Intradermal alpha 1 antitrypsin therapy avoids fatal anaphylaxis, and prevents and reverses type 1 diabetes in NOD mice

Abstract

Aims/hypothesis

Human alpha 1 antitrypsin (hAAT) gene therapy prevents type 1 diabetes in NOD mice. However, repeated intraperitoneal (IP) 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 in the NOD.

Methods

To evaluate hAAT pharmacokinetics, serum hAAT levels were monitored in NOD mice receiving a single dose (2mg) of hAAT by IP, subcutaneous (SC) or intradermal (ID) injection. For studies of type 1 diabetes prevention and reversal, mice received ID hAAT (2mg/mouse/3days) for 8 or 10 weeks or hAAT and G-CSF (IP, 6μg/day) for 6 weeks. Blood glucose determinations, glucose tolerance testing, and insulin tolerance tests were performed.

Results

IP and SC injections resulted in fatal anaphylaxis. ID injection avoided anaphylaxis. ID injection of hAAT into 11-week-old NOD mice prevented diabetes (P=0.005, AAT vs. PBS at 40 week 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 does not enhance the reversal rates of hAAT. Glucose tolerance and insulin levels were normalized in hAAT prevented and reversed mice.

Conclusions/interpretation

Intradermal hAAT prevents and reverses diabetes in NOD mice without inducing anaphylaxis.

Introduction

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 [1]. While recent studies have shown promising results in terms of reversing type 1 diabetes, many of these treatments can lead to detrimental side effects [2–4]. As such, the exploration of therapies with more tolerable adverse event profiles is urgently needed.

Alpha 1 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 type 1 diabetes. We demonstrated that human AAT (hAAT) gene therapy prevented type 1 diabetes in NOD mice [5, 6]. Follow up investigations showed that AAT protein therapy protected beta cells from apoptosis [7]. Work performed by Lewis et al demonstrated that AAT therapy induced immune tolerance and prolonged survival of transplanted islets [8, 9]. Studies of Koulmanda et al demonstrated a profound ability for hAAT to reverse type 1 diabetes in NOD mice through a combination of beneficial mechanisms [10]. However, our own attempts designed to assess the therapeutic effects of AAT protein therapy in NOD mice demonstrated that repeated intraperitoneal (IP) administration of hAAT led to fatal anaphylaxis [11]. The reasons for the discrepancy (in terms of anaphylaxis induction) between ours and the aforementioned studies remain unclear. Here, we report that introdermal administration of hAAT avoids fatal anaphylaxis and show that hAAT has the ability to both prevent as well as reverse type 1 diabetes in NOD mice.

Granulocyte-colony stimulating factor (G-CSF) is another relatively low risk agent that has the potential to be utilized in type 1 diabetes immunotherapies. G-CSF induces an immunoregulatory shift from a T_H1 to a T_H2 cytokine phenotype, increases tolerogenic dendritic cells and mobilize T_reg cells [12–14]. G-CSF has successfully prevented the onset of disease in the NOD mouse and cyclophosphamide-mediated acceleration of diabetes [15, 16]. Parker et al has recently shown G-CSF enhanced the long-term reversal of diabetes afforded by murine antithymocyte globulin (ATG) [4]. In order 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.

Methods

Animals

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.

Kinetics studies

Eight week old female NOD mice (n=6) received intraperitoneal (IP), subcutaneous (SC), intradermal (ID) injection or an osmotic pump (Alzet® Osmotic Pumps, Cupertino, CA, USA), which was implanted subcutaneously. All mice received a single administration of clinical grade human alpha one antitrypsin (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 hr, 1.5hr, 2 hr, 4 hr, 8 hr, and daily thereafter until 7 days after hAAT administration. Serum hAAT levels were detected by a hAAT specific ELISA.

Area under-the-curve of serum hAAT concentration in the first 7 days (AUC_0–7day) was calculated by WinNonlin 5.2 (Pharsight Inc., Moutain View, CA, USA) using the linear trapezoidal rule.

Prevention studies

Cohorts of 11week old female NOD mice were ID injected with hAAT (2 mg/mouse/3 days, for 10 weeks), hAAT (2 mg/mouse/3 days, for 10 weeks) plus G-CSF (Neupogen®, IP, 6□g daily, for 8 weeks), G-CSF alone or saline. Blood glucose was monitored weekly. Serum hAAT levels and anti-hAAT antibodies were analyzed biweekly until 20 weeks of age.

Reversal studies

Mice used in this study were monitored 3 times per week for hyperglycemia 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 ID injection of hAAT (Prolastin®, 2mg/3days, for 8 weeks), hAAT plus G-CSF (IP, 6 μg daily for 8weeks), G-CSF alone or saline. All mice at the onset of diabetes received insulin treatment by subcutaneously insulin pellets (LinβiT™, LinShin Canada, Inc). The dose of insulin was adjusted to achieve a blood glucose level between 5.55 to 11.1 mmol/L in the first three days after the treatment._Blood glucose levels in all animals were continually monitored.

Intraperitoneal glucose tolerance test (IPGTT)

Mice were fasted for six hours 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 IP 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 around 2 pm. The mice were intraperitoneally injected with insulin (0.75 U/kg) in ~ 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 inmmunohistochemistry

Insulitis was evaluated and scored on hematoxylin and eosin stained pancreatic sections as described previously [4–6]. Briefly, the degree of lymphocytic infiltration in each islet was scored according to the following scale: 0, none; 1, periislet infiltrates; 2, <50% intraislet infiltrates; 3, >50% intraislet infiltrates. Insulin immunohistochemistry was performed as previously described [4–6]. Fractional insulin area was determined from whole digital slide scans using a positive pixel count algorithm (Spectrum, Aperio, Vista CA).

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 [17]. Purified hAAT was used as standard (Athens Research & Technology, Athens, GA). Detection of BAFF in serum was performed according to manufactures instructions (R&D systems, Inc. Minneapolis, MN).

Results

Slow release of hAAT prevents fatal anaphylaxis in NOD mice.

In a pharmacokinetics study, cohorts of 8 week old NOD mice (n=6) received 2 mg of hAAT (Prolastin®) by an intraperitoneal (IP), subcutaneous (SC), intradermal (ID) injection, or an osmotic pump (Alzet® Pump). As shown in Figure 1a, administration of this protein by different routes resulted in distinct kinetics with IP resulting in the highest levels for the entire 60 minutes. At 30 min after injection, serum hAAT levels in SC, ID or Pump group was 20%, 5% and 2% respectively of that in the IP group. IP, SC, and ID injections resulted in similar area under-the-curve of serum concentration in the first 7 days (AUC _{0–7 day}), while Alzet® Pump resulted in 2-fold increase of AUC _{0–7 day} (Fig. 1b and 1c). Since serum hAAT levels in the SC injected group were dramatically lower than that in the IP injected group, we performed repeated SC injections of hAAT (2 mg per 3 days) to NOD mice (4 week old). Unfortunately, 90% (9/10) of mice died after the 4^th or 5^th injection. We next performed repeated ID injections of hAAT, which resulted in 4 fold lower levels of hAAT at 30 min when compared to SC injection (Figure 1a). Surprisingly, no animal died after up to 15 injections (Table 1, group 2 and 3). Furthermore, animals that received more than 9 ID injections also survived from 7 additional IP injections of hAAT (Table 1, group 3), indicating that the animals developed tolerance to hAAT after 9 ID injections. These results demonstrated that the fatal anaphylaxis can be avoided by controlling release of hAAT in the first 30 to 40 min of administration. Therefore, we chose to use ID injections for the following studies.

Figure 1: Pharmacokinetics of hAAT in NOD mice.

Cohorts of 8 week old NOD mice (n=6) were injected with 2 mg of hAAT (Prolastin®). Serum hAAT levels were detected by hAAT specific ELISA. Filled diamond, intraperitoneal injection; open square, subcutaneous injection; filled triangle, intradermal injection; open circle, using an osmotic pump (Alzet® Pump). a, hAAT levels within 60 minutes of administration. b, Long term (7 days) hAAT levels after single administration. c, Area under-the-curve of hAAT levels in the first 7 days (AUC_{0–7 day}) of hAAT levels.

Table 1.

Fatal anaphylaxis rate in mice receiving ID injections of hAAT followed by IP injection of hAAT.

Groups	1	2	3
Number of mice	8	7	7
Number of ID injections	0	1–7	9–15
Death (%)	0	0	0
Number of IP injections	8	7	7
Death (%)	90	70	0

AAT protein therapy prevents type 1 diabetes development in NOD mice.

In order to determine whether ID hAAT protein therapy prevents the development of type 1 diabetes, cohorts of 11 week-old female NOD mice were ID injected with hAAT, hAAT plus G-CSF, G-CSF alone or saline. G-CSF treatment lasted 8 weeks (11 to 18 weeks of age) and hAAT treatment lasted 10 weeks (11 to 20 weeks of age). Injection of hAAT resulted in high serum levels of hAAT and anti-hAAT antibodies (Figs. 2a and 2b). All mice in hAAT and hAAT plus G-CSF treated groups remained diabetes free until 25 weeks of age, while 67 % of G-CSF and saline groups developed diabetes at 21 weeks (Figure 2c). At 40 weeks of age (the end of the experiment), 63 % of mice in the AAT group were diabetes free (P=0.005, AAT vs. PBS). These data clearly demonstrated that hAAT protein therapy significantly prevented type 1 diabetes in NOD mice, while G-CSF had no enhancing effect.

Figure 2: Human AAT (hAAT) protein therapy prevents type 1 diabetes development in NOD mice.

Cohorts of 11 week-old female NOD mice were ID injected with hAAT (2 mg/mouse, every 3 days) for 10 weeks (indicated by a gray bar), and/or G-CSF (6 mg/mouse, daily) for 8 weeks (indicated by open bar). Open diamond, hAAT (n=8); filled diamond, hAAT plus G-CSF (n=7); filled triangle, G-CSF alone (n=8); open triangle, saline (n=9). a, Serum levels of hAAT. b, Levels of anti-hAAT antibodies detected by ELISA. c, Life table analysis for diabetes development. At 25 weeks of age, all mice in hAAT and hAAT plus G-CSF treated groups remained diabetes free. At 40 weeks of age (the end of the experiment), 63 % of mice in AAT group were diabetes free (P=0.005, AAT vs PBS).

AAT reversed type 1 diabetes in NOD mice.

To test the feasibility of hAAT in reversing type 1 diabetes, newly diagnosed diabetic mice were treated with hAAT, hAAT plus G-CSF, G-CSF alone or saline. Both hAAT and G-CSF treatments were continued for 8 weeks. All mice were given insulin pellets which provided a source of exogenous insulin for 10 to 15 days. Blood glucose levels of all saline and G-CSF alone injected mice were greater than 16.65 mmol/L after exhaustion of the insulin pellets (Figures 3a and 3b). In the AAT treated group, more than 50% of mice (4/7) remained diabetes free for at least 100 days (Figure 3c). In hAAT plus G-CSF treated group, 50% of the mice similarly remained diabetes free (Figures 4a and 4b). Blood glucose in two hAAT plus G-CSF-treated mice oscillated considerably for 8 weeks, remained below 13.32 mg/dL for 2 more weeks and finally rose to 27.75 mmol/L (Figure 4b) indicating that the treatment has a partial effect on the intervention of diabetes in these mice. Notably, the starting blood glucose levels in mice successfully reversed by AAT plus G-CSF therapy were significantly lower than that in mice who failed therapy (Figure 4c). A similar trend can be observed in hAAT treated mice with exception of one mouse with a very high starting blood glucose level that was successfully reversed. Consistent with the observations in a previous study using antithymocyte globulin (ATG) and G-CSF for reversal of type 1 diabetes, these results indicate that the severity of disease at the onset or before treatment affects the efficacy of the treatment.

Figure 3. Reversal of type 1 diabetes by hAAT protein therapy.

New onset diabetic mice were treated with hAAT (2mg/mouse), G-CSF (6mg/mouse) alone or saline. Both hAAT and G-CSF treatments were administered for 8 weeks. Each line represents data from an individual animal. a, Blood glucose levels in saline treated group (n=5). b, Blood glucose levels in G-CSF treated group (n=6). c, Blood glucose levels in mice treated with hAAT and successfully reversed from type 1 diabetes (n=4). d. Blood glucose levels in mice treated with hAAT that failed to reverse type 1 diabetes (n=3).

Figure 4. Reversal of type 1 diabetes by hAAT and G-CSF therapy.

New onset diabetic mice (n=6) were treated with hAAT (2mg/mouse) and G-CSF (6mg/mouse) for 8 weeks. Each line represents data from an individual animal. a, Blood glucose in long term reversed mice. b, Blood glucose in mice who failed to reverse type 1 diabetes. c, Effect of disease severity on the efficacy of treatments. Starting blood glucose levels (at the onset of diabetes) in different groups are plotted. **, P=0.0044.

AAT treatment enhanced islet function and decreased B cell activating factor (BAFF) levels.

In addition to monitoring blood glucose, we performed glucose tolerance tests and insulin resistance tests. As shown in Figure 5a, mice that survived in the prevention and reversal studies responded to glucose similarly to normal mice indicating islet function was retained in these mice. The AAT treated mice also responded to insulin challenge similarly to normal mice, indicating that no insulin resistance developed in these mice (Figure 5b). Insulitis was evaluated as previously described. As shown in Figure 5c, more islets were found in hAAT prevented and reversed mice compared to those in new onset diabetic mice. Similarly, more insulin positive area was found in those mice compared to control type 1 diabetes.

Figure 5. The effects of hAAT treatment on islet function, insulin resistance and immune system.

a, Intraperitoneal glucose tolerance test (IPGTT). Open diamond, diabetic mice (n=4); filled square, hAAT prevented mice (40 weeks of age, n=4); open triangle, hAAT reversed mice (euglycemic for more that 100 days, n=3); filled circle, normal diabetic free mice (n=4). b, Insulin tolerance test (ITT). c, The effects of hAAT on insulitis. Insulitis was evaluated in AAT prevented mice (40 weeks of age, n=4) and reversed mice (euglycemic for more that 100 days, n=3). The new onset diabetic mice served as control (T1D, n=2).

In order to further understand the effects of AAT on immune system, we measured BAFF levels in both control and AAT treated mice. We showed that AAT significantly decreased serum BAFF levels 2 weeks after treatment (Figure 6) suggesting an effect of hAAT on B cell mediated autoimmunity.

Figure 6. The effect of hAAT on serum BAFF levels.

BAFF levels in hAAT treated group (n=8) and saline treated mice (PBS, n=10) was detected by ELISA at 2 weeks after the treatment. P=0.023 (AAT vs. PBS)

Discussion:

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 [11]. The immune response is not AAT specific, but is instead due to the over reactive immune system of the NOD mice. This high death rate has been the major hurdle for the further investigation of AAT for treatment of type 1 diabetes [11]. In the present study, we showed that ID 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 ID injection were 20 and 4 fold lower than IP and SC injection, 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 [18]. These results suggest that controlled interaction of AAT and AAT-specific IgE on the mast cells is critical to avoid anaphylaxis. Although IP, SC and ID injections of AAT displayed distinct kinetics in the first 2 hours, the areas under-the-cure of the first 7 days (AUC_{0–7 day}) were similar in these groups. Delivery of AAT by an osmotic pump resulted in a 2-fold increase of the AUC_{0–7 day} suggesting a possible clinical application in large animals and in humans. These results not only revealed the kinetics of AAT administrations by various routes, but also enabled us to further investigate the therapeutic effect of AAT.

Previously, we showed that AAT gene therapy prevented type 1 diabetes in NOD mice [5, 6]. AAT inhibited caspase-3 activity and protected against islet cell death [7]. 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 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 rational of using G-CSF in this study was to employ its immunoregulatory properties including its ability to mobilize Treg cells, induce tolerogenic dendritic cells and shift cytokine phenotype from a T_H1 to a T_H2 [13, 14]. Previously we demonstrated that G-CSF enhanced the efficacy of anti-thymocye globulin (ATG)-mediated reversal of type 1 diabetes in NOD mice, but as in the present study G-CSF monotherapy did not prevent or reverse type 1 diabetes [4]. Unfortunately, in the present study, we did not observe an enhancing effect when G-CSF was used in combination with AAT. In fact, while 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 remains further investigation. Future studies will also focus upon the development of combination therapies of AAT and 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 euglycemic. 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 3 groups: reversal, partial-reversal, and non-reversal groups. The partial-reversal and non-reversal groups may require dose optimization and/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 [4]. These results suggest that the severity or beta-cell mass remaining at the onset of diabetes is likely 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 to 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: 1) the protective effect was AAT-dependent; and/or 2) 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 [6]. 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 serine proteinase inhibitor (SERPIN) 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 β-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 B cell activating factor (BAFF). These results are consistent with our previous observations that AAT gene therapy reduced insulin autoantibody (IAA) levels and that AAT protein and gene therapy reduced serum levels of BAFF and autoantibodies in collagen induced arthritis mouse model. These results strongly suggest that hAAT reduces autoantibody levels through inhibition of BAFF production. Since 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 investigated in the 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: 1) NOD specific anaphylaxis can be avoided by controlling the drug’s release within 30 minutes after the injection; and 2) AAT protein therapy effectively prevents and reverses diabetes in NOD mice. These results are consistent with previous observations, demonstrate the therapeutic effect of AAT and support the possible clinical application of AAT in patient with type 1 diabetes.

Abbreviations:

hAAT

human alpha 1 antitrypsin

G-CSF

granulocyte colony stimulating factor

ID

intradermal

BAFF

B cell activating factor