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Treg Cells in Inflammation Experiment

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Ulcerative colitis (UC) and Crohn disease (CD) are chronic relapsing-remitting inflammatory disorders of the gut that afflicts millions of the individuals throughout the world with symptoms which impair performance and quality of life [1, 2]. The etiology of UC is still not fully understood, although some studies suggest that loss of tolerance toward endoluminal microbes result in dysregulated immune activity of the intestinal mucosa[2-4]. Both the innate and acquired immune systems are involved in excessive inflammatory reaction of the gut[5].

Human Regulatory T cells (Treg Cells) play a crucial role in the maintenance of T cell homeostasis and immunological tolerance[6]. This ability of Treg cells ascribes to them a key role in the pathophysiology of autoimmune diseases. Thereby, deficiencies in Treg cells frequency and function lead to the development of autoimmune disorders [6, 7]. Tregs are now identified as CD4+T cells with high expression of CD25 and low expression of IL-7 receptor (CD127). However, the expression of lineage-specific transcription factor, Forkhead box P3 (FoxP3) is crucial for regulatory function of Treg cells and defining the Treg populations[8-10].Mutations of FoxP3 in humans causes development of immunodysregulation polyendocrinopathy and enteropathy X-linked syndrome (IPEX)[11]. Intestine is the most frequently affected organ in IPEX, which exemplifies the importance of FoxP3 in gut homeostasis[12].

Although some clinical studies have shown decreased numbers of Treg cells in the active state of UC [13-17], conflicting results have also been found [18, 19]. Reinvestigation using more comprehensive panels of Treg cell markers may answer the question of whether or not Treg cell numbers are deficient in UC. In our present study, we assessed the frequency of CD4+ CD25+ CD127low FoxP3+ regulatory T cells and evaluated Treg cells suppressive function in the peripheral blood of untreated newly diagnosed UC. Finally, we compared the results with those of age-matched healthy population.

Material and Methods

Study subjects

We studied 32 patients (16 men and 16 women) aged 37.94 ± 10.37 years (range 19 - 63 years) with Ulcerative Colitis. The control group (HC) consists of 31 age- and gender-matched healthy volunteers. All patients were newly diagnosed cases that were on no medications and recruited from Tooba Clinic at Mazandaran University of Medical Sciences. The mean UC clinical activity index (CAI) was 13.06 ± 2.01 (range 10 - 17) measured according to Lichtiger et al[20]. UC patients were excluded for other acute or chronic disorders with possible effects on the immune function such as autoimmune diseases other than UC. Our study protocol was approved by the Ethics Committee of the Tarbiat Modares University. All participants signed written informed consents.

Preparation of peripheral blood mononuclear cells (PBMCs)

PBMCs were isolated from fresh whole blood samples using density gradient centrifugation (Biowest, Nuaille, France) at 400g for 25 min at room temperature. Cells at the interphase were collected and suspended (1 × 106 Cells/mL) in RPMI 1640 medium supplemented with 100 μg/mL streptomycin, 100 U/mL penicillin, 10% heat-inactivated fetal calf serum (FCS) and 2mM L-glutamine (Gibco, Life Technologies, CA, USA).

Flow cytometry

We didn’t use FC-receptor blockers, instead removed monocytes from PBMCs. To removing monocytes, PBMCs were cultured in 75 cm2 Flasks (SPL Life Sciences, Gyeonggi-do, South Korea) for one hour and non-adherent cells (mostly lymphocytes) collected for staining by conjugated antibodies and other assessments. Lymphocytes were washed twice and re-suspended in staining buffer consisting of phosphate buffer saline (PBS) with 1% FCS. Multiparameter flow cytometry was used to identify the Treg cells and expression of cell markers. All monoclonal antibodies (mAbs) were purchased from eBioscience. Immunostaining was done according to manufacturer’s protocol. Briefly, 106 lymphocytes were stained for 30 min at dark and 4°C with allophycocyanin (APC)-conjugated mAb against CD4 (Clone RPA-T4), phycoerythrin-cyanine (PE-Cy)-7-conjugated mAb against CD25 (Clone BC96) and fluorescein isothiocyanate (FITC)-conjugated mAb against CD127 (Clone eBioRDR5, eBioscience, San Diego, CA, USA). After cell surface staining, the lymphocytes were washed by staining buffer. For intracellular FoxP3 staining, cells were fixed and permeabilised with FoxP3 staining buffer set (eBioscience) according to the manufacturer’s instructions. Then, cells were stained with PE-conjugated mAb against FoxP3 (Clone 236A/E7). Fluorochrome-matched mAbs with appropriate isotypes were used as negative control to assess the background fluorescent. Flow cytometric analysis was done by FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and results aere analyzed using CellQuest (BD Biosciences) and FlowJo (Tree Star, Ashland, Ore, USA) softwares. A minimum of 105 events at gate of lymphocytes were analyzed.

To determine absolute numbers of Treg cells, a small volume of peripheral blood was analyzed for complete blood examination with a differential. Absolute counts of Treg cells were calculated as the product of the total lymphocytes (× 103/μl) count from complete blood examination and frequency of target cells determined in the cytometric analysis. Mean fluorescence intensity (MFI) of FoxP3+-expressing cells gated on CD4+ CD25+ CD127low Tregs was analyzed.

Cell separation

CD4+ CD25high CD127low Treg cells and CD4+ CD25- Tres cells were isolated by MACS method using the CD4+ CD25+ CD127low regulatory T cell isolation kit II and CD4+ CD25+ regulatory T cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) respectively. Briefly, CD4+ CD127low T cells were isolated from single-cell suspension of lymphocytes by MACS-negative selection using a cocktail of biotin-conjugated antibodies and anti-biotin microbeads. In the second step, CD4+ CD25high CD127low regulatory T cells were labeled with anti-CD25 microbeads and isolated by positive selection. For increasing the purity of CD4+ CD25high CD127low Treg cells, 50% of recommended amounts of anti-CD25 microbeads were used. Two consecutive negative selections were done for isolation of CD4+ CD25- Tres cells. First, CD4+ T cells were isolated by using a cocktail of biotinylated antibodies and anti-biotin microbeads. Then, CD4+ CD25- Tres cells were purified using anti-CD25 microbeads. The purity of isolated CD4+ CD25high CD127low and CD4+ CD25- cells were >90% and >92% respectively.

Treg suppression assays

Responder T cells were re-suspended in PBS (0.1% BSA) at 106 cells/ml and incubated with 2 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen, Life Technologies, CA, USA) for 10 min at 37°C. Then, cells were washed twice with pre-chilled RPMI medium and re-suspended in culture medium at the indicated cell concentrations. 2 × 104 CFSE labeled Tres cells were cocultured in the absence or presence of Treg cells at 1:1 to 1:8 Treg:Tres ratios. The ratio of 1:1 was selected using different ratios of Treg:Tres in healthy controls. All suppression assays were done in 96-well round bottom plates (SPL Life Sciences) in duplicates. Anti-CD2/CD3/CD28 conjugated macsibeads (Miltenyi Biotech) were used as stimulators of proliferation according to manufacturer’s instructions. The suppressive capacity of Treg cells was expressed as the difference between the percent proliferation of Tres cells in the absence of Tregs and the percentage of Tres cells proliferation in Treg:Tres cocultures by means of following formula (1) × 100[21]. In addition, we used Flow Jo software for analyzing proliferation index (PI) of the CD4+ T cells.

Statistical analysis

The data were analyzed by GraphPad Prism software version 6.00 for Windows (GraphPad software, La Jolla, CA, USA). The variables are presented as mean ± standard deviation (SD) and comparison between two independent groups were done using the student t test (unpaired, two-tailed). Correlation analysis was performed by the pearson correlation coefficient. P values < 0.05 were considered significant. values less than 0.05, 0.01, 0.001, and 0.0001 were indicated with *, **, ***, and****, respectively.


Clinical characteristics of subjects

Some demographic and clinical data of UC patients and healthy controls were presented in table 1. All patients of UC had active disease at entry and didn’t use any medications. CAI score in the patients was 13.06 ± 2.01, range 10-17. Pancolitis (25 subjects) was the most common location involved that followed by left-sided colitis (8 subjects). Total white blood cell counts (×103/μl) in UC patients group (9.69 ± 1.69) was significantly higher than its level in healthy control group (8.43 ± 1.03, P = 0.0007). There was no significant difference in the absolute counts of lymphocytes between two groups (2634 ± 529 vs 2536 ± 315), although the percentage of lymphocytes was higher in UC patients (27.41 ± 4.75) compared to healthy controls (30.42 ± 4.63, P = 0.0135).

Frequency of CD4+ T cell subpopulations

Typical data on CD4+ T cell subpopulations in a healthy control and a single UC patient are presented in Fig. 1. Additionally, in Table 2, frequencies of CD4+ T cell subpopulations from the two groups are shown as mean ± SD. We found no significant difference in the percentage of CD4+ T cells between patients with UC and control subjects. Similar results were observed when absolute numbers of these cells were analyzed. Neither the percentage nor the absolute counts of CD4+ CD25+ T cells in total lymphocytes were significantly different between patients and controls.

To determine the frequencies of CD4+ CD25+ T effector cells and CD4+ CD25+ T regulatory cells, CD4+ CD25+ subpopulation was divided into two groups according to the expression of intracellular protein FoxP3. Our results showed no significant differences in the percentage and absolute counts of CD4+ CD25+ FoxP3+ Treg cells in patients and controls. However, the percentage and absolute numbers of CD4+ CD25+ FoxP3- Teff cells were significantly increased in patients compared with controls (P = 0.0452 and P = 0.0377, respectively). We also determined the percentage and absolute numbers of CD4+ CD25+ CD127low Treg cells in healthy control subjects and UC patients and observed no significant differences between groups. The analysis of CD4+ CD25+ CD127low FoxP3+ Treg cells in newly diagnosed patients with UC disease revealed a significant reduction in the percentage of these cells as compared to the healthy controls (P =0.0379). No difference was observed in the absolute numbers of CD4+ CD25+ CD127low FoxP3+ Treg cells between patients and controls. The amount of FoxP3 expression level was determined by MFI in CD4+ CD25+ CD127low FoxP3+ Treg cells. The patients showed decreased expression level of FoxP3 protein in gated Tregs (P = 0.029).

Suppressive function of CD4+ CD25high CD127low Treg cells

The main function of Treg cells is their remarkable ability to suppress immune reactions driven by Teff cells. In order to investigate suppressive function of Treg cells on Teff cells, we carried out a Treg-Tres coculture experiment using CD4+ CD25high CD127low cells as Tregs and CD4+ CD25- cells as Tres cells and CFSE dilution assay. When CFSE-labeled cells proliferate, the CFSE is equally distributed to their daughter cells. Thus, each peak in the histogram of flow cytometry corresponds to cells from one cell division cycle. As predicted, suppressive capacity was stronger when the Treg:Tres ratio was 1:1, because the PI was much lower than that observed for the 1:2, 1:4, 1:8 and 1:16 ratios of Treg:Tres in both healthy controls and UC patients. We analyzed percent of Treg suppression and PI of Tres in both groups. When we calculated the percent of Treg suppression, we observed a decreased suppressive capacity of Treg cells in patients compared with controls (32.73 ± 9.21 vs 37.03 ± 7.39, respectively, P = 0.0458). However, no significant difference was found in PI comparison between UC patients (1.83 ± 0.43) and healthy controls (1.67 ± 0.32).

Correlating the frequency and function of Treg cells with disease activity

We observed a significant inverse correlation between the percentage of CD4+ CD25+ CD127low FoxP3+ Treg cells and disease activity of UC using CAI (r = -0.853, P < 0.0001). Moreover, a statistically significant negative correlation was found between the MFI of FoxP3 in Treg cells and CAI (r = -0.385, P = 0.03). No significant correlation was noted between Treg cells frequency and age of patients. Finally, a negative correlation was detected between CAI and the suppressive capacity (% suppression) of Tregs (r = -0.576, P = 0.001).

Our study indicated that CD4+ CD25+ CD127low FoxP3+ Treg might be superior to CD4+ CD25+ CD127low and CD4+ CD25+ FoxP3+ Tregs.

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