Institute for Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Strasse 2, 97080, Würzburg, Germany.
Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Würzburg, Germany.
Fluids Barriers CNS. 2022 Oct 26;19(1):81. doi: 10.1186/s12987-022-00379-z.
Bacterial meningitis is a life-threatening disease that occurs when pathogens such as Neisseria meningitidis cross the meningeal blood cerebrospinal fluid barrier (mBCSFB) and infect the meninges. Due to the human-specific nature of N. meningitidis, previous research investigating this complex host-pathogen interaction has mostly been done in vitro using immortalized brain endothelial cells (BECs) alone, which often do not retain relevant barrier properties in culture. Here, we developed physiologically relevant mBCSFB models using BECs in co-culture with leptomeningeal cells (LMCs) to examine N. meningitidis interaction.
We used BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in co-culture with LMCs derived from tumor biopsies. We employed TEM and structured illumination microscopy to characterize the models as well as bacterial interaction. We measured TEER and sodium fluorescein (NaF) permeability to determine barrier tightness and integrity. We then analyzed bacterial adherence and penetration of the cell barrier and examined changes in host gene expression of tight junctions as well as chemokines and cytokines in response to infection.
Both cell types remained distinct in co-culture and iBECs showed characteristic expression of BEC markers including tight junction proteins and endothelial markers. iBEC barrier function as determined by TEER and NaF permeability was improved by LMC co-culture and remained stable for seven days. BEC response to N. meningitidis infection was not affected by LMC co-culture. We detected considerable amounts of BEC-adherent meningococci and a relatively small number of intracellular bacteria. Interestingly, we discovered bacteria traversing the BEC-LMC barrier within the first 24 h post-infection, when barrier integrity was still high, suggesting a transcellular route for N. meningitidis into the CNS. Finally, we observed deterioration of barrier properties including loss of TEER and reduced expression of cell-junction components at late time points of infection.
Here, we report, for the first time, on co-culture of human iPSC derived BECs or hCMEC/D3 with meningioma derived LMCs and find that LMC co-culture improves barrier properties of iBECs. These novel models allow for a better understanding of N. meningitidis interaction at the mBCSFB in a physiologically relevant setting.
细菌性脑膜炎是一种危及生命的疾病，当奈瑟脑膜炎球菌等病原体穿过血脑脊液屏障（mBCSFB）并感染脑膜时就会发生。由于奈瑟脑膜炎球菌具有人类特异性，因此之前研究这种复杂的宿主-病原体相互作用的研究大多是在体外使用永生化脑内皮细胞（BEC）单独进行的，而这些细胞在培养中往往无法保留相关的屏障特性。在这里，我们使用 BEC 与脑膜瘤衍生的软膜细胞（LMC）共培养来建立生理相关的 mBCSFB 模型，以研究奈瑟脑膜炎球菌的相互作用。
我们使用诱导多能干细胞（iPSC）衍生的 BEC 样细胞或 hCMEC/D3 细胞与肿瘤活检衍生的 LMC 共培养。我们使用 TEM 和结构光照明显微镜来对模型以及细菌相互作用进行特征描述。我们测量跨上皮电阻（TEER）和钠荧光素（NaF）通透性来确定屏障的紧密性和完整性。然后，我们分析了细菌对细胞屏障的粘附和渗透，并研究了宿主紧密连接蛋白和内皮标志物等基因表达的变化，以及对感染的趋化因子和细胞因子的反应。
两种细胞类型在共培养中仍然保持明显的特征，iPSC 衍生的 BEC 样细胞表现出 BEC 标志物的特征性表达，包括紧密连接蛋白和内皮标志物。由 LMC 共培养确定的 iBEC 屏障功能通过 TEER 和 NaF 通透性得到改善，并且在七天内保持稳定。LMC 共培养对 BEC 对奈瑟脑膜炎球菌感染的反应没有影响。我们检测到大量粘附在 BEC 上的脑膜炎球菌和相对较少的细胞内细菌。有趣的是，我们发现细菌在感染后 24 小时内就穿过了 BEC-LMC 屏障，而此时屏障的完整性仍然很高，这表明奈瑟脑膜炎球菌进入中枢神经系统的途径是细胞间的。最后，我们观察到屏障特性的恶化，包括 TEER 的丧失和细胞连接成分表达的减少，这些都发生在感染的晚期。
在这里，我们首次报告了人类 iPSC 衍生的 BEC 或 hCMEC/D3 与脑膜瘤衍生的 LMC 的共培养，并发现 LMC 共培养可改善 iPSC 衍生的 BEC 的屏障特性。这些新型模型可在生理相关的环境下更好地理解 mBCSFB 中奈瑟脑膜炎球菌的相互作用。
