Bronchoalveolar lavage fluid IFN-γ+ Th17 cells and regulatory T cells in pulmonary sarcoidosis

Abstract

In sarcoidosis, increased Th17 cell fractions have been reported in bronchoalveolar lavage fluid, and elevated numbers of Th17 cells producing IFN- γ have been observed in peripheral blood. The balance between Th1, Th17, and FoxP3(+) CD4(+) T cell subsets in sarcoidosis remains unclear. Bronchoalveolar lavage fluid cells, from 30 patients with sarcoidosis, 18 patients with other diffuse parenchymal lung diseases, and 15 healthy controls, were investigated with flow cytometry for intracellular expression of FoxP3. In a subset of the patients, expression of the cytokines IL17A and IFN- γ was investigated. The fractions of FoxP3(+) CD4(+) T cells and Th17 cells were both lower in sarcoidosis compared to controls (P = 0.017 and P = 0.011, resp.). The proportion of Th17 cells positive for IFN- γ was greater in sarcoidosis than controls (median 72.4% versus 31%, P = 0.0005) and increased with radiologic stage (N = 23, rho = 0.45, and P = 0.03). IFN- γ (+) Th17 cells were highly correlated with Th1 cells (N = 23, rho = 0.64, and P = 0.001), and the ratio of IFN- γ (+) Th17/FoxP3(+) CD4(+) T cells was prominently increased in sarcoidosis. IFN- γ (+) Th17 cells may represent a pathogenic subset of Th17 cells, yet their expression of IFN- γ could be a consequence of a Th1-polarized cytokine milieu. Our results indicate a possible immune cell imbalance in sarcoidosis.

Figure 1

T helper cells in bronchoalveolar lavage fluid: regulatory T cells, Th1, Th17, and IFN-γ ⁺ Th17 cells. Flow cytometry dot plots of bronchoalveolar lavage fluid CD4⁺ T cells gated by expression of FoxP3, IL17 (synonymous to IL-17A), and IFN-γ from a healthy control subject ((a)–(c)) and a patient with sarcoidosis ((d)–(f)). The T cell subsets are defined as follows: Th1 cells: IFN-γ ⁺ CD4⁺; Th17 cells: IL17⁺ CD4⁺; regulatory T cells: FoxP3⁺ CD4⁺ and IFN-γ ⁺; Th17 cells: IFN-γ ⁺ IL-17⁺ CD4⁺. Thus, the Th17 cells include the cells in both upper quadrants of plots (c) and (f). The patient and healthy control are representative examples. Gates for IL17 and IFN-γ were set according to unstimulated CD4⁺ T cells. Very few cells express intracellular IL-17 or IFN-γ prior to stimulation (median 0.0% in fractions of CD4⁺ T cells for both IL-17 and IFN-γ).

Figure 2

Bronchoalveolar lavage fluid fractions of FoxP3⁺ CD4⁺ T cells, Th17, and IFN-γ ⁺Th17 in sarcoidosis, other DPLDs, and healthy control subjects. (a) Lower fractions of regulatory T cells, defined as FoxP3⁺ CD4⁺ T cells, in sarcoidosis compared to other DPLDs and HC. (b) Lower Th17 cell fractions of CD4⁺ T cells in sarcoidosis compared to HC (median: 2.5% versus 4.5%, P = 0.011). (c) Higher IFN-γ ⁺ fraction of Th17 cells in sarcoidosis compared to HC (median: 72.4% versus 31%, P = 0.0005). Pairwise comparisons were done with Mann-Whitney U test. DPLD: Diffuse parenchymal lung disease; HC: healthy controls.

Figure 3

Fractions of IFN-γ ⁺ Th17 cells increase with radiological stage in sarcoidosis. The relationship of IFN-γ ⁺ Th17 cells and radiological stage in patients with sarcoidosis (N = 23) and healthy control subjects (N = 15). Fractions of IFN-γ ⁺ Th17 cells increase with advancing stage (N = 23, Spearman's rho = 0.454, and P = 0.03). Bars represent median. HC: Healthy control subjects.

Figure 4

The relationship of Th1, IFN-γ ⁺ Th17, and FoxP3⁺ CD4⁺ T cells in bronchoalveolar lavage fluid. (a) There was a highly significant correlation between fractions of IFN-γ ⁺ Th17 cells and Th1 cells (IFN-γ ⁺ CD4⁺) (n = 48, Spearman's rho = 0.75, and P < 0.0001). Black and grey lines represent linear regression lines with 95% CI. (b) The IFN-γ ⁺ Th17/FoxP3⁺ CD4⁺ T cell ratio was markedly elevated in patients with sarcoidosis compared to other DPLDs or healthy control subjects. DPLD: diffuse parenchymal lung disease; HC: healthy control subjects.