Intradermal α1-antitrypsin therapy avoids fatal anaphylaxis, prevents type 1 diabetes and reverses hyperglycaemia in the NOD mouse model of the disease
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Human α1-antitrypsin (hAAT) gene therapy prevents type 1 diabetes in a NOD mouse model of diabetes. However, repeated i.p. injections of hAAT into NOD mice leads to fatal anaphylaxis. The aim of the study was to determine if an alternative route of administration avoids anaphylaxis and allows evaluation of hAAT’s potential for diabetes prevention and reversal. We also sought to determine if the addition of granulocyte colony-stimulating factor (G-CSF), augments hAAT’s capacity to prevent or reverse disease in the NOD mice.
To evaluate hAAT pharmacokinetics, serum hAAT levels were monitored in NOD mice receiving a single dose (2 mg) of hAAT by i.p., s.c. or i.d. injection. For studies of type 1 diabetes prevention and reversal, mice received i.d. hAAT (2 mg/mouse/3 days) for 8 or 10 weeks or hAAT and G-CSF (i.p., 6 μg/day) for 6 weeks. Blood glucose determinations, glucose tolerance testing and insulin tolerance tests were performed.
Both i.p. and s.c. injections resulted in fatal anaphylaxis. The i.d. injection avoided anaphylaxis and i.d. injection of hAAT into 11-week-old NOD mice prevented disease (p = 0.005, AAT vs PBS at 40 weeks of age). Treatment of diabetic NOD mice with hAAT or hAAT plus G-CSF provided long-term (at least 100 days) reversal of diabetes in 50% of treated animals. G-CSF did not enhance the reversal rates of hAAT. Glucose tolerance and insulin levels were normalised in mice with hAAT prevention and reversal.
Intradermal hAAT prevents and reverses disease in a NOD mouse model of type 1 diabetes without inducing anaphylaxis.
Keywordsα1-Antitrypsin Type 1 diabetes Non-obese diabetic mice Disease prevention
B cell activating factor
Granulocyte colony-stimulating factor
Intraperitoneal glucose tolerance test
Insulin tolerance test
Type 1 diabetes is an autoimmune disease that results from an imbalanced and over-reactive immune response, resulting in the destruction of insulin-producing pancreatic beta cells. Due to the pathogenic complexity of this disease, development of an effective method for late prevention or reversal post-onset of the disorder has been remarkably challenging . Although recent studies have shown promising results in terms of reversing type 1 diabetes, many of these treatments can lead to detrimental side effects [2, 3, 4]. As such, the exploration of therapies with more tolerable adverse event profiles is urgently needed.
The α1-antitrypsin (AAT) is a multifunctional protein with both proteinase inhibitor and anti-inflammatory activities. These facets render it a potential therapeutic candidate for immune disease intervention including in type 1 diabetes. We demonstrated that human AAT (hAAT) gene therapy prevented type 1 diabetes in NOD mice as a model of diabetes [5, 6]. Follow-up investigations showed that AAT protein therapy protected beta cells from apoptosis . Work performed by Lewis et al. demonstrated that AAT therapy induced immune tolerance and prolonged survival of transplanted islets [8, 9] and Koulmanda et al. demonstrated a profound ability for hAAT to reverse type 1 diabetes in a NOD mouse model through a combination of beneficial mechanisms . However, our own attempts, designed to assess the therapeutic effects of AAT protein therapy in NOD mice, demonstrated that repeated i.p. administration of hAAT led to fatal anaphylaxis . The reasons for the discrepancy (in terms of anaphylaxis induction) between ours and the aforementioned studies remain unclear. Here, we report that i.d. administration of hAAT avoids fatal anaphylaxis and show that hAAT has the ability to both prevent as well as reverse disease in a NOD mouse model of type 1 diabetes.
Granulocyte-colony stimulating factor (G-CSF) is another relatively low-risk agent that has the potential for use in type 1 diabetes immunotherapies. G-CSF induces an immunoregulatory shift from a T-helper type 1 to a T-helper type 2 cytokine phenotype, increases tolerogenic dendritic cells and mobilises regulatory T cells [12, 13, 14]. G-CSF has successfully prevented both the onset of disease in the NOD mouse and cyclophosphamide-mediated acceleration of diabetes [15, 16]. Parker et al. have recently shown that G-CSF enhances the long-term reversal of diabetes afforded by murine antithymocyte globulin (ATG) . To determine if the combination of G-CSF and hAAT would enhance the protective effect of hAAT alone on type 1 diabetes prevention and reversal, we included G-CSF as a second drug in the present study.
Female NOD/LtJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and housed in specific-pathogen-free facilities at the University of Florida. The Institutional Animal Care and Use Committee at the University of Florida approved all animal manipulations.
Eight-week-old female NOD mice (n = 6) received i.p., s.c. or i.d. injections or s.c. implantation of an osmotic pump (Alzet Osmotic Pumps, Cupertino, CA, USA). All mice received a single administration of clinical-grade hAAT (Prolastin, Bayer, Elkhart, IN, USA) at a dose of 2 mg/mouse. All mice were bled at 0 min, 5 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 4 h, 8 h, and daily thereafter until 7 days after hAAT administration. Serum hAAT levels were detected using a hAAT-specific ELISA.
Area under the curve of serum hAAT concentration in the first 7 days (AUC0–7 days) was calculated by WinNonlin 5.2 (Pharsight Inc., Mountain View, CA, USA) using the linear trapezoidal rule.
Cohorts of 11-week-old female NOD mice were injected i.d. with hAAT (2 mg/mouse/3 days, for 10 weeks), with hAAT (2 mg/mouse/3 days, for 10 weeks) plus G-CSF (Neupogen, i.p., 6 μg daily, for 8 weeks), with G-CSF alone or with saline. Blood glucose was monitored weekly. Serum hAAT levels and anti-hAAT antibodies were analysed biweekly until 20 weeks of age.
Mice used in this study were monitored three times per week for hyperglycaemia, defined as a blood glucose >13.32 mmol/l, by tail bleed. Animals measuring above this threshold on two consecutive days were considered diabetic. At the onset of diabetes, mice received i.d. injection of hAAT (Prolastin, 2 mg/3 days, for 8 weeks), hAAT plus G-CSF (i.p., 6 μg daily for 8 weeks), G-CSF alone or saline. All mice at the onset of diabetes received insulin treatment with s.c. insulin pellets (LinBit; LinShin Canada, Inc., Toronto, ON, Canada). The dose of insulin was adjusted to achieve a blood glucose level between 5.55 and 11.1 mmol/l in the first 3 days after the treatment. Blood glucose levels in all animals were continually monitored.
Intraperitoneal glucose tolerance test (IPGTT)
Mice were fasted for 6 h by removal to a clean cage without food at the end of their dark (feeding) cycle. A fasting glucose level was obtained from tail venous blood. Mice were weighed and i.p. injected with glucose (1 mg/g bodyweight). Blood glucose values were obtained at 5, 15 30, 60, 120 and 240 min after glucose challenge.
Insulin tolerance test (ITT)
The test was performed on random-fed mice at about 14:00 hours. The mice were i.p. injected with insulin (0.75 U/kg) in approximately 0.1 ml 0.9% NaCl. Blood glucose was detected at 0, 15, 30, 45 and 60 min after the injection of insulin.
Histology and immunohistochemistry
Insulitis was evaluated and scored on pancreatic sections stained with haematoxylin and eosin, as described previously [4, 5, 6]. Briefly, the degree of lymphocytic infiltration in each islet was scored according to the following scale: 0, none; 1, peri-islet infiltrates; 2, <50% intra-islet infiltrates; 3, >50% intra-islet infiltrates. Insulin immunohistochemistry was performed as previously described [4, 5, 6]. Fractional insulin area was determined from whole digital slide scans using a positive pixel count algorithm (Spectrum, Aperio, Vista, CA, USA).
ELISA for the detection of serum hAAT and BAFF levels and antibodies against hAAT
Detection of hAAT and anti-hAAT antibodies in mouse serum was performed as previously described . Purified hAAT was used as standard (Athens Research & Technology, Athens, GA, USA). Detection of B cell activating factor (BAFF) in serum was performed according to the manufacturer’s instructions (R&D Systems, Inc. Minneapolis, MN, USA).
Slow release of hAAT prevents fatal anaphylaxis in NOD mice
Fatal anaphylaxis rate in mice receiving i.d. injections of hAAT followed by i.p. injection of hAAT
Number of mice
Number of i.d. injections
Number of i.p. injections
AAT protein therapy prevents development of type 1 diabetes in a NOD mouse model
AAT reversed type 1 diabetes in NOD mice
AAT treatment enhanced islet function and decreased BAFF levels
Although hAAT has potential in the prevention and reversal of type 1 diabetes [5, 6, 8, 9], repeated administration of hAAT may lead to fatal anaphylaxis in NOD mice . The immune response is not AAT specific, but is instead the result of the over-reactive immune system of the NOD mice. This high death rate has been a major hurdle for the further investigation of AAT for treatment of type 1 diabetes . In the present study, we showed that i.d. injection of hAAT resulted in slow release in NOD mice and effectively avoided fatal anaphylaxis. Given the fact that the serum hAAT levels at 30 min after i.d. injection were 20-fold and fourfold lower than i.p. and s.c. injections, respectively, the most important factor for avoiding fatal anaphylaxis appears to be controlling the serum levels of AAT in the first 30 min following injection . These results suggest that controlled interaction of AAT and AAT-specific IgE on the mast cells is critical to avoid anaphylaxis. Although i.p., s.c. and i.d. injections of AAT displayed distinct kinetics in the first 2 h, the AUC0–7days were similar in these groups. Delivery of AAT by an osmotic pump resulted in a twofold increase of the AUC0–7days suggesting a possible clinical application in large animals and in humans. These results not only revealed the kinetics of AAT administration by various routes, but also enabled us to further investigate the therapeutic effect of AAT.
Previously, we showed that AAT gene therapy prevented disease in a NOD mouse model of type 1 diabetes [5, 6]. AAT inhibited caspase-3 activity and protected against islet cell death . In the present study, we showed that AAT protein therapy partially prevented and reversed type 1 diabetes in NOD mice. Consistent with our observations and those of other groups, these results support our hypothesis that AAT is beneficial for the treatment of type 1 diabetes [8, 9]. However, partial prevention (i.e. delay of diabetes development) and reversal (50%) of type 1 diabetes by hAAT monotherapy indicate that hAAT therapy has the potential for further improvement. Therefore, a combination therapy using drugs targeting different pathways may improve the treatment effect compared with hAAT monotherapy. The rationale of using G-CSF in this study was to employ its immunoregulatory properties, including its ability to mobilise regulatory T cells, induce tolerogenic dendritic cells and shift cytokine phenotype from T-helper type 1 to type 2 [13, 14]. Previously we demonstrated that G-CSF enhanced the efficacy of ATG-mediated reversal of type 1 diabetes in the NOD mouse model, but as in the present study G-CSF monotherapy did not prevent or reverse disease . Unfortunately, in the present study, we did not observe an enhancing effect when G-CSF was used in combination with AAT. In fact, although there was no statistical difference, we noticed that the addition of G-CSF to hAAT decreased the preventive effect of hAAT alone. These results indicated that G-CSF in combination with different drugs could lead to different outcomes. The possible mechanism behind this observation requires further investigation. Future studies will also focus upon the development of combination therapies of AAT with other drugs.
It is interesting to note that blood glucose levels oscillated in some of the AAT and G-CSF-treated animals. Although these mice eventually developed diabetes, they survived for 10 weeks post diabetes onset, during which time they were mostly euglycaemic. The results indicate that the treatment was partially effective in these mice and provides strong evidence of considerable individual differences in NOD mice despite being an inbred strain. Based on this treatment effect, one can classify NOD mice into three groups: reversal, partial-reversal, and non-reversal groups. The partial-reversal and non-reversal groups may require dose optimisation or additional treatments. It is notable that the reversal efficacy of hAAT therapy appears to be inversely correlated with the blood glucose levels at the onset of diabetes. This tendency is consistent and similar with the observations in a previous study using ATG and G-CSF for reversal of type 1 diabetes . These results suggest that the severity or beta cell mass remaining at the onset of diabetes is probably a major determinant of the success of a treatment.
In the prevention studies, we observed that late AAT treatment (at 10 weeks of age) resulted in the complete prevention of type 1 diabetes up to 25 weeks of age. These results, when compared with controls, clearly demonstrated the powerful protective effect of AAT on disease development. On the other hand, some AAT-treated mice developed diabetes after withdrawal of AAT treatment. This delayed diabetes development suggests that the protective effect was AAT-dependent and/or longer-term treatment with AAT may be required to induce long-term prevention. In fact, our previous observation that gene therapy mediated long-term AAT expression and resulted in long-term prevention supports the latter notion . Together, these results imply that AAT gene therapy rather than AAT protein therapy may one day be useful in preventing type 1 diabetes.
AAT is a multifunctional protein. In addition to acting as a serine proteinase inhibitor in the circulation, AAT can inhibit production of major inflammatory cytokines, such as IL-6 and TNF-α, and enhance the production of the anti-inflammatory cytokine IL-10 through increasing cellular cAMP levels [19, 20]. Evidence from previous studies has shown that AAT treatment may enhance pancreatic beta cell function [7, 8]. In the present studies, we showed that AAT-treated mice responded to glucose challenge similarly to the normal mice. Interestingly, AAT treatment also decreased the serum levels of BAFF. These results are consistent with our previous observations that AAT gene therapy reduced insulin autoantibody levels and that AAT protein and gene therapy reduced serum levels of BAFF and autoantibodies in a collagen-induced arthritis mouse model. These results strongly suggest that hAAT reduces autoantibody levels through inhibition of BAFF production. As BAFF is a B cell activator, it is possible that this reduction may contribute to the inhibition of other B cell-mediated immunity. Detailed molecular mechanism(s) underlying the effect of hAAT on B cell immunity remains to be investigated in future studies. Together, these results indicate that AAT may play an important role in controlling B cell-mediated immunity and imply a new function of AAT.
In summary, we have shown that NOD-specific anaphylaxis can be avoided by controlling the drug’s release within 30 min after the injection and that AAT protein therapy effectively prevents and reverses disease in a NOD mouse model of type 1 diabetes. These results are consistent with previous observations, demonstrate the therapeutic effect of AAT and support the possible clinical application of AAT in patients with type 1 diabetes.
This work was supported by grants from NIH (HL079132, DK062652), Juvenile Diabetes Research Foundation and University of Florida Office of Research. H. Ma was supported by China Scholarship Council.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.