Interplay of T Helper 17 Cells with CD4+CD25high FOXP3+ Tregs in Regulation of Allergic Asthma in Pediatric Patients

Background. There is evidence that Tregs are important to prevent allergic diseases like asthma but limited literature exists on role of TH17 cells in allergic diseases. Methods. Fifty children with asthma and respiratory allergy (study group) and twenty healthy children (control group) were recruited in this study. Total IgE levels and pulmonary function tests were assessed. The expression of Tregs and cytokines was determined by flow cytometry. Results. The average level of total IgE in study group (316.8 ± 189.8 IU/mL) was significantly higher than controls (50 ± 17.5 IU/mL, P < 0.0001). The frequency of TH17 cells and culture supernatant level of IL-17 in study group (12.09 ± 8.67 pg/mL) was significantly higher than control group (2.01 ± 1.27 pg/mL, P < 0.001). Alternatively, the frequency of FOXP3 level was significantly lower in study group [(49.00 ± 13.47)%] than in control group [(95.91 ± 2.63)%] and CD4+CD25+FOXP3+ to CD4+CD25+ ratio was also significantly decreased in study group [(6.33 ± 2.18)%] compared to control group [(38.61 ± 11.04)%]. The total serum IgE level is negatively correlated with FOXP3 level (r = −0.5273, P < 0.0001). The FOXP3 expression is negatively correlated with the IL-17 levels (r = −0.5631, P < 0.0001) and IL-4 levels (r = −0.2836, P = 0.0460). Conclusions. Imbalance in TH17/Tregs, elevated IL-17, and IL-4 response and downregulation of FOXP3 were associated with allergic asthma.


Introduction
Asthma, characterized by T H 2 immune response, is a chronic inflammatory disorder, affecting children worldwide [1]. It is now universally accepted that T H 2 cytokines play a critical role in amplifying asthma [2] whereas T H 1 cytokines prevent this allergic inflammation [3,4]. In recent years, it has been shown that the manifestation of asthma in humans is beyond the control of T H 1 and T H 2 cells. Some studies have suggested that other T cell subsets like T H 17 and Treg also play a role in regulating asthma [5]. Treg cells play a key role in the maintenance and tolerance of immune regulation [6] by suppression of T H 1, T H 2, T H 17, and allergen specific IgE. They have also been found to suppress basophils, eosinophils, and mast cells but induce levels of specific IgG4 [7]. Different types of Treg cells are classified as natural and adaptive [8]. Natural Treg cells possess high levels of CD25 (CD25 high ) present on the surface of T cells and the expression of FOXP3 required for the generation and maintenance of their suppressive activity [6,8,9]. FOXP3 appears to be a key marker for CD4 + CD25 + T cells and is considered as a master switch for development and function of natural Treg cells [10][11][12][13]. Recent studies suggest that Treg cells adopt different mechanisms to suppress immune responses: directly via cell contact and indirectly via reducing the capacity of antigen presentation on antigen presenting cells [14] or via anti-inflammatory cytokines [15,16]. Some studies have suggested that pulmonary CD4 + CD25 high Tregs are impaired in pediatric asthma [17]. A new subset of CD4 + T cells, termed as T H 17, produces IL-17 [18]. T H 17 cells are now considered the key mediator in development of asthma [19]. T H 17 cells enhance both neutrophilic and eosinophilic 2 International Journal of Pediatrics airway inflammation in mouse model of asthma [20,21]. T H 17 cells play a key role in filling the gap between T H 1 and T H 2 by secreting IL-17A and IL-17F and also contributing to immunity against certain extracellular bacteria and fungi [22]. IL-17, a proinflammatory cytokine mainly derived from CD4 + T cells and also from monocytes, mast cells, macrophages, and neutrophils [23,24], has been suggested in modulating various inflammatory diseases like asthma in humans [24][25][26]. T H 1 and T H 2 cells as well as T H 17 differentiation are suppressed by Tregs [27]. However, Treg cells do not suppress T H 17 cells in vitro [28,29]. Recent evidence indicates that CD4 + CD25 high FOXP3 + Tregs and T H 17 cells play an important role in mediating asthma.
Hypothesis. The null hypothesis states that T regulatory cells do not play any role in bronchial asthma. We hypothesize that T regulatory cells play a protective role in asthma. T regulatory cells, which regulate the balance between T H 1 and T H 2 cells, are downregulated in cases of asthma and allergy.

Sample Preparation.
Five milliliters of heparinized blood was obtained from 20 healthy subjects and 50 asthmatic patients. For cytokine analysis, plasma was isolated from peripheral blood and stored at −80 ∘ C until it was used. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood sample by density gradient centrifugation (250 g for 20 minutes at room temperature) using Histopaque (Sigma-Aldrich, Saint Louis, MO, USA). Th1 (IFN-), Th2 (IL-2, IL-4, IL-6, IL-10, IL-12, and IL-13), and Th17 (IL-17) were assessed using BD CBA flex set. Tests were performed according to manufacturer's instructions (BD Cytometric Bead Array, San Diego, CA). The analysis was carried out using flow cytometry (FACSCanto (Becton Dickinson, Mountain View, CA, USA) with FACS Diva Software).

Flow Cytometric Analysis. The serum levels of cytokines
For analysis of Treg cells, the buffy coat (lymphocytes and monocytes) was separated. The cell pellet washed with PBS (Phosphate Buffer Saline) was centrifuged at 200 g for 15 minutes. PBMCs were cultured in a petri dish containing 5% CO 2 at 37 ∘ C for one and half hour. Surface phenotyping (CD4 and CD25) of the cells (peripheral blood lymphocytes) and intracellular phenotyping (FOXP3) were performed by staining, paraformaldehyde fixation, and permeabilization according to the manufacturer's instructions (BD biosciences San Diego, CA). PBMCs were determined using forward and side scatter properties based on size and granularity by FAC-SCanto (Becton Dickinson, Mountain View, CA, USA) with FACS Diva Software. The following mAbs (BD biosciences) were used: APC antihuman CD4, PE-Cy antihuman CD25, and PE antihuman FOXP3. To correct nonspecific binding, matched isotype controls were used.

Results
Total IgE and FEV1 levels were tested in all children diagnosed with asthma. The difference between IgE levels in study and control group was analyzed using Graphpad Prism software. The nonparametric Student's t-test was applied between study group and control group for total IgE level. The average level of total IgE in study group (316.8±189.8 IU/mL, range 80-720 IU/mL) was significantly higher than in control group (50.3 ± 17.5 IU/mL, range 10-80 IU/mL, < 0.001) (Figure 1(a)). FEV1 (% predicted) was significantly lower in study group (75.36 ± 14.45) compared to control group (102.3 ± 8.97, < 0.0001) ( Table 1). Total serum IgE and FEV1 levels were also analyzed for their correlation studies. This was analyzed using Spearman's correlation coefficient in study group only. There was a negative correlation between total IgE and FEV1% levels ( = −0.4820, = 0.0004) (Figure 1(b)).  ( Figure 2(a)) but values of IFN-were significantly lower in study group (12.08 ± 8.67 pg/mL) compared to control group (21.00 ± 7.53 pg/mL, = 0.009) (Figure 2(b) Table 2).

Expression of Cytokines in Asthmatic
The Student's t-test was done to analyze IL-17 in control and study group. The results depict a significant difference between the two groups ( < 0.0001). Similarly, there was also significant difference between study group and control group for IFN-( = 0.009).

Discussion
The present work demonstrates the relationship between T H 17 and Treg cells. It is universally accepted that total IgE level is directly correlated with allergy and asthma. In our study, the average level of total IgE was significantly higher in children with bronchial asthma compared to healthy subjects. On the basis of available studies, we had hypothesized that Treg cells would be associated with lower levels of allergy markers such as IgE and T H 2 cytokines. Most studies of Treg activity come from immunotherapy studies in allergic diseases [30]. FOXP3 transcription factor has been shown as a key regulator for development of Treg cells and is expressed by these cells [10,11,31]. In our study, we found that FOXP3 level is significantly lower in study group compared to control group. Furthermore, there was a negative correlation between total IgE and FOXP3 expression. In this study, we also demonstrated a T H 17/Treg cytokine profile in study group. Studies have suggested that asthma is associated with chronic and recurrent inflammation [32]. T H 2 cells are associated directly with inflammation whereas T H 17 cells behave primarily as proinflammatory markers [33]. Studies suggested  that transcription factors and cytokines are involved in generation, differentiation, and expansion of T H 17 cells. The interaction between T H 17 cells and Tregs in various inflammatory diseases needs to be further defined [34]. The knowledge of suppressive activity of Treg cells in atopic disease is still contradictory and limited. This study supports the notion that function of Tregs is altered or impaired in allergic patients compared to healthy individuals [13,17,[35][36][37][38][39][40]. However, there are some studies that have shown results going the opposite way [41][42][43]. These alterations may be related to different allergic diseases, different environmental influences, and differences in methodology for identification of cell markers that are used in proper identification of Tregs. In our study, we found significantly higher IL-17 level in  asthmatic patients compared to controls. Previous studies showed that IL-17 is elevated in sputum samples of patients with asthma compared to healthy controls [44][45][46]. Another study showed that patients with asthma had elevated IL-17 levels in serum compared with control subjects [47]. It has been suggested that IL-17 plays an important role in inflammatory and autoimmune diseases [48]. In patients with asthma, IL-17 level was significantly increased and T cell population was skewed toward T H 17 phenotype. Thus, there is a correlation of increase in IL-17 levels in patients with asthma coupled with a significant decrease in transcription factor FOXP3 Treg level when compared to controls. We could not find a study in children with asthma reporting the relationship of T H 17 with Treg response in the milieu of T H 2 activity. These results show that there is a correlation between FOXP3 and IL-17 level and also a functional imbalance in T H 17/Treg in children with asthma. In this study, we demonstrated that IL-17 and FOXP3 are reciprocally interconnected with each other. It has already been shown that CD4 + CD25 + FOXP3 + play a protective role in autoimmune disease [12]. We found that the suppressive activity of CD4 + CD25 high T cells was variable, which is already reported in previous studies [49,50]. FOXP3 transcription factor plays a key role in regulation and development of CD4 + CD25 + T cells and is expressed by these cells [10,11,31]. Our study also shows that there is a significant negative correlation between IL-4 and FOXP3.
In conclusion, the present study demonstrates that there is an imbalance between T H 17 and Tregs associated with asthma, which may play a potential role in development of asthma. Our study also shows inverse correlation between IL-17 and FOXP3. Future studies are needed to clarify these findings.