En Årehinden Plexus epitelcelle-baserede Model af Menneskeblod-cerebrospinalvæske Barrier at studere bakteriel infektion fra den basolaterale side
Summary
The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.
Abstract
The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.
Introduction
Blod-cerebrospinalvæske barriere (BCSFB) er en af de tre barriere sites mellem blodet og hjernen 1. Dens morfologiske korrelat er de epitelceller i choroid plexus (CP) 2,3, en endotel-epitel sammenfoldede, som er stærkt vaskulariseret og placeret i ventriklerne i hjernen. CP tjener til frembringelse cerebrospinalvæsken (CSF) samt at adskille dem fra blodet. For at opnå barrierefunktion, CP epitelceller viser en lav pinocytotic aktivitet, udtrykke specifikke transportører, og er tæt forbundet med en kontinuerligt netværk af tight junctions (TJs) 2,3.
Human choroid plexus papillom (HIBCPP) celler, der er afledt af en malign choroid plexus papilloma af en japansk kvinde 4, blev anvendt til at konstruere en funktionel in vitro model af BCSFB. HIBCPP celler viser et par karakteristika for en funktionel BCSFB dannelsen af TJtråde, udvikling af en høj transepithelial membran potentiale, der kan bestemmes som transepithelial elektriske modstand (TEER), og mindre permeabiliteter til makromolekyler. Desuden HIBCPP celler udtrykker karakteristiske transportører, der kan tjene til at regulere den ioniske mikromiljø, og viser apikale / basolaterale polaritet 5,6,7.
Den BCSFB har vist sig at fungere som en indgang site for patogener (bakterier, virus og svampe) i centralnervesystemet (CNS) 8. Invasionen af patogener, herunder Neisseria meningitidis (N. meningitidis), en Gram-negativ bakterie, kan forårsage alvorlige sygdomme som meningitis. Bevis for, at den overvinder den beskyttende epitel barriere af CP er støttet af histopatologiske observationer i patienter med meningokoksygdom udviser forøgede mængder af meningokokker i skibene og CP epitelceller 9,10. For at få adgang i værtsceller bacteria ofte kapre endocytotiske mekanismer, som medieres eller udløses af specifikke overfladereceptorer placeret på værtscellerne. Da interaktioner af patogener med disse receptorer kan være artsspecifikke 11, dyremodeller kan kun høres i begrænset omfang. Den HIBCPP cellelinien giver mulighed for at studere invasionen proces samt de underliggende molekylære mekanismer i et humant modelsystem. Anvender cellekultur indsætter kan vi analysere interaktioner af patogener med værtsceller fra to forskellige celle sider. Mange bakterier, herunder N. meningitidis, er stærkt underlagt virkningen af tyngdekraften under infektion analyser. For optimal interaktion af patogener med HIBCPP celler under assayene bakterierne oprindeligt tilsat til det øvre kammer i cellekultur filterindsatsen system. At muliggøre infektion fra det apikale eller den basolaterale celle side, henholdsvis har to variationer af in vitro system været established: I standardsystem HIBCPP celler podes ind i det øvre kammer af filterindsatsen, efterligne situationen, når mikroorganismer er placeret på CSF-side og komme i kontakt med den apikale side af cellerne (figur 1A, C). I modsætning hertil ved hjælp af de HIBCPP celler i et omvendt cellekultur filterindsats systemet afspejler vilkårene når bakterier har indtastet blodbanen. Mikroorganismer formidler i blodet og støder CP epitelceller fra den basolaterale side (figur 1B, D). Bemærkelsesværdigt, i dette modelsystem er det blevet vist, at bakterier invaderer HIBCPP celler i et polært mode specifikt fra den basolaterale celleside 5,7.
Efterfølgende til infektion af CP, kan de invaderede patogener genkendes af det medfødte immunsystem gennem ligation til mønster-genkendelse receptorer (PRRS). Velbeskrevne medlemmer af PRRS tilhører Toll-lignende receptor (TLR) -familien. TLRs kan bind til karakteristiske strukturer af infektiøse mikroorganismer, der betegnes patogen-associeret molekylære mønstre (PAMPs). Ligering af receptorerne resulterer i aktivering af værtscelle signaleringskaskader, der udløser ekspressionen af cytokiner og chemokiner 12, som igen stimulerer transmigration af immunceller over BCSFB 13,14. Det er blevet vist, at HIBCPP celler udtrykker adskillige TLR'er på mRNA-niveau og at infektion med N. meningitidis resulterer i sekretion af adskillige cytokiner og chemokiner, herunder CXCL1-3, IL6, IL8 og TNFa 15,16.
Her beskriver vi dyrkning og infektion af den humane cellelinie HIBCPP i en omvendt celledyrkningsinsert system, der efterligner BCSFB. Denne model system muliggør at studere interaktioner af patogener med in vivo relevante basolaterale celle side samt den efterfølgende cellulære respons.
Protocol
Representative Results
Discussion
Epitelcellerne af CP danne BCSFB der adskiller CSF fra blodet 2,3. Vi har for nylig etableret HIBCPP cellelinie som en funktionel menneskelig model af BCSFB. Cellerne vise vigtige barriere funktioner BCSFB in vitro, herunder udvikling af en høj membranpotentiale, en lav permeabilitet for makromolekyler, såvel som tilstedeværelse af kontinuerlige strenge af TJs 5. TJ proteiner bidrager til en apikal / basolaterale polaritet af cellerne. Polariteten er af stor betydning for en rettet loka…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Prof. Hartwig Wolburg for performing the electron microscopy.
Materials
| 0.25% Trypsin-EDTA | Gibco | 25200-056 | |
| 4´,6 diamidino-2-phenylindole (DAPI) | Life Technologies | D1306 | |
| 12-well plates | Starlab | CC7682-7512 | |
| 24-well plates | Starlab | CC7682-7524 | |
| Anti Neisseria meningitidis α-OMP | This antibody was a gift from Drs. H. Claus and U. Vogel (University of Würzburg, Germany) | ||
| Alexa Fluor 488 (chicken anti rabbit) | Invitrogen | A21441 | |
| Alexa Fluor 594 (chicken anti rabbit) | Invitrogen | A21442 | |
| Alexa Fluor 660 Phalloidin | Invitrogen | A22285 | |
| Bovine serum albumine (BSA) | Calbiochem | 12659 | |
| Chocolate agar plates | Biomerieux | 43109 | |
| Cytochalasin D | Sigma | C8273 | |
| DMEM/F12 + L-Glut + 15 mM HEPES | Gibco | 31330-095 | |
| DMEM/F12 + L-Glut + 15 mM HEPES w/o Phenolred | Gibco | 11039-047 | |
| Dimethyl sulfoxide | Sigma | D2650 | |
| Fetal calf serum (FCS) | Life Technologies | 10270106 | |
| FITC-Inulin | Sigma | F3272 | |
| Insulin | Sigma | 19278 | |
| MgCl2 | Sigma | 2393 | |
| NaHCO3 | Sigma | 55761 | |
| PBS + Mg +Ca | Gibco | 14040-174 | |
| Penicillin/Streptomycin | MP Biomedicals | 1670049 | |
| Polyvitex | Biomerieux | 55651 | |
| Proteose peptone | BD | 211684 | |
| Serum-free medium | Gibco | 10902-096 | |
| Thincert cell culture inserts for 24-well plates, pore size 3 µm | Greiner | 662630 | |
| Tissue culture flask 75 cm² red cap sterile | Greiner | 658175 | |
| Triton X-100 | Sigma | T8787 | |
| Volt-Ohm Meter Millicell-ERS2 with MERSSTX01 electrode | Millipore | MERSSTX00 |
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