Platelet association with leukocytes in active eosinophilic esophagitis

Abstract

We previously demonstrated that the percentage of blood eosinophils that are associated with platelets and thus positive for CD41 (integrin αIIb-subunit) correlates with and predicts peak eosinophil count (PEC) in biopsies of eosinophilic esophagitis (EoE) patients after treatment. Thus, flow cytometric determination of CD41+ eosinophils is a potential measure of EoE disease activity. Determinants of association of platelets with eosinophils and other leukocytes in EoE are largely unknown. The objectives of this study were to test the hypotheses that platelets associate with blood leukocytes other than eosinophils in EoE and that such associations also predict EoE activity. Whole blood flow cytometry was performed on samples from 25 subjects before and after two months of standard of care EoE treatment. CD41 positivity of cells within gates for eosinophils, neutrophils, monocytes, lymphocytes, and natural killer cells was compared. We found that percent CD41+ neutrophils, monocytes, and eosinophils correlated with one another such that principal component analysis of the five cell types identified "myeloid" and "lymphoid" factors. Percent CD41+ neutrophils or monocytes, or the myeloid factor, like CD41+ eosinophils, correlated with PEC after treatment, and CD41+ neutrophils or the myeloid factor predicted PEC < 6/high power field after treatment, albeit with lower area under the curve than for CD41+ eosinophils. We conclude that the processes driving platelets to associate with eosinophils in EoE also drive association of platelets with neutrophils and monocytes and that association of platelets with all three cell types is related to disease activity. Clinicaltrials.gov identifier: NCT02775045.

Conflict of interest statement

The authors have read the journal’s policy and identified the following competing interests: SKM reports receiving consulting and speaker fees from Astra-Zeneca and Glaxo-Smith-Kline, consulting fee from Boehringer-Ingelheim, and data safety monitoring board fee from TEVA, all unrelated to this work. MWJ received fees for consulting from Guidepoint Global and funds for research from Hoffmann-La Roche, all unrelated to this work. This does not alter our adherence to PLoS One policies on sharing data and materials. The remaining authors declare no conflict of interest.

Figures

Fig 1. Examples of flow cytometry histogram for blood leukocyte CD41 at V2.

Red, CD41; blue, isotype control. A and B) Neutrophils. C and D) Monocytes. E and F) Lymphocytes. G and H) Natural killer (NK) cells. A, C, E, and G) Subject No. 8 with 0.2% CD41-positive neutrophils, monocytes, and NK cells; 0.1% CD41-positive lymphocytes; and PEC = 0/HPF. B, D, F, and H) Subject No. 13 with 34% CD41-positive neutrophils, 96% CD41-positive monocytes, 19% CD41-positive lymphocytes, 14% CD41-positive NK cells, and PEC = 85/HPF. Note that CD41+ cells in B, D, F, and H form a distinct peak to the right well separated from the CD41- cells to the left that overlay cells stained with non-immune control immunoglobulin. The location of the marker was set in the “valley” between the negative and positive peaks or subpopulations, as described in Methods section.

Fig 2. Blood leukocyte CD41 and correlations among leukocytes at V1 and V2.

A and B) CD41-positive leukocytes at V1 (A) and V2 (B), median and quartiles. C and D) Correlations among the various leukocytes at V1 (C) and V2 (D). Numbers in colored lower left area represent Spearman rank (in C) and Pearson (in D) correlation coefficient values, respectively. Numbers in white upper right area represent p values. Eos, eosinophils; lymph, lymphocytes; monos, monocytes; neuts, neutrophils; NK, natural killer cells.

Fig 3. Principal component analysis (PCA) plots.

Principal component (PC) 2 (lymphoid factor) (on y axis) versus PC2 (myeloid factor) (on x axis). A) At V1. B) At V2, empty symbols, PEC

Fig 4. Odds ratios of CD41-positive leukocytes or PCA factors for PEC

Odds ratios and 95% confidence intervals from logistic regression. A) With V2 data, p for eosinophils = 0.02, neutrophils = 0.03, monocytes = 0.18, lymphocytes = 0.48, NK cells = 0.52, myeloid factor = 0.04, lymphoid factor = 0.34. B) With V2-V1 data, p for eosinophils = 0.04, neutrophils = 0.08, monocytes = 0.16, lymphocytes = 0.28, NK cells = 0.43, myeloid factor = 0.06, lymphoid factor = 0.30. Eos, eosinophils; lymph, lymphocytes; monos, monocytes; neuts, neutrophils; NK, natural killer cells; PCA, principal component analysis.

Fig 5. Receiver operating characteristic (ROC) curves for CD41-positive leukocytes to predict PEC

A) Eosinophils, B) neutrophils, C) mean of eosinophil and neutrophil values. AUC, area under curve.