New insights into immunotherapy strategies for treating autoimmune diabetes

Type 1 diabetes mellitus (T1D) is an autoimmune illness that affects millions of patients worldwide. The main characteristic of this disease is the destruction of pancreatic insulin-producing beta cells that occurs due to the aberrant activation of different immune effector cells. Currently, T1D is treated by lifelong administration of novel versions of insulin that have been developed recently; however, new approaches that could address the underlying mechanisms responsible for beta cell destruction have been extensively investigated. The strategies based on immunotherapies have recently been incorporated into a panel of existing treatments for T1D, in order to block T-cell responses against beta cell antigens that are very common during the onset and development of T1D. However, a complete preservation of beta cell mass as well as insulin independency is still elusive. As a result, there is no existing T1D targeted immunotherapy able to replace standard insulin administration. Presently, a number of novel therapy strategies are pursuing the goals of beta cell protection and normoglycemia. In the present review we explore the current state of immunotherapy in T1D by highlighting the most important studies in this field, and envision novel strategies that could be used to treat T1D in the future.Miriam Cabello-Olmo was granted by the Industrial PhD program (Navarre Government) (Ref 001114082016000011)

Preclinical characterization of recombinant human tissue kallikrein-1 as a novel treatment for type 2 diabetes mellitus.

Modulation of the kallikrein-kinin system (KKS) has been shown to have beneficial effects on glucose homeostasis and several other physiological responses relevant to the progression of type 2 diabetes mellitus (T2D). The importance of bradykinin and its receptors in mediating these responses is well documented, but the role of tissue kallikrein-1, the protease that generates bradykinin in situ, is much less understood. We developed and tested DM199, recombinant human tissue kallikrein-1 protein (rhKLK-1), as a potential novel therapeutic for T2D. Hyperinsulinemic-euglycemic clamp studies suggest that DM199 increases whole body glucose disposal in non-diabetic rats. Single-dose administration of DM199 in obese db/db mice and ZDF rats, showed an acute, dose-dependent improvement in whole-body glucose utilization. Sub-acute dosing for a week in ZDF rats improved glucose utilization, with a concomitant rise in fasting insulin levels and HOMA1-%B scores. After cessation of sub-acute dosing, fasting blood glucose levels were significantly lower in ZDF rats during a drug wash-out period. Our studies show for the first time that DM199 administration results in acute anti-hyperglycemic effects in several preclinical models, and demonstrate the potential for further development of DM199 as a novel therapeutic for T2D

Autoimmune Diabetes Is Suppressed by Treatment with Recombinant Human Tissue Kallikrein-1

<div><p>The kallikrein-kinin system (KKS) comprises a cascade of proteolytic enzymes and biogenic peptides that regulate several physiological processes. Over-expression of tissue kallikrein-1 and modulation of the KKS shows beneficial effects on insulin sensitivity and other parameters relevant to type 2 diabetes mellitus. However, much less is known about the role of kallikreins, in particular tissue kallikrein-1, in type 1 diabetes mellitus (T1D). We report that chronic administration of recombinant human tissue kallikrein-1 protein (DM199) to non-obese diabetic mice delayed the onset of T1D, attenuated the degree of insulitis, and improved pancreatic beta cell mass in a dose- and treatment frequency-dependent manner. Suppression of the autoimmune reaction against pancreatic beta cells was evidenced by a reduction in the relative numbers of infiltrating cytotoxic lymphocytes and an increase in the relative numbers of regulatory T cells in the pancreas and pancreatic lymph nodes. These effects may be due in part to a DM199 treatment-dependent increase in active TGF-beta1. Treatment with DM199 also resulted in elevated C-peptide levels, elevated glucagon like peptide-1 levels and a reduction in dipeptidyl peptidase-4 activity. Overall, the data suggest that DM199 may have a beneficial effect on T1D by attenuating the autoimmune reaction and improving beta cell health.</p></div

The effect of DM199 on glucose infusion rate during a hyperinsulinemic-euglycemic clamp.

<p>Catheterized Sprague-Dawley rats (n = 4; age 11–12 weeks) were injected s.c. with either PBS (Control), or DM199 (7 and 35 µg/kg) 30 minutes prior to commencement of a 120-minute hyperinsulinemic-euglycemic clamp (HEC). (A) Profile of glucose infusion rates during HEC. (B) Glucose infusion rate total AUC. Data are presented as the mean ± SEM, **<i>p<</i>0.01 vs. control.</p

Effects of DM199 administration on insulitis and beta cell mass.

<p>Insulitis in pancreatic tissue sections from non-diabetic animals after the indicated treatment periods was graded in a blinded fashion and scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107213#s2" target="_blank">Materials and Methods</a>. Beta cell masses were evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107213#s2" target="_blank">Materials and Methods</a>. (A) The proportions of islets in each treatment group with an observed grade; n, number of mice in each treatment group. (B) Average insulitis scores +/− SEM for the two study cohorts. (C) Histomorphometric quantification of pancreatic beta cell mass. *<i>P</i><0.05 <i>vs.</i> control using a one-way ANOVA analysis with Tukey post-hoc multiple comparisons test.</p

Treatment-dependent anti-diabetic responses.

<p>(A) Glucose area under the curves (AUC) during IPGTT (n = 4–7 mice/group) at the indicated time points were calculated from blood glucose readings corrected for fasting blood glucose, and are plotted as the mean +/− SEM. (B) Dose-dependent increase in C-peptide levels. Fasting serum C-peptide concentrations were measured by ELISA (n = 3–11 mice/group) in triplicate at each time point and are represented as mean +/− SEM. (C) Replicating β cell counts in pancreatic islets at 10 weeks. Cells double-positive for insulin and EdU were manually counted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107213#s2" target="_blank">Materials and Methods</a>. Data are represented as mean number of replicating β cells per islet ± SEM. (D) DM199 treatment enhances serum GLP-1 levels. Blood samples were collected in triplicate (n = 6–7 mice/group) in the presence of DPP-4 inhibitor and active GLP-1 levels in sera were determined by ELISA. Data represent mean ± SEM. (E) High-dose treatment-dependent reduction in DPP-4 activity. Serum DPP-4 activity was determined by monitoring kinetics of formation of a fluorogenic peptide by proteolysis of H-Gly-Pro-AMC synthetic substrate. Data are represented in arbitrary units (AU) mean ± SEM; (n = 6–7 mice/group). *<i>P</i><0.05 <i>vs.</i> control using a one-way ANOVA analysis with Tukey post-hoc multiple comparisons test.</p

Modulation of CD8⁺ T cell populations.

<p>(A) Upper panels: representative images of infiltrated islets stained for CD4 (red), CD8 (green) and DNA (blue) on pancreatic sections from mice treated with either vehicle or 100 U/kg/day of DM199 for 18 weeks. Lower panels: representative images of islets immuno-stained for insulin (brown) and counterstained with H&E. (B) CD4⁺/CD8⁺ ratios in islets. Pancreatic sections from all treatment groups at the indicated time points were fluorescently stained for CD4 and CD8; positive cells were manually counted in a blinded fashion (15 islets/mouse; n = 3–10 mice/group). Data are presented as the mean +/− SEM of the CD4⁺/CD8⁺ ratios for each treatment group. (C) DM199 treatment reduces activated CD8⁺ T cells (CTLs) in pancreatic lymph nodes (PLN). Single cell suspensions of PLN from treated mice were stained against CD3, CD8 and CD44, analyzed by FACS and the percentages of CD44⁺CD8⁺ cells within the CD3⁺ T cell population were calculated. Data are mean ± SEM (n = 4–10 mice/group). *<i>P</i><0.05 <i>vs.</i> control, ***<i>P</i><0.001 <i>vs.</i> control, using a one-way ANOVA with Tukey post-hoc multiple comparisons test.</p

Purified DM199.

<p>1, 2 and 4 µg of purified DM199 were either non-reduced (lanes 2, 3 & 4) or reduced (lanes 6, 7 & 8) with NuPAGE Sample Reducing Agent (Life Technologies, Carlsbad, CA) and loaded on to a NuPage Novex 4–12% BIS-TRIS pre-cast polyacrylamide gel (Life Technologies). The gel was stained with Colloidal Blue Staining (Life Technologies, Carlsbad, CA). Lanes 1, 5 and 9: molecular weight marker (SeeBlue Plus2 Standard, Life Technologies, Carlsbad CA).</p

Effect of DM199 on TGF-β1 levels.

<p>TGF-β1 levels in sera of mice treated with the indicated DM199 doses were measured by ELISA. (A) Total TGF-β1 from Cohort 1 mice treated for up to 18 weeks. (B) Active TGF-β1 from Cohort 2 mice treated for up to 10 weeks. Data are mean ± SEM (n = 5–7 mice/group). *<i>P</i><0.05 <i>vs.</i> controls, using one-way ANOVA with Tukey post-hoc multiple comparisons test.</p