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

Abstract 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 evalu...

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Autores principales:	Alexander J. Dwyer, Jacob M. Ritz, Jason S. Mitchell, Tijana Martinov, Mohannad Alkhatib, Nubia Silva, Christopher G. Tucker, Brian T. Fife
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/6ed0acc70c384d58b92cd026ebe5a4ee
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spelling	oai:doaj.org-article:6ed0acc70c384d58b92cd026ebe5a4ee2021-12-02T18:53:14ZEnhanced CD4+ and CD8+ T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses10.1038/s41598-021-96327-22045-2322https://doaj.org/article/6ed0acc70c384d58b92cd026ebe5a4ee2021-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-96327-2https://doaj.org/toc/2045-2322Abstract 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.Alexander J. DwyerJacob M. RitzJason S. MitchellTijana MartinovMohannad AlkhatibNubia SilvaChristopher G. TuckerBrian T. FifeNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-17 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Alexander J. Dwyer Jacob M. Ritz Jason S. Mitchell Tijana Martinov Mohannad Alkhatib Nubia Silva Christopher G. Tucker Brian T. Fife Enhanced CD4+ and CD8+ T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses
description	Abstract 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.
format	article
author	Alexander J. Dwyer Jacob M. Ritz Jason S. Mitchell Tijana Martinov Mohannad Alkhatib Nubia Silva Christopher G. Tucker Brian T. Fife
author_facet	Alexander J. Dwyer Jacob M. Ritz Jason S. Mitchell Tijana Martinov Mohannad Alkhatib Nubia Silva Christopher G. Tucker Brian T. Fife
author_sort	Alexander J. Dwyer
title	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_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_sort	enhanced cd4+ and cd8+ t cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/6ed0acc70c384d58b92cd026ebe5a4ee
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Enhanced CD4+ and CD8+ T cell infiltrate within convex hull defined pancreatic islet borders as autoimmune diabetes progresses

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