SQSTM1/p62/A170 regulates the severity of Legionella pneumophila pneumonia by modulating inflammasome activity

Sequestosome1/A170/p62 (SQSTM1) is a scaffold multifunctional protein involved in several cellular events, such as signal transduction, cell survival, cell death, and inflammation. SQSTM1 expression by macrophages is induced in response to environmental stresses; however, its role in macrophage‐mediated host responses to environmental stimuli, such as infectious pathogens, remains unclear. In this study, we investigated the role of SQSTM1 in host responses to Legionella pneumophila, an intra‐cellular pathogen that infects macrophages, in both an SQSTM1‐deficient (SQSTM1^?/?) mouse model and macrophages from these mice. Compared with wild‐type (WT) macrophages, the production and secretion of the proinflammatory cytokine IL‐1β was significantly enhanced in SQSTM1^?/? macrophages after infection with L. pneumophila. Inflammasome activity, indicated by the level of IL‐18 and caspase‐1 activity, was also elevated in SQSTM1^?/? macrophages after infection with L. pneumophila. SQSTM1 may interact with nucleotide‐binding oligomerization domain‐like receptor family, caspase recruitment domain‐containing 4 and nucleotide‐binding oligomerization domain like receptor family, pyrin domain containing 3 proteins to inhibit their self‐dimerization. Acute pulmonary inflammation induced by L. pneumophila and silica was enhanced in SQSTM1^?/? mice with an increase in IL‐1β levels in the bronchoalveolar lavage fluids. These findings suggest that SQSTM1 is a negative regulator of acute pulmonary inflammation, possibly by regulating inflammasome activity and subsequent proinflammatory cytokine production.     

Toll样受体4抑制过氧化物酶体增殖物激活受体γ加重血脂蓄积的分子机制

目的 选取高脂饮食模型大鼠和氧化低密度脂蛋白(oxLDL)暴露下巨噬细胞模型,验证在血脂蓄积过程中Toll样受体4(TLR4)和过氧化物酶体增殖物激活受体γ(PPARγ)的具体干预机制。 方法 健康雄性Wistar大鼠20只,随机分为对照组和高脂组,每组10只。测定各组大鼠总胆固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白水平,采用苏木精-伊红染色检测颈动脉血管内膜中膜厚度比,采用Western blotting法检测TLR4、PPARγ蛋白表达水平。体外培养小鼠巨噬细胞RAW264.7,以oxLDL(50 mg/L)刺激巨噬细胞制备模型,且应用siRNA-TLR4沉默巨噬细胞内的TLR4因子制备TLR4沉默模型,将细胞分为空白组(A组)、oxLDL组(B组)、oxLDL+siRNA组(C组)、oxLDL+siRNA-TLR4组(D组)、oxLDL+siRNA-TLR4+PPARγ激动剂组(E组)、oxLDL+siRNA-TLR4+PPARγ抑制剂组(F组)。采用油红O染色法观察巨噬细胞内血脂蓄积情况,定量检测巨噬细胞内胆固醇含量,采用Western blotting法检测 TLR4、PPARγ蛋白表达水平。 结果 在动物模型实验中,与对照组相比,高脂组总胆固醇、甘油三酯、低密度脂蛋白水平、颈动脉内膜中膜厚度比、TLR4蛋白相对表达含量明显增高(P<0.01),血清高密度脂蛋白水平、PPARγ蛋白相对表达含量明显降低(P<0.01),且TLR4与PPARγ呈负相关性(r=-0.928 1,P<0.001)。在oxLDL暴露巨噬细胞实验中,与A组比较,B、C组巨噬细胞内胆固醇含量、油红O颗粒、光密度值及TLR4蛋白相对表达含量明显增多(P<0.01),PPARγ蛋白相对表达含量明显减少(P<0.05),且B组TLR4与PPARγ呈负相关性(r=-0.986 7,P<0.001)。与B组相比,C组巨噬细胞内胆固醇含量、油红O颗粒、光密度值及TLR4、PPARγ蛋白相对表达含量未见明显改变(P>0.05)。与B组相比,D组巨噬细胞内胆固醇含量、油红O颗粒及光密度值及TLR4蛋白相对表达含量明显减少(P<0.01),PPARγ蛋白相对表达含量明显增多(P<0.05)。与D组相比,E组巨噬细胞内胆固醇含量、油红O颗粒及光密度值明显减少(P<0.01),PPARγ蛋白相对表达含量明显增多(P<0.05),TLR4蛋白相对表达含量未见明显改变(P>0.05)。与D组相比,F组巨噬细胞内胆固醇含量、油红O颗粒及光密度值明显增多(P<0.01),PPARγ蛋白相对表达含量明显减少(P<0.05),TLR4蛋白相对表达含量未见明显改变(P>0.05)。 结论 细胞内的血脂蓄积是动脉粥样硬化形成的机制之一,PPARγ可以抑制巨噬细胞血脂蓄积进而参与动脉粥样硬化调节,是巨噬细胞血脂蓄积过程的保护性因子。而TLR4作为PPARγ上游调控位点,通过抑制PPARγ的表达,加重血脂蓄积的过程,进而干预动脉粥样硬化进程。     

巨噬细胞的极化分型在慢性阻塞性肺疾病中的研究进展

巨噬细胞是单核巨噬细胞系统的终末分化细胞,由单核细胞分化而来。巨噬细胞通过吞噬作用参与非特异性免疫,在适应性免疫中主要发挥免疫调节及抗原递呈功能,从而影响机体全身代谢、造血、血管生成、凋亡、肿瘤和生殖等多种进程。COPD是一种机体对吸烟或吸入其他有害气体、颗粒物引起的异常炎症反应,病理学定义为持续存在的气流受限。它包括一系列病理过程,如大气道的炎症（慢性支气管炎）和小气道的气道重塑、弹性减退和肺间质的破坏造成的肺气肿。尽管引起炎症和肺部组织损伤的原因没有完全研究清楚,但单核巨噬细胞通过释放炎性介质在引起肺部炎症和肺气肿的过程中起到了关键的作用。巨噬细胞因其所处微环境的不同存在不同的分型,而且这种分型在COPD中也有了一定的进展,但吸烟对于巨噬细胞极化分型的影响仍颇有争议。现就巨噬细胞的极化分型在COPD中的研究进展综述如下。     

Use of chemically extracted muscle grafts to repair extended nerve defects in rats

Nerve regeneration, measured as axonal outgrowth, Schwann cell migration, macrophage invasion, and neovascularisation, was compared after repair of a 15 mm gap in rats' sciatic nerves using autologous muscle grafts made acellular either by freezing and thawing or by chemical extraction. Both extracted and freeze-thawed acellular muscle grafts could be used to bridge the defect. However, axons and Schwann cells, as shown by immunohistochemical staining for neurofilaments and S-100 protein, respectively, grew faster into the extracted muscle grafts than into the freeze-thawed acellular muscle grafts and somewhat more axons were observed in the former graft. There were no significant differences between the two graft types with respect to neovascularisation as showed by staining for endothelial alkaline phosphatase, and limited differences concerning invasion of macrophages (ED1 and ED2) as detected by immunocytochemistry. The results showed that chemically extracted muscle grafts could be used to bridge an extended nerve defect and that such grafts in some aspects were superior to freeze-thawed muscle grafts for extended gaps.     

Serum soluble CD163 levels in patients with influenza-associated encephalopathy

Background: Influenza-associated encephalopathy (IE) is a serious complication during influenza viral infection. Common clinical symptoms of IE include seizures and progressive coma with high-grade fever. We previously reported that hypercytokinemia and monocyte/macrophage activation may play an important role in the pathogenesis of IE. CD163 is a scavenger receptor for hemoglobin–haptoglobin complexes and is expressed by monocytes/macrophages. Proteolytic cleavage of monocyte-bound CD163 by matrix metalloproteinases releases soluble CD163 (sCD163). However, there have been no reports regarding serum sCD163 levels in IE patients. Methods: We measured serum levels of sCD163 as a marker of monocyte/macrophage activation in IE patients with poor outcomes, those without neurological sequelae, influenza patients without IE, and control subjects. Results: Serum sCD163 levels were significantly higher in IE patients with poor outcomes than in those without neurological sequelae. In particular, sCD163 levels in cases of death were significantly higher than those in other cases. Conclusions: Our results suggest that monocyte/macrophage activation is related to the pathogenesis of severe IE.     

桃红四物汤调控巨噬细胞极化对肩袖损伤的机制研究

目的基于白介素(IL)-4/信号传导及转录激活蛋白(STAT)6信号通路调控巨噬细胞极化,利用小鼠动物模型探讨桃红四物汤对肩袖撕裂术后愈合的影响及其作用机制。 方法将50只C57BL/6J小鼠随机分为空白对照组、肩袖损伤组(模型组)及桃红四物汤低、中、高剂量组,每组10只。除对照组外,其他组小鼠均构建肩袖撕裂重建模型。术后预防感染并正常饲养12周,桃红四物汤组小鼠灌胃相应药液,每次每只小鼠灌胃量均为2 ml;对照组每日清晨予等体积生理盐水;连续给药12周,1次/d。末次给药24 h后,进行冈上肌腱生物力学刚度测试;采用酶联免疫吸附测定法(ELISA)检测小鼠血清一氧化氮合酶(iNOS)、IL-6、甘露糖受体(CD206)的含量;采用实时定量PCR(RT-PCR)技术检测小鼠腱骨界面组织IL-1β、肿瘤坏死因(TNF)子-α、IL-10、炎症区域分子1(Fizz1)mRNA表达;采用Western Blot法检测腱骨界面组织iNOS、精氨酸酶-1(Arg-1)、IL-4、STAT6、磷酸化- STAT 6(p-STAT6)蛋白含量。多组间数据比较采用单因素方差分析,组间两两比较采用LSD-t检验。 结果用药干预后与肩袖损伤组比较,桃红四物汤各个剂量组拉断载荷、刚度测试数值上调(F＝64.822、58.431,均为P<0.05);iNOS、IL-6(M1极化标志物)水平下调(F＝82.618、61.372,均为P<0.05),CD206(M2极化标志物)水平上调(F＝58.942,P<0.05);桃红四物汤中、高剂量组IL-1β、TNF-mRNA(M1极化标志物)的表达下调(F＝38.631、33.714,均为P<0.05),而IL-10、Fizz1 mRNA(M2极化标志物)的表达上调(F＝41.731、67.431,均为P<0.05)。桃红四物汤低、中、高剂量组Arg1、IL-4、p-STAT6的蛋白表达上调(F＝26.841、29.750、26.863,均为P<0.05)。 结论桃红四物汤可促进肩袖撕裂小鼠的肌腱愈合,其机制可能与IL-4/STAT6信号通路调控巨噬细胞极化有关。     

氯膦酸二钠脂质体对大鼠重症急性胰腺炎肺损伤的影响及与Akt、MAPK（ERK1/2）通路的关系

目的:探讨氯膦酸二钠脂质体(LC)对大鼠重症急性胰腺炎(SAP)肺损伤的影响及与Akt、MAPK(ERK1/2)通路的关系。方法:将48只SD大鼠随机均分为假手术组、SAP模型组(模型组)、SAP模型+LC处理组(LC组),后两组采用膜下注射5%牛磺胆酸钠制作SAP模型,并分别于造模后尾静脉注射空白脂质体与LC。各组分别于术后2、6h后检测血清淀粉酶(AMS)、IL-6、TNF-α的含量,观察肺组织病理学变化,及肺组织中Akt和MAPK(ERK1/2)的表达。结果:与假手术组比较,模型组与LC组血清AMS、IL-6及TNF-α含量、肺组织病理学评分,肺组织Akt和MAPK(ERK1/2)表达水平均明显升高,且均随时间延长而更加明显(均P0.05),但LC组的上述指标在各时间点上均明显低于模型组(均P0.05)。结论:LC有减轻大鼠SAP肺损伤的作用,机制可能与肺泡巨噬细胞吞噬LC后,Akt和MAPK(ERK1/2)信号通路抑制,从而减少炎症细胞因子的释放有关。     

Antigen presentation and transfer between B cells and macrophages

B cells play an active role in directing immunity against specific proteins in part because of their capacity to sequester antigen via B cell receptor (BCR). Our prior findings indicate that B cells can initiate an immune response in vivo to self proteins independent of other antigen-presenting cells (APC). However, these studies also demonstrated that both dendritic cells and macrophages are important in the ongoing immune response. The present work illustrates a mechanism by which antigen acquired by B cells through BCR is specifically transferred to other APC, in particular, macrophages. The transfer of antigen is dependent on the specificity of BCR and requires direct contact between the cells, but does not require MHC compatibility between the cells and is independent of the activation state of macrophages. Antigen transfer is functional, in that macrophages, which received B cell derived-antigen, can activate CD4 T cells. Overall, these results define a novel mechanism by which B cells can focus immunity toward a specific antigen and transfer the ability to activate CD4 T cells to other APC.     

乳腺癌新辅助化疗前后肿瘤相关巨噬细胞相关基因变化的生物信息学分析

背景与目的 乳腺癌是全球女性发病率最高的恶性肿瘤,化疗是乳腺癌最重要的治疗方式之一,最近的研究表明,化疗可能通过增强肿瘤微环境中的抗肿瘤免疫力来发挥抗肿瘤效应。因此,本研究通过生物信息学分析明确乳腺癌患者新辅助化疗（NAC）前后肿瘤相关巨噬细胞（TAMs）及相关基因的变化,评估NAC对乳腺癌患者免疫影响。方法 GEO数据库输入“Breast Cancer”,“TAMs”,“Chemotherapy”进行检索,选择人乳腺癌组织的GSE134600数据集进行分析。通过R包（limma函数）筛选乳腺癌患者NAC前后组织样本中差异表达基因（DEGs）。对所有DEGs进行GO功能富集和KEGG通路分析。通过Cytoscape软件对DEGs进行蛋白互作网络可视化,并筛选关键核心基因,通过cBioPortal对10个关键基因进行突变分析。使用R包（CIBERSORT）对GSE134600数据中的免疫细胞分布及相关性进行评估。结果 鉴定出751个乳腺癌NAC前后DEGs（409个上调基因和342个下调基因）。通过GO富集分析DEGs的生物过程（BP）、细胞组分（CC）和分子功能（MF）。在BP中主要富集在I型干扰素（IFN-I）信号通路/病毒应答与防御和病毒生命周期方面;在CC中主要富集在细胞膜的外在成分和细胞膜的细胞质侧方面;在MF中主要富集在细胞因子受体结合、双链RNA结合和脂肽结合方面。KEGG通路富集分析中,DEGs主要富集在甲型H1N1流感、麻疹、丙型肝炎、冠状病毒病COVID-19、NF-κB信号通路、EBV病毒感染、NOD样受体信号通路和阿米巴病信号通路。通过CytoHubba插件筛选出乳腺癌NAC前后TAMs相互作用程度最高的前10个关键基因：IFIT1、ISG15、MX1、MX2、IRF7、RSAD2、IFIT3、IFI35、IFI6、IFITM1。多组学分析发现IFIT1、MX1和MX2主要发生缺失突变,IFIT1主要发生基因深度删除,而MX1和MX2主要发生基因扩增。NAC后乳腺癌组织中M0巨噬细胞、CD8⁺T细胞及M2巨噬细胞含量减少,M0巨噬细胞与记忆性B细胞成正相关（r=0.64）,与未活化的CD4⁺记忆性T细胞呈负相关（r=-0.66）。结论 所发现的乳腺癌患者NAC前后TAMs相关的DEGs与干扰素信号通路密切相关,提示干扰素信号通路在NAC可能通过改变TAMs而发挥重要作用。同时NAC前后M0巨噬细胞发生明显改变,提示化疗可能通过改变M0巨噬细胞分布及免疫功能调节对肿瘤的免疫应答。     

Atorvastatin inhibits CXCR7 induction to reduce macrophage migration

We have recently reported that CXCR7, the alternate high affinity SDF-1 receptor, is induced during monocyte-to-macrophage differentiation, leading to increased macrophage phagocytosis linked to atherosclerosis. Statins, the most widely used medications for atherosclerosis, were shown to have pleiotropic beneficial effects independent of their cholesterol-lowering activity. This study aimed to determine whether induction of CXCR7 during macrophage differentiation is inhibited by statins and its significance on macrophage physiology. Here we show for the first time that atorvastatin dose-dependently inhibited CXCR7 mRNA and protein expression in THP-1 macrophages, without affecting the other SDF-1 receptor, CXCR4. Pharmacotherapy relevant dose of atorvastatin affected neither cell viability nor macrophage differentiation. Suppression of CXCR7 expression was completely reversed by supplementation with mevalonate. Inhibition of squalene synthase, the enzyme committed to cholesterol biosynthesis, also decreased CXCR7 induction, albeit not as efficacious as atorvastatin. However, the geranylgeranyl transferase inhibitor, GGTI-286, the farnesyl transferase inhibitor, FTI-276, and the Rho kinase inhibitor, Y-27632, all failed to mimic the effect of atorvastatin, suggesting that the protein prenylation pathways are not critical for atorvastatin inhibition of CXCR7 induction. Interestingly, the dramatic effect of atorvastatin was only partially mimicked by other statins including pravastatin, fluvastatin, mevastatin, and simvastatin. Furthermore, activation of CXCR7 by SDF-1, TC14012, or I-TAC all prompted macrophage migration, which was significantly suppressed by atorvastatin treatment, but not by the CXCR4 antagonist. We conclude that atorvastatin modulates macrophage migration by down-regulating CXCR7 expression, suggesting a new CXCR7-dependent mechanism of atorvastatin to benefit atherosclerosis treatment beyond its lipid lowering effect.