Asthma is a most frequent chronic inflammatory disorder in childhood worldwide and characterized by TH2 immune response. It is universally accepted now that TH2 cytokines play critical role in amplifying the asthma(2) and TH1 cytokines prevent this allergic inflammation(3, 4). In recent years it was shown that TH1 and TH2 do not fully control the asthmatic symptoms in human. Some studies have suggested that other T cell subsets like TH17 and Treg also play a role in regulating asthma(5). T-regulatory (Treg) cells play a key role in maintenance and tolerance of immune regulation(6) by suppression of TH1, TH2, TH17 and allergen specific IgE, Suppression of basophils, eosinophils and mast cells and induction of specific IgG4 level(7). Different type of Treg cells have been identified as adaptive and natural Treg cells(8). Natural Treg cells posses high levels of CD25 (CD25high) which is present on the surface of T cells and the expression of a forkhead /winged helix transcription factor box protein 3(FoxP3) required for the generation and maintenance of their suppressive activity (6, 8-10). FOXP3 appears to be a key marker for CD4+CD25+ T cells and FOXP3 is considered as master switch for natural Treg development and function (11-14). Recent studies suggest that Treg cells employ different mechanisms to suppress immune responses: directly via cell contact and indirectly via reducing the capacity of antigen presenting of antigen presenting cells (15) or via suppressive cytokines (16, 17). Some studies have suggested that pulmonary CD4+ CD25hi Tregs are impaired in pediatric asthma (18). A new subset of CD4+ T cells, termed as TH17, produce IL-17(19). IL-17 is a pro-inflammatory cytokine which is mainly derived from T cells, and from monocytes, mast cells, macrophages and neutrophills(20, 21) has been suggested in modulating various inflammatory diseases in human like asthma(22). TH17 cells have now become the key mediator and play a key role in asthma(23). TH17 cells enhance both in neutrophilic and eosinophilic airway inflammation in mouse model of asthma (24, 25). Th17 cells play a role in filling the gap between TH1 and TH2 by secretion IL-17A, IL-17F and also contributing to immunity against certain extracellular bacteria and fungi (26). There is strong evidence that an increase level of IL-17 is associated with different inflammatory disease (21, 27). It has been shown that TH17 differentiation is inhibited by TH1 and TH2 cells. TH1 and TH2 cell immune response has been suppressed by Tregs (28). However, TH17cells has not been suppressed by Treg in vitro (29, 30). Recent evidences indicated that CD4+CD25high Foxp3+ Tregs and TH17 cells play a important role in patients with asthma.
Material and Methods-
Subjects
Fifty patient's, who attended Advanced Pediatric Centre in PGIMER (Post Graduate Institute of Medical Education and Research), Chandigarh, reported their allergic symptoms were recruited in this study with their informed consent. The sera of age and sex matched non-allergic patients were taken as control. The Ethics Committee of PGIMER approved this study (Micro / 2006 / 754 / 8th May 2006).
Methods
The diagnosis of asthma was done by skin prick tests to specific food allergens, total IgE level and FEV1 measurement. Blood samples were collected for evaluation of Th1, Th2 and Th17 expression.
Table 1 Patients characteristics
Study Group
Control Group
Total subjects
50
20
Age (Months)(Mean±SD)
88.86±38.67
85.95±35.55
Male; n (%)
40(80%)
14(70%)
Total IgE (IU/ml)
Mean±SD
316.8±189.8
50.3±17.5
Estimation of total IgE
The total IgE of allergic patients was measured using PATHOZYME immunoglobulin E OD 417 kit. Specific monoclonal anti-IgE antibodies were coated on microtiter wells. Test sera were applied and incubated with zero buffer (phosphate based buffer containing stabilizing proteins). After washing with distilled water IgE-HRP conjugate was added to the wells. The absorbance was measured at 450 nm after addition of tetramethyl benzedine hydrochloride (TMB) substrate and dilute hydrochloric acid. The concentration of IgE is directly proportional to the colour intensity of the test sample. This test was calibrated to WHO 2nd International Reference Preparation 75/502 (1981).
Sample Preparation
Ten milliliters of heparinized blood was obtained from 20 healthy subjects and 50 asthmatic patients. For cytokine analysis plasma was isolated stored at -80oC until used from peripheral blood. Peripheral Blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (250 g for 20 minutes at room temperature) using Histopaque (Sigma-Aldrich, Saint Louis, MO, USA).
Flow cytometric analysis
The serum level of the cytokines Th1 (IFN-γ), Th2 (IL-2, IL-4, IL-6, IL-10, IL-12, IL-13) and Th17 (IL-17) were assessed using BD CBA flex set. Tests were performed according to manufacturer's instructions (BDTM Cytometric Bead Array, San Diego, CA). The analysis was carried out in a flow cytometry [FACSCanto (Becton Dickinson, Mountain View, CA, USA) with FACS DivaTM Software].
For analysis of Treg cells, the buffy coat (lymphocytes and monocytes) was separated. The cell pellet washed with PBS (Phosphate Buffer Saline), centrifuged at 200 g for 15 minutes. PBMC's were cultured in petri dish at 37oC at 5 CO2%, for one and half hour. Surface phenotyping (CD4 and CD25) of the cells (peripheral blood lymphocytes) and intracellular phenotyping (FoxP3) was performed by staining, paraformaldehyde fixation and permeabilization according to the manufacturer's instruction (BD biosciences San Diego, CA). PBMC's were determined using Forward and Side scatter properties based on size and granularity by FACSCanto (Becton Dickinson, Mountain View, CA, USA) with FACS DivaTM Software. The following mAbs (BD biosciences) were used: APC antihuman CD4, PE-CyTM antihuman CD25 and PE antihuman FoxP3. To correct nonspecific binding, matched isotype controls were used.
Statistical analysis
Data were analyzed with SPSS (v16.0; SPSS Inc, Chicago, IL, USA) and Graphpad prism (v5.0; Graphpad software Inc, Le Jolla, CA, USA). The mean values and their internal differentiation with standard deviation were calculated. The spearman's r rank correlation coefficients were used to evaluate relationship between variables. When assessing the flow cytometric data, Student t-test was used. p values < 0.05 were considered statistically significant.
RESULTS
Total IgE
The average level of total IgE in the studied group was 88.86±38.67 and in control group was 50.3±17.5.
Expression of Cytokines in asthmatic children
Cytokine level in sera of IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IFN-γ and IL-17 were expressed as mean ± standard deviation. The average level of IL-17 expression in the study group was 12.09±8.67pg/mL and significantly higher than corresponding values in control group (2.01±1.27pg/mL, p=0.001) but values of IFN-γ were significantly lower in all asthmatic patients 12.08±8.67pg/mL compared to control patients 21.00±7.53pg/mL (p=0.02). No significant differences were observed between asthmatic and control group (IL-2: 20.78±9.22pg/mL vs 18.93±13.73pg/mL [p>0.05]; IL-4: 21.88±10.35pg/mL vs 19.79±12.38pg/mL [p>0.05]; IL-6: 18.17±10.49pg/mL vs 15.11±9.79pg/mL [p>0.05]; IL-10: 22.82±19.16pg/mL vs 18.62±5.31pg/mL [p>0.05]; IL-12: 17.58±9.27pg/mL vs 16.94±11.00 [p>0.05]; IL-13: 34.55±17.51pg/mL vs 29.39±10.12pg/mL [p>0.05]).
Flow cytometric analysis of FOXP3 was performed in asthmatic group (n=50) and in control group (n=20) in CD4+CD25+ cells. The percentages of FOXP3 expressing cells were significantly lower in asthmatic group [(49.00±13.47) %] than in the control group [(95.91±2.63) %, p<0.0001].
Relationship of Total IgE and FOXP3 expression-
There was a significantly negative correlation between %FOXP3/CD4+CD25high and total IgE level (r= -0.5273 p<0.0001).
Interaction between FOXP3 expression and level of IL-17 and IL-4-
FOXP3 percentage and IL-17 level were significantly inversely correlated to each other (r= -0.5631, p< 0.0001). There was also a significant but loosely correlation between %FOXP3 and IL-4 (r= -0.2836 p=0.0460).
Discussion-
The present work demonstrates relation between TH17 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 compare to healthy subjects. On the basis of studies we hypothesize that Treg cells would be associated with allergy and asthma. Greater numbers of Treg cells with lower rates of allergy markers. Most studies of Tregs activity come from immunotherapy studies in allergic diseases (31). FOXP3 transcription factor has been shown a key regulator for the development of Treg cells and expressed by these cells (11, 12, 32). In our study we found that FOXP3 level is significantly lower in asthmatic patients compare to controls. Furthermore, there was a negative correlation between total IgE and FOXP3. In this study we also demonstrated a TH17/Treg cytokine profile in asthmatic patients. Recent studies suggested that asthma developed by chronic and recurrent inflammation(33). It has been suggested that TH2 cells are associated with inflammation, TH17 has also been behaving as proinflammatory(34). The knowledge of the suppressive activity of Treg cells in atopic disease is still contradictory and limited. There are several studies which suggest that the function of Tregs altered or impaired in allergic patients compared with healthy individuals (14, 18, 35-45). Our study also suggested the similar thing. But there are some studies which have shown the opposite result (46-48). There are variation of results in number and function of Tregs in pathogenesis of allergic diseases. These alterations may be related to different allergic diseases, differential environment influences, difficulty in cell markers which are used in proper identification of Tregs. To find out whether the TH17 and Treg balance impaired in asthmatic patients, we measured the level of Treg and TH17 cell. In our study we found significantly higher IL-17 level in asthmatic patients compare to controls. There is significant evidence to support this; existing literature showed that in sputum samples from asthmatic patients IL17 is elevated compared to control subjects (49-51). Another study showed that asthmatic patients had elevated IL-17 level compared with control subjects(52). It has been suggested that IL-17 play important roles in inflammatory and autoimmune disease(53). In asthmatic patients IL-17 level was significantly increased, it shows that T cells population was skewed toward TH17 phenotype. Increased IL-17 level in asthmatic patients coupled with a significantly decrease in transcription factor FOXP3 Treg level compare to control subjects. These result shows that there was a correlation between FOXP3 and IL-17 level also functional imbalance in TH17/Treg in patients 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(13). We found the suppressive activity of CD4+CD25high T cells was variable, already been reported in previous studies (54, 55). FOXP3 transcription factor is a key factor in regulation and development of CD4+CD25+ T cells and expressed by these cells. (11, 12, 32). Tregs also strongly associated with TH2 responses (39). Our study also shows that there was a significantly negative correlation between IL-4 and FOXP3.
In conclusion, the present study demonstrated that there was imbalance between TH17 and Tregs associated with asthma and this imbalance may play a potential role in developing asthma. Our study also showed inverse correlation between IL-17 and FOXP3. Future studies also been needed to clarify these findings. There is a limitation in our study that the study population was small and heterogeneous so it is possible that some effects could be excluded or not reliably detected due to low statistical power. In future prospective these results may provide valuable information for the new therapeutic approach to treat asthma
