Sarcoidosis Th17 cells are ESAT-6 antigen specific but demonstrate reduced IFN-γ expression

Abstract

Rationale: Sarcoidosis is a granulomatous disease of unknown etiology. Many patients with sarcoidosis demonstrate antigen-specific immunity to mycobacterial virulence factors. Th-17 cells are crucial to the immune response in granulomatous inflammation, and have recently been shown to be present in greater numbers in the peripheral blood and bronchoalveolar lavage (BAL) fluid (BALF) of sarcoidosis patients than healthy controls. It is unclear whether Th-17 cells in sarcoidosis are specific for mycobacterial antigens, or whether they have similar functionality to control Th-17 cells.

Methods: Flow cytometry was used to determine the numbers of Th-17 cells present in the peripheral blood and BALF of patients with sarcoidosis, the percentage of Th-17 cells that were specific to the mycobacterial virulence factor ESAT-6, and as well as to assess IFN-γ expression in Th-17 cells following polyclonal stimulation.

Results: Patients with sarcoidosis had greater numbers of Th-17 cells in the peripheral blood and BALF than controls and produced significantly more extracellular IL-17A (p = 0.03 and p = 0.02, respectively). ESAT-6 specific Th-17 cells were present in both peripheral blood and BALF of sarcoidosis patients (p < 0.001 and p = 0.03, respectively). After polyclonal stimulation, Th-17 cells from sarcoidosis patients produced less IFN-γ than healthy controls.

Conclusions: Patients with sarcoidosis have mycobacterial antigen-specific Th-17 cells peripherally and in sites of active sarcoidosis involvement. Despite the Th1 immunophenotype of sarcoidosis immunology, the Th-17 cells have reduced IFN-γ expression, compared to healthy controls. This reduction in immunity may contribute to sarcoidosis pathogenesis.

Fig. 1. Patients with sarcoidosis have a greater frequency of Th-17 cells in the peripheral blood compared to healthy controls

a Analysis of the mRNA expression of the master transcription factor retinoic acid-related orphan receptor RORγt from CD4+ T cells isolated from the peripheral blood of healthy controls, (n=6) and sarcoidosis patients (n=10). b CBA analysis of IL-17A production from polyclonally activated CD4+ T cells isolated from the peripheral blood of healthy controls (diamonds, n=11) and patients with sarcoidosis (squares, n=28). c Representative plots of flow cytometric detection of intracellular IL-17A and surface expression of IL-23R on CD4+ T cells from polyclonally activated PBMC. d Frequencies of IL-17A+CD4+ and IL-23R+CD4+ from polyclonally activated PBMC of healthy controls (diamonds, n=11) and patients with sarcoidosis (square, n=25). P values are indicated

Fig. 2. Patients with sarcoidosis have a greater frequency of IL-17A+and IFN-γ+TCR γδ cells in the peripheral blood as compared to healthy controls

a Representative plots of intracellular IL-17A and IFN-γ in TCR γδ cells from polyclonally activated PBMC of healthy controls and patients with sarcoidosis. b Frequencies of IL-17A+TCR γδ+and IFN-γ+TCR γδ+from polyclonally activated PBMC of healthy controls (diamonds, n=9) and patients with sarcoidosis (squares, n=16). P values are indicated

Fig. 3. Patients with sarcoidosis exhibit an ESAT-6 specific Th-1/Th-17 response

a Representative plots of flow cytometric detection of production of intracellular IL-17A, surface expression of IL-23R and production of intracellular IFN-γ in CD4+ T cells from ESAT-6 stimulated PBMC of healthy controls and patients with sarcoidosis. b Frequencies of IL-17A+CD4+ and IL-23R+CD4+ from ESAT-6 stimulated PBMC of healthy controls (diamonds, n=9) and patients with sarcoidosis (squares, n=24). c Representative plots of flow cytometric detection of production of intracellular IFN-γ in CD4+ T cells from ESAT-6 stimulated PBMC of healthy controls and patients with sarcoidosis. d Frequencies of CD4+ T cells expressing IFN-γ from ESAT-6 stimulated PBMC of healthy controls (diamonds, n=9) and patients with sarcoidosis (squares, n=24). P values are indicated

Fig. 4. Sarcoidosis BAL cells exhibit an ESAT-6 specific Th-1/Th-17 response

a Representative plots of flow cytometric detection of production of intracellular IL-17A and production of intracellular IFN-γ in BAL CD4+ T cells from polyclonally activated BAL cells of disease controls and patients with sarcoidosis. b Frequencies of IL-17A+CD4+ and IFN-γ+ CD4+ T cells from polyclonally activated BAL of disease controls (diamonds, n=5) and patients with sarcoidosis (square, n=10). c Representative plots of flow cytometric detection of production of intracellular IL-17A and production of intracellular IFN-γ in BAL CD4+ T cells from ESAT-6 stimulated BAL cells of disease controls and patients with sarcoidosis. d Frequencies of IL-17A+CD4+ and IFN-γ+CD4+ from ESAT-6 stimulated BAL cells of disease controls (diamonds, n=5) and patients with sarcoidosis (squares, n=10). P values are indicated

Fig. 5. Sarcoidosis BAL Tregs do not produce IL-17

a Frequencies of IL-17A+CD4+FoxP3+ and IFN-γ+CD4+FoxP3+ cells from polyclonally activated BAL of disease controls (diamonds, n=5) and patients with sarcoidosis (square, n=10). FoxP3 used a marker for Tregs. b Frequencies of IL-17A+CD4+FoxP3+ and IFN-γ+CD4+FoxP3+ cells from ESAT-6 stimulated BAL from disease controls (diamonds, n=5) and patients with sarcoidosis (squares, n=10). P values are indicated

Fig. 6. Sarcoidosis Th-17 cells demonstrate reduced IFNγ expression

a Representative gating strategy used for identification of the IFN-γ producing CD4+IL-17A+cells. b Percent of Th-17 cells producing IFN-γ following polyclonal activation of PBMC from healthy controls (triangles, n=9) and sarcoidosis patients (squares, n=24). c Median fluorescent intensity (MFI) of IFN-γ expression on CD4+IL-17A+cells following polyclonal activation of PBMC of healthy controls (triangles, n=9) and sarcoidosis patients (circles, n=24). P values are indicated