Diabetologia

, Volume 56, Issue 12, pp 2669–2678 | Cite as

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

  • Anja Schmidt-Christensen
  • Lisbeth Hansen
  • Erwin Ilegems
  • Nina Fransén-Pettersson
  • Ulf Dahl
  • Shashank Gupta
  • Åsa Larefalk
  • Tine D. Hannibal
  • Alexander Schulz
  • Per-Olof Berggren
  • Dan Holmberg
Article

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.

Keywords

Animal–mouse Imaging Islet degeneration and damage Islet transplantation 

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

Supplementary material

125_2013_3024_MOESM1_ESM.pdf (1.3 mb)
ESM Methods(PDF 1347 kb)
125_2013_3024_MOESM2_ESM.pdf (77 kb)
ESM Table 1(PDF 76 kb)
125_2013_3024_MOESM3_ESM.pdf (144 kb)
ESM Table 2(PDF 144 kb)
125_2013_3024_MOESM4_ESM.pdf (4.8 mb)
ESM Fig. 1(PDF 4930 kb)
125_2013_3024_MOESM5_ESM.pdf (4 mb)
ESM Fig. 2(PDF 4128 kb)
125_2013_3024_MOESM6_ESM.pdf (5.8 mb)
ESM Fig. 3(PDF 5979 kb)
125_2013_3024_MOESM7_ESM.pdf (8.3 mb)
ESM Fig. 4(PDF 8510 kb)
125_2013_3024_MOESM8_ESM.pdf (2.1 mb)
ESM Fig. 5(PDF 2127 kb)
125_2013_3024_MOESM9_ESM.pdf (4.5 mb)
ESM Fig. 6(PDF 4609 kb)
125_2013_3024_MOESM10_ESM.pdf (7.2 mb)
ESM Fig. 7(PDF 7348 kb)
125_2013_3024_MOESM11_ESM.mov (1.4 mb)
ESM Video 1Time-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)
125_2013_3024_MOESM12_ESM.mov (5.3 mb)
ESM Video 2Time-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)
125_2013_3024_MOESM13_ESM.mov (5.2 mb)
ESM Video 3Three 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)

125_2013_3024_MOESM15_ESM.mov (6.3 mb)
ESM Video 5Non-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)
125_2013_3024_MOESM16_ESM.mov (7.4 mb)
ESM Video 6Non-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)
125_2013_3024_MOESM17_ESM.mov (1.6 mb)
ESM Video 7Non-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)

125_2013_3024_MOESM20_ESM.mov (3.2 mb)
ESM Video 10Non-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)
125_2013_3024_MOESM21_ESM.mov (4.1 mb)
ESM Video 11Non-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)
125_2013_3024_MOESM22_ESM.mov (7.4 mb)
ESM Video 12Non-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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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Anja Schmidt-Christensen
    • 1
    • 2
  • Lisbeth Hansen
    • 1
    • 2
  • Erwin Ilegems
    • 3
  • Nina Fransén-Pettersson
    • 1
    • 2
  • Ulf Dahl
    • 4
  • Shashank Gupta
    • 1
    • 2
  • Åsa Larefalk
    • 4
  • Tine D. Hannibal
    • 1
    • 2
  • Alexander Schulz
    • 5
  • Per-Olof Berggren
    • 3
  • Dan Holmberg
    • 1
    • 2
    • 4
  1. 1.ISIM–Immunology, Faculty of Health and Medical SciencesCopenhagen UniversityCopenhagenDenmark
  2. 2.EMV–ImmunologyLund UniversityLundSweden
  3. 3.The Rolf Luft Research Center for Diabetes and EndocrinologyKarolinska InstitutetStockholmSweden
  4. 4.Department of Clinical Microbiology, Immunology, Faculty of MedicineUmeå UniversityUmeåSweden
  5. 5.Imaging Core Facility, Faculty of Health and Medical SciencesCopenhagen UniversityFrederiksbergDenmark

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