ANALYSIS of EARLY STROKE-INDUCED CHANGES in CIRCULATING LEUKOCYTE COUNTS USING TRANSCRIPTOMIC Grant C

Research Article • DOI: 10.1515/tnsci-2018-0024 • Translational Neuroscience • 9 • 2018 • 161-166 Translational Neuroscience ANALYSIS OF EARLY STROKE-INDUCED CHANGES IN CIRCULATING LEUKOCYTE COUNTS USING TRANSCRIPTOMIC Grant C. O’Connell*, DECONVOLUTION Julia H.C. Chang Abstract School of Nursing, Growing evidence suggests that stroke alters the phenotype of the peripheral immune system; better Case Western Reserve University, Cleveland, OH characterization of this response could provide new insights into stroke pathophysiology. In this investigation, we employed a deconvolution approach to informatically infer the cellular composition of the circulating leukocyte pool at multiple timepoints following stroke onset based on whole blood mRNA expression. Microarray data generated from the peripheral blood of 23 cardiovascular disease controls and 23 ischemic stroke patients at 3, 5, and 24 hours post-symptom onset were obtained from a public repository. Transcriptomic deconvolution was used to estimate the relative counts of nine leukocyte populations based on the expression of cell-specific transcripts, and cell counts were compared between groups across timepoints. Inferred counts of lymphoid cell populations including B-cells, CD4+ T-cells, CD8+ T-cells, γδ T-cells, and NK-cells were significantly lower in stroke samples relative to control samples. With respect to myeloid cell populations, inferred counts of neutrophils and monocytes were significantly higher in stroke samples compared to control samples, however inferred counts of eosinophils and dendritic cells were significantly lower. These collective differences were most dramatic in samples collected at 5 and 24 hours post-symptom onset. Findings were subsequently confirmed in a second dataset generated from an independent population of 24 controls and 39 ischemic stroke patients. Collectively, these results offer a comprehensive picture of the early stroke-induced changes to the complexion of the circulating leukocyte pool, and provide some of the first evidence that stroke triggers an acute decrease in eosinophil counts. Keywords Received 09 August 2018 Complete blood count • CBC • NLR • Neutrophil lymphocyte ratio • Immune suppression accepted 14 October 2018 • WBC • White blood cell • WBC Differential • Eosinophil • Infection Introduction routine clinical evaluation; unfortunately, the [2–6]. Recent work by our group suggests clinical white blood cell differential provides that similar to other conditions [7], several It is becoming increasingly evident that the quantification of a limited number of cell of the gene expression changes observed peripheral immune system responds robustly populations, often only total neutrophils, between stroke patients and controls in to stroke, and that this response influences monocytes, and lymphocytes, and thus these investigations were likely artifacts of clinical outcome. For example, peripheral provides a relatively low-detail picture underlying changes in leukocyte counts, and immune changes triggered by stroke are regarding peripheral immune status. Multi- not true changes in transcription at the cellular believed to contribute to the pathogenesis color flow cytometry experiments have been level [8,9]. Transcriptomic deconvolution is a of adverse complications such as secondary used to examine more discrete subpopulations process which leverages such phenomena to tissue damage, hemorrhagic transformation, of leukocytes, however they have often only informatically infer the cellular composition and post-stroke infection [1]. Thus, better focused on small numbers of cell types in a single of complex biological samples based on characterization of the stroke-induced analysis. Thus, more detailed characterization aggregate gene expression through the peripheral immune response could provide of the stroke-induced changes to the cellular analysis of cell-specific transcripts [10]. In this novel insights into stroke pathophysiology and complexion of the peripheral immune system study, in an attempt to better characterize the open new avenues for immunotherapeutic could reveal nuanced alterations which are stroke-induced peripheral immune response, intervention. pathologically relevant. we employed a deconvolution approach to Many studies which have investigated Several prior studies have performed infer the counts of nine major circulating the peripheral immune response to stroke genome-wide transcriptomic profiling of leukocyte populations at multiple timepoints in humans have done so using the standard peripheral whole blood with the goal of following stroke onset using publicly available white blood cell differential collected as part of identifying clinically-useful stroke biomarkers human whole blood gene expression data. * E-mail: [email protected] © 2018 Grant C. O’Connell, Julia H.C. Chang, published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License. 161 Translational Neuroscience Methods null hypothesis was rejected when p<0.05. immune response to stroke could provide novel insights into stroke pathophysiology and Microarray data processing Validation of results identify new targets for immunotherapeutic Raw microarray data generated from the Findings were subsequently confirmed via intervention. In this study, we employed a peripheral whole blood of 23 controls, as well as deconvolution of a second publically available transcriptomic deconvolution approach to infer 23 ischemic stroke patients at 3, 5, and 24 hours microarray dataset generated from an the relative counts of nine major circulating post-symptom onset, were downloaded as .CEL independent population of 24 controls and leukocyte populations in blood samples files from the National Center for Biotechnology 39 ischemic stroke patients (GEO accession collected at multiple timepoints over the first Information (NCBI) Gene Expression Omnibus number GSE16561) [14]. 24 hours following stroke onset. With respect (GEO) via accession number GSE58294. to several cell types, our results confirmed the Probe annotations were updated via the Results findings of prior cytometric studies, however, ‘annotate’ package for R (R project for our analysis also revealed changes in other Statistical Computing). Raw perfect match Clinical and demographic leukocyte populations that have yet to be probe intensities were background corrected, characteristics widely reported on in human stroke. quantile normalized, and summarized at the set Stroke samples originated from patients Our observations suggest that the level via robust multi-array averaging using the which were signiﬁcantly older than control circulating counts of lymphoid cell populations rma() function of the ‘affy’ package. Data were counterparts, but were well matched in are ubiquitously suppressed in response to further summarized at the gene level via max terms of sex and ethnicity. In terms of risk stroke. This is consistent with prior cytometric intensity using the collapserows() function of factors for cardiovascular disease, groups investigations which have reported stroke- the ‘WGCNA’ package. were well matched with regards to rates of induced decreases in total lymphocyte counts hypertension and diabetes, however control [15–18], as well as reductions in counts of more Deconvolution subjects displayed a signiﬁcantly higher discrete lymphoid subpopulations such as Estimated counts of B-cells, CD4+ T-cells, CD8+ prevalence of dyslipidemia relative to stroke B-cells, CD4+ T-cells, CD8+ T-cells, and NK-cells T-cells, gamma delta (γδ) T-cells, natural killer patients. All stroke patients had received [15,16,18]. To our knowledge, only three other (NK) cells, monocytes, neutrophils, eosinophils, thrombolytic intervention via recombinant human studies have reported on circulating γδ and dendritic cells were generated from tissue plasminogen activator (rtPA) following 3 T-cell counts in stroke; one reporting that γδ normalized expression data using a list of 226 hour blood collection (Table 1). T-cell counts are elevated [19], one reporting cell-specific genes (Figure 1) aggregated from that they are unaffected [20], and one reporting a compendium of immune cell microarray Inferred leukocyte counts that they are decreased [21]. Our results provide data compiled by Newman et al. [11]. Inferred counts of lymphoid populations additional evidence suggesting that stroke Weighted correlation network analysis was including B-cells, CD4+ T-cells, CD8+ T-cells, triggers an acute decrease in circulating γδ used to produce a relative count for each cell γδ T-cell and NK-cells were all significantly T-cell numbers similar to that which is observed population based on the expression levels of reduced in stroke samples relative to controls in other lymphoid populations. its associated genes using the collapserows() (Figure 2A-E); this effect was most pronounced Our results are also consistent with those of function of the ‘WGCNA’ package according in samples collected at 5 and 24 hours following several prior cytometry-based investigations to the method described by Miller et al. [12]. onset of symptoms. With regards to myeloid reporting that counts of the two most abundant Relative counts of each cell population were populations, inferred counts of neutrophils and peripheral blood myeloid cell populations, arbitrarily scaled from zero to one using unity- monocytes were significantly higher in stroke neutrophils and monocytes, become robustly based normalization.

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