Interleukin 1 receptor type 1 gene variants associate with disease susceptibility to IgG4-related periaortitis/periarteritis in IgG4-related disease

Norihiro Ashihara Shinshu Daigaku Daigakuin Igakukei Kenkyuka Igakubu Takeji Umemura (  tumemura@shinshu-u.ac.jp ) Shinshu Daigaku Daigakuin Igakukei Kenkyuka Igakubu https://orcid.org/0000-0001-7985-919X Yasunari Fujinaga Shinshu Daigaku Daigakuin Igakukei Kenkyuka Igakubu Makiko Ozawa Shinshu Daigaku Daigakuin Sogo Kogakukei Kenkyuka Kogakubu Yasuhiro Kuraishi Shinshu Daigaku Daigakuin Igakukei Kenkyuka Igakubu Takayuki Watanabe Shinshu Daigaku Daigakuin Igakukei Kenkyuka Igakubu Hideaki Hamano Shinshu Daigaku Daigakuin Igakukei Kenkyuka Igakubu Akira Meguro Yokohama Shiritsu Daigaku Igakubu Daigakuin Igaku Kenkyuka Shigeyuki Kawa Matsumoto Shika Daigaku Masao Ota Shinshu Daigaku Daigakuin Igakukei Kenkyuka Igakubu


Conclusions
This study indicated that IL1R1 SNPs contributed to IgG4-related periaortitis/periarteritis, but not IgG4-RD, onset. Our ndings raise the possibility of certain genetic factors in uencing the risk of speci c IgG4-RD manifestations.

Background
IgG4-related disease (IgG4-RD) is an immune-mediated disorder characterized by high serum IgG4 concentration and IgG4-bearing plasma cell in ltration in affected organs in association with lymphoplasmacytic in ltration, storiform brosis, and obliterative phlebitis [1]. In 2001, these clinical and pathological features were rst identi ed in patients with autoimmune pancreatitis (AIP) [2], now referred to as type 1 AIP. Since then, simultaneous or metachronous lesions with distinguishing common pathological features have been found in nearly every organ system.
Recently, IgG4-RD has been recognized to also affect the cardiovascular system as retroperitoneal brosis, in ammatory aneurysms, lymphoplasmacytic aortitis, and aortic dissection, which has been newly termed IgG4-related periaortitis/periarteritis [3][4][5]. Although the clinical features of IgG4-related periaortitis/periarteritis are now being identi ed, the contributing factors to disease onset remain unclear.
IgG4-related periaortitis/periarteritis tends to occur in patients with an older IgG4-RD onset age and in those with a highly active disease state displaying elevated serum IgG, IgG4, circulating immune complexes, and soluble interleukin (IL)2 receptor [3,4]. The elevation of serum IgG4 is considered to be under the control of cytokine signals, including IL-4, IL-5, IL-10, IL13, IL12, transforming growth factor (TGF)-β, and B-cell activating factor [6]. In turn, serum IgG4 concentration is correlated with the number of pathologic T follicular helper type 2 cells. Therefore, high concentrations of both serum IgG4 and circulating T follicular helper type 2 cells may be involved in the pathogenesis of IgG4-related periaortitis/periarteritis. Signi cant associations for HLA haplotype, Fc receptor-like protein 3, cytotoxic T-lymphocyte antigen 4, and other genetic factors with type 1 AIP have been reported [7][8][9]. A recent genome-wide association study (GWAS) showed that HLA-DRB1 and the Fc fragment of IgG receptor IIb regions were possible susceptibility loci for IgG4-RD [10], although no genetic studies of patients with IgG4-related periaortitis/periarteritis exist to date. We therefore sought to determine the susceptibility genes to IgG4related periaortitis/periarteritis using GWAS-based, ne-tuned mapping of candidate genes in a Japanese population.

Subjects.
Between 2002 and 2015, a total of 223 Japanese patients were diagnosed as having IgG4-RD at Shinshu University Hospital or its a liated institutions. Among them, 99 patients were excluded for the reason of no stored DNA samples (n = 55) or (2) no contrast-enhanced computed tomography (CT) analysis due to renal impairment or allergy (n = 44). The remaining 124 patients with IgG4-RD (median age: 67 years, 75% male) were included in this retrospective study along with 344 previously reported volunteer healthy subjects [11]. IgG4-RD was diagnosed based on the Japanese Comprehensive Diagnostic Criteria for IgG4-RD [12]. Contrast-enhanced CT ndings were reviewed by an experienced radiologist (Y.F.).
Periaortitis and periarteritis were de ned as vessel wall thickness and wall enhancement either partially or circumferentially on CT images, as reported previously [4]. The protocol of this study was reviewed and approved by the Institutional Review Board of Shinshu University School of Medicine in Matsumoto, Japan (approval number: 571). Written informed consent was obtained from all participating patients. The investigation was conducted according to the principals of the Declaration of Helsinki.
Genomic DNA was isolated from whole-blood samples using Quick Gene-800 assays (Kurabo, Osaka, Japan). Genotyping was done with the GeneChip Human Mapping 500K Array Set (Affymetrix, CA, USA) according to the manufacturer's protocol in the rst stage of this analysis. Samples with a < 93% genotype call rate were excluded from the study, as were SNPs with a call rate of < 95% or a minor allele frequency of < 5% overall. From a prior genome-wide association analysis between patients with IgG4related periaortitis/periarteritis (n = 28) and those without (n = 47), we extracted 3 candidate genes (interleukin 1 receptor type 1 [IL1R1]; 2q11.2-q12.1, calcium/calmodulin-dependent protein kinase II alpha [CAMK2A]; 5q32, and vacuolar protein sorting 13 homolog B [VPS13B]; 8q22.2) containing highly signi cant SNPs (i.e., P < 10 − 4 ). SNPs in the IL1R1 gene were examined to identify possible susceptibility loci to IgG4-related periaortitis/periarteritis since this gene has been established as an important mediator involved in many cytokine-induced immune and in ammatory responses [13]. Three SNPs that showed a signi cant association with IgG4-related periaortitis/periarteritis by a GWAS were selected along with 5 additional SNPs. In total, 8 SNPs (rs3917225, rs2287049, rs2287048, rs3917273, rs2160227, rs951192, rs3917318, and rs7582198) having minor allele frequencies of > 5% in the ILR1 gene were evaluated. The genotyping of all SNPs was performed using the ABI TaqMan allelic discrimination kit and the StepOnePlus™ Real-Time PCR System (Thermo Fisher Scienti c, MA, USA) following the manufacturer's instructions.
Measurement of serum IL-1β levels.
Serum samples were obtained and stored below − 20 °C until measurements. Serum IL-1β levels were examined by a commercially available ELISA kit (Proteintech, IL, USA) following the manufacturer's instructions.

Statistical analysis.
We assessed the signi cance of allele distribution among patients using the chi-square test by means of 2 × 2 comparisons (df = 1). Differences between groups were examined by Mann-Whitney U-test. Pvalues were subjected to Bonferroni's correction by multiplication by the number of different alleles observed in each locus (Pc). Association strength was estimated by calculating the odds ratio (OR) and 95% con dence interval (CI). A P < 0.05 was considered statistically signi cant. Statistical analyses were performed using IBM SPSS statistics software version 24 (IBM, IL, USA). Genetic power was calculated using G*Power version 3.1.9.6 [14].

Discussion
IgG4-related periaortitis/periarteritis is a recently identi ed new disease entity candidate in IgG4-RD. As investigations on the clinical features of this disease have only just commenced, the exact pathogenesis of IgG4-related periaortitis/periarteritis is unknown. This study is the rst to identify genetic factors related to IgG4-related periaortitis/periarteritis. While IL1R1 gene polymorphisms were not linked to susceptibility in IgG4-RD involving other organs in a prior GWAS and this study [10], our data uncovered intriguing associations for 6 IL1R1 SNPs and IgG4-related periaortitis/periarteritis. IL1R1 is a member of the IL-1 receptor family. This membrane receptor binds to IL-1 ligands (IL-1α and IL-1β cytokines) involved in the initiation of IL-1-mediated immune and in ammatory responses [13]. In spite of their identical activities, IL-1α is biologically active as a precursor molecule, whereas IL-1β is secreted and circulates systemically following in ammasome activation. Therefore, we also investigated whether relationships between serum IL-1β levels and IL1R1 gene polymorphisms affected the development of IgG4-related periaortitis/periarteritis.
Several reports have shown that CD4 + cytotoxic T cells (CTLs) are clonally expanded both in the peripheral blood and in in ammatory tissues to secrete IL-1β, TGF-β, and interferon-γ [15]. Moreover, CTLs were decreased after successful therapy, suggesting that IL-1β-, interferon-γ-, and TGF-β-secreting CTLs were related to IgG4-RD and other brotic in ammatory disorders. However, our study showed that serum IL-1β levels were lower in the periaortitis/periarteritis positive group than in the negative group among patients with IgG4-RD. This may have been due to increased levels of soluble sIL-1R1 receptors in the vasculitis positive group. Soluble sIL-1R1 and sIL-1R2 receptors were found to be signi cantly increased in IgG4-RD sera as compared with healthy controls [16]. Elevated sIL-1R1 binds serum IL-1β and may inhibit excess IL-1β activity to protect ongoing IL-1-mediated in ammation. However, no signi cant correlations were observed between serum IL-1β levels and IL1R1 genotypes.
The limitations of this study are lack of disease controls, validation cohort, limited sample size, and no pathological specimens; thus, the possibility of type I error cannot be excluded. Additional series are needed to validate our newly discovered associations in a larger number of IgG4-related periaortitis/periarteritis subjects of other ethnicities. However, power calculations based on the study subjects of 43 patients with IgG4-related periaortitis/periarteritis and 81 IgG4-RD patients without IgG4related periaortitis/periarteritis and an effect size of 0.44 at rs3917318 showed su cient detection power (0.97) at the 0.05 level of signi cance.
In summary, the preset study revealed signi cant associations of IL1R1 SNPs with the onset of IgG4related periaortitis/periarteritis, but not with IgG4-RD onset, in a Japanese population. Our ndings raise the possibility of certain genetic factors in uencing the risk of speci c IgG4-RD manifestations and require further study.
Abbreviations AIP: autoimmune pancreatitis; CAMK2A: Calcium/calmodulin-dependent protein kinase II alpha; CI: con dence interval; CT: Computed tomography; CTLs: cytotoxic T cells; GWAS: genome-wide association study; IgG4: Immunoglobulin G4; IgG4-RD: Immunoglobulin G4-related disease; IL: interleukin; IL1R1: interleukin 1 receptor type 1; OR: odds ratio; Pc: corrected P; SNPs: single nucleotide polymorphisms; TGFβ: transforming growth factor-β; VPS13B: vacuolar protein sorting 13 homolog B; sIL-2R: Soluble interleukin-2 receptor Declarations Ethics approval and consent to participate The protocol of this study was reviewed and approved by the Institutional Review Board of Shinshu University School of Medicine in Matsumoto, Japan (approval number: 571). Written informed consent was obtained from all participating patients. The investigation was conducted according to the principals of the Declaration of Helsinki.

Consent for publication
Not applicable Availability of data and materials The dataset analyzed in this paper is available from the corresponding author on reasonable request, and with appropriate additional ethical approvals, where necessary.
T.U., and M.Ota designed the study, performed the data analysis, and wrote the manuscript. N.O., and A.M. conducted experiments and data analysis. Y.F. performed data collection, data analysis, and data interpretation. M.Ozawa, Y.K., T.W., and H.H. participated in the case and data collection. S.K. designed the study, and performed data interpretation. The authors read and approved the nal manuscript.