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Revista chilena de pediatría

versión impresa ISSN 0370-4106

Rev. chil. pediatr. vol.91 no.3 Santiago jun. 2020

http://dx.doi.org/10.32641/rchped.v91i3.1431 

ORIGINAL ARTICLE

Immune response Th1/Th2 to Helicobacter pylori and Helminths in co-infected patients

Luis Eduardo Bravo1 

Andrés Jenuer Matta1  2 

José Manuel Restrepo-Avenia1 

1 Registro Poblacional de Cancer de Cali, Department of Pathology, School of Medicine, Universidad del Valle, Cali, Colombia.

2 Faculty of Education and Sport Sciences, Escuela Nacional del Deporte, Cali, Colombia.

Abstract:

Introduction:

Inflammation associated with Helicobacter pylori (H. pylori) infection is linked to the development of a gastric precancerous lesion. Helminth infections could influence the pro-inflam matory response to such infection from LTCD4+ Th1 to a less harmful LTCD4+ Th2 response.

Ob jective:

To characterize the polarization of the LTCD4+ Th2 immune response in co-infected pa tients with H. pylori and helminths from low-risk areas for developing gastric cancer.

Patients and Method:

We analyzed 63 patients infected by H. pylori (40 adults and 23 children). Through the Multiplex Analysis technology (xMAP), we determined the serum profiles of the interleukins asso ciated with the polarization of the immune response of LTCD4+ Th1 (IL-1Β, INF-γ, TNF-α) as well as the LTCD4+ Th2 (IL-4, IL-10, and IL-13). The ratio between helminths co-infection status in H. pylori-infected patients and the polarization of the immune response mediated by LTCD4+ Th1 and LTCD4+ Th2 was assessed using a Mixed Effects Logistic Regression Model.

Results:

The frequency of helminths was similar between adults (15%) and children (17%). The polarization of the immu ne response was more prevalent in LTCD4+ Th1. Serum values of interleukins associated with the immune response polarization of LTCD4+ Th1 (IL-1Β, INF-γ, and TNF-α) and LTCD4+ Th2 (IL-4, IL-10, and IL-13) were independent of helminths infection status.

Conclusion:

The prevalence of in testinal parasitic infection was high and the immune response polarization was mainly LTCD4 + Th1.

Keywords: Infection; Helicobacter pylori; Immune Response; helminths

What do we know about the subject matter of this study?

The inflammation associated with Helicobacter pylori infection is related to gastric lesions. Such inflammation is caused by the Th1- type CD4+ immune response in the gastric mucosa on the inflam matory cells of the host.

What does this study contribute to what is already known?

Helminth infections cause the polarization of the Th2-type CD4+ immune response and can influence the H. pylori pro-inflamma tory response from a Th1-type CD4+ profile to a less harmful Th2- type CD4+ response.

Introduction

Since its discovery in 19831, Helicobacter pylo ri (H. pylori) has been recognized as an etiological agent of acute and chronic gastritis as well as an im portant risk factor in the predisposition to diseases such as peptic ulcer, the appearance of MALT (Mu cous Associated Lymphoid Tissue) type lymphoma, and is one of the main determinants of gastric cancer2, the third cause of cancer mortality in Colombia3.

It is estimated that more than 50% of the world’s population is infected with H. pylori, however, a few of them develop a more serious gastrointestinal disease4. In most cases, the infection is acquired in childhood and persists throughout life, causing chronic inflam mation and development of lesions in the gastric mu cosa, since the host’s immune response is not capable of removing this bacterium5,6.

The immune response to pathogens varies ac cording to the pattern of interleukin secretion. In general, the polarization of that response towards Thl-type CD4+ profile is related to intracellular microorganisms including bacteria, protozoa and fungi, production of interleukin-1 beta (IL-1Β), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α); while helminths induce pola rization of the immune response towards Th2-type CD4+ with production of interleukin-4 (IL-4), inter leukin-5 (IL-5), interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-13 (IL-13), and induce activation of B-lymphocytes and their differentiation. The degree of polarization may vary depending on the nature of the infection and its persistence in the host7,8.

The inflammation associated with H. pylori infec tion is caused by Th1-type CD4+ in the gastric muco sa through the release of IFN-γ by host inflammatory cells9,10.

Since concurrent helminth infections induce pola rization of the Th2-type CD4+ immune response11 and are endemic in developing countries with high preva lence of H. pylori infection, helminth infections may modulate the pro-inflammatory response to H. pylori Th1-type CD4+ infection to a less harmful Th2-type CD4+ response.

The objective of this study is to determine the im mune response profile in people with H. pylori and helminthiasis coinfection in Tumaco, Nariño, Co lombia, where the prevalence of infection is > 80%12. This new knowledge will allow defining more fre quent interleukin profiles in populations with high prevalence of H. pylori and at low risk of developing gastric cancer, which may show great differences re gionally.

Patients and Method

Type of study and population

A descriptive study was conducted in the Tuma- co region including 63 people, 40 adults with a histo- pathological diagnosis of chronic H. pylori-associated gastritis and 23 children in the Growth and Develop ment program at Tumaco health centers who tested positive for H. pylori through the [13C]-Urea breath test, to compare the Th1- and Th2-type CD4+ immune response.

A blood sample (15 ml) for immune profile eva luation and stool samples for intestinal parasites detection were collected from each participant during three different cycles. The cycle is defined as the time at which the antiparasitic therapy was administered and the respective blood and stool samples were collected, each with three months apart. In each cycle, a control coprological examination was performed. Patients with a positive diagnosis of intestinal parasite infection received antiparasitic treatment with Albendazole (two doses of 400 mg each 15 days apart) and Tinidazole (2 g per day for two days) within 15 days after obtaining the coprological results.

The research was approved by the Institutional Committee for the Review of Human Ethics (CIREH) of the Universidad del Valle. All adult participants and the child’s parents or legal guardians gave informed consent, as did the children’s assent.

Histopathology procedures

Three gastric mucosa fragments were obtained from each participant, one from the body mucosa of the anterior middle wall and two from the antral re gion, one in the lesser curvature adjacent to the angu lar incisure and the other one in the anterior face 5 cm from the pylorus. Each fragment obtained was fixed, processed, and examined separately, with a 2-hour de hydration protocol, followed by paraffin embedding in the first 24 hours. Histological sections were cut into microtomes (Accu-Cut® SRM) and stained with he- matoxylin-eosin (H & amp; E). To determine the pre sence of H. pylori, modified Giemsa staining was used to detect curved and spiral bacilli13.

[13C]-Urea Breath Test (UBT)

The first sample of exhaled air was collected in a 10 ml Exetainer® vial. A glucose solution containing 100 mg of [13C]-Urea (Cambridge Isotope Laboratories, USA) was immediately administered for ingestion. Another breath sample was collected 30 min after the ingestion of the solution. The concentration of stable [13C]-Urea radioisotope gas was determined at baseli ne and 30 min post-ingestion through the Iris®-Doc Infra-Red Isotope analyzer (Wagner Analyzen Technik, Bremen, Germany)14. The 13CO2/12CO2 ratio mea surement was compared with a reference gas of known isotope ratio.

Diagnosis of intestinal parasite infection

Two aliquots of each stool sample were emulsified in 10% neutral formalin for direct examination by flotation in zinc sulfate solution15. A third aliquot was immersed in Sheather’s sugar16, for trichrome staining as a control and comparison gold standard15. The pa rasite identification was carried out by morphology fo llowing the Guidelines of the American Society of Pa rasitology. Patients diagnosed with intestinal parasite infection were treated with Albendazole (two doses of 400 mg each 15 days apart) and Tinidazole (2 g per day for two days) within 15 days after obtaining the results.

Characterization of the Th1- and Th2-type CD4+ immune response

Serum profiles determination of interleukins asso ciated with Th1-type CD4+ (IL-1Β, INF-γ, and TNF-α) and Th2-type CD4+ (IL-4, IL-10, and IL-13) immune response polarization was established using xMAP® Technology according to the manufacturer’s instruc tions.

Statistical analysis

The normality assumption for the value distribu tion of each interleukin was addressed by analyzing the residuals in ANOVA of repeated measurements through the Shapiro-Wilk test. We used the Friedman test to determine if there were significant differences in each interleukin in the study’s cycles. A mixed-effects logistic regression model was developed to study the relationship between helminthiasis coinfection status in H. pylori-infected patients and the polarization of the Th1-type CD4+ and Th2-type CD4+ -mediated im mune response. The response variable was the presen ce or absence of helminths and the predictor variables, age, sex, and interleukin measurements that characte rize the Th1-type CD4+ and Th2-type CD4+ immune response.

Results

The average age (average ± standard deviation) in the adult participants was 49.6 ± 5.0 years and were mostly women (80%). The average age of the children was 11.0 ± 2.4 years and also most of them were female patients (57%). The adults and children patients were colonized by H. pylori according to the results of the histopathology and Urea (UBT) tests.

Intestinal parasite infection

The frequency of intestinal parasite infection was higher in adults than in children (67% and 48% res pectively) with a p = 0.001 value and helminth infesta tion occurred regardless of age.

Protozoa were more frequent in adults, p = 0.058 (Table 1). Trichocephalus (T. trichiura) and Ascaris (A. lumbricoides) were the most common helminth species in adults and children. In children, there were no En- terobius, Strongyloides, and Ancylostoma; and in adults, no Giardia (Figure 1).

Table 1 Prevalence of protozoa and helminths. 

Figure 1 Parasite species detected in stool samples according to the study cycle. 

Adult and child cytokine profile

Adults presented high serum levels of IFN-γ and TNF-α, however, these interleukins related to the Th1- type CD4+ response profile showed no variation in the different cycles. In contrast, serum values of IL-1ß, IFN-γ, and TNF-α in children were significantly hig her in cycle III (Table 2). When studying the interleu kins related to the Th2-type CD4+ response profile, an important increase in the serum levels of IL4-, IL-10, and IL-13 was observed in children through the diffe rent cycles. In adults, the behavior was similar only in IL-4 values. The other interleukins (IL-10 and IL-13) showed a significant decrease in serum concentrations in cycle III, compared with the values of the cycle I (Fi gure 2).

Table 2 Descriptive Analysis for helminths per cycle. 

Figure 2 LT CD4+ Th1 cytokine profile (pg/ml) in adults and children, by cycle. 

Th1- and Th2-type CD4+ immune response in Helicobacter pylori and helminthiasis coinfected

In children and adults, IFN-γ and TNF-α interleu kins have the highest serum values, which suggests a predominance of the Th1-type CD4+ immune respon se profile, however, serum determinations of the diffe rent interleukins related to the Th1- Th2-type CD4+ immune response do not depend on the intestinal hel minth infestation status (Table 2).

Mixed-effects logistic regression model

All variables were included (cycles, IL-1Β, IFN-γ, TNF-α, IL-4, IL-10, and IL-13) adding the randomized intercept to assess the influence of each patient throug hout the study. The model was adjusted individually in both adults and children. There was no relationship between H. pylori and helminthiasis coinfection status and Th1- and Th2-type CD4+ immune response pro files (Table 3).

Table 3 Ratio of the interleukins profile associated with polarization of immune response type LTCD4+ Th1/Th2 and the state of co-infection by Helicobacter pylori and helminths. 

Discussion

During childhood, H. pylori infection is acquired and its prevalence increases dramatically during the first four years of life11,17. Some studies suggest that during the first years of life, it is at home that the infection is contracted, although there are still large gaps in knowledge regarding the predictors of initial acqui sition18,19.

This study was conducted with participants with positive infection by H. pylori, both adults and child population, from Tumaco, Colombia, a place located at sea level, where sanitary and environmental conditions (heat and humidity) favor the life cycle and transmission of geo-parasites such as helminths20. This study did not evaluate the presence of common comorbidities in tropical areas, such as malaria or leis hmaniasis, as other authors have done21.

The protozoa (Blastocystis hominis and Endolimax nana) were the most prevalent intestinal parasite infec tion in the adult and child population, both considered non-pathogenic17,20 and the most frequent helminths in the pediatric population were Ascaris lumbricoides and Trichuris trichiura.

An outstanding point was the techniques used to collect, process, store, and transport the coprological samples since, despite the distance between the collec tion site (Tumaco) and the processing one (Cali), cys tic forms, trophozoites, and adult parasitic forms were identified which was also favored by the use of analysis techniques (direct, trichrome, and Sheather’s sugar).

In searching for models of the host immune res ponse to H. pylori infection, it has been suggested that the polarization of the immune response towards a Th1-type CD4+ profile contributes to gastric mucosal injury2,5. However, other authors find no association between H. pylori infection and circulating pro-in flammatory interleukins, which has been specifically identified in children and associated with presumed H. pylori inactivity during childhood when there is no ulcerated tissue21.

In our study, we found that adult and pediatric participants infected with H. pylori, explain the polarization of the immune response towards Th1-type CD4+ in both populations. In adults, there was a higher concentration of IFN-γ and TNF-α in all cycles, but no statistically significant difference. This may be related to the chronic nature of H. pylori infection, triggering an immune response that mainly produces an inflam matory reaction6. A similar finding was observed in the pediatric population, where higher concentrations of pro-inflammatory interleukins were associated with the polarization of the immune response towards a Th1-type CD4+ profile, with higher IFN-γ and TNF-α levels, in contrast to what has been published21. We must be careful in interpreting the results because these immune response models (Th1- and Th2-type CD4+) are theoretical in humans and most of the evidence for these models come from murine ones.

Helminths are the best example of a stimulus to in duce a reaction in Th2-type CD4+ cells in humans and experimental models, as eosinophilia, mastocytosis, and high IgE production have been observed in affec ted tissues22,23.

On the one hand, in our case, we found in adult patients high concentrations of IL-10 and IL-13 at the beginning of the study, but there was a slight increa se of IL-4 at the end when they had already received two doses of antiparasitic medication. This may reflect the easy reinfection of the population living in risk areas22. On the other hand, we found in the pediatric population higher levels of IL-13 at the beginning of the study; however, in the final stage, the most pro- inflammatory interleukins were higher than the anti inflammatory ones, therefore, we found that there was a partial relationship between co-infection status (H. pylori-helminths) and the polarization of the immune response towards a Th2-type CD4+ profile, especially in children.

In other investigations, in adult and pediatric pa tients with gastritis or ulcer, the expression of the Th1 response against Th2 has been compared with normal patients, showing that patients infected with H. pylori have higher concentrations of Th1 against Th2, howe ver, in non-infected patients, a higher expression of Th2 against Th1 was observed24, suggesting that the immune response of children and adults against an H. pylori infection shows mainly Th1 concentrations.

This increase in Th1 is directly proportional to the degree of gastritis and, therefore, to the inflammation of the gastric mucosa. This difference in secretion aga inst patients with positive H. pylori infection compared with the negative ones may indicate a key role for this cytosine against gastritis caused by H. pylori24.

This study contrasts with previous publications on the modulation of host immune response in H. pylori and helminthiasis coinfection. Although there are re ports in the literature that in murine models the pola rization of the immune response is evident for a Th2- type CD4+ profile25 and the immune basis confirms a cellular response mediated by Th2-type CD4+ in the presence of intestinal parasites associated with high levels of IgE and IgG126 also evident in certain popu lations, our study found no such association or at least it was only partially so during the bivariate analysis as explained above.

Ethical Responsibilities

Human Beings and animals protection: Disclo sure the authors state that the procedures were fo llowed according to the Declaration of Helsinki and the World Medical Association regarding human experimentation developed for the medical commu nity.

Data confidentiality: The authors state that they have followed the protocols of their Center and Local regu lations on the publication of patient data.

Rights to privacy and informed consent: The authors have obtained the informed consent of the patients and/or subjects referred to in the article. This docu ment is in the possession of the correspondence author.

Financial Disclosure: This research study was sponsored by Universidad del Valle:1106-40820552; COLCIENCIAS Young resear chers and innovators program: No.645-2014; Univer sidad del Valle -Universidad de Vandervilt- NIH-USA (Prime Award No.2 P01 CA028842-24. Subaward No. VUMC3239, 2009-2015).

Acknowledgment: These authors express heartfelt thanks to the Micro biology, Histopathology, and Molecular Biology La boratory of the Department of Pathology of the Uni versidad del Valle, which offered its facilities to carry out this research study. Thanks also go to the technical personnel at the Registro Poblacional de Cancer of Cali and to the Department of Pathology of the School of Medicine of the Universidad del Valle.

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Received: October 01, 2019; Accepted: March 16, 2020

Correspondence: Luis Eduardo Bravo. E-mail: luis.bravo@correounivalle.edu.co.

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