Combination Therapy Reverses Hyperglycemia in NOD Mice With Established Type 1 Diabetes

Abstract

An increasing number of therapies have proven effective at reversing hyperglycemia in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D), yet situations of successful translation to human T1D are limited. This may be partly due to evaluating the effect of treating immediately at diagnosis in mice, which may not be reflective of the advanced disease state in humans at disease onset. In this study, we treated NOD mice with new-onset as well as established disease using various combinations of four drugs: antithymocyte globulin (ATG), granulocyte-colony stimulating factor (G-CSF), a dipeptidyl peptidase IV inhibitor (DPP-4i), and a proton pump inhibitor (PPI). Therapy with all four drugs induced remission in 83% of new-onset mice and, remarkably, in 50% of NOD mice with established disease. Also noteworthy, disease remission occurred irrespective of initial blood glucose values and mechanistically was characterized by enhanced immunoregulation involving alterations in CD4+ T cells, CD8+ T cells, and natural killer cells. This combination therapy also allowed for effective treatment at reduced drug doses (compared with effective monotherapy), thereby minimizing potential adverse effects while retaining efficacy. This combination of approved drugs demonstrates a novel ability to reverse T1D, thereby warranting translational consideration.

Figure 1

AGDP combination therapy induces long-term euglycemia in NOD mice with new-onset and established T1D. A: At diabetes onset, mice were implanted with an insulin pellet and treatment was initiated (n = 11–13 per group). AGDP therapy induced durable remission in 83% of mice (P < 0.0001 vs. control, Kaplan-Meier survival test), compared with 54% of mice treated with DPP-4i+PPI and 50% of mice treated with ATG+G-CSF. B: Mice were implanted with an insulin pellet at onset, with treatment delayed 2 weeks (n = 11–13 per group). AGDP therapy induced remission in 50% of mice (P < 0.0013 vs. control) compared with 42% of mice treated with DPP-4i+PPI and 33% of mice treated with ATG+G-CSF. C–K: Nonfasting blood glucose curves are shown for individual mice in each treatment group. The day of T1D onset was set to day 0 (C–G) or day −14 (H–K) for the new-onset and established (delay) treatment groups, respectively, so that curves reflect blood glucose values after the initiation of treatment. L: Insulitis scoring of remitted mice showed that despite euglycemia at 120 days, the numbers of remaining islets with no to minimal inflammation (scores 0–2) were significantly reduced in all treatment groups compared with age-matched control nondiabetic NOD mice (P < 0.0001).

Figure 1

AGDP combination therapy induces long-term euglycemia in NOD mice with new-onset and established T1D. A: At diabetes onset, mice were implanted with an insulin pellet and treatment was initiated (n = 11–13 per group). AGDP therapy induced durable remission in 83% of mice (P < 0.0001 vs. control, Kaplan-Meier survival test), compared with 54% of mice treated with DPP-4i+PPI and 50% of mice treated with ATG+G-CSF. B: Mice were implanted with an insulin pellet at onset, with treatment delayed 2 weeks (n = 11–13 per group). AGDP therapy induced remission in 50% of mice (P < 0.0013 vs. control) compared with 42% of mice treated with DPP-4i+PPI and 33% of mice treated with ATG+G-CSF. C–K: Nonfasting blood glucose curves are shown for individual mice in each treatment group. The day of T1D onset was set to day 0 (C–G) or day −14 (H–K) for the new-onset and established (delay) treatment groups, respectively, so that curves reflect blood glucose values after the initiation of treatment. L: Insulitis scoring of remitted mice showed that despite euglycemia at 120 days, the numbers of remaining islets with no to minimal inflammation (scores 0–2) were significantly reduced in all treatment groups compared with age-matched control nondiabetic NOD mice (P < 0.0001).

Figure 2

AGDP combination therapy and pancreatic insulin content. In a cross-sectional study, NOD mice with new-onset and established disease were implanted with a subcutaneous insulin pellet and treated with ATG+G-CSF, DPP-4i+PPI, or AGDP or not treated (control), and tissues were harvested 30 days after the initiation of treatment. A: Serum C-peptide (n = 6–7 per group), as measured via ELISA, trended higher in new-onset animals that received ATG+G-CSF and AGDP therapy compared with insulin-only controls (P = 0.1115, Kruskal-Wallis test). Estab., established. Pancreata (n = 4 per group) were stained for insulin via IHC and analyzed for the number of insulin-positive (B) islets and fractional insulin area (C). The difference between treatment groups was not significant, but there was a trend toward increased insulin-positive islets and insulin fractional area in new-onset mice treated with ATG+G-CSF and AGDP therapy (P = 0.7776 and P = 0.7237, respectively; one-way ANOVA). Pancreata (n = 2–3 per treatment group and age-matched nondiabetic NOD controls) were processed for total protein via acid-ethanol extraction and analyzed for total proinsulin (D), C-peptide (E), and insulin (F) via ELISA. Compared with insulin-treated animals, there was a trend toward increased proinsulin in new-onset mice treated with ATG+G-CSF and AGDP therapies, but all treatment groups demonstrated significantly reduced proinsuiln, C-peptide, and insulin compared with nondiabetic controls. Data are presented as mean ± SEM. ****P < 0.0001, all (one-way ANOVA).

Figure 3

AGDP combination therapy and initial blood glucose. A: Initial blood glucose values were similar between mice in all therapy groups (n = 11–13 per group). P = NS, one-way ANOVA. In new-onset mice (B) and established T1D mice (C), ATG+G-CSF therapy was less effective at reversing diabetes in those with higher initial blood glucose. Data are presented as scatter with mean ± SD. *P < 0.05 (Student t test).

Figure 4

Combination therapy induces T-cell and NK-cell immunomodulation. T-cell subsets were analyzed by flow cytometry on treated mice (n = 11–13 per treatment group) or unmanipulated nondiabetic NOD mice (n = 9) at the study end point (therapy failure or 120 days). In new-onset (A) and established (B) T1D mice, AGDP therapy increased the CD4⁺-to-CD8⁺ T-cell ratio compared with control mice and mice treated with DPP-4i+PPI therapy, whereas ATG+G-CSF therapy had an increased ratio compared with DPP-4i+PPI therapy. These data were validated in a cross-sectional study of NOD mice with new-onset (C) and established (D) disease, where tissues were harvested 30 days after the initiation of treatment (n = 6–11 per group). ATG+G-CSF and AGDP therapies increased the CD4⁺-to-CD8⁺ T-cell ratio compared with control mice and mice treated with DPP-4i+PPI therapy (P < 0.0001 and P = 0.0016, respectively; one-way ANOVA). E: Splenocyte CD8⁺ T-cell frequency within the lymphocyte population was significantly reduced by ATG+G-CSF and AGDP therapy in mice with new-onset as well as established T1D, relative to insulin pellet controls (P < 0.0001, one-way ANOVA). F: Compared with control mice, splenic CD4⁺ T-cell frequency was increased in new-onset AGDP-treated animals but decreased with ATG+G-CSF treatment in mice with established disease (P < 0.05). In NOD mice with new-onset T1D (G), AGDP therapy decreased the splenic NK-cell frequency compared with insulin-treated control mice (P = 0.0015; one-way ANOVA), whereas in NOD mice with established disease (H), ATG+G-CSF and AGDP therapies decreased the NK-cell frequency (P = 0.005). Data are presented as scatter with mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

At day 30 after initiation of treatment, pancreata were fixed and embedded in paraffin. Serial sections were stained via IHC for CD4 (A) and CD8 (B). The scanned images were annotated for quantification of the percentage of cells staining positive for CD4 (C) and CD8 (D) within the islet area using CytoNuclear IHC Quantification software. (E) The CD4⁺-to-CD8⁺ T-cell ratio within the pancreatic islets was not significantly different across treatment groups.

Figure 6

Combination therapy induces T-cell immunoregulation. In new-onset (A) and established (B) T1D mice, remitted mice receiving AGDP, ATG+G-CSF, and DPP-4i+PPI therapies had elevated Tregs compared with failures. B: With the exception of ATG+G-CSF therapy in established disease, remitted mice from all three therapies had elevated Tregs compared with control nondiabetic mice. In new-onset (C) and established (D) T1D mice, remitted AGDP animals exhibited increased CD8⁺ Tregs at 120 days compared with nondiabetic controls (C) or failed AGDP (D) mice, respectively. *P < 0.05 (one-way ANOVA).

Figure 7

Naïve and memory CD4⁺ T-cell subsets modulation by combination therapy. T-cell subsets were analyzed by flow cytometry in treated mice (n = 11–13 per treatment group) or unmanipulated nondiabetic NOD mice (n = 9) at the study end point. A: At 120 days, mice in all new-onset therapy groups, regardless of ability to reverse, had higher CD4⁺ naïve cell frequency than controls. B: In mice with established disease, only remitted AGDP animals corresponded to higher naïve T cells compared with nondiabetic controls, whereas naïve T-cell levels were higher in failed vs. reversed animals treated with DPP-4i+PPI. CD4⁺ memory T cells were not different between control and treated animals with new-onset (C) or established (D) T1D. However, successfully treated mice did show differences, as indicated, vs. mice that failed to remit. Data are presented as scatter with mean ± SD. Remitted vs. control mice analyzed by one-way ANOVA; remitted mice vs. failures by Student t test. *P < 0.05.

Figure 8

Memory and naïve CD8⁺ T-cell analysis of treatment groups. Naïve CD8⁺ T cells were elevated at 120 days with ATG+GSCF therapy in new-onset (A) but not in established (B) T1D mice vs. nondiabetic mice (one-way ANOVA). C: Mice successfully treated with ATG+G-CSF or DPP-4i+PPI had lower levels of memory CD8⁺ cells than nondiabetic NOD mice at 120 days but higher levels of memory CD8 T cells than failed animals, as indicated. *P < 0.05 (one-way ANOVA). D: Memory CD8⁺ cells were elevated in remitted mice compared with failures for all treatment conditions in the group with established disease. Data are presented as mean ± SD. Differences between remitted and failed mice within treatment groups were analyzed by Student t test. *P < 0.05.