Abstract:
Despite the established concept of the human mammary gland (MG) as a habitat with its own microbiota, the exact mechanism of MG colonization is still elusive and a well-characterized in vitro model would reinforce studies of the MG microbiota development. We aimed to establish and characterize an in vitro cell model for studying MAmmary Gland mIcrobial Colonization (MAGIC) model. We used the immortalized cell line MCF10A, which expresses the strong polarized phenotype similar to MG ductal epithelium when cultured on a permeable support (Transwell). We analyzed the surface properties of the MAGIC model by gene expression analysis of E-cadherin, tight junction proteins, and mucins and by scanning electron microscopy. To demonstrate the applicability of the model, we tested the adhesion capability of the whole human milk (HM) microbial community and the cellular response of the model when challenged directly with raw HM samples. MCF10A on permeable supports differentiated and formed a tight barrier, by upregulation of CLDN8, MUC1, MUC4, and MUC20 genes. The surface of the model was covered with mucins and morphologically diverse with at least two cell types and two types of microvilli. Cells in the MAGIC model withstood the challenge with heat-treated HM samples and responded differently to the imbalanced HM microbiota by distinctive cytokine response. The microbial profile of the bacteria adhered on the MAGIC model reflected the microbiological profile of the input HM samples. The well-studied MAGIC model could be useful for studies of bacterial attachment to the MG and for in vitro studies of biofilm formation and microbiota development.IMPORTANCEThe MAGIC model may be particularly useful for studies of bacterial attachment to the surface of the mammary ducts and for in vitro studies of biofilm formation and the development of the human mammary gland (MG) microbiota. The model is also useful for immunological studies of the interaction between bacteria and MG cells. We obtained pioneering information on which of the bacteria present in the raw human milk (HM) were able to attach to the epithelium treated directly with raw HM, as well as on the effects of bacteria on the MG epithelial cells. The MAGIC cell model also offers new opportunities for research in other areas of MG physiology, such as the effects of bioactive milk components on microbial colonization of the MG, mastitis prevention, and studies of probiotic development. Since resident MG bacteria may be an important factor in breast cancer development, the MAGIC in vitro tool also offers new opportunities for cancer research.
摘要:
尽管已经确立了人类乳腺(MG)作为具有自身微生物群的栖息地的概念,MG定植的确切机制仍然难以捉摸,并且充分表征的体外模型将加强对MG微生物群发育的研究。我们旨在建立和表征体外细胞模型,以研究乳腺胫骨定植(MAGIC)模型。我们使用永生化细胞系MCF10A,当在可渗透支持物(Transwell)上培养时,其表达类似于MG导管上皮的强极化表型。我们通过E-cadherin的基因表达分析分析了MAGIC模型的表面特性,紧密连接蛋白,和粘蛋白和扫描电子显微镜。为了证明模型的适用性,我们测试了全人乳(HM)微生物群落的粘附能力,以及直接用原始HM样品攻击时模型的细胞反应。MCF10A在可渗透的支持物上分化并形成紧密的屏障,通过上调CLDN8、MUC1、MUC4和MUC20基因。模型的表面被粘蛋白覆盖,并且在形态上具有至少两种细胞类型和两种类型的微绒毛。MAGIC模型中的细胞经受了热处理的HM样品的挑战,并通过独特的细胞因子反应对不平衡的HM微生物群产生不同的反应。粘附在MAGIC模型上的细菌的微生物谱反映了输入HM样品的微生物谱。经过充分研究的MAGIC模型可用于研究细菌与MG的附着以及生物膜形成和微生物群发育的体外研究。重要性MAGIC模型可能特别适用于研究细菌附着在乳腺导管表面的研究,以及对生物膜形成和人类乳腺(MG)微生物群发育的体外研究。该模型还可用于细菌和MG细胞之间相互作用的免疫学研究。我们获得了关于原人乳(HM)中存在的哪些细菌能够附着于用原始HM直接处理的上皮的开创性信息,以及细菌对MG上皮细胞的影响。MAGIC细胞模型还为MG生理学其他领域的研究提供了新的机会,如生物活性乳成分对MG微生物定植的影响,预防乳腺炎,和益生菌发育的研究。由于居住的MG细菌可能是乳腺癌发展的重要因素,MAGIC体外工具也为癌症研究提供了新的机会.