Mycoplasma pneumoniae (M. pneumoniae, Mp) is an intracellular pathogen that causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans and can infect and survive in the host cells leading to excessive immune responses. Extracellular vesicles (EVs) from host cells carry components of pathogens to recipient cells and play a role in intercellular communication during infection. However, there is limited knowledge on whether EVs derived from M. pneumoniae-infected macrophages play as intercellular messengers and functional mechanisms. In this study, we establish a cell model of M. pneumoniae-infected macrophages that continuously secrete EVs to further asses their role as intercellular messengers and their functional mechanisms. Based on this model, we determined a method for isolating the pure EVs from M. pneumoniae-infected macrophages, which employs a sequence of operations, including differential centrifugation, filtering, and ultracentrifugation. We identified EVs and their purity using multiple methods, including electron microscopy, nanoparticle tracking analysis, Western blot, bacteria culture, and nucleic acid detection. EVs from M. pneumoniae-infected macrophages are pure, with a 30–200 nm diameter. These EVs can be taken up by uninfected macrophages and induce the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 through the nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPK) signals pathway. Moreover, the expression of inflammatory cytokines induced by EVs relies on TLR2-NF-κB/JNK signal pathways. These findings will help us better understand a persistent inflammatory response and cell-to-cell immune modulation in the context of M. pneumoniae infection.

Human endometrial epithelia undergo injury repair and regeneration with the menstrual cycle; however, mechanisms underpinning the roles of endometrial epithelial cells in endometrial lesions and regeneration remain incompletely understood, mainly owing to the difficulty in the isolation and expansion of primary endometrial epithelial cells and the lack of reliable models for in vitro and in vivo studies. In this report, we sought to improve methods for the isolation and expansion of human endometrial epithelial cells with a Rho-associated protein kinase (ROCK) inhibitor–modified medium and subsequently characterize endometrial epithelium generated with primary cells cultured in an air–liquid interface (ALI) state. Immunocytochemistry staining revealed the expression of epithelial cellular adhesion molecule (EpCam) and stage-specific embryonic antigen-1 (SSEA-1) but a lack of CD13 in endometrial epithelial cells. Meanwhile, a large number of proliferative Ki67+ cells were observed in isolated epithelial cells. Importantly, the EpCam+/CD13− cells were capable of forming spheroids, a characteristic of epithelial stem/progenitor cells. Interestingly, these cells also exhibited a capacity to reconstitute epithelial layers in an ALI state. Morphological analysis revealed mucosal secretion of differentiated epithelial cells with cilia and microvilli in ALI epithelial cells as determined by electronic microscopy. Immunoblotting assay further demonstrated the expression of endometrial epithelial cell markers keratin 17/19 and EpCam and stem cell marker OCT3/4 but not stromal cell marker Vimentin protein and CD13 in cell expansions. Furthermore, molecular analysis also showed that the exposure of cells to estrogen elevated the expression of estrogen receptor and progesterone receptors in ALI cultures. Our results shed light on the possibility of expanding sufficient numbers of endometrial epithelial cells for stem cell biology studies, and they provide a feasible and alternative model that can recapitulate the characteristics and physiology of endometrial epithelium in vivo.

Lung cancer remains one of the most common cancer-related deaths worldwide. The cigarette smoking is a risk factor for lung cancer development. Interestingly, the cystic fibrosis transmembrane conductance regulator encoded by CFTR gene, an ATP-binding cassette transporter-class ion channel that conducts chloride and bicarbonate anions across membrane of epithelial cells, has recently been suggested to play a role in the development and progression of many types of cancer. It has been well-documented that mutations of CFTR gene are the cause of cystic fibrosis, the most common fatal hereditary lung disease in Caucasian population; the function of cystic fibrosis transmembrane conductance regulator in the development of lung cancer however has not yet been established. In the present study, we aimed to interrogate the impact of cystic fibrosis transmembrane conductance regulator on the nicotine-promoted progressive potency in lung adenocarcinoma cells by assessing capacities of cystic fibrosis transmembrane conductance regulator to cell migration, invasion, and clonogenicity and the expression of markers of cell proliferation and lung stem cell–related transcription factors in lung adenocarcinoma A549 cells. The exposure of nicotine exhibited an ability to enhance progressive properties of adenocarcinoma cells including A549 cells, HCC827 cells, and PC-9 cells, alone with an inhibition of cystic fibrosis transmembrane conductance regulator protein expression. Remarkably, an overexpression of cystic fibrosis transmembrane conductance regulator significantly inhibited the progressive potency of A549 cells, including capacity of cell migration and invasion and clonogenicity, along with a decreased expression of cell proliferative markers Ki67, p63, and proliferating cell nuclear antigen, and cancer stem cell marker CD133, stem cell pluripotency-related transcription factors octamer-binding transcription factor ¾, and sex-determining region Y-box 2, regardless of the presence of nicotine. In contrast, opposite effects were observed in A549 cells that the cystic fibrosis transmembrane conductance regulator was knockdown by short hairpin RNA to cystic fibrosis transmembrane conductance regulator. This study thus suggests that cystic fibrosis transmembrane conductance regulator may play a tumor suppressor role in lung cancer cells, which may be a novel therapeutic target warranted for further investigation.

肺癌发病率逐年上升，有必要寻找新型的治疗靶点，而最新研究发现囊状纤维化跨膜转导调节子（cystic fibrosis transmembrane conductance regulator, CFTR）与多种肿瘤的发生和恶性转化有关。本研究探讨CFTR对肺癌A549细胞恶性特性的影响。 应用CCK8细胞增殖实验、细胞划痕实验、Transwell细胞侵袭实验以及克隆形成实验等方法分别检测CFTR的表达对非小细胞肺癌A549细胞的增殖、迁移、侵袭等细胞恶性特性的影响。同时通过免疫印迹（Western blot）分析CFTR基因表达对肿瘤干细胞相关转录因子表达的影响。 过表达CFTR基因显著抑制A549细胞的增殖、迁移、侵袭和克隆形成等肿瘤恶性特征，而RNA干扰A549细胞CFTR的表达导致细胞上述特征的明显增强。免疫印迹实验进一步发现CFTR基因过表达抑制A549细胞中干细胞相关转录因子SOX2和OCT3/4，以及细胞表面CD133蛋白的表达；相反，RNA干扰A549细胞中CFTR基因的表达增加SOX2、OCT4和CD133的表达。然而，免疫印迹和流式细胞术发现CFTR基因表达对A549细胞肺癌干细胞标志乙醛脱氢酶1的表达和阳性细胞数量无显著影响。 CFTR基因在肺癌A549细胞中具有抑制细胞恶性特征的作用，提示其可能是肺腺癌治疗的一个新的靶点，但其对其他肺腺癌细胞的作用与分子机制还有待进一步研究。