Modulation of the Immunological Milieu in Acute Aneurysmal Subarachnoid Hemorrhage: The Potential Role of Monocytes Through CXCL10 Secretion

Modulation of the Immunological Milieu in Acute Aneurysmal Subarachnoid Hemorrhage: The Potential Role of Monocytes Through CXCL10 Secretion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Modulation of the Immunological Milieu in Acute Aneurysmal Subarachnoid Hemorrhage: The Potential Role of Monocytes Through CXCL10 Secretion Sebastian Sanchez, Michael S Chimenti, Yongjun Lu, Elena Sagues, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4259692/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Introduction Emerging evidence indicates that aneurysmal subarachnoid hemorrhage (aSAH) elicits a response from both innate and adaptive immune systems. An upregulation of CD8 + CD161 + cells has been observed after aSAH, yet the precise role of these cells in the context of aSAH is yet to be elucidated. Methods CSF samples from patients aSAH and non-aneurysmal SAH (naSAH) were analyzed. Single-cell RNA sequencing (scRNAseq) was performed on CD8 + CD161 + sorted samples from aSAH patients. Cell populations were identified using “clustering”. Gene expression levels of ten previously described genes involved in inflammation were quantified from aSAH and naSAH samples using RT-qPCR. The study focused on the following genes: CCL5, CCL7, APOE, SPP1, CXCL8, CXCL10, HMOX1, LTB, MAL, and HLA-DRB1. Results Genes clustering analysis revealed that monocytes, NK cells, and T cells expressed CD8 + CD161 + in the CSF of patients with aSAH. In comparison to naSAH samples, aSAH samples exhibited higher mRNA levels of CXCL10 (median, IQR = 90, 16–149 vs 0.5, 0-6.75, p = 0.02). A trend towards higher HMOX1 levels was also observed in aSAH (median, IQR = 12.6, 9-17.6 vs 2.55, 1.68–5.7, p = 0.076). Specifically, CXCL10 and HMOX1 were expressed by the monocyte subpopulation. conclusion Monocytes, NK cells and T cells can potentially expressed CD8 + CD161 + in patients with aSAH. Notably, monocytes show high levels CXCL10. The elevated expression of CXCL10 in aSAH compared to non-aneurysmal SAH naSAH indicates its potential significance as a target for future studies. Monocytes Hemorrhage Subarachnoid Aneurysm Figures Figure 1 Figure 2 Figure 3 Introduction Delayed cerebral ischemia (DCI) is a devastating complication that ocurrs in a subset of patients with aneurysmal subarachnoid hemorrhage (aSAH). 1 DCI is a complex process involving a activation of the innate and adaptive immunological responses.[ 1 , 2 ] Various cytokines such as CXCL-10 have been implicated in neurotoxicity after aSAH.[ 3 – 5 ] These mediators can promote recruitment and activation of macrophages, monocytes and neutrophils in the subarachnoid space.[ 3 , 4 ] Ultimately, inflammation may result in vasospasm, ischemia and neuronal damage. [ 6 ] In this prospective pilot study, our objective was to investigate potential elements of the immune response following aSAH. Our previous data suggested that CD8 + CD161 + cells increase in patients who developed vasospasm after aSAH. Moreover, confocal immunostaining of unruptured intracranial aneurysms demonstrated the presence of CD8 + CD161 + cells in the aneurysm wall.[ 7 ] The role of CD8 + CD161 + cells in the response that follows aSAH is unclear. In this study we characterized their possible role in aSAH. Consequently, we employed single cell RNA sequencing (scRNAseq) using human CSF samples. We conducted our analysis specifically on CD8 + CD161 + cells from aSAH samples. Messenger RNA levels of ten candidate genes previously associated with the immune response following aSAH were quantified using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in samples from patients with aSAH and non-aneurysmal subarachoid hemorrhage (naSAH). Our hypothesis centers around the potential role of CD8 + CD161 + cells in orchestrating immune mechanisms that could contribute to the DCI following aSAH. Methods Patient Population: After Institutional Review Board approval by the University of Iowa, patients with subarachnoid hemorrhage (SAH), or their legal representatives, provided written consent to participate in this study. SAH was confirmed by computed tomography. Subsequently, patients with suspicion of SAH underwent digital subtraction angiography to confirm the presence of an aneurysm.Both patients with aSAH and naSAH were included. Patients with active central nervous system infections and autoimmune disorders were excluded. Experimental Approach: Two experiments were conducted to characterize the immunological reponse of patients with SAH (Fig. 1). The first experiement used scRNA-seq to investigate the transcriptosome of different cellular populations in human CSF from patients with aSAH. The second experiment quantified messenger ribonucleic acid (mRNA) levels of aSAH and naSAH samples using Reverse-transcriptase quantitative PCR (RT-qPCR). CSF processing CSF samples were drawn from extra-ventricular drains of patients with SAH. Five mL samples were centrifuged at 2,000 rpm for 10 min. The supernatants were aliquoted into 1.5 mL tubes and stored at -80 o C. Experiment 1: scRNA analysis of aSAH and naSAH CD8 + 161 + cells aSAH and naSAH samples were sorted for CD8 + CD161 + cells. Details of the methods for sorting the samples are found in the supplementary methods. CD8 + CD161 + sorted cells were prepared as described above for scRNAseq with a10X Chromium system (10XGenomics). The cells were partitioned into droplet-based Gel Bead‑In‑Emulsions for reverse transcription and to generate 10X barcoded cDNA libraries. These libraries were then sequenced with an Illumina NovaSeq 6000 at the Genomics Division of the Iowa Institute of Human Genetics at the University of Iowa. The reads files for each sample were pre-processed with Cell Ranger Single-Cell Software (v.4.0.0), which was used to perform sample demultiplexing, barcode processing, read alignment and single-cell counting (10X genome reference ‘refdata-gex-GRCh38-2020-A’ was used for the reference alignment). Downstream analysis was performed in R using the ‘seurat’ package.[ 8 ] [ 9 ] Quality control filtering of cells was done to exclude cells containing less than 500 or more than 2,500 genes detected. Gene expression values were normalized and scaled. A nearest neighbor graph was computed with “FindNeighbors” and clusters were detected with “FindClusters” with resolution = 0.1. A Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction was calculated on the resulting seurat object using the calculated PCA components to obtain a two-dimensional representation of the cell states. Cluster markers were calculated with seurat “FindMarkers” using the ‘wilcoxon’ test. Two different analyses were performed: whole CSF unsorted aSAH and naSAH samples, and sorted CD8 + CD161 + aSAH samples. Cellular types were identified for each cluster classified using the PangalaoDB database.[ 10 ] Cell populations were identified based on the expression the top 10 expressed cellular markers (supplementary table 1 ). After identifying the cell types, the expression of ten target genes of clinical interest in each cellular population were analyzed. The following genes were selected after a thorough review of the literature: CCL5, [ 11 , 5 , 12 ] CCL7, [ 13 , 14 ] APOE, [ 13 , 14 ], SPP1, [ 15 ], CXCL8, [ 16 ] CXCL10,[ 17 , 18 ] HMOX1,[ 17 , 18 ] LTB, [ 19 ] MAL,[ 20 ] and HLA-DRB1.[ 21 ] We compared the transcriptosomes of CD8 + CD161 + cells from two aSAH patients. These cells were sorted by flow cytometry based on a previous observation that CD8 + CD161 + cells are elevated during the acute phase of aSAH in patients who developed vasospasm.[ 7 ] Finally, the expression of previously described microglial markers was analyzed in the aSAH samples. [ 22 ] This subanalysis was perform to analyze the origin of the cells that were clustered by scRNA analysis. Experiment N2: RT-qPCR Analysis of Gene Targets mRNA expression levels of these previously described 10 genes were quantifed in 13 CSF samples of patients with aSAH and naSAH. This included the samples used for the scRNA-seq analysis in the first experiment. Detailed primers are found on supplementary table 2. Statistical Analysis: Statistical analysis was performed using SPSS Version 27.0 (IBM, Aarmonk, NY). The Shapiro-Wilk test was used to evaluate normality in our sample. Normally distributed variables are reported as mean ± SD and non-normally distributed variables as median (IQR). The Mann Whitney U or the student t test were used to compare variables depeding upon distribution. Statistical significance is expressed by p-values at an α = 0.05. Results A total of 13 patients were included through all experiments conducted in this study. Patient demographics are described in Table 1 . Specific causes of SAH for each naSAH patient are described in supplementary table 3. Table 1 Patient demographics. Demographics Aneurysmal SAH (N = 9) Nonaneurysmal SAH (N = 4) Age median (IQR) 56 (49, 66) 67 (54,73) Female % (n) 66 (6) 50 (2) Sample day posthemorrhage median (IQR) 3 (1,4) 3 (2,4) SAH: subarachnoid hemorrhage. Experiment 1: scRNAseq of CD8 + CD161 + aSAH CSF Samples Two CD8 + CD161 + sorted aSAH samples were analyzed with scRNAseq. The first sample was collected at day 3 and the other at day 6. The total number of sorted cells was 3,921 and 5,738 CD8 + CD161 + cells. Distinct cell populations of NK cells, monocytes and T cells were identified as expressing CD8 + CD161 + markers (Fig. 1). CCL5 was expressed throughout all cellular populations. CCL7, HLA-DRB1, APOE, SPP1, CXCL8, HMOX1 were primarily expressed by monocytes (supplementary Fig. 1). Monocytes also were the main cell type expressing CXCL10 (supplementary Fig. 2). LTB and MAL were mainly expressed in T cells (supplementary Fig. 1). Most of the monocytes co-expressed CD14 and CD16 suggesting that these cells were in an intermediate stage of maturation (Fig. 2 ). Additionally, these cells expressed peripheral monocyte markers such CD14, FCER1G, CD86, CD68, TMEM 119 and CD44, suggesting a peripheral origin for these cells. Furthermore, the monocyte cluster in the CD8 + CD161 + aSAH samples had a very weak expression of microglial specific markers such as TMEM119, CD80 and MYB (Fig. 3). Experiment 2: RT-qPCR Analysis of Gene Targets Thirteen CSF samples were analyzed using RT-qPCR: 9 patients with aSAH, and 4 patients with naSAH. aSAH samples were collected at a median day 3 (1,4) while naSAH samples were collected at a median day 3 (2,4). Of the 10 gene targets that were analyzed, CXCL10 levels were higher in aSAH samples than in naSAH (median, IQR = 90, 16–149 vs 0.5, 0-6.75 p = 0.02) (Table 2 ). Similarly, patients with aSAH had higher levels of HMOX1 compared to patients with naSAH (median, IQR = 12.6, 9-17.6 vs 2.55, 1.68–5.7 p = 0.076). Table 2 Genes analyzed using RT-qPCR. RNA levels of ten genes of interest in the CSF of patients with aSAH (Aneurysmal) and naSAH (Non-aneurysmal). Data are represented as a median (IQR). *indicates a significant difference between gene expression in the Non-aneurysmal and Aneurysmal groups (Mann-Whitney U; p < 0.05). Gene Aneurysmal median (IQR) Non-aneurysmal median (IQR) P value CCL5 4.76 (2.95, 6.8) 4.5 (2.95, 9) 1 CCL7 20 (10, 29.6) 19.2 (10, 34.1) 1 HLA-DRB1 9.89 (6.44, 11,11) 7.89 (6, 8,86) 0.604 APOE 9.02 (2.87, 18.42) 9.73 (2.87, 18,59) 0.825 SPP1 3.81 (1.13, 9.85) 5.52 (2.99, 7.84) 1 CXCL8 2.31 (1.15, 2.56) 1.19 (1.15, 3,46) 0.604 CXCL10 90 (16, 139) 0.5 (0, 6.75) 0.02* HMOX1 12.6 (9, 17.6) 2.55 (1.68, 5.7) 0.076 LTB 1.15 (1.04, 2.13) 1.08 (0,75, 1.89) 0.710 MAL 0.76 (0.68, 1.88) 1.23 (1.13, 1.41) 0.710 * p < 0.05 Discussion We previously described that CD8 + CD161 + cells are elevated in the acute phase of aSAH in patients that developed DCI.[ 7 ] In the present study, we observed that monocytes, NK cells and T cells expressing CD8 + CD161 + in aSAH samples. Among these cells types, we identified a subpopulation of monocytes that expressed high levels of CXCL10. Microglial marker analysis suggested a peripheral origin for this subpopulation. Furthermore, we observed that CXCL10 was significantly elevated in aSAH compared to naSAH at the mRNA level. Aneurysmal rupture leads to the release of various signals that increase the permeability of the blood brain barrier (BBB) by inducing endothelial injury.[ 23 ] A damaged BBB favors migration of activated neutrophils, T cells and monocytes into the CNS.[ 24 ] Hemoglobin deposition in the subarachnoid space promotes inflammation, increased BBB dysfunction and activation of peripheral inflammatory cells and microglia.[ 25 ] scRNAseq of CD8 + CD161 + cells in the CSF of patients with aSAH led to the profiling of three different cells populations: monocytes, NK and T cells. Monocytes are especially relevant to the immune response in aSAH as multiple studies have shown increased monocytes counts in the CSF of patients with aSAH.[ 2 ] Roa et al identified that blockage of monocyte infiltration in the CNS has prevented vasospasm in animal models.[ 7 ] The clinical significance of CD8 + CD161 + monocytes in aSAH CSF samples is unclear, but it is highly suggestive of monocyte activation. Monocytes can be activated through direct cellular interaction or by circulating cytokines.[ 26 ] An altered BBB may allow peripheral monocyte infiltration. CXCL10 production by infiltrating monocytes can recruit more monocytes and amplify the immune response. We observed that mRNA levels of CXCL10 were higher in aSAH compared to naSAH CSF. CXCL10 is strong chemoattract for T cells, NK cells and monocytes leading to recruitment and migration.[ 27 , 28 ] It has been postulated that increased leukocyte counts lead to late complications of aSAH.[ 29 ] [ 30 ] In our study, CXCL10 was mainly expressed in the CD8 + CD161 + samples. The main cell type expressing CXCL10 were monocytes. Although CXCL10 can be expressed by various cell types,[ 31 ] previous studies have demonstrated secretion of CXCL10 mainly by human monocytes.[ 27 , 28 ] In our samples, monocytes expressed markers that suggested a peripheral origin (Fig. 3). CD44 was widely expressed, this marker is known to be expressed in the peripheral leukocytes but not by the microglia.[ 22 ] Moreover, intermediate monocytes (CD14 + CD16+, Fig. 2 ), the main subtype present in our CD8 + CD161 + samples, actively secrete cytokines that favor inflammation.[ 32 ] Increased levels of intermediate monocytes have also been associated with a worse neuropsychiatric outcomes during inflammatory states.[ 33 ] This finding supports the potential role of monocytes in generating signals for leukocyte recruitment in aSAH. CXCL10 may also facilitate cellular injury through direct neurotoxicity. CXCL10 signaling through CXCR3 can induce apoptosis in fetal neurons through intracellular calcium dysregulation.[ 34 ] Moreover, CXCL10 blockage results in improved neurologic function and halting of disease progression in mice with induced multiple sclerosis.[ 30 ] However, further research is needed considering most studies are animal based and were not done in aSAH. CXCL10 may also mediate the cellular environment in the unruptured aneurysm. Prior to rupture, CXCL10 is elevated in the unruptured aneurysmal sac.[ 3 ] Moreover, CXCL10 was identified in a group of genes that predicted the presence of unruptured aneurysms.[ 35 ] CD8 + CD161 + cells are present in the aneurysmal wall of unruptured aneurysms.[ 7 ] It is unclear if CD14 + monocytes expressing CD8 + CD161 + may play a role in aneurysm formation. Further molecular profiling of CXCL10 in aSAH could help in the identification of potential therapeutic targets. LIMITATIONS The present study has several limitations that should be acknowledged. The scRNAseq analysis in this study was conducted on a small sample size. Although this sample size is typical for scRNAseq studies, it may limit the generalizability of the findings. Additionaly, the CSF samples were collected at different time points during the posthemorrhage phase. The study's focus was not on examining the chronological changes in the immune response over time but rather providing a broad characterization of the role of CD8 + CD161 + cells. Future studies should investigate how cellular subpopulations mature and evolve over time. The study primarily focused on CD8 + CD161 + cells in aSAH, given previous research suggesting their activation. However, multiple cell lineages are involved in the immune response following aSAH, and the characterization of all these cell lineages was beyond the scope of this pilot study. Lastly, the analysis was limited to ten genes that were previously described in the literature as potential players in the immune response following aSAH. It is important to explore other genes that may be activated or involved in aSAH to gain a comprehensive understanding of the immunological response. CONCLUSIONS Monocytes, NK cells and T cells may express CD8 + CD161 + in patients with aSAH. CXCL10 was primarily expressed by monocytes. CXCL10 is a potential target for future studies due to its higher levels of expression in aSAH compared to naSAH patients. Declarations ACKNOWLEDGEMENTS None. Funding: Internal Grant from the Department of Neurology at the Univeristy of Iowa. Conflicts of interest: All authors have no conflicts of interest to disclose. Authors Contribution: Supervision and concept of the study: EAS, DH and SS. Acquisition and analysis of data: MSC, YL, ES, AG and CD. Manuscript drafting and final approval: all authors. Ethics approval: The Institutional Review Board of the University of Iowa approved this study. Under IRASH (IRB ID number: 201902739) Informed consent: Informed Consent was aquired for every patient under supervision of the Institutional Review Board of the University of Iowa. Data availability: Data is available upon reasonable request to the corresponding author. 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Supplementary Files Supplementalmaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 02 May, 2024 Reviews received at journal 02 May, 2024 Reviews received at journal 01 May, 2024 Reviewers agreed at journal 22 Apr, 2024 Reviewers agreed at journal 22 Apr, 2024 Reviewers invited by journal 15 Apr, 2024 Editor assigned by journal 15 Apr, 2024 Submission checks completed at journal 12 Apr, 2024 First submitted to journal 12 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4259692","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":291886516,"identity":"2f9ac549-8533-41bf-ba09-a70082a3b8e5","order_by":0,"name":"Sebastian Sanchez","email":"","orcid":"","institution":"Yale University","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Sanchez","suffix":""},{"id":291886517,"identity":"5eb4e073-8b1f-4c7e-ab31-e503f68734f8","order_by":1,"name":"Michael S Chimenti","email":"","orcid":"","institution":"University of Iowa","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"S","lastName":"Chimenti","suffix":""},{"id":291886518,"identity":"e0cf76cb-67a3-4ea7-babd-2ab8a379cdb3","order_by":2,"name":"Yongjun Lu","email":"","orcid":"","institution":"University of Iowa","correspondingAuthor":false,"prefix":"","firstName":"Yongjun","middleName":"","lastName":"Lu","suffix":""},{"id":291886519,"identity":"a19e3eed-c013-45f3-a084-a460243ce04a","order_by":3,"name":"Elena Sagues","email":"","orcid":"","institution":"University of Iowa","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Sagues","suffix":""},{"id":291886520,"identity":"36991fdf-18dc-4a60-ae8c-7d1ace02188b","order_by":4,"name":"Andres Gudino","email":"","orcid":"","institution":"University of Iowa","correspondingAuthor":false,"prefix":"","firstName":"Andres","middleName":"","lastName":"Gudino","suffix":""},{"id":291886521,"identity":"1bc1f5ec-2eb2-4a57-8ac7-2c5efeca75c0","order_by":5,"name":"Carlos Dier","email":"","orcid":"","institution":"University of Iowa","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Dier","suffix":""},{"id":291886522,"identity":"c1fda5b6-a7f4-4fdd-87b5-71944b0ea247","order_by":6,"name":"David Hasan","email":"","orcid":"","institution":"Duke University","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Hasan","suffix":""},{"id":291886523,"identity":"afa824f3-36f7-408e-8507-60ebfe1254af","order_by":7,"name":"Edgar A. Samaniego","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIie3OMUvDQBTA8RcC6RLtmoC1n6BwISAI0X6VhEBuiS4unUpc7BK751tkuvnCg7pEgluKi+CaoeCSaqomiNbBE90E7w8H9+D9uAOQyf5k7vbK27MP6tugRD8l9u9Il/e+KST9Hr15mMB0OJpdZ3z16ND0Sl0YNTiDlH9NzLg6M3NAi+WnbpbMg5MUtcCMIbBFhJSha54/c4XxkOBOjC3RD0od0BORcRn66wimY1ZUBDcxUtKSZQMvQkIMujAjUD1Wtq9AjW5HbnXgQmLklXYYAfqsrEh2GQVWghp92iO+nQhIf0bvl+3HjlgR2qu6cYa7BaJVTY4HcwEB0Mn2rlx8fFi03tW7+zQ0323KZDLZf+0VxUFqjCFSUZUAAAAASUVORK5CYII=","orcid":"","institution":"University of Iowa","correspondingAuthor":true,"prefix":"","firstName":"Edgar","middleName":"A.","lastName":"Samaniego","suffix":""}],"badges":[],"createdAt":"2024-04-12 21:14:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4259692/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4259692/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55006158,"identity":"b3032584-2096-4453-90c7-e1e867f422c5","added_by":"auto","created_at":"2024-04-19 18:57:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":369619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe experimental approach in our study involved the following steps: \u003c/strong\u003e(a) Following aneurysmal subarachnoid hemorrhage (aSAH), an external ventricular device was inserted to drain cerebrospinal fluid (CSF) from the ventricles. The CSF collected through this device contained blood due to the hemorrhage.\u003cstrong\u003e \u003c/strong\u003e(b) To investigate the cellular composition and gene expression patterns, single-cell RNA sequencing (scRNAseq) was performed on the collected CSF samples. Prior to sequencing, the cells were sorted based on the expression of CD8+ CD161+ markers. This sorting allowed us to focus on a specific subset of cells for analysis.\u003cstrong\u003e \u003c/strong\u003e(c) We then performed RT-qPCR in samples from aSAH and naSAH. In this experiment we observed elevated levels of two molecules, CXCL10, in the CSF of patients with aSAH compared to individuals with non-aneurysmal subarachnoid hemorrhage. These markers were particularly expressed in monocytes (represented as cluster 0).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eaSAH: aneurysmal subarahnoid hemorrhage. RT-qPCR: reverse transcriptase quantitative polymerase chain reaction. scRNAseq: single- cell RNA sequencing.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4259692/v1/fd46ecff57031826306b9764.png"},{"id":55006159,"identity":"ed79507f-72cb-420e-a126-d0683f86073b","added_by":"auto","created_at":"2024-04-19 18:57:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":226352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMonocyte markers expressed in aSAH samples\u003c/strong\u003e\u003cem\u003e. \u003c/em\u003eWe observed the widespread expression of commonly used markers associated with monocytes in the monocyte cluster (a). Specifically, CD14, a marker commonly found on immature and intermediate monocytes, exhibited a strong signal within the monocyte population. Additionally, markers of monocyte activation, such as CD86 and CD68, showed significant expression within the monocyte cluster. The coexpression of these markers further supports the notion that the monocytes in our study were in an activated state.(b) Similarly, CD16, although not limited to the monocyte cluster, was strongly expressed in the cluster cluster. This suggests that the intermediate phenotype of monocytes is predominant in our sample.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eaSAH: aneurysmal subarahnoid hemorrhage\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4259692/v1/a8a2f070d6de57e19727b4f9.png"},{"id":55006157,"identity":"22d41352-775a-40dd-86ac-b30e1d3d9c11","added_by":"auto","created_at":"2024-04-19 18:57:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":138949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroglial markers in aSAH. \u003c/strong\u003eVarious shared markers of peripheral monocytes and microglial cells were expressed in monocytes during aSAH. Microglial specific markers including TMEM119, MYB and CD80 were not widely expressed suggesting a peripheral origin for monocytes. A peripheral origin is feasible in the setting of damaged blood brain barrier that favors the infiltration of peripheral cells that can modify the local immunological environment of aSAH.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eaSAH: aneurysmal subarahnoid hemorrhage\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4259692/v1/7fb4d7d6f13fef78184d5bd3.png"},{"id":55008481,"identity":"b802fb37-16cd-45de-a5c8-637b1b62c4fb","added_by":"auto","created_at":"2024-04-19 19:05:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":851091,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4259692/v1/89b08840-7728-4e54-aa0e-9b788d167777.pdf"},{"id":55006160,"identity":"5211b64e-15ad-416b-ba8d-5366d34b8d4f","added_by":"auto","created_at":"2024-04-19 18:57:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1448320,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4259692/v1/d6660926064d99a39490d89b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modulation of the Immunological Milieu in Acute Aneurysmal Subarachnoid Hemorrhage: The Potential Role of Monocytes Through CXCL10 Secretion","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDelayed cerebral ischemia (DCI) is a devastating complication that ocurrs in a subset of patients with aneurysmal subarachnoid hemorrhage (aSAH).\u003csup\u003e1\u003c/sup\u003e DCI is a complex process involving a activation of the innate and adaptive immunological responses.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Various cytokines such as CXCL-10 have been implicated in neurotoxicity after aSAH.[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] These mediators can promote recruitment and activation of macrophages, monocytes and neutrophils in the subarachnoid space.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Ultimately, inflammation may result in vasospasm, ischemia and neuronal damage. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn this prospective pilot study, our objective was to investigate potential elements of the immune response following aSAH. Our previous data suggested that CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells increase in patients who developed vasospasm after aSAH. Moreover, confocal immunostaining of unruptured intracranial aneurysms demonstrated the presence of CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells in the aneurysm wall.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] The role of CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells in the response that follows aSAH is unclear. In this study we characterized their possible role in aSAH. Consequently, we employed single cell RNA sequencing (scRNAseq) using human CSF samples. We conducted our analysis specifically on CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells from aSAH samples. Messenger RNA levels of ten candidate genes previously associated with the immune response following aSAH were quantified using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in samples from patients with aSAH and non-aneurysmal subarachoid hemorrhage (naSAH). Our hypothesis centers around the potential role of CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells in orchestrating immune mechanisms that could contribute to the DCI following aSAH.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient Population:\u003c/h2\u003e \u003cp\u003e After Institutional Review Board approval by the University of Iowa, patients with subarachnoid hemorrhage (SAH), or their legal representatives, provided written consent to participate in this study. SAH was confirmed by computed tomography. Subsequently, patients with suspicion of SAH underwent digital subtraction angiography to confirm the presence of an aneurysm.Both patients with aSAH and naSAH were included. Patients with active central nervous system infections and autoimmune disorders were excluded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Approach:\u003c/h2\u003e \u003cp\u003eTwo experiments were conducted to characterize the immunological reponse of patients with SAH (Fig.\u0026nbsp;1). The first experiement used scRNA-seq to investigate the transcriptosome of different cellular populations in human CSF from patients with aSAH. The second experiment quantified messenger ribonucleic acid (mRNA) levels of aSAH and naSAH samples using Reverse-transcriptase quantitative PCR (RT-qPCR).\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCSF processing\u003c/h2\u003e \u003cp\u003eCSF samples were drawn from extra-ventricular drains of patients with SAH. Five mL samples were centrifuged at 2,000 rpm for 10 min. The supernatants were aliquoted into 1.5 mL tubes and stored at -80\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 1: scRNA analysis of aSAH and naSAH CD8\u0026thinsp;+\u0026thinsp;161\u0026thinsp;+\u0026thinsp;cells\u003c/h2\u003e \u003cp\u003eaSAH and naSAH samples were sorted for CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells. Details of the methods for sorting the samples are found in the supplementary methods. CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;sorted cells were prepared as described above for scRNAseq with a10X Chromium system (10XGenomics). The cells were partitioned into droplet-based Gel Bead‑In‑Emulsions for reverse transcription and to generate 10X barcoded cDNA libraries. These libraries were then sequenced with an Illumina NovaSeq 6000 at the Genomics Division of the Iowa Institute of Human Genetics at the University of Iowa. The reads files for each sample were pre-processed with Cell Ranger Single-Cell Software (v.4.0.0), which was used to perform sample demultiplexing, barcode processing, read alignment and single-cell counting (10X genome reference \u0026lsquo;refdata-gex-GRCh38-2020-A\u0026rsquo; was used for the reference alignment). Downstream analysis was performed in R using the \u0026lsquo;seurat\u0026rsquo; package.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] Quality control filtering of cells was done to exclude cells containing less than 500 or more than 2,500 genes detected. Gene expression values were normalized and scaled. A nearest neighbor graph was computed with \u0026ldquo;FindNeighbors\u0026rdquo; and clusters were detected with \u0026ldquo;FindClusters\u0026rdquo; with resolution\u0026thinsp;=\u0026thinsp;0.1. A Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction was calculated on the resulting seurat object using the calculated PCA components to obtain a two-dimensional representation of the cell states. Cluster markers were calculated with seurat \u0026ldquo;FindMarkers\u0026rdquo; using the \u0026lsquo;wilcoxon\u0026rsquo; test. Two different analyses were performed: whole CSF unsorted aSAH and naSAH samples, and sorted CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;aSAH samples. Cellular types were identified for each cluster classified using the PangalaoDB database.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Cell populations were identified based on the expression the top 10 expressed cellular markers (supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfter identifying the cell types, the expression of ten target genes of clinical interest in each cellular population were analyzed. The following genes were selected after a thorough review of the literature: CCL5, [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] CCL7, [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] APOE, [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], SPP1, [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], CXCL8, [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] CXCL10,[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] HMOX1,[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] LTB, [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e19\u003c/span\u003e] MAL,[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and HLA-DRB1.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e21\u003c/span\u003e] We compared the transcriptosomes of CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells from two aSAH patients. These cells were sorted by flow cytometry based on a previous observation that CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells are elevated during the acute phase of aSAH in patients who developed vasospasm.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] Finally, the expression of previously described microglial markers was analyzed in the aSAH samples. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e22\u003c/span\u003e] This subanalysis was perform to analyze the origin of the cells that were clustered by scRNA analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExperiment N2: RT-qPCR Analysis of Gene Targets\u003c/h2\u003e \u003cp\u003emRNA expression levels of these previously described 10 genes were quantifed in 13 CSF samples of patients with aSAH and naSAH. This included the samples used for the scRNA-seq analysis in the first experiment. Detailed primers are found on supplementary table 2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis:\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS Version 27.0 (IBM, Aarmonk, NY). The Shapiro-Wilk test was used to evaluate normality in our sample. Normally distributed variables are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and non-normally distributed variables as median (IQR). The Mann Whitney U or the student t test were used to compare variables depeding upon distribution. Statistical significance is expressed by p-values at an α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 13 patients were included through all experiments conducted in this study. Patient demographics are described in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Specific causes of SAH for each naSAH patient are described in supplementary table 3.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient demographics.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eDemographics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAneurysmal SAH\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(N\u0026thinsp;=\u0026thinsp;9)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eNonaneurysmal SAH\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(N\u0026thinsp;=\u0026thinsp;4)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge median (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56 (49, 66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67 (54,73)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale % (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50 (2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample day posthemorrhage\u003c/p\u003e \u003cp\u003emedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (1,4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (2,4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSAH: subarachnoid hemorrhage.\u003c/em\u003e \u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 1: scRNAseq of CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;aSAH CSF Samples\u003c/h2\u003e \u003cp\u003eTwo CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;sorted aSAH samples were analyzed with scRNAseq.\u0026nbsp;The first sample was collected at day 3 and the other at day 6. The total number of sorted cells was 3,921 and 5,738 CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells. Distinct cell populations of NK cells, monocytes and T cells were identified as expressing CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;markers (Fig.\u0026nbsp;1). CCL5 was expressed throughout all cellular populations. CCL7, HLA-DRB1, APOE, SPP1, CXCL8, HMOX1 were primarily expressed by monocytes (supplementary Fig.\u0026nbsp;1). Monocytes also were the main cell type expressing CXCL10 (supplementary Fig.\u0026nbsp;2). LTB and MAL were mainly expressed in T cells (supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eMost of the monocytes co-expressed CD14 and CD16 suggesting that these cells were in an intermediate stage of maturation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, these cells expressed peripheral monocyte markers such CD14, FCER1G, CD86, CD68, TMEM 119 and CD44, suggesting a peripheral origin for these cells. Furthermore, the monocyte cluster in the CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;aSAH samples had a very weak expression of microglial specific markers such as TMEM119, CD80 and MYB (Fig.\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 2: RT-qPCR Analysis of Gene Targets\u003c/h2\u003e \u003cp\u003eThirteen CSF samples were analyzed using RT-qPCR: 9 patients with aSAH, and 4 patients with naSAH. aSAH samples were collected at a median day 3 (1,4) while naSAH samples were collected at a median day 3 (2,4). Of the 10 gene targets that were analyzed, CXCL10 levels were higher in aSAH samples than in naSAH (median, IQR\u0026thinsp;=\u0026thinsp;90, 16\u0026ndash;149 vs 0.5, 0-6.75 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, patients with aSAH had higher levels of HMOX1 compared to patients with naSAH (median, IQR\u0026thinsp;=\u0026thinsp;12.6, 9-17.6 vs 2.55, 1.68\u0026ndash;5.7 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.076).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eGenes analyzed using RT-qPCR.\u003c/b\u003e RNA levels of ten genes of interest in the CSF of patients with aSAH (Aneurysmal) and naSAH (Non-aneurysmal). Data are represented as a median (IQR). *indicates a significant difference between gene expression in the Non-aneurysmal and Aneurysmal groups (Mann-Whitney U; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAneurysmal\u003c/p\u003e \u003cp\u003emedian (IQR)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNon-aneurysmal median (IQR)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCL5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.76\u0026nbsp; (2.95, 6.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.5 (2.95, 9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCL7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 (10, 29.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19.2 (10, 34.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHLA-DRB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.89 (6.44, 11,11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.89 (6, 8,86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.604\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAPOE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.02 (2.87, 18.42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.73 (2.87, 18,59)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.825\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSPP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.81 (1.13, 9.85)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.52 (2.99, 7.84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCXCL8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.31 (1.15, 2.56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.19 (1.15, 3,46)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.604\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCXCL10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90 (16, 139)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5 (0, 6.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.02*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHMOX1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.6 (9, 17.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.55 (1.68, 5.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.076\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLTB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.15 (1.04, 2.13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.08 (0,75, 1.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.710\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMAL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.76 (0.68, 1.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.23 (1.13, 1.41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.710\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe previously described that CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells are elevated in the acute phase of aSAH in patients that developed DCI.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] In the present study, we observed that monocytes, NK cells and T cells expressing CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;in aSAH samples. Among these cells types, we identified a subpopulation of monocytes that expressed high levels of CXCL10. Microglial marker analysis suggested a peripheral origin for this subpopulation. Furthermore, we observed that CXCL10 was significantly elevated in aSAH compared to naSAH at the mRNA level.\u003c/p\u003e \u003cp\u003eAneurysmal rupture leads to the release of various signals that increase the permeability of the blood brain barrier (BBB) by inducing endothelial injury.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e23\u003c/span\u003e] A damaged BBB favors migration of activated neutrophils, T cells and monocytes into the CNS.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e24\u003c/span\u003e] Hemoglobin deposition in the subarachnoid space promotes inflammation, increased BBB dysfunction and activation of peripheral inflammatory cells and microglia.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e25\u003c/span\u003e] scRNAseq of CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells in the CSF of patients with aSAH led to the profiling of three different cells populations: monocytes, NK and T cells. Monocytes are especially relevant to the immune response in aSAH as multiple studies have shown increased monocytes counts in the CSF of patients with aSAH.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Roa et al identified that blockage of monocyte infiltration in the CNS has prevented vasospasm in animal models.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] The clinical significance of CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;monocytes in aSAH CSF samples is unclear, but it is highly suggestive of monocyte activation.\u003c/p\u003e \u003cp\u003eMonocytes can be activated through direct cellular interaction or by circulating cytokines.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e26\u003c/span\u003e] An altered BBB may allow peripheral monocyte infiltration. CXCL10 production by infiltrating monocytes can recruit more monocytes and amplify the immune response. We observed that mRNA levels of CXCL10 were higher in aSAH compared to naSAH CSF. CXCL10 is strong chemoattract for T cells, NK cells and monocytes leading to recruitment and migration.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e28\u003c/span\u003e] It has been postulated that increased leukocyte counts lead to late complications of aSAH.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e29\u003c/span\u003e] [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e30\u003c/span\u003e] In our study, CXCL10 was mainly expressed in the CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;samples. The main cell type expressing CXCL10 were monocytes. Although CXCL10 can be expressed by various cell types,[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e31\u003c/span\u003e] previous studies have demonstrated secretion of CXCL10 mainly by human monocytes.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e28\u003c/span\u003e] In our samples, monocytes expressed markers that suggested a peripheral origin (Fig.\u0026nbsp;3). CD44 was widely expressed, this marker is known to be expressed in the peripheral leukocytes but not by the microglia.[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e22\u003c/span\u003e] Moreover, intermediate monocytes (CD14\u0026thinsp;+\u0026thinsp;CD16+, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the main subtype present in our CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;samples, actively secrete cytokines that favor inflammation.[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e] Increased levels of intermediate monocytes have also been associated with a worse neuropsychiatric outcomes during inflammatory states.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e33\u003c/span\u003e] This finding supports the potential role of monocytes in generating signals for leukocyte recruitment in aSAH.\u003c/p\u003e \u003cp\u003eCXCL10 may also facilitate cellular injury through direct neurotoxicity. CXCL10 signaling through CXCR3 can induce apoptosis in fetal neurons through intracellular calcium dysregulation.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e34\u003c/span\u003e] Moreover, CXCL10 blockage results in improved neurologic function and halting of disease progression in mice with induced multiple sclerosis.[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e30\u003c/span\u003e] However, further research is needed considering most studies are animal based and were not done in aSAH.\u003c/p\u003e \u003cp\u003eCXCL10 may also mediate the cellular environment in the unruptured aneurysm. Prior to rupture, CXCL10 is elevated in the unruptured aneurysmal sac.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] Moreover, CXCL10 was identified in a group of genes that predicted the presence of unruptured aneurysms.[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e35\u003c/span\u003e] CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells are present in the aneurysmal wall of unruptured aneurysms.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] It is unclear if CD14\u0026thinsp;+\u0026thinsp;monocytes expressing CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;may play a role in aneurysm formation. Further molecular profiling of CXCL10 in aSAH could help in the identification of potential therapeutic targets.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLIMITATIONS\u003c/h2\u003e \u003cp\u003eThe present study has several limitations that should be acknowledged. The scRNAseq analysis in this study was conducted on a small sample size. Although this sample size is typical for scRNAseq studies, it may limit the generalizability of the findings. Additionaly, the CSF samples were collected at different time points during the posthemorrhage phase. The study's focus was not on examining the chronological changes in the immune response over time but rather providing a broad characterization of the role of CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells. Future studies should investigate how cellular subpopulations mature and evolve over time. The study primarily focused on CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells in aSAH, given previous research suggesting their activation. However, multiple cell lineages are involved in the immune response following aSAH, and the characterization of all these cell lineages was beyond the scope of this pilot study. Lastly, the analysis was limited to ten genes that were previously described in the literature as potential players in the immune response following aSAH. It is important to explore other genes that may be activated or involved in aSAH to gain a comprehensive understanding of the immunological response.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eMonocytes, NK cells and T cells may express CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;in patients with aSAH. CXCL10 was primarily expressed by monocytes. CXCL10 is a potential target for future studies due to its higher levels of expression in aSAH compared to naSAH patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003eFunding: Internal Grant from the Department of Neurology at the Univeristy of Iowa.\u003c/p\u003e\n\u003cp\u003eConflicts of interest: All authors have no conflicts of interest to disclose.\u003c/p\u003e\n\u003cp\u003eAuthors Contribution: Supervision and concept of the study: EAS, DH and SS. Acquisition and analysis of data: MSC, YL, ES, AG and CD. Manuscript drafting and final approval: all authors. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthics approval: \u0026nbsp;The Institutional Review Board of the University of Iowa approved this study. Under IRASH (IRB ID number: 201902739)\u003c/p\u003e\n\u003cp\u003eInformed consent: Informed Consent was aquired for every patient under supervision of the Institutional Review Board of the University of Iowa.\u003c/p\u003e\n\u003cp\u003eData availability: Data is available upon reasonable request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGris T, Laplante P, Thebault P, Cayrol R, Najjar A, Joannette-Pilon B et al. Innate immunity activation in the early brain injury period following subarachnoid hemorrhage. J Neuroinflammation. 2019;16(1):253. doi:10.1186/s12974-019-1629-7.\u003c/li\u003e\n\u003cli\u003eMoraes L, Grille S, Morelli P, Mila R, Trias N, Brugnini A et al. Immune cells subpopulations in cerebrospinal fluid and peripheral blood of patients with Aneurysmal Subarachnoid Hemorrhage. 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Nat Med. 2019;25(3):496-506. doi:10.1038/s41591-018-0336-8.\u003c/li\u003e\n\u003cli\u003eLi X, O\u0026apos;Regan AW, Berman JS. IFN-gamma induction of osteopontin expression in human monocytoid cells. J Interferon Cytokine Res. 2003;23(5):259-65. doi:10.1089/107999003321829971.\u003c/li\u003e\n\u003cli\u003eDas ST, Rajagopalan L, Guerrero-Plata A, Sai J, Richmond A, Garofalo RP et al. Monomeric and Dimeric CXCL8 Are Both Essential for In Vivo Neutrophil Recruitment. PLOS ONE. 2010;5(7):e11754. doi:10.1371/journal.pone.0011754.\u003c/li\u003e\n\u003cli\u003eDunn LL, Midwinter RG, Ni J, Hamid HA, Parish CR, Stocker R. New insights into intracellular locations and functions of heme oxygenase-1. Antioxid Redox Signal. 2014;20(11):1723-42. doi:10.1089/ars.2013.5675.\u003c/li\u003e\n\u003cli\u003eHofmann A, Muglich M, Wolk S, Khorzom Y, Sabarstinski P, Kopaliani I et al. Induction of Heme Oxygenase-1 Is Linked to the Severity of Disease in Human Abdominal Aortic Aneurysm. 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Overview of General and Discriminating Markers of Differential Microglia Phenotypes. Front Cell Neurosci. 2020;14:198. doi:10.3389/fncel.2020.00198.\u003c/li\u003e\n\u003cli\u003eLublinsky S, Major S, Kola V, Horst V, Santos E, Platz J et al. Early blood-brain barrier dysfunction predicts neurological outcome following aneurysmal subarachnoid hemorrhage. EBioMedicine. 2019;43:460-72. doi:10.1016/j.ebiom.2019.04.054.\u003c/li\u003e\n\u003cli\u003eMacdonald RL. Delayed neurological deterioration after subarachnoid haemorrhage. Nat Rev Neurol. 2014;10(1):44-58. doi:10.1038/nrneurol.2013.246.\u003c/li\u003e\n\u003cli\u003eWu F, Liu Z, Li G, Zhou L, Huang K, Wu Z et al. Inflammation and Oxidative Stress: Potential Targets for Improving Prognosis After Subarachnoid Hemorrhage. Front Cell Neurosci. 2021;15:739506. doi:10.3389/fncel.2021.739506.\u003c/li\u003e\n\u003cli\u003eLiang H, Xie Z, Shen T. Monocyte activation and cardiovascular disease in HIV infection. Cellular \u0026amp; Molecular Immunology. 2017;14(12):960-2. doi:10.1038/cmi.2017.109.\u003c/li\u003e\n\u003cli\u003eVargas-Inchaustegui DA, Hogg AE, Tulliano G, Llanos-Cuentas A, Arevalo J, Endsley JJ et al. CXCL10 production by human monocytes in response to Leishmania braziliensis infection. Infect Immun. 2010;78(1):301-8. doi:10.1128/IAI.00959-09.\u003c/li\u003e\n\u003cli\u003eSorensen EW, Lian J, Ozga AJ, Miyabe Y, Ji SW, Bromley SK et al. CXCL10 stabilizes T cell-brain endothelial cell adhesion leading to the induction of cerebral malaria. JCI Insight. 2018;3(8). doi:10.1172/jci.insight.98911.\u003c/li\u003e\n\u003cli\u003evan den Borne P, Quax PH, Hoefer IE, Pasterkamp G. The multifaceted functions of CXCL10 in cardiovascular disease. Biomed Res Int. 2014;2014:893106. doi:10.1155/2014/893106.\u003c/li\u003e\n\u003cli\u003eLiu MT, Keirstead HS, Lane TE. Neutralization of the Chemokine CXCL10 Reduces Inflammatory Cell Invasion and Demyelination and Improves Neurological Function in a Viral Model of Multiple Sclerosis. The Journal of Immunology. 2001;167(7):4091-7. doi:10.4049/jimmunol.167.7.4091.\u003c/li\u003e\n\u003cli\u003eVazirinejad R, Ahmadi Z, Kazemi Arababadi M, Hassanshahi G, Kennedy D. The Biological Functions, Structure and Sources of CXCL10 and Its Outstanding Part in the Pathophysiology of Multiple Sclerosis. Neuroimmunomodulation. 2014;21(6):322-30. doi:10.1159/000357780.\u003c/li\u003e\n\u003cli\u003eKim YS, Yang HJ, Kee S-J, Choi I, Ha K, Ki KK et al. The \u0026ldquo;Intermediate\u0026rdquo; CD14\u0026thinsp;+\u0026thinsp;CD16\u0026thinsp;+\u0026thinsp;monocyte subpopulation plays a role in IVIG responsiveness of children with Kawasaki disease. Pediatric Rheumatology. 2021;19(1):76. doi:10.1186/s12969-021-00573-7.\u003c/li\u003e\n\u003cli\u003eVeenhuis RT, Williams DW, Shirk EN, Abreu CM, Ferreira EA, Coughlin JM et al. Higher circulating intermediate monocytes are associated with cognitive function in women with HIV. JCI Insight. 2021;6(11). doi:10.1172/jci.insight.146215.\u003c/li\u003e\n\u003cli\u003eSui Y, Stehno-Bittel L, Li S, Loganathan R, Dhillon NK, Pinson D et al. CXCL10-induced cell death in neurons: role of calcium dysregulation. Eur J Neurosci. 2006;23(4):957-64. doi:10.1111/j.1460-9568.2006.04631.x.\u003c/li\u003e\n\u003cli\u003ePoppenberg KE, Li L, Waqas M, Paliwal N, Jiang K, Jarvis JN et al. Whole blood transcriptome biomarkers of unruptured intracranial aneurysm. PLoS One. 2020;15(11):e0241838. doi:10.1371/journal.pone.0241838.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-stroke-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trsr","sideBox":"Learn more about [Translational Stroke Research](http://jcmr-online.biomedcentral.com)","snPcode":"12975","submissionUrl":"https://submission.nature.com/new-submission/12975/3","title":"Translational Stroke Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Monocytes, Hemorrhage, Subarachnoid, Aneurysm","lastPublishedDoi":"10.21203/rs.3.rs-4259692/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4259692/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction\u003c/h2\u003e \u003cp\u003eEmerging evidence indicates that aneurysmal subarachnoid hemorrhage (aSAH) elicits a response from both innate and adaptive immune systems. An upregulation of CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;cells has been observed after aSAH, yet the precise role of these cells in the context of aSAH is yet to be elucidated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eCSF samples from patients aSAH and non-aneurysmal SAH (naSAH) were analyzed. Single-cell RNA sequencing (scRNAseq) was performed on CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;sorted samples from aSAH patients. Cell populations were identified using \u0026ldquo;clustering\u0026rdquo;. Gene expression levels of ten previously described genes involved in inflammation were quantified from aSAH and naSAH samples using RT-qPCR. The study focused on the following genes: CCL5, CCL7, APOE, SPP1, CXCL8, CXCL10, HMOX1, LTB, MAL, and HLA-DRB1.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eGenes clustering analysis revealed that monocytes, NK cells, and T cells expressed CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;in the CSF of patients with aSAH. In comparison to naSAH samples, aSAH samples exhibited higher mRNA levels of CXCL10 (median, IQR\u0026thinsp;=\u0026thinsp;90, 16\u0026ndash;149 vs 0.5, 0-6.75, p\u0026thinsp;=\u0026thinsp;0.02). A trend towards higher HMOX1 levels was also observed in aSAH (median, IQR\u0026thinsp;=\u0026thinsp;12.6, 9-17.6 vs 2.55, 1.68\u0026ndash;5.7, p\u0026thinsp;=\u0026thinsp;0.076). Specifically, CXCL10 and HMOX1 were expressed by the monocyte subpopulation.\u003c/p\u003e\u003ch2\u003econclusion\u003c/h2\u003e \u003cp\u003eMonocytes, NK cells and T cells can potentially expressed CD8\u0026thinsp;+\u0026thinsp;CD161\u0026thinsp;+\u0026thinsp;in patients with aSAH. Notably, monocytes show high levels CXCL10. The elevated expression of CXCL10 in aSAH compared to non-aneurysmal SAH naSAH indicates its potential significance as a target for future studies.\u003c/p\u003e","manuscriptTitle":"Modulation of the Immunological Milieu in Acute Aneurysmal Subarachnoid Hemorrhage: The Potential Role of Monocytes Through CXCL10 Secretion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-19 18:57:14","doi":"10.21203/rs.3.rs-4259692/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-02T15:33:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-02T09:39:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-02T02:16:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"d692721b-ba55-45d8-b505-185180ae527c","date":"2024-04-23T02:40:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5fb187af-6db7-471a-a5d0-a044c70e7d69","date":"2024-04-22T15:01:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-15T16:05:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-15T15:31:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-12T22:33:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Stroke Research","date":"2024-04-12T20:59:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-stroke-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trsr","sideBox":"Learn more about [Translational Stroke Research](http://jcmr-online.biomedcentral.com)","snPcode":"12975","submissionUrl":"https://submission.nature.com/new-submission/12975/3","title":"Translational Stroke Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"77bf1e59-3db8-4d6a-b638-e3e6910877ec","owner":[],"postedDate":"April 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-07T23:24:50+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-19 18:57:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4259692","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4259692","identity":"rs-4259692","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}