Inhaled corticosteroid use is associated with increased circulating T regulatory cells in children with asthma

Anne Marie Singh, Paul Dahlberg, Kristjan Burmeister, Michael D Evans, Ronald Gangnon, Kathy A Roberg, Christopher Tisler, Douglas Dasilva, Tressa Pappas, Lisa Salazar, Robert F Lemanske Jr, James E Gern, Christine M Seroogy, Anne Marie Singh, Paul Dahlberg, Kristjan Burmeister, Michael D Evans, Ronald Gangnon, Kathy A Roberg, Christopher Tisler, Douglas Dasilva, Tressa Pappas, Lisa Salazar, Robert F Lemanske Jr, James E Gern, Christine M Seroogy

Abstract

Background: T regulatory (Treg) cells are important in balancing immune responses and dysregulation of Treg cells has been implicated in the pathogenesis of multiple disease states including asthma. In this study, our primary aim was to determine Treg cell frequency in the peripheral blood of children with and without asthma. The secondary aim was to explore the association between Treg cell frequency with allergen sensitization, disease severity and medication use.

Methods: Peripheral blood mononuclear cells from healthy control subjects (N = 93) and asthmatic children of varying disease severity (N = 66) were characterized by multi-parameter flow cytometry.

Results: Our findings demonstrate that children with asthma had a significantly increased frequency of Treg cells compared to children without asthma. Using a multivariate model, increased Treg cell frequency in children with asthma was most directly associated with inhaled corticosteroid use, and not asthma severity, allergic sensitization, or atopic status of the asthma.

Conclusion: We conclude that low dose, local airway administration of corticosteroids is sufficient to impact the frequency of Treg cells in the peripheral blood. These data highlight the importance of considering medication exposure when studying Treg cells and suggest inhaled corticosteroid use in asthmatics may improve disease control through increased Treg cell frequency.

Figures

Figure 1
Figure 1
CD4 + CD25 + CD127lo/- T cells express the majority of high level Foxp3 protein. PBMCs were isolated and stained for flow cytometry as described in the methods section. Lymphocytes were gated using forward and side scatter profiles followed by CD3 and CD4 gating. A. Within this defined gate, CD127 and CD25 staining was determined using fluorescence minus one (FMO) controls. One representative subject is shown out of 159 assayed with the percentage of each population in upper right corner. B. Foxp3 staining was determined using an isotype control. The percentage of Foxp3 staining within each defined quadrant is plotted for the entire cohort (mean% of Foxp3+ cells within CD3 + CD4 + CD25 + CD127lo/-: 71.1%; SD 14.7%). The CD127 + CD25+ T cells have a subset of low expressing Foxp3+ cells (mean% of Foxp3+ within the CD3 + CD4 + CD25 + CD127+: 20.8%; SD 11.1%). C. Mean Fluorescent Intensity (MFI) for Foxp3 is shown. CD25 + CD127lo/- cells display the highest amount of Foxp3 per cell. The mean MFI for Foxp3 in CD3 + CD4 + CD25 + CD127lo/- T cells: 377 SD 137.8 vs. CD3 + CD4 + CD25 + CD127+ T cells: 146 SD 49.4.
Figure 2
Figure 2
Peripheral blood Treg cells are increased in children with asthma. The percentage CD3 + CD4 + CD25 + CD127lo/- T cells in the peripheral blood of asthmatics (6.91% [6.52, 7.3]) versus control (6.4% [6.07, 6.73]) children. Data are expressed as the mean percentage of Treg cells within gated CD3 + CD4+ T cells with 95% CI.
Figure 3
Figure 3
Peripheral blood Treg cells are increased in asthmatic children using inhaled corticosteroids independent of asthma severity. The percentage of Treg cells in the peripheral blood is elevated in asthmatics treated intermittently (Int) or daily with ICS. Data are expressed as the percentage of Treg cells within gated CD3 + CD4+ T cells with 95% CI. No asthma: 6.4% (6.08, 6.73); Asthma no ICS: 6.24% (5.64, 6.84); Asthma Int ICS: 7.42% (6.54, 8.31); Asthma daily ICS: 7.34% (6.74, 7.94).

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