Indian Journal of Pathology and Microbiology
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Ahead Of Print Login 
Users Online: 39003
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size


 
  Table of Contents    
ORIGINAL ARTICLE  
Year : 2023  |  Volume : 66  |  Issue : 3  |  Page : 556-559
Natural regulatory T cells increase significantly in pediatric patients with parasitic infections: Flow cytometry study


1 Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, Northern Border University, Arar, Saudi Arabia
2 Department of Microbiology, Faculty of Medicine, Northern Border University, Arar, Saudi Arabia

Click here for correspondence address and email

Date of Submission29-Dec-2021
Date of Decision14-Feb-2022
Date of Acceptance14-Feb-2022
Date of Web Publication27-Jan-2023
 

   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. Materials and Methods: 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 article:
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 2023;66:556-9

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 [serial online] 2023 [cited 2023 Sep 23];66:556-9. Available from: https://www.ijpmonline.org/text.asp?2023/66/3/556/368577





   Introduction Top


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.[1],[2],[3] Tregs decrease immune reactivity through cytokines production.[4] Natural regulatory T cells (nTregs) and inducible regulatory T cells (iTregs) are two types of Tregs (iTregs).[5] 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.[6] Tregs display the biomarkers CD4, Foxp3, and CD25 and are believed to be related to natural CD4 cells.[7]

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.[8] 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.[9] Additionally, Tregs can release IL-10, IL-35, or TGF, which elicit cell cycle arrest in surrounding effector T cells.[10] 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 Top


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.[11]

Weber's trichrome stain was used for Microsporidia analysis.[12] Strongyloides stercoralis parasites were grown on Agar plates for analysis.[13] 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 Top


[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 1: Clinical data of pediatric patients

Click here to view
Table 2. Comparison of CD4+, CD25+and Foxp3+ levels of different groups according to the types of parasites

Click here to view
Table 3: Relationship between the percentages of biomarkers, Foxp3+, CD25+ and CD4+, and the degree of infection in pediatric subjects

Click here to view


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

Click here to view



   Discussion Top


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.[14],[15] When Tregs maintain host homeostasis by regulating the immune response, the result is greater pathogen survival.[16] Tregs aggregate at the site of infection with Leishmania and control the function of the localized effector cells, preventing the parasite from being killed.[17]

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.[18] 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.[19] Indeed, Giardia acts as a suppressor of the inflammatory response.[20]

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.[21],[22]

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.[15] 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.[23] 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.

Author's contributions

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.

Acknowledgements

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.



 
   References Top

1.
Roncarolo MG, Gregori S, Bacchetta R, Battaglia M, Gagliani N. The biology of T regulatory type 1 cells and their therapeutic application in immune-mediated diseases. Immunity 2018;49:1004-19.  Back to cited text no. 1
    
2.
S. Gahlot GP, Das P, Baloda V, Singh A, Vishnubhatla S, Gupta SD, et al. Duodenal mucosal immune cells in treatment-naive adult patients with celiac disease having different histological grades and controls. Indian J Pathol Microbiol 2019;62:399-404.  Back to cited text no. 2
    
3.
Murphy KM, Stockinger B. Effector T cell plasticity: Flexibility in the face of changing circumstances. Nat Immunol 2010;11:674-80.  Back to cited text no. 3
    
4.
Sojka DK, Huang Y-H, Fowell DJ. Mechanisms of regulatory T-cell suppression – A diverse arsenal for a moving target. Immunology 2008;124:13-22.  Back to cited text no. 4
    
5.
Gückel E, Frey S, Zaiss M, Schett G, Ghosh S, Voll R. Cell-intrinsic NF-κB activation is critical for the development of natural regulatory T cells in mice. PLoS One 2011;6:e20003.  Back to cited text no. 5
    
6.
Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006;441:235-8.  Back to cited text no. 6
    
7.
Curiel TJ. Tregs and rethinking cancer immunotherapy. J Clin Invest 2007;117:1167-74.  Back to cited text no. 7
    
8.
Maizels RM, Balic A, Gomez-Escobar N, Nair M, Taylor MD, Allen JE. Helminth parasites – masters of regulation. Immunol Rev 2004;201:89-116.  Back to cited text no. 8
    
9.
Nakamura K, Kitani A, Strober W. Cell contact-dependent immunosuppression by CD4+CD25+regulatory T cells is mediated by cell surface-bound transforming growth factor beta. J Exp Med 2001;194:629-44.  Back to cited text no. 9
    
10.
Sakaguchi S, Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T. Regulatory T cells: How do they suppress immune responses? Int Immunol 2009;21:1105-11.  Back to cited text no. 10
    
11.
Cossarizza A, Chang H-D, Radbruch A, Akdis M, Andrä I, Annunziato F, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol 2017;47:1584-797.  Back to cited text no. 11
    
12.
Weber R, Bryan RT, Owen RL, Wilcox CM, Gorelkin L, Visvesvara GS. Improved light-microscopical detection of microsporidia spores in stool and duodenal aspirates. N Engl J Med 1992;326:161-6.  Back to cited text no. 12
    
13.
Arakaki T, Hasegawa H, Asato R, Ikeshiro T, Kinjo F, Saito A, et al. A new method to detect strongyloides stercoralis from human stool. Japanese J Trop Med Hyg 1988;16:11-7.  Back to cited text no. 13
    
14.
Thakur S, Singla A, Chawla Y, Rajwanshi A, Kalra N, Arora SK. Expansion of peripheral and intratumoral regulatory T-cells in hepatocellular carcinoma: A case-control study. Indian J Pathol Microbiol 2011;54:448-53.  Back to cited text no. 14
[PUBMED]  [Full text]  
15.
Hesse M, Piccirillo CA, Belkaid Y, Prufer J, Mentink-Kane M, Leusink M, et al. The pathogenesis of schistosomiasis is controlled by cooperating IL-10-producing innate effector and regulatory T cells. J Immunol 2004;172:3157-66.  Back to cited text no. 15
    
16.
Scott-Browne JP, Shafiani S, Tucker-Heard GS, Ishida-Tsubota K, Fontenot JD, Rudensky AY, et al. Expansion and function of Foxp3-expressing T regulatory cells during tuberculosis. J Exp Med 2007;204:2159-69.  Back to cited text no. 16
    
17.
Belkaid Y, Piccirillo CA, Mendez S, Shevach EM, Sacks DL. CD4+CD25+regulatory T cells control Leishmania major persistence and immunity. Nature 2002;420:502-7.  Back to cited text no. 17
    
18.
Shevach E. Mechanisms of Foxp3+T regulatory cell-mediated suppression. Immunity 2009;30:636-45.  Back to cited text no. 18
    
19.
Oberhuber G, Kastner N, Stolte M. Giardiasis: A histologic analysis of 567 cases. Scand J Gastroenterol 1997;32:48-51.  Back to cited text no. 19
    
20.
Roxström-Lindquist K, Palm D, Reiner D, Ringqvist E, Svärd S. Giardia immunity-An update. Trends Parasitol 2006;22:26-31.  Back to cited text no. 20
    
21.
Jung HC, Eckmann L, Yang SK, Panja A, Fierer J, Morzycka-Wroblewska E, et al. A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clin Invest 1995;95:55-65.  Back to cited text no. 21
    
22.
Roxström-Lindquist K, Ringqvist E, Palm D, Svärd S. Giardia lamblia-induced changes in gene expression in differentiated Caco-2 human intestinal epithelial cells. Infect Immun 2005;73:8204-8.  Back to cited text no. 22
    
23.
Hams E, Aviello G, Fallon PG. The schistosoma granuloma: Friend or foe? Front Immunol 2013;4:89.  Back to cited text no. 23
    

Top
Correspondence Address:
Nida Suhail
Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, Northern Border University, Arar
Saudi Arabia
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_1262_21

Rights and Permissions



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
    References
    Article Tables

 Article Access Statistics
    Viewed1040    
    Printed46    
    Emailed0    
    PDF Downloaded30    
    Comments [Add]    

Recommend this journal