In vivo labeling of epithelial cell–associated antigen passages in the murine intestine

Abstract

The intestinal immune system samples luminal contents to induce adaptive immune responses that include tolerance in the steady state and protective immunity during infection. How luminal substances are delivered to the immune system has not been fully investigated. Goblet cells have an important role in this process by delivering luminal substances to the immune system through the formation of goblet cell–associated antigen passages (GAPs). Soluble antigens in the intestinal lumen are transported across the epithelium transcellularly through GAPs and delivered to dendritic cells for presentation to T cells and induction of immune responses. GAPs can be identified and quantified by using the ability of GAP-forming goblet cells to take up fluorescently labeled dextran. Here, we describe a method to visualize GAPs and other cells that have the capacity to take up luminal substances by intraluminal injection of fluorescent dextran in mice under anesthesia, tissue sectioning for slide preparation and imaging with fluorescence microscopy. In contrast to in vivo two-photon imaging previously used to identify GAPs, this technique is not limited by anatomical constraints and can be used to visualize GAP formation throughout the length of the intestine. In addition, this method can be combined with common immunohistochemistry protocols to visualize other cell types. This approach can be used to compare GAP formation following different treatments or changes to the luminal environment and to uncover how sampling of luminal substances is altered in pathophysiological conditions. This protocol requires 8 working hours over 2–3 d to be completed.

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Fig. 1
Fig. 2: GAP formation in the small intestine.
Fig. 3: GAP formation per goblet cell.
Fig. 4: GAP formation in the colon.

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Acknowledgements

This work was supported by grants AI136515, AI140755, AI12626, AI131342 and DK097317 (to R.D.N.); grants DK052574, DK09789 and AI095542 (to K.A.K.); Swedish Research Council International Postdoc Award 2014-00366 (to J.K.G.); and Crohn’s and Colitis Foundation 610605 (to D.H.K.).

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Contributions

All authors conceived the protocol and reviewed and discussed the manuscript. K.A.K. and D.H.K. prepared the figures. K.A.K., J.E.D. and A.N.F. prepared the supplementary videos.

Corresponding author

Correspondence to Rodney D. Newberry.

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Competing interests

R.D.N., K.A.K. and K.G.M. are inventors on the US Non-provisional Application Serial No. 15/880,658: Compositions and methods for modulation of dietary and microbial exposure.

Supplementary information

Reporting Summary

Supplementary Video 1

Mouse surgery and intraluminal dextran administration

Supplementary Video 2

Embedding and freezing of tissue

Supplementary Video 3

Sectioning of tissue for slide preparation

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Knoop, K.A., Kulkarni, D.H., McDonald, K.G. et al. In vivo labeling of epithelial cell–associated antigen passages in the murine intestine. Lab Anim 49, 79–88 (2020). https://doi.org/10.1038/s41684-019-0438-z

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