Increased Interleukin-6 Levels in Neuron-Derived Plasma Small Extracellular Vesicles of Subarachnoid Haemorrhage Patients

Background: Aneurismal subarachnoid hemorrhage (aSAH) is a serious type of stroke with high mortality and disability. Identifying circulating biomarkers is helpful to improve theranostics of aSAH. In this study, we are for the rst time to report circulating interleukin-6(IL-6) in neuron-derived small extracellular vesicles(NDSEVs) were identied to be the potential biomarkers in prognosis of aSAH. Methods: We extracted small extracellular vesicles from the plasma of aSAH patients and healthy controls and were enriched by sequential precipitation and anti-L1CAM antibody immunoabsorption. Subsequently,we determined IL-6 levels by an enzyme-linked immunosorbent assay (ELISA). Result: Plasma IL-6 NDSEVs showed distinct pattern differences between aSAH patients and healthy controls. The IL-6 NDSEVs levels were increased and positively associated with disease monitoring and prognosis of aSAH patients. These data suggest an elevated neuroinammatory cascade in aSAH patients. Conclusion: The IL-6 NDSEVs maybe prospective biomarkers to indicate its progression, and thus may own great potential in applications such as prognostic evaluation of aSAH in the near future.

Therefore, we hypothesize that aSAH leads to changes in the expression of IL-6 in sEVs in the brain and that these NDSEVs are secreted into circulating where they may serve as biomarkers for aSAH.In the present study, we extracted NDSEVs from the plasma of aSAH patients and healthy controls to determine the expression of IL-6 levels in NDSEVs and, ultimately, we detected increased IL-6 levels in NDSEVs of aSAH patients, which evaluated possible associations between the markers of early in ammatory response and disease progression.

Ethics
Study participants were recruited from the Department of Neurosurgery, The First A liated Hospital of Wannan Medical College, Wuhu City, China. The study was performed in accordance with the Declaration of Helsinki. Written informed consent was received from participants or valid proxies (family or a professional not directly involved in the study) prior to inclusion in the study. All experiments were approved by the Ethics Committee of the First A liated Hospital of Wannan Medical College.

Study design
The aSAH patients included in the present study were admitted from May 2016 to March 2017. One hundred -seventeen patients with aSAH within the 24 h after SAH, out of a total of 224 patients with SAH during this period, were collected plasma. The mean age ± SD was 59.91 ± 9.65 years (range, 40-83 year); there were 72 women and 45 men.Exclusion criteria were as follows: Admission later than 72 h after onset of bleeding; non-aneurysmal SAH; with liver, kidney, heart or lung insu ciency or infectious diseases; poor prognosis upon admission without any intervention. Age-matched health persons in the fasting state were used as controls(n = 40).
Initial clinical status was accessed was using the World Federation of Neurological Surgeons (WFNS) grade [28]. The amount of blood in computed tomography (CT) was assessed using the modi ed Fisher' scale. All patients were treated with endovascular coiling. Neurologic outcome was assessed using the Modi ed Rankin Scale (mRS) at 1-year post-aSAH by structured telephone interview [29]. If no contact was obtained after this procedure, the patient was declared lost to follow-up. At the end of the follow-up period, patients with a mRS score of 0-2 were classi ed as having a good outcome, and those with a mRS score of 3-6 were classi ed as having a poor outcome. Table 1and Table 2 summarizes the basic characteristics of the population.

Sample processing
The blood samples were processed for plasma isolation within 2 h after withdrawal using BD EDTAtreated plasma separator tubes. Whole blood was centrifuged at 500 rpm and 4 °C for 10 min. And the upper-layer was transferred into a RNase/DNase -free 1.5 mL EP tubes followed by further centrifugation at 3000 rpm and 4 °C for 10 min. Plasma was divided into aliquots and stored at -80ºC for further analysis.
Isolation of NDSEVs from plasma samples sEVs were isolated using miRCURY Exosome Kits (from Cell/Urine/CSF; Qiagen, Valencia, CA) following the manufacturerʼs protocol. Brie y, 400µL of the Precipitation Buffer B was added to 1 mL of plasma samples and mixed well for 60 minutes at 4 ºC, followed by centrifugation at 10,000 g for 30 minutes at room temperature. The sEVs were harvested by removing the supernatant. The sEVs were resuspended in 100 µL of Resuspension Buffer. To enrich NDSEVs, total sEVs were harvested by immunoabsorption enrichment with mouse anti-human CD171(L1CAM neural adhesion protein) biotinylated antibody ( eBiosciences, San Diego, CA, USA) as described [11]. Then NDSEVs were lysed by 1% Triton X-100 lysis buffer(Beyotime, P0013) that contained protease and phosphatase inhibitor cocktails. The lysates were stored at -80ºC .

Exosomes identi cation
For NDSEVs identi cation, transmission electron microscopy (TEM, Hitachi HT7700, Tokyo, Japan) was performed to observe the morphology of precipitated particles. Brie y, we extracted previous eluates of 5 µl and diluted them to 10 µl. Then, we extracted 10 µl sample onto copper disks for 1 min and use lter paper to remove the oating material. Subsequently, 10 µl phosphotungstic acid was dropped on copper disks for 1 min and the oating material was removed using lter paper. After drying for severe minutes and nally examined with a JEOL JEM-1400 transmission electron microscope at 80 kV. The size distribution and concentration of the particles was measured and analysed using NanoFCM instrument (NanoFCM Inc. China). Biomarkers for exosomes CD9, CD81 were also measured using the NanoFCM instrument.
Isolation and measure concentration of exosomes protein The bicinchoninic acid (BCA) method was used to measure the concentration of exosomes using the enhanced BCA protein assay kit (Beyotime, P0010). Brie y, the isolated NDSEVs were melted at 37 o C. We quickly added equal volumes of RIPA lysis buffer, then mixed and split on ice for 30 min. A standard sample of BCA method protein concentration was formulated and then added to the BCA mixture and

Flow nano analyser studies
We diluted 20 µL NDSEVs to 60 µL, then added 20 µL uorescent markers antibodies (CD9, CD81) to 30 µL dilution. We incubated for 30 min at 37 o C after mixing. We added 1 ml pre-cooled PBS, then ultracentrifuged for 70 min with 110,000 x g at 4 o C. After removing supernatants, we repeated centrifugation. We removed supernatant and resuspended in 50 µL pre-cooled 1 x PBS and analysed using the NanoFCM as per manufacturer's instructions.
Enzyme-linked immunosorbent assay (ELISA) The concentrations of IL-6, and tetra-spanning exosome marker CD81 in NDSEVs were quanti ed using speci c ELISA kits (Elabscience Biotechnology, China) according to the manufacturer's instructions. The mean value for all determinations of CD81 in the patient cohort was set at 1.00 and relative values for each specimen were used to normalize their recovery.The nal concentration of cytokines was measured using OD values. One laboratory technicians measured all ELISAs without knowledge of the clinical information.

Statistical analysis
All data were analyzed using MedCalc version 15.0.0 (Medcalc Software bvba, Ostend, Belgium). Data were presented as mean ± standard deviation (SD). The Mann-Whitney U test was used to assess the differences between two groups and the Kruskal-Wallis test for differences between more than two groups. The correlations among the variables were calculated using Spearman rank correlation coe cient analysis. For comparison of levels of IL-6 over time between patients with poor and good outcome, a generalized linear relationship model with Spearman's correlation coe cient was used.
Receiver operating characteristic(ROC) curves were constructed to determine the optimal thresholds of IL-6 for aSAH. A logistic regression model was analyzed to determine factors that predicted the mRS after adjusting for risk factors that reached P < 0.1 in the univariate analyses.A P-value of < 0.05 was considered signi cant.

Patient characteristics
In this pilot study, a total of 117 aSAH patients and 40 health controls were recruited. Study design was followed as Fig. 1.Plasma was taken at 24 h after SAH, and obtained in the fasting state in each healthy control. The detailed characteristics for these two groups are summarized in Table 2.

NDSEVs characterization
The morphology of NDSEVs was evaluated using TEM. Exosomes had spherical shapes with sizes of 86.71 ± 16.82 nm surrounded by membranes ( Fig. 2A and 2B). The STD concentration of exosomes was 6.09E + 8 particles/mL (Fig. 2C). Exosome protein concentration was determined using the BCA method. The OD 560 nm was 0.231 and the protein concentration was 1.23 µg/µL according to the standard curve (Fig. 2D). The identity of exosomes was further validated by quantitating the exosome membraneassociated markers CD9 and CD81 using the NanoFCM. The positivity rates were 5.4% and5.7% ( Fig. 2E  and 2F). There were no apparent differences in size or shape of exosomes between the control and aSAH samples.

Relationships of IL-6 NDSEVs with aSAH severity
The WFNS grade is a tool that is used to assess the level of early brain injury after SAH. The modi ed Fisher' scale was used to assess the amount of blood in CT images of aSAH patients. At the beginning of the acute period, the patients with a WFNS grade of I-III were classi ed as mildly (mild aSAH), and those with a WFNS grade of -V were classi ed as severely (severe aSAH). As illustrated in Fig. 4A, comparison of the severe aSAH patients with the mild revealed that the IL-6 levels signi cantly increased(Severe aSAH: 409.14 ± 27.06 pg/ml; mild aSAH: 338.56 ± 29.15 pg/ml) (P < 0.001). To evaluate the utility of IL-6 NDSEVs for discrimination of severe aSAH from mild aSAH, we performed ROC curve analysis and found that the area under curves(AUCs) of IL-6 NDSEVs was 0.961 (95% CI: 0.909-0.988; P < 0.001) (Fig. 4B) To further investigate the relationships between the expression levels of IL-6 NDSEVs and WFNS grade, Spearman's correlation coe cient analysis was performed. The results revealed that in the aSAH patients, the level of IL-6 NDSEVs (ρ = 0.845; 95% CI: 0.753 to 0.890; P < 0.001) was closely correlated with aSAH severity as scored by WFNS grade.

Relationships of IL-6 NDSEVs with clinical outcomes of aSAH patients
It is important to determine the potential outcomes of aSAH patients at the earliest stage to optimize the treatment. Thus, the patients were divided into two groups according to their clinical outcomes. At the end of the follow-up period, patients with a mRS score of 0-2 were classi ed as having a good outcome, and those with a mRS score of 3-6 were classi ed as having a poor outcome. The mRS scores were lower in the patients with lower expression of IL-6 NDSEVs than in those with higher expression (P < 0.001; Fig. 5 ).
On univariate analysis, age,WFNS grade, modi ed Fisher score and IL-6 NDSEVs levels were identi ed as prognostic factors 1 year post-SAH (P < 0.1).In the logistic regression models,the levels of IL-6 NDSEVs were signi cantly correlated with mRS score at 1 year post-SAH (P < 0.001; Table 3).Therefore, outcome prediction may be improved by determination of the expression of IL-6 in.

Discussion
In the present study, rstly, the circulating contains in aSAH patients and healthy controls. Secondly, we found that the circulating IL-6 NDSEVs showed distinct pattern differences between aSAH patients and healthy controls. Thirdly, the circulating IL-6 NDSEVs were higher in patients with higher WFNS grade than in those with lower WFNS grade. Finally, the circulating IL-6 NDSEVs were associated with prognosis in aSAH patients.
Several in ammatory cytokines have been found to be linked to aSAH [21]. As a well-known cytokine, IL-6 play an important role in brain injury, and associated with aSAH in numerous studies [23,26,30]. Previous study showed that IL-6 in cerebrospinal uid (CSF) seem to be a biomarker for predicting vasospasm after SAH [31]. Previous studies also showed that IL-6 levels in CSF was associated with prognosis in aSAH patients [21,27,30]. Additionally, elevated IL-6 levels in CSF may induce neuroin ammation and may be closely associated with the progression of delayed ischemic neurological de cits after SAH [25]. Furthermore, IL-6 and TNF-α in CSF could be important biomarkers for early diagnosis and disease monitoring in SAH patients [22]. However, another study reported that CSF IL-6 could be a biomarker of the ventricular infection [32]. In addition, previous study showed that IL-6 in CSF could be a useful diagnostic tool for predicting shunt dependency in aSAH patients with acute hydrocephalus [33]. Serum IL-6 was elevated, and was associated with prognosis in aSAH patients [26,34]. However, many previous studies showed that determining the IL-6 levels in blood circulation may not be a good way [30,35]. The circulating could directly re ect the situation of the brain, and peripheral factors might have little effect on cytokines in NDSEVs. Therefore, the measurement of IL-6 levels in CNS-derived sEVs, compared with blood or CSF, may be better to illuminate the actual role of IL-6 in aSAH. Hence, in our study, We analysed whether IL-6 NDSEVs could serve as biomarkers between aSAH patients and healthy controls.
The important ndings of our study were that IL-6 NDSEVs levels elevated in aSAH patients and could be a biomarker for the prognosis of aSAH. IL-6 NDSEVs expression may be used to discriminate severe aSAH from mild aSAH. ROC curves with AUC values were constructed. The sensitivity and speci city shown in ROC curve were actually high for IL-6 NDSEVs levels. Furthermore, the WFNS grade and IL-6 NDSEVs were good predictors of neurologic outcome, whereas age or modi ed Fisher score were not. These results suggest that IL-6 may participate in the occurrence, development and repair progression of aSAH. However, the others CNS diseases is also associated with the neuroin ammation. Actually, it has been reported that in ammatory cytokines in NDSEVs elevated in traumatic brain injury [11]. Additionally, increased IL-6 Levels in the astrocyte-derived exosomes could be useful to reveal neuroin ammation in sporadic amyotrophic lateral sclerosis patients [36]. Thus, the IL-6 NDSEVs may not be suitable to help distinguish aSAH from other CNS diseases.
Although the present study aimed at investigating the IL-6 NDSEVs levels as potential biomarkers correlated with disease severity and prognosis in aSAH patients, there were a few methodological limitations. First, these was a one single-center study with a wide age range, a small number of patients, and a diverse grade of severity of aSAH with WFNS grades I-V. Second, the samples were collected from patients after surgery and during drug therapy. The surgical treatment and drugs may induce changes in the expression levels of IL-6 NDSEVs .

Conclusion
In summary, IL-6 NDSEVs may be a reliable biomarker associated with EBI of aSAH. IL-6 NDSEVs could be another interesting predictor with clinical outcomes. The indicator may be an early outcome predictor for patients with aSAH,but not the independent one. Future studies are critical to determine if IL-6 NDSEVs plays a regulatory role in disease progression of aSAH and evaluate its potential as a therapeutic target. Flow chart of the study design.

Figure 5
Relative levels of IL-6NDSEVs in aSAH patients with good outcome and those with poor outcome. ***P<0.001.