Transgenic expression of haem oxygenase-1 in pancreatic beta cells protects non-obese mice used as a model of diabetes from autoimmune destruction and prolongs graft survival following islet transplantation
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- Huang, S.H., Chu, C.H., Yu, J.C. et al. Diabetologia (2010) 53: 2389. doi:10.1007/s00125-010-1858-x
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Haem oxygenase 1 (HO-1) has strong anti-apoptotic, anti-inflammatory and antioxidative effects that help protect cells against various forms of immune attack. We investigated whether transgenic expression of Ho-1 (also known as Hmox1) in pancreatic beta cells would protect NOD mice from autoimmune damage and prolong graft survival following islet transplantation.
To evaluate the protective effect of beta cell-specific HO-1 in autoimmune diabetes, we used an insulin promoter-driven murine Ho-1 construct (pIns-mHo-1) to generate a transgenic NOD mouse. Transgene expression, insulitis and the incidence of diabetes in mice were characterised. Lymphocyte composition, the development of T helper (Th)1, Th2 and T regulatory (Treg) cells, T cell proliferation and lymphocyte-mediated disease transfer were analysed. The potential effects of transgenic islets and islet transplantation on apoptosis, inflammation and the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) were evaluated.
Transgenic mice showed less severe insulitis and a lower incidence of diabetes than non-transgenic control littermates. Lymphocyte composition and functions were not affected. Islets from transgenic mice expressed lower levels of proinflammatory cytokines/chemokines, proapoptotic gene expression and amounts of ROS/RNS, and were more resistant to TNF-α- and IFN-γ-induced apoptosis. Islet grafts from transgenic mice also survived longer in diabetic recipients than control islets.
Transgenic overexpression of Ho-1 in beta cells protected NOD mice from diabetes and delayed the autoimmune destruction of islet grafts, providing valuable insight into the development of better strategies for clinical islet transplantation in patients with type 1 diabetes.
KeywordsHeme oxygenase 1 NOD mice Type 1 diabetes
Forkhead box P3
Green fluorescent protein
Haem oxygenase 1
Immature dendritic cells
Inhibitory protein of NF-κB
National Defense Medical Center
Nuclear factor kappa-light-chain-enhancer of activated B cells
Insulin promoter-driven murine HO-1 construct
Reactive oxygen species
Reactive nitrogen species
Signal transducer and activator of transcription-1
Human Thy-1 cell surface antigen
Mouse thymus cell antigen 1, theta
Autoimmune destruction of beta cells in the pancreatic islets of Langerhans leads to type 1 diabetes mellitus . The NOD mouse is an inbred strain that spontaneously develops autoimmune diabetes resembling human type 1 diabetes [2, 3]. Destruction of beta cells is caused by the release of inflammatory cytokines and cytotoxic molecules, such as IL-1β, IFN-γ, TNF-α, granzyme B and perforin, or by directly inducing downstream cell death signals of the Fas–Fas ligand pathway through natural killer cells, macrophages, pathogenic T helper (Th)1 cells and cytotoxic T cells. In addition, levels of intracellular nitric oxide, reactive oxygen species (ROS) and reactive nitrogen species (RNS) can be induced by these different reactive pathways and also damage beta cells [4, 5, 6, 7].
Haem oxygenase-1 (HO-1) is an inducible intracellular enzyme, which is produced at high levels in the spleen, liver and kidney, and catabolises the haem component of haemoglobin from senescent erythrocytes. HO-1 can break the porphyrin ring of haem to yield equal molar amounts of biliverdin, free iron and carbon monoxide . HO-1 also possesses critical cytoprotective functions that are activated under cellular stress situations, such as inflammation, ischaemia, hypoxia, hyperoxia, hyperthermia or radiation . HO-1 exerts major cytoprotective functions against inflammation, apoptosis and oxidative damage, and acts in the maintenance of microcirculation . Accumulating evidence indicates that HO-1 plays an important role in immune regulation. Thus, immature dendritic cells (iDCs) spontaneously produce HO-1, which is downregulated by maturation stimuli such as lipopolysaccharide. Induction of HO-1 production rendered iDCs refractory to lipopolysaccharide-induced maturation, but preserved IL-10 secretion, suggesting that HO-1 plays an important role in the maturation and function of iDCs, and could be used to modulate the immune response . Splenocytes from Ho-1 (also known as Hmox1) knockout mice secreted disproportionately high levels of Th1 cell-associated and proinflammatory cytokines on stimulation, implying a critical regulatory role of HO-1 in Th1/Th2 balance and early inflammatory responses . In addition, Foxp3 and Ho-1 are coexpressed in human peripheral CD4+CD25+ T regulatory (Treg) cells and the suppressive function of the cells is abrogated by inhibition of HO-1 activity . Moreover, adeno-associated virus-mediated overexpression of Ho-1 protected NOD mice from autoimmune diabetes by reducing the population of mature dendritic cells and autoreactive T lymphocytes, providing a successful preventive strategy for systemic Ho-1 expression in this disease . Induction or overexpression of Ho-1 also successfully prolonged survival of transplanted grafts following allotransplantation of the heart , liver , thyroid  and islets . However, it remains unclear whether HO-1 has a protective effect on pancreatic beta cells in NOD mice.
To investigate the protective potential of beta cell-specific overexpression of Ho-1 in NOD mice and its ability to counter autoimmune attack in syngeneic islet transplantation, we generated murine Ho-1 (mHo-1)-transgenic NOD mice, which overproduce HO-1 under the control of the human insulin promoter. The expression of transgenic Ho-1 in beta cells significantly ameliorated the severity of insulitis and the incidence of diabetes in NOD mice, and increased survival of islet grafts. Although local and persistent HO-1 production did not alter systemic immunity, it mediated against inflammation and apoptosis, and reduced levels of ROS/RNS in islets. Furthermore, transgenic islet grafts successfully delayed recurrence of autoimmunity. Thus, for the first time, we have demonstrated the protective potential of transgenic Ho-1 in islets in this animal model of autoimmune diabetes, providing a potential therapeutic strategy using tissue-specific genetic manipulation.
Cells and animals
NIT-1 is an insulinoma cell derived from NOD mice and was purchased from the American Type Culture Collection (Manassas, VA, USA). The NOD/Sytwu (Kd, Db, Ld, I-Ag7) mice were originally purchased from Jackson Laboratory (Bar Harbor, ME, USA). NOD.CB17-Prkdcscid/J (NOD/SCID) mice were provided by the National Laboratory Animal Center (Taipei, Taiwan). All mice were bred and maintained under specific pathogen-free conditions at the Animal Center of the National Defense Medical Center (NDMC) (Taipei, Taiwan), which is accredited by Association for Assessment and Accreditation of Laboratory Animal Care International. Experiments were conducted in accordance with institutional guidelines and were approved by NDMC’s Institutional Animal Care and Use Committee.
Generation and detection of transgenic NOD mice
To generate transgenic mice, we used an insulin promoter-driven mHo-1 construct (pIns-mHo-1) that was created by inserting cDNA into the pIns-plasmid under the control of a modified human insulin promoter.
Tissue sections were probed with a rat anti-mouse HO-1 monoclonal antibody (eBioscience, San Diego, CA, USA), an anti-insulin monoclonal antibody (eBioscience) and an anti-Ki67 antibody (Abcam, Cambridge, UK), followed by a horseradish peroxidase-conjugated secondary antibody. Aminoethyl-carbazole reagent (DAKO, Carpinteria, CA, USA) was added for enzymatic stain development and Mayer’s haematoxylin was applied as a counterstain.
Assessment of insulitis and diabetes
Pancreatic tissues were obtained from 14-week-old transgenic or non-transgenic mice and the severity of insulitis was scored on haematoxylin–eosin stained sections and classified as described . Urine glucose concentration was measured weekly using Chemstrips (Boehringer Mannheim, Indianapolis, IN, USA). Mice with urine glucose concentration >27.75 mmol/l at two consecutive tests were defined as diabetic.
Islet isolation and transplantation
Pancreatic islets were isolated and transplanted into recipients as described in previous reports [20, 21, 22, 23]. The success rate for transplantation, any recurrence of diabetes or loss of graft function were defined as described .
T cell proliferation
Splenocytes of female mHo-1-transgenic or non-transgenic donor mice (12-week-old) were treated with Tris-buffered ammonium chloride for erythrocyte depletion and 2 × 107 cells were injected into female NOD/SCID mice (6-week-old) via the retro-orbital plexus. Diabetes was assessed as described above.
Real-time RT-PCR was performed using PCR supermix (iQ SYBR Green; Bio-Rad, Hercules, CA, USA) in an iCycler (Bio-Rad) as previously described .
Sections were probed with rabbit anti-GLUT2 primary antibody (Millipore, Billerica, MA, USA). The secondary antibody used was a Cy5-conjugated goat anti-rabbit antibody (Jackson Immunoresearch, West Grove, PA, USA). TUNEL staining was used to detect apoptosis with an in situ cell death detection kit (Roche, Indianapolis, IN, USA). Propidium iodide (2 μg/ml) was used as the nuclear counterstain. Images were captured on a confocal microscope (LSM510; Zeiss, Thornwood, NY, USA).
Islets were stimulated with IFN-γ plus TNF-α (1,000 U/ml or 2,000 U/ml) for 24 h and viability of islets was tested by the MTT assay (Sigma-Aldrich, Saint Louis, MO, USA) .
Measurements of intracellular peroxides
The isolated islets were incubated for 30 min at 37°C with 10 μmol/l dichlorodihydrofluorescein diacetate (Molecular Probes, Eugene, OR, USA). Islets were then dispersed using trypsin treatment and levels of intracellular peroxide were analysed using a FACSCaliber (BD, Franklin Lakes, NJ, USA).
Islets isolated from non-transgenic or mHo-1-transgenic mice were treated with 2,000 U/ml TNF-α plus 2,000 U/ml IFN-γ or 20 ng/ml IL-1β for 24 h. At the end of treatment, islets were washed and dispersed by cell dissociation buffer. Beta cells were stained with 7-amino-actinomycin D (AAD) and FITC-conjugated annexin-V. Apoptotic cells were determined by annexin-V-FITC positive cells.
Differences in islet graft survival time in mHo-1-transgenic and non-transgenic groups were assessed using Kaplan–Meier survival analysis. For the other experiments, differences were compared using Student’s one-tailed unpaired and paired t tests. Differences were considered significant at p < 0.05.
Expression of mHo-1 in pIns-mHo-1-transfected NIT-1 cells
Generation of pIns-mHo-1-transgenic NOD mice
Characterisation of the diabetogenic process in mHo-1-transgenic NOD mice and evaluation of mHo-1-transgenic islets following syngeneic islet transplantation
Transplantation of pancreatic islets into a diabetic recipient is a potential way to cure individuals with type 1 diabetes. To investigate whether transgenic expression of mHo-1 in transplanted islets could reverse diabetes in recent-onset mouse recipients and protect beta cells against immune attack, we performed islet transplantation. We isolated islets from mHo-1-transgenic or non-transgenic mice and implanted them into the left kidney capsule of newly diabetic female NOD recipients. In most recipients implanted with control islets, hyperglycaemia recurred within 7 days after transplantation; the mean graft survival time was 6.643 days. All recipients grafted with transgenic islets maintained them for at least 8 days and the mean graft survival time was 10.875 days (Fig. 3c). These results indicate that the transgenic expression of mHo-1 in grafted islets significantly prolonged survival of cells in diabetic recipients (p < 0.01). To further investigate whether transplantation disturbed transgene expression and how long mHo-1-transgenic islets continue to produce HO-1, we examined the production of HO-1 in graft islets by IHC staining. The mHo-1-transgenic NOD islet grafts at day 8 after transplantation still produced HO-1, with preservation of the islet architecture, and also showed more intact islets with insulin-secreting function (Fig. 3d). In summary, expression of transgenic mHo-1 was effective in prolonging islet graft survival, but did not provide permanent protection from recurrence of diabetes.
Lymphocyte and dendritic cell development in mHo-1-transgenic NOD mice
To further dissect the protective mechanisms in mHo-1-transgenic mice, we characterised the pathogenicity of T lymphocytes and performed adoptive islet transfer experiments. Splenocytes from transgenic or control mice proliferated equally well upon stimulation with anti-CD3 antibody, concanavalin A or NOD islet antigens (Fig. 4d, e), indicating that the transgenic expression of mHo-1 did not affect the proliferative ability of lymphocytes in antigen-specific or non-specific manners. These results also suggest that transgenic Ho-1 did not interfere with the function of antigen-presenting cells (Fig. 4d). To further evaluate the pathogenic ability of lymphocytes of mHo-1-transgenic mice, we injected NOD/SCID recipients intravenously with splenocytes from mHo-1-transgenic or non-transgenic mice and compared the progress of diabetes in the two groups. No significant differences were observed between the two groups of recipients (Fig. 4f), indicating that the expression of transgenic Ho-1 in beta cells did not affect systemic immunity in the NOD mice.
Inflammation and apoptosis in mHo-1-transgenic NOD islets
In NOD mice, transient and systemic overexpression of mHo-1 by viral transduction or using CoPP induction can successfully reduce the degree of insulitis and decrease the frequency of spontaneous diabetes because both systemic autoimmunity and ROS production by the pancreas are suppressed [14, 30]. However, it is unclear whether constitutive production of HO-1 in a beta cell-specific manner could prevent autoimmune diabetes and prolong graft survival following syngeneic islet transplantation. To test this idea, we first established mHo-1-transgenic NOD mice under control of the insulin promoter . These animals produce high levels of HO-1 in pancreatic beta cells from birth. The degree of insulitis was milder, and disease kinetics and incidence in mHo-1-transgenic mice were ameliorated compared with non-transgenic littermates. These results demonstrate that the local and persistent expression of mHo-1 in pancreatic beta cells offers protective effects against autoimmune diabetes.
Islet transplantation is a better therapeutic strategy than administration of exogenous insulin for the treatment of patients with type 1 diabetes, as it can adjust blood glucose to an adequate level in ‘real time’, avoiding secondary complications . However, autoimmune attack and allograft rejection are major problems leading to destruction of islet grafts. Autoimmune attack occurred faster and was more severe than allograft rejection . Induction of HO-1 by viral transduction or drugs can alleviate allograft rejection following islet transplantation [18, 34], but the potential for beta cell-specific production of HO-1 in protecting against autoimmune attack has not yet been evaluated. Our results here are the first to demonstrate that HO-1 overproduction in beta cells effectively prolongs graft survival. This suggests that local production of HO-1 helps protect against recurrence of autoimmune diabetes. However, this protective effect is not complete or lifelong; this may be because expression of the Ho-1 transgene is varied and insufficient. This point is supported by some evidence that the expression level of HO-1 in cells affects its cytoprotective effects .
Accumulating evidence has shown that not only Th1 cells, but also mature dendritic cells and Treg cells are related to the development of autoimmune diabetes in NOD mice. Dendritic cells in NOD mice have abnormally high immunostimulatory and Th1-inducing abilities. In addition, inhibition of iDC maturation can also suppress immune response and induce peripheral tolerance in NOD mice. A decline in the Treg cell population was also noted in NOD mice and transfer of polyclonal CD4+CD25+FOXP3+ Treg cells has been demonstrated to prevent diabetes in NOD mice . A previous study has reported that systemic expression of Ho-1 by AAV-HO-1 transduction suppressed the population and activities of systemic Th1 cells by decreasing the population of mature dendritic cells in NOD mice, but this did not affect systemic Th2 and Treg cells . However, the populations of lymphocytes such as CD4 including Th1 and Th2, CD8, Treg cells and mature dendritic cells in spleen or pancreatic lymph nodes were indistinguishable between transgenic and control mice in our study. We further evaluated the diabetogenic ability of lymphocytes in transgenic mice by adoptive transfer experiments. The result indicated an equal diabetogenic effect of lymphocytes from both mouse strains. These data suggest that transgenic Ho-1-mediated protection may not act by modulating systemic autoimmunity.
Previous studies have indicated that HO-1 counteracts inflammation, including reduction of inducible nitric oxide synthase, chemokines and cytokine levels in islets and other cells [36, 37, 38, 39]. Using real-time RT-PCR, we found lower expression of inflammatory chemokines in islets from transgenic mice. Our results support the idea that overexpression of Ho-1 in beta cells decreases the secretion of inflammatory chemokines in islets and hence reduces the number of lymphocytes attacking the islets. The insulitis score of transgenic mice further supports this conclusion. Besides, HO-1 also contributes to cytoprotection by reducing apoptosis. Tobiasch et al. demonstrated that CoPP-induced βTC3 cells (an insulinoma cell line) with high HO-1 levels were able to counteract the apoptosis of beta cells caused by various stimuli through activation of the p38 mitogen-activated protein kinase pathway . Our TUNEL assay data apparently support those findings. Moreover, human islets highly expressing Ho-1 can resist apoptosis induced by TNF-α and cycloheximide  through downregulation of the proapoptotic proteins caspase-3 and -8, and by upregulation of the antiapoptotic proteins apoptosis regulator Bcl-2 (BCL-2) and apoptosis regulator Bcl-xL (BCL-XL) [41, 42]. Similarly, caspase-3 and -8 were also suppressed in mHo-1-transgenic islets in our results. NIT-1 cells with CoPP treatment or transduced with lenti-Ho-1 showed better protection against TNF-α-mediated cell death. Similar results were also observed in mHo-1-transgenic islets, further demonstrating the antiapoptotic effect of transgenic Ho-1.
In this study, we have demonstrated that transgenic Ho-1 in pancreatic beta cells protected against autoimmune diabetes in NOD mice by increasing the ability of islets to counter apoptosis and inflammation without changing the status of systemic immunity. These findings further suggest that genetic manipulation of HO-1 levels in islets could be a potential therapeutic strategy to treat type 1 diabetes and prevent disease recurrence following islet transplantation.
This work was supported by the National Science Council, Taiwan, Republic of China (NSC-96-2628-B-016-002-MY3, NSC98-3112-B-016-002 and NSC99-3112-B-016-001 to H.-K. Sytwu) and research grant from Tri-Service General Hospital, Taiwan, Republic of China (TSGH-C98-12-S01 and TSGH-C99-011-12-S01 to H.-K. Sytwu). It was also supported in part by the C.Y. Foundation for Advancement of Education, Sciences and Medicine.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.