Abstract: Amoebiasis, caused by Entamoeba histolytica, is still considered a major health problem in developing countries. Since the immune response during human amoebiasis has not been clearly defined, we chose to evaluate cytokine production in patients suffering from amoebic colitis. A case-control association study was carried out on 62 subjects, including 31 patients with amoebic colitis and 31 healthy controls (age, sex and geographic region-matched). Serum levels of IL-12, IFN-γ, IL-13 and IL-5 were measured by solid-phase sandwich enzyme linked immunosorbant assay. Serum levels of IFN-γ, IL-12, IL-13 and IL-5 were higher in the patients with amoebic colitis than in healthy controls, but were only statistically increased for IL-5 (p = 0.04) and IL-13 (p = 0.014). Stratification of patients according to gender revealed that IL-13 was significantly elevated in men as compared to levels measured in women (p = 0.04). These findings suggest that E. histolytica induce a mixed Th-1/Th-2 response with a polarization toward Th-2 during the early stage of amoebiasis, which may aide in developing a clinical illness.
INTRODUCTION
Entamoeba histolytica is an enteric dwelling human protozoan parasite that causes the disease amoebiasis, which is endemic in the developing world. Estimates suggest that 40-50 million cases of amebic colitis and liver abscess occur annually in the world, which result in 40,000-110,000 deaths (WHO/PAHO/UNESCO, 1997). Intestinal amoebiasis is the most common disease caused by E. histolytica. It is characterized by invasion of the intestinal mucosa, which causes ulcerative colitis.
Both humoral and Cell-Mediated Immunity (CMI) have been implicated in the pathogenesis of amoebiasis, the latter playing the dominant role (Trissl, 1982; Guo et al., 2009). It has been shown that cytokines orchestrate the immune response and influence the outcome of the disease. Therefore, much attention is now focused on the role of cytokines, which modify the immune response to various infections (Denis and Chadee, 1989; Tallima et al., 2009). Studies examining the immune response during human amoebiasis are lacking (Gonzalez-Amaro et al., 1994).
Despite the importance of amoebiasis in terms of public health burden, the lack of integrated data on immunopathogenesis means that we have only an incomplete description of the initiation and development of amoebic colitis. To date, most studies determining host resistance mechanisms to E. histolytica have been done using a hamster model of disease. Hamsters with an amoeba infection mount a vigorous Th-1 response, driven by high levels of IL-12, IL-2 and IFN-γ. However, a Th-2 type immune response deteriorates the disease and leads to chronic disease. Previous studies have indicated that CMI mediated by IFN-γ plays a major role in immunity against amoebiasis (Guo et al., 2009) and may predict future susceptibility to symptomatic amoebiasis (Haque et al., 2007). During invasive amoebiasis, studies of cellular immune responses have shown decreases of CD4+ cells, increases of CD8+ cells and decreases in the in vitro proliferation of T lymphocytes against amoebic antigen, indicating a suppression of the immune response (Salata et al., 1990). Previous research has shown that antigenic epitopes of E. histolytica can stimulate either the Th-1 or Th-2 immune responses (Mann et al., 1993). In humans, the mechanisms are less well-defined and it appears that a balance between the Th-1 and Th-2 responses might present the optimal response.
The present study was conducted to evaluate IL-12, IFN-γ, IL-5 and IL-13 production in individuals suffering from amoebic colitis to demonstrate the role of Th-1 and Th-2 responses in immunity during human amoebiasis.
MATERIALS AND METHODS
The study consisted of 31 (10 females and 21 males, mean age 23.7±15.2 years) patients with clinical and laboratory diagnosis of amoebic colitis from areas of Northern Iran, who referred to the Razi Hospital of Mazandaran University of Medical Sciences during September 2007 and June 2008. Detailed medical records, including demographic data, disease status, clinical presentation and laboratory findings were reviewed. Control subjects included 35 clinically healthy individuals residing in the same geographic area and were age- and sex-matched subjects without inflammatory or autoimmune diseases. Controls were also CRP and stool exam negative and did not receive any medication. Serum from each individual was obtained by centrifugation at 1600 g for 15 min. Serum samples were stored at -70°C until analyzed. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by Mazandaran University of Medical Sciences medical research committee. Informed consent was obtained from all patients and controls enrolled in the study.
Parasitological examination: Three consecutive stool samples were withdrawn from each individual and examined for the presence of Entamoeba by saline preparation and iodine staining. All negative stool samples were subjected to the zinc sulphate floatation concentration technique (Campbell and Chadee, 1997) and examined for Entamoeba trophozoite and cysts. Patients were considered to have amoebic colitis if trophozoites were found in stool samples (Kretschmer and López- Osuna, 1990). Since, E. histolytica and E. dispar cannot be differentiated microscopically, all microscopy positive samples were evaluated by a TechLab E. histolytica stool antigen detection ELISA test (TechLab, Inc., Blacksburg, VA).
Cytokine assays: Cytokines were detected in the serum by using commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kits with paired cytokine-specific monoclonal antibodies according to the manufacturer's recommended procedure (Bender Med Systems; Vienna, Austria). The detection limit for IL-5, IL-10, IL-12 and IFN-γ were 1.46, 0.99, 2.1 and 0.99 pg mL-1, respectively.
Statistical analysis: Statistical analyses were performed using SPSS 10.0 for Windows (SPSS, Chicago, Illinois). Statistical significance of continuous variables was determined by using a Student's T test. Continuous variables were reported as Mean±standard deviation (SD). All tests were two-tailed and p<0.05 was considered statistically significant.
RESULTS
Study population: The χ2 test demonstrated that the number of males and females were not significantly different in the patients and controls (21 versus 10 and 16 versus 15, p = 0.3, respectively). The ages of the two groups were also not significantly different (23.7±15.2 and 28.35±11.97, p = 019). No significant differences in white blood cells and platelet counts or hemoglobin concentration between the groups were observed (Table 1). The most common symptoms among amoebisis patients were abdominal pain, dysentery and abdominal distention.
| Table 1: | Characteristics of study population |
| ND: Non detectable | |
| Table 2: | Cytokine levels in serum of patients with amoebic colitis and healthy controls |
| Table 3: | Gender differences in cytokine levels in serum of patients with amoebiasis |
None of the patients had peritonitis, empyema, pericarditis, chronic colitis and liver or brain abscess complications.
Serum levels of IL-5, IL-12, IL-13 and IFN-γ in amoebiasis patients and controls: To determine the role of Th-1/Th-2 responses in human amoebiasis, we studied serum concentrations of IL-5 and IL-13 (Th-2-type), IFN-γ (Th-1-type) and also IL-12 in patients with amoebic colitis and healthy controls. As shown in Table 2, the serum levels of all cytokines tested were higher in patients than in controls, but only IL-5 and IL-13 levels were statistically significant.
It was previously reported that prevalence of amoebiasis is far more common in males than females (Denis and Chadee, 1989). However, further analysis was conducted comparing cytokine levels in male and female patients. As shown in Table 3, only serum levels of IL-13 were significantly higher in infected males than in females.
DISCUSSION
In the present study, the serum levels of IL-5 and IL-13 were significantly higher in patients with amoebic colitis as compared to healthy controls. Resistance to amoebiasis requires an effective cell-mediated immune response against E. histolytica, which is mediated by nitric oxide released from activated macrophages. The pathogenicity of E. histolytica results from its adhesion to the intestinal epithelium followed by its penetration into the mucosa (Kretschmer and López-Osuna, 1990). During mucosal invasion, the activation of the immune response is induced via the release of inflammatory cytokines by host epithelial cells that initiate an acute inflammatory process (Seydel et al., 1997). However, inflammation induced by amoeba infection in the intestine, is driven by a Th-2 cell phenotype. Polarization of the immune response toward Th-2 type, which presents by production of IL-5, IL-13 and IL-4 (Chatelain et al., 1999), has various effects on amoeba pathogenesis. First, production of these cytokines can down regulate macrophage parasitocidal activity, which may result in tissue damage by the release of necrotizing enzymes (Virk et al., 1990). This phenomenon helps to an invasive effort in predisposed individuals (Murray, 1998). It has been reported that parasite secreted products are likely to regulate macrophage mediated responses to E. histolytica trophozoites (Salata et al., 1989). Therefore, E. histolytica suppresses both the macrophage respiratory burst and antigen presentation by class II Major-Histocompatibility-Complex (MHC) molecules (Haque et al., 2003). Second, the induction of mucosal inflammation driven by Th-2 cytokines helps to contain the amoebic infection at an early stage, at the cost of extensive mucosal destruction. Third, high production of IL-5 in combination with IL-10 promotes IgA class switching and maturation in the late stage of amoebic colitis and thus, induction of protective mucosal immunity (Corthesy, 2007). It has been suggested that intestinal antilectin IgA antibodies could prevent parasite colonization of the gut (Ravdin et al., 2003). It has also been demonstrated that depletion of CD4+ T lymphocytes in susceptible mice results in a reduction of mastocyte infiltration to the submucosa and a diminished secretion of IL-4 and IL-13 (Houpt et al., 2002). Consequently, a reduction in parasite burden was also observed. On the contrary, occurrence of an acute inflammatory reaction in the early stage of intestinal amoebiasis, which orchestrates with Th-2 cytokines, is a double edged sword.
The Th1/Th2 polarization paradigm, yet biologically questionable in humans (Rook, 2001) follows many complex and changing responses to environmental factors and, on the other hand, depends on the genetic background of the cells. Th-1 driven responses appear to be responsible for the outcome and the protective immunity in resolved invasive amoebiasis (Kretschmer and López-Osuna, 1990). Indeed, activation of CD4+ T cells by macrophages is pivotal for an effective specific immune response, requiring mainly a Th-1 type cytokine secretion, such as IFN-γ and IL-12 [5, 6]. Decreases in IFN-γ and IL-12 in patients with amoebiasis could represent suppression of the protective Th-1 responses in acute amoeba colitis. Otherwise, amoebic components suppress NO production by macrophages during a primary infection (Seguin et al., 1997) and cytokine induction in the late phase of amebic abscess of the liver (Bekker-Mendez et al., 2006). Previous research determined that failure of macrophage activation due to suppression of Th-1 responses causes rapid spread of E. histolytica, as well as increased multiplication (Campbell and Chadee, 1997). This interpretation was confirmed by detecting increased IFN-γ in the patients with amoeba liver abscess after treatment (Sanchez-Guillen et al., 2002).
Interleukin-13 has been shown to have potent anti-inflammatory properties and can down-regulate macrophage functions such as production of IL-12, expression of inducible nitric oxide synthase, as well as up-regulation of IL-10 production (Wills-Karp et al., 1998). Significant increases in IL-13 production in amoeba patients may explain the low IL-12 production. Indeed, high production of IL-5 and IL-13 in patients with intestinal amoebiasis is in agreement with a previous study that reported increases in IL-4 and IL-10 production in symptomatic patients (Bansal et al., 2005). Paradoxically, IL-13 and also IL-4 may have the potential to systemically induce immune suppression in E. histolytica infected patients and thereby aide in developing a clinical illness. These findings are in agreement with the results of an in vitro study that demonstrated that E. histolytica induces increases in IL-1β, IL-2, IL-5, IL-6 and IL-10 production in axenic culture (Salata et al., 1989). In addition, high IL-13 levels in male patients compared to females may be partially responsible for the high prevalence of disease in these patients (Acuna-Soto et al., 2000).
ACKNOWLEDGMENTS
We are grateful to Dr Alian, Department of Infectious Diseases, Razi Hospital, Ghaemshar, Iran; for their help in providing samples.