Andrew Ziolkowski

Heparan sulfate and heparanase play key roles in mouse β cell survival and autoimmune diabetes

The autoimmune type 1 diabetes (T1D) that arises spontaneously in NOD mice is considered to be a model of T1D in humans. It is characterized by the invasion of pancreatic islets by mononuclear cells (MNCs), which ultimately leads to destruction of insulin-producing β cells. Although T cell dependent, the molecular mechanisms triggering β cell death have not been fully elucidated. Here, we report that a glycosaminoglycan, heparan sulfate (HS), is expressed at extraordinarily high levels within mouse islets and is essential for β cell survival. In vitro, β cells rapidly lost their HS and died. β Cell death was prevented by HS replacement, a treatment that also rendered the β cells resistant to damage from ROS. In vivo, autoimmune destruction of islets in NOD mice was associated with production of catalytically active heparanase, an HS-degrading enzyme, by islet-infiltrating MNCs and loss of islet HS. Furthermore, in vivo treatment with the heparanase inhibitor PI-88 preserved intraislet HS and protected NOD mice from T1D. Our results identified HS as a critical molecular requirement for islet β cell survival and HS degradation as a mechanism for β cell destruction. Our findings suggest that preservation of islet HS could be a therapeutic strategy for preventing T1D.

Unexpected new roles for heparanase in Type 1 diabetes and immune gene regulation

Heparanase (Hpse) is an endo-β-d-glucuronidase that degrades the glycosaminoglycan heparan sulfate (HS) in basement membranes (BMs) to facilitate leukocyte migration into tissues. Heparanase activity also releases HS-bound growth factors from the extracellular matrix (ECM), a function that aids wound healing and angiogenesis. In disease states, the degradation of HS in BMs by heparanase is well recognized as an invasive property of metastatic cancer cells. Recent studies by our group, however, have identified unexpected new roles for heparanase and HS. First, we discovered that in Type 1 diabetes (T1D) (i) HS in the pancreatic islet BM acts as a barrier to invading cells and (ii) high levels of HS within the insulin-producing islet beta cells themselves are critical for beta cell survival, protecting the cells from free radical-mediated damage. Furthermore, catalytically active heparanase produced by autoreactive T cells and other insulitis mononuclear cells was shown to degrade intra-islet HS, increasing the susceptibility of islet beta cells to free radical damage and death. This totally novel molecular explanation for the onset of T1D diabetes opens up new therapeutic approaches for preventing disease progression. Indeed, administration of the heparanase inhibitor, PI-88, dramatically reduced T1D incidence in diabetes-prone NOD mice, preserved islet beta cell HS and reduced islet inflammation. Second, in parallel studies it has been shown that heparanase and HS can be transported to the nucleus of cells where they impact directly or indirectly on gene transcription. Based on ChIP-on-chip studies heparanase was found to interact with the promoters and transcribed regions of several hundred genes and micro-RNAs in activated Jurkat T cells and up-regulate transcription, with many of the target genes/micro-RNAs being involved in T cell differentiation. At the molecular level, nuclear heparanase appears to regulate histone 3 lysine 4 (H3K4) methylation by influencing the recruitment of demethylases to transcriptionally active genes. These studies have unveiled new functions for heparanase produced by T lymphocytes, with the enzyme mediating unexpected intracellular effects on T cell differentiation and insulin-producing beta cell survival in T cell-dependent autoimmune T1D.

Heparanase and autoimmune diabetes

Heparanase (Hpse) is the only known mammalian endo-β-d-glucuronidase that degrades the glycosaminoglycan heparan sulfate (HS), found attached to the core proteins of heparan sulfate proteoglycans (HSPGs). Hpse plays a homeostatic role in regulating the turnover of cell-associated HS and also degrades extracellular HS in basement membranes (BMs) and the extracellular matrix (ECM), where HSPGs function as a barrier to cell migration. Secreted Hpse is harnessed by leukocytes to facilitate their migration from the blood to sites of inflammation. In the non-obese diabetic (NOD) model of autoimmune Type 1 diabetes (T1D), Hpse is also used by insulitis leukocytes to solubilize the islet BM to enable intra-islet entry of leukocytes and to degrade intracellular HS, an essential component for the survival of insulin-producing islet beta cells. Treatment of pre-diabetic adult NOD mice with the Hpse inhibitor PI-88 significantly reduced the incidence of T1D by ~50% and preserved islet HS. Hpse therefore acts as a novel immune effector mechanism in T1D. Our studies have identified T1D as a Hpse-dependent disease and Hpse inhibitors as novel therapeutics for preventing T1D progression and possibly the development of T1D vascular complications.

Prolonged Xenograft Survival Induced by Inducible Costimulator-Ig Is Associated With Increased Forkhead Box P3+ Cells

Background. Blockade of the inducible costimulator (ICOS) pathway has been shown to prolong allograft survival; however, its utility in xenotransplantation is unknown. We hypothesize that local expression of ICOS-Ig by the xenograft will suppress the T-cell response resulting in significant prolonged graft survival. Methods. Pig iliac artery endothelial cells (PIEC) secreting ICOS-Ig were generated and examined for the following: (1) inhibition of allogeneic and xenogeneic proliferation of primed T cells in vitro and (2) prolongation of xenograft survival in vivo. Grafts were examined for Tregs by flow cytometry and cytokine levels determined by quantitative reverse-transcriptase polymerase chain reaction. Results. Soluble ICOS-Ig markedly decreased allogeneic and xenogeneic primed T-cell proliferation in a dose-dependent manner. PIEC-ICOS-Ig grafts were significantly prolonged compared with wild-type grafts (median survival, 34 and 12 days, respectively) with 20% of PIEC-ICOS-Ig grafts surviving more than 170 days. Histological examination showed a perigraft cellular accumulation of Forkhead box P3 (Foxp3+) cells in the PIEC-ICOS-Ig grafts, these were also shown to be CD3+CD4+CD25+. Survival of wild-type PIEC grafts in a recipient simultaneously transplanted with PIEC-ICOS-Ig were also prolonged, with a similar accumulation of Foxp3+ cells at the periphery of the graft demonstrating ICOS-Ig induces systemic graft prolongation. However, this prolongation was specific for the priming xenograft. Intragraft cytokine analysis showed an increase in interleukin-10 levels, suggesting a potential role in induction/function of CD4+CD25+Foxp3+ cells. Conclusions. This study demonstrates prolonged xenograft survival by local expression of ICOS-Ig, we propose that the accumulation of CD4+CD25+Foxp3+ cells at the periphery of the graft and secretion of interleukin-10 is responsible for this novel observation.

Porcine endogenous retrovirus encodes xenoantigens involved in porcine cellular xenograft rejection by mice.

Background. Identification of the antigens that stimulate transplant rejection can help develop graft-specific antirejection strategies. The xenoantigens recognized during rejection of porcine cellular xenografts have not been clearly defined, but it has been assumed that major histocompatibility complex (MHC) xenoantigens are involved. Methods. The role of porcine endogenous retrovirus (PERV) as a source of xenoantigens was examined. The authors used morphometry to compare the kinetics of swine leukocyte antigen (SLA)dd pig thyroid xenograft rejection in control mice and mice immunized with PERV+ PK15 cells (porcine kidney epithelial cells), PERV− SLAdd pig peripheral blood lymphocytes (PBL), PERV virions purified from PK15 cells, and PERV or PERV A pseudotypes produced from infected human 293 cells. The tempo of rejection for cellular xenografts of PERV A pseudotype-producing human 293 cells, uninfected human 293 cells, and PK15 cells in PERV-preimmunized and control mice was also compared. Results. Mice immunized with PK15 cells rejected pig thyroid xenografts significantly faster at day 5 than control mice and mice immunized with pig PBL. This correlated with the amount of PERV RNA and virions produced, but not with the amount of SLAdd class I MHC expressed by PK15 cells. Immunization of mice with PERV virions purified from porcine PK15 cells and with PERV virions or PERV A pseudotypes produced by human 293 cells also induced accelerated xenograft rejection by 5 days. Accelerated rejection induced by virus pretreatment was CD4 T-cell dependent and restricted to PERV-expressing cellular xenografts of porcine or nonporcine origin. Conclusions. PERV acts as a significant source of xenoantigens that target porcine cellular xenografts for rejection.

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

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.

PROLONGED XENOGRAFT SURVIVAL INDUCED BY ICOS-IG IS ASSOCIATED WITH INCREASED FOXP3+ CELLS: 2087

R. Hodgson1, A. Ziolkowski2, D. Christiansen3, E. Mouhtouris4, C.J. Simeonovic2, F. Ierino5, M.S. Sandrin6 1Department Of Surgery (austin), University of Melbourne, Heidelberg/ VIC/AUSTRALIA, 2Immunology, The John Curtin School of Medical Research, Canberra/ACT/AUSTRALIA, 3Surgery (ah/nh), The University of Melbourne, Heidelberg/AUSTRALIA, 4Department Of Surgery, University of Melbourne, Heidelberg/AUSTRALIA, 5Director Of Renal Transplantation, Austin Health, Heidelberg/VIC/AUSTRALIA, 6Surgery (ah/nh), The University of Melbourne, Heidelberg/VIC/ AUSTRALIA

PANCREATIC ISLETS UNDERGO SUBSTANTIAL MOLECULAR REMODELLING AFTER ISLET ISOLATION AND TRANSPLANTATION, RESULTING IN HEPARANASE-MEDIATED DAMAGE DURING ALLOGRAFT REJECTION: 1065

Introduction: In situ pancreatic islets are surrounded by a basement membrane (BM) that acts as a barrier to invading leukocytes. This function is partly due to the HS proteoglycan (HSPG) perlecan in the BM. HS is also heavily distributed within islet beta cells and is required for their survival. Our study investigated the status of the islet BM, and expression of islet HS, heparanase (the endoglycosidase that degrades HS) and cathepsin L (activates proheparanase) in islets before and after islet isotransplantation and allotransplantation to mice. Methods: Islets were isolated by collagenase digestion of mouse pancreas and beta cells by dispase dispersion of isolated islets. Matrix proteins, HSPG core proteins, HS and PECAM-1 were localised in mouse islets, the islet BM or in beta cells by histochemistry (Alcian Blue) or by in situ immunofluorescence staining, immunohistochemistry and/or flow cytometry using anti-collagen type IV, -laminin, -nidogen, -HSPGs (perlecan, syndecan, glypican, agrin, collagen type XVIII, CD44), -HS (10E4, HepSS-1) and anti-PECAM-1 antibodies. Heparanase and cathepsin L were localised in isolated islets, islet isografts and allografts by immunohistochemistry using HP130 and MAB9521 mAbs. Heparanase, CD45R (common leukocyte marker) and UBC (house-keeping gene) transcripts were analysed quantitatively by real-time RT-PCR in isolated C57BL/6J(H-2b) and CBA/H(H-2k) islets and at 3-7 days after isoand allotransplantation, respectively, to C57BL/6J mice. Western blotting was used to compare the relative levels of active heparanase in islet isografts and allografts. Results: Compared to islets in situ, isolated islets showed loss of the islet BM matrix proteins collagen type IV, laminin, nidogen and perlecan, indicating that BM degradation had occurred. In parallel, intra-islet HS was substantially reduced but HSPG core proteins remained largely unchanged. Islet BM and HS were restored after islet isotransplantation and peri-islet PECAM-1 staining was observed. HS and HSPGs but not islet BM matrix proteins were localised within islet beta cells, suggesting that beta cells produce HS and HSPGs endogenously. Compared to islet isografts, heparanase and CD45R transcripts increased significantly by day 5 (P<0.05) in islet allografts, and cathepsin L transcripts decreased significantly by day 6 (P<0.05). Immunohistochemistry revealed strong expression of heparanase protein by infiltrating mononuclear cells in rejecting islet allografts and strong expression of cathepsin L protein in donor islets before and after transplantation. Rejecting islet allografts at day 7 expressed 19-fold higher levels of active heparanase than corresponding islet isografts. Conclusion: Isolated islets are transiently depleted of vital molecular components necessary for their integrity. An intact islet BM may be important for maintaining intra-islet HS. Following transplantation, beta cells replenish their lost HS and the islet BM is restored, possibly by endothelial cells. Islets undergo substantial remodelling post-transplantation which renders them susceptible to heparanase-mediated damage. Heparanase produced by infiltrating leukocytes (and possibly activated by islet-derived cathepsin L) probably contributes to allograft rejection by degrading HS in the reconstituted islet BM and in the intra-islet cell mass, thereby inducing islet beta cell death. Inhibitors of heparanase may represent a novel anti-rejection therapy.