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Prevention of diabetes by FTY720-mediated stabilization of peri-islet tertiary lymphoid organs.

Penaranda C, Tang Q, Ruddle NH, Bluestone JA - Diabetes (2010)

Bottom Line: Treatment withdrawal led to accelerated disease independent of the PLN.Interestingly, naive T-cells trafficked to and proliferated in the TLOs.In addition, morphological changes were observed that occurred during the development of the disease.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center and Department of Medicine, University of California, San Francisco, San Francisco, California, USA.

ABSTRACT

Objective: The nonobese diabetic (NOD) mouse is a well-established mouse model of spontaneous type 1 diabetes, which is characterized by an autoimmune destruction of the insulin-secreting pancreatic beta-cells. In this study, we address the role of tertiary lymphoid organs (TLOs) that form in the pancreas of NOD mice during disease progression.

Methods: We developed a model designed to "lock" lymphocytes in the pancreatic lymph node (PLN) and pancreas by the use of FTY720, which blocks the exit of lymphocytes from lymph nodes. A combination of flow cytometry, immunofluorescence, and analysis of clinical scores was used to study the effects of long-term FTY720 treatment on TLO development and development of diabetes.

Results: Continuous treatment of NOD mice with FTY720 prevented diabetes development even at a time of significant insulitis. Treatment withdrawal led to accelerated disease independent of the PLN. Interestingly, naive T-cells trafficked to and proliferated in the TLOs. In addition, morphological changes were observed that occurred during the development of the disease. Remarkably, although the infiltrates are not organized into T/B-cell compartments in 8-week-old mice, by 20 weeks of age, and in age-matched mice undergoing FTY720 treatment, the infiltrates showed a high degree of organization. However, in naturally and FTY720-induced diabetic mice, T/B-cell compartmentalization was lost.

Conclusion: Our data show that TLOs are established during diabetes development and suggest that islet destruction is due to a loss of TLO integrity, which may be prevented by FTY720 treatment.

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Related in: MedlinePlus

Analysis of TLOs in the pancreas of NOD mice. Pancreas sections from (A) 20-week-old, (B) naturally diabetic, and (C) Rag2 knockout NOD mice were costained with anti-insulin and anti-CD31, anti-ERTR7 and anti-insulin, anti–Lyve-1 and anti-insulin, anti-MAdCAM and anti-insulin, anti-CD4 and anti-B220, or anti–Lyve-1 and MECA79 antibodies. Representative immunofluorescence images of consecutive sections (except for [C]) stained with the indicated antibodies are shown on the top and are superimposed on hematoxylin counterstain bright-field images on the bottom (green fluorophore appears as pink, red fluorophore appears as blue). (D–H) Infiltrates were scored based on their association with insulin-producing islets (column 1), whether there was T/B-cell zone compartmentalization (column 2), and association with Lyve-1+ (column 3), MECA79/PNAd+ (column 4), or MAdCAM+ (column 5) vessels. Each row indicates a separate infiltrate/islet analyzed. Each block indicates a separate mouse. Bar represents 100 μm. *Infiltrates considered to be true TLOs based on T/B compartmentalization and association with at least two of the vascular markers analyzed. (A high-quality digital representation of this figure is available in the online issue).
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Figure 2: Analysis of TLOs in the pancreas of NOD mice. Pancreas sections from (A) 20-week-old, (B) naturally diabetic, and (C) Rag2 knockout NOD mice were costained with anti-insulin and anti-CD31, anti-ERTR7 and anti-insulin, anti–Lyve-1 and anti-insulin, anti-MAdCAM and anti-insulin, anti-CD4 and anti-B220, or anti–Lyve-1 and MECA79 antibodies. Representative immunofluorescence images of consecutive sections (except for [C]) stained with the indicated antibodies are shown on the top and are superimposed on hematoxylin counterstain bright-field images on the bottom (green fluorophore appears as pink, red fluorophore appears as blue). (D–H) Infiltrates were scored based on their association with insulin-producing islets (column 1), whether there was T/B-cell zone compartmentalization (column 2), and association with Lyve-1+ (column 3), MECA79/PNAd+ (column 4), or MAdCAM+ (column 5) vessels. Each row indicates a separate infiltrate/islet analyzed. Each block indicates a separate mouse. Bar represents 100 μm. *Infiltrates considered to be true TLOs based on T/B compartmentalization and association with at least two of the vascular markers analyzed. (A high-quality digital representation of this figure is available in the online issue).

Mentions: ERTR7 reactivity and CD31+ vessels were observed surrounding all islets and throughout the acinar tissue at all time points analyzed (Fig. 2A and data not shown). In addition, ERTR7 reactivity was found within the infiltrate, suggesting that these structures have a supporting stromal network, as is seen in T-cell zones in the spleen and lymph nodes (14). However, although the results trend toward an increase in the association of the presence of MAdCAM+ or Lyve+ vessels and disease progression, these differences were not statistically significant (supplementary Fig. 2B). In addition, most infiltrates in both pre-diabetic and naturally diabetic mice contained PNAd+ HEVs (Fig. 2D–G), suggesting that, like secondary lymphoid organs, these structures are capable of recruiting naive T-cells. Analysis of at least three consecutive sections showed that infiltrates of pre-diabetic 20-week-old mice were usually, but not always, associated with insulin-producing islets (Fig. 2A and E). Although T- and B-cells were present in the pancreas of 8-week-old mice, only 15% (3/20) of infiltrates showed clear compartmentalization, whereas 89% (24/27) of infiltrates in 20-week-old mice showed distinct T- and B-cell zones. In the majority of these, the T-cell zone was closer to the islet, whereas the B-cell zone was on the edge of the infiltrate, reminiscent of lymph nodes (Fig. 2A). Only 15% of infiltrates (3/19) in naturally diabetic mice showed T/B-cell compartmentalization, which correlated with an absence of insulin-positive islets that we attribute to destroyed β-cells (Fig. 2B and G). However, β-cells may have been present but not producing insulin because of inflammation (15). True TLOs, defined as infiltrates with T/B compartmentalization and association with at least two of the vascular markers analyzed, were present in 15% (3/20) of infiltrates in 8-week-old mice, increasing to 81% (22/27) of infiltrates in 20-week-old mice but decreasing to only 26% (5/19) of infiltrates in naturally diabetic mice (Fig. 2D–F). Therefore, TLOs peaked as a proportion of the infiltrate at the “peak” of the inflammatory response before diabetes onset.


Prevention of diabetes by FTY720-mediated stabilization of peri-islet tertiary lymphoid organs.

Penaranda C, Tang Q, Ruddle NH, Bluestone JA - Diabetes (2010)

Analysis of TLOs in the pancreas of NOD mice. Pancreas sections from (A) 20-week-old, (B) naturally diabetic, and (C) Rag2 knockout NOD mice were costained with anti-insulin and anti-CD31, anti-ERTR7 and anti-insulin, anti–Lyve-1 and anti-insulin, anti-MAdCAM and anti-insulin, anti-CD4 and anti-B220, or anti–Lyve-1 and MECA79 antibodies. Representative immunofluorescence images of consecutive sections (except for [C]) stained with the indicated antibodies are shown on the top and are superimposed on hematoxylin counterstain bright-field images on the bottom (green fluorophore appears as pink, red fluorophore appears as blue). (D–H) Infiltrates were scored based on their association with insulin-producing islets (column 1), whether there was T/B-cell zone compartmentalization (column 2), and association with Lyve-1+ (column 3), MECA79/PNAd+ (column 4), or MAdCAM+ (column 5) vessels. Each row indicates a separate infiltrate/islet analyzed. Each block indicates a separate mouse. Bar represents 100 μm. *Infiltrates considered to be true TLOs based on T/B compartmentalization and association with at least two of the vascular markers analyzed. (A high-quality digital representation of this figure is available in the online issue).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Analysis of TLOs in the pancreas of NOD mice. Pancreas sections from (A) 20-week-old, (B) naturally diabetic, and (C) Rag2 knockout NOD mice were costained with anti-insulin and anti-CD31, anti-ERTR7 and anti-insulin, anti–Lyve-1 and anti-insulin, anti-MAdCAM and anti-insulin, anti-CD4 and anti-B220, or anti–Lyve-1 and MECA79 antibodies. Representative immunofluorescence images of consecutive sections (except for [C]) stained with the indicated antibodies are shown on the top and are superimposed on hematoxylin counterstain bright-field images on the bottom (green fluorophore appears as pink, red fluorophore appears as blue). (D–H) Infiltrates were scored based on their association with insulin-producing islets (column 1), whether there was T/B-cell zone compartmentalization (column 2), and association with Lyve-1+ (column 3), MECA79/PNAd+ (column 4), or MAdCAM+ (column 5) vessels. Each row indicates a separate infiltrate/islet analyzed. Each block indicates a separate mouse. Bar represents 100 μm. *Infiltrates considered to be true TLOs based on T/B compartmentalization and association with at least two of the vascular markers analyzed. (A high-quality digital representation of this figure is available in the online issue).
Mentions: ERTR7 reactivity and CD31+ vessels were observed surrounding all islets and throughout the acinar tissue at all time points analyzed (Fig. 2A and data not shown). In addition, ERTR7 reactivity was found within the infiltrate, suggesting that these structures have a supporting stromal network, as is seen in T-cell zones in the spleen and lymph nodes (14). However, although the results trend toward an increase in the association of the presence of MAdCAM+ or Lyve+ vessels and disease progression, these differences were not statistically significant (supplementary Fig. 2B). In addition, most infiltrates in both pre-diabetic and naturally diabetic mice contained PNAd+ HEVs (Fig. 2D–G), suggesting that, like secondary lymphoid organs, these structures are capable of recruiting naive T-cells. Analysis of at least three consecutive sections showed that infiltrates of pre-diabetic 20-week-old mice were usually, but not always, associated with insulin-producing islets (Fig. 2A and E). Although T- and B-cells were present in the pancreas of 8-week-old mice, only 15% (3/20) of infiltrates showed clear compartmentalization, whereas 89% (24/27) of infiltrates in 20-week-old mice showed distinct T- and B-cell zones. In the majority of these, the T-cell zone was closer to the islet, whereas the B-cell zone was on the edge of the infiltrate, reminiscent of lymph nodes (Fig. 2A). Only 15% of infiltrates (3/19) in naturally diabetic mice showed T/B-cell compartmentalization, which correlated with an absence of insulin-positive islets that we attribute to destroyed β-cells (Fig. 2B and G). However, β-cells may have been present but not producing insulin because of inflammation (15). True TLOs, defined as infiltrates with T/B compartmentalization and association with at least two of the vascular markers analyzed, were present in 15% (3/20) of infiltrates in 8-week-old mice, increasing to 81% (22/27) of infiltrates in 20-week-old mice but decreasing to only 26% (5/19) of infiltrates in naturally diabetic mice (Fig. 2D–F). Therefore, TLOs peaked as a proportion of the infiltrate at the “peak” of the inflammatory response before diabetes onset.

Bottom Line: Treatment withdrawal led to accelerated disease independent of the PLN.Interestingly, naive T-cells trafficked to and proliferated in the TLOs.In addition, morphological changes were observed that occurred during the development of the disease.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center and Department of Medicine, University of California, San Francisco, San Francisco, California, USA.

ABSTRACT

Objective: The nonobese diabetic (NOD) mouse is a well-established mouse model of spontaneous type 1 diabetes, which is characterized by an autoimmune destruction of the insulin-secreting pancreatic beta-cells. In this study, we address the role of tertiary lymphoid organs (TLOs) that form in the pancreas of NOD mice during disease progression.

Methods: We developed a model designed to "lock" lymphocytes in the pancreatic lymph node (PLN) and pancreas by the use of FTY720, which blocks the exit of lymphocytes from lymph nodes. A combination of flow cytometry, immunofluorescence, and analysis of clinical scores was used to study the effects of long-term FTY720 treatment on TLO development and development of diabetes.

Results: Continuous treatment of NOD mice with FTY720 prevented diabetes development even at a time of significant insulitis. Treatment withdrawal led to accelerated disease independent of the PLN. Interestingly, naive T-cells trafficked to and proliferated in the TLOs. In addition, morphological changes were observed that occurred during the development of the disease. Remarkably, although the infiltrates are not organized into T/B-cell compartments in 8-week-old mice, by 20 weeks of age, and in age-matched mice undergoing FTY720 treatment, the infiltrates showed a high degree of organization. However, in naturally and FTY720-induced diabetic mice, T/B-cell compartmentalization was lost.

Conclusion: Our data show that TLOs are established during diabetes development and suggest that islet destruction is due to a loss of TLO integrity, which may be prevented by FTY720 treatment.

Show MeSH
Related in: MedlinePlus