播散性暗色丝孢霉病患者CARD9突变及相关免疫学研究

暗色丝孢霉病是指由暗色真菌引起的皮肤、皮下组织乃至深部组织脏器的感染。本研究探讨1例由皮肤及皮下组织暗色丝孢霉病逐渐进展为播散性暗色丝孢霉病患者的遗传背景及其抗真菌免疫功能。收集患者10余年病情进展的临床资料及真菌检查结果,进行临床资料及真菌学研究;对外周血DNA采用Sanger测序,进行遗传学研究;提取患者及正常人外周血单个核细胞(peripheral blood mononuclear cell,PBMC),采用蛋白免疫印迹法、流式细胞术、化学发光法、酶联免疫吸附试验等进行免疫学研究。结果显示,患者胱天蛋白酶募集域蛋白9(caspase recruitment domain-containing protein 9,CARD9)基因存在新的复合杂合错义突变(p.R35Q和p.E81K),CARD9蛋白表达正常,外周血淋巴细胞(peripheral blood lymphocyte,PBL)Th1和Th17细胞比例均降低;PBMC抗疣状瓶霉感染存在天然及适应性免疫缺陷。本研究首次报道1例播散性暗色丝孢霉病患者存在CARD9基因新发错义突变,同时发现患者PBMC抗真菌的天然及适应性免疫存在缺陷,提示CARD9蛋白在暗色丝孢霉病发病中可能发挥重要作用。     

凋亡相关斑点样蛋白的研究进展

凋亡相关斑点样蛋白（apoptosis-associated speck-like protein containing a CARD,ASC）是一种含有N端热蛋白样结构域和C端胱天氨酸募集结构域的接头分子。ASC可以通过它含有的同源蛋白互作结构域PYD和CARD的寡聚化来募集上下游与其含有同源结构域的其他蛋白,从而参与多条信号转导途径,在炎症反应、肿瘤发生、细胞凋亡和NF-κB信号通路的调节方面发挥重要的生物学作用。     

黄芩汤对糖尿病肾病大鼠肾脏NF-κB/NLRP3/Caspase-1细胞焦亡通路的影响*

目的:研究黄芩汤对糖尿病肾病(diabetic nephropathy,DN)大鼠肾组织核因子κB(nuclear factor kappa-B,NF-κB)/NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)/胱天蛋白酶-1(cysteinyl aspartate specific proteinase-1,Caspase-1)细胞焦亡通路的影响。方法: 将SD大鼠随机分为空白组、模型组、厄贝沙坦组(27 mg/kg)和黄芩汤低、高剂量组(5 g/kg和20 g/kg),高脂饲料喂养6周联合一次性腹腔注射链脲佐菌素(35 mg/kg)诱导DN大鼠模型,每组9只。灌胃给药6周后检测大鼠血清空腹血糖(fasting blood glucose,FBG)、总胆固醇(total cholesterol,TC)、甘油三酯(triacylglycerol,TG)、尿蛋白(urine protein,UP)、尿素氮(blood urea nitrogen,BUN)、血肌酐(serum creatinine,Scr)、白介素-1β(interleukin 1β,IL-1β)和IL-18水平;HE染色和Masson染色观察大鼠肾脏病理变化;Western blot和免疫组化检测肾脏NF-κB/NLRP3/Caspase-1细胞焦亡通路相关蛋白及阳性细胞表达。结果: 与空白组比较,模型组大鼠FBG、TC、TG、UP、BUN、Scr、IL-1β和IL-18水平明显升高(P<0.01);肾脏出现肾小球体积增大及基底膜增厚,肾小管管腔扩张,炎性浸润及纤维化明显等病理变化;肾脏组织NF-κB的磷酸化水平,以及NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、Caspase-1、IL-1β和消皮素D(gasdermin D,GSDMD)的蛋白质表达明显升高(P<0.01);肾脏组织NLRP3和GSDMD阳性细胞表达水平明显升高(P<0.01)。与模型组比较,黄芩汤组大鼠上述血糖、血脂、肾功能及炎性因子水平均得到明显改善(P<0.05,P<0.01);肾脏肾小球及肾小管结构趋于正常,炎性浸润及纤维化程度得到改善;肾脏组织NF-κB的磷酸化水平,以及NLRP3、ASC、Caspase-1、IL-1β和GSDMD的蛋白质表达水平明显降低(P<0.05,P<0.01);肾脏组织NLRP3和GSDMD阳性细胞表达明显降低(P<0.05,P<0.01)。结论: 黄芩汤对DN大鼠具有确切的疗效,机制可能与抑制NF-κB/NLRP3/Caspase-1细胞焦亡通路有关。     

RKIP低表达与鼻咽癌侵袭转移和NF-κB信号通路活化相关

为筛选鼻咽癌(nasopharyngeal carcinoma,NPC)发病相关的蛋白质,采用双向凝胶电泳(two-dimensional electrophoresis,2-DE)和质谱(mass spectrometry,MS)技术比较NPC组织与癌旁正常鼻咽上皮组织(adjacent normal nasopharyngeal epithelial tissue,ANNET)蛋白质表达的差异,鉴定了21个差异蛋白,其中Raf 激酶抑制蛋白(Raf kinase inhibitor protein,RKIP)等9个蛋白质在NPC组织中的表达水平低于ANNET．为探讨RKIP在NPC转移中的作用和机制,采用Western blot检测RKIP在不同转移潜能的5-8F和6-10B NPC细胞中的表达水平,采用免疫组化方法检查RKIP在石蜡包埋NPC组织、正常鼻咽上皮组织(normal nasopharyngeal epithelial tissue,NNET))及颈淋巴结转移NPC组织(lymphnode metastatic NPC,LMNPC)中的表达水平,采用脂质体转染方法将正义、反义RKIP表达质粒及其相应空白载体分别转染5-8F和6-10B细胞,建立相应的稳定转染细胞系,分析RKIP表达水平改变对NPC细胞体外侵袭能力和NF-κB信号通路活性的影响．结果显示：RKIP在高转移5-8F细胞中的表达水平低于非转移6-10B细胞、在NPC组织中的表达水平低于NNET、在转移癌中表达缺失．上调RKIP表达能抑制5-8F细胞的侵袭能力,而下调RKIP表达能增强6-10B细胞的侵袭能力;上调RKIP表达能降低5-8F细胞的p-IκB-α水平和NF-κB的转录活性,而下调RKIP表达能增加6-10B细胞的p-IκB-α水平和NF-κB的转录活性．研究结果提示,RKIP 可能是NPC的一个转移抑制蛋白,RKIP表达下调可能通过活化NF-κB信号通路促进NPC细胞侵袭和转移．     

Immunity:抗真菌感染的天然免疫信号新发现

正CARD9蛋白是天然免疫系统中C-type lectin受体下游的结合蛋白,负责传递上游受体识别特定物质后产生的信号。C-type lectin受体主要包括dectin-1,dectin-2等,它们主要识别真菌或细菌细胞壁的组分。C-type lectin受体激活后会介导胞内的免疫信号,最终诱导NF-k B的激活与炎性因子的释放。最初时CARD9蛋白被发现与自身免疫病[比如IBD(immune bowel disease),强直性脊柱炎等等]有关,CARD9蛋白相关基因突变的患者中     

GST pull-down联合质谱筛选(肌)营养不良短小蛋白结合蛋白1在小鼠睾丸组织中的相互作用蛋白质北大核心CSCD

(肌)营养不良短小蛋白结合蛋白1(dystrobrevin binding protein 1,dysbindin-1)是溶酶体相关细胞器生物发生复合体-1(biogenesis of lysosome-related organelles complex 1,BLOC-1)的1个亚基,在多种组织细胞中广泛表达;然而,其在睾丸组织中的作用至今尚不明确。为寻找(肌)营养不良短小蛋白结合蛋白1在睾丸组织中的相互作用蛋白质,以进一步研究(肌)营养不良短小蛋白结合蛋白1在睾丸中的作用,本研究首先在Rosetta(DE3)菌种中表达可溶性GST-dysbindin-1融合蛋白,经谷胱甘肽-琼脂糖珠亲和纯化后,与小鼠的睾丸组织蛋白质孵育进行GST pull-down实验,并通过液相色谱串联质谱(LC MS/MS)分析筛选(肌)营养不良短小蛋白结合蛋白1在睾丸组织中的相互作用蛋白质。利用Bio GPS数据库聚类在睾丸组织中高表达和特异性表达的互作蛋白质,运用DAVID6.8在线分析工具从细胞组分、分子功能、生物学过程和KEGG通路等方面对筛选出的互作蛋白质进行GO(gene ontology)富集分析。本实验共筛选出108个(肌)营养不良短小蛋白结合蛋白1在睾丸组织中的潜在互作蛋白质,其中98个为尚未报道的(肌)营养不良短小蛋白结合蛋白1相互作用蛋白质,7个为睾丸高表达蛋白质,5个为睾丸特异性表达的蛋白质。这些候选蛋白质主要分布在细胞质、细胞核、细胞膜、细胞外泌体等细胞组分中,通过与蛋白质、核酸等分子结合参与蛋白质翻译和转运、囊泡运输及凋亡等生物学过程以及氨基酸生物合成、溶酶体及蛋白酶体等生物学通路。我们推测,在睾丸组织中(肌)营养不良短小蛋白结合蛋白1可能通过与多种蛋白质相互作用参与精子的发生和受精等过程。     

AICAR通过激活Foxc1信号来调节小鼠F9细胞的增殖

为了探索5-氨基咪唑-4-氨甲酰核糖核苷（AICAR）抑制小鼠F9 细胞（F9 embryonal carcinoma cells）增殖的作用机制,本文构建了Foxc1的慢病毒真核表达载体,通过实时定量PCR、免疫荧光染色、双荧光素酶报告基因检测系统以及细胞增殖检测试验,探索AICAR抑制小鼠F9细胞的增殖作用机制. 结果发现,AICAR可以在RNA和蛋白水平促进Foxc1的基因表达,并可以作用于核转录因子κB通路. 另外在培养液中添加AICAR或过表达Foxc1都能抑制F9细胞的增殖. 信号通路报告载体检测发现Foxc1可以激活核转录因子κB通路以及细胞周期相关的通路. 总之,本研究证明,AICAR 通过激活Foxc1通路及其下游多条信号通路来抑制F9细胞增殖.     

SIP蛋白调节p65核转位的作用机制

核因子κB（nuclear factor-κB, NF-κB）参与转录调控许多与细胞生长、凋亡、肿瘤形成和转移、胚胎发育及炎症反应相关的基因.它的二聚体与抑制蛋白结合,如抑制蛋白κB（IκBα,β或γ）,而被滞留在细胞质中处于失活状态.然而NF-κB是否还有其它的抑制因子目前还不清楚.本研究结果表明,SIP（steroid receptor coactivator, SRC,SRC-interacting protein）是NF-κB家族的一个新抑制因子,它通过PEST结构域与NF-κB家族的p65蛋白相互作用.当细胞处于静息状态时,SIP将p65蛋白隔离于细胞质中;当有刺激因子TNFα或IL-1作用时,SIP与p65解离继而使p65进入细胞核启动下游靶基因转录激活.该研究为进一步认识NF-κB介导基因转录调控机制和相关疾病的发生发展提供了重要的理论依据.     

有氧运动降低胰岛素抵抗小鼠海马细胞焦亡相关蛋白及炎症因子的表达

本研究通过观察高脂饮食及有氧运动干预后小鼠海马细胞焦亡及炎症相关蛋白的表达情况,探讨运动改善胰岛素抵抗(insulin resistance, IR)的可能机制。选取6周龄C57BL/6J雄性小鼠38只,随机以正常饮食(n=12)或高脂饮食(n=26)喂养12周后进行葡萄糖和胰岛素耐量实验,根据结果将小鼠分为对照组(n=12)、IR组(n=10)和IR有氧运动组(n=10)。IR有氧运动组进行12周的渐增跑台训练。干预结束后麻醉处死小鼠并剥离海马组织,提取蛋白进行Western blot实验,检测细胞焦亡及炎症相关蛋白的表达情况。结果显示:(1)与对照组相比,IR小鼠海马NFκB,炎症小体相关蛋白Nod样受体蛋白3 (Nod-like receptor protein 3, NLRP3)、斑点样蛋白(apoptosis-associated speck-like protein containing CARD, ASC),细胞焦亡相关蛋白proCaspase-1、gasdermin D (GSDMD)、GSDMD-N及炎症因子IL-1β、IL-18均显著升高,而炎症小体相关蛋白NIMA相关蛋白激酶7 (NIMA-related kinase 7, NEK7)及焦亡相关蛋白Caspase-1有升高趋势,但无显著差异。(2)与IR组相比,渐增跑台训练能够显著降低IR小鼠海马NFκB、NLRP3、NEK7、ASC、pro-Caspase-1、GSDMD、GSDMD-N、IL-1β、IL-18的表达。以上结果提示,12周渐增跑台训练能够显著降低IR小鼠海马焦亡相关蛋白及炎症因子表达,抑制细胞焦亡。     

沙门菌毒力效应蛋白对宿主NF-κB信号通路的调控

沙门菌(Salmonella)通过向宿主细胞分泌毒力效应蛋白(effector protein)来调控细胞内一系列的信号传导通路,从而有利于沙门菌的侵染和繁殖。NF-κB信号通路在宿主对病原菌的炎症反应及免疫应答中发挥着重要的作用,也是很多毒力效应蛋白调控的靶点。沙门菌致病岛(Salmonella pathogenicity island,SPI)-1上的毒力效应蛋白Sip A、Sop E、Sop E2和Sop B都能激活宿主细胞的NF-κB信号通路,而毒力效应蛋白Spt P、Avr A、Ssp H1以及SPI-2上的Sse L能有效地抑制NF-κB信号通路。研究这些毒力效应蛋白对NF-κB信号通路的时相调控和协同作用,将进一步揭示沙门菌的致病机制。     

CARD9 Regulation and its Role in Cardiovascular Diseases

Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor protein expressed on myeloid cells and located downstream of pattern recognition receptors (PRRs), which transduces signals involved in innate immunity. CARD9 deficiency is associated with increased susceptibility to various fungal diseases. Increasing evidence shows that CARD9 mediates the activation of p38 MAPK, NF-κB, and NLRP3 inflammasome in various CVDs and then promotes the production of proinflammatory cytokines and chemokines, which contribute to cardiac remodeling and cardiac dysfunction in certain cardiovascular diseases (CVDs). Moreover, CARD9-mediated anti-apoptosis and autophagy are implicated in the progression of CVDs. Here, we summarize the structure and function of CARD9 in innate immunity and its various roles in inflammation, apoptosis, and autophagy in the pathogenesis of CVDs. Furthermore, we discuss the potential therapies targeting CARD9 to prevent CVDs and raise some issues for further exploring the role of CARD9 in CVDs.     

Candidiasis of the Central Nervous System in Neonates and Children With Primary Immunodeficiencies

Purpose of review

Candida infections of the central nervous system (CNS) are a life-threatening complication of invasive infections that most often affect vulnerable groups of patients, including neonates and children with primary immunodeficiency disorders (PID). Here, we review the currently known risk factors for CNS candidiasis, focusing predominantly on the PID caused by biallelic mutations in CARD9.

Recent findings

How the CNS is protected itself against fungal invasion is poorly understood. CARD9 promotes neutrophil recruitment and function, and is the only molecule shown to be critical for protection against CNS candidiasis in humans thus far.

Summary

Fundamental insights into the pathogenesis of CNS candidiasis gained from studying rare CARD9-deficient patients has significant implications for other patients at risk for this disease, such as CARD9-sufficient neonates. These findings will be important for the development of adjunctive immune-based therapies, which are urgently needed to tackle the global burden of invasive fungal diseases.

     

CARD9 Mediates Dectin-2-induced IκBα Kinase Ubiquitination Leading to Activation of NF-κB in Response to Stimulation by the Hyphal Form of Candida albicans

The scaffold protein CARD9 plays an essential role in anti-fungus immunity and is implicated in mediating Dectin-1/Syk-induced NF-κB activation in response to Candida albicans infection. However, the molecular mechanism by which CARD9 mediates C. albicans-induced NF-κB activation is not fully characterized. Here we demonstrate that CARD9 is involved in mediating NF-κB activation induced by the hyphal form of C. albicans hyphae (Hyphae) but not by its heat-inactivated unicellular form. Our data show that inhibiting Dectin-2 expression selectively blocked Hyphae-induced NF-κB, whereas inhibiting Dectin-1 mainly suppressed zymosan-induced NF-κB, indicating that Hyphae-induced NF-κB activation is mainly through Dectin-2 and not Dectin-1. Consistently, we find that the hyphae stimulation induces CARD9 association with Bcl10, an adaptor protein that functions downstream of CARD9 and is also involved in C. albicans-induced NF-κB activation. This association is dependent on Dectin-2 but not Dectin-1 following the hyphae stimulation. Finally, we find that although both CARD9 and Syk are required for Hyphae-induced NF-κB activation, they regulate different signaling events in which CARD9 mediates IκBα kinase ubiquitination, whereas Syk regulates IκBα kinase phosphorylation. Together, our data demonstrated that CARD9 is selectively involved in Dectin-2-induced NF-κB activation in response to C. albicans hyphae challenging.     

A critical role for CARD9 in pneumocystis pneumonia host defence

Caspase recruitment domains‐containing protein 9 (CARD9) is an adaptor molecule critical for key signalling pathways initiated through C‐type lectin receptors (CLRs). Previous studies demonstrated that Pneumocystis organisms are recognised through a variety of CLRs. However, the role of the downstream CARD9 adaptor signalling protein in host defence against Pneumocystis infection remains to be elucidated. Herein, we analysed the role of CARD9 in host defence against Pneumocystis both in CD4‐depleted CARD9^?/? and immunocompetent hosts. Card9 gene‐disrupted (CARD9^?/?) mice were more susceptible to Pneumocystis, as evidenced by reduced fungal clearance in infected lungs compared to wild‐type (WT) infected mice. Our data suggests that this defect was due to impaired proinflammatory responses. Furthermore, CARD9^?/? macrophages were severely compromised in their ability to differentiate and express M1 and M2 macrophage polarisation markers, to enhanced mRNA expression for Dectin‐1 and Mincle, and most importantly, to kill Pneumocystis in vitro. Remarkably, compared to WT mice, and despite markedly increased organism burdens, CARD9^?/? animals did not exhibit worsened survival during pneumocystis pneumonia (PCP), perhaps related to decreased lung injury due to altered influx of inflammatory cells and decreased levels of proinflammatory cytokines in response to the organism. Finally, although innate phase cytokines were impaired in the CARD9^?/? animals during PCP, T‐helper cell cytokines were normal in immunocompetent CARD9^?/? animals infected with Pneumocystis. Taken together, our data demonstrate that CARD9 has a critical function in innate immune responses against Pneumocystis.     

CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 ^-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 ^-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.     

Role of CARD9 in inflammatory signal pathway of peritoneal macrophages in severe acute pancreatitis

Previous studies revealed that caspase recruitment domain protein 9 (CARD9) was involved in severe acute pancreatitis (SAP) inflammation and that interfering with its expression in vivo could inhibit inflammation. However, the specific mechanism is unknown. This study aimed to discover the related signal pathways of CARD9 in macrophages. SiRNA interference technology was used in vivo and in vitro to detect CARD9‐related signal pathways in peritoneal macrophages. Furthermore, Toll‐like receptor 4 (TLR4) and membrane‐associated C‐type lectin‐1 (Dectin‐1) pathways in macrophages were activated specially to looking for the upstream signal path of CARD9. Results showed up‐regulation of CARD9 expression in peritoneal macrophages of SAP rats (P < .05). CARD9 siRNA alleviated inflammatory cytokines, and inhibited the phosphorylation of NF‐κB and p38MAPK in peritoneal macrophages in vivo or in vitro. Meanwhile, CARD9 siRNA reduced the concentration of CARD9 and Bcl10 in peritoneal macrophages, and TLR4 and Dectin‐1 took part in CARD9 signal pathways in macrophages. In conclusion, there is an inflammation signal pathway comprised of TLR4/Dectin‐1‐CARD9‐NF‐κB/p38MAPK activated in macrophages in SAP. Blockade of CARD9 expression in macrophages can effectively alleviate SAP inflammation.     

Insights from human studies into the host defense against candidiasis

Candida spp. are the most common cause of mucosal and disseminated fungal infections in humans. Studies using mutant strains of mice have provided initial information about the roles of dectin-1, CARD9, and Th17 cytokines in the host defense against candidiasis. Recent technological advances have resulted in the identification of mutations in specific genes that predispose humans to develop candidal infection. The analysis of individuals with these mutations demonstrates that dectin-1 is critical for the host defense against vulvovaginal candidiasis and candidal colonization of the gastrointestinal tract. They also indicate that CARD9 is important for preventing both mucosal and disseminated candidiasis, whereas the Th17 response is necessary for the defense against mucocutaneous candidiasis. This article reviews the recent studies of genetic defects in humans that result in an increased susceptibility to candidiasis and discusses how these studies provide new insight into the host defense against different types of candidal infections.     

Invasive Fungal Infection in Primary Immunodeficiencies Other Than Chronic Granulomatous Disease

Purpose of review

We aimed to review invasive fungal infections complicating primary immunodeficiencies (PID).

Recent findings

Several PID predisposing to fungal infections were recently deciphered. CARD9 deficiency selectively predisposes to fungal infections including candidiasis, aspergillosis, deep dermatophytosis, and phaeohyphomycosis, with frequent central nervous system location, especially after Candida infection. Patients with heterozygous STAT1 gain-of-function mutations are mostly predisposed to chronic mucocutaneous candidiasis but may also display, even though less frequently, invasive fungal infections. Aspergillosis complicating STAT3 deficiency is also a major concern in patients with lung cavities. Antifungal prophylaxis is recommended in this first group of patients. Previously well-reported PID are known to predispose to fungal infections, such as genetic defects impairing the IL-12/IFN-γ axis can predispose to cryptococcosis, and dimorphic fungal infections.

Summary

Patients developing invasive fungal infections including candidiasis, aspergillosis, cryptococcosis, phaeohyphomycosis, pneumocystosis, or disseminated infections caused by dimorphic fungi, without known underlying risk factors, should be explored immunogenetically in order to diagnose primary immunodeficiencies, even in the absence of previous other infectious episodes.

     

Rad50 promotes ovarian cancer progression through NF-κB activation

Rad50 is a component of MRN (Mre11-Rad50-Nbs1), which participates in DNA double-strand break repair and DNA-damage checkpoint activation. Here, we sought to investigate the clinical and functional significance of Rad50 in high-grade serous ovarian cancer (HGSOC). We found that Rad50 was frequently upregulated in HGSOCs and enhanced Rad50 expression inversely correlated with patient survival. In addition, ectopic expression of Rad50 promoted proliferation/invasion and induced EMT of ovarian cancer cells, whereas knockdown of Rad50 led to decreased aggressive behaviors. Mechanistic investigations revealed that Rad50 induced aggressiveness in HGSOC via activation of NF-κB signaling pathway. Moreover, we identified CARD9 as an interacting protein of Rad50 in ovarian cancer cells and the activation of NF-κB pathway by Rad50 is CARD9 dependent. Our findings provide evidence that Rad50 exhibits oncogenic property via NF-κB activation in HGSOC.