Assessment of interleukin-10 promoter variant (-1082A/G) and cytokine production in patients with hemolytic uremic syndrome

Abstract

Introduction: Hemolytic uremic syndrome (HUS) is a condition that results in acute kidney failure mainly in children, which is caused by Shiga toxin-producing Escherichia coli and inflammatory response. Although anti-inflammatory mechanisms are triggered, studies on the implication in HUS are scarce. Interleukin-10 (IL-10) regulates inflammation in vivo, and the interindividual differences in its expression are related to genetic variants. Notably, the single nucleotide polymorphism (SNP) rs1800896 -1082 (A/G), located in the IL-10 promoter, regulates cytokine expression.

Methods: Plasma and peripheral blood mononuclear cells (PBMC) were collected from healthy children and HUS patients exhibiting hemolytic anemia, thrombocytopenia, and kidney damage. Monocytes identified as CD14⁺ cells were analyzed within PBMC by flow cytometry. IL-10 levels were quantified by ELISA, and SNP -1082 (A/G) was analyzed by allele-specific PCR.

Results: Circulating IL-10 levels were increased in HUS patients, but PBMC from these patients exhibited a lower capacity to secrete this cytokine compared with those from healthy children. Interestingly, there was a negative association between the circulating levels of IL-10 and inflammatory cytokine IL-8. We observed that circulating IL-10 levels were threefold higher in HUS patients with -1082G allele in comparison to AA genotype. Moreover, there was relative enrichment of GG/AG genotypes in HUS patients with severe kidney failure.

Discussion: Our results suggest a possible contribution of SNP -1082 (A/G) to the severity of kidney failure in HUS patients that should be further evaluated in a larger cohort.

Keywords: HUS; IL-10; SNP; monocytes; renal failure.

Figure 1

Plasma IL-10 levels. IL-10 levels were analyzed and compared between the control group HC (n = 18), HUS patients (n = 17), HUS I + II group (n = 6), and HUS III group (n = 11). Bars represent the median ± IR. *p < 0.05 and **p < 0.0001 in comparison to HC. The Kruskal–Wallis test followed by Dunn’s post-hoc test. IR, interquartile range; HUS, hemolytic uremic syndrome; HC, healthy children.

Figure 2

Plasma IL-10 levels and SNP −1082A>G. IL-10 levels in HUS patients and HC according to −1082A>G genotypes were compared by applying the dominant model of inheritance. Bars represent the median ± IR. *p < 0.05 and **p < 0.005 in comparison to the same genotype in HC. The Kruskal–Wallis test followed by Dunn’s post-hoc test. IL-10, interleukin 10; IR, interquartile range; HUS, hemolytic uremic syndrome; HC, healthy children.

Figure 3

Circulating Mo. (A) Mo absolute numbers in HUS patients (n = 16) and HC (n = 13), *p < 0.05, Mann–Whitney test. (B) Inflammatory Mo (CD14⁺CD16⁺) absolute number in HUS patients (n = 13) and HC (n = 9). IL-10 production in basal condition or after LPS stimulation in Mo from HUS (C) or HC (D). *p < 0.05, Wilcoxon matched-paired test. Comparison of IL-10 concentrations produced in (E) basal condition and (F) after stimulation with LPS between both clinical groups. ***p < 0.05 in comparison to basal treatment in each clinical group, Mann–Whitney test. Mo, monocytes; HUS, hemolytic uremic syndrome; IL-10, interleukin 10; LPS, lipopolysaccharide; HC, healthy children.

Figure 4

IL-10 levels according to SNP rs1800896 genotypes. IL-10 concentrations produced by Mo from HUS patients and HC, segregated according to their genotypes, after LPS stimulation. Mo, monocytes; HUS, hemolytic uremic syndrome; IL-10, interleukin 10; LPS, lipopolysaccharide; HC, healthy children.

Figure 5

Evaluation of peripheral inflammatory factors in HUS patients. (A) IL-8 and (B) cf-DNA levels and (C) elastase activity were evaluated in plasma from HUS and HC. *p < 0.05 in comparison to HC, Mann–Whitney test. HUS, hemolytic uremic syndrome; IL-8, interleukin 8; HC, healthy children.