Loss of intra-islet heparan sulfate is a highly sensitive marker of type 1 diabetes progression in humans

Abstract

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstrated that heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shown to be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intra-islet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxide-induced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.

Fig 1. Intra-islet HS and HSPG core proteins in human pancreases correlate with insulin-positive beta cells.

Immunohistochemical analyses of (A-E) a normal human pancreas (nPOD #6134) and (F-J) a pancreas with insulin-containing (Ins+) islets from a donor with T1D (nPOD #6084, 4 years post-T1D onset) show the distribution of (B,G) insulin-positive beta cells, intra-islet (C,H) HS, (D,I) Col18 core protein and (E,J) Sdc1 core protein. (A,F), H&E. Scale bar = 100 μm.

Fig 2. Immunohistochemical localization of intra-islet HS and HSPG core proteins in Ins+ T1D human pancreas with insulitis.

Insulitis in the pancreas of nPOD #6070 (7 years post-T1D onset) (A) is adjacent to (B) residual insulin-positive beta cells which show staining for (C) Col18 and (D) Sdc1 core proteins but (E) little HS (*). (F) Glucagon staining is distinct from HSPGs (C,D) and HS (E). (A) H&E. Scale bar = 100 μm.

Fig 3. Intra-islet HS colocalizes with insulin not glucagon staining in normal human pancreas.

Immunofluorescence staining of (A) insulin (INS), (B,F) HS and (E) glucagon (GLUC), in normal human pancreas (nPOD #6134). Nuclei were stained with DAPI (D,H). (A) anti-insulin Ab; (B,F) 10E4 anti-HS mAb; (E) anti-glucagon Ab; (C,G) merged (excluding DAPI); (D,H) merged (including DAPI). Scale bar = 20 μm.

Fig 4. Intra-islet HS and HSPG core protein levels decline in Ins+ islets in human T1D.

Morphometric analysis of the (A) HS-positive, (B) Col18-positive, (C) Sdc1-positive, (D) insulin-positive and (E) glucagon-positive islet area in normal human pancreases (black bars) and Ins+ T1D human pancreases (open bars). Data shows mean ± SEM; n = 6–8 pancreases (n = 56–80 islets) examined/group for normal controls and n = 6–8 pancreases (n = 52–66 islets)/group for Ins+ diabetic donors, except for analyses of Sdc1 where n = 4–5 pancreases (n = 40–42 islets) were examined/group. Significance was determined using unpaired Student’s t test, * = P<0.0001, ** = P = 0.0217 and Mann-Whitney test, *** P = 0.0025.

Fig 5. HS and HSPG core protein are localized inside isolated human islet beta cells.

Immunofluorescence staining of 1 day-cultured isolated human islets show co-localization of (A) insulin (INS) and (B) HS. Nuclei were stained with DAPI (D). (A) anti-insulin Ab; (B) 10E4 anti-HS mAb; (C) merged (excluding DAPI); (D) merged (including DAPI). Scale bar = 20 μm. (E) Representative single color flow cytometry histograms of freshly isolated human islet cells (89% were NG-positive beta cells) show staining for intracellular HS (pink solid line) and Col18 (blue solid line) compared to background staining with corresponding isotype control Ig (dotted lines) and the autofluorescence of unstained cells (black solid line).

Fig 6. HS optimizes human beta cell survival in vitro.

Flow cytometry analyses of the viability of freshly isolated human islet cells (Con) on day 0 and after culture for 2 days with or without heparin, PI-88 or BT548 at 50 μg/ml. (A-C) Islet cells were stained with Newport Green (NG) to identify beta cells and with 7AAD (B and C) to label non-viable cells; NG+ve, 7AAD-ve staining identified viable beta cells (C). Con, control; Hep, Heparin; BT548, chemically modified LMWH. Data (% islet cells) shows mean ± SEM; n = 8–10 independent experiments and significance was measured by non-parametric ANOVA (Kruskal-Wallis test) with Dunn’s Multiple Comparisons test. * P<0.001, **P<0.01, ***P<0.05.

Fig 7. HS mimetics protect human beta cells from oxidative damage.

Flow cytometric analyses of isolated human islet cells cultured with or without HS mimetics for 2 days and then treated acutely with hydrogen peroxide. Islet cell death/damage was measured by Sytox green fluorescence. Con, control; Hep, Heparin; BT548, chemically modified LMWH. Data shows mean ± SEM; n = 11–12 independent experiments. Con d0 versus Con d0 + hydrogen peroxide, Unpaired t-test, *P<0.0001; Con d2 + hydrogen peroxide versus PI-88 d2 + hydrogen peroxide, non-parametric ANOVA with Dunn’s Multiple Comparisons test, ** P<0.05.

Fig 8. Heparanase is expressed by human insulitis leukocytes.

(A) An islet (I) with insulitis in nPOD pancreas #6084 (4 years post-T1D onset) shows (B) leukocytes strongly expressing heparanase (Hpse) and (C) glucagon staining. Normal human islet cells (nPOD #6012) (D) showed negligible heparanase expression compared to pancreatic lymph node (PLN) from the same donor (E). Islet cells in Ins+ T1D pancreas (nPOD #6070) weakly expressed heparanase (arrowhead), compared to nearby infiltrating leukocytes (arrow) (G) and host PLN (H). Background staining with isotype control Ig was absent in PLNs (F and I). (A) H&E; (B, D, E, G, H) anti-Hpse HP130 mAb; (C) anti-glucagon mAb; (F, I) mouse IgM. Scale bar = 100 μm.