Analysis of cell‐specific peripheral blood biomarkers in severe allergic asthma identifies innate immune dysfunction

Asthma is a disease of complex origin and multiple pathologies. There are currently very few biomarkers of proven utility in its diagnosis, management or response to treatment. Recent studies have identified multiple asthma phenotypes following biofluid analysis; however, such findings may be driven by the well-characterised alterations in immune cell populations in asthma. We present a study designed to identify cell type-specific gene signatures of severe allergic asthma in peripheral blood samples. Using transcriptomic profiling of four magnetically purified peripheral blood cell types, we identify significant gene expression changes in monocytes and NK cells but not T lymphocytes in severe asthmatics. Pathway analysis indicates dysfunction of immune cell regulation and bacterial suppression in the NK cells. These gene expression changes may be useful on their own as prognostic peripheral blood cell markers of severe asthma, but also may indicate novel cell pathways for therapeutic intervention.


Analysis of cell-specific peripheral blood biomarkers in severe allergic asthma identifies innate immune dysfunction
To the editor, Asthma is a common chronic airways disease with complex aetiology, having no single causative genetic trigger and with multiple factors appearing to contribute to disease pathogenesis. 1 Thus, asthma can manifest in numerous different pathologies related to severity and the degree of allergy or response to therapeutic intervention. Currently, the principal clinical biomarkers for initial diagnosis and staging of disease severity include lung function and hyper-responsiveness tests. Sometimes fractional exhaled nitric oxide (FeNO) is used as a surrogate for direct measurement of eosinophils. Improvement of clinical symptoms following therapeutic intervention and assessment of atopy and a history of wheeze are also useful.
Monitoring of the disease is generally through symptom assessment and therapeutic dose requirements combined with periodic lung function tests. Novel approaches, such as biologics targeting key disease pathways, have identified the utility of additional biomarkers such as IgE or sputum inflammatory cell counts as inclusion criteria for initiating treatment; however, in clinical practice, often the principal read-outs remain the generalized clinical ones. No single test provides definitive evidence of asthma, its severity or therapeutic efficacy of drugs.
The aim of this study was to identify markers of cell type and asthma using gene expression profiles to identify patterns of disease in the periphery where they may most easily be examined. Recent reports have identified multiple disease phenotypes using unbiased clustering of genomic and proteomic data from airway samples. 2 Likewise, mRNA signatures of severe asthma have been identified in whole blood. 3 The importance of such studies may be confounded by known changes in inflammatory cell profiles in asthma, thus, missing changes in blood cell subtypes by defining the phenotypic clusters of subjects based on inflammatory cell predominance in biofluids. Our approach in the current study was to examine gene expression markers of cell type and asthma in four peripheral blood cell populations that have been a focus of interest for some time but have not yet been analysed in the level of detail as done in this study.
We recruited 11 severe allergic asthmatics on step 4 of BTS/Sign management (none on oral corticosteroids), and 10 healthy control subjects (see Table 1  Fresh blood was drawn and PBMCs were isolated from buffy coats. Flow cytometry determined no significant differences in the relative proportions of four immune cell populations, monocytes, NK cells, CD4 + and CD8 + T cells in the peripheral blood of healthy and severe asthmatic subjects ( Table 1). For example, a major component of the monocyte cluster was CD14, which was to be expected since it was the target isolation molecule, but also included 425 other genes contributing to this cluster, inclusive of the well-characterized monocyte-specific proteins such as CD68, CD163, L-selectin and S100A12.
T cells exhibited a well-defined cluster; however, a small degree of overlap between the CD4 + and CD8 + T-cell clusters, reflects the phenotypic similarity between these two cell types.
Similarly, our top-ranked genes contributing to the NK cell cluster were well-known phenotypic markers of NK cells, inclusive of KLRD1, NCAM-1 and EOMES. Clusters were driven primarily by cell type.
We then identified individual differentially expressed genes appropriate. Volcano plots ( Figure 1B,C) indicated two genes upregulated in asthmatic monocytes compared to healthy controls (IGF2R and C3AR1). IGF2R is known to be upregulated in monocytes during differentiation into macrophages, 4 but changes in IGF2R expression by monocytes in asthma have not previously been observed.
C3AR1 has been previously identified as a susceptibility gene for bronchial asthma. 5 Although C3AR1 has been previously associated with severe asthma in whole blood, 3 expression was not localized to monocytes.
We found six upregulated (LILRA5, LILRB2 and SLC11A1, PDGFRB, LYN, CD97) and eight downregulated (LTB, RORC, GZMK, DPP4, RPS6, FLT3LG, DOCK9 and CD28) genes in CD56 + cells from asthmatics ( Figure 1B,C). LILRB2 has been previously associated with severe asthma and CD28 and DPP4 were both associated with health in peripheral blood cells in a previous study. 3 Our data confirms these findings, adds further to the list of DEGs, and also indicates the cellular source of these gene expression patterns.

Key messages
• Our study sought cell type-specific gene signatures of severe allergic asthma in peripheral blood.
• We identified changes in monocytes and NK cells, but not T lymphocytes in severe asthmatics.
• Gene expression changes may serve as prognostic markers of severe asthma or as therapeutic targets.  them has yet been assigned. Our study suggests that further investigation of these cells is warranted.
Additionally, monocytes in asthma showed increases in gene expression associated with differentiation and chemotaxis. Thus, we found innate but not adaptive cell phenotypic changes in peripheral blood cells from severe asthmatics.  Table of significantly upregulated genes corresponding to red points in volcano plots. Genes previously observed as altered in peripheral blood in a severe asthma cohort 3 are highlighted with an asterisk. (D) Summary of GO terms associated with differential gene expression in NK cells in asthma.