创伤弧菌LuxS对其所致急性肺损伤小鼠肺组织固有免疫细胞稳态的影响  

Effects of Vibrio vulnificus LuxS on the homeostasis of murine pulmonary innate immune cells during acute lung injury

作　　者：林皓楠 蒋烨琳 施晓锋 唐露[3] 陈筑[4] 黄显辉[1] 楼永良[1] 谢旦立[1] Lin Haonan;Jiang Yelin;Shi Xiaofeng;Tang Lu;Chen Zhu;Huang Xianhui;Lou Yongliang;Xie Danli(Key Laboratory of Laboratory Medicine,Ministry of Education of China,School of Laboratory Medicine and Life Science,Wenzhou Medical University,Wenzhou 325035,China;Department of Laboratory Medicine,Traditional Chinese Medicine Hospital of Changxing,Changxing 313100,China;Department of Urology,the Third Medical Center of Chinese PLA General Hospital,Beijing 100039,China;Department of Laboratory Medicine,Ningbo No.2 Hospital,Ningbo 315000,China)

机构地区：[1]温州医科大学检验医学院,生命科学学院,检验医学教育部重点实验室,温州325035 [2]长兴县中医院检验科,湖州313100 [3]中国人民解放军第三医学中心泌尿外科医学部,北京100039 [4]宁波市第二医院检验科,宁波315000

出　　处：《中华微生物学和免疫学杂志》2025年第3期214-222,共9页Chinese Journal of Microbiology and Immunology

基　　金：国家重点研发计划(2021YFC2300300);浙江省自然科学基金(LY22H190002);浙江省医药卫生科技计划项目(2022RC045)。

摘　　要：目的探讨创伤弧菌群体感应蛋白LuxS介导的肺部固有免疫细胞稳态改变对脓毒症所致的急性肺损伤的影响。方法构建luxS基因敲除创伤弧菌菌株及回补菌株,使用分光光度计检测创伤弧菌野生株、luxS敲除株和luxS回补株在液体培养基中生长至600 nm处的吸光度所需的时间;利用1×10^(5)CFU的创伤弧菌野生株、luxS敲除株、luxS回补株腹腔注射感染铁过载小鼠,根据脓毒症呼吸困难临床评分评价小鼠呼吸质量;感染7 h后,HE染色观察小鼠肺组织病理变化,测定肺组织菌载量,并对肺组织单细胞悬液进行流式细胞染色;利用统一流形近似与投影(uniform manifold approximation and projection,UMAP)对PBS对照组和不同菌株感染组的小鼠肺部CD45+免疫细胞分布进行降维分析;使用多色流式细胞术检测上述各组肺组织中固有免疫细胞比例和数量变化情况。采用单因素方差分析和t检验进行统计学分析。结果创伤弧菌野生株、luxS敲除株和luxS回补株在液体培养基中的生长速度无差异。与野生株和回补株比较,luxS敲除株感染后,小鼠整体呼吸困难程度减轻,且肺组织炎症细胞浸润和细菌载量也有所下降(P<0.05),而回补菌株感染组与野生株感染组结果相似。UMAP结果显示,与luxS敲除株感染组比较,野生株和回补株感染组小鼠肺组织中均出现中性粒细胞和嗜酸性粒细胞比例的增多。多色流式细胞术结果进一步验证luxS敲除株感染后,小鼠中性粒细胞、嗜酸性粒细胞比例较野生株和回补株感染组均显著下降(P<0.01和P<0.0001),肺泡巨噬细胞比例较野生株和回补株感染组显著上升(P<0.01)。结论在创伤弧菌感染过程中,LuxS可能通过影响宿主肺部中性粒细胞、嗜酸性粒细胞和肺组织常驻巨噬细胞的稳态进而促进肺部的急性损伤。ObjectiveTo investigate the effects of Vibrio vulnificus(Vv)quorum-sensing protein LuxS on the homeostasis of pulmonary innate immune cells in sepsis-induced acute lung injury.MethodsThis study constructed luxS knockout and complemented Vv strains.The time required for wild type,luxS knockout,and complemented Vv strains to grow to an absorbance of 600 nm in liquid medium was measured using a spectrophotometer.Iron-overloaded mice were intraperitoneally infected with 1×10^(5)CFU of the above three kinds of Vv strains,respectively.Clinical scoring for sepsis-induced dyspnea was used to evaluate the respiratory quality in mice.At 7 h after infection,the pathological changes in lung tissues were observed by HE staining;the bacterial loads in lung tissues were measured;the single-cell suspension of lung tissues was analyzed by flow cytometry.Uniform manifold approximation and projection(UMAP)was used to reduce the dimension of the distribution of CD45+immune cells in lung tissues of mice in the PBS control group and infection groups with different strains.The frequency and absolute number of innate immune cells in lung tissues were analyzed by multicolor flow cytometry.One-way analysis of variance and t test were used for statistical analysis.ResultsThere was no significant difference in the growth rate of wild type,luxS knockout,and complemented Vv strains in liquid medium.Compared with the mice infected with the wild type or complemented strain,the mice infected with the luxS knockout strain exhibited overall alleviated respiratory difficulty,decreased inflammatory cell infiltration in lung tissues,and reduced bacterial load in lung tissues(P<0.05).Besides,there was no significant difference in clinical respiratory scores,inflammatory cell infiltration,or bacterial loads between the mice infected with the complemented strain and wild type strain.UMAP analysis showed that compared with the mice infected with the luxS knockout strain,the mice infected with the wild type or complemented strain showed increased proport

关 键 词：创伤弧菌 群体感应 LUXS 脓毒症 急性肺损伤 

分 类 号：R563[医药卫生—呼吸系统]