In this study, we characterized the frequency of Treg cells in the peripheral blood of 159 children. To the best of our knowledge, this is the largest number of allergic asthmatic children analyzed using multi-parameter flow cytometry for Treg cell frequency determination. Our findings demonstrated a statistically significant association between increased frequency of Treg cells and intermittent or chronic ICS use. This association was independent of asthma disease severity, allergic sensitization, gender, BMI, or other atopic disease status. These findings indicate that ICS use is associated with an elevation in Treg cell frequency and that intermittent ICS use is sufficient for this systemic change to occur. This increase in Treg cell frequency observed with ICS use represents 1% of the total CD4+ cells and approximately 17% of the Treg cells. Moreover, these data suggest that inhaled corticosteroids may work by shifting the balance of adaptive immune responses toward Treg cell predominance. Lastly, our findings suggest that ICS treatment is an important covariate to consider in studies of Treg cells in asthma.
In this study, CD4 + CD25 + CD127lo/- T cells were used to define Treg cells. Recent work from our group utilizing multi-parameter flow cytometry alongside the demethylation status of a region of the Foxp3 promoter (Treg specific demethylated region,TSDR) found the strongest positive relationship using CD4 + CD25 + CD127lo/- T cells as the Treg cell defining phenotype when compared to CD4 + CD25 + CD127lo/-Foxp3+ T cells. This finding suggests Foxp3 protein levels may not be constantly detectable using flow cytometry in bona fide Treg cells. Thus, multi-parameter flow cytometry using CD25 and CD127 to define Treg cells is reliable for quantification of CD4+ T cells expressing high levels of Foxp3 protein and demethylation at TSDR.
The impact of corticosteroids on Treg cells in asthma has not been extensively studied. It has been reported that corticosteroid exposure leads to increased Foxp3 mRNA expression in peripheral blood CD4+ T cells from adult asthmatics treated with oral and inhaled corticosteroids. Smyth et al. demonstrated an increased frequency of CD4 + Foxp3+ T cells in the bronchial alveolar lavage fluid (BALF) in adult patients with moderate-to-severe asthma compared to mild asthmatics and healthy controls, but did not account for medication use or atopic status of the patients. In our study, all of the moderate-to-severe asthmatics were on ICS, and a subset of the study subjects (16%) had one or more bursts of oral corticosteroids (OCS) during the study period. In contrast to ICS, the use of OCS was not significantly associated with increased Treg cell numbers (p = 0.80, data not shown). Our findings in peripheral blood suggest that the increased frequency of BALF Treg cells in the study by Smyth et al. might also be secondary to ICS use. Hartl et al. studied a small number of children with symptomatic asthma (n = 18) demonstrating diminished BALF Treg cell, defined as CD4 + CD25hi, frequency with normalization after initiation of ICS use compared to control groups. Yuksek et al. noted an increase in peripheral blood Treg cells, defined as CD4 + CD25hiFoxp3+, in a small number of asthmatic children (n = 16) after initiation of inhaled corticosteroids. In general, our data are in agreement with the observation that ICS use increases Treg cell frequency, and indicate that there are similar changes in the blood and airway. Our data further clarifies the relationship of ICS and Treg cells using a more rigorous definition of Treg cells with multi-parameter flow cytometry, an increased sample size, and presents a more thorough characterization of other atopic characteristics (allergic sensitization, asthma severity, atopic status).
Several mechanisms could contribute to a positive association between ICS use and increased Treg cells. For example, corticosteroids enhance Foxp3 transcription[37, 39]. Alternatively, ICS could alter homing characteristics of Treg cells, or represent a compensatory response to dampen ongoing lung inflammation. Similarly, the positive association between serum IgE and Treg cell frequency could represent a compensatory response to allergic inflammation. Additional studies are warranted to investigate longitudinal changes in Treg cells before and after ICS use. These lines of investigation would help support a mechanism of action for ICS and may serve as a prognostic indicator of response to therapy.
Recently, several studies have implicated Treg cell dysfunction in allergic asthmatics[4, 19]. Further analysis of Treg cells in our cohort, including quantifying the percentage of Foxp3+ T cells within the defined Treg cell population and MFI of Foxp3, did not reveal any significant differences with these indirect measurements of Treg cell function. This remained consistent when subjects were segregated by asthma, disease severity, or ICS use (data not shown). Studies further phenotyping the Treg cell compartment and analyzing functional characteristics are ongoing in our laboratory.