Abstract：
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
摘要：
背景：细菌性脑膜炎是一种威胁生命的疾病，当病原体如脑膜炎奈瑟氏球菌穿过脑膜血脑脊液屏障（mBCSFB）并感染脑膜时发生。由于脑膜炎奈瑟氏球菌的人类特异性，以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单独使用永生化脑内皮细胞（BECs）进行的，在文化中通常不保留相关的屏障特性。这里,我们使用BECs与软脑膜细胞（LMCs）共培养建立了生理相关的mBCSFB模型，以检查脑膜炎奈瑟球菌的相互作用。
方法：我们使用源自诱导多能干细胞（iBEC）的BEC样细胞或hCMEC/D3细胞与源自肿瘤活检的LMC共培养。我们采用TEM和结构化照明显微镜来表征模型以及细菌相互作用。我们测量了TEER和荧光素钠（NaF）的渗透性，以确定屏障的密封性和完整性。然后，我们分析了细菌粘附和细胞屏障的穿透，并检查了紧密连接的宿主基因表达以及趋化因子和细胞因子对感染的反应。
结果：两种细胞类型在共培养中仍然不同，iBECs显示出BEC标记的特征性表达，包括紧密连接蛋白和内皮标记。由TEER和NaF通透性确定的iBEC屏障功能通过LMC共培养得到改善,并保持稳定7天。BEC对脑膜炎奈瑟球菌感染的反应不受LMC共培养的影响。我们检测到大量粘附BEC的脑膜炎球菌和相对少量的细胞内细菌。有趣的是,我们发现细菌在感染后的第一个24小时内穿越BEC-LMC屏障，当屏障完整性仍然很高时，提示脑膜炎奈瑟球菌进入中枢神经系统的跨细胞途径。最后,我们观察到屏障特性的恶化,包括TEER的丧失和在感染后期细胞连接成分的表达降低.
结论：这里，我们报告,第一次,关于人iPSC衍生的BEC或hCMEC/D3与脑膜瘤衍生的LMC的共培养,发现LMC共培养改善iBEC的屏障特性。这些新的模型允许更好地理解在生理相关环境中mBCSFB处的脑膜炎奈瑟球菌相互作用。
方法：我们使用源自诱导多能干细胞（iBEC）的BEC样细胞或hCMEC/D3细胞与源自肿瘤活检的LMC共培养。我们采用TEM和结构化照明显微镜来表征模型以及细菌相互作用。我们测量了TEER和荧光素钠（NaF）的渗透性，以确定屏障的密封性和完整性。然后，我们分析了细菌粘附和细胞屏障的穿透，并检查了紧密连接的宿主基因表达以及趋化因子和细胞因子对感染的反应。
结果：两种细胞类型在共培养中仍然不同，iBECs显示出BEC标记的特征性表达，包括紧密连接蛋白和内皮标记。由TEER和NaF通透性确定的iBEC屏障功能通过LMC共培养得到改善,并保持稳定7天。BEC对脑膜炎奈瑟球菌感染的反应不受LMC共培养的影响。我们检测到大量粘附BEC的脑膜炎球菌和相对少量的细胞内细菌。有趣的是,我们发现细菌在感染后的第一个24小时内穿越BEC-LMC屏障，当屏障完整性仍然很高时，提示脑膜炎奈瑟球菌进入中枢神经系统的跨细胞途径。最后,我们观察到屏障特性的恶化,包括TEER的丧失和在感染后期细胞连接成分的表达降低.
结论：这里，我们报告,第一次,关于人iPSC衍生的BEC或hCMEC/D3与脑膜瘤衍生的LMC的共培养,发现LMC共培养改善iBEC的屏障特性。这些新的模型允许更好地理解在生理相关环境中mBCSFB处的脑膜炎奈瑟球菌相互作用。
