Subsets of airway myeloid-derived regulatory cells distinguish mild asthma from chronic obstructive pulmonary disease

Abstract

Background: Subsets of myeloid-derived regulatory cells (MDRCs), which are phenotypically similar to the myeloid-derived suppressor cells found in patients with cancer, have recently been appreciated as critical regulators of airway inflammation in mouse models of asthma.

Objective: We test the hypothesis that subsets of airway MDRCs contribute differentially to the inflammatory milieu in human asthma and chronic obstructive pulmonary disease (COPD).

Methods: We used bronchoalveolar lavage to identify and characterize human airway MDRCs from 10 healthy subjects, 9 patients with mild asthma, and 8 patients with COPD, none of whom were treated with inhaled or systemic corticosteroids. We defined subsets of airway MDRCs using flow cytometry, the molecular mediators they produce, and their abilities to regulate proliferation of polyclonally activated autologous T lymphocytes.

Results: We found substantial differences in the functional potential of MDRC subsets in healthy subjects, patients with asthma, and patients with COPD, with these differences regulated by the nitrosative and oxidative free radicals and cytokines they produced. Nitric oxide-producing MDRCs suppressed and superoxide-producing MDRCs enhanced proliferation of polyclonally activated autologous CD4 T cells. HLA-DR(+)CD11b(+)CD11c(+)CD163(-) superoxide-producing MDRCs, which stimulated proliferation of autologous T cells, comprised a high fraction of MDRCs in the airways of patients with mild asthma or COPD but not those of healthy control subjects. CD11b(+)CD14(+)CD16(-)HLA-DR(-) nitric oxide-producing MDRCs, which suppressed T-cell proliferation, were present in high numbers in airways of patients with mild asthma but not patients with COPD or healthy control subjects.

Conclusion: Subsets of airway MDRCs conclusively discriminate patients with mild asthma, patients with COPD, and healthy subjects from each other. The distinctive activities of these MDRCs in patients with asthma or COPD might provide novel targets for new therapeutics for these common disorders. [Corrected]

Keywords: Myeloid cell; macrophage; nitric oxide; regulatory T cell; superoxide.

Figure 1. Different profiles of free radical-producing subsets of myeloid-derived regulatory cell subsets are recovered from the airways of normal subjects and subjects with asthma or COPD

Cells obtained by BAL were stained with fluorescently labeled antibodies and with the free radical-reactive fluorescent indicator dyes DAF-FM-DA or DHE prior to analysis by flow cytometry. (A) FACS plots of MDRC recovered from BAL fluid demonstrating that the NO-producing CD11b⁺DAF-FM-DA⁺ population can be fractionated into three subsets based on differential expression of CD14 and CD16. These subsets are labeled B (CD11b⁺DAF-FM-DA⁺CD14⁺CD16⁺), C (CD11b⁺DAF-FM-DA⁺CD14⁺CD16⁻), & D (CD11b⁺DAF-FM-DA⁺CD14⁻CD16⁺). The CD11b⁻DAF-FM-DA⁺ population is designated A. (B) ROS-producing MDRC, identified by staining with DHE, can be fractionated into CD163⁺ subsets (labeled E) and CD163⁻ subsets (labeled F).

Figure 2. Absolute numbers of the different MDRC subsets recovered from the airways of subjects with asthma or COPD compared to normal controls

BAL cells were stained and analyzed as in Figure 1. MDRC populations were identified as A–F, as described in Figures 1A & 1B. The absolute numbers of each of the MDRC subsets present in the total BAL fluids were calculated for each study subject as the product of total BAL cell numbers and the proportions of each (open symbols, COPD; grey filled symbols, asthma; and black filled symbols, normal). Numeric data represent pooled means. For A: **p=0.046 comparing normals versus asthmatic subjects; for B: **p=0.024 comparing COPD versus normal and asthmatic subjects; for C: **p<0.001 comparing asthmatic versus COPD and normal subjects; for D: **p<0.001 comparing normal versus asthmatic and COPD subjects; for E: **p=0.0023 comparing asthmatic versus normal and COPD subjects; for F: **p<0.001 comparing asthmatic and COPD versus normal subjects.

Figure 3. MDRC subsets show different abilities to modulate T-cell proliferation using the iNOS, arginase and NADPH oxidase pathways

MDRC subsets (10⁵ each; populations A–F as defined in Figures 1A and 1B) purified by FACS from the BAL fluid of study subjects were cultured at a 1:1 ratio with autologous CD4⁺ T-cells purified from peripheral blood and activated using plate bound anti-CD3 with or without 1400 w, SOD or nor-NOHA, as indicated. After 48h, ³H-thymidine was added and incorporated ³H was determined 16h later. Co-culture of anti-CD3-activated autologous T-cells with the indicated MDRC subsets purified from BAL fluid of normal subjects (panel A), asthmatic subjects (panel B), and subjects with COPD (panel C). Means of replicate cpm data for each sample and treatment group for each individual for each study group were pooled and the averages of the means are presented as mean cpm ± SD.

Figure 4. Multivariate analysis of MDRC subsets discriminates and predicts the group status of the normal, asthmatic and COPD study groups

A 2-dimensional Principal Component Analysis Plot using the proportions of cells with surface phenotypes defined by flow cytometry, free radical production defined by DAF-FM-DA- or DHE-staining, and ability to regulate T-cell proliferation shows the variance in principal components one and two and separation of asthmatic subjects (grey filled symbols) from COPD subjects (open symbols) and normals (black filled symbols).