Laboratory for Epithelial Immunobiology, RIKEN Research Center for Allergy and Immunology
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Fukuda, S., Hase, K., Ohno, H. Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells. J. Vis. Exp. (58), e3225, doi:10.3791/3225 (2011).
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.
1. Preparation of bacterial cells
3. Ligated Peyer's patch loop assay
4. Whole mount staining and confocal microscopic analysis of Peyer's patch
5. Representative Results:
The specimen example of a mouse ligated Peyer's patch loop assay with GFP-S. Typhimurium was observed with a DeltaVision Restoration deconvolution microscope (Figure 4). GFP-S. Typhimurium is transcytosed by GP2+M cells. A mouse Peyer's patch intestinal loop assay can identify the kind of commensal or pathogenic bacteria that can be transcytosed by M cells.
Figure 1. Anesthesia of mice under vaporized isoflurane condition. Vaporized isoflurane was supplied by an anesthesia-dedicated apparatus (left side).
Figure 2. Summary of ligated Peyer's patch intestinal loop assay. The fluorescent bacterial suspension was injected by syringe into ligated Peyer's patch loop on the loose side of the intestine.
Figure 3. Mount of the specimens. The specimens were embedded in a 30% solution of glycerol in PBS on a special slide in which a perforated plastic plate (1mm thick) was bound by adhesive agent on a glass slide.
Figure 4. Example of a mouse ligated Peyer's patch loop assay with GFP-S. Typhimurium. GP2, which is reported as M cell specific marker5, was stained with anti-mouse GP2 antibody (Red).The specimen was observed with a DeltaVision Restoration deconvolution microscope. GFP-expressing S. Typhimurium was co-localized with GP2 on the apical plasma membrane of M cell and in the subapical cytoplasmic vesicles (arrowheads). Top, apical view. Bottom and side, lateral views. Scale bar: 10 μm.
The incubation time of ligated Peyer's patch with bacterial suspension is usually for 1 hr to observe the bacterial incorporation into M cells. In case of 4 hrs incubation, the bacteria are often detected in the T cell zone of Peyer's patches. As the vaporized isoflurane inhalant anesthesia could keep mice stable, the incubation time of ligated Peyer's patch with bacterial suspension is extendable to observe the bacteria which move into T-cell zone in Peyer's patch. The important point of this experiment is to take care not to scratch the blood vessel when ligating the intestine. In the case of not to use GFP-expressed bacteria, we could alternatively use fluorescently labeled bacteria by commercial fluorescence labeling reagent. If the reagent possibly inhibits the bacterial, which you want to use, attachment to the molecule expressed on M cell, you should use the specific primary antibody, if available, to detect the incorporated bacteria.
The ligated loop assay method was previously described6, 7. We improve some points of this method as follows: 1. Isoflurane is used as an anesthetic agent for keeping the mice steady. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch is set up. 3. Bacteria taken up by M cells are fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as GFP. 4. M cells in the follicle-associated epithelium covering Peyer's patch are detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells are observed by confocal microscopic analysis.
We and others have demonstrated that diverse heterogenous antigens actively gain entry into M cells. Substantial numbers of pathogens including Salmonella spp., Shigella spp., Yersinia spp. exploit M cells for host invasion8-10. Furthermore, the coccoid form of Helicobacter pylori can potentially translocate into Peyer's patch via M cells, and this translocation is essential for the induction of chronic inflammation in the stomach11. On the other hand, the mechanisms by which these pathogens invade M cells remain to be fully elucidated. We suggest that the ligated Peyer's patch loop assay is an appropriate experimental method for dissecting the interplay between M cells and pathogenic agents in vivo.
No conflicts of interest declared.
The authors would like to thank all EIB members for developing this technique. This research was supported in part by Grant-in-Aid for Scientific Research on Priority Areas "Membrane Traffic" (H.O.), and "Matrix of Infectious Phenomena" (K.H.), Young Scientists (S.F. and K. H.), and Scientific Research (H.O.), and Scientific Research on Innovative Areas "Intracellular Logistics" (H.O.), from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
|LB Agar Ampicillin- 100||SIGMA||L5667|
|Cy3 mono-Reactive Dye Pack||GE Healthcare||PA23001|
|Alexa Fluor 350 carboxylic acid, succinimidyl ester||Invitrogen||A10168|
|Inhalation anesthesia apparatus||Shinano Seisakusho||SN-487|
|Fixation and Permeabilization Solution||BD||554722|
|Anti mouse Glycoprotein 2 antibody||MBL||D278-3||200-fold dilution (5μg/ml)|
|Goat Anti-Rat IgG, F(ab’)2 fragment specific||Jackson ImmunoResearch||112-505-006||200-fold dilution (20μg/ml)|
|Alexa Fluor 633 Phalloidin||Molecular Probes||A22284||50-fold dilution|
|Confocal laser scanning microscope||Leica Microsystems||DMIRE2|
|DeltaVision Restoration deconvolution microscope||Applied Precision||DeltaVision Core|