chip (Illumina, San Diego, CA; Geńome NIK (MAP3K14) rs2074293 0.818 1.000 Que´bec Innovation Centre, Montreál, Que´bec, Canada). Microarray raw rs7222094 0.00024 0.014 data were normalized, and fold change was calculated using the Flexarray rs9972936 0.883 1.000 (version 1.4.1) package available from Geńome Que´bec (http://genome- CHUK rs11190421 0.050 1.000 quebec.mcgill.ca/FlexArray/license.php). MIAME compliant microarray rs11591741 0.234 1.000 data are publically available at GEO (GSE25543; http://www.ncbi.nlm.nih. rs3818411 0.658 1.000 gov/geo/). rs7903344 0.644 1.000 ELISA protein expression analysis in vitro. Twenty-six CEPH population NFKB2 rs1409312 0.442 1.000 lymphoblastoid cell lines of known genotype for NIK rs7222094 were rs3802681 0.867 1.000 cultured in RPMI 1640 and subsequently stimulated by the addition of rs7086205 0.226 1.000 The Journal of Immunology 2323

Table II. Hazard ratios of 28-d mortality in Caucasian patients with septic shock (Cox regression)

SPH Cohort (n = 453) VASST Cohort (n = 517) Parameter HR (95% CI) p Value HR (95% CI) p Value Age, per year 1.006 (1.003–1.009) 0.0036 1.019 (1.008–1.030) 0.0003 Female 0.9092 (0.670–1.123) 0.530 0.987 (0.717–1.348) 0.930 Surgical diagnosis 0.8244 (0.600–1.118) 0.220 0.859 (0.583–1.233) 0.420 NIK rs7222094 C allele 1.35 (1.116–1.635) 0.002 1.24 (1.002–1.524) 0.048

ELISA protein expression analysis in vivo. Briefly, whole-blood samples regression to test an additive model in the SPH septic shock cohort were drawn into chilled 7-ml EDTA Vacutainer tubes (BD, Mississauga, and then used the same analysis in the VASST septic shock cohort. Ontario, Canada), put on ice immediately, then spun at 3000 rpm for 15 min Patients in the SPH cohort who had the CC genotype of NIK 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 had a significantly increased hazard of 28-d mortality rs7222094 (30 TT and 30 CC) were randomly selected and assayed for compared with that of patients having the CT or TT genotypes of CXCL10 concentrations in duplicate by ELISA (R&D Systems). rs7222094 (hazard ratio [HR], 1.35; 95% confidence interval [CI], 1.12–1.64; p = 0.002 Caucasian only) (Table II). This finding was Statistical analysis replicated in the VASST cohort (HR, 1.24; 95% CI, 1.00–1.52; p = We assessed baseline characteristics using a x2 test for categorical data and 0.048 Caucasian only) (Table II). The results were similar for all a Kruskal–Wallis test for continuous data and then reported the median and patients with ethnicity included as a covariate in a Cox regression Downloaded from interquartile ranges. We then tested for association between SNP genotype model (Fig. 1, Table III). and 28-d mortality in the SPH discovery cohort using an Armitage trend Similarly, in an unadjusted univariate analysis, the C allele of test, as is commonly used for initial discovery surveys in genome-wide association studies (Table I). One SNP emerged as statistically significant rs7222094 TC was associated with mortality in SPH (mortality, after a Bonferroni correction for multiple comparisons. We then tested for Caucasian only: TT, 33.8%; CT, 42.4%; CC, 53.7%; p = 0.005; replication of this finding in the VASST cohort of septic shock patients. To mortality, all ethnicities: TT, 33.8%; CT, 43.2%; CC, 53.0%; p = correct for potentially confounding variables, including age, gender, an- 0.001), and a similar trend was observed in VASST (mortality, http://www.jimmunol.org/ cestry, and surgical versus medical diagnostic category, we used Cox regression. We then tested for association between secondary outcome Caucasian only: TT, 26.3%; CT, 34.7%; CC, 38.4%; p = 0.09; measures of days alive and free of organ failure using Kruskal–Wallis tests mortality, all ethnicities: TT, 27.5%; CT, 33.2%; CC, 40.8%; p = in both SPH and VASST cohorts. Vasopressin treatment by genotype (NIK 0.03). Allele frequencies of survivors versus nonsurvivors in both rs7222094 CT) interaction was assessed using logistic regression analysis Caucasian and all ethnicities are reported in Table IV. 3 (interaction statistics): P(Death) vasopressin + genotype + vasopressin In the SPH Caucasian cohort, patients having the CC genotype genotype. A Student t test was performed to test for differences between genetic of rs7222094 had greater baseline creatinine concentrations groups (rs7222094 CC versus TT) of CXCL10 ELISA concentrations. (p = 0.04) and significantly higher PaO2/FIO2 at baseline (p = Analyses used SPSS (version 16; SPSS, Chicago, IL), R statistical soft- 0.002) than patients having the CT or TT genotypes of rs7222094 ware package, and GraphPad Prism (version 5.02; GraphPad, La Jolla, (Table V). The only difference at baseline among VASST Cau- by guest on September 30, 2021 CA). casian patients was that patients having the CC genotype of rs7222094 had significantly lower platelet counts than those of Results patients having the CT or TT genotypes of rs7222094 (p = 0.02) Twenty-eight–day mortality in septic shock patients (Table V). Because the VASST cohort was a clinical trial com- Of the 59 tagSNPs in 19 genes in canonical and noncanonical NF- paring efficacy of vasopressin versus norepinephrine in septic kB pathways, one SNP, rs7222094 in NIK, was significantly as- shock, in a secondary analysis we tested for an interaction by sociated with 28-d mortality in the SPH discovery cohort (un- logistic regression between NIK rs7222094 and vasopressin corrected p = 0.00024, Bonferroni corrected p = 0.014) (Sup- treatment in Caucasian patients. We found no significant in- plemental Fig. 1, Table I). teraction (interaction statistic p = 0.462). Hardy–Weinberg equilibrium and minor allele frequencies of Organ failure in septic shock patients all SNPs genotyped are presented along with literature based minor allele frequencies in Supplemental Table I. Allele fre- Patients homozygous for the C allele of rs7222094 in both the SPH quencies of rs7222094 differed between ethnic groups within the and VASST Caucasian cohorts had more organ dysfunction as SPH and VASST cohorts (Supplemental Table II). Therefore, our defined by Brussels criteria compared with that in patients having primary analysis was limited to Caucasians only (SPH, n = 453, the CT or TT genotypes of rs7222094 (15). SPH patients with VASST, n = 517), and our secondary analysis of all patients in- rs7222094 CC genotype had significantly fewer days alive and cluded ethnicity as a covariate (SPH, N = 589; VASST, N = 616). free of renal dysfunction (p = 0.003) and fewer days alive and free To consider and correct for potential confounding variables due of acute renal replacement therapy (p = 0.008) as well as fewer to differences at baseline in septic shock patients, we used Cox days alive and free of hematological dysfunction (p = 0.011)

Table III. Hazard ratios of 28-d mortality in patients with septic shock (Cox regression)

SPH Cohort (N = 589) VASST Cohort (N =616) Parameter HR (95% CI) p Value HR (95% CI) p Value Age, per year 1.006 (1.003–1.009) 9.0 3 1024 1.017 (1.008–1.026) 2.0 3 1024 Female 0.868 (0.668–1.122) 0.28 1.004 (0.756–1.328) 0.98 European ancestry 0.962 (0.717–1.290) 0.76 0.870 (0.609–1.272) 0.46 Surgical diagnosis 0.937 (0.712–1.220) 0.63 0.800 (0.557–1.123) 0.20 NIK rs7222094 C allele 1.33 (1.126–1.573) 8.0 3 1024 1.25 (1.030–1.521) 0.024 2324 VARIANT IN NIK IS ASSOCIATED WITH SEPTIC SHOCK MORTALITY

Table IV. Allele frequencies in both SPH and VASST cohorts of survivors and nonsurvivors

VASST SPH Cohort Cohort

Group TCp Value TCp Value Survivors 0.57 0.43 0.005 0.52 0.48 0.09 (Caucasian) Nonsurvivors 0.45 0.55 0.45 0.55 (Caucasian) Survivors 0.52 0.48 0.001 0.49 0.51 0.03 (all ethnicities) Nonsurvivors 0.40 0.60 0.41 0.59 (all ethnicities)

CXCL10 mRNA production by lymphoblastoid cell lines in vitro The gene with the greatest difference (D) in fold change between Downloaded from 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 genotype compared with that of TT or TC (Supplemental Table III). CXCL10 protein production by lymphoblastoid cell lines

in vitro http://www.jimmunol.org/ Protein levels of CXCL10 were measured in 26 cell lines of known genotype for rs7222094. Cell lines homozygous for the C allele of FIGURE 1. Survival curves for patients with septic shock in the SPH rs7222094 produced less CXCL10 at both baseline and after in- and VASST cohorts by NIK genotype of rs7222094. Patients who were flammatory stimulation than that of cell lines homozygous for the homozygous minor (CC) (gray dashed line) experienced increased mor- T allele (p = 0.032 and p = 0.050, respectively) (Fig. 2). tality in both the SPH and VASST cohorts. CXCL10 protein production in VASST plasma samples Baseline plasma specimens of a random sample of patients with by guest on September 30, 2021 during the 28-d study period than those of patients having the septic shock from the VASST cohort were assayed in duplicate for CT or TT genotypes of rs7222094 (Table VI). Patients of the CXCL10 by ELISA, and as was found in both the control and VASST cohort with the rs7222094 CC genotype show the same stimulated lymphoblastoid cell lines, patients of the CC genotype trend toward more renal dysfunction (Table VI). The number of had significantly lower CXCL10 than that of the TT genotype patients affected by each type of organ dysfunction by genotypic (p = 0.017) (Fig. 3). group is outlined in Table VII. In the SPH cohort, patients with the rs7222094 CC genotype Discussion also had significantly fewer days alive and free of cardiovascular We found that patients of the CC genotype of NIK rs7222094 had dysfunction (p = 0.008), with correspondingly fewer days alive significantly increased mortality compared with that of patients and free of vasopressor support (p = 0.01), which was also seen as having the CT or TT genotypes of rs7222094 in two cohorts of a trend in the VASST cohort (Table VI). SPH cohort patients also patients who had septic shock. Specifically, Caucasian patients in experienced more hepatic and neurologic dysfunction (p = 0.007 the SPH cohort who had the CC genotype of NIK rs7222094 and p = 0.006, respectively) (Table VI). 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

FIGURE 2. CXCL10 protein concentrations in lymphoblastoid cell FIGURE 3. CXCL10 protein concentrations in septic shock patients of lines. Twenty-six lymphoblastoid cell lines of known genotype (13 TT and the VASST replication cohort. Baseline plasma samples from 60 VASST 13 CC) for rs7222094 were cultured and stimulated with Cytomix (2.5 ng/ septic shock patients of known genotype (30 TT and 30 CC) for rs7222094 ml of each TNF-a, IL-1b, IFN-g and 12.5 mM CpG) for 24 h. Supernatant were randomly selected and assayed for CXCL10 concentrations by was collected, and CXCL10 concentrations were measured by ELISA. Cell ELISA. Patients homozygous for the C allele had signiﬁcantly lower lines homozygous for the C allele produce signiﬁcantly less CXCL10 at concentrations of CXCL10 than those of patients who were homozygous both baseline (*p = 0.032) and under stimulation conditions (*p = 0.050). for the T allele. *p = 0.017. The Journal of Immunology 2325

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

Value with ethnicity included as a covariate in a Cox regression model. 0.73 0.67 0.005 0.52 0.92 0.68 0.27 p Also, patients of the CC genotype of rs7222094 of SPH experienced 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

= 133) cohort with the rs7222094 CC genotype showed the same trend 6.0 n 20.3 18.8 72.2 11.3 12.0 14.3 toward more renal dysfunction as found in the SPH cohort. 63 (50–73) 0.77 27 (22–32) 0.76 56 (51–62) 0.44 14 (10–17) 0.11 CC( NIK was ﬁrst discovered in human B cells, and hence we chose

a 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 canonical pathway stimuli (21, 22). In a recent study by Zarnegar VASST Cohort

=251) et al. (23), CXCL10 levels were dependent on NIK suggesting 8.0 9.6 n 22.7 22.3 58.6 10.0 18.7 noncanonical signaling. CXCL10 is transcribed in response to NF- 63 (51–72) 26 (22–31) 55 (50–61) 14 (12–18) CT( 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 Downloaded from supernatant of lymphoblastoid cell lines at baseline and after Cytomix stimulation (p = 0.032 and p = 0.050). Similarly,

=133) CXCL10 levels were signiﬁcantly lower in septic shock patients 9.0 9.8 19.6 19.6 54.1 10.5 21.8 of the CC genotype (p = 0.017), suggesting a biologically plau- 64 (51–74) 26 (22–32) 55 (48–61) 14 (11–17) sible explanation for our observations.

It was interesting to find that the patients with the NIK rs7222094 http://www.jimmunol.org/ 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 Value TT ( n 0.79 0.77 0.12 p 0.66 0.74 0.54 0.19 was measured in VASSTpatient samples. As mentioned previously, CXCL10 is a proinflammatory chemokine released during inflammatory 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. = 123) by guest on September 30, 2021 4.9 8.1 4.9 4.9 33.3 57.7 12.2 CXCL10 is a chemokine that instigates chemotaxis of activated 11 (8–15) 0.71 61 (49–73) 0.34 26 (20–33) 0.61 54 (49–59) 0.41 T cells and NK cells (24). We speculate that without enough CC ( n CXCL10 to drive recruitment of inflammatory cells, it is possible

a that these patients (who had the CC genotype of NIK rs7222094) have an immunological disadvantage and so have increased mortality of septic shock.

SPH Cohort The original characterization of NIK indicated that NIK was = 191) 6.8 3.1 7.9 n a powerful effector of canonical NF-kB signaling under TNF-a 29.8 69.1 10.0 19.9

11 (6–15) and IL-1b stimulation (20). Later studies were unable to conﬁrm 62 (48–73) 26 (20–31) 55 (50–58) CT ( 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 = 139) 5.8 3.6 5.8 (23, 28, 29). It is possible that the timing of experimental proce- n 30.2 66.2 11.5 17.3 dures is critical to our understanding of NIK, as many studies that 59 (46–71) 25 (20–30) TT ( 1.4 (0.0–2.9) 1.3 (0.0–3.0) 1.8 (0.0–4.9) 0.09 1.7 (0.8–3.6) 1.7 (1.0–3.3) 2.1 (1.2–4.3) 0.17 125 (80–194) 138 (84–196) 170 (109–232) 0.0015 193 (150–255) 197 (140–261) 185 (126–254) 0.70 157 (84–234) 178 (103–245) 144 (76–255) 0.13 168 (75–256) 174 (93–276) 136 (81–199) 0.02 125 (80–219) 148 (91–245) 180 (94–329) 0.04 141 (83–269) 150 (90–238) 167 (94–294) 0.35 115 (95–130) 114 (95–130) 115 (95–131) 0.86 124 (112–135) 129 (108–140) 126 (108–140) 0.95

13.7 (9.0–19.0) 15.0 (11.1–21.0) 14.0 (9.3–20.0)separated 0.055 the 14.6 (9.0–23.7) two pathways 13.8 (7.3–20.6) 11.8 and (7.0–19.4) excluded 0.07 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, 3

3 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 mol/l m

, torr degradation of p100 to p52 and consequently translocation of 2 Characteristic heterodimers to the nucleus (6, 30). It is conceivable that accu- /FiO

2 mulation of NIK over time is a key facet to its mechanism. Because NIK has been implicated in the host response to in- Mean arterial pressure, mm Hg 55 (50–60) Heart rate, bpm Central venous pressure, mm Hg 12 (8–15) WBC count, 103/mm Creatinine, Lactate, mmol/l Chronic pulmonary disease Chronic liver disease Chronic renal failure Chronic corticosteroid use Chronic heart failure Platelet count, 103/mm PaO Values shown are median and interquartile range. Cardiovascular variables day 1 Laboratory variables day 1 Surgical percentage Preexisting conditions, % Age, y Gender, percentage male APACHE II

a fection, we speculate that NIK could modulate the immune re-

Table V. Baseline characteristics by NIK genotype rs7222094 for Caucasian patients with septic shock in both SPH and VASST cohorts sponse during septic shock. NIK is important in the host response 2326 VARIANT IN NIK IS ASSOCIATED WITH SEPTIC SHOCK MORTALITY

Table VI. Days alive and free of organ dysfunction in Caucasian septic shock patients according to NIK rs7222094 genotype

SPH Cohorta VASST Cohorta

Parameter TT (n = 139) CT (n = 191) CC (n = 123) p Value TT (n = 133) CT (n = 251) CC (n = 133) p Value Organ dysfunction Cardiovascular 15 (13–16) 13 (12–15) 11 (9–13) 0.008 15 (14–17) 14 (12–15) 13 (11–15) 0.227 Respiratory 12 (10–14) 11 (10–13) 10 (8–12) 0.320 9 (7–10) 8 (7–9) 7 (6–9) 0.554 Renal 17 (15–19) 16 (14–17) 13 (11–15) 0.003 19 (17–21) 17 (16–18) 15 (14–17) 0.067 Hematologic 18 (16–20) 18 (17–20) 15 (13–17) 0.011 19 (18–21) 19 (17–20) 18 (16–20) 0.391 Hepatic 19 (17–21) 17 (16–19) 14 (12–17) 0.007 19 (17–21) 18 (17–20) 18 (16–20) 0.856 Neurologic 19 (17–20) 18 (16–19) 15 (13–17) 0.006 15 (13–17) 14 (13–15) 13 (11–15) 0.203 Artificial organ support Vasopressor 17 (15–19) 16 (14–17) 13 (11–15) 0.010 15 (14–17) 14 (12–15) 14 (12–15) 0.253 Ventilator 12 (10–14) 10 (9–12) 9 (7–11) 0.230 12 (10–13) 10 (9–12) 10 (8–12) 0.328 Renal replacement therapy 18 (16–20) 17 (15–18) 14 (12–16) 0.008 20 (18–22) 18 (17–19) 18 (16–20) 0.215 aValues shown are median and interquartile range. to numerous infections including respiratory syncytial virus [with Inhibitors of NIK have been synthesized for diseases such as evidence to suggest activation of both noncanonical and canonical multiple myeloma and other cancers, as anti-inflammatory agents Downloaded from signaling (31)], HIV (32), hepatitis B (33), Escherichia coli (34), for inflammatory diseases (41), and as a vaccine adjuvant (42). as well as the response to LPS (35). Our data suggest that rs7222094 may be of interest in randomized Several lines of evidence suggest why polymorphisms of NIK controlled trials of therapies for septic shock by defining risk may be predictors of outcome in septic shock (1). First, NIK stim- categories of patients and perhaps defining response to anti- ulates inflammation by upregulating the noncanonical (and possi- inflammatory agents.

bly the canonical) pathway of NF-kB activation. Second, NIK is This study has several limitations. The analysis of the SPH and http://www.jimmunol.org/ required for optimal IgG production by lymphocytes (36). Third, VASST cohorts was performed retrospectively. The association NIK may modulate blood pressure: NIK has a role in the mechanism of rs7222094 with mortality, organ dysfunction, and CXCL10 of action of the calcium channel blocker nifedipine (37), and angio- levels does not prove a causal link. Furthermore, CXCL10 was used tensin II induces inflammation through NIK activation of the non- as a marker for differences in NIK-induced NF-kB signaling. We canonical NF-kB pathway (38). However, polymorphisms of NIK do not currently know of, nor did we test for, the influence of have not been widely studied. In a survey of 181 SNPs of 17 genes CXCL10 itself on organ dysfunction or mortality. In view of the in the NF-kB pathway, polymorphisms of NIK were not associated large number of genes connected in some way to NF-kB signaling, with rheumatoid arthritis susceptibility (39). Notably, rs4792847 of we chose to limit our analysis to genes of the cytosolic members NIK was found to be significantly associated with response to anti- of the NF-kB pathway, excluding receptors and downstream tar- by guest on September 30, 2021 TNF treatment in rheumatoid arthritis. Patients with GG genotype gets. Therefore, this analysis does not include all potentially had the greatest improvement at the 6-mo mark in a discovery co- functional variants, in particular those recently published after the hort; however, this effect was not found in a replication cohort (40). design of this study (43–45). The GG genotype of rs4792847 (patients who may have had a more In conclusion, we found that NIK rs7222094 was consistently favorable response to anti-TNF) is in high linkage disequilibrium and significantly associated with mortality in two independent with the TT genotype of rs7222094 (r2 = 1.0). This is consistent cohorts of patients who had septic shock. Patients homozygous with our observation of a protective effect of the TT genotype for the CC genotype of rs7222094 had increased mortality and (i.e., lower mortality and higher CXCL10 production) seen in our also experienced more renal and hematological failure in the study of patients who had septic shock and replicated in our in vitro SPH cohort of Caucasian patients with a similar trend in VASST experiments. Caucasian patients. Furthermore, we found that lymphoblastoid To our knowledge, NIK has not been implicated in septic shock cell lines homozygous for CC of rs7222094 produced less CXCL10 to date. However, NIK is a drug target for other diseases (5). 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) TT (n = 133) CT (n = 251) CC (n = 133) Parameter N (%) N (%) N (%) p Value N (%) N (%) N (%) p Value Organ dysfunction Cardiovascular 139 (100) 191 (100) 123 (100) NA 133 (100) 251 (100) 133 (100) NA Respiratory 132 (95.0) 183 (95.8) 116 (94.3) 0.830 130 (97.7) 246 (98.0) 133 (100) 0.240 Renal 81 (58.3) 133 (69.6) 97 (78.9) 0.002 76 (57.1) 160 (63.7) 92 (69.2) 0.124 Hematologic 71 (51.1) 119 (62.3) 86 (69.9) 0.007 74 (55.6) 139 (55.4) 87 (65.4) 0.134 Hepatic 65 (46.8) 108 (56.5) 81 (65.9) 0.008 71 (53.4) 139 (55.4) 72 (54.1) 0.927 Neurologic 95 (68.3) 139 (72.8) 96 (78.0) 0.212 110 (82.7) 217 (86.5) 123 (92.5) 0.060 Artificial organ support Vasopressor 125 (89.9) 178 (93.2) 114 (92.7) 0.531 133 (100) 251 (100) 133 (100) NA Ventilator 134 (96.4) 185 (96.9) 116 (94.3) 0.509 128 (96.2) 241 (96.0) 128 (96.2) 0.991 Renal replacement therapy 62 (44.6) 112 (58.6) 83 (67.5) 0.0007 59 (44.4) 134 (53.4) 75 (56.4) 0.115 NA, The p value was not calculated because all patients in each genotypic group are affected. 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