| Background and ObjectivesIntracerebral hemorrhage(ICH) is an important subtype of stroke that has been the second most important cause of death worldwide. There are high mortality and disability rate in acute ICH that lacking of effective treatments. Most of ICH patients have different degrees of neural disfunction and often need long-term rehabilitation training and life care, which brings about heavy economic burden to the society and family.The ultimate pathological impairment of ICH is determined by primary injury and secondary injury. The primary injuries of ICH are hematoma enlargement and compression that causing brain parenchymal lesions and raised intracranial pressure. ICH-induced secondary injuries involve a series of complex events including immune response, clot dissolution and the subsequent production and release of various harmful substances. The secondary injuries of ICH are the major reasons of neural dysfunctions by inducing cerebral edema, neuronal apoptosis, degeneration and necrosis that eventually cause the interruption of neural circuity.Increasing evidences have indicated that immune response plays a very important role in the secondary injuries of brain tissue after ICH. The systemic immune responses after acute ICH include immune activation and subsequent immunosuppression. The former mainly involves microglial activation, intracephalic infiltration of inflammatory-related cells such as neutrophil, mononuclear-macrophage and lymphocyte, and pro-inflammatory cytokine secretion. The latter mainly refers to stroke-induced immunodepression syndrome(SIDS). After bleeding in acute ICH, brain antigens are released into blood circulation as a result of the disruption of blood brain barrier(BBB). Brain antigens are closely related to the severity and prognosis of stroke. Post-ICH immunosuppression can avoid immune overreaction against brain antigen, but adversely undermines the host resistance to pathogens and results in various infections, which is an important cause of death post stroke. Altered immune status and time-dependent recruitment of different inflammatory cells imply that most of the inflammatory cell types display beneficial and adverse effects following ICH. It is well documented about the biological characteristics and actions of neutrophil and microglia in the innate immune response of acute ICH, but little is known about the specific subsets of lymphocytes in adaptive immunity to ICH, especially T-subsets.CD4+CD25+Foxp3+ regulatory T cells(Tregs) are generally considered as the crucial regulator in maintaining immunologic homeostasis and resisting the resulting tissue damages from various immune imbalance-mediated disorders via inhibition of varied immunocytes which mainly including effector T cells, as well as other cells such as B cells, neutrophils and mononuclear-macrophages. Recent research progresses on post-stroke immune response have shown that Tregs have an important and complicated role in regulating stroke-induced inflammatory damage as well as mediating sequent immunosuppression of ischemic stroke with undefined immunoregulatory mechanisms.At present, considerable progress on Tregs has been made in ischemic stroke rather than hemorrhagic stroke. Based on latest developments in the complex and multiphasic roles of immune-regulating Tregs in the pathphysiology of acute stroke, this study therefore tested the ongoing changes of ICH patients’ Tregs, as well as neural disfunction, brain antigens and pneumonia, and their possible relations in this study, looking for evidence of possible immunotherapeutic strategies through targeting Tregs in patients with acute hemorrhagic stroke. 1 Materials and methods 1.1 Subjects and GroupsAcute ICH patients were enrolled from the Neurological Departments and Intensive Care Units of the First Affiliated Hospitals of Zhengzhou University and the First Affiliated Hospitals of Xinxiang Medical University, and age and sex-matched 60 healthy persons who had no history of stroke that were served as control group. All seventy patients were confirmed by head CT or MRI and excluded with subarachnoid hemorrhage, epidural and subdural hemorrhage, brain tumor or trauma hemorrhage, surgery and acute infection at onset of disease. During the study, the managements and treatments for ICH patients were carried out under the guideline of AHA/ASA. The clinical data of the patients and the controls were collected, including hypertension, diabetes and hyperlipidemia. The experimental protocols were approved by the ethics committees of the First Affiliated Hospitals of Zhengzhou University and Xinxiang Medical University.NIHSS was used to evaluate the degrees of ICH patients’ neurological impairments. According to the scores of NIHSS, ICH patients were subdivided into the mild to moderate groups and severe groups at day 3 and day 7 after bleeding. 1.2 Blood sample collectionTen ml of EDTA-anticoagulant peripheral blood were collected from patients on the morning of 3 and 7 days post ICH. The same amounts of blood sample were collected from controls. Seven ml of blood was used for flow cytometric analysis and another 3 ml was for plasma ELISA test. 1.3 Determination of ICH patients with pneumonia after strokeAt day 3 and day 7 after ICH onset, we assess whether ICH patients have stroke-associated pneumonia that based on the result of diagnostic analysis on the clinical manifestations and medical examinations including lung auscultation and percussion, purulent respiratory secretions, fever, sputum culture, and the results of chest X-ray or CT scan of the lung. 1.4 Flow CytometryFor flow cytometry, EDTA blood was treated with RBC lysing buffer to obtain peripheral blood mononuclear cells(PBMCs). Then PBMCs were stained with mouse-anti-human, fluorescent dye-conjugated monoclonal antibodies(m Abs) against respective surface antigens. The m Abs used for surface staining included CD3-APC, CD4-PE-cy7, CD69-FITC, CD25-PE, CD127-FITC and the appropriate isotype-matched Abs respectively. To confirming the consistency of CD4+CD25highCD127low/- cells with CD4+CD25+Foxp3+ Tregs, Foxp3-Alexa Fluor 647 m Ab was used for intracellular staining to identify most CD4+CD25high CD127low/- cells were CD4+CD25+Foxp3+ Tregs. 1.5 Plasma ELISA TestThe levels of transforming growth factor(TGF-β), interleukin-10(IL-10), NSE, S100 B, and MBP in plasma were measured by enzyme-linked immunosorbent assay(ELISA) according to the protocols from supplier. The sensitivities for targeted analytes were 1.0 ng/ml for NSE and TGF-β, 0.1pg/ml for IL-10, 1.0 pg/ml for S100 B, and 0.1 ng/ml for MBP. 2 Results 2.1 Lymphocyte subpopulationThe frequency of circulating CD3+ cells in lymphocytes of ICH patients at day 3(67.96±7.331%) and day 7(66.19±7.439%) after ICH onset had no statistically difference with that of controls(69.22±7.166%)(P>0.05).The frequency of CD3+CD4+ cells in lymphocytes of ICH patients at day 3(44.32±8.614%) and day 7(42.24±7.653%) after ICH onset had no statistically difference with that of controls(44.52±7.247%)(P>0.05).The frequency of CD3+CD69+ cells in lymphocytes of ICH patients at day 3(4.669±2.842%) and day 7(5.739±2.954%) after ICH onset were obviously higher than that of controls(2.622±2.159%) and had apparent significant difference(P<0.01, respectively). 2.2 TregsCompared with the control group(4.199±1.869%), the frequency of circulating CD4+CD25highCD127low/- cells(Tregs) in CD4+ cells of ICH patients were increased significantly at day 3(5.569±2.597%) and day 7(6.653±2.924%) after ICH onset(P<0.05, P<0.01). 2.3 The effects of disease severity and gender on TregsThe frequency of circulating Tregs in the severe group(6.301±2.654%) was higher than that in the mild to moderate group(4.793±2.329%) at day 3 after ICH onset(P<0.05). Furthermore, The frequency of Tregs in the severe group(7.641±2.950%) were increased obviously in comparison to the mild to moderate group(5.634±2.560%) at day 7 after ICH onset(P<0.01).The frequency of circulating Tregs in the female group(5.268±2.648% and 6.292±2.798%) had no statistically difference with male group(5.807±2.566% and 6.944±3.029%) at day 3 and at day 7 after ICH onset(P>0.05, rspectively). 2.4 The frequency of Tregs in ICH patients with pneumoniaThe frequency of circulating Tregs in ICH patients with pneumonia(6.945±2.553% and 7.823 ± 2.771%) were higher than that of ICH patients without pneumonia(5.111±2.468% and 6.205±2.884%) at day 3 and at day 7 after ICH onset(P<0.01, rspectively). 2.5 Tregs-associated cytokines TGF- β and IL-10 in plasmaCompared with controls(14.18±2.17 ng/ml), the plasma levels of TGF-β in ICH patients were increased significantly at day 3(22.82±3.11 ng/ml) and day 7(26.01±3.05 ng/ml) after ICH onset(P<0.01, rspectively).The plasma levels of IL-10 in ICH patients were also increased significantly at day 3(21.34±2.98 pg/ml) and day 7(22.91±3.48 pg/ml) after ICH onset compared with the control group(14.15±2.59 pg/ml)(P<0.01, rspectively). 2.6 Brain antigens in plasmaCompared with the control group(14.33±2.428 ng/m L), the plasma levels of NSE in ICH patients were significantly increased at day 3(23.57±2.897 ng/m L) and day 7(22.49±2.956 ng/m L) after ICH onset(P<0.01, rspectively).Compared with the control group(3.401±0.755 ng/m L), the plasma levels of MBP in ICH patients were significantly increased at day 3(6.713±1.232 ng/m L) and day 7(7.828±1.394 ng/m L) after ICH onset(P<0.01, rspectively).Compared with the control group(289.9±45.89 pg/m L), the plasma levels of S100 B in ICH patients were significantly increased at day 3(377.6±56.74 pg/m L)(P<0.01) and day 7(319.9±58.16 pg/m L)(P<0.05) after ICH onset. Furthermore, the plasma levels of S100 B in the severe group(398.65±55.26 pg/m L and 339.424±62.008 pg/m L) were higher than that in the mild to moderate group(355.21±49.92 pg/m L and 299.750±46.783 pg/m L) at day 3 and day 7 post ICH onset(P<0.01, rspectively). 2.7 Correlation analysis of circulating Tregs and brain related antigensThe frequency of circulating Tregs and the plasma level of S100 B were positively related at day 3 and day 7 after ICH onset(P<0.01, respectively).The frequency of Tregs and the plasma levels of NSE and MBP had no obvious correlation at day 3 and day 7 after ICH onset(P> 0.05, respectively). 2.8 Correlation analysis of plasma S100 B and cytokinesThe plasma levels of S100 B and TGF-β in ICH patients were positively related at day 3 and day 7 after ICH onset(P<0.01, respectively), while not with IL-10 at day 3 and day 7 after ICH onset(P>0.05, respectively). 3 Conclusions 3.1 T cells are activated in the peripheral blood of ICH patients, which as a result of systematic and peripheral immune reaction following ICH. 3.2 The frequency of circulating Tregs, as well as plasma TGF-β and IL-10, are significantly increased in ICH patients, suggesting Tregs may involve in the immune response after ICH. 3.3 The disease severity affects the frequencies of circulationg Tregs in ICH patients with different degree of neural dysfunction. 3.4 Infection affects the frequencies of circulationg Tregs in ICH patients with pneumonia. 3.5 The plasma levels of brain related antigens S100 B, NSE and MBP increase in ICH patients. The disease severity affects the plasma S100 B in ICH patients with different degree of neural dysfunction. 3.6 Plasma S100 B of ICH patients, but not NSE and MBP, has correlation with Tregs, which imply that S100 B, as an activator, may amplify circulating Tregs of ICH patients in antigen-specific manner. 3.7 The correlation of plasma S100 B and TGF-β suggests that S100 B might through affecting plasma TGF-β to boost the frequency of circulating Tregs in ICH patients to involve in Treg-mediated immunesuppression reaction after stroke. |
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