BTLA在重症社区获得性肺炎患者中的表达及糖皮质激素干预动物模型的影响
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摘要
目的本研究通过观察BT淋巴衰减子(B and T lymphocyte attenuator,BTLA)在重症CAP患者外周血淋巴细胞及支气管黏膜上的表达,外源性干预LPS-诱导的急性肺部炎症小鼠模型BTLA表达对肺部炎症的作用以及给予糖皮质激素对BTLA表达的影响。方法利用流式细胞学分析重症CAP患者外周血BTLA~+CD4~+淋巴细胞比例,另外对重症CAP患者支气管黏膜常规行HE染色和免疫组织化学检测BTLA表达情况。利用流式细胞学分析LPS诱导的急性肺部炎症模型右心血BTLA~+CD4~+淋巴细胞比例。收集肺泡灌洗液(bronchoalveolar lavage fluid,BALF)行炎症细胞分类计数,并利用ELISA检测炎症因子。取肺组织分别行HE染色、免疫组织化学染色和蛋白印迹。结果流式细胞学分析重症肺炎患者外周血中BTLA~+CD4~+淋巴细胞比例明显高于正常对照组。此外,支气管黏膜HE染色结果显示重症CAP患者支气管黏膜有大量炎症细胞浸润,并且伴有支气管壁增厚;进一步用免疫组织化学检测支气管黏膜BTLA表达,结果提示重症肺炎患者支气管黏膜BTLA表达明显增高,浸润的炎症细胞BTLA表达增高明显。流式细胞学分析LPS诱导的急性肺部炎症模型小鼠右心血中BTLA~+CD4~+淋巴细胞比例明显高于正常对照组。利用激动性抗-BTLA抗体6A6干预急性肺部炎症模型后淋巴细胞BTLA表达较模型小鼠明显增高,同时,激动性抗-BTLA抗体6A6能减轻肺部炎症反应和抑制NF-κB信号通路激活。利用糖皮质激素干预急性肺部炎症模型后淋巴细胞BTLA表达较模型小鼠明显增高,糖皮质激素能减轻肺部炎症反应和抑制NF-κB信号通路激活。结论通过本研究我们推测BTLA信号可能参与了重症CAP患者的免疫调节过程,BTLA可能成为重症CAP治疗的一个新靶点。同时,BTLA的表达可能反应患者机体的免疫状态,为临床使用糖皮质激素治疗提供指导作用。
In this study, we investigated potential changes in B and T lymphocyte attenuator(BTLA) expressedon lymphocytes and bronchial mucosa in patients with severe community acquired pneumonia(CAP), and toevaluate the effects of BTLA on a model of LPS-induced acute lung inflammation. The ratio of BTLA~+CD4~+lymphocytes in human subject was analyzed by flow cytometry; HE staining and immunohistochemical staining wereemployed to detecl the BTLA in bronchial mucosa from patients with severe CAP. The percentages ofcirculating BTLA~+CD4~+lymphocytes were determined in an LPS-induced acute lung inflammation model.Bronchoalveolar lavage fluid(BALF) was collected from mice and analyzed for leukocyte counts by enzyme-linkedwere collected and assessed via Western blotting,immunohistochemistry and HE staining. Dataowedthat the percentage of circulating BTLA~+ shCD4~+lymphocytes in patients with severe CAP wassignificantly higher than that in control groups; HE staining showed that there were inflammatory cell infiltration inbronchial mucosa and submucosa, as well as thickening of bronchial walls in patients with severe CAP.Immunohistochemistry for BTLA in bronchial mucosa showed that BTLA protein expression was upregulated inbronchial epithelium and inflammatory cells of patients with severe CAP. The percentage of circulating BTLA~+CD4~+lymphocytes were significantly higher in the LPS-induced acute lung inflammation model than that in control groups.Agonistic anti-BTLA antibody 6A6 could upregulate BTLA expression significantly in LPS-induced acute lunginflammation mice. Moreover, the agonistic anti-BTLA antibody 6A6 attenuated inflammatory responses and NF-κBpathway activation in model mice. These results demonstrated that BTLA play an important role in immunoregulationof severe CAP, which may be a therapeutic target for the treatment of severe CAP. Meanwhile, BTLA expression mayreflect the body's immune status and guide decisions regarding steroid therapy for treating severe CAP.
In this study, we investigated potential changes in B and T lymphocyte attenuator(BTLA) expressedon lymphocytes and bronchial mucosa in patients with severe community acquired pneumonia(CAP), and toevaluate the effects of BTLA on a model of LPS-induced acute lung inflammation. The ratio of BTLA~+CD4~+lymphocytes in human subject was analyzed by flow cytometry; HE staining and immunohistochemical staining wereemployed to detecl the BTLA in bronchial mucosa from patients with severe CAP. The percentages ofcirculating BTLA~+CD4~+lymphocytes were determined in an LPS-induced acute lung inflammation model.Bronchoalveolar lavage fluid(BALF) was collected from mice and analyzed for leukocyte counts by enzyme-linkedwere collected and assessed via Western blotting,immunohistochemistry and HE staining. Dataowedthat the percentage of circulating BTLA~+ shCD4~+lymphocytes in patients with severe CAP wassignificantly higher than that in control groups; HE staining showed that there were inflammatory cell infiltration inbronchial mucosa and submucosa, as well as thickening of bronchial walls in patients with severe CAP.Immunohistochemistry for BTLA in bronchial mucosa showed that BTLA protein expression was upregulated inbronchial epithelium and inflammatory cells of patients with severe CAP. The percentage of circulating BTLA~+CD4~+lymphocytes were significantly higher in the LPS-induced acute lung inflammation model than that in control groups.Agonistic anti-BTLA antibody 6A6 could upregulate BTLA expression significantly in LPS-induced acute lunginflammation mice. Moreover, the agonistic anti-BTLA antibody 6A6 attenuated inflammatory responses and NF-κBpathway activation in model mice. These results demonstrated that BTLA play an important role in immunoregulationof severe CAP, which may be a therapeutic target for the treatment of severe CAP. Meanwhile, BTLA expression mayreflect the body's immune status and guide decisions regarding steroid therapy for treating severe CAP.
引文
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[2]Tamada K,Shimozaki K,Chapoval AI,et al.LIGHT,a TNF like molecule,costimulates T cell proliferation and is required for dendritic cell-mediated allogeneic T cell response[J].J Immunol,2000,164(8):4105-4110.
[3]Del Rio ML,Lucas CL,Buhler,et al.HVEM/LIGHT/BTLA/CD160 cosignaling pathways as targets for immune regulation[J].J Leukoc Biol,2010,87(2):223-235.
[4]Shui JW,Steinberg MW,Kronenberg M.Regulation of inflammation,autoimmunity,and infection immunity by HVEM-BTLA signaling[J].J Leukoc Biol,2011,89(4):517-523.
[5]Akram AR,Chalmers JD,Taylor JK,et al.An evaluation of clinical stability criteria to predict hospital course in community-acquired pneumonia[J].Clin Microbiol Infect,2013,19(12):1174-1180.
[6]Zhao C,Su EM,Yang X,et al.Important role of platelets in modulating endotoxin-induced lung inflammation in CFTR-deficient mice[J].PLo S One,2013,8(12):e82683.
[7]Kobayashi Y,Iwata A,Suzuki K,et al.B and T lymphocyte attenuator inhibits LPS-induced endotoxic shock by suppressing Toll-like receptor 4 signaling in innate immune cells[J].Proc Natl Acad Sci U S A,2013,110(13):5121-5126.
[8]D′Addio F,Yuan X,Habicht A,et al.A novel clinically relevant approach to tip the balance toward regulation in stringent transplant model.Transplantation,2010,90(3):260-269.
[9]De Pascale G,Bello G,Antonelli M.Steroids in severe pneumonia:a literature review[J].Minerva Anestesiol,2011,77(9):902-910.
[10]Lim WS,van der Eerden MM,Laing R,et al.Defining community acquired pneumonia severity on presentation to hospital:an international derivation and validation study[J].Thorax,2003,58(5):377-382.
[11]Zhao C,Su EM,Yang X,et al.Important role of platelets in modulating endotoxin-induced lung inflammation in CFTR-deficient mice[J].PLo S One,2013,8(12):e82683.
[12]程圆圆,刘健,万磊,等.新风胶囊对膝骨关节炎患者B、T淋巴衰减因子及氧化应激的影响[J].免疫学杂志,2013,29(5):416-421.
[13]Shubin NJ,Monaghan SF,Heffernan DS,et al.B and T lymphocyte attenuator expression on CD4+T-cells associates with sepsis and subsequent infections in ICU patients[J].Crit Care,2013,17(6):R276.
[14]Steinberg MW,Cheung TC,Ware CF.The signaling networks of the herpesvirus entry mediator(TNFRSF14)in immune regulation[J].Immunol Rev,2011,244(1):169-187.
[15]Shui JW,Larange A,Kim G,et al.HVEM signalling at mucosal barriers provides host defence against pathogenic bacteria[J].Nature,2012,488(7410):222-225.
[16]Deppong C,Degnan JM,Murphy TL,et al.B and T lymphocyte attenuator regulates T cell survival in the lung[J].J Immunol,2008,181(5):2973-2979.
[17]Kawai T,Akira S.The role of pattern-recognition receptors in innate immunity:Update on Toll-like receptors[J].Nat Immunol,2010,11(5):373-384.
[18]Seifart C,Dempfle A,Plagens A,et al.TNF-α,TNF-β,IL-6-,and IL-10-promoter polymorphisms in patients with chronic obstructive pulmonary disease[J].Tissue Antigens,2005,65(1):93-100.
[19]Barnes PJ,Karin M.Nuclear factor-κB-a pivotal transcription factor in chronic inflammatory diseases[J].N Engl J Med,1997,336(15):1066-1071.
[20]Patel BV,Wilson MR,O'Dea K P,et al.TNF-induced death signaling triggers alveolar epithelial dysfunction in acute lung injury[J].J Immunol,2013,190(8):4274-4282.
[21]Xing K,Gu B,Zhang P,et al.Dexamethasone enhances programmed cell death 1(PD-1)expression during T cell activation:an insight into the optimum application of glucocorticoids in anti-cancer therapy[J].BMC Immunol,2015,16:39.
[22]da Silva Antunes R,Madge L,Soroosh P,et al.The TNF family molecules LIGHT and lymphotoxinαβinduce a distinct steroid-resistant inflammatory phenotype in human lung epithelial Cells[J].J Immunol,2015,195(5):2429-2441.
[23]Catley MC,Sukkar MB,Chung KF,et al.Validation of the anti-inflammatory properties of small molecule IKK2inhibitors by comparison to adenoviral-mediated delivery of dominant negative IKK1 and IKK2 in human airways smooth muscle[J].Mol Pharmacol,2006,70(2):697-705.
[24]Birrell MA,Hardaker E,Wong S,et al.Ikappa-B kinase-2inhibitor blocks inflammation in human airway smooth muscle and a rat model of asthma[J].Am J Respir Crit Care Med,2005,172(8):962-971.
[25]Newton R,Hart LA,Stevens DA,et al.Effect of dexamethasone on interleukin-1beta-(IL-1beta)-induced nuclear factor-kappa B(NF-kappa B)and kappa Bdependent transcription in epithelial cells[J].Eur J Biochem,1998,254(1):81-89.