Name: enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model
Uploaded: 2013-10-22T14:00:00
Description: Watch this Scientific Journal Video about Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model at JoVE.com

Dominic Mills was supported by a Bloomsbury Colleges PhD Studentship (2007-2010). The authors would like to thank both Ozan Gundogdu and Abdi Elmi for their assistance in developing the VDC model.

1. Growth of IEC Monolayers on Special 0.4 &#956;m&#160;Filters
<ol>
	<li>Culture Caco-2 cells in Dulbecco&#39;s modified essential media (DMEM) supplemented with 10% (v/v) fetal calf serum, 100 U/ml penicillin, 100 &#956;g/ml streptomycin and 1% (v/v) nonessential amino acids in a standard tissue culture incubator at 37 &#176;C in 5% CO2 and 95% air. Seed 4 x 105 Caco-2 IECs per 0.4 &#956;m filter and grow to polarization over 21 days, changing the media every 2-3 days.</li>
	<li>Measure the TEER...

<ol>
	<li>Mills, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+Campylobacter+jejuni+invasion+of+intestinal+epithelial+cells+under+low-oxygen+coculture+conditions+that+reflect+the+in+vivo+environment.">Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment.</a> Infect. Immun. 80, 1690-1698 (2012).</li><li>Marteyn, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Shigella+virulence+in+response+to+available+oxygen+in+vivo.">Modulation of Shigella virulence in response to available oxygen in vivo.</a> Nature. 465, 355-358 (2010).</li><li>Dorrell, N., Wren, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+century+of+Campylobacter+research:+recent+advances,+new+opportunities+and+old+problems.">The second century of Campylobacter research: recent advances, new opportunities and old problems.</a> Curr. Opin. Infect. Dis. 20, 514-518 (2007).</li><li>Young, K. T., Davis, L. M., Dirita, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Campylobacter+jejuni:+molecular+biology+and+pathogenesis.">Campylobacter jejuni: molecular biology and pathogenesis.</a> Nat. Rev. Microbiol. 5, 665-679 (2007).</li><li>Hu, L., Kopecko, D. J., Nachamkin, I., Szymanski, C. M., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+17.">Chapter 17.</a> Campylobacter. , 297-313 (2008).</li><li>Everest, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+Caco-2+cells+as+a+model+for+enteric+invasion+by+Campylobacter+jejuni+and+C.+coli.">Differentiated Caco-2 cells as a model for enteric invasion by Campylobacter jejuni and C. coli.</a> J. Med. Microbiol. 37, 319-325 (1992).</li><li>Konkel, M. E., Hayes, S. F., Joens, L. A., Cieplak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+the+internalization+and+intracellular+survival+of+Campylobacter+jejuni+in+human+epithelial+cell+cultures.">Characteristics of the internalization and intracellular survival of Campylobacter jejuni in human epithelial cell cultures.</a> Microb. Pathog. 13, 357-370 (1992).</li><li>Monteville, M. R., Konkel, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin-facilitated+invasion+of+T84+eukaryotic+cells+by+Campylobacter+jejuni+occurs+preferentially+at+the+basolateral+cell+surface.">Fibronectin-facilitated invasion of T84 eukaryotic cells by Campylobacter jejuni occurs preferentially at the basolateral cell surface.</a> Infect. Immun. 70, 6665-6671 (2002).</li><li>Friis, L. M., Pin, C., Pearson, B. M., Wells, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+culture+methods+for+investigating+Campylobacter+invasion+mechanisms.">In vitro cell culture methods for investigating Campylobacter invasion mechanisms.</a> J. Microbiol. Methods. 61, 145-160 (2005).</li><li>Schuller, S., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microaerobic+conditions+enhance+type+III+secretion+and+adherence+of+enterohaemorrhagic+Escherichia+coli+to+polarized+human+intestinal+epithelial+cells.">Microaerobic conditions enhance type III secretion and adherence of enterohaemorrhagic Escherichia coli to polarized human intestinal epithelial cells.</a> Environ. Microbiol. 12, 2426-2435 (2010).</li><li>Cottet, S., Corthesy-Theulaz, I., Spertini, F., Corthesy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microaerophilic+conditions+permit+to+mimic+in+vitro+events+occurring+during+in+vivo+Helicobacter+pylori+infection+and+to+identify+Rho/Ras-associated+proteins+in+cellular+signaling.">Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling.</a> J. Biol. Chem. 277, 33978-33986 (2002).</li></ol>

The use of in vitro cell culture methods to study the interactions of bacterial pathogens with host cells is a technique widely employed in many research laboratories. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. The widely used in vitro infection models are especially inappropriate for studying microaerophilic C. jejuni ...

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. Using such cell culture assays, bacterial adhesion to host cells, identification of host cell receptors, host cell signaling pathways and bacterial invasion of host cells have all been studied in detail, resulting in many important observations. However such cell culture assays are performed under aerobic conditions that may not be representative of the in vivo environment. A major limitation of in vitro models used to study gastrointestinal infections is that culture conditions including high oxygen levels generally favor eukaryotic cell survival. However conditions in the intestinal lumen will be almost anaerobic. Enteric pathogens in a very low oxygen environment express virulence genes whose expression changes under aerobic conditions2. As such, data obtained using standard cell culture models may give an inaccurate indication of bacterial interactions with host cells.
Campylobacter jejuni is the leading causative agent of bacterial acute gastroenteritis worldwide, with symptoms that range from mild diarrhea to severe inflammatory enteritis. The majority of C. jejuni infections result in uncomplicated gastroenteritis, however C. jejuni is also the most commonly identified infectious agent in peripheral neuropathies including Guillain-Barr&#233; syndrome (GBS). In the UK, it is estimated that there are over 500,000 cases of enteritis caused by C. jejuni infection each year with a predicted cost to the UK economy of &#163;580 million. In the developing world, C. jejuni is a leading cause of mortality among children. Despite the undoubted importance of Campylobacter infection and decades of research, including thorough genomics-based analysis, C. jejuni pathogenesis is still poorly understood, in contrast to other enteric pathogens such as Salmonella, Escherichia coli, Shigella, and Vibrio cholerae. The lack of a convenient small animal model is one major reason for this3. Also the widely used in vitro infection models are more inappropriate for studying microaerophilic C. jejuni than for other enteric pathogens that are facultative anaerobes. Whilst C. jejuni is recognized as an invasive pathogen, the mechanisms of C. jejuni invasion of intestinal epithelial cells (IECs) are still unclear4,5. C. jejuni invasion has been shown to be dependent on either microfilaments, microtubules, a combination of both or neither5. The confusion in this area is most probably due to the use of inappropriate in vitro assay conditions.
A number of different cell culture assays have been used to investigate the interactions of C. jejuni with host cells. Caco-26, INT 4077, and T848 cell lines have all been used to study the adhesion and invasion capabilities of different C. jejuni strains. However, the levels of bacterial adhesion and invasion for C. jejuni with IECs are dramatically lower than for other enteric pathogens9. The coculturing of C. jejuni with IECs is normally performed in a CO2 incubator under conditions close to atmospheric oxygen levels, required for the survival of IECs. C. jejuni gene expression will be very different in the low oxygen environment of the intestinal lumen compared to atmospheric oxygen conditions.
The use of a Vertical Diffusion Chamber (VDC) system has been developed which permits the coculture of bacteria and host cells under different medium and gas conditions1,10,11. This system mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Polarized IEC monolayers grown in special 0.4 &#956;m filters were placed into a VDC creating an apical and basolateral compartment, which were individually filled with bacterial broth and cell culture medium respectively (Figure 1). The VDC was placed into the variable atmosphere incubator containing 85% N2, 5% O2, and&#160;10% CO2 at 37 &#176;C, representing optimum conditions for C. jejuni. The apical compartment was left open and exposed to the microaerobic atmosphere within the variable atmosphere incubator, whilst the sealed basolateral compartment was supplied with oxygen by constant administration of 5% CO2/95% O2 gas mixture with an outlet tube preventing accumulation of pressure. Caco-2 cell survival and monolayer integrity under these conditions were confirmed by monitoring the transepithelial electrical resistance (TEER) across the monolayer over 24 hr to demonstrate survival of the Caco-2 cell monolayer and physical separation of the apical and basolateral compartments under low oxygen conditions in the apical compartment. The TEER of monolayers in VDCs maintained in the variable atmosphere incubator (microaerobic conditions) and in a standard cell culture CO2 incubator (aerobic conditions) showed no significant differences, indicating integrity of the cell monolayer under different atmospheric conditions1. Under microaerobic conditions, tight junctions remained present and evenly distributed between the cell borders with an occludin staining pattern similar to cells maintained under aerobic conditions1.
The interactions of C. jejuni with Caco-2 and T84 cells in the VDC were investigated by assessing bacterial interaction (adhesion and invasion) and invasion. Two different C. jejuni wild-type strains were used1. C. jejuni 11168H is a hypermotile derivative of the original sequence strain NCTC11168. The 11168H strain shows much higher colonization levels in a chick colonization model and is thus considered a suitable strain to use for host-pathogen interaction studies. 81-176 is a human isolate and is one of the most invasive widely studied laboratory strains. C. jejuni strains were added to the apical compartment of a VDC under either microaerobic or aerobic conditions. We observed higher rates for both interaction and invasion were recorded for C. jejuni under microaerobic conditions1. The increased C. jejuni interactions were not due to an increase in bacterial numbers under microaerobic conditions1. This data supports our hypothesis that the low oxygen environment in the apical compartment of the VDC enhances bacteria-host interactions and indicates that the invasive properties of C. jejuni are increased under these conditions. This was the first report of the use of the VDC model to study an invasive bacterial pathogen and highlights the significance of performing in vitro infection assays under conditions that more closely mimic the in vivo situation. The VDC model could be used to study the host-pathogen interactions for many different bacterial species.

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Speciality vertical diffusion system for use with Snapwell inserts</td>
			<td>Harvard Apparatus</td>
			<td>66-0001</td>
			<td>Manifold &#38; six Snapwell chambers</td>
		</tr>
		<tr>
			<td>Caps</td>
			<td>Harvard Apparatus</td>
			<td>66-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>O-rings</td>
			<td>Harvard Apparatus</td>
			<td>66-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamps</td>
			<td>Harvard Apparatus</td>
			<td>66-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Snapwell filters (pore size 0.4 &#956;m)</td>
			<td>Corning Costar</td>
			<td>3407</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell ERS-2 Volt-Ohm resistance meter</td>
			<td>Millipore</td>
			<td>MERS00002</td>
			<td></td>
		</tr>
		<tr>
			<td>WPA Lightwave II spectrophotometer</td>
			<td>Biochrom</td>
			<td>80-3003-72</td>
			<td></td>
		</tr>
	</tbody>
</table>

enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Coculturing experiments performed with a C. jejuni wild-type strain and IECs in the VDC model with microaerobic conditions in the apical compartment have demonstrated an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions in a time-dependent manner1. This observation was reproducible using two different C. jejuni wild-type strains (11168H and 81-176) and two different IEC lines (Caco-2 and T84), highlightin...

Watch this Scientific Journal Video about Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model at JoVE.com

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

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