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IL17A genetic variation is associated with altered susceptibility to Gram-positive infection and mortality… Nakada, Taka-aki; Russell, James A; Boyd, John H; Walley, Keith R Oct 25, 2011

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RESEARCH Open AccessIL17A genetic variation is associated with alteredsusceptibility to Gram-positive infection andmortality of severe sepsisTaka-aki Nakada, James A Russell, John H Boyd and Keith R Walley*AbstractIntroduction: Interleukin 17A (IL17A) plays a key role in host defense against microbial infection including Gram-positive bacteria. Genetic factors contribute to the host defense, but the role of IL17A single nucleotidepolymorphisms (SNPs) has not yet been investigated in severe sepsis. Therefore, we hypothesized that SNPs in theIL17A gene alter susceptibility to infection and clinical outcome of severe sepsis.Methods: We tested for the association of IL17A SNPs with susceptibility to infection and clinical outcome ofsevere sepsis using two cohorts of European ancestry (derivation cohort, St Paul’s Hospital (SPH), n = 679;validation cohort, Vasopressin and Septic Shock Trial (VASST), n = 517). The primary outcome variable wassusceptibility to Gram-positive bacterial infection. The secondary outcome variable was 28-day mortality.Results: Of four tested IL17A tag SNPs (rs4711998, rs8193036, rs2275913, rs1974226), rs1974226 SNP was associatedwith altered susceptibility to Gram-positive infection in the derivation SPH cohort (corrected P = 0.014). Patientshaving the rs1974226 GG genotype were more susceptible to Gram-positive infection, compared to AG/AAgenotype in the two cohorts of severe sepsis (SPH, P = 0.0036, odds ratio (OR) 2.19, 95% confidence interval (CI)1.28-3.72; VASST, P = 0.011, OR 1.95, 95%CI 1.16-3.27) and in the subgroup having lung infection (P = 0.017, OR1.90, 95%CI 1.12-3.21). Furthermore, the IL17A rs1974226 G allele was associated with increased 28-day mortality intwo cohorts (SPH, adjusted OR 1.44, 95%CI 1.04-2.02, P = 0.029; VASST, adjusted OR 1.67, 95%CI 1.17-2.40, P =0.0052).Conclusions: IL17A genetic variation is associated with altered susceptibility to Gram-positive infection and 28-daymortality of severe sepsis.IntroductionInterleukin 17A (IL17A) plays a key role in host defenseagainst infection and development of inflammatory dis-eases [1-5]. IL17A production is increased as an innateresponse to bacterial infection in human immune cells[6,7], and elevated serum IL17A levels are observed inhuman [8] and animal models of sepsis [9,10]. In con-trast, deficiency of the IL17 response impairs bacterialclearance, delays recovery from infection [1,11] andincreases susceptibility to infection [12-15].Recently two autosomal mutations of IL17 pathwaygenes were identified in patients with chronicmucocutaneous candidiasis disease, which is character-ized by recurrent or persistent mucocutaneous infec-tions by Candida albicans and Staphylococcus aureus[16]. The mutations, single nucleotide substitutions inthe coding region of IL17 genes, abolish the IL17immune response leading to persistent infections [16].IL17 deficiency appears to differentially increase sus-ceptibility to infection including S. aureus [12] and C.albicans [13-15]. These discoveries highlight the impor-tance of the IL17A gene on infection and, in particular,on Gram-positive and fungal infections. Severe sepsis isa leading cause of death in intensive care units (ICUs)[17]. Multiple studies have shown that single nucleotidepolymorphisms (SNPs) in important immune responsegenes alter susceptibility to infection and/or outcome ofsevere sepsis [18-21], but the role of IL17A SNPs has* Correspondence: Keith.Walley@hli.ubc.caUniversity of British Columbia, Critical Care Research Laboratories, Heart +Lung Institute, St. Paul’s Hospital, 1081 Burrard Street, Vancouver, BC, V6Z1Y6, CanadaNakada et al. Critical Care 2011, 15:R254http://ccforum.com/content/15/5/R254© 2011 Nakada et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.not yet been investigated in severe sepsis. Gram-positiveinfections, including S. aureus, are very common patho-gens isolated from severe sepsis patients [22,23], whilefungal infections are somewhat less common. Thus, wetested the hypothesis that IL17A genetic polymorphismsalter susceptibility of patients to Gram-positive infectionusing isolated pathogens from patients in two largesevere sepsis cohorts of European ancestry. We subse-quently tested for association of IL17A genotype withsurvival from severe sepsis.Materials and methodsPatientsSt Paul’s Hospital (SPH) CohortSevere sepsis was defined by the presence of two ormore diagnostic criteria for the systemic inflammatoryresponse syndrome [24], proven or suspected infection,and at least one new organ dysfunction by Brussels cri-teria [25]. Septic shock was defined by severe sepsis plushypotension [25] despite adequate fluid resuscitation.All patients admitted to the ICU at St Paul’s Hospital(SPH) in Vancouver, Canada between July 2000 and Jan-uary 2004 were screened (n = 1, 626). Of these, 691patients of European ancestry had severe sepsis, wereextensively phenotyped [26], and had DNA available.Twelve patients, who were also enrolled in the Vaso-pressin and Septic Shock Trial (VASST) [25] wereexcluded from this cohort to avoid duplication. Thus,679 severe sepsis patients, including 453 septic shockpatients, were analyzed in this study. The InstitutionalReview Board at SPH and the University of BritishColumbia (UBC) approved the study. For this fullyanonymized analysis the UBC and SPH Review Boardswaived the need for informed patient consent.Vasopressin and Septic Shock Trial (VASST) CohortVASST was a multicenter, randomized, double blind,and controlled trial evaluating the efficacy of vasopressinversus norepinephrine in a total of 778 septic shockpatients [25]. Of these, 517 patients of European ances-try had DNA available and were included in the analysis.The research ethics boards of all participating institu-tions approved this trial and written informed consentwas obtained from all patients or their authorized repre-sentatives. The research ethics board at the coordinatingcenter (UBC) approved the genetic analysis.MicrobiologyWe assessed all microbiological cultures taken at thetime of admission to the ICU for patients in the SPHcohort. Results from cultures that were collected from 48hours prior to enrollment to 48 hours after enrollment inthe VASST cohort [25] were analyzed. We defined bac-terial infection in this study as clinical evidence of infec-tion associated with positive microbiological culture,excluding the cultures judged to be positive due to con-tamination or colonization by the attending physician.We categorized positive microbial cultures into Gram-positive bacteria, Gram-negative bacteria or fungus sincethese are the three major pathogen categories indentifiedin previous studies of sepsis in ICUs [23,27], andincluded in the analysis. The source of a positive culturewas categorized as lung (sputum), blood, abdomen (peri-toneal fluid, abscess drainage, biliary tract), skin (soft tis-sues or wounds), genitourinary system (urine) or other.Selection of SNPs and genotypingTag SNPs for IL17A gene were identified using a multi-marker tagging algorithm of Tagger software [28]. Toidentify tag SNPs, we used the sequence of the IL17Agene plus 2, 000 bp of 5’ upstream sequence and consid-ered all SNPs with a minor-allele frequency (MAF) >10% in the HapMap Phase 2+3 Utah residents withNorthern and Western European ancestry from theCentre d’Etude du Polymorphisme Humain (CEPH) col-lection (CEU) data [29] composed of 174 individualsand chose an r2 threshold of 0.5. This identified four tagSNPs (rs4711998, rs8193036, rs2275913, rs1974226),which were genotyped in the SPH derivation cohort.IL17A rs1974226 was genotyped in the VASST replica-tion cohort. DNA was extracted from the buffy coat ofdiscarded blood samples using a QIAamp DNA maxi kit(Qiagen, Mississauga, ON, Canada) and genotyped usingthe Illumina Golden Gate assay (Illumina, San Diego,CA). We performed quality control of the genotypingusing 5% repeat and HapMap Coriell DNA controls. For5% repeat genotyping and Coriell controls, the concor-dance rate was > 99%. We did not discard any of theIL17A SNPs based on the quality control metrics.Statistical analysisThe primary outcome variable was susceptibility toGram-positive infection. To screen the four SNPs of theIL17A gene in the derivation cohort we used an Armi-tage’s trend test followed by Bonferroni correction formultiple comparisons. Power for the genetic associationof susceptibility to infection by Gram-positive bacteriawas calculated using the Genetic Power Calculator [30].We tested a joint analysis across the SPH and VASSTcohorts on genetic susceptibility to Gram-positive,Gram-negative infection and Gram-positive infection bysite of infection using a logistic regression controlling forthe cohort. For the secondary analysis, we chose logisticregression to test for the genetic effect on 28-day mortal-ity to allow for correction of potential confounding fac-tors, including age, gender, surgical versus medicalprimary diagnosis and septic shock as covariates.We tested for differences in baseline characteristics bygenotype using a chi-square test for categorical data andNakada et al. Critical Care 2011, 15:R254http://ccforum.com/content/15/5/R254Page 2 of 9a Kruskal Wallis test for continuous data. We tested forHardy-Weinberg equilibrium using a chi-square test(threshold P-value < 0.05 was considered significant).We tested for differences in survival curves using a log-rank test for trend. R2 measure of linkage disequilibriumwas calculated using HapMap Phase 3 genotyping data(Han Chinese in Beijing (HCB), Japanese in Tokyo(JPT), CEU) in the genome variation server [31]. Differ-ences were considered significant using a two-tailed P <0.05. Analyses were performed using R (version 2.8.1)[32] and SPSS (SPSS, version 16, Chicago, IL) statisticalsoftware packages.ResultsA total of 679 severe sepsis patients of European ances-try in the derivation SPH cohort were successfully geno-typed for four tag SNPs of IL17A. Patients with apositive microbiological culture (n = 301) had similarbaseline characteristics to the entire population in thederivation cohort (Table 1) and had similar allele fre-quencies to those of HapMap European ancestry data(Table 2). IL17A is reported to be essential for hostdefense against Gram-positive bacterial infection [1].We first tested for the association of four SNPs withsusceptibility to Gram-positive infection using the Armi-tage trend test for the additive model in the derivationcohort (Table 2). Of the four SNPs, the major G alleleof IL17A rs1974226 G/A was significantly associatedwith increased susceptibility to infection by Gram-positive bacteria (percentages of positive culture: AA38.5%, AG 46.4%, GG 64.2%, uncorrected P = 0.0037,Bonferroni corrected P = 0.014) (Table 2).Whether the observation that IL17A rs1974226 geno-type altered susceptibility to infection, is specific toGram-positive bacteria is unknown. Therefore, we nexttested for genetic association of altered susceptibility toinfection by three pathogen categories including Gram-positive bacteria, Gram-negative bacteria and fungus inthe SPH derivation cohort. Due to the small sample sizeof the minor homozygote AA genotype patients whohad a positive culture (SPH AA genotype; Gram-positiven = 5, Gram-negative n = 8, fungus n = 0, mixed n = 1,total n = 14), we compared AA+AG genotype versusGG genotype in this analysis. In the derivation SPHcohort, the GG genotype patients had increased suscept-ibility to Gram-positive bacterial infection (AA+AG vs.GG, Gram-positive, P = 0.0036, odds ratio (OR) 2.19,95% confidence interval (CI) 1.28-3.72) and decreasedsusceptibility to Gram-negative bacterial infection (AA+AG vs. GG, Gram-negative, P = 0.0086, OR 0.49 95%CI 0.29-0.84) (Figure 1). We subsequently tested forreplication using the validation VASST cohort of Eur-opean ancestry, which was successfully genotyped forIL17A rs1974226. VASST patients with a positive micro-biological culture (n = 282) had a similar allele fre-quency (MAF = 0.195) compared to HapMap and SPH(Table 1) and were in Hardy-Weinberg equilibrium (P =0.63). In accord with the observation in the derivationTable 1 Characteristics of culture-positive patients in two cohorts of severe sepsisSPH VASSTCulture Positive All Culture Positive All(n = 301) (n = 679) (n = 282) (n = 517)Age-years 58 (45-71) 59 (46-72) 63 (50-73) 63 (51-73)Gender -% male 65.4 66.0 60.3 60.9APACHE II 24 (18-30) 23 (18-29) 26 (21-31) 26 (21-32)Surgical -% 26.6 28.7 19.1 21.3Septic shock-number (%) 208 (69.1) 453 (66.7) 282 (100) 517 (100)Pathogens-number (%)Gram-positive bacteria alone 152 (50.5) 115 (40.8)Gram-negative bacteria alone 100 (33.2) 82 (29.1)Fungus alone 9 (3.0) 37 (13.1)Mixed 40 (13.3) 48 (17.0)Source of a positive culture-number (%)Lung 126 (41.9) 140 (49.6)Blood 115 (38.2) 131 (46.5)Abdomen 20 (6.6) 53 (18.8)Skin and soft tissue 21 (7.0) 36 (12.8)Genitourinary system 16 (5.3) 17 (6.0)Other 3 (1.0) 29 (10.3)APACHE, Acute Physiology and Chronic Health Evaluation; SPH, St. Paul’s Hospital; VASST, Vasopressin and Septic Shock TrialData are median (interquartile range) for continuous variables.Nakada et al. Critical Care 2011, 15:R254http://ccforum.com/content/15/5/R254Page 3 of 9cohort, patients having the GG genotype had increasedsusceptibility to Gram-positive bacterial infection com-pared to those with the AA+AG genotype in the replica-tion cohort (P = 0.011, OR 1.95, 95%CI 1.16-3.27)(Figure 1). When we calculated power to detect an asso-ciation between IL17A rs1974226 genotype (GG versusAG/AA) and Gram-positive infection, we used theobserved prevalences (Gram-positive bacteria, 0.505(SPH), 0.408 (VASST)), observed allele frequencies anda relative risk = 2.0. We found that our study had a99.8% power for SPH and 98.0% power for VASST(alpha = 0.05) of detecting a genotype effect. Joint analy-sis across the SPH and VASST cohorts using a logisticregression controlling for the cohorts yields the sameconclusion for Gram-positive bacterial infection (AA+AG versus GG, P = 1.4 × 10-4, OR 2.06, 95%CI 1.42-2.99). A non-significant trend in the same direction ofaltered susceptibility to Gram-negative infection wasobserved in the VASST cohort (P = 0.13, OR 0.66, 95%CI 0.39-1.13). In the joint analysis across the SPH andVASST cohorts, the GG genotype patients haddecreased susceptibility to Gram-negative bacterialinfection (AA+AG versus GG, P = 0.0035, OR 0.57, 95%CI 0.39-0.83). There was no difference of genetic sus-ceptibility to fungal infection in the two cohorts (AA/AG versus. GG, SPH, 4.9% versus 2.8%, P = 0.33;VASST, 15.3% versus 12.0%, P = 0.43), however thenumber of patients having a fungal infection was low sothat this negative result has limited statistical power.We further tested for the altered genetic susceptibilityto Gram-positive bacteria by site of infection (Table 1).We combined two cohorts due to smaller subset samplesize and analyzed using a logistic regression controllingfor the cohort. The GG genotype patients had signifi-cantly increased Gram-positive bacterial infections com-pared to the AA/AG genotype in the lung (P = 0.017, ORTable 2 Allele frequency and association of interleukin17A polymorphisms with Gram-positive bacterial infection inthe derivation cohort of severe sepsisLocationabase pairMajor/minor allele MAF (HapMapb) HWEP valuecOdds Ratio (95% CI) P valued(Corrected P valuee)rs4711998 -832 G/A 0.326 (0.217) 0.025 1.13 (0.80-1.60) 0.48rs8193036 -692 T/C 0.287 (0.241) 0.53 1.41 (0.97-2.05) 0.073rs2275913 -399 G/A 0.331 (0.383) 0.79 1.45 (1.01-2.10) 0.045 (0.18)rs1974226 +4150 (3’ UTR) G/A 0.182 (0.179) 0.10 1.89 (1.22-2.92) 0.0037 (0.015)HWE, Hardy-Weinberg equilibrium; MAF, minor allele frequency; UTR, untranslated region.aRelative distance to transcription start site; bMinor allele frequency of HapMap Utah residents with Northern and Western European ancestry from the Centred’Etude du Polymorphisme Humain collection (CEU) data; cHardy-Weinberg equilibrium P values for culture positive patients were calculated using the chi-squaretest; dP values were calculated using Armitage’s test and corrected for multiple comparison by a Bonferroni teste.Figure 1 Frequency of patients who had Gram-positive or Gram-negative bacterial culture-positive by IL17A rs1974226 genotype intwo cohorts of severe sepsis. IL17A rs1974226 GG genotype patients had an increased culture-positive rate of Gram-positive bacteriacompared to the AA/AG genotype patients and a decreased rate of Gram-negative bacteria in two cohorts (AA/AG versus GG, SPH, Gram-positive P = 0.0036, Gram-negative P = 0.0086; VASST, Gram-positive P = 0.011, Gram-negative P = 0.13). SPH, St Paul’s Hospital; VASST,Vasopressin and Septic Shock Trial. P values were calculated using chi-square test. *P < 0.05Nakada et al. Critical Care 2011, 15:R254http://ccforum.com/content/15/5/R254Page 4 of 91.90, 95%CI 1.12-3.21), and non-significant trends in thesame direction were observed in the other sites (blood,P = 0.53; abdomen, P = 0.91; skin and soft tissue (SST),P = 0.22; genitourinary system (GU), P = 0.25) (Figure 2).We next tested whether the IL17A rs1974226 alteredthe 28-day mortality of septic shock in the two cohorts.Because VASST only included septic shock patients, welimited our analysis of the SPH cohort to septic shockin order to examine cohorts of similar overall severity ofillness. There was no baseline difference by the genotypeof rs1974226 in age, gender, Acute Physiology andChronic Health Evaluation (APACHE) II, surgical versusmedical, pre-existing conditions, physiological or labora-tory variables in the SPH cohort (Table 3). In theVASST cohort, patients who had rs1974226 GG or AGgenotype had a decreased PaO2/FIO2 ratio compared toAA genotype in parallel with an increased rate of GG orAG genotype patients of chronic lung disease (Table 3).The GG genotype patients had increased mortality over28 days compared to the AG or AA genotype in the twocohorts of septic shock (Figure 3, SPH, P = 0.029;VASST, P = 0.010, log-rank test for trend: Table 4, GFigure 2 Frequency of Gram-positive bacteria infection by site.Patients who had the GG genotype of IL17A rs1974226 SNP hadsignificantly increased Gram-positive infection compared to AA/AGgenotype in the lung, and non-significant trends in the samedirection were observed in other sites (AA/AG versus GG, SPH+VASST, lung, P = 0.017; blood, P = 0.53; abdomen, P = 0.91; skinand soft tissue [SST], P = 0.22; genitourinary system [GU], P = 0.25).SPH, St Paul’s Hospital; VASST, Vasopressin and Septic Shock Trial. Pvalues were calculated using a logistic regression controlling for thecohort. *P < 0.05Table 3 Baseline characteristics in two cohorts of severe sepsis patients by the genotype of IL17A rs1974226polymorphismSPH (n = 679) VASST (n = 517)IL17A rs1974226 G/A AA AG GG AA AG GG(n = 39) (n = 174) (n = 466) P (n = 19) (n = 162) (n = 336) PAge-years 60(47-73) 60(47-71) 59(46-72) 0.96 60(47-71) 66(51-74) 63(51-72) 0.57Gender -% male 76.9 60.3 67.2 0.089 57.9 55.6 63.7 0.21APACHE II 21(17-28) 23(17-29) 23(18-29) 0.74 25(19-35) 26(21-32) 27(22-31) 0.63Surgical -% 30.8 32.2 27.3 0.45 21.1 22.8 20.5 0.84Septic shock-n(%) 26 (5.7) 125 (27.6) 302 (66.7) 0.24 19 (3.7) 162 (31.3) 336 (65.0) NAPreexisting disease-n(%)Chronic heart failure 2(5.1) 13(7.5) 25(5.4) 0.59 1(5.3) 13(8.0) 27(8.0) 0.91Chronic lung disease 5(12.8) 41(23.6) 79(17.0) 0.10 1(5.3) 39(24.1) 55(16.4) 0.037Chronic liver disease 4(10.3) 16(9.2) 49(10.5) 0.89 1(5.3) 15(9.3) 36(10.7) 0.69Chronic renal failure 1(2.6) 9(5.2) 15(3.2) 0.47 2(10.5) 16(9.9) 36(10.7) 0.96Vasopressin-n(%) 3(7.7) 27(15.5) 63(13.5) 0.43 9(47.4) 86(53.1) 168(50.0) 0.77Corticosteroidsa -n(%) 9(23.1) 68(39.1) 156(33.5) 0.13 11(57.9) 85(52.5) 184(54.8) 0.84Corticosteroids-day 4(1-20) 6(2-16) 4(2-12) 0.61 10(6-21) 8(5-16) 8(4-14) 0.64Activated protein C-n(%) 1(2.6) 4(2.3) 22(4.7) 0.34 0(0) 27(16.7) 43(12.8) 0.11Variables-Day1Body temperature-C° 37.8(37.1-38.5) 37.9(37.0-38.6) 37.9(36.6-38.6) 0.73 38.8(37.6-39.3) 38.5(37.5-39.1) 38.5(37.6-39.3) 0.85Heart rate -/min 115(105-120) 106(95-126) 112(95-130) 0.67 126(107-140) 128(115-140) 125(106-137) 0.12MAP-mmHg 59(52-71) 57(51-65) 58(52-68) 0.34 58(51-63) 56(50-61) 55(50-61) 0.60WBC-103/mm3 12.9(9.0-17.1) 15.0(9.3-21.7) 13.5(9.3-18.9) 0.075 14.3(4.6-19.6) 13.4(8.3-19.9) 13.9(7.7-21.3) 0.69Platelet-103/mm3 149(97-183) 191(113-261) 171(95-259) 0.091 150(123-224) 145(74-246) 165(86-263) 0.44PaO2/FIO2 ratio-mmHg 162(106-234) 154(106-224) 158(98-229) 0.84 233(182-317) 198(153-263) 190(131-251) 0.040Creatinine -μmol/L 165(73-295) 112(73-240) 126(80-231) 0.50 190(96-297) 149(90-228) 154(90-270) 0.38APACHE, Acute Physiology and Chronic Health Evaluation; MAP, mean arterial pressure; n, number; NA, not applicable; SPH, St Paul’s Hospital; VASST, Vasopressinand Septic Shock Trial; WBC, white blood cell count.aCorticosteroids, low dose corticosteroid.Data are median (interquartile range) for continuous variables. P values were calculated with the use of chi-square test and Kruskal Wallis test.Nakada et al. Critical Care 2011, 15:R254http://ccforum.com/content/15/5/R254Page 5 of 9allele; SPH (septic shock), adjusted OR 1.44, 95%CI1.04-2.02, P = 0.029; VASST, adjusted OR 1.67, 95% CI1.17-2.40, P = 0.0052, logistic regression). In addition,repeating the analysis of the SPH cohort including allpatients in a logistic regression analysis adjusted byincluding septic shock as a covariate yielded the sameconclusions (SPH (severe sepsis), adjusted OR 1.35, 95%CI 1.01-1.80 P = 0.042).DiscussionWe found that patients who had the GG genotype ofIL17A rs1974226 G/A SNP had increased susceptibilityto Gram-positive bacterial infection in the derivationcohort and this result was replicated in the validationcohort. In addition, this genotype was significantly asso-ciated with adverse clinical outcome of severe sepsis.We found that the G allele of the IL17A SNP was asso-ciated with increased 28-day mortality in both cohortsof severe sepsis/septic shock patients.IL17A is an essential cytokine for host defense againstbacteria [1,9], which is produced by a variety of cells[2,3] including T cells (T helper 17, gδ T, NKT cell),lymphoid-tissue inducer-like cells [33], neutrophils [34]and intestinal Paneth cell [35]. Cells triggered bymicrobes secrete IL17A, which is then recognized by anIL17 receptor [2-4]. Subsequent cellular signaling acti-vates inflammatory pathways including NF-kB andMAPK/AP-1, which invoke production of pro-inflamma-tory cytokines, chemokines and antimicrobial peptides,which induce inflammation required for host defense[2-4]. Human IL17A-producing cells increase IL17Aproduction against bacteria or their toxins [6,7]. SerumIL17A levels were elevated in bacterial sepsis patientswith acute respiratory distress syndrome (ARDS) [8] andin animal models of abdominal bacterial infection [9,10].IL17 deficiency in mice impairs microbial clearance,delays recovery from infection [1,11] and increases sus-ceptibility to infection including S. aureus [12] and C.albicans [13-15]. In accord with the observations inIL17 deficient mice, low birth weight infants who hadblood stream infections had deceased blood IL17 levelscompared to those without blood stream infections [36].These results are in accord with our current observa-tions that severe sepsis patients who had the IL17Ars1974226 GG genotype had increased susceptibility toGram-positive infection.The human IL17A gene is composed of three exons(two introns) and located on chromosome 6p12 cover-ing 4, 252 bases of genomic DNA. The transcript (1,859 bp) has a relatively long 3’UTR region (1, 345 bp),where the rs1974226 SNP is located. Since the 3’UTR isinvolved in regulation of gene expression, such asmRNA stability and/or degradation as well as translationefficiency [37,38], a potential mechanism of thers1974226 effect is alteration of gene regulation. Alter-natively, other SNPs in high linkage disequilibrium withthis SNP may have a biological function. In the Cauca-sian population the at-risk allele (G) is the major alleleof rs1974226 G/A, potentially marking a haplotype thatincludes many rare functional SNPs that increase risk,or the rarer A allele may actually be protective. Of thefour IL17A SNPs screened in this study, two IL17AFigure 3 Survival curves over 28 days by IL17A rs1974226genotype in two cohorts of septic shock. Patients with IL17Ars1974226 GG genotype had increased mortality in the SPH andVASST cohort of septic shock compared to the AG or AA genotypepatients (SPH P = 0.029, VASST, P = 0.010). SPH, St Paul’s Hospital;VASST, Vasopressin and Septic Shock Trial. P values were calculatedusing a log-rank test for trend.Nakada et al. Critical Care 2011, 15:R254http://ccforum.com/content/15/5/R254Page 6 of 9SNPs (rs8193036 and rs2275913) were associated withsusceptibility to inflammatory diseases, such as pediatricasthma (rs8193036, risk CC genotype, Taiwan) [39],ulcerative colitis (rs2275913, risk A allele, Japan) [40]and rheumatoid arthritis (rs2275913, risk GG allele,Norway and New Zealand) [41]. These reported SNPsare not in high linkage disequilibrium with rs1974226(rs8193036 r2 = 0.028 (HapMap HCB), rs2275913, r2 =0.028 (HapMap JPT), r2 = 0.135 (HapMap CEU)). Thus,our finding regarding rs1974226 is not identical to theseprevious reports but similarly leads to the conclusionthat genetic variation in the IL17A gene alters outcomefrom a variety of inflammatory disorders.The GG genotype was associated with increased sus-ceptibility to Gram-positive bacterial infection in twocohorts of severe sepsis patients, whereas a trendtowards decreased susceptibility to Gram-negative bac-teria was also observed. Genetic associations of patho-gen specific susceptibility have been documented inpattern recognition receptors (PRRs) such as Toll-likereceptors (TLRs) [42]. The potential mechanism of thedifferent pathogen-specific susceptibility may be differ-ence of PRRs.IL17 induces neutrophil recruitment in the airways [43],which is an important mechanism of host defense for thelung. In accordance with this, we found that thers1974226 GG genotype patients had significantlyincreased susceptibility to Gram-positive infection in thelung. While this association was significant for the lung,associations were not significant for other sites. Thismight be due to the small sample sizes of infections foundin other sites. Other limitations of this study are, first, thatwe did not investigate mechanisms of action such asaltered IL17A gene expression or alternative splicing bythe IL17A rs1974226 SNP. Thus, further investigationregarding the genetic effect on IL17A mRNA structureand mRNA expression levels and protein levels wouldstrengthen the results of this study. Second, the GG geno-type had increased susceptibility to Gram-positive infec-tion and 28-day mortality, but the causal link is notproven in this study. Third, this study finding of an asso-ciation of the IL17A rs174226 GG genotype with increasedsusceptibility to a category of Gram-positive bacteriawould be strengthened by testing for an association with aspecific Gram-positive bacterial pathogen, such as S. aur-eus, in further larger studies.Decreased susceptibility to Gram-negative bacteriaobserved in the GG genotype patients in the derivationcohort did not replicate; this needs further replicationtests in larger cohorts.ConclusionsTo conclude, the IL17A rs1974226 GG genotype is asso-ciated with increased numbers of Gram-positive infec-tions and increased 28-day mortality in severe sepsispatients.Key messages• Patients of European ancestry having the IL17Ars1974226 GG genotype were more susceptible toGram-positive infection, compared to those havingthe AG/AA genotype in the derivation and valida-tion cohorts of severe sepsis.• The IL17A rs1974226 GG genotype patients hadsignificantly increased Gram-positive bacterial infec-tion compared to the AA/AG genotype in the sub-group having lung infection.• The IL17A rs1974226 G allele was associated withincreased 28-day mortality in both cohorts of severesepsis/septic shock patients.AbbreviationsAPACHE: Acute Physiology and Chronic Health Evaluation; ARDS: Acuterespiratory distress syndrome; GU: genitourinary system; HWE: Hardy-Weinberg equilibrium; IL17A: Interleukin 17A; MAF: Minor-allele frequency;SST: Skin and soft tissue; SNPs: Single nucleotide polymorphisms; SPH: StPaul’s Hospital; UTR: Untranslated region; VASST: Vasopressin and SepticShock Trial.AcknowledgementsThis work was supported by the Heart and Stroke Foundation. TN is aCanadian Institute of Health Research IMPACT Postdoctoral Fellow. JHB is aNational Sanitarium Association Scholar.Authors’ contributionsTN and KRW contributed to study conception and design, acquisition ofdata, statistical analysis, interpretation of data, and drafting of themanuscript. JAR and JHB contributed to study conception and design,acquisition of data, interpretation of data, and drafting of the manuscript. Allthe authors read and approved the final manuscript.Table 4 Logistic regression analysis of 28-day mortalitySPH VASSTOdds Ratio (95% CI) P Odds Ratio (95% CI) PAge-yearsr 1.029 (1.016-1.042) 1.4 × 10-5 1.021 (1.008-1.034) 0.0011Female 0.99 (0.66-1.49) 0.99 0.92 (0.62-1.35) 0.67Surgical 0.70 (0.45-1.07) 0.10 0.84 (0.53-1.33) 0.46IL17A rs1974226 G allele 1.44 (1.04-2.02) 0.029 1.67 (1.17-2.40) 0.0052SPH, St. Paul’s Hospital; VASST, Vasopressin and Septic Shock Trial.Odds ratio was calculated for patients with septic shock using a logistic regression including potential cofounding factors: age, gender (female versus male),surgical versus medical primary diagnosis, and IL17A rs1974226 G allele (GG versus GA versus AA) as covariates.Nakada et al. Critical Care 2011, 15:R254http://ccforum.com/content/15/5/R254Page 7 of 9Competing interestsTN and JHB have no potential conflicts of interest to disclose. JAR and KRWhold shares in Sirius Genomics Inc., which has submitted patents owned byUBC and licensed to Sirius Genomics Inc., that are related to the genetics ofvasopressin and protein C. JAR has received consulting fees from Ferring,which manufactures vasopressin, from Astra Zeneca which manufacturesanti-TNF, and from Sirius Genomics Inc, and has received grant support fromSirius Genomics, Novartis, Ferring, and Eli Lilly, and has received speakinghonoraria from Pfizer and Eli Lilly.Received: 27 July 2011 Revised: 29 September 2011Accepted: 25 October 2011 Published: 25 October 2011References1. Cho JS, Pietras EM, Garcia NC, Ramos RI, Farzam DM, Monroe HR,Magorien JE, Blauvelt A, Kolls JK, Cheung AL, Cheng G, Modlin RL, Miller LS:IL-17 is essential for host defense against cutaneous Staphylococcusaureus infection in mice. J Clin Invest 2010, 120:1762-1773.2. Iwakura Y, Ishigame H, Saijo S, Nakae S: Functional specialization ofinterleukin-17 family members. 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J Immunol 1999, 162:2347-2352.doi:10.1186/cc10515Cite this article as: Nakada et al.: IL17A genetic variation is associatedwith altered susceptibility to Gram-positive infection and mortality ofsevere sepsis. Critical Care 2011 15:R254.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitNakada et al. Critical Care 2011, 15:R254http://ccforum.com/content/15/5/R254Page 9 of 9


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