Interleukin-17 production and T helper 17 cells in peripheral blood mononuclear cells in response to ocular lysate in patients with birdshot chorioretinopathy

Abstract

Purpose: To determine the cytokine response to ocular lysates of peripheral blood mononuclear cells (PBMCs) from patients with birdshot chorioretinopathy (BSCR).

Methods: In the PBMCs of 19 patients with BSCR, T cell cytokine production in response to human retina and choroid lysates was analyzed with flow cytometry and compared to the responses against skin lysates. Five patients had active disease and had not yet been treated (naïve to systemic therapy); 14 patients had either immunomodulatory therapy (IMT) or inactive disease (referred as inactive/IMT). The PBMCs of 11 HLA-A29-positive healthy individuals were used as controls.

Results: The levels of interleukin-17 (IL-17) in supernatant of cultures stimulated with retina lysate were higher in patients with active BSCR compared to the HLA-A29 positive controls. The levels of other T cell cytokines (IL-10 and interferon-γ [IFN-γ]) in PBMC cultures did not change significantly after stimulation with ocular lysate. The frequency of CD4(+) IL-17(+) (T helper 17 [Th17]) T cells but not of CD4(+) IFN-γ (Th1) T cells was elevated in the PBMCs of patients with active BSCR stimulated by retina lysates compared to skin lysates.

Conclusions: Our data demonstrate that PBMCs exhibit an IL-17-mediated immune response to retina lysate in patients with active disease naïve to systemic therapy. This is accompanied by the enrichment of IL-17-producing CD4(+) T cells. These findings support the current concept of chronic Th17-cell mediated inflammation and provide evidence that links the Th17 signatures to ocular-specific immune responses in BSCR.

Figure 1

Scatterplots showing the levels of interleukin (IL)-10, IL-17, and interferon (IFN)-γ in supernatant (pg/ml) of peripheral blood mononuclear cell cultures stimulated for 6 days with ocular and control lysates, for each birdshot chorioretinopathy subgroup and the control group. Each circle represents a patient or control as indicated in the upper right legend. Horizontal lines indicate the geometric mean per group. Active=patients with active disease naïve to immunomodulatory therapy (IMT). Inactive=patients with a current or past history of IMT or inactive disease. Skin=protein lysate derived from a skin biopsy of two healthy controls. SEB=Staphylococcal enterotoxin B. Retina=protein lysate derived from retinas of two healthy controls. Choroid=protein lysate derived from choroid of two healthy controls. Limit for detection of cytokines (pg/ml); interleukin (IL)-10, >1.7; IL-17, >8.7; interferon (IFN)-γ, >0.8. Statistical analysis was performed using Kruskal-Wallis test with Dunn's multiple-comparison post-hoc test. *=p<0.05, **=p<0.01, ***=p<0.001.

Figure 2

Th1 and Th17 cells in peripheral blood mononuclear cells stimulated with ocular and control lysates. A: Scatterplots showing the percentages of interleukin (IL)-17 CD4⁺, CD3⁺, and interferon (IFN)-γ⁺ CD4⁺, CD3⁺ T cells in peripheral blood monocytes cultures stimulated for 6 days with ocular and control lysates, for each birdshot chorioretinopathy (BSCR) subgroup and the control group. Each circle represents a patient or control as indicated in the upper right legend. Horizontal lines indicate the geometric mean. B: Representative flow cytometric plots of a healthy control, a patient with a current or past history of immunomodulatory therapy (IMT), or inactive disease (birdshot inactive/IMT) and a patient naïve to immunomodulatory therapy (birdshot active/naïve). Cells were gated on CD3⁺, CD4⁺ lymphocytes. Skin=protein lysate derived from a skin biopsy of two healthy unrelated controls. Retina=protein lysate derived from retinas of two healthy unrelated controls. Choroid=protein lysate derived from choroid of two healthy unrelated controls. Statistical analysis was performed using Kruskal-Wallis test with Dunn's multiple-comparison post-hoc test. *=p<0.05, **=p<0.01, ***=p<0.001.