Hostname: page-component-586b7cd67f-2brh9 Total loading time: 0 Render date: 2024-11-28T08:53:37.876Z Has data issue: false hasContentIssue false
  • English
  • Français

Three-Dimensional Reconstruction of Murine Peyer's Patches from Immunostained Cryosections

Published online by Cambridge University Press:  04 November 2013

Sarita Ahlawat ,
Magdia De Jesus ,
Kedar Khare ,
Richard A. Cole  and
Nicholas J. Mantis
Sarita Ahlawat
Affiliation:
New York State Department of Health, Wadsworth Center, Division of Infectious Diseases, Albany, NY 12208, USA
Magdia De Jesus
Affiliation:
New York State Department of Health, Wadsworth Center, Division of Infectious Diseases, Albany, NY 12208, USA
Kedar Khare
Affiliation:
Department of Physics, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India
Richard A. Cole
Affiliation:
Advanced Light Microscopy Core, New York State Department of Health, Wadsworth Center, Albany, NY 12201, USA
Nicholas J. Mantis*
Affiliation:
New York State Department of Health, Wadsworth Center, Division of Infectious Diseases, Albany, NY 12208, USA Department of Biomedical Sciences, University at Albany, Albany, NY 12208, USA
*
*Corresponding author. E-mail: [email protected]
Get access

Abstract

Peyer's patches, macroscopic aggregates of lymphoid follicles present throughout the small intestines of humans and other mammals, are considered the gateway through which luminal dietary antigens and microbes are sampled by the mucosal immune system. The cellular make-up of Peyer's patch lymphoid follicles is not only complex, but highly dynamic, as there are at least four major cell types that are known to migrate in response to antigenic stimulation. In an effort to capture the complexity and dynamic nature of this specialized tissue, here we report the three-dimensional (3D) reconstruction of immunofluorescent-labeled mouse Peyer's patch cryosections. The technology that enabled the stacking and linear blending of serial cryosections was a novel macro for Fiji, the open source image-processing package based on ImageJ. By simultaneously labeling cryosections for surface markers CD45R, CD3, and CD11c, we provide a 3D image as well as quantitative measures of B-cell, T-cell, and dendritic cell populations at steady state and following exposure to the mucosal adjuvant cholera toxin.

Keywords

lymphoid tissueintestineimmunostainingFijilinear blendingthree-dimensional reconstruction
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 1 , February 2014 , pp. 198 - 205
Copyright
Copyright © Microscopy Society of America 2014 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

†

These authors contributed equally to this work.

References

Anosova, N.G., Chabot, S., Shreedhar, V., Borawski, J.A., Dickinson, B.L. & Neutra, M.R. (2008). Cholera toxin, E. coli heat-labile toxin, and non-toxic derivatives induce dendritic cell migration into the follicle-associated epithelium of Peyer's patches. Mucosal Immunol 1(1), 59–67.CrossRefGoogle ScholarPubMed
Bergqvist, P., Stensson, A., Hazanov, L., Holmberg, A., Mattsson, J., Mehr, R., Bemark, M. & Lycke, N.Y. (2013). Re-utilization of germinal centers in multiple Peyer's patches results in highly synchronized, oligoclonal, and affinity-matured gut IgA responses. Mucosal Immunol 6(1), 122–135.CrossRefGoogle ScholarPubMed
Blumberg, R.S. (2009). Inflammation in the intestinal tract: Pathogenesis and treatment. Dig Dis 27(4), 455–464.CrossRefGoogle ScholarPubMed
Brandtzaeg, P. (2010). Function of mucosa-associated lymphoid tissue in antibody formation. Immunol Invest 39(4-5), 303–355.CrossRefGoogle ScholarPubMed
Chieppa, M., Rescigno, M., Huang, A.Y. & Germain, R.N. (2006). Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement. J Exp Med 203(13), 2841–2852.CrossRefGoogle ScholarPubMed
De Jesus, M., Ahlawat, S. & Mantis, N.J. (2013). Isolating and immunostaining lymphocytes and dendritic cells from murine Peyer's patches. J Vis Exp 73, e50167. Google Scholar
Ebisawa, M., Hase, K., Takahashi, D., Kitamura, H., Knoop, K.A., Williams, I.R. & Ohno, H. (2011). CCR6hiCD11c(int) B-cells promote M-cell differentiation in Peyer's patch. Int Immunol 23(4), 261–269.CrossRefGoogle ScholarPubMed
Ermak, T. & Owen, R.L. (1986). Differential distribution of lymphocytes and accessory cells in mouse Peyer's patches. Anat Rec 215, 144–152.CrossRefGoogle ScholarPubMed
Farstad, I.N., Halstensen, T.S., Kvale, D., Fausa, O. & Brandtzaeg, P. (1997). Topographic distribution of homing receptors on B and T-cells in human gut-associated lymphoid tissue: Relation of L-selectin and integrin alpha 4 beta 7 to naive and memory phenotypes. Am J Pathol 150(1), 187–199.Google Scholar
Iwasaki, A. & Kelsall, B. (2001). Unique functions of CD11b+, CD8a+, and double negative Peyer's patch dendritic cells. J Immunol 166, 4884–4890.CrossRefGoogle Scholar
Iwasaki, A. & Kelsall, B.L. (2000). Localization of distinct Peyer's patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3alpha, MIP-3beta, and secondary lymphoid organ chemokine. J Exp Med 191(8), 1381–1394.CrossRefGoogle ScholarPubMed
Kelsall, B.L. & Strober, W. (1996). Distinct populations of dendritic cells are present in the subepithelial dome and T cell regions of the murine Peyer's patch. J Exp Med 183(1), 237–247.Google Scholar
Lelouard, H., Fallet, M., de Bovis, B., Meresse, S. & Gorvel, J.P. (2011). Peyer's patch dendritic cells sample antigens by extending dendrites through M-cell-specific transcellular pores. Gastroenterology 142, 592–601.CrossRefGoogle ScholarPubMed
Ma, B., Wang, L., von Wasielewski, R., Lindenmaier, W. & Dittmar, K.E. (2008). Serial sectioning and three-dimensional reconstruction of mouse Peyer's patch. Micron 39(7), 967–975.Google Scholar
Mantis, N.J., Rol, N. & Corthesy, B. (2011). Secretory IgA's complex roles in immunity and mucosal homeostasis in the gut. Mucosal Immunol 4(6), 603–611.Google Scholar
Neutra, M., Mantis, N. & Kraehenbuhl, J.P. (2001). Collaboration of epithelial cells with organized mucosal lymphoid tissue. Nat Immunol 2, 1004–1009.CrossRefGoogle Scholar
Pittet, M.J. & Weissleder, R. (2011). Intravital imaging. Cell 147(5), 983–991.Google Scholar
Regoli, M., Bertelli, E., Borghesi, C. & Nicoletti, C. (1995). Three-dimensional (3D-) reconstruction of M cells in rabbit Peyer's patches: Definition of the intraepithelial compartment of the follicle-associated epithelium. Anat Rec 243(1), 19–26.Google Scholar
Rescigno, M. & Di Sabatino, A. (2009). Dendritic cells in intestinal homeostasis and disease. J Clin Invest 119(9), 2441–2450.Google Scholar
Sanchez, J. & Holmgren, J. (2011). Cholera toxin—A foe & a friend. Indian J Med Res 133, 153–163.Google Scholar
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.Y., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P. & Cardona, A. (2012). Fiji: An open-source platform for biological-image analysis. Nat Methods 9(7), 676–682.CrossRefGoogle ScholarPubMed
Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. (2012). NIH image to ImageJ: 25 years of image analysis. Nat Methods 9(7), 671–675.CrossRefGoogle ScholarPubMed
Seymour, R., Shirley, B.J., Hogenesch, H., Shultz, L.D. & Sundberg, J.P. (2013). Loss of function of the mouse sharpin gene results in Peyer's patch regression. PLoS One 8(2), e55224. Google Scholar
Shreedhar, V.K., Kelsall, B.L. & Neutra, M.R. (2003). Cholera toxin induces migration of dendritic cells from the subepithelial dome region to T- and B-cell areas of Peyer's patches. Infect Immun 71(1), 504–509.Google Scholar
Thevenaz, P., Ruttimann, U.E. & Unser, M. (1998). A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process 7(1), 27–41.CrossRefGoogle ScholarPubMed
Xu, C., Shen, Y., Littman, D.R., Dustin, M.L. & Velazquez, P. (2012). Visualization of mucosal homeostasis via single- and multiphoton intravital fluorescence microscopy. J Leukoc Biol 92(3), 413–419.CrossRefGoogle ScholarPubMed

Ahlawat et al. Supplementary Material

3D reconstruction of mouse PP in the presence or absence of CT. Shown are full rotations of 3D structures of mouse PP obtained from a (A) control or (B) CT-treated mouse, as described in the legend to Figure 3. B cells are labeled in blue, T-cells in green and DCs in magenta.

Download Ahlawat et al. Supplementary Material(Video)
Video 6.6 MB

Ahlawat et al. Supplementary Material

3D reconstruction from optical Z sections of PP GCs following challenge with CT. The three images (left to right) each represent 3D reconstructions of approximately 40 optical Z sections corresponding to the proximal, middle and distal segments of a single Peyer’s patch take from a mouse treated with CT, as described in the text. B cells are labeled in magenta and CD3+ T-cells are labeled in blue. These data reveal the migration of B cells from the GC toward the FAE.

Download Ahlawat et al. Supplementary Material(Video)
Video 5.5 MB
Supplementary material: PDF

Ahlawat et al. Supplementary Material

Supplementary Material

Download Ahlawat et al. Supplementary Material(PDF)
PDF 69.8 KB
Supplementary material: File

Ahlawat et al. Supplementary Material

Supplementary Material

Download Ahlawat et al. Supplementary Material(File)
File 1.7 MB