Insulitis and aging: Immune cell dynamics in Langerhans islets

Abstract

With increasing age, the risk for age-related type-2-diabetes also increases due to impaired glucose tolerance and insulin secretion. This disease process may be influenced by various factors, including immune cell triggered inflammation and fibrosis. Although immune cells are a necessary component of islets, little is known about immune cell accumulation, immune cell subtype shifts and subsequent influence on glucose metabolism in healthy aging. However, this is critical for understanding the mechanisms that influence β-cell health. Therefore, we studied young and old male C57BL/6J mice, focusing on immune cell composition, patterns of accumulation, and the presence of fibrosis within the pancreatic islets. Our findings demonstrate that insulitis occurs in healthy aged mice without immediate development of a diabetic phenotype. Aged islets exhibited an increase in leukocytes and a shift in immune cell composition. While insulitis typically involves excessive immune cell accumulation, we observed a moderate increase in macrophages and T-cells during aging, which may support β-cell proliferation via cytokine secretion. In fact, aged mice in our study showed an increase in β-cell mass as well as a partially higher insulin secretory capacity, which compensated for the loss of β-cell functionality in insulitic islets and led to improved glucose tolerance. Furthermore, fibrosis which is normally triggered by immune cells, increased with age but appears to reach a steady state, emphasizing the importance of counter-regulatory mechanisms and immune system regulation. Our results suggest, that immune cell subtypes change with age and that non-pathological accumulation of immune-cells may regulate glucose metabolism through secretion of cytokines.

Keywords: Aging; Fibrosis; Immune cells; Insulitis; Langerhans islets.

Graphical abstract

Fig. 1

Glucose utilization and glucose-stimulated insulin secretion as a function of age. A. Bodyweight of young and old mice, n = 12. B. Pancreas weight, n = 6–8. C. Blood glucose during oral glucose tolerance test (oGTT), n = 8–10. D. Fasting blood glucose levels of young and old mice, n = 8–10. E. Area under the curve of the oGTT from young and old mice, n = 8–10. F. Random plasma insulin measured by insulin ELISA from young and old mice, n = 9–12. G. Calculated β-cell mass, n = 6–8. H. Ratio between random insulin plasma and calculated β-cell mass, n = 6. I. Glucose stimulated insulin secretion of isolated Langerhans islets from young and old mice, n = 10–11. Data are presented as Mean±SD, Student's t-test or Whitney U for comparison between two groups or ordinary two-way ANOVA with post-hoc Sidaks multiple comparison test for more variables; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig. 2

Insulitis in young and old CD57BL/6J mice. A. Representative images of insulitis grades 0–4. B. Insulitis Score. C. Insulitis score distribution between young and old mice and healthy islets (grade 0) scored in two to three different sectional levels, n = 6–8, Mean± SD, Unpaired Students t-test; ∗∗p < 0.01.

Fig. 3

Characterization of immune cell subtypes in Langerhans islets. A. Illustration of sample preparation for flow cytometry: Langerhans islets were isolated from the pancreas of young and old CD57BL/6J mice, separated into individual cells with accutase and stained with fluorescent antibodies for immune cell markers, followed by flow cytometric analysis of the immune cells. Created in BioRender. Jelleschitz, J. (2025) https://BioRender.com/x91t850 B. Gating strategy to identify leukocytes, which were separated to subpopulations of CD20⁺ (B-lymphocytes), CD68⁺ (macrophages), CD3⁺ (T-lymphocytes), CD3⁺CD4⁺ (T-helper cells) and CD3⁺CD8a⁺ (cytotoxic T-lymphocytes). C. Analysis of immune cell subpopulations, results presented as ratio of immune cell types of total infiltrated immune cells (CD45⁺, leukocytes), n = 4–7. D. Gating strategy to identify polarized macrophages. CD68⁺ cells were additionally stained for pro-inflammatory markers (CD80⁺, CD86⁺) or anti-inflammatory markers (CD163⁺, CD206⁺). E. Pro- and anti-inflammatory ratios of total viable macrophages (CD68⁺), n = 3–4. Data are presented as Mean± SD, Unpaired Students t-test or Whitney U test; ∗p < 0.05; ∗∗p < 0.01.

Fig. 4

Characterization of immune cells in pancreatic paraffin sections. A. Representative images of leukocytes (CD45⁺) in paraffin embedded pancreatic tissue section. Intra-islet CD45⁺ cells in young and old Langerhans islets. Peri-islet associated CD45⁺ cells in young and old Langerhans islets. B. Representative image of macrophages (CD68⁺) in paraffin embedded pancreatic tissue section. Intra-islet CD68⁺ cells in young and old Langerhans islets. Peri-islet associated CD68⁺ cells in young and old Langerhans islets. C. Representative image of T-lymphocytes (CD3⁺) in paraffin embedded pancreatic tissue section. Intra-islet CD3⁺ cells in young and old Langerhans islets. Peri-islet associated CD3⁺ cells in young and old Langerhans islets. D. Representative image of cytotoxic T-lymphocytes (CD3⁺CD8a⁺) in paraffin embedded pancreatic tissue section. Intra-islet CD3⁺CD8a⁺ cells in young and old Langerhans islets. Peri-islet associated CD3⁺CD8a⁺ cells in young and old Langerhans islets. Data are presented as Mean± SD, n = 6–8, unpaired Students t-test or Whitney U test; ∗∗p < 0.01.

Fig. 5

Correlation between insulitis and immune cell subtypes. A. Non-linear fit (second order polynomial) between insulitis (Grade 1–3) and CD45⁺ cells (intra- and peri-islet), n = 14, R² = 0.6660. B. Non-linear fit (second order polynomial) between insulitis (Grade 1–3) and CD68⁺ cells (intra- and peri-islet), n = 13, R² = 0.6064. C Non-linear fit (second order polynomial) between insulitis (Grade 1–3) and CD3⁺ cells (intra- and peri-islet), n = 14, R² = 0.9047. D. Non-linear fit (second order polynomial) between insulitis (Grade 1–3) and CD3⁺CD8a⁺ cells (intra- and peri-islet), n = 14, R² = 0.9059.

Fig. 6

Age related induction of fibrotic structures in Langerhans islets. A. Representative images for scoring grade 0–4 of fibrotic Langerhans islets. B. Criteria for grading fibrotic islets. C. Fibrosis scoring of young and old C57Bl/6J mice and non-fibrotic islets (grade 0) of young and old mice, n = 6–8. D. Protein carbonyls derivatized with DNPH and quantified using a DNP antibody, as well as 3-nitrotyrosine levels, in lysed islets from young and old mice, normalized to total protein values determined by Ponceau staining. n = 3–5. E. Representative images for α-smooth muscle actin (SMA) staining in young and old pancreatic tissue samples and αSMA positive area/insulin positive area in young and old mice., n = 6–8. Data represented as Mean± SD, unpaired Students t-test was performed, ∗p < 0.05. ∗∗∗p < 0.001.