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Imaging dynamics of CD11c+ cells and Foxp3+ cells in progressive autoimmune insulitis in the NOD mouse model of type 1 diabetes

Abstract

Aims/hypothesis

The aim of this study was to visualise the dynamics and interactions of the cells involved in autoimmune-driven inflammation in type 1 diabetes.

Methods

We adopted the anterior chamber of the eye (ACE) transplantation model to perform non-invasive imaging of leucocytes infiltrating the endocrine pancreas during initiation and progression of insulitis in the NOD mouse. Individual, ACE-transplanted islets of Langerhans were longitudinally and repetitively imaged by stereomicroscopy and two-photon microscopy to follow fluorescently labelled leucocyte subsets.

Results

We demonstrate that, in spite of the immune privileged status of the eye, the ACE-transplanted islets develop infiltration and beta cell destruction, recapitulating the autoimmune insulitis of the pancreas, and exemplify this by analysing reporter cell populations expressing green fluorescent protein under the Cd11c or Foxp3 promoters. We also provide evidence that differences in morphological appearance of subpopulations of infiltrating leucocytes can be correlated to their distinct dynamic behaviour.

Conclusions/interpretation

Together, these findings demonstrate that the kinetics and dynamics of these key cellular components of autoimmune diabetes can be elucidated using this imaging platform for single cell resolution, non-invasive and repetitive monitoring of the individual islets of Langerhans during the natural development of autoimmune diabetes.

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Abbreviations

ACE:

Anterior chamber of the eye

B6:

C57BL/6

DC:

Dendritic cell

GFP:

Green fluorescent protein

IHC:

Immunohistochemical

LYVE-1:

Lymphatic vessel endothelial hyaluronan receptor 1

NK:

Natural killer

pDC:

Plasmacytoid dendritic cell

TBS:

TRIS-buffered saline

Treg:

Regulatory T cell

TxRed:

Texas Red

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Acknowledgements

Imaging data were collected at the Center for Advanced Bioimaging (CAB) Denmark, University of Copenhagen, and we would like to thank Michael Hansen (CAB, University of Copenhagen, Denmark) for his excellent technical assistance. B6 Rag2 −/− was a kind gift from Fred W. Alt (Boston Children’s Hospital, Boston, MA, USA).

Funding

This work was supported by grants from the European Commission (VIBRANT CP-IP 228933-2), the Danish Research Council, Lundbeckfonden and the Swedish Research Council. LH was supported by a PhD fellowship from the Lundbeck foundation.

Duality of interest

P-OB is founder and CEO of the Biotech Company Biocrine AB. He is also a member of the board of that company. EI is a consultant of Biocrine AB.

Contribution statement

AS-C, LH and DH contributed to the conception and design of the experiments; AS-C and LH collected the data; and AS-C, LH, EI, NF-P, UD, SG, ÅL, TDH, AS, P-OB and DH contributed to the analysis and interpretation of the data. All authors contributed to the drafting of the article and revising it critically, and gave final approval of the version to be published.

Author information

Correspondence to Dan Holmberg.

Additional information

Anja Schmidt-Christensen and Lisbeth Hansen contributed equally to this study.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Foxp3-GFP+ cell exiting blood vessel into pancreatic parenchyma. Sequential three-dimensional rendering of snapshots shown in Figure 4b and ESM video 3. Each time point is shown in a three dimensional rotation. (MOV 6243 kb)

Repeated non-invasive 2-photon imaging of Foxp3-GFP cells. Time-lapse recordings (26 min, 18 min and 30 min) of the same intraocular islet showing graft-infiltrating GFP-labeled Foxp3+ cells at 3 weeks, 4 weeks and 5 weeks post adoptive transfer. Video is shown as maximum projection and blood vessels are shown in red. Time resolution: 25 sec, scale bar: 100 μm. Related images are shown in ESM Fig. 5. (MOV 5692 kb)

Foxp3-GFP cells moving along blood vessels in the pancreatic parenchyma. Time-lapse recording (40 min) of Foxp3-GFP+ cells moving along blood vessels in the pancreatic parenchyma. Video (shown as maximum projection) was cropped to area of interest (x,y) from ESM video 5. Foxp3-GFP+ cells show the typical elongated shape of fast moving cells. It also shows short-term interaction between GFP-labeled cells in the beginning and the end of the video. Blood vessels are visualized in red. (MOV 2495 kb)

ESM Methods

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ESM Table 1

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ESM Table 2

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ESM Fig. 1

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ESM Fig. 2

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ESM Fig. 3

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ESM Fig. 4

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ESM Fig. 5

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ESM Fig. 6

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ESM Fig. 7

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ESM Video 1

Time-lapse recording (1 min 24 sec) of an intraocular islet showing graft-infiltrating Foxp3-GFP+ cells rolling within blood vessel indicated by arrows. Snapshots are shown in Fig. 4a. Experimental setup: islets isolated from immunodeficient NOD Rag2 -/- mice were transplanted into the ACE of a recipient NOD Foxp3-Gfp reporter mouse. Engrafted islet was imaged 8 days post transplantation. Blood vessels are shown in red. Time resolution: 2 seconds, stack size 10 μm. Video is shown as maximum projection of image z-stacks. (MOV 1432 kb)

ESM Video 2

Time-lapse recording (1 min 15 sec) of an intraocular islet showing graft-infiltrating Foxp3-GFP+ cells rolling within blood vessel, indicated by arrows. Experimental setup and image acquisition: same as in ESM video 1. (MOV 5429 kb)

ESM Video 3

Three dimensional rendering of a recording (7:30 min) showing a Foxp3-GFP+ cell exciting a blood vessel into the pancreatic parenchyma. Video xy-dimensions and timeframe (20:00:45 = 0 min) were cropped to the area of interest from ESM video 5. Snapshots with GFP+ cell of interest are shown in Figure 4b. (MOV 5320 kb)

ESM Video 4

Foxp3-GFP+ cell exiting blood vessel into pancreatic parenchyma. Sequential three-dimensional rendering of snapshots shown in Figure 4b and ESM video 3. Each time point is shown in a three dimensional rotation. (MOV 6243 kb)

ESM Video 5

Non-invasive 2-photon imaging and tracking of Foxp3-GFP+ cells. Time-lapse recording (40 min) of an intraocular islet showing graft-infiltrating Foxp3-GFP+ cells patrolling the site of inflammation. Three different Foxp3-GFP+ cell morphologies could be distinguished: round, ruffled and elongated, indicated by red, orange and purple trajectories. Experimental setup: islets were transplanted into the ACE of recipient NOD Rag2 -/- mice followed by adoptive transfer with spleen cells from 3w-old NOD Foxp3-Gfp reporter mice. Engrafted islet was imaged 8 weeks post adoptive transfer. Video is shown as maximum projection of image z-stacks. Blood vessels are shown in red. Time resolution: 25 sec, scale bar: 100 μm. Related images are shown in Fig. 5. A previous 2-photon time-lapse recording was performed 7 weeks post adoptive transfer and is seen in ESM video 6 and analyzed in EMS Fig. 4. (MOV 6410 kb)

ESM Video 6

Non-invasive 2-photon imaging and tracking of Foxp3+ cells. Time-lapse recording (40 min) of an intraocular islet (same as in ESM video 5) showing graft-infiltrating GFP-labeled Foxp3+ cells. Round, ruffled and elongated Foxp3-GFP+ cell morphologies are indicated by red, orange and purple trajectories, respectively. Engrafted islet was imaged 7 weeks post adoptive transfer. Video is shown as maximum projection and blood vessels are shown in red. Time resolution: 25 sec, scale bar: 100 μm. Related images and tracking analysis are shown in ESM Fig. 4. (MOV 7601 kb)

ESM Video 7

Non-invasive 2-photon imaging and tracking of Foxp3-GFP+ cells. Time-lapse recording (15 min) of an intraocular islet (same recipient mouse as in ESM movie 5) showing graft-infiltrating Foxp3-GFP+ cells. Round, ruffled and elongated Foxp3-GFP+ cell morphologies are indicated by red, orange and purple trajectories, respectively. Engrafted islet was imaged 7 weeks post adoptive transfer. Video is shown as maximum projection and blood vessels are shown in red. Time resolution: 25 sec, scale bar: 100 μm. Related images as well as tracking analysis are shown in ESM Fig. 4. (MOV 1658 kb)

ESM Video 8

Repeated non-invasive 2-photon imaging of Foxp3-GFP cells. Time-lapse recordings (26 min, 18 min and 30 min) of the same intraocular islet showing graft-infiltrating GFP-labeled Foxp3+ cells at 3 weeks, 4 weeks and 5 weeks post adoptive transfer. Video is shown as maximum projection and blood vessels are shown in red. Time resolution: 25 sec, scale bar: 100 μm. Related images are shown in ESM Fig. 5. (MOV 5692 kb)

ESM Video 9

Foxp3-GFP cells moving along blood vessels in the pancreatic parenchyma. Time-lapse recording (40 min) of Foxp3-GFP+ cells moving along blood vessels in the pancreatic parenchyma. Video (shown as maximum projection) was cropped to area of interest (x,y) from ESM video 5. Foxp3-GFP+ cells show the typical elongated shape of fast moving cells. It also shows short-term interaction between GFP-labeled cells in the beginning and the end of the video. Blood vessels are visualized in red. (MOV 2495 kb)

ESM Video 10

Non-invasive 2-photon imaging and tracking of CD11c-GFP+ cells. Time-lapse recording (20 min) of an intraocular islet showing a major population of graft-infiltrated CD11c-GFP+ cells with typical DC morphology and a minor population of CD11c-GFP cells with round cell morphology moving in the periphery and in close association to the islet. Experimental setup: nondiabetic NOD Cd11c-Gfp recipient mouse previously transplanted with NOD Rag2 -/- islets into the ACE was imaged using 2-photon laser microscopy 7 days post transplantation. Blood vessels are shown in red. Time resolution: 30 sec, scale bar = 100 μm. Related images are shown in Fig. 6 and a subsequent time-lapse recording of the same intraocular islet at 14 days post transplantation is shown in ESM video 11. (MOV 3270 kb)

ESM Video 11

Non-invasive 2-photon imaging and tracking of CD11c-GFP+ cells. Time-lapse recording (20 min) of the same intraocular islet as ESM video 10 and Fig. 6 but 14 days post transplantation. Experimental setup: nondiabetic NOD Cd11c-Gfp recipient mouse previously transplanted with NOD Rag2 -/- islets into the ACE was imaged using 2-photon laser microscopy 14 days post transplantation. Blood vessels are shown in red. Time resolution: 30 sec, scale bar = 100 μm. Related images and tracking analysis are shown in ESM Fig. 6. (MOV 4210 kb)

ESM Video 12

Non-invasive 2-photon imaging and tracking of CD11c-GFP+ cells. Time-lapse recording (20 min) of a second islet, but in the same mouse as ESM video 11. Experimental setup: nondiabetic NOD Cd11c-Gfp recipient mouse previously transplanted with NOD Rag2 -/- islets into the ACE was imaged using 2-photon laser microscopy 14 days post transplantation. Blood vessels are shown in red. Time resolution: 30 sec, scale bar = 100 μm. Related images and tracking analysis are shown in ESM Fig. 6. (MOV 7551 kb)

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Schmidt-Christensen, A., Hansen, L., Ilegems, E. et al. Imaging dynamics of CD11c+ cells and Foxp3+ cells in progressive autoimmune insulitis in the NOD mouse model of type 1 diabetes. Diabetologia 56, 2669–2678 (2013) doi:10.1007/s00125-013-3024-8

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Keywords

  • Animal–mouse
  • Imaging
  • Islet degeneration and damage
  • Islet transplantation