The Effect Of Lymphocytes Co-cultured With Human Cord Blood-derived Multipotent Stem Cells On Inflammation Response After Ischemic Stroke

BackgroundStroke is a leading cause of death and permanent disability worldwide.Due to the growing and ageing population and increased prevalence of risk factors,there are excessive stroke burden globally.Currently,intravenous thrombolysis and thrombectomy have shown clear efficacy for ischemic stroke patients,and advances in recanalization therapies have extended the time window for the intervention of ischemic stroke.Whereas,a low number of stroke patients are eligible for recanalization treatment,and cerebral injury caused by ischemia-reperfusion is still a burning problem.Together,currently available therapies are insufficient for cerebral injury after stroke,strongly suggesting novel treatment options.Inflammatory events are currently known to play critical roles in the progression of cerebral injury after stroke.Growing evidence suggests the manipulation of lymphocytes in ischemic stroke as a potential therapeutic strategy for the management of stroke.Regulatory T lymphocytes(Tregs)have been characterized as disease-limiting protective cells,and play a key part in controlling immune responses in ischemic stroke.Therefore,increasing the proportion of Tregs is conducive in the immunomodulatory therapy for stroke.Stem cell therapy is a promising and attractive alternative for repairing stroke-induced neurological damage.During the past years,human cord blood-derived multipotent stem cells(HCB-SCs)have emerged as a highly promising source for cell therapy in stroke therapy,including improved neurobehavioral functions,reduced infarct volume,and prolonged survival.Moreover,increasing evidence has shown that HCB-SCs can modulate immunologic responses by altering Tregs.30,31 Our previous study discovered that HCB-SCs could increase the proportion of Tregs in peripheral lymphocytes,and lymphocytes co-cultured with HCB-SCs could improve pathological impairment of APP/PS1 mice via an immunomodulatory effect.25 Given the potency of HCB-SCs and Tregs under cerebral ischemic condition,rat spleen lymphocytes were co-cultured with HCB-SCs in this study,and then co-cultured lymphocytes were intravenously transplanted into the rat stroke model of middle cerebral artery occlusion and reperfusion(MCAO).Meanwhile,we observed the modulating effect of HCB-SCs on lymphocytes,the neuroprotective effect of cocultured lymphocytes as well as the underlying mechanism in modulating immune responses in ischemic stroke.Method1.Experimental AnimalsMale Wistar rats(weighing 250?300 g)we re obtained from Beijing HFK Bioscience Co.,LTD(Beijing,China).All rats were kept under a 12:12 hrs light/dark cycle at a constant temperature of 22? and were free access to food and water.This study was approved by the Ethical Committee of,Shandong University.All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Shandong University.The rats were randomly divided into 3 groups as follows:sham-operated group(Sham,n=10),transient middle cerebral artery occlusion and reperfusion group(tMCAO,n=12),and tMCAO treated with the transplantation of lymphocytes co-cultured with HCB-SCs(tMCAO+LT,n=12).2.Transient Middle Cerebral Artery Occlusion(tMCAO)ModelThe rats were anesthetized with 10%chloral hydrate(3 mL/kg)by intraperitoneal injection.The left common carotid artery was identified and isolated through a ventral midline cervical incision.A nylon filament(0.28 mm in diameter)with a rounded tip was introduced from the common carotid artery lumen into the internal carotid artery to block the origin of the left middle cerebral artery(MCA).The left MCA was occluded with the filament for 90 min,and the filament was withdrawn to allow MCA reperfusion.In the sham-operated group,the left neck incision was made to expose the arteries,but the nylon filament was not inserted into internal carotid artery.3.Lymphocyte Co-Culture and TransplantationThe preparation and co-culture of HCB-SCs and spleen lymphocytes were conducted according to our previous report.Human cord blood collection for research was approved by the institutional review board of Shandong University and Jinan Central Hospital.Cord blood samples were collected from the placenta of healthy donors in Department of Obstetrics,Jinan Central Hospital with donors' written informed consent.The rat mononuclear lymphocytes were co-cultured with HCB-SCs at a ratio of 10:1 in vitro for 3 days.The rats were anesthetized with isoflurane(2?5%in 02 delivered at 2 L/min).The spleen was taken and the isolated cells were ground in a nylon mesh.Cells were adjusted to a concentration of 2×107 cells/mL.The rats received administration of lymphocytes co-cultured with HCB-SCs through tail vein injection at a final dose of 1×107 cells/500 ?L saline once at 2 hrs and 24 hrs after reperfusion.The rats in the tMCAO group received saline injection without lymphocytes.An immunosuppressant was not given to the rats.4.Neurological Deficits MeasurementBederson's Scale Scores-The behavioral assay was scored using a modified Bederson's test.At 48 hrs after reperfusion,the rats were examined by suspending 20?30 cm above the testing platform.The neurological deficits were scored according to the following criteria:0,rat extends both forelimbs straight;1,rat keeps one forelimb to the breast and extends the other forelimb straight;2,rat shows decreased resistance to a lateral push without circling;3,rat shows decreased resistance to a lateral push with circling;and 4,rat loses spontaneous walking and consciousness.Rats scoring 0 or 4 were not used in the evaluation of this experiment.Tape Removal Test-The tape removal test was performed to assess forepaw sensorimotor deficits at 48 hrs after tMCAO.Rats were placed into a box(60 cm×50 cm).Two adhesive tapes with equal length(113 mm2)were quickly attached at the distal radial region of 2 forelimbs;then,the rats were placed into the box.We took and recorded the time that for each animal of removing the tapes from each forelimb.A rat was tested for three times,and the median was taken for statistics.If an animal could not remove the tapes within 180s,then time was noted as 180s.Rats were trained before surgery for 3 days.5.Measurement of Infarct VolumeAfter 48 hrs of reperfusion,the rats were deeply anesthetized with 10%chloral hydrate.The brains were removed and sliced into 2.0 mm thick coronal sections.The brain slices were immediately incubated in 0.9%saline with 2%triphenyl tetrazolium chloride(TTC,Solarbio,China)at 37? for 30 min.Then the slices were transferred into 4%paraformaldehyde solution and fixed for 12 hrs.The photographs were taken by the scanner.Areas of the infarct regions were analyzed in Image-Pro Plus 6.0.The infarct areas of each section were the average of the sum of two sides.The volume of infarction for each animal was calculated by taking the product of the average slice thickness(2 mm)and summing the infarct areas in all brain slices.The results were represented as the percentage of the total volume.6.Flow CytometryPeripheral blood was harvested in tubes with heparin sodium through angular veins.After centrifugation at 40 C(1000 rpm,30 min),the supernatant plasma was collected and stored at-80? for further detection.For flow cytometry,the resuspended lymphocytes were isolated byrat lymphocyte separation medium(Solarbio,China)according to the manual.After washing,the cells were incubated with antibodies at 4? for 30 min as recommended by the manufacturer.The antibodies included antirat FITC-conjugated CD4,anti-rat AF647-conjugated CD25 and anti-rat PE-conjugated Foxp3.These antibodies were purchased from BioLegend Inc.Cell fixation and staining methods referred to the manufacturer's instruction.All samples were analyzed by a flow cytometer(Beckman CytoFLEX)using FlowJo VX software.7.TUNEL StainingTUNEL staining was performed using a situ one-step immunofluorescence TUNEL apoptosis assay kit(Beyotime Biotechnology,China)according to the manufacturer's instructions.Cy3-labeled TUNEL positive cells were observed under fluorescence microscope(Olympus IX710 Camera,Japan).Red fluorescent cells were defined as apoptotic cells.For quantification of apoptotic cells,three slices of the ischemic cerebral cortex in each animal were analyzed.The number of apoptosis cells in five fields of each slice at 200 × magnification was measured using the Image Pro Plus software.8.Western BlottingBrain samples(n=4 for each group)were separated from the cortex including both the penumbra and ischemic core regions,and protein was extracted with RIPA lysis buffer(Beyotime Biotechnology)containing 1%protease inhibitors(sodium fluoride)and 1%phosphatase inhibitors(sodium ortho vanadate).Protein concentration was determined using a BCA protein assay kit.The protein from each sample was separated on 10%sodium dodecyl sulfate polyacrylamide gel electrophoresis gels,then transferred onto a PVDF membrane.The membrane was incubated in blocking buffer for 1 hr at room temperature,and was then incubated overnight at 4? with primary antibodies against NF-?B p-P65(Cell Signaling),NF-?B P65(Cell Signaling),p-ERK(Cell Signaling),ERK(Cell Signaling),NLRP1(Cell Signaling),NLRP3(Cell Signaling),caspase-1(Abcam),IL-1?(Abcam),Cleaved caspase-3(Cell Signaling),and?-actin(Cell Signaling).After washing three times(5 min per wash)with Tris-buffered saline,the membrane was incubated with secondary antibodies against the primary antibody for 1 hr at room temperature.The membrane was washed with Tris-buffered saline and scanned using the gel imaging system(Protein Simple,FluorChem Q).Quantification of protein levels was achieved by densitometry analysis using Image Pro Plus 6.0 software.In detail,the densitometry of p-P65 NF-?B and p-ERK(MAPK)signaling proteins divided by the densitometry of its corresponding"total protein" indicated the activation of NF-?B and ERK signaling.The densitometry of proteins caspase-1,mature IL-1?,NLRP3 inflammasome,NLRP1 inflammasome and cleaved caspase-3 was determined by the densitometry of its corresponding ?-actin,which was used as a loading control.9.Statistical Analysis Data analysis was performed using the GraphPad Prism 6 software.The data were represented as mean ± SEM.Statistical analysis of the data was performed using oneway ANOVA followed by Student-Newman-Keuls post hoc comparisons.P<0.05 weas considered significant.Results1.The Lymphocytes Transplantation(LT)Treatment Alleviates Neurological DeficitsNeurological deficits were assessed by two observers in a blind manner at 48 hrs after reperfusion.Conformity assessment certificated that there was no significant difference in behavior scores of the experimental animals at 2 hrs after the tMCAO operation.At 48 hrs after reperfusion,the rats received tMCAO operation showed severe neurologic impairment,relative to rats in the Sham group(P<0.05,Figure 1A).The transplantation of lymphocytes co-cultured with HCB-SCs(LT)treatment significantly reduced the neurology deficits score in tMCAO rats at 48 hrs after reperfusion(P<0.05,Figure 1A).The sensorimotor function of rats was assessed by tape removal test following neurology deficits score.As shown in Figure 1B,the tape removal latency time in the Sham group was 9.9±0.924 s at 48 hrs after sham operation.The tape removal latency time increased significantly in the tMCAO(139.3±9.996 s)and tMCAO+LT(103.7± 11.87 s)groups,respectively,at 48 hrs after ischemia and reperfusion operation,compared with that in the Sham group(P<0.05).LT treatment significantly decreased the latency time of tMCAO rats(P<0.05).2.LT Treatment Reduces Infarct VolumeInfarct volume was calculated using TTC-stained brain sections.Representative images of TTC staining of experimental rats are shown in Figure 2A.Compared with rats in the Sham group,rats in the tMCAO group showed a significantly larger infarct volume at 48 hrs after reperfusion(P<0.05,Figure 2B).The rats in the tMCAO+LT group exhibited a significant reduction of infarct volume,compared to rats in the tMCAO group(P<0.05,Figure 2B).3.LT Treatment Modulates the Differentiation of Tregs SubsetsIn order to evaluate the effects of LT treatment on the immune response of tMCAO rats,we separated monocytes from peripheral blood and analyzed the proportion of CD4+CD25+and CD4+CD25+Foxp3+Tregs by flow cytometry.As shown in Figure 3A and C,we observed that the percentage of CD4+CD25+ Tregs significantly increased at 48 hrs after tMCAO(P<0.05),and LT treatment did not change the percentage of CD4+CD25+Tregs in tMCAO rats.The proportion of CD4+CD25+Foxp3+Tregs was markedly increased at 48 hrs after tMCAO in the tMCAO group relative to that in the Sham group,and LT treatment further increased the percentage of CD4+CD25+Foxp3+Tregs in tMCAO rats(P<0.05,Figure 3B and D).4.LT Treatment Alleviates the Inflammasome ActivityIn this study,we investigated the effect of LT treatment on immune-inflammatory activity,and the expression of several inflammasome proteins(caspase-1,mature IL-1?,NLRP1 and NLRP3)was detected in brain tissues(cerebral cortex)following 48 hrs of tMCAO.Western blot results demonstrated that protein levels of caspase-1,mature IL-1?,NLRP1 and NLRP3 were significantly increased at 48 hrs after tMCAO,compared with that in the Sham group(P<0.05,Figure 4A-D).LT treatment could remarkably reduce the protein levels of caspase-1,mature IL-1?and NLRP3 in tMCAO rats(P<0.05,Figure 4A-C).LT treatment had no significant effect on the expression of NLRP1 in tMCAO rats(P>0.05,Figure 4D).5.LT Treatment Modulates the Immune Response by ERK and NF-?B Signal PathwayWe investigated the effect of LT treatment on the activation of intracellular NF-?B and ERK signaling pathways.Our results demonstrated that the ratio of p-ERK/total ERK and p-NF-KB(P65)/total NF-?B(P65)were significantly increased in the tMCAO group than that in the Sham group(P<0.05,Figure 4E and F).LT treatment reduced the ratio of p-ERK/total ERK and p-NF-KB(P65)/total NF-?B(P65)in tMCAO rats(P<0.05,Figure 4E and F).The results suggested that LT treatment could reduce the phosphorylation of NF-?B(P65)and ERK,and inhibit the activation of ERK and NF-?B signaling pathways.6.LT Treatment Protects Neurons Against tMCAO-Induced ApoptosisTo elucidate the anti-apoptotic effect of LT treatment,the expression level of cleaved caspase-3 was determined by Western blot.The result showed that the protein level of cleaved caspase-3 in the tMCAO group was higher than that in the Sham group(P<0.05,Figure 4G).LT treatment could significantly decrease the level of cleaved caspase-3 in tMCAO rats(P<0.05,Figure 4G).To further study the effect of LT treatment on neuron apoptosis,TNEL staining was performed to analyze the number of apoptotic cells in ischemic cerebral cortex.The results showed that the number of apoptosis cells in cortex were markedly increased in mice with tMCAO compared with that with Sham operation(P<0.05,Figure 5).LT treatment significantly decreased the number of apoptosis cells in ischemic cerebral cortex(P<0.05,Figure 5),suggesting that the transplantation of lymphocytes co-cultured with HCB-SCs might protect neurons against tMCAO induced apoptosis.ConclusionOur study has proved that HCB-SCs can regulate the proportion of Tregs and modulate the immune inflammatory response.Therefore,the study of transplantation of lymphocytes co-cultured with HCB-SCs can explore a new approach for brain protection treatment for ischemic stroke.Our research shows that:1.The transplantation of lymphocytes co-cultured with HCB-SCs could improve the neurological deficit symptoms of ischemic stroke in tMCAO rats and reduce the cerebral infarct volume.2.The transplantation of lymphocytes co-cultured with HCB-SCs could increase the proportion of CD4+CD25+Foxp3+Tregs in peripheral blood of tMCAO rat model and inhibit the inflammatory response after ischemic stroke.3.The transplantation of lymphocytes co-cultured with HCB-SCs could regulate the inflammatory response in the brain after ischemic stroke by inhibiting the activation of NLRP3 inflammasome signaling pathway and the production of IL-1? in tMCAO rats.4.The transplantation of lymphocytes co-cultured with HCB-SCs could play a neuroprotective role in alleviating the apoptosis by inhibiting the production of apoptotic protein caspase-3 in the ischemic brain tissues of tMCAO rats.In summary,the transplantation of lymphocytes co-cultured with HCB-SCs has the potential to intervene the changes of neuroinflammatory response after cerebral ischemia in tMCAO rats,which is a new approach for neuroprotective treatment after ischemic stroke.