Circulating Human Eosinophils Share a Similar Transcriptional Profile in Asthma and Other Hypereosinophilic Disorders

Abstract

Eosinophils are leukocytes that are released into the peripheral blood in a phenotypically mature state and are capable of being recruited into tissues in response to appropriate stimuli. Eosinophils, traditionally considered cytotoxic effector cells, are leukocytes recruited into the airways of asthma patients where they are believed to contribute to the development of many features of the disease. This perception, however, has been challenged by recent findings suggesting that eosinophils have also immunomodulatory functions and may be involved in tissue homeostasis and wound healing. Here we describe a transcriptome-based approach-in a limited number of patients and controls-to investigate the activation state of circulating human eosinophils isolated by flow cytometry. We provide an overview of the global expression pattern in eosinophils in various relevant conditions, e.g., eosinophilic asthma, hypereosinophilic dermatological diseases, parasitosis and pulmonary aspergillosis. Compared to healthy subjects, circulating eosinophils isolated from asthma patients differed in their gene expression profile which is marked by downregulation of transcripts involved in antigen presentation, pathogen recognition and mucosal innate immunity, whereas up-regulated genes were involved in response to non-specific stimulation, wounding and maintenance of homeostasis. Eosinophils from other hypereosinophilic disorders displayed a very similar transcriptional profile. Taken together, these observations seem to indicate that eosinophils exhibit non-specific immunomodulatory functions important for tissue repair and homeostasis and suggest new roles for these cells in asthma immunobiology.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Eosinophil isolation by FACS generates high quality RNA.

(A) Flow cytometry gating strategy for the identification of eosinophils. Eosinophils were identified among a granulocyte suspension (see Methods) as a CD16 fluorescence negative cell population (B) Purity of sorted eosinophils as assessed by cytospin preparation was close to 100%. (C) Bioanalyzer RNA profile with RNA integrity number (RIN) of an eosinophil sample. All RNA samples included in the expression analysis had a RIN > 8.

Fig 2. Circulating eosinophils in asthma differ in their gene expression profile when compared to healthy subjects.

Heat map of hierarchical clustering of the top expressed genes of circulating eosinophils from subjects with asthma (n = 4) vs healthy controls (n = 3). The horizontal dendrogram represents the relationship between asthmatic and healthy subjects. The vertical dendrogram represents the relationship between the expression levels of each gene across all the samples. Over-expressed genes are shown in red and under-expressed genes are depicted in green.

Fig 3. RT-qPCR of selected genes.

Gene expression was determined by RT-qPCR in eosinophils isolated from subjects with asthma and healthy controls. Results were normalized to β-actin and expressed as fold change compared with samples from healthy controls. Results are presented as means and SEM. *p<0.05; **p<0.01 (2-tailed non parametric Mann-Whitney).

Fig 4. Differentially expressed genes in unrelated hypereosinophilic diseases.

Volcano plot of genes differentially expressed between subjects with peripheral hypereosinophilia (i.e. asthma, dermatological disease, parasitosis and pulmonary aspergillosis) vs. healthy controls. Genes with a p-value <0.01 are depicted in red.

Fig 5. Eosinophils from asthmatic subjects display a similar transcriptional profile as eosinophils from other hypereosinophilic conditions.

Scatterplots are based on fold changes of selected genes that are differentially expressed at least in one hypereosinophilic disease (p