The primary study endpoint was the modulation of TNFα by SNPs of rs34436714. As it can be seen in Figure 2A, circulating concentrations of TNFα were greater among patients carrying at least 1 T allele of rs34436714. In order to elaborate this further, we isolated PBMCs from 20 patients, 10 with UC and 10 with CD. After stimulation with bacterial LPS, PBMCs of patients carrying T alleles of rs34436714 produced more TNFα than PBMCs of patients carrying only the major frequency allele (Fig. 2B). However, it is suggested that treatment with chemical or biological disease response modifiers may modulate the ability of the host for TNFα production. To this end, circulating concentrations of TNFα were compared separately between patients carrying major and minor frequency alleles of rs34436714 in relation to treatment with at least 1 drug and with at least 2 or more drugs. Circulating TNFα was greater among patients under treatment with 2 or 3 drugs who were carriers of at least 1 minor frequency T allele of rs34436714 (Fig. 2C).
Expression levels of sTREM-1, IL-6, and IL-12 did not differ between carriers of at least 1 T allele of rs34436714 and carriers of only major frequency alleles (Fig. 3).
We hypothesized that carriage of minor frequency T alleles that was associated with greater production capacity for TNFα might also be associated with the clinical phenotypes of CD. Analysis showed that carriage of GT and TT genotypes was associated with greater frequency of CD with fistulizing behavior (Fig. 4A) and with CD located only at the small intestine (Fig. 4B).
Among the comparators, 66 (67.3%), 28 (29.5%), and 4 (4.1%) had GG, GT, and TT genotypes, respectively. Among IBD patients, 59 (65.6%), 28 (31.1%), and 3 (3.3%) had GG, GT, and TT genotypes, respectively. No deviation from Hardy–Weinberg equilibrium was detected in either comparators (HWE: 0.01; P: .976) or patients (HWE: 0.02; P: .884). No difference in the genotype distribution was found between patients and comparators (P: .907); 81.6% (n = 160) of comparators were carriers of major frequency G alleles and 18.4% (n = 36) were carriers of minor frequency T alleles; this distribution was 81.8% (n = 146) and 80.2% (n = 34), respectively among patients.
Our study shows how patients suffering with IBD who are carrying minor frequency T alleles of rs34436714 of NLRP12 have greater circulating levels of TNFα and greater potential for the production of TNFα after PBMCs stimulation. It also shows how carriage of these alleles may have an impact on the phenotype of patients with CD.
A thorough literature search identified few studies on the impact of the function of NLRP12 gene for the pathogenesis of IBD. To our knowledge there is only clinical study trying to investigate the role of the expression of NLRP12 in IBD: in a paired comparison that involved 10 pairs of monozygotic UC twins, healthy twins, and 7 UC patients, gene-profiling showed reduced expression of NLRP12 in active UC. However, the role of rs34436714 was not explored.
Our results point towards an impact of SNPs of rs34436714 on the production of TNFα. Indeed, IBD has been associated with increased expression of TNFα in intestinal tissues.[7,8] The lack of measurements of TNFα in at the gut tissue level may, at first glance, appear as a limitation of our study. However, 2 points may be argued to strengthen the impact of the analyzed SNPs of rs34436714 on TNFα expression as performed in this study through the use of serum TNFα measurements:
- increased serum TNFα has been described by others in patients with IBD[8,9]; and
- carriage of minor frequency T alleles of rs34436714 was associated with greater serum TNFα but not with other circulating cytokines.
This is in accordance with the current knowledge that IBD is a disease hallmarked by elevated TNFα but not by elevated IL-6 and IL-12.
It has been shown that circulating monocytes of patients with CD produce more TNFα than healthy volunteers or patients with UC. Although it is anticipated that greater production of TNFα may be associated with the behavior of CD and refractoriness to treatment, such data are not published. To this end, only indirect evidence is available. More precisely, it has been shown in patients with IBD that carriage of minor frequency A alleles at the -308 gene position increases by 6- to 7-fold the gene transcription of TNFα and the subsequent TNFα deposition at the tissue level. This influences the behavior of CD towards less steroid-dependent disease.[12–14] To this end, it was interesting to find that carriage of minor frequency T alleles of rs34436714 that are associated with elevated circulating TNFα and with high stimulated production of TNFα by PBMCs was also associated with the behavior of CD and with response to treatment. These carriers presented commonly with CD with fistulizing behavior involving the small intestine and necessitated 2 or 3 drugs. Although this may be considered of importance from a pharmacogenomics perspective, a recent genome-wide association study of 359 patients using the Illumina immunochip and covering 196,524 SNPs did not report any association between durability of response to anti-TNFs and rs34436714.
The production of TNFα in the gut is stimulated by microbiota where NLRP12 may function as a modulator. This is supported by studies describing NLRP12 as an antagonist of pro-inflammatory signals induced by Toll-like receptor ligands and Mycobacterium tuberculosis and downregulation of TLR-dependent NFκB after gene silencing of NLRP12. The diversity of gut microbiota is decreased when mice that are deficient for NLRP12 become obese after being fed high-fat diet; this is associated with greater systemic inflammation. Similar findings were shown in mice subject to experimental colitis.
The increased production of sTREM-1 that was found in the IBD patients of our study is in agreement with the findings of 1 previous study of our group that identified sTREM-1 as a mediator in IBD positively correlated with IBD activity. Despite the elevation of sTREM-1 in CD, this was not affected by the carriage of minor frequency alleles of NLRP12. This outscores the importance of the pathway linking NLRP12 to TNFα and not to other pro-inflammatory mediators.
The small study population is also a limitation of our study. However, the primary endpoint, that is, the association of serum TNFα with the carriage of minor frequency T alleles of re34436714 was shown even under that limitation.
Our findings showed that carriage of minor frequency alleles of rs34436714 of NRLP12 was accompanied by greater circulating levels of TNFα and by greater capacity for stimulated TNFα production by PBMCs. These alleles had an impact on the phenotype of patients with CD. Although our results pinpoint some significance of SNPs of rs34436714 in the pathogenesis of IBD, they cannot prove if they drive or not a direct modulation of the production of blood TNFα.
Conceptualization: Mihai G. Netea, Evangelos Giamarellos-Bourboulis, Konstantinos Triantafyllou.
Data curation: Erminia Dellaporta, Lazaros-Dimitrios Lazaridis, Vasilleios Koussoulas.
Formal analysis: Evangelos Giamarellos-Bourboulis, Konstantinos Triantafyllou.
Funding acquisition: Evangelos Giamarellos-Bourboulis.
Methodology: Mihai G. Netea, Evangelos Giamarellos-Bourboulis.
Software: Erminia Dellaporta.
Supervision: Konstantinos Triantafyllou.
Writing – original draft: Evangelos Giamarellos-Bourboulis, Konstantinos Triantafyllou.
Writing – review and editing: Erminia Dellaporta, Lazaros-Dimitrios Lazaridis, Vasilleios Koussoulas, Mihai G. Netea, Evangelos Giamarellos-Bourboulis, Konstantinos Triantafyllou.
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Keywords:Copyright © 2019 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.
inflammatory bowel disease; NLRP12; single nucleotide polymorphisms; TNFα