We thank Jenny-Lee Thomassin for her help in editing the manuscript. The work was supported by the ANR grants MITOPATHO and PATHIMMUN, grants from the Labex Memolife and PSL IDEX Shigaforce. Chunhui Sun is a recipient of a Ph.D. grant from the China Scholarship Council. Laurent Combettes and Guy Tran Van Nhieu are recipients of a WBI-France exchange Tournesol program N&#176;31268YG (Wallonie-Bruxelles International, Fonds de la Recherche Scientifique, Minist&#232;re Fran&#231;ais des Affaires &#233;trang&#232;res et europ&#233;ennes, Minist&#232;re de l&#39;Enseignement sup&#233;rieur et de la Recherche dans le cadre des Partenariats Hubert Curien).

1. Preparations
<ol>
	<li>Bacteria preparation
	<ol>
		<li>Plate the bacteria&#8212;Shigella wild-type strain expressing the AfaE adhesin (M90T-AfaE)&#8212;onto a trypticase soy (TCS) agar plate containing 0.01% Congo red (CR) and incubate them for 18 h at 37 &#176;C. 
		Note: To increase their reproducibility, the Shigella plates obtained in step 1.1.1 are stored at 4 &#176;C and should be used within a week, because all colonies on CR medium will eventually turn red over time.<...

<ol>
	<li>Ashida, H., Ogawa, M., Kim, M., Mimuro, H., Sasakawa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+and+host+interactions+in+the+gut+epithelial+barrier.">Bacteria and host interactions in the gut epithelial barrier.</a> Nature Chemical Biology. 8 (1), 36-45 (2012).</li><li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signaling.">The versatility and universality of calcium signaling.</a> Nature Reviews Molecular Cell Biology. 1 (1), 11-21 (2000).</li><li>Strehler, E. 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A., 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=Shigella+induces+mitochondrial+dysfunction+and+cell+death+in+nonmyleoid+cells.">Shigella induces mitochondrial dysfunction and cell death in nonmyleoid cells.</a> Cell Host &amp; Microbe. 5 (2), 123-136 (2009).</li><li>Horng, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+and+mitochondrial+destabilization+in+the+triggering+of+the+NLRP3+inflammasome.">Calcium signaling and mitochondrial destabilization in the triggering of the NLRP3 inflammasome.</a> Trends in Immunology. 35 (6), 253-261 (2014).</li><li>Galan, J. E., Lara-Tejero, M., Marlovits, T. C., Wagner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+type+III+secretion+systems:+specialized+nanomachines+for+protein+delivery+into+target+cells.">Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells.</a> Annual Review of Microbiology. 68, 415-438 (2014).</li><li>Ashida, H., Mimuro, H., Sasakawa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shigella+manipulates+host+immune+responses+by+delivering+effector+proteins+with+specific+roles.">Shigella manipulates host immune responses by delivering effector proteins with specific roles.</a> Frontiers in Immunology. 6, 219 (2015).</li><li>Tran Van Nhieu, G., 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=Actin-based+confinement+of+calcium+responses+during+Shigella+invasion.">Actin-based confinement of calcium responses during Shigella invasion.</a> Nature Communications. 4, 1567 (2013).</li><li>Niebuhr, K., 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=Conversion+of+PtdIns(4,5)P(2)+into+PtdIns(5)P+by+the+S.+flexneri+effector+IpgD+reorganizes+host+cell+morphology.">Conversion of PtdIns(4,5)P(2) into PtdIns(5)P by the S. flexneri effector IpgD reorganizes host cell morphology.</a> The EMBO Journal. 21 (19), 5069-5078 (2002).</li><li>Konradt, 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=The+Shigella+flexneri+type+three+secretion+system+effector+IpgD+inhibits+T+cell+migration+by+manipulating+host+phosphoinositide+metabolism.">The Shigella flexneri type three secretion system effector IpgD inhibits T cell migration by manipulating host phosphoinositide metabolism.</a> Cell Host &amp; Microbe. 9 (4), 263-272 (2011).</li><li>Friedrich, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intriguing+Ca2++requirement+of+calpain+activation.">The intriguing Ca2+ requirement of calpain activation.</a> Biochemical and Biophysical Research Communications. 323 (4), 1131-1133 (2004).</li><li>Thomas, D., 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=A+comparison+of+fluorescent+Ca2++indicator+properties+and+their+use+in+measuring+elementary+and+global+Ca2++signals.">A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals.</a> Cell Calcium. 28 (4), 213-223 (2000).</li><li>Sun, C. 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=The+Shigella+type+III+effector+IpgD+recodes+Ca2++signals+during+invasion+of+epithelial+cells.">The Shigella type III effector IpgD recodes Ca2+ signals during invasion of epithelial cells.</a> The EMBO Journal. 36 (17), 2567-2580 (2017).</li><li>Allaoui, A., Menard, R., Sansonetti, P. J., Parsot, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Shigella+flexneri+IpgD+and+IpgF+genes,+which+are+located+in+the+proximal+part+of+the+mxi+locus.">Characterization of the Shigella flexneri IpgD and IpgF genes, which are located in the proximal part of the mxi locus.</a> Infection and Immunity. 61 (5), 1707-1714 (1993).</li></ol>

This manuscript describes the protocol that we engineered to follow local Ca2+ signals during the relatively short kinetics of a Shigella invasion, as well as global Ca2+ responses during the extended kinetics of Shigella. Below, key issues can be found that need to be addressed to optimize the detection of Ca2+ signals while minimizing any interference with the biological processes.
Chemical vs. genetically encoded Ca2+ probe...

Ca2+ regulates all known cell processes, including cytoskeletal reorganization, inflammatory responses, and cell death pathways related to host-pathogen interactions1,2,3. Under physiological conditions, basal cytosolic Ca2+ concentrations are low, in the hundreds of nM range, but can be subjected to transient increases upon agonist stimulation. These variations often show oscillatory behavior through the action of pumps and channels at the plasma and endoplasmic reticulum membranes. The pattern of these oscillations is characterized by the period, duration, and amplitude of Ca2+ increases, and is decrypted by cells which, in turn, trigger specific responses in what is known as the Ca2+ code4,5. A sustained increase in the cytosolic Ca2+ concentration under pathological conditions may lead to cell death associated with the permeabilization of mitochondrial membranes and the release of pro-apoptotic or necrotic factors6,7.
Shigella, the causative agent of bacillary dysentery, invades epithelial cells by injecting effectors into host cells using a type III secretion system (T3SS)8,9. A Shigella invasion of host cells is associated with local and global Ca2+ signals elicited by the T3SS. As for pore-forming toxins, the T3SS translocon that inserts into host cell membranes and is required for the injection of T3SS effectors is likely responsible for the activation of PLC and the inositol (1, 4, 5) trisphosphate (InsP3)-dependent Ca2+ release. The combination of the localized PLC stimulation and the accumulation of polymerized actin at sites of a Shigella invasion result in an atypically long-lasting InsP3-dependent Ca2+ release10. The type III effector IpgD, a phosphatidyl 4,5 bisphosphate (PIP2)-4-phosphatase, limits the local amount of PIP2, thereby controlling the quantity of available substrate for PLC to generate InsP3, which contributes to the confinement of local Ca2+ responses at bacterial invasion sites11,12. These local Ca2+ responses likely contribute to the actin polymerization at Shigella invasion sites10. Global Ca2+ responses that are also elicited by Shigella, however, are dispensable for the bacterial invasion process but trigger the opening of connexin hemichannels at the plasma membrane and the release of ATP in the extracellular compartment. Released ATP acting in a paracrine manner, in turn, stimulates Ca2+ oscillatory responses in cells next to the infected cell. IpgD is also responsible for shaping global Ca2+ responses into erratic isolated responses with slow dynamics. Eventually, upon a prolonged bacterial infection, IpgD leads to the inhibition of InsP3-mediated Ca2+ signals. Through its interference with Ca2+ signaling, IpgD delays a Ca2+-dependent calpain activation leading to the disassembly of focal adhesion structures and the premature detachment of infected cells13.
While Ca2+ signals are involved in critical aspects of pathogenesis, the use of a microorganism raises a number of technical challenges that are not encountered in classical agonist studies. The protocols described here use the commonly used fluorescent Ca2+ chemical indicator Fluo-4 that we engineered to characterize local Ca2+ signals during a Shigella infection. Steps critical for the detection of these signals are discussed, as well as procedures implemented for their quantitative analysis that is required to characterize the role of bacterial effectors in Ca2+ signaling.

<table border="0" cellpadding="0" cellspacing="0" style="width:693px;" width="694">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fluo-4 AM</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:136px;">F14201</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Metamorph version 7.7</td>
			<td style="width:120px;">Universal Imaging</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CoolLED illumination system pE-2</td>
			<td style="width:120px;">Roper Scientific</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">micro-dish 35 mm, high&#160;</td>
			<td style="width:120px;">IBIDI</td>
			<td>81156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Trypticase Soy (TCS) broth</td>
			<td style="width:120px;">Thermofisher</td>
			<td style="width:136px;"></td>
			<td>B11768</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">TCS agar</td>
			<td style="width:120px;">Thermofisher</td>
			<td style="width:136px;"></td>
			<td>B11043</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Congo red</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:136px;"></td>
			<td>75768</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">M90T-AfaE</td>
			<td style="width:120px;">Sun et al. 2017</td>
			<td style="width:136px;">Shigella flexneri serotype V. expressing the AfaE adhesin</td>
			<td></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">ipgD-AfaE</td>
			<td style="width:120px;">Sun et al. 2017</td>
			<td style="width:136px;">isogenic ipgD mutant strain expressing the AfaE adhesin</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging ca2+ responses during shigella infection of epithelial cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Shigella invasion is associated with atypical long-lasting local Ca2+ responses:
Following the protocol mentioned above, Fluo-4-loaded HeLa cells were challenged with WT Shigella and stream acquisitions were performed to analyze Ca2+ signals. A representative experiment is shown in Figure 1, with time-lapse images series of the fluorescence intensity of the Fluo-...

Watch this Scientific Journal Video about Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells at JoVE.com

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

The overall goal of this fluorescent microscopy analysis using chemical probes is to analyze local and global calcium responses elicited by host cells during infection by pathogenic Shigella. This method can help answer key questions in the field of cellular microbiology such as pathogen-induced signaling leading to cytoskeletal reorganization or cell death. The main advantage of this technique is that by using chemical probes one can follow local calcium signals associated with local processes diverted by the pathogen.Generally individuals new to this method will struggle because of the setting of parameters that enable to follow local calcium signals while preserving the integrity of biological samples. To begin, plate wild-type Shigella expressing the AfaE adhesion onto a trypticase soy or TCS agar plate containing 0.01%Congo red and incubate the plates at 37 degrees Celsius for 18 hours. Prepare pre-cultures from the streaked plates by picking three red colonies and inoculating TCS broth with 75 micrograms per milliliter of ampicillin.Grow the liquid cultures in a shaking incubator at 37 degrees Celsius and 200 rpm for 16 hours. Next, inoculate TCS with the bacterial pre-culture at a 1:100 dilution. Incubate the culture at 37 degrees Celsius in a shaking incubator at 200 rpm for two hours.Ensure that the optical density at 600 nanometers is 0.2 to 0.4. Centrifuge the culture at 13, 000 g and 21 degrees Celsius for two minutes and re-suspend the pellet in an equivalent volume of EM medium. Dilute the bacterial suspension in EM buffer at a final OD 600 of 0.1 and use it immediately or store it at 21 degrees Celsius and use it within the next 60 minutes.Grow HeLa cells in DMEM with one gram per liter of glucose supplemented with 10%FCS and grow the culture at 37 degrees Celsius with 10%CO2. Grow TC7 cells in DMEM with 4.5 grams per liter of glucose, 10%FCS, and non-essential amino acids and culture at 37 degrees Celsius and 10%CO2. For cell maintenance, split the cells regularly to avoid growth contact inhibition linked to confluent states.This is critical for high calcium probe loading and transfection efficiency. Trypsinize and count the TC7 cells. The day before the experiment with HeLa cells, plate the cells onto sterile, 25-millimeter diameter circular cover slips in six-well plates at a density of three times 10 to the five cells per well.Then, to allow for polarization before the experiments, seed the cells at four times 10 to the five cells per well and incubate them for five to seven days, replacing the medium every day. On the day of the experiment, remove the medium and use one milliliter of EM medium to wash the cells three times. Load the cells with three micromolar Fluo-4 AM and EM buffer and incubate the plates at 21 degrees Celsius for 30 minutes.With EM buffer, wash the samples three times, then add one milliliter of EM buffer, and incubate the cells at 21 degrees Celsius to allow for the hydrolysis of the AM moiety. Place the coverslip containing the Fluo-4 loaded cells in an imaging or glass-bottom chamber. Use EM buffer to wash the sample three times to remove compounds potentially resulting from cell lysis.Then, add one milliliter of EM buffer. Place the chamber on an inverted fluorescence microscope stage heated at 33 degrees Celsius. Select a microscopy field and set up acquisition parameters including exposure time and binning if necessary to optimize the Fluo-4 fluorescent signal.To add bacteria to the sample, carefully remove 500 microliters of EM buffer from the chamber and add 500 microliters of the bacterial suspension to obtain a final OD 600 of 0.05. Immediately perform an acquisition or wait 10 minutes to allow the bacteria to sediment onto the cells. At the end of the acquisition stream, acquire a phase-contrast image of the selected field to visualize the bacteria contacting the cells, and the membrane ruffles associated with bacterial invasion sites.Repeat the acquisition procedure at intervals that are amenable to the duration of the image acquisitions, file savings, and selection of a new field to cover the whole process. At the end of the acquisition procedure, add to the sample two micromolar of the calcium ionophore ionomycin to determine the maximal amplitude of the calcium signals. Acquire images every three to five seconds until the signal stabilizes, usually for less than 10 minutes.Follow by adding the calcium chelator EGTA to a final concentration of 10 millimolar to determine the fluorescent signal in the absence of calcium. Acquire images every three to five seconds until signal stabilization, usually for less than 10 minutes. Carry out image analysis according to the text protocol.In this experiment, Fluo-4 loaded HeLa cells were challenged with wild-type Shigella and stream acquisitions were performed to analyze calcium signals. A time-lapse image series of the fluorescence intensity of the Fluo-4 probe is averaged in a region of interest for a single cell, and the corresponding phase-contrast image is shown. Atypical local increases in free cytosolic calcium are observed at the Shigella invasion site with a varying amplitude and durations ranging from 2.5 to five seconds, followed by global increases in the infected cell.In this example, an isogenic mutant strain, deficient for the type III effector IpgD, a phosphatidyl 4, 5-bisphosphate phosphatase, induced more global and less atypical local responses with long durations as compared to the wild-type Shigella strain. As seen in this experiment, the imaging of global calcium responses over extended infection kinetics shows a decrease in the frequency of responses 30 minutes following the challenge with wild-type Shigella. Finally, the quantification of dose-dependent cell responses to the calcium agonist histamine illustrates the inhibition of an inositol triphosphate dependent calcium release at late stages of a Shigella infection.Following this procedure, all the methods simultaneously combining compatible fluids and imaging can be performed in order to address the role of calcium signals in a given process. Don't forget that working with pathogens can be extremely hazardous, and precautions such as waste disposal, cleaning of contaminated surfaces or equipments should always be taken while performing this procedure.

imaging-ca2-responses-during-shigella-infection-of-epithelial-cells

Research

JoVE Journal

Immunology and Infection

5.4K Views.  Collège de France. The overall goal of this fluorescent microscopy analysis using chemical probes is to analyze local and global calcium responses elicited by host cells during infection by pathogenic Shigella. This method can help answer key questions in the field of cellular microbiology such as pathogen-induced signaling leading to cytoskeletal reorganization or cell death. The main advantage of this technique is that by using chemical probes one can follow local calcium signals associated with local process...

Video: Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are...

an imaging technique to study the calcium response in bacteria-infected cells

This video demonstrates a technique for detecting local and global calcium ion response upon Shigella infection of human epithelial cells. Shigella invades host cells and releases invasion effectors, impacting intracellular calcium levels. The amplitude and frequency of the local and global cytosolic calcium are studied using a fluorescent...

An Imaging Technique to Study the Calcium Response in Bacteria-Infected Cells

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characterization of inflammatory responses during intranasal colonization with streptococcus pneumoniae

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...