Association of Ocular Surface Immune Cells With Dry Eye Signs and Symptoms in the Dry Eye Assessment and Management (DREAM) Study

Abstract

Purpose: Dry eye disease (DED) is a multifactorial, heterogeneous disease of the ocular surface with one etiology being ocular surface inflammation. Studies using animal models demonstrate the role of ocular surface immune cells in the inflammatory pathway leading to DED, but few have evaluated humans. This study described the white blood cell population from the ocular surface of patients with DED and assessed its association with DED signs and symptoms in participants of the Dry Eye Assessment and Management (DREAM) study.

Methods: Participants were assessed for symptoms using the Ocular Surface Disease Index, signs via corneal staining, conjunctival staining, tear break-up time, and Schirmer test, and Sjögren's syndrome (SS) based on the 2012 American College of Rheumatology classification criteria. Impression cytology of conjunctival cells from each eye was evaluated using flow cytometry: T cells, helper T cells (Th), regulatory T cells (Tregs), cytotoxic T cells, and dendritic cells.

Results: We assessed 1049 eyes from 527 participants. White blood cell subtype percentages varied widely across participants. Significant positive associations were found for Th and conjunctival staining (mean score of 2.8 for 0% Th and 3.1 for >4.0% Th; P = 0.007), and corneal staining (mean score of 3.5 for 0% Th and 4.3 for >4.0% Th; P = 0.01). SS was associated with higher percent of Tregs (median 0.1 vs. 0.0; P = 0.01).

Conclusions: Th were associated with more severe conjunctival and corneal staining, possibly indicating their role in inflammation leading to damage of the ocular surface. There is no consistent conclusion about Tregs in SS, but these results support that Tregs are elevated in SS.

Figure 1.

Flow cytometry gating strategy. Flow cytometer analysis. Dot plots of representative sample from the study. Total analyzable cells (B) were gated out from a scatter plot of forward scatter-width (FCS-W) vs. side scatter-area (SSC-A). (A) Sequential gating of CD45⁺ expressing cells (C) from the total population yielded total WBC from the sample. CD3⁺ and CD3^– (D) were gated from the total WBCs. CD3^– cells were gated for CD11c (E) to give total DCs. CD3⁺ cells were gated for CD8⁺ CD4^– to give Ttox, and CD4⁺ CD8^– for Th (F). CD4⁺ cells were gated for CD25⁺ and CD127^– for Tregs (G). CD4⁺ were further gated to determine Th subtypes (H). CD4⁺ CXCR3^– CCR4⁺ CCR6⁺ were gated for Th17 (I), and CD4⁺ CXCR3⁺ CCR4^– CCR6^– were gated for Th1 cells (J).

Figure 2.

Association between corneal staining score and Th. The 75th and 25th percentiles make up the upper and lower boundaries of the boxes. Within the box, the diamond represents the mean and the horizontal line in the box represents the median. The vertical lines issuing from the box extend to the highest value within 1.5× the interquartile range of the 75th percentile and the lowest value within 1.5× the interquartile range of the 25th percentile. Each dot outside the upper and lower fence corresponds with an outlier value.

Figure 3.

Association between conjunctiva staining score and Th. The 75th and 25th percentiles make up the upper and lower boundaries of the boxes. Within the box, the diamond represents the mean and the horizontal line in the box represents the median (the median is the same as the first quartile for the 0% group). The vertical lines issuing from the box extend to the highest value within 1.5× the interquartile range of the 75th percentile and the lowest value within 1.5× the interquartile range of the 25th percentile. Each dot outside the upper and lower fence corresponds to an outlier value.

Figure 4.

Association between Sjögren's syndrome and Treg cells. The 75th and 25th percentiles make up the upper and lower boundaries of the boxes. Within the box, the diamond represents the mean and the horizontal line in the box represents the median. The vertical lines issuing from the box extend to the highest value within 1.5× the interquartile range of the 75th percentile and the lowest value within 1.5× the interquartile range of the 25th percentile. Each dot outside the upper and lower fence corresponds with an outlier value.