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Enhanced CD4(+) and CD8(+) T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses

The notion that T cell insulitis increases as type 1 diabetes (T1D) develops is unsurprising, however, the quantitative analysis of CD4(+) and CD8(+) T cells within the islet mass is complex and limited with standard approaches. Optical microscopy is an important and widely used method to evaluate i...

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Autores principales: Dwyer, Alexander J., Ritz, Jacob M., Mitchell, Jason S., Martinov, Tijana, Alkhatib, Mohannad, Silva, Nubia, Tucker, Christopher G., Fife, Brian T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387412/
https://www.ncbi.nlm.nih.gov/pubmed/34433860
http://dx.doi.org/10.1038/s41598-021-96327-2
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author Dwyer, Alexander J.
Ritz, Jacob M.
Mitchell, Jason S.
Martinov, Tijana
Alkhatib, Mohannad
Silva, Nubia
Tucker, Christopher G.
Fife, Brian T.
author_facet Dwyer, Alexander J.
Ritz, Jacob M.
Mitchell, Jason S.
Martinov, Tijana
Alkhatib, Mohannad
Silva, Nubia
Tucker, Christopher G.
Fife, Brian T.
author_sort Dwyer, Alexander J.
collection PubMed
description The notion that T cell insulitis increases as type 1 diabetes (T1D) develops is unsurprising, however, the quantitative analysis of CD4(+) and CD8(+) T cells within the islet mass is complex and limited with standard approaches. Optical microscopy is an important and widely used method to evaluate immune cell infiltration into pancreatic islets of Langerhans for the study of disease progression or therapeutic efficacy in murine T1D. However, the accuracy of this approach is often limited by subjective and potentially biased qualitative assessment of immune cell subsets. In addition, attempts at quantitative measurements require significant time for manual analysis and often involve sophisticated and expensive imaging software. In this study, we developed and illustrate here a streamlined analytical strategy for the rapid, automated and unbiased investigation of islet area and immune cell infiltration within (insulitis) and around (peri-insulitis) pancreatic islets. To this end, we demonstrate swift and accurate detection of islet borders by modeling cross-sectional islet areas with convex polygons (convex hulls) surrounding islet-associated insulin-producing β cell and glucagon-producing α cell fluorescent signals. To accomplish this, we used a macro produced with the freeware software ImageJ equipped with the Fiji Is Just ImageJ (FIJI) image processing package. Our image analysis procedure allows for direct quantification and statistical determination of islet area and infiltration in a reproducible manner, with location-specific data that more accurately reflect islet areas as insulitis proceeds throughout T1D. Using this approach, we quantified the islet area infiltrated with CD4(+) and CD8(+) T cells allowing statistical comparison between different age groups of non-obese diabetic (NOD) mice progressing towards T1D. We found significantly more CD4(+) and CD8(+) T cells infiltrating the convex hull-defined islet mass of 13-week-old non-diabetic and 17-week-old diabetic NOD mice compared to 4-week-old NOD mice. We also determined a significant and measurable loss of islet mass in mice that developed T1D. This approach will be helpful for the location-dependent quantitative calculation of islet mass and cellular infiltration during T1D pathogenesis and can be combined with other markers of inflammation or activation in future studies.
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spelling pubmed-83874122021-09-01 Enhanced CD4(+) and CD8(+) T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses Dwyer, Alexander J. Ritz, Jacob M. Mitchell, Jason S. Martinov, Tijana Alkhatib, Mohannad Silva, Nubia Tucker, Christopher G. Fife, Brian T. Sci Rep Article The notion that T cell insulitis increases as type 1 diabetes (T1D) develops is unsurprising, however, the quantitative analysis of CD4(+) and CD8(+) T cells within the islet mass is complex and limited with standard approaches. Optical microscopy is an important and widely used method to evaluate immune cell infiltration into pancreatic islets of Langerhans for the study of disease progression or therapeutic efficacy in murine T1D. However, the accuracy of this approach is often limited by subjective and potentially biased qualitative assessment of immune cell subsets. In addition, attempts at quantitative measurements require significant time for manual analysis and often involve sophisticated and expensive imaging software. In this study, we developed and illustrate here a streamlined analytical strategy for the rapid, automated and unbiased investigation of islet area and immune cell infiltration within (insulitis) and around (peri-insulitis) pancreatic islets. To this end, we demonstrate swift and accurate detection of islet borders by modeling cross-sectional islet areas with convex polygons (convex hulls) surrounding islet-associated insulin-producing β cell and glucagon-producing α cell fluorescent signals. To accomplish this, we used a macro produced with the freeware software ImageJ equipped with the Fiji Is Just ImageJ (FIJI) image processing package. Our image analysis procedure allows for direct quantification and statistical determination of islet area and infiltration in a reproducible manner, with location-specific data that more accurately reflect islet areas as insulitis proceeds throughout T1D. Using this approach, we quantified the islet area infiltrated with CD4(+) and CD8(+) T cells allowing statistical comparison between different age groups of non-obese diabetic (NOD) mice progressing towards T1D. We found significantly more CD4(+) and CD8(+) T cells infiltrating the convex hull-defined islet mass of 13-week-old non-diabetic and 17-week-old diabetic NOD mice compared to 4-week-old NOD mice. We also determined a significant and measurable loss of islet mass in mice that developed T1D. This approach will be helpful for the location-dependent quantitative calculation of islet mass and cellular infiltration during T1D pathogenesis and can be combined with other markers of inflammation or activation in future studies. Nature Publishing Group UK 2021-08-25 /pmc/articles/PMC8387412/ /pubmed/34433860 http://dx.doi.org/10.1038/s41598-021-96327-2 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Dwyer, Alexander J.
Ritz, Jacob M.
Mitchell, Jason S.
Martinov, Tijana
Alkhatib, Mohannad
Silva, Nubia
Tucker, Christopher G.
Fife, Brian T.
Enhanced CD4(+) and CD8(+) T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses
title Enhanced CD4(+) and CD8(+) T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses
title_full Enhanced CD4(+) and CD8(+) T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses
title_fullStr Enhanced CD4(+) and CD8(+) T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses
title_full_unstemmed Enhanced CD4(+) and CD8(+) T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses
title_short Enhanced CD4(+) and CD8(+) T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses
title_sort enhanced cd4(+) and cd8(+) t cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387412/
https://www.ncbi.nlm.nih.gov/pubmed/34433860
http://dx.doi.org/10.1038/s41598-021-96327-2
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