Introduction

Blastocystis is a parasite associated to the development of cutaneous and intestinal disorders as irritable bowel syndrome (IBS) (Yakoob et al. 2004, 2010; Tan 2008; Stensvold et al. 2009a; Tan et al. 2010; Jimenez-Gonzalez et al. 2011). IBS is one of the most common gastrointestinal diagnoses seen by primary care providers and gastroenterologists, in which abdominal pain or discomfort is associated with a change in bowel habits and disordered defecation (Longstreth et al. 2006). Although the definition of IBS states that symptoms are not due to active inflammation, transient mucosal inflammation is considered to be an important factor for the manifestation of this syndrome (Hotoleanu et al. 2008). In addition, some experimental infections in rats and in vitro assays have shown that Blastocystis can elicit a proinflammatory response, upregulating the expression of interferon-γ, interleukin (IL)-12, and tumor necrosis factor-alpha (TNF-α) (Iguchi et al. 2009), as well as an early production of IL-8 (Long et al. 2001; Puthia et al. 2008).

Studies performed in Dutch and Iranian IBS patients demonstrated that certain genetic polymorphisms related to some proinflammatory ILs were associated with susceptibility to this disease (van der Veek et al. 2005; Barkhordari et al. 2010a, b). In a recent study, our group found that Blastocystis carriers have twofold increased relative risks for IBS, while gene promoter single nucleotide polymorphisms (SNPs) of IL-6 and TNF-α genes had similar frequencies in patients and controls; thus, we decided to assess the role of IL-8 and IL-10 SNPs.

Material and methods

Subjects

Forty-five IBS patients with mean age of 46.5 ± 14.3, diagnosed according to Rome III criteria and 45 unrelated controls with mean age of 52.2 ± 14.4, referred to the gastroenterology area of the Hospital General “Dr. Manuel Gea Gonzalez” between 2008 and 2009 were studied. Exclusion criteria for both groups were the presence of intestinal viruses or bacteria that cause diarrhea, parasites other than Blastocystis such as Entamoeba histolytica/dispar, Giardia, Endolimax, and Hymenolepis and the presence of an organic cause of disease, evident psychological alterations, social problems, pregnancy, dependence on analgesics, ulcerative colitis, tumors, or diverticulitis. Blastocystis diagnosis by PCR (Stensvold et al. 2006) was performed in all participants and showed that 14 patients (31%) and 6 controls (13%) were positive. Blastocystis subtypes (ST) detected were ST1 and ST3 similarly distributed between the IBS and control groups; the presence of IBS did not correlate to any particular ST (Jimenez-Gonzalez et al. 2011). This study was approved by the ethical and research committee of our hospital and a written informed consent was obtained from each participant, who afterwards, underwent a full physical examination and provided biological samples.

Genotyping

DNA was obtained from 10 mL EDTA-treated peripheral blood using proteinase K and phenol/chloroform extraction (Sambrook et al. 2001). Typing of the IL8 polymorphism was achieved by PCR with the primers IL8(−251)F (5′-TCTAACACCTGCCACTCTAG-3′), IL8(−251)R (5′-CCTGAGTCATCACACTTCCT-3′), IL8(+396)F (5′-CTCTGTGTGAAGGTAAGCAC-3′), IL8(+396)R (5′-TATCAACAGGCACAGCTCTG-3′), IL8(+1633)F (5′-GGAAGAGAGCTCTGTCTGGA-3′), and IL8(+1633)R (5′-GATCCTGGCTAGCAGACTAG-3′) that generate fragments of 211, 798, and 988 bp, respectively. For IL8(−251) polymorphism, the probes IL8(−251)A (5′-CATACATTTGATAATTCA-3′) and IL8(−251)T (5′-CATACAATTGATAATTCA-3′) were used. The primer IL8(+396) amplified two polymorphisms that may be defined with the probes IL8(+396)G (5′-ATGCATGCTACATGGTATAA-3′), IL8(+396)T (5′-ATGCATGCTAAATGGTATAA-3′), IL8(+781)C (5′-ACATTGAACGACTTCCTAT-3′), and IL8(+781)T (5′-ACATTGAACAACTTCCTAT-3′). Alleles at position 1633 were defined with the probes IL8(+1633)T (5′-GACATGAGTACAACAAACT-3′) and IL8(+1633)C (5′-GACATGGGTACAACAAACT-3′). For the IL-10 polymorphisms, primers and probes for SNPs at positions −592, −819, and −1082 described by Meenagh et al. (2002) were used. IL-8 and IL-10 probes were labeled with digoxigenin-11-ddUTP, and alleles were identified by dot blot analysis with chemiluminescence as in Fajardo-Dolci et al. (2006).

Statistical analysis

Allele and genotype frequencies were calculated by direct counting and were compared between patients and controls; etiologic fraction (EF) was calculated according to Olivo-Díaz et al. (2004). Frequencies between groups were compared using Mantel–Haenszel test and Fisher’s exact test when appropriate. Odds ratio (OR) and 95% confidence intervals were calculated to estimate the relative risk conferred by a particular allele and genotype using Epi-Info version 6.0 (Centers for Disease Control and Prevention, Atlanta, GA).

Results

Table 1 summarizes the significant associations found between alleles or genotypes and other comparisons observed in patients with IBS and/or Blastocystis or without IBS and/or Blastocystis vs. IL-8 and IL-10 SNPs. Five of seven SNPs showed some association with either IBS or Blastocystis; thus, polymorphisms for IL-8 at positions +396 and +781, as well as IL-10 at position −1082 were SNPs associated to the development of IBS, while IL-8+781 (T) and IL-10-592 (C) alleles were associated to being a Blastocystis carrier, the former in all participants and the latter only IBS group, respectively. No association was found regarding Blastocystis and control group, although IL-10-819 (CC) genotype showed some tendency to this regard. Interestingly, participants harboring IL-10-592 (C) allele or IL-8+396(GG) genotype showed an EF >42%, meaning that these variants have a high contribution to the development of IBS in Blastocystis carriers; while other alleles and genotypes for IBS or for the presence of Blastocystis, showed, independently, low values of EF (<35%).

Table 1 Association between IBS, Blastocystis, and IL-8 and IL-10 gene polymorphisms
Full size table

Discussion

Despite the fact that Blastocystis was discovered almost 100 years ago, its clinical significance and many aspects of its biology remain unresolved. There are arguments for and against its pathogenicity, some being based on parasite loads, genetic type, or subtype and the usefulness of chemotherapeutic intervention (Boorom et al. 2008; Tan 2008; Stensvold et al. 2009b).

The pathophysiology of IBS remains elusive and no single mechanism explains entirely the clinical manifestations of IBS. There are probably several interconnected factors occurring at different level in the patient that consequently may conduce to clinical symptoms of this syndrome (Stark et al. 2007). Thus, some studies focused on the potential role of Blastocystis have found this parasite associated to the development of IBS (Yakoob et al. 2004, 2010; Stensvold et al. 2009a; Jimenez-Gonzalez et al. 2011). In addition, SNPs of cytokine genes involved in the regulation of the immune and inflammatory reaction, such as TNF-α, IL-4, IL-6, and IL-10, have been studied in IBS, suggesting that these SNPs might have a role in differential expression, regulating the pathophysiology of the disease (van der Veek et al. 2005; Wang et al. 2006; Barkhordari et al. 2010a, b). However, the concurrent role of IL-8 and IL-10 SNPs and the presence of Blastocystis carriers in IBS patients have not been previously evaluated. IL-8 is a cytokine involved in the initiation and amplification of acute inflammatory responses and in chronic inflammation (Modi et al. 1990). In contrast, IL-10 limits and may ultimately cease inflammatory responses (Moore et al. 2001); its production has been associated to some SNPs in the promoter gene region (Turner et al. 1997; Moore et al. 2001).

In the present study, we found that alleles G and A, as well as the homozygous variant for IL-8 at position +396 and IL-10 at position −1082, respectively, were relevant to develop IBS. A case–cohort study performed in a population from North Central China revealed that IL-8+396(T/T) genotype has twofold increased relative risks for gastric adenocarcinoma (Savage et al. 2004), while the same SNPs in Dutch IBS patients were similarly distributed in patients and controls (van der Veek et al. 2005); Gonsalkorale et al. (2003) showed that the high producer IL-10-1082(G) allele and the homozygous variant were slightly higher in healthy controls compared with English IBS patients; and in the present study, we found the low producers IL-10-1082 (A) allele and IL-10-1082(AA) genotype were associated to IBS patients. All these data point to the absence or decrease of IL-10 production in patients, which seems to be logical, since IBS patients have an inflammatory processes and IL-10 is related to regulation of inflammation.

The IL-8+396 (GG) genotype was relevant because it was only found associated to Blastocystis carriers and in IBS–Blastocystis carrier; in the latter, the influence of the genotype was higher, considering the OR and EF values. IL-8+781 (T) and IL-10-592 (C) alleles were also associated to Blastocystis carriers and to IBS–Blastocystis carrier, respectively. Puthia et al. (2008) demonstrated that cysteine proteases of Blastocystis ratti induce IL-8 production in colonic epithelial T84 cells and that an NF-κB-dependent transcriptional process is involved. Another study reported that after 24 h incubation of Blastocystis with colonic epithelial cells HT-29 and T-84, the production of inflammatory cytokines IL-8 and granulocyte–macrophage colony-stimulating factor, were induced by the presence of this parasite (Long et al. 2001). Besides, in rats infected with Blastocystis, a significant upregulation of the expression of interferon-γ, IL-12, and tumor necrosis factor alpha, but not IL-6 or granulocyte–macrophage colony-stimulating factor, was seen in the cecal mucosa at 2 and/or 3 weeks post-infection (Iguchi et al. 2009). Thus, because Blastocystis is capable of inducing proinflammatory responses and some SNPs of cytokine genes might have a role in the pathophysiology of IBS, when Blastocystis carriers harbor relevant SNPs of IL-8 or IL-10 (particularly for IL-8+396(G) or IL-10-1082 (A) allele), the relative risks for IBS could be >threefold increased. Finally, association between IL-8 and IL-10 gene polymorphisms to the pair IBS–Blastocystis carrier suggests either their individual or additive role as relevant factors in the etiology of this disease.