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Autoreactive T cells induce necrosis and not BCL-2-regulated or death receptor-mediated apoptosis or RIPK3-dependent necroptosis of transplanted islets in a mouse model of type 1 diabetes

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Abstract

Aims/hypothesis

Type 1 diabetes results from T cell-mediated destruction of pancreatic beta cells. The mechanisms of beta cell destruction in vivo, however, remain unclear. We aimed to test the relative roles of the main cell death pathways: apoptosis, necrosis and necroptosis, in beta cell death in the development of CD4+ T cell-mediated autoimmune diabetes.

Methods

We altered expression levels of critical cell death proteins in mouse islets and tested their ability to survive CD4+ T cell-mediated attack using an in vivo graft model.

Results

Loss of the B cell leukaemia/lymphoma 2 (BCL-2) homology domain 3-only proteins BIM, PUMA or BID did not protect beta cells from this death. Overexpression of the anti-apoptotic protein BCL-2 or combined deficiency of the pro-apoptotic multi-BCL2 homology domain proteins BAX and BAK also failed to prevent beta cell destruction. Furthermore, loss of function of the death receptor Fas or its essential downstream signalling molecule Fas-associated death domain (FADD) in islets was also not protective. Using electron microscopy we observed that dying beta cells showed features of necrosis. However, islets deficient in receptor-interacting serine/threonine protein kinase 3 (RIPK3), a critical initiator of necroptosis, were still normally susceptible to CD4+ T cell-mediated destruction. Remarkably, simultaneous inhibition of apoptosis and necroptosis by combining loss of RIPK3 and overexpression of BCL-2 in islets did not protect them against immune attack either.

Conclusions/interpretation

Collectively, our data indicate that beta cells die by necrosis in autoimmune diabetes and that the programmed cell death pathways apoptosis and necroptosis are both dispensable for this process.

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Abbreviations

BAK:

BCL-2 antagonist/killer

BAX:

BCL-2-associated X protein

BCL-2:

B cell leukaemia/lymphoma 2

BH:

BCL-2 homology domain

BID:

BH3-interacting domain death agonist

BIM:

BCL-2-like 11 (apoptosis facilitator)

ER:

Endoplasmic reticulum

FADD:

Fas-associated death domain

FasL:

Fas ligand

IVIS:

In vivo imaging system

MLKL:

Mixed lineage kinase domain-like

PUMA:

BCL-2-binding component 3

RIP:

Rat insulin promoter

RIPK:

Receptor-interacting serine/threonine protein kinase

ROS:

Reactive oxygen species

TEM:

Transmission electron microscopy

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Acknowledgements

We thank the following people: J. Allison (St Vincent’s Institute, Fitzroy, VIC), J. Silke, P. Bouillet, S. Cory, J. M. Adams, W. Alexander, J. Murphy (all WEHI, Parkville, VIC), A. Villunger (University of Innsbruck, Innsbruck, Austria), S Hedrick (UCSD, San Diego, CA), A Powers (Vanderbilt University, Nashville, TN), D. Mathis (Joslin Diabetes Center, Boston, MA) and K. Newton (Genentech, South San Francisco, CA) for gifts of mice; O. Kanagawa (Osaka University, Osaka, Japan) for BDC2.5 antibody; S. Thorburn, T. Smith, E. Duff, D. Novembre-Cycon, R. Branch and A. Gomes (St Vincent’s Institute, Fitzroy, VIC) for genotyping and animal husbandry and S. Ellis, S. Asquith and J. Danne (all Peter MacCallum Cancer Center, East Melbourne, VIC) for electron microscopy. These data were presented as an abstract at the Immunology of Diabetes Society meeting in 2013.

Funding

This work was funded by a National Health and Medical Research Council of Australia (NHMRC) and Juvenile Diabetes Research Foundation (JDRF) joint special program grant in type 1 diabetes, program grants (HET, TWHK and AS) and fellowships from the NHMRC (HET and AS), a Leukemia and Lymphoma Society SCOR grant (AS) and a Postdoctoral Fellowship from the JDRF (YZ). The St Vincent’s Institute and The Walter and Eliza Hall Institute receive support from the Operational Infrastructure Support Scheme of the Government of Victoria. This work was made possible through a Victorian State Government OIS grant and an Australian NHMRC IRIIS grant.

Contribution statement

YZ, NAS, SF and LE performed experiments, analysed data and critically revised the manuscript. YZ, NAS and HET designed the study and wrote the manuscript. WWW, KDM, AS, DCH and TWHK contributed reagents, contributed to conception, design and interpretation of this work and critically revised the manuscript. All authors approved the final version of the manuscript. HET is responsible for the integrity of the work as a whole.

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

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Author notes

  1. W. Wei-Lynn Wong

    Present address: Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland

Authors and Affiliations

  1. St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Melbourne, VIC, 3065, Australia

    Yuxing Zhao, Nicholas A. Scott, Stacey Fynch, Lorraine Elkerbout, Thomas W. H. Kay & Helen E. Thomas

  2. The University of Melbourne Department of Medicine, St Vincent’s Hospital, Fitzroy, Melbourne, VIC, Australia

    Nicholas A. Scott, Thomas W. H. Kay & Helen E. Thomas

  3. The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia

    W. Wei-Lynn Wong, Kylie D. Mason, Andreas Strasser & David C. Huang

  4. Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia

    Kylie D. Mason, Andreas Strasser & David C. Huang

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  1. Yuxing Zhao
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Correspondence to Helen E. Thomas.

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Zhao, Y., Scott, N.A., Fynch, S. et al. Autoreactive T cells induce necrosis and not BCL-2-regulated or death receptor-mediated apoptosis or RIPK3-dependent necroptosis of transplanted islets in a mouse model of type 1 diabetes. Diabetologia 58, 140–148 (2015). https://doi.org/10.1007/s00125-014-3407-5

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