Impact of hyperglycemia on autoimmune pancreatitis and regulatory T-cells

Abstract

Aim: To evaluate the influence of hyperglycemia on the progression of autoimmune pancreatitis.

Methods: We induced hyperglycemia by repetitive intraperitoneal (ip) injection of 50 mg/kg streptozotocin in MRL/MpJ mice, which develop autoimmune pancreatitis due to a genetic predisposition. We compared the extent of inflammation (histological score, CD3⁺ lymphocytes, CD8⁺ T-cells, CD4⁺ T-cells, Foxp3⁺ T-helper cells) in the pancreas of hyperglycemic and normoglycemic mice. We also analyzed the number of leukocytes, lymphocytes, granulocytes and monocytes in the blood. In addition, we determined the percentage of CD3⁺ lymphocytes, CD8⁺ T-cells, CD4⁺ T-cells, Foxp3⁺ T-helper cells, Foxp3⁺ CD25⁺ T-helper and Foxp3^- T-helper cells in the spleen by flow cytometry.

Results: Treatment with streptozotocin caused a strong induction of hyperglycemia and a reduction in body weight (P < 0.001). Severe hyperglycemia did not, however, lead to an aggravation, but rather to a slight attenuation of autoimmune pancreatitis. In the pancreas, both the histological score of the pancreas as well as the number of CD3⁺ lymphocytes (P < 0.053) were decreased by hyperglycemia. No major changes in the percentage of CD8⁺ T-cells, CD4⁺ T-cells, Foxp3⁺ T-helper cells were observed between hyperglycemic and normoglycemic mice. Hyperglycemia increased the numbers of leukocytes (P < 0.001), lymphocytes (P = 0.016), granulocytes and monocytes (P = 0.001) in the blood. Hyperglycemia also moderately reduced the percentage of CD3⁺ lymphocytes (P = 0.057), significantly increased the percentage of Foxp3⁺ T-helper cells (P = 0.018) and Foxp3⁺ CD25⁺ T-helper cells (P = 0.021) and reduced the percentage of Foxp3^- T-helper cells (P = 0.034) in the spleen.

Conclusion: Hyperglycemia does not aggravate but moderately attenuates autoimmune pancreatitis, possibly by increasing the percentage of regulatory T-cells in the spleen.

Keywords: Autoimmune disease; Autoimmune pancreatitis; Diabetes; FoxP3; MRL/MpJ mice; Treg.

Figure 1

Experimental protocol of the hyperglycemic autoimmune pancreatitis model. 28-40 wk old- MRL/MpJ mice were intraperitoneally (ip) injected with streptozotocin on day 1-5 (group: STZ), while one age-matched control cohort was ip injected with the appropriate vehicle (group: Sham). The tissue was harvested on day 113-116; (A) Treatment with STZ increased the blood glucose concentration; (B) and reduced the body weight on day 113-116; (C) Box plots indicate the median, the 25^th and 75^th percentiles in the form of a box, and the 10^th and 90^th percentiles as whiskers. The number of animals evaluated was n = 19 (Sham) and n = 17 (STZ). Differences between the indicated cohorts are indicated as significant. ^aP < 0.001; ^bP = 0.009. STZ: Streptozotocin-treated.

Figure 2

Evaluation of autoimmune pancreatitis. A: Representative images of the histological scores of autoimmune pancreatitis. Arrows point at small inflammatory infiltrates embedded in healthy surrounding tissue; B: hyperglycemia (STZ) reduced the histological score slightly compared to normoglycemic controls (sham); little differences are observed in the pancreas to body weight ratio (C), lipase activity (D), and amylase activity (E). The number of animals evaluated was n = 19 (sham) and n = 17 (STZ) in Panel B and C and n = 19 (Sham) and n = 15 (STZ) in D and E. Scale bar = 50 μm. STZ: Streptozotocin-treated.

Figure 3

Evaluation of the local pancreatic inflammation during autoimmune pancreatitis. Representative images of T-helper cells (CD3⁺CD4⁺cells) (A); and cytotoxic T-cells (CD3⁺CD8⁺ cells) (B); hyperglycemia induced by STZ decreased the number of T-lymphocytes (defined as number of CD3⁺ per field) (C); little differences are observed in the percentage of cytotoxic T-lymphocytes (CD8⁺CD3⁺ cells × 100 divided by the number of CD3⁺cells) (D); T-helper cells (CD4⁺CD3⁺ cells × 100 divided by the number of CD3⁺cells) (E); and regulatory T-cells (FoxP3⁺CD4⁺cells × 100 divided by the number of CD4⁺ cells) (F). The number of animals evaluated was n = 19 (Sham) and n = 17 (STZ). Differences between the indicated cohorts are indicated as tendency, ^aP = 0.053. STZ: Streptozotocin-treated.

Figure 4

Diabetes increased the number of leukocytes in the blood. Treatment with STZ raised the number of leukocytes (A); lymphocytes (B); as well as granulocytes and monocytes (C). The number of animals evaluated was n = 19 (Sham) and n = 14 (STZ). Differences between the indicated cohorts are indicated as significant, (A) ^aP < 0.001; (B) ^bP = 0.016; (C) ^cP = 0.001. STZ: Streptozotocin-treated.

Figure 5

Evaluation of leukocytes in the spleen during autoimmune pancreatitis. Representative images of stained T-helper cells and their distinction between regulatory T-cells (FoxP3⁺CD4⁺ cells), CD25 expressing regulatory T-cells (CD4⁺CD25⁺ FoxP3⁺ cells) and FoxP3- T-cells (FoxP3^- CD4⁺ cells) (A); application of STZ decreased the percentage of T-lymphocytes (defined as number of CD3⁺ cells × 100 divided by the number of lymphocytes) (B); little differences are observed in the percentage of cytotoxic T-cells (CD8⁺CD3⁺ cells × 100 divided by the number of CD3⁺cells) (C); and T-helper cells (CD4⁺CD3⁺ cells × 100 divided by the number of CD3⁺cells) (D); STZ increased the percentage of regulatory T-cells (FoxP3⁺CD4⁺cells × 100 divided by the number of CD4⁺ cells) (E); and CD25 expressing regulatory T-cells (FoxP3⁺ CD25⁺ CD4⁺ cells × 100 divided by the number of CD4⁺cells) (F); the percentage of FoxP3^- T-cells (FoxP3^-CD4⁺cells × 100 divided by the number of CD4⁺ cells) was decreased (G). The number of animals evaluated was n = 19 (sham) and n = 17 (STZ). Differences between the indicated cohorts are indicated as tendency, (B) ^aP = 0.057 or as significant, (E) ^bP = 0.018; (F) ^cP = 0.021; (G) ^dP = 0.034. STZ: Streptozotocin-treated.