Predictive Factors Of Stroke-associated Pneumonia And Macrophage M2 Polarization In Stroke-induced Immunodepression

Background: Stroke-associated pneumonia(SAP)is the common complication after acute stroke and leads to poor prognosis,so the early diagnosis and treatment of SAP is essential.Many studies have explored the risk factors of SAP,and some indicated that some routine examinations on admission,which could reflect the inflammatory response and the immune function of the patients such as neutrophil lymphocyte ratio(NLR),albumin,globulin,hemoglobin(HGB),high-sensitivity C-reactive protein(hs-CRP),may also be related to SAP risk.The function of the immune system plays an important role in the pathogenesis of SAP and the stroke-induced immunodepression is mainly manifested as the dysfunction of immune cells and the massive release of anti-inflammatory cytokines.Therefore,cytokines reflecting peripheral immune function status could be used as new biological markers for SAP.Up to now,the mechanism of the stroke-induced immunodepression is still unclear.Studies suggested that the inhibition of immunocyte function by adrenaline acting on the β2-adrenergic receptor(β2AR)of the immunocytes was the main mechanism of immunosuppression after stroke.It is known that monocyte-macrophages play an important role in SAP,and some researchers suggested that the immunodepression after stroke may also be related to the anti-inflammatory transformation of macrophages,however,the specific molecular mechanism remains unclear and deserves further study.This study was divided into three parts.Part Ⅰ Risk factors of stroke-associated pneumoniaObjective: To analyze the risk factors of SAP and to investigate the relationship between NLR,albumin globulin ratio(A/G),HGB,hs-CRP,meteorological factors at admission and SAP in patients with acute ischemic stroke.Methods: The basic information,clinical data,laboratory indexes at admission(neutrophil and lymphocyte count,A/G,hs-CRP,HGB,etc.)of 2032 patients with acute ischemic stroke were collected.At the same time,the meteorological data(daily average temperature,air pressure,relative humidity,wind speed and air quality index)were provided.Cold spell was defined by the daily average temperature.Results: 288(14.2%)patients with acute ischemic stroke were diagnosed as SAP after admission.Multivariate logistic regression analysis showed that the increased levels of NLR(OR=1.58,95% CI: 1.33-1.87)and hs-CRP(OR=1.89,95%CI: 1.56-2.25)and the decreased levels of A/G(OR=0.73,95% CI: 0.62-0.87)and HGB(OR=0.99,95% CI: 0.98-0.99)at admission were independent risk factors for SAP.The area under the ROC curve(AUC)of NLR was 0.746,and the cut-off value was 4.64,and the sensitivity and specificity was 0.585 and 0.821 respectively.The AUC of hs-CRP was 0.769,and the cut-off value was 15mg/L,and the sensitivity and specificity was 0.642 and 0.789 respectively.The AUC of A/G was 0.683,and the cut-off value was 1.59,and the sensitivity and specificity was 0.671 and 0.637 respectively.The diagnostic efficiency of HGB for SAP was low(AUC=0.583).Other factors including age,hyperlipidemia,stroke history,smoking,dysphagia,unconsciousness,insular infarct,NIHSS score and A2DS2 score at admission were all independent risk factors for SAP(OR>1).Winter was also a risk factor for SAP(OR=1.68,95% CI: 1.14-2.50).While cold wave had no effect on SAP,the general linear model showed that cold spell was associated with increased systolic blood pressure(β=7.34,95 % CI: 3.20-11.49),diastolic blood pressure(β=3.61,95 % CI:0.91-6.32),white blood cell count(β=0.75,95 %CI: 0.27-1.23)and decreased thrombin time(β=-0.62,95 % CI:-0.97--0.27)at admission.Conclusion: The levels of NLR,hs-CRP,A/G and HGB at admission were related to the incidence of SAP in patients with acute ischemic stroke.These indicators cost low and easy to implement which deserves attention in clinical practice.Seasons are also associated with SAP.The effects of cold wave on blood pressure,inflammation and coagulation system may be the potential biological mechanism underlying the cerebrovascular effects of exposure to extreme temperatures.Part Ⅱ The association between peripheral IL-10 and IL12 levels and stroke-associated pneumoniaObjective: To determine the levels of peripheral anti-inflammatory cytokine IL-10 and pro-inflammatory cytokine IL-12 in patients with acute ischemic stroke,to explore the peripheral immune status of the patients and analyze their relationship with the onset and prognosis of SAP and find new biological markers for SAP.Methods: The basic information,medical history data,neurological impairment at admission and discharge were collected among 98 patients with acute ischemic stroke.Venous blood was collected within 24 hours after admission.Plasma IL-10 and IL-12 levels were determined by enzyme-linked immunosorbent assay.Results: 17(17.3%)patients were diagnosed as SAP after admission.In the SAP group,the peripheral level of IL-10 was higher and the level of IL-12 was lower at admission.Multivariate logistic regression showed that the level of IL-10(OR=1.11,95% CI: 1.04-1.18),IL-12(OR=0.89,95% CI: 0.81-0.97)and IL-10/IL-12(OR=3.11,95% CI: 1.65-5.86)were independent risk factors for SAP.The area under the ROC curve(AUC)of IL-10 was 0.804,and the cut-off value was 11.76pg/ml,and the sensitivity and specificity was 0.706 and 0.827 respectively.The AUC of IL-12 was0.773,and the cut-off value was 11.01pg/ml,and the sensitivity and specificity was0.778 and 0.647 respectively.The AUC of IL-10/IL-12 was 0.888,and the cut-off value was 1.95,and the sensitivity and specificity was 0.765 and 0.938 respectively.And patients with higher discharge NIHSS score had lower IL-12 level at admission.Conclusion: The peripheral immune function of patients with SAP was inhibited in early stage.The levels of IL-10,IL-12,and IL-10/IL-12 indicating immunodepression might be a useful biomarker to predict SAP.Part Ⅲ Adrenaline promoted the M2 regulatory macrophages throughβ2AR-β-arrestin2-NFκB pathway in stroke-induced immunodepressionObjective: To explore the M2 polarization of macrophages induced by adrenaline after acute stroke and the underlying mechanism.Methods: The cultured macrophages were treated with adrenaline and lipopolysaccharide(LPS)to establish an in vitro cell model of neural immune regulation after acute stroke,and the effect of adrenaline on the phenotypic transformation of macrophages was observed.Then macrophages were treated withβ2AR agonists and inhibitors.Macrophage polarization marker IL-10 was detected by immunofluorescence and the m RNA levels of IL-10,IL-12 and CD206 were detected by Q-PCR.The protein levels of downstream β-arrestin2 and NFκB pathways were detected by western-blot to determine whether adrenaline regulated immune function through β2AR.Cells were then treated with β-arrestin2 si RNA and NFκB inhibitor to explore the downstream pathways of the effects of adrenaline on macrophage phenotype transformation.Results: Adrenaline inhibited the expression of inflammatory cytokine IL-12 induced by LPS,and up-regulated the expression of the m RNA levels of anti-inflammatory molecules such as IL-10,IL-1RA and CD206 in concentration and time dependent manner.Adrenaline and β2AR agonists promoted the M2 polarization of macrophages which was manifested as the up-regulation of IL-10,CD206 and the down-regulation of IL-12,up-regulated the downstream β-arrestin2 and inhibited IκBα phosphorylation and NFκB pathway activation.The antagonist of β2AR ICI118551 could reverse the M2 polarization of macrophages induced by adrenaline,as well as the up-regulation of the downstream β-arrestin2 and the inhibition of NFκB pathway.The knocking down of β-arrestin2 reversed the M2 polarization of macrophages and the inhibition of downstream IκBα phosphorylation which was manifested as the as the up-regulation of IL-2,the down-regulation of IL-10 and CD206 and the up-regulation of the protein level of p-IκBα,p-NFκB and NFκB.The IκBα phosphorylation inhibitor sauchinone also inhibited the expression of inflammatory cytokines induced by LPS and promoted the expression of anti-inflammatory molecules in macrophages.Conclusion: Adrenaline acting on β2AR promoted the M2 polarization of macrophage through the downstream β-arrestin2.The inhibition of IκBαphosphorylation and NFκB pathway activation by β-arrestin2 may be one of the mechanisms of the anti-inflammatory transformation of macrophage induced by adrenaline.