Immune depletion with cellular mobilization imparts immunoregulation and reverses autoimmune diabetes in nonobese diabetic mice

Matthew J Parker, Song Xue, John J Alexander, Clive H Wasserfall, Martha L Campbell-Thompson, Manuela Battaglia, Silvia Gregori, Clayton E Mathews, Sihong Song, Misty Troutt, Scott Eisenbeis, John Williams, Desmond A Schatz, Michael J Haller, Mark A Atkinson, Matthew J Parker, Song Xue, John J Alexander, Clive H Wasserfall, Martha L Campbell-Thompson, Manuela Battaglia, Silvia Gregori, Clayton E Mathews, Sihong Song, Misty Troutt, Scott Eisenbeis, John Williams, Desmond A Schatz, Michael J Haller, Mark A Atkinson

Abstract

Objective: The autoimmune destruction of beta-cells in type 1 diabetes results in a loss of insulin production and glucose homeostasis. As such, an immense interest exists for the development of therapies capable of attenuating this destructive process through restoration of proper immune recognition. Therefore, we investigated the ability of the immune-depleting agent antithymocyte globulin (ATG), as well as the mobilization agent granulocyte colony-stimulating factor (GCSF), to reverse overt hyperglycemia in the nonobese diabetic (NOD) mouse model of type 1 diabetes.

Research design and methods: Effects of each therapy were tested in pre-diabetic and diabetic female NOD mice using measurements of glycemia, regulatory T-cell (CD4+CD25+Foxp3+) frequency, insulitis, and/or beta-cell area.

Results: Here, we show that combination therapy of murine ATG and GCSF was remarkably effective at reversing new-onset diabetes in NOD mice and more efficacious than either agent alone. This combination also afforded durable reversal from disease (>180 days postonset) in animals having pronounced hyperglycemia (i.e., up to 500 mg/dl). Additionally, glucose control improved over time in mice subject to remission from type 1 diabetes. Mechanistically, this combination therapy resulted in both immunological (increases in CD4-to-CD8 ratios and splenic regulatory T-cell frequencies) and physiological (increase in the pancreatic beta-cell area, attenuation of pancreatic inflammation) benefits.

Conclusions: In addition to lending further credence to the notion that combination therapies can enhance efficacy in addressing autoimmune disease, these studies also support the concept for utilizing agents designed for other clinical applications as a means to expedite efforts involving therapeutic translation.

Figures

FIG. 1.
FIG. 1.
Blood glucose values were obtained for up to 180 days postonset in NOD mice treated with control (A), GCSF (B), ATG (C), or ATG + GCSF (D).
FIG. 2.
FIG. 2.
Long-term diabetes reversal was achieved in 75% (12 of 16; P = 0.0000006 vs. control) of murine ATG + GCSF–treated mice, which was significantly improved versus murine ATG monotherapy (P = 0.013), which reversed 33% (5 of 15) mice. Long-term remission was observed in neither GCSF (0 of 14) nor control-treated (0 of 15) mice. ♦, control; ▲, GCSF; ■, ATG; ●, ATG + GCSF.
FIG. 3.
FIG. 3.
GCSF enhances the ability of murine ATG to reverse mice with greater hyperglycemia at diabetes onset. Therapeutic success of murine ATG therapy (●) was largely limited to starting blood glucose values of 380 mg/dl and below with an average of 317.2 mg/dl. The addition of GCSF to murine ATG treatment significantly raised the starting average blood glucose of successful therapy (▲) to 401.8/mg/dl (P = 0.019).
FIG. 4.
FIG. 4.
IPGTT indicates improving glucose control from 60 to 120 days postonset in combination therapy-treated NOD mice. NOD mice remitted from diabetes using combination therapy were administered an IPGTT a 60 (A), 90 (B), and 120 (C) days postonset. An area under the curve analysis revealed a significant improvement at 120 (21,940 ± 1,250, n = 7) days compared with 60 (26,840 ± 1,068, n = 7) days (P = 0.0235, unpaired t test).
FIG. 5.
FIG. 5.
Murine ATG and GCSF combination therapy in NOD mice induces immunomodulation. A: An alteration in peripheral blood leukocyte counts was observed only 2 weeks after initiation of therapy, with murine ATG + GCSF–treated mice exhibiting a significantly greater number (P = 0.0129, unpaired t test) than murine ATG monotherapy. No differences were seen at later time points. GCSF and murine ATG + GCSF–treated mice exhibited significant increases in splenic macrophages (B) (P < 0.0001, P = 0.0027, respectively; unpaired t test) and neutrophils (C) (P < 0.0001, P < 0.0001, respectively; unpaired t test) 2 weeks after initiation of therapy with no differences observed thereafter. D: As expressed out of total splenocytes, the percentage of splenic Treg was reduced in all treatments versus control at 2 weeks due to murine ATG–mediated depletion and/or the mobilization effect of GCSF but was significantly increased versus control at 4 weeks in GCSF-treated mice (P = 0.003, unpaired t test) and at 8 weeks in murine ATG + GCSF–treated mice (P < 0.0001, unpaired t test). ●, control; ■, ATG; ▲, GCSF; ▼, ATG + GCSF.
FIG. 6.
FIG. 6.
Administration of mATG induces an increase in the splenic CD4-to-CD8 ratio. ATG-treated mice exhibited a significantly higher CD4-to-CD8 ratio than both controls and GCSF-treated mice at 2 (P = 0.003, P = 0.0003, respectively, unpaired t test), 4 (P < 0.0001, P < 0.0001, respectively, unpaired t test), and 8 (P = 0.0004, P = 0.0003, respectively, unpaired t test) weeks postinitiation. Combination-treated mice also exhibited significantly higher ratios versus controls and GCSF-treated mice at 2 (P < 0.0001, P < 0.0001, respectively, unpaired t test), 4 (P = 0.0012, P = 0.0056, respectively, unpaired t test), and 8 (P = 0.0002, P = 0.0001, respectively, unpaired t test) weeks postinitiation. ●, control; ■, ATG; ▲, GCSF; ▼, ATG + GCSF.
FIG. 7.
FIG. 7.
Murine ATG and GCSF combination therapy in NOD mice protects pancreatic islets from autoimmune destruction. Insulitis scoring (A) of islets (B) 8 weeks after initiation of therapy indicated that combination therapy skewed scoring toward healthy islets, with significant improvement versus control in the number of islets with a score of 1 (P = 0.0055, unpaired t test). C: Insulin staining at the 8-week time point revealed that combination therapy afforded the greatest β-cell/acinar area and was significantly improved versus murine ATG monotherapy (P = 0.0359, unpaired t test). (A high-quality digital representation of this figure is available in the online issue.)

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