Membrane Inflammasome Activation by Choriodecidual Ureaplasma parvum Infection without Intra-Amniotic Infection in an NHP Model.
Membrane Inflammasome Activation by Choriodecidual Ureaplasma parvum Infection without Intra-Amniotic Infection in an NHP Model.
复制标题
NHP 模型中无羊膜内感染的绒毛膜蜕膜解脲支原体感染激活膜炎性体。
DOI：
10.1101/2023.09.18.557989
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Kelleher,MeredithA
中科院分区：
文献类型：
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作者：
Tripathy,Sudeshna;Burd,Irina;Kelleher,MeredithA
宫内感染是早产和新生儿发病率和死亡率的重要原因。微小脲原体是最常从早产和早产胎膜早破（pPROM）病例中分离出的微生物。然而，上行生殖道感染早期阶段启动母胎炎症途径、早产和胎膜早破的机制仍知之甚少。为了检测胎儿（绒毛膜）膜对微小脲原体感染的反应，我们利用了一种新的早期绒毛膜蜕膜感染的体内非人灵长类动物模型。对8只长期置管的恒河猴孕105-112天进行母-胎置管手术，并于绒毛膜蜕膜接种微小脲原体（105 cfu/mL低传代临床分离株，血清型1; n= 4）或盐水/无菌培养基（对照组; n= 4）从妊娠115-119天开始，每5天重复一次，直到妊娠136- 140天（足月= 167天）计划剖腹产。平均接种至分娩间隔为21天，所有动物的羊水培养和PCR均未检测到支原体感染。采用Luminex、ELISA和多重检测法对羊水（AF）中的炎症介质进行评估。从绒毛膜和绒毛膜提取RNA用于单基因分析（qRT-PCR），并通过Western印迹和免疫组织化学测定蛋白质表达。我们的绒毛膜蜕膜脲原体感染的NHP模型，代表了早期上行生殖道感染，没有微生物侵入羊膜腔，导致胎膜蛋白和MMP-9和PTGS 2的基因表达增加，但没有导致早产（子宫收缩力未增加）或羊水促炎细胞因子（IL-1β、IL-6、IL-8、IL-18、TNF-α）浓度增加。然而，与未感染的对照组相比，在脲原体组中炎性体传感器分子NLRP 3、NLRC 4、AIM 2和NOD 2的膜表达以及接头蛋白ASC（PYCARD）基因表达显著增加。在支原体感染的胎膜中，IL-1β、IL-18、IL-18 R1受体、Caspase-1和pro-caspase-1蛋白的基因表达也增加。下游炎症信号基因MyD 88在羊膜和绒毛膜中也显著上调，沿着绒毛膜中NF-κ B的显著增加。这些结果表明，即使在上行生殖道脲原体感染的早期阶段，也存在炎性小体复合物的激活和与绒毛膜完整性降解相关的途径。因此，本研究提供了实验证据的重要性，早期阶段的上行支原体感染的启动过程中的pPROM和早产。这些发现对宫内感染引起的早产和胎儿损伤的时机和潜在治疗以及支原体感染介导这些过程的机制具有意义。
Intrauterine infection is a significant cause of preterm labor and neonatal morbidity and mortality. Ureaplasma parvum is the micro-organism most commonly isolated from cases of preterm birth and preterm premature rupture of membranes (pPROM). However, the mechanisms during the early stages of ascending reproductive tract infection that initiate maternal-fetal inflammatory pathways, preterm birth and pPROM remain poorly understood. To examine inflammation in fetal (chorioamnionic) membranes in response to Ureaplasma parvum infection, we utilized a novel in vivo non-human primate model of early choriodecidual infection. Eight chronically catheterized pregnant rhesus macaques underwent maternal-fetal catheterization surgery at 105-112 days gestation and choriodecidual inoculation with Ureaplasma parvum (105cfu/mL of a low passaged clinical isolate, serovar 1; n= 4) or saline/sterile media (Controls; n= 4) starting at 115-119 days gestation, repeated every 5 days until scheduled cesarean-section at 136-140d gestation (term= 167d). The average inoculation to delivery interval was 21 days and Ureaplasma infection of the amniotic fluid was undetectable by culture and PCR in all animals. Inflammatory mediators in amniotic fluid (AF) were assessed by Luminex, ELISA and multiplex assays. RNA was extracted from the chorion and amnionic membranes for single gene analysis (qRT-PCR) and protein expression was determined by Western blot and immunohistochemistry. Our NHP model of choriodecidual Ureaplasma infection, representing an early-stage ascending reproductive tract infection without microbial invasion of the amniotic cavity, resulted in increased fetal membrane protein and gene expression of MMP-9 and PTGS2, but did not result in preterm labor (no increase in uterine contractility) or increased concentrations of amniotic fluid pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-18, TNF-α). However, membrane expression of inflammasome sensor molecules, NLRP3, NLRC4, AIM2 and NOD2, and the adaptor protein ASC (PYCARD) gene expression were significantly increased in the Ureaplasma group when compared to non-infected controls. Gene expression of IL-1β, IL-18, the IL-18R1 receptor, Caspase-1 and pro-caspase-1 protein were also increased in the fetal membranes with Ureaplasma infection. Downstream inflammatory signaling genes MyD88 was also significantly upregulated in both the amnion and chorion, along with a significant increase in NF-kappaB in the chorion. These results demonstrate that even at the early stages of ascending reproductive tract Ureaplasma infection, activation of inflammasome complexes and pathways associated with degradation of chorioamnionic membrane integrity are present. This study therefore provides experimental evidence for the importance of the early stages of ascending Ureaplasma infection in initiating processes of pPROM and preterm labor. These findings have implications for the timing and potential treatments of preterm labor and fetal injury caused by intrauterine infection and the mechanisms by which Ureaplasma infection may mediate these processes.