A cytokine-based evaluation of T lymphocyte proliferation and differentiation into T-helper 1 and T-helper 2 subsets in patients with chronic renal failure at the Yaounde General Hospital

TITUS DESTERNE NDZANA NGUEME (DESTERNE@yahoo.com)
Microbiology parasitology hematology Immunology, University of Yaounde I
July, 2015
 

Abstract

Background and Rationale
Patients with Chronic Renal Failure (CRF) are at high risk of infections. Infections represent 20% of mortality in this group of patients and this in part due to the alteration of the cellular immune system. Studies in vitro have shown that CRF patients suffer from lymphopenia. In addition, the clonal expansion of CD4 T cells after activation and their differentiation into effector subsets have also been found to be altered in different cohorts of CKD patients. However, the variation of this dysfunction with the stages of Chronic Kidney Disease (CKD) is not well known. Moreover, very few studies have concomitantly investigated T cell proliferation and differentiation within the sample set.
Objectives
Our main objective was to determine if there is a stage-dependent variation of CD4 T cell function in patients with CKD. Specifically, we sought to: 1) Determine basal Peripheral Blood Mononuclear Cell (PBMC) count at various stages of CKD; 2) Evaluate in vitro mitogen-induced production of the T cell proliferative cytokine IL2 at various stages of CKD; 3) Assess in vitro mitogen-induced production of the T-helper 1 cytokine 1L1b and T-helper 2 cytokine IL10 at various stages of CKD;
Materials and Methods
In order to attain our objectives, we carried out a cross-sectional study. We did a convenience sampling and recruited 15 Cameroonian CKD predialysis patients (5 CKD stage 3 (CKD G3), 5 CKD stage 4 (CKD G4), and 5 CKD stage 5 non-dialysed (CKD G5ND)), 8 CKD G5 patients on maintenance haemodialysis (CKDG5D) and 10 healthy controls (HC). Patients with diabetes, HIV-AIDS, patients >60years old, those taking steroids as well as those who did not give an informed consent where excluded from the study. Density gradient separation was used to isolate Peripheral Blood Mononuclear Cells (PBMC) from collected whole blood sample. The isolated PBMCs were then washed, counted and seeded in 96-well culture plates at a concentration of 200,000 cells per well of 200uL supplemented Roswell Park Memorial Institute(RPMI) 1640 medium. For every sample, cells were treated with the T-cell mitogen phytohemagglutinin (PHA) or left untreated. The treatment and no treatment wells were each incubated in triplicates for 3 days. On the third day of culture, supernatants were harvested and stored at -80°C until analyses. Levels of the cytokines IL2, IL1b and IL10 were quantified in the culture supernatants using the MagPix Multiplex Analyte Platform (MAP) assay (Luminex ® Corporation). Our data was analysed using Graph Pad Prism 5.0. The statistical tests Kruskal-Wallis and Mann Whitney U were used to analyse our data.
Results
Generally CKD patients had lower PBMC counts compared to healthy controls and patients on maintenance haemodialysis (p<0.05). From stage 3 to stage 5, we observed a decrease in the PBMC count (p<0.05). The IL2 culture supernatant levels post PHA stimulation, were higher in healthy controls compared to CKD predialysis patients (p = 0.2117). There was no relation between the stages of CKD and the culture supernatant levels of IL2. However, we noticed that CKD G5ND patients had the lowest levels of IL2 post PHA stimulation compared to CKD G3 and CKD G4. There was a non-significant increase of the culture supernatant levels of IL2 in haemodialysis patients compared to the non-dialysed patients (p=1.0000). Patients with CKD had higher culture supernatant levels of IL10 compared to healthy controls (p = 0.0795). Patients on maintenance haemodialysis had lower culture supernatant levels of IL10 compared to non-dialysed patients (p = 0.3543). There was no relation between the stage of the disease and the culture supernatant levels of IL10. Patients with CKD had lower culture supernatant levels of IL1b compared to healthy controls (0.1499). The IL1b/IL10 ratio was lower in CKD patients when compared to healthy controls but higher in haemodialysis patients.
Conclusion
Thus, there is an altered cellular immune function in CRF. Specifically, 1) CKD is associated with decreased PBMC counts, which worsens with the severity of the disease; 2) CKD is associated with decreased clonal expansion of CD4 T cells; 3) CKD is associated with a differentiation bias of CD4 T cells towards T helper 2, which is least in CKD G3; 4) Haemodialysis tends to improve CD4 T cell clonal expansion and is associated with a differentiation bias towards Th1.


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