The Effect Of Adrenergic-triggered NF-?B Pathway In Monocyte Dysfunction After Stroke And Its Related Mechanism

Part 1 Immunological potential predictive indicators for stroke associated infectionBackground and Objective: Stroke associated infection(SAI)is one of the most common complications of acute stroke.And,the contribution of stroke-induced immunosuppression on SAI has received increasing attention.The aim of this study is to systematically evaluate the difference of plasma immunological indicator(s)levels between SAI patients and no-infection stroke patients,in order to explore the immunological potential predictive indicators for SAI.Methods: A systematic search for eligible clinical studies was applied to pool the differences in peripheral cytokine levels between infection and no-infection stroke patients.The standardized mean difference(SMD)was calculated using meta-analysis,and a subgroup analysis was conducted based on time-points after stroke onset.Results: Eleven articles involving 781 patients were identified for the pooled analyses.SAI patients had significantly higher IL-6 and IL-10 levels and lower HLA-DR levels than no-infection stroke patients within one week after stroke onset.However,there were no significant differences in TNF-? and IFN-? levels between SAI and no-infection stroke patients.The results of subgroup analysis were in agreement with the total results except a significant difference of IFN-? expression between the subgroup of day 7 and total group.The Egger's test showed no significant publication bias among the included studies.Conclusion: IL-6,IL-10 and HLA-DR are potential candidate indicators for SAI prediction.Monocyte dysfunction may be an important contributing factor of SAI.Part 2 Effects of the adrenergic signaling in the immune activity of monocytesBackground and Objective: Monocyte dysfunction contributes to the occurrence of SAI after stroke,although the underlying mechanism remains largely elusive.Studies demonstrated that the adrenergic signaling is hyperactivated after stroke,which results in SAI.Hence,we aim to explore the effects of the adrenergic signaling in monocyte dysfunction in-vitro.Methods: Human monocytes line THP-1 was applied as experimental subject,and was stimulated by lipopolysaccharide(LPS)for immune activation.Cytokine levels were detected by ELISA analysis after norepinephrine(NA)interventions at different concentrations to select the minimum effective concentration for follow-up experiments.After that,NA and propranolol(PROP)was applied to simulate sympathetic pathway activation or not.The immune activation of monocytes was assessed by the detection of cytokine expression using ELISA and RT-qPCR and the detection of HLA-DR expression using flow cytometry.Results: NA inhibited LPS-induced pro-inflammatory cytokine expression with concentration,and 100?M was its lowest effective concentration.100?M of NA significantly decreased LPS-induced pro-inflammatory cytokines and HLA-DR expression as well as increased anti-inflammatory cytokine IL-10 expression.However,PROP significantly reversed this effect of NA.Conclusion: The adrenergic signaling plays an important role in modulating monocyte dysfunction.Part 3 Monocyte dysfunction after stroke and its adrenergic signaling pathwayBackground and Objective: Stroke-induced hyperactivated adrenergic pathway facilitates immunodepression of lymphocytes and NK cells.However,it remains poorly understood whether the adrenergic pathway modulate monocyte dysfunction after stroke.Thus,we aim to investigate the effect of the adrenergic pathway in monocyte dysfunction after stroke in vivo.Methods: Transient models of middle cerebral artery occlusion(MCAO)were established and three groups(Sham + Saline,MCAO + Saline and MCAO + PROP)were randomly assigned.Three days after MCAO,neurological deficit scores and infarct volume were tested to assess neurological outcome.And,monocytes / macrophages were isolated from spleen to detect the expression of cytokines and MHC-? after LPS stimulation.Results: MCAO establishment significantly increased infarct volume and neurological deficit.Blockade of adrenergic-activity by PROP markedly improved MCAO-induced neurological results.On the other hand,MCAO establishment decreased LPS-induced pro-inflammatory cytokines and HLA-DR expression as well as increased IL-10 expression.PROP significantly reversed the alteration in spite of MCAO establishment.Conclusion: Stroke induces peripheral monocyte dysfunction via the the adrenergic pathway.Part 4 Monocyte dysfunction after stroke and its adrenergic signaling pathwayBackground and Objective: The underlying mechanism of adrenergic-triggered monocyte dysfunction is largely elusive.?-arrestin2(ARRB2)is a signal transducing protein downstream of G protein coupled receptors(GPCRs).And,the NF-?B pathway modulates the expression of various immunological factors.Hence,we aim to investigate the effect of adrenergic activation in ARRB2 expression and NF-?B pathway activity in monocytes.Methods: MCAO models were established and three groups(Sham + Saline,MCAO + Saline and MCAO + PROP)were randomly assigned.Three days after MCAO,Splenic monocytes / macrophages were isolated to detect ARRB2 expression and NF-?B pathway activity.In vitro,THP-1 cells were stimulated by NA and PROP to simulate adrenergic activity or not.After immune activation by LPS,ARRB2 expression and NF-?B pathway activation were detected by western blot and RT-qPCR.Results: MCAO establishment significantly increased ARRB2 expression as well as decreased LPS-induced NF-?B pathway activation.PROP significantly reversed the alteration in spite of MCAO establishment.In vitro,adrenergic activity increased ARRB2 expression as well as decreased LPS-induced NF-?B pathway activation.Also,PROP significantly reversed the alteration in spite of adrenergic activity.Conclusion: Adrenergic activity increased monocytic ARRB2 expression as well as decreased LPS-induced NF-?B pathway activation in monocyte after stroke.Part 5 ?-arrestin2 is a key regulator in the adrenergic-triggered monocyte dysfunctionBackground and Objective: Adrenergic activity increased monocytic ARRB2 expression,and decreased LPS-induced NF-?B pathway activation and pro-inflammatory cytokine expression.However,it remains unclear whether ARRB2 is a key regulator in the adrenergic-triggered monocyte dysfunction.Hence,we aim to explore the effect of ARRB2 in the adrenergic-triggered monocyte dysfunction and NF-?B pathway inactivation.Methods: siRNAs were applied for the deficiency of ARRB2 in vivo.After that THP-1 cells were stimulated by NA and LPS to simulate adrenergic activity and immune activation.ARRB2 expression and NF-?B pathway activation were detected by western blot and RT-qPCR.And,cytokine expression was detected by ELISA and RT-qPCR.Results: ARRB2 expression in both protein and gene levels were dramatically decreased.Deficiency of ARRB2 significantly repealed adrenergic-triggered inactivation of NF-?B pathway.Moreover,deficiency of ARRB2 signally reversed adrenergic-triggered decrease of pro-inflammatory cytokine(IL-6,TNF-?,IL-1?)levels and increase of anti-inflammatory cytokine(IL-10)level.Conclusion: ARRB2 is implicated in the adrenergic-triggered monocyte dysfunction.