| Abstract|| |
Background: The most accepted definition of regulatory T cells (Tregs) relies on the expression of several biomarkers, including CD4, CD25, and transcription factor, Foxp3. The Tregs maintain tolerance to self-antigens and prevent autoimmune diseases. Aim: The purpose of this study was to determine the difference in natural Treg levels in Entamoeba histolytica, Schistosoma mansoni, Giardia lamblia, Enterobius vermicularis, and Hymenolepis nana infected patients. Setting and Design: Fifty-one pediatric subjects (29 males and 22 females) were recruited from a tertiary care hospital, and were divided into infected and non-infected (control) groups. The mean age of the subjects was 8.7 years. Methods and Material: Blood samples were collected from infected and non-infected groups, and change in the level of Tregs in these subjects was investigated by flow cytometry. Statistical Analysis Used: The statistical analysis of data was performed by SPSS software. Quantitative data used in this study included mean and standard deviation. Data from the two groups were compared by the Student's t-test. The age of the patient and infection status were used for multivariate logistic regression analysis. Odds ratios (ORs) were estimated within a 95% confidence interval, and a P value of <0.05 was considered significant. Results and Conclusions: The levels of natural regulatory T cells, indicated by the biomarkers, CD4+, CD25+, and Foxp3+, increase significantly in patients infected by Entamoeba histolytica, Schistosoma mansoni, Giardia lamblia, Enterobius vermicularis, and Hymenolepis nana as compared to controls. They also increase in cases of mixed infection as compared to infection by a single parasite.
Keywords: Flow cytometry, parasitic infection, pediatric patients, regulatory T cells
|How to cite this URL:|
Kizilbash N, Suhail N, Alzahrani A K, Basha W J, Soliman M. Natural regulatory T cells increase significantly in pediatric patients with parasitic infections: Flow cytometry study. Indian J Pathol Microbiol [Epub ahead of print] [cited 2023 Mar 28]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=368577
| Introduction|| |
Immunological tolerance is maintained by regulatory T cells (Tregs). Down-modulated anti-tumor immunity, autoimmunity, and constrained allergic diseases are restricted by suppressing multiple immune system cell types for maintaining dominant tolerance. The effector's response to pathogens can be influenced by Tregs. Their function is to inhibit T cell-mediated immunity at the termination of an immune response and to reduce auto-reactive T cells that evade the thymic negative selection process.,, Tregs decrease immune reactivity through cytokines production. Natural regulatory T cells (nTregs) and inducible regulatory T cells (iTregs) are two types of Tregs (iTregs). Natural regulatory T cells (nTregs) are responsible for self-antigen tolerance. nTregs grow in the thymus in response to self-antigen exposure. Tregs appear to be immunosuppressive and primarily inhibit or down-regulate effector T cell activation and proliferation. Tregs display the biomarkers CD4, Foxp3, and CD25 and are believed to be related to natural CD4 cells.
Many mechanisms have been developed by pathogens to indirectly exploit the regulatory machinery of the host to secure their survival by the production of Tregs. Tregs are primarily responsible for maintaining immune cell homeostasis. This is accomplished through four distinct processes that suppress conventional T cells and dendritic cells (DC), as well as other important types of immune cells such as natural killer (NK) cells. One such strategy is the usage of suppressive cytokines or other substances released or expressed on the surface of Tregs cells. Through exposure to TGF- β linked to the Tregs cell membrane, contact-dependent suppression of CD4+ or CD8+ effector T cells is obtained. Additionally, Tregs can release IL-10, IL-35, or TGF, which elicit cell cycle arrest in surrounding effector T cells. Tregs can produce galectin-1 to cause death in nearby T cells positive for CD45 and CD43 glycoprotein receptors expression. The purpose of this study was to determine the difference in natural Tregs levels in Entamoeba histolytica, Schistosoma mansoni, Giardia lamblia, Enterobius vermicularis, and Hymenolepis nana affected patients.
| Materials and Methods|| |
Fifty-one pediatric research subjects (29 males and 22 females) were recruited from a tertiary care hospital. The mean age of the subjects was 8.7 years, and they were divided into infected and non-infected (control) groups. The exclusion criteria included malnutrition, immune deficiency, inflammatory diseases, diabetes mellitus, infection by the malarial parasite, and any anti-parasitic treatment in the previous three months.
The procedures followed were in accordance with the ethical standards of the Institutional Ethical Committee and with the Helsinki Declaration of 1975, as revised in 2000. Written consent was obtained from the parents of all children prior to enrollment in this study. All the information collected was kept confidential. The study was approved by the institutional ethical committee (dated: 12/5/2021).
Blood samples were collected from healthy controls and research subjects. RBCs were isolated after centrifugation and cultured in RPMI 1640 medium. They were stimulated by the addition of Phorbol myristate acetate (PMA) (50 ng/mL), Ionomycin (1 μg/mL), and Brefeldin A (BFA) (10 μg/mL). They were then further centrifuged at 1200g and washed and stained by the use of anti-CD3 and anti-CD4 antibodies. To stain cells intracellularly, cells were transfected with chemicals according to conventional methods.
The cells were incubated for 30 minutes with a fluorescence-labeled anti-Foxp3+. Finally, the cells were resuspended in PBS and examined using an EPICS XL Flow Cytometer. Cell sorting was utilized in this work to classify cells according to the presence or lack of specified physical parameters. The Flow Cytometer detected cells based on size, morphology, and protein expression level.
Weber's trichrome stain was used for Microsporidia analysis. Strongyloides stercoralis parasites were grown on Agar plates for analysis. Phycoerythrin (PE)–anti-CD4, fluorescein isothiocyanate–anti-CD25, Phycoerythrin-cyanine 5, and (RPE-CY5)–anti-Foxp3 were utilized as monoclonal antibodies for Flow Cytometry. The fixation and permeabilization of cells were accomplished by the use of Intraprep kits.
The statistical analysis of data was performed by SPSS software (Chicago, USA). Quantitative data used in this study included median, range, mean, and standard deviation. Data from the two groups were compared by the Student's t-test. The age of the patient and infection status were used for multivariate logistic regression analysis. Odds ratios (ORs) were estimated within a 95% confidence interval, and a P value of <0.05 was considered significant.
| Results|| |
[Table 1] and [Table 2] show that the research subjects had significantly higher levels of natural regulatory T cells (CD4+, CD25+, and Foxp3+) when compared to healthy controls (P < 0.001). Additionally, a contrast of the CD4+, CD25+, and Foxp3+ proportions in the examined group according to the type of parasites versus control [Table 2] reveals that patients infected with each type of parasite have a higher level of CD4+, CD25+, and Foxp3+ population than those with no infection at all (P = 0.001). Giardia infection resulted in significantly higher CD4+, CD25+, and Foxp3+ levels in subjects than infection with other parasites (P = 0.001). Subjects infected with Schistosoma mansoni, on the other hand, had substantially reduced CD4+, CD25+, and Foxp3+ levels than those infected with other parasites (P = 0.001). Also, subjects with mixed infection showed substantially greater levels of CD4+, CD25+, and Foxp3+ than those with a single infection (P = 0.028) [Table 3].
|Table 2. Comparison of CD4+, CD25+and Foxp3+ levels of different groups according to the types of parasites|
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|Table 3: Relationship between the percentages of biomarkers, Foxp3+, CD25+ and CD4+, and the degree of infection in pediatric subjects|
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Statistical analysis was performed using the age of the subjects and infection status to obtain CD4+, CD25+, and Foxp3+ percentages as dependent variables [Table 4]. It showed that parasitic infection is a risk factor for higher CD4+, CD25+, and Foxp3+ levels (P < 0.001). The data also showed that adults have higher CD4+, CD25+, and Foxp3+ levels (P < 0.001).
|Table 4: Multivariate analysis of Foxp3+, CD25+ and CD4+ regulatory T cell levels as a dependent variable in all subjects|
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| Discussion|| |
Tregs exercise their function by a variety of processes, including regulation of antigen-presenting cell (APC) development and function, target cell death, impairment of metabolic pathways, and generation of anti-inflammatory cytokines. Experimental data from prior studies suggest that Treg-mediated immune system modulation may be especially critical for guarding tissues with highly specific functions, including the liver or the eyes., When Tregs maintain host homeostasis by regulating the immune response, the result is greater pathogen survival. Tregs aggregate at the site of infection with Leishmania and control the function of the localized effector cells, preventing the parasite from being killed.
The results from this study showed that CD4+, CD25+, and Foxp3 + levels increased significantly in infected patients as compared to controls. Also, parasitic infection status was found to be a risk factor for higher CD4+, CD25+, and Foxp3+ levels. During chronic phases of the disease, E. histolytica induces the formation of Treg populations that suppress the other T cells. Giardia lamblia infection resulted in significantly greater CD4+, CD25+, and Foxp3+ levels than infection with other parasites (P = 0.001). The comparatively high CD4+, CD25+, and Foxp3+ levels observed in G. lamblia infection could be explained by the fact that G. lamblia induces very little or no inflammation. Indeed, Giardia acts as a suppressor of the inflammatory response.
However, investigation of the gene expression of several cytokines in human intestinal epithelial cells following G. lamblia infection revealed no evidence of a significant increase in the generated cytokines.,
The study found that Schistosomiasis patients had significantly reduced CD4+, CD25+, and Foxp3+ levels than those infected with other parasites (P = 0.001). Additionally, Hesse et al. discovered that not all Schistosoma mansoni infections result in elevated Tregs levels. The medicinal treatment resulted in a drop in the percentage of Tregs. Hans and coworkers discovered that S. granuloma infection also increased Foxp3+ expression. Also, mixed infections showed significantly higher CD4+, CD25+, and Foxp3+ levels as compared to infection by a single parasite (P = 0.028). Strong infections showed higher CD4+, CD25+, and Foxp3+ levels as compared to mild infections (P = 0.003).
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
NK designed the study, performed literature search, and prepared the original draft of the manuscript. NS and AKA helped in data interpretation, reviewed, and edited the final manuscript. WJB and MS helped in data acquisition, processing, and analysis.
The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia for funding this research work through project number 1112-AMS-2019-1-F.
Financial support and sponsorship
This work was funded by the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia (Project number 1112-AMS-2019-1-F).
Conflicts of interest
There are no conflicts of interest.
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Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, Northern Border University, Arar
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]