Polymorphisms of Interlukin-1β rs16944 confer susceptibility to myelodysplastic syndromes

Life Sciences 165 (2016) 109–112

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Polymorphisms of Interlukin-1β rs16944 confer susceptibility to myelodysplastic syndromes Congcong Yin a,1, Na He a,1, Peng Li a,1, Chen Zhang a, Jie Yu a,b, Mingqiang Hua a, Chunyan Ji a, Daoxin Ma a,⁎ a b

Department of Hematology, Qilu Hospital, Shandong University, Jinan 250012, China Department of Hematology, Weihai Municipal Hospital, Weihai, 264200, China

a r t i c l e

i n f o

Article history: Received 10 July 2016 Received in revised form 17 September 2016 Accepted 24 September 2016 Available online 29 September 2016 Keywords: Myelodysplastic syndromes Inflammasome polymorphisms Susceptibility

a b s t r a c t Genetic factors have been shown to be associated with Myelodysplastic syndromes (MDS) susceptibility. In recent years, the role of inflammation in the promotion of tumor growth is supported by a broad range of experimental and clinical evidence. But the relationship between polymorphisms in NOD-like receptor protein 3 (NLRP3) inflammasome and MDS is rarely reported. Thus, we conducted a case-control study, and genotyped five single nucleotide polymorphisms (SNPs) (NLRP3, IL-1β, IL-18, CARD8, and NF-κB) in MDS patients and healthy controls. The association of different genotypes with patient characteristics was analyzed. Comparing MDS patients with controls, GG genotype of IL-1β (rs16944) was observed to be associated with a significantly increased risk of MDS 78/166 (48.8%) vs 26/96 (27.0%), OR = 2.1, CI (1.0–4.4). No significant association was identified regarding the rest of investigated polymorphisms and MDS susceptibility. Complex karyotypes were more frequent in patients with GG genotype of IL-1β (rs16944). Patients with IL-1β polymorphisms (rs16944) GG and GA had lower hemoglobin than those without. Patients with IL-1β polymorphisms (rs16944) GG had higher IPSS scores than those without IL-1β polymorphisms. In conclusion, our present data shows that the IL1β polymorphisms (rs16944) GG were frequently occurred in MDS. IL-1β (rs16944) GG genotype might serve as a novel biomarker and potential targets for MDS. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Myelodysplastic syndromes (MDS) are a collection of myeloid malignancies characterized by ineffective hematopoiesis and peripheral cytopenias, and an increased propensity to evolve to acute myeloid leukemia [1–3]. Immunological mechanisms are more and more recognized in the progression of MDS [4–6]. Inflammasomes are molecular platforms activated upon cellular infection or stress that trigger the maturation of proinflammatory cytokines such as interleukin-1β to engage innate immune defenses [7]. NLRP3 inflammasome, the best-studied inflammasome, is composed of NLRP3 scaffold protein, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 and caspase recruitment domain-containing protein 8 (CARD8) [8]. To be activated, the inflammasome requires two distinct signals, and the first step involves the assembly of the multiprotein complex that cleaves and activates caspase-1, resulting in cleavage of pro-IL-1β and pro-IL-18. The second procedure is mediated by NF-κB activation, and the activation of NF-κB can activate downstream of numerous receptors [9]. NF-κB activation will also induce the expression ⁎ Corresponding author at: Department of Hematology, Qilu Hospital, Shandong University, 107 West Wenhua Road, Jinan, 250012, P. R. China. E-mail address: [email protected] (D. Ma). 1 #Congcong Yin, Na He and Peng Li contributed equally to this work.

http://dx.doi.org/10.1016/j.lfs.2016.09.019 0024-3205/© 2016 Elsevier Inc. All rights reserved.

of inflammasome components, including NLRP3, pro-IL-1β and pro-IL18[10]. However, CARD-8 interacts physically with caspase-1 through the CARD-CARD homophilic interaction and negatively regulates caspase-1 dependent IL-1β generation [11]. Previous studies have identified that inflammasome activation has potent antitumorigenic effects [12]. However, the role of inflammation in the promotion of tumor growth is supported by a broad range of experimental and clinical evidence in recent years. NLRP3 has been shown to activate the pro-IL-1 and pro-IL-18, which have been implicated in the relationship between tumor genesis/progression and inflammation [13]. Genetic variations leading to the altered production or function of inflammasome and inflammatory cytokines were linked to various diseases. NLRP3 rs35829419 polymorphisms are associated with increased susceptibility to multiple diseases, including colorectal cancer, rheumatoid arthritis and abdominal aortic aneurysms [14]. IL-1β polymorphisms have also been linked to several kinds of malignant tumors, such as gastric cancer, hepatocellular cancer and lung cancer [15–17]. For IL-18, the strongest evidence for an involvement in carcinogenesis is derived from colitis-associated cancer [18,19]. In contrast to IL-1, it seems that IL-18 functions as a factor to maintain tissue homeostasis and thereby reduces tumorigenesis [19]. RNAi-mediated knockdown of IL-18 in tumors, or its systemic depletion by IL-18–binding protein, is sufficient to stimulate NK cell-dependent immunosurveillance in various tumor models [20,21]. Specifically, the NF-KB 94-ins/del ATTG

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polymorphisms (rs28362491) is involved in susceptibility to inflammatory colorectal diseases and related to an increased cancer risk [22,23]. Thus, inflammasomes are promising diagnostic and therapeutic targets in cancer-related clinical conditions. To our knowledge, there is no data reporting a relation between MDS and NLRP3 inflammasome polymorphisms. Here, the purpose of this study was to determine whether polymorphisms of genes involved in NLRP3 inflammatory signaling (NLRP3 rs35829419, IL-1β rs16944, IL-18 rs1946518, CARD8-C10X rs2043211, and NF-κB-94 rs28362491 ins/del) were associated with the susceptibility of myelodysplastic syndromes.

2.3. Statistical analyses Continuous variables were shown as mean ± SD, and categorical variables were shown as frequencies and percentage (%). Standard chi-square test was used to test genotype frequencies for Hardy-Weinberg equilibrium. Associations between genotype and disease risk were assessed by calculating odds ratios (OR) and corresponding 95% confidence intervals (CI). Statistical analyses were conducted using SPSS software version 18.0 (SPSS, Inc., Chicago, USA) and Stata 11 (StataCorp LP, Lakeway Drive, TX). A two-tailed p value of 0.05 was considered significant.

2. Methods 3. Results 2.1. Patients 3.1. Samples characteristics All participants signed an informed consent and the study was approved by the Institutional Review Boards of Qilu Hospital of Shandong University. A total of 160 consecutive patients with MDS diagnosed according to WHO classification criteria were enrolled from January 2014 to June 2016. Clinical characteristics are summarized in Table 1. Ninetysix healthy donors without any evidence of hematological disease served as the control group. 2.2. Genotyping Blood or bone marrow samples were collected from each MDS case and control subject after being enrolled into our study. Genomic DNA (gDNA) was extracted using a TIANamp blood DNA kit (Tiangen Biotech, Beijing, China) according to manufacturer’ instructions. Four SNPs, NLRP3-Q750K (rs35829419, IL-1β (rs16944), IL-18 (rs1946518) and CARD8-C10X (rs2043211), were purchased from Thermo Fisher Scientific company (Cat.# 4,351,379). NFκB −94 ins/del ATTG promoter polymorphism was detected using the forward primer: 5′- CCG TGC TGC CTG CGT T − 3′, reverse primer: 5′- GCT GGA GCC GGT AGG GAA − 3′ as well as probe 1: 5′-VIC- ACC ATT GAT TGG GCC -MGB-3′ and probe 2: 5′-FAM- CGA CCA TTG GGC C -MGB-3′. All PCR reactions were run in triplicate, and contained 1ul of DNA, 0.15 ul of TaqMan Universal PCR Master Mix, 1.85ul H2O and 3 ul of Allelic Discrimination Mix. Real-time PCR was performed on an ABI 7500 real-time PCR System (SDS, PE Biosystems) using the following conditions: 50 °C for 2 min, 95 °C for 10 min, and then 40 cycles of amplification (92 °C denaturation for 15 s, 62 °C annealing/extension for 60 s). Genotypes were analyzed using ABI 7500 Sequence Detection System (SDS) 1.3.1. Table 1 Samples characteristics Parameter

Male:female (ratio) Median age (years)

Number MDS (n = 160)

Control (n = 96)

1.25 56 (16–95)

1.13 42(17–85)

MDS subtypes (WHO) RCUD RAS RCMD (RS) RAEB-1 RAEB-2 MDS-U MDS with isolated 5q−

24 (15.0%) 12(7.5%) 71(44.3%) 12(7.5%) 30(18.8%) 8(5.0%) 3(1.9%)

Cytogenetics Normal karyotypes Aberrant karyotypes Not available

64(40.0%) 75(46.9%) 21(13.1%)

IPSS risk group Lower risk Higher risk Not available

53(33.1%) 86(53.8%) 21(13.1%)

SNPs of five genes in 160 patients and 96 controls were genotyped by TaqMan assays. As shown in Table 1, the gender or age of MDS patients was similar to that of controls, and no significant difference was found(p N 0.05). WHO MDS subtype was available for 160 MDS cases, which included 24 (15.0%) RCUD (refractory cytopenia with unilineage dysplasia), 12(7.5%) RAS (refractory anemia with ringed sideroblasts), 71(44.3%) RCMD (RCMD-ring sideroblasts (RS)), 12(7.5%) RAEB-1 (refractory anemia with excess blasts-1), 30(18.8%) RAEB-2(refractory anemia with excess blasts-2), 8(5.0%) MDS-U (MDS unclassified) and 3(1.9%) MDS with isolated 5q−. Of the 160 MDS cases with International Prognostic Scoring System (IPSS) data, 53(33.1%) were grouped as lower risk, which included both low-risk and intermediate-1 IPSS values, whereas 86(53.8%) were grouped as higher risk, which included intermediate- 2 and high-risk patients. (Table 1). 4. SNPs and MDS susceptibility SNPs genotypic frequencies were in the Hardy–Weinberg equilibrium in both patients and control groups. No NLRP3-Q750K (rs35829419)-A polymorphisms were observed in 160 MDS patients,

Table 2 Genotype distribution of the different polymorphisms in MDS patients and normal control population investigated in the study and their association with MDS susceptibility Polymorphisms

MDS n (%)

Controls n (%)

NLRP3 (rs35829419) CA10 (0%) AA CC CA + AA

0 (0%) 0 (0%) 160(100%) 0 (0%)

0 (0%) 0 (0%) 96(100%) 0 (0%)

IL-1β (rs16944) GG AG AA GG + AG

78(48.8%) 56(35.0%) 26(16.3%) 134(83.8%)

26(27.0%) 52(54.2%) 18(18.7%) 78(81.2%)

IL-18 (rs1946518) GG GT TT GT + GG

54(33.8%) 70(43.8%) 36 (22.5%) 124(77.5%)

27(28.1%) 44 (45.8%) 25(26.0%) 71(73.9%)

CARD8-C10X (rs2043211) AA 42(26.3%) AT 80(50.0%) TT 38(23.8%) AA + AT 122(76.3%)

33(34.3%) 45(46.9%) 18(18.7%) 78 (81.2%)

NF-kB −94 ins/del (rs28362491 Ins/ins 63(39.4%) Ins/del 70(43.8%) del/del 27(16.9%) ins/ins + Ins/del 133(83.1%)

39(40.6%) 43(44.8%) 14(14.6%) 82(85.4%)

OR (95% CI)

P value

2.1(1.0–4.4) 0.7(0.4–1.5)

0.045 0.42

1.2(0.6–2.3)

0.61

1.4(0.7–2.8) 1.0(0.6–2.4)

0.35 0.76

1.2(0.7–2.2)

0.52

0.6(0.3–1.2) 0.8(0.4–1.6)

0.17 0.36

0.7(0.4–1.4)

0.35

0.8 (0.4–1.8) 0.8(0.4–1.8)

0.65 0.46

0.8(0.4–1.7)

0.63

Statistical significant P values and corresponding lines are displayed in bold.

C. Yin et al. / Life Sciences 165 (2016) 109–112 Table 3 Comparison of gender and age between MDS patients with or without IL-1β polymorphisms Characteristic

GG

GA

AA

p1

p2

Age, y median (range) Male:female

54(21–81) 42:33

57(16–83) 34:27

58(18–95) 13:13

0.62 0.38

0.90 0.39

p1 P values of homozygous polymorphisms,p2 P values of heterozygous polymorphisms

as well as 96 normal subjects. Other four polymorphisms IL-1β (rs16944)-G, IL-18 (rs1946518)-G, CARD8-C10X (rs2043211)-A, and NF-kB 94 ins/del (rs28362491)-ins were differently distributed between patients and controls. Only rs16944 in IL-1β gene resulted to be significantly associated with MDS (OR = 2.1, 95% CI: 1.0–4.4, P = 0.045). No significant association was identified regarding the rest of investigated polymorphisms and MDS susceptibility (Table 2). 4.1. Patient characteristics in relation to IL-1β polymorphisms status Our data showed that there was no significant difference in age or gender between patients with IL-1β (rs16944) GG or GA and IL-1β (rs16944) AA (p N 0.05) (Table 3). IL-1β (rs16944) polymorphisms were more frequently found in MDS subtypes (RCMD or RS) (p1 = 0, p2 = 0). IL-1β (rs16944) GG closely correlated with karyotypes. Complex karyotypes were more often observed in patients with IL-1β (rs16944) GG than those with IL-1β (rs16944) AA. Among the 78 cases with IL-1β GG, 25 (32.1%) presented with a complex karyotype, while 2 (7.7%) presented with a complex karyotype in 21 IL-1β AA cases (p = 0.03) (Table 4). Moreover, IL-1β polymorphisms GG occurred less frequently in association with del (20q) (p1 = 0.04). We also found IL-1β polymorphisms GG were more common in high-risk subtypes (RAEB-1/− 2) than in RCUD/RARS/MDS-U cases (Table 4). Patients with IL-1β polymorphisms GG had higher IPSS scores than those with IL-1β (rs16944) AA (p = 0.03) (Table 4). In addition, IL1β polymorphisms closely correlated with hemogram change. Patients with IL-1β (rs16944) GG had lower hemoglobin than those IL-1β (rs16944) AA (p1 = 0.04) (Table 5). 5. Discussion Epidemiological, genetic and molecular studies have shown the strong relation between inflammation and cancer [12]. A significant

Table 4 The correlations of IL-1β polymorphisms with types of MDS patients GA

AA

p1

p2

WHO classification, n (%) RCUD 6(7.7%) RARS 7(9.0%) RCMD (RS) 32(41.0%) RAEB1/2 26(33.3%) MDS-U 4(5.1%) MDS with isolated 5q− 3(3.8%)

6(10.7%) 2(3.6%) 36(64.3%) 12(21.4.4%) 0 0

12 (46.2%)) 3(11.5%) 3(11.5%) 4(15.4%)) 4(15.4%) 0

0.00 0.70 0.00 0.08 0.08 −

0.00 0.16 0.00 0.57 − −

Karyotype Normal −5/5q− −7/7q− +8 20q− Complex Other abnormalities Not available

31 (39.7%) 2 (2.6%) 4 (5.1%) 6 (7.7%) 3(3.8%) 25(32.1%) 0 7 (9.0%)

26(46.4%) 1(1.8%) 2(3.6%) 7 (12.5%) 1 (1.8%) 4(7.1%) 4(7.1%) 11(19.6%)

8(26.9%) 0 2 (7.7%) 5 (29.2%) 4(15.4%) 2(7.7%) 2(7.7%) 3(11.5%)

0.41 − 0.67 0.09 0.04 0.03 −

0.05 − 0.42 0.42 0.05 0.93 0.93

IPSS risk group, n (%) Lower risk Higher risk Not available

22(28.2%) 49(62.8%) 7(9.0%)

18(32.1%) 27(48.2%) 11(19.6%)

13(50.0%) 10(38.4%) 3(11.5%)

0.03

019

Characteristic

GG

p1 P values of homozygous polymorphisms,p2 P values of heterozygous polymorphisms

111

Table 5 The correlations of IL-1β polymorphisms with hemogram change of MDS patients p2

Characteristic

GG

GA

AA

p1

Neutrophils, ×109/l, mean ±

2.27 ± 3.17

2.17 ±

3.72 ±

0.19 0.24

SD Hemoglobin, g/l, mean ± SD

65.2 ± 13.0

4.08 69.3 ±

7.38 72.7 ±

0.04 0.54

93.1 ±

22.3 78.4 ±

15.6 68.6 ±

0.09 0.44

116.7

86.6

18.1

Platelet, ×109/l, mean ± SD

p1 P values of homozygous polymorphisms,p2 P values of heterozygous polymorphisms

relationship was found between immune environment and MDS progression [4–6]. In previous study we have shown that the frequency of Th17 cells was profoundly increased in MDS patients compared to healthy controls [24]. Inflammasomes activation is responsible for IL-1 release and the consequent production of Th17 [25]. Thus, it is reasonable to assume that variants in genes coding pro-inflammatory and anti-inflammatory cytokines have effect on MDS risk. In the present study, we detected polymorphisms in NLRP3-inflammasome signaling genes including NLRP3-Q750K (rs35829419), IL-1β (rs16944), IL-18 (rs1946518), CARD8-C10X (rs2043211), and NF-B-94 ins/del (rs28362491) in 160 patients with MDS and 96 healthy controls. Our results suggest that polymorphisms GG in IL-1β at position rs16944 is significantly associated with the risk of MDS. IL-1β gene is located on chromosome 2q14 and its expression has been found to be associated with various cancer types [26]. IL-1 gene cluster polymorphisms is in near-complete linkage disequilibrium and is a TATA-box polymorphism that markedly affects DNA protein interactions, which enhances production of IL-1β [15]. In addition, IL-1β polymorphisms were associated with several kinds of malignant tumors, such as gastric cancer, hepatocellular cancer and lung cancer [15–17]. Here, we report that the homozygous polymorphisms in IL-1β at position-G/A (rs16944) are new candidate loci for susceptibility to MDS. We also found that IL-1β (rs16944) heterozygote polymorphisms were not significantly associated with MDS. Our study also showed that IL-1β polymorphisms were more common in high-risk subtypes RAEB)-1/− 2 than in RCUD/RARS/MDS-U cases. Patients with IL-1β polymorphisms GG had higher IPSS scores than those IL-1β AA. Therefore, IL-1β (rs16944) polymorphisms may be used as a new diagnostic and therapeutic target in the future. The spectrum of IL-1β polymorphisms closely correlated with WHO subtypes. IL-1β polymorphisms were frequently confirmed in MDS subtypes with RCMD or RCMD-RS. IL-1β (rs16944) polymorphisms closely correlated with karyotypes. Complex karyotypes were more frequent in patients with IL-1β (rs16944) GG than IL-1β (rs16944) AA. Therefore, the detection of IL-1β (rs16944) polymorphisms may contribute to establishing a diagnosis and classifying into subtypes for MDS in patients. IL-1β polymorphisms closely correlated with hemogram change. Patients with IL-1β homozygous G-allele had lower hemoglobin than those with wild types. We found that CARD8-C10X (rs2043211) polymorphisms were marginally significant under homozygote and heterozygote models in MDS. Regarding the variants of the CARD8 gene, the C10X variant (rs2043211) was found to be associated with complex diseases with inflammatory background, like inflammatory bowel disease [27,28], cardiovascular diseases [29,30], rheumatoid arthritis [31], and type 1 diabetes [32]. From our results, the frequencies of the CARD8-C10X (rs2043211) AA and AT genotypes were found to be lower in patients compared to controls. CARD8-C10X (rs2043211)-AA seemed to be a protective factor for MDS. Although a trend for relation between CARD8-C10X (rs2043211) polymorphisms and MDS was observed, this difference was not significant. However, further studies on more patients are needed to substantiate the relation between CARD8-C10X (rs2043211) polymorphisms and MDS.

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In summary, for the first time we showed that individuals carrying the IL-1β (rs16944) GG genotypes were at an increased risk for developing MDS. IL-1β (rs16944)-GG polymorphisms occur much more frequently in MDS, which may favor the development of the disease. Patients with IL-1β (rs16944)-GG polymorphisms had a higher IPSS scores suggest that shifty of the IL-1β (rs16944) could be a parameter affecting disease progression. Thus, IL-1β (rs16944) polymorphisms could be used as diagnostic markers to identify patients at high risk of rapid MDS progression. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments This work was supported by grants from National Natural Science Foundation of China (No. 81470319) and Nature Science Foundation of Shandong Province (No. ZR2015PH060). References [1] D.P. Steensma, The spectrum of molecular aberrations in myelodysplastic syndromes: in the shadow of acute myeloid leukemia, Haematologica 92 (6) (2007) 723–727. [2] S.D. Nimer, Myelodysplastic syndromes, Blood 111 (10) (2008) 4841–4851, http:// dx.doi.org/10.1182/blood-2007-08-078139. [3] A. Orazi, M.B. Czader, Myelodysplastic syndromes, Am. J. Clin. Pathol. 132 (2) (2009) 290–305, http://dx.doi.org/10.1309/AJCPRCXX4R0YHKWV. [4] J.J. Molldrem, Y.Z. Jiang, M. Stetler-Stevenson, D. Mavroudis, N. Hensel, A.J. Barrett, Haematological response of patients with myelodysplastic syndrome to antithymocyte globulin is associated with a loss of lymphocyte-mediated inhibition of CFU-GM and alterations in T-cell receptor Vbeta profiles, Br. J. Haematol. 102 (5) (1998) 1314–1322. [5] J.N. Kochenderfer, S. Kobayashi, E.D. Wieder, C. Su, J.J. Molldrem, Loss of T-lymphocyte clonal dominance in patients with myelodysplastic syndrome responsive to immunosuppression, Blood 100 (10) (2002) 3639–3645, http://dx.doi.org/10.1182/ blood-2002-01-0155. [6] C. Fozza, S. Contini, A. Galleu, M.P. Simula, P. Virdis, S. Bonfigli, et al., Patients with myelodysplastic syndromes display several T-cell expansions, which are mostly polyclonal in the CD4(+) subset and oligoclonal in the CD8(+) subset, Exp. Hematol. 37 (8) (2009) 947–955, http://dx.doi.org/10.1016/j.exphem.2009.04.009. [7] K. Schroder, J. Tschopp, The inflammasomes, Cell 140 (6) (2010) 821–832, http://dx. doi.org/10.1016/j.cell.2010.01.040. [8] L. Agostini, F. Martinon, K. Burns, M.F. McDermott, P.N. Hawkins, J. Tschopp, NALP3 forms an IL-1beta-processing inflammasome with increased activity in MuckleWells autoinflammatory disorder, Immunity 20 (3) (2004) 319–325. [9] T.D. Kanneganti, M. Lamkanfi, G. Nunez, Intracellular NOD-like receptors in host defense and disease, Immunity 27 (4) (2007) 549–559, http://dx.doi.org/10.1016/j. immuni.2007.10.002. [10] F.G. Bauernfeind, G. Horvath, A. Stutz, E.S. Alnemri, K. MacDonald, D. Speert, et al., Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression, J. Immunol. 183 (2) (2009) 787–791, http://dx.doi.org/10.4049/jimmunol.0901363. [11] M. Razmara, S.M. Srinivasula, L. Wang, J.L. Poyet, B.J. Geddes, P.S. DiStefano, et al., CARD-8 protein, a new CARD family member that regulates caspase-1 activation and apoptosis, J. Biol. Chem. 277 (16) (2002) 13952–13958, http://dx.doi.org/10. 1074/jbc.M107811200. [12] S.K. Drexler, A.S. Yazdi, Complex roles of inflammasomes in carcinogenesis, Cancer J. 19 (6) (2013) 468–472, http://dx.doi.org/10.1097/PPO.0000000000000004.

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