Tellurium Compounds Prevent and Reverse Type-1 Diabetes in NOD Mice by Modulating α4β7 Integrin Activity, IL-1β, and T Regulatory Cells

Abstract

The study shows that treatment of NOD mice with either of two tellurium-based small molecules, AS101 [ammonium trichloro(dioxoethylene-o,o')tellurate] or SAS [octa-O-bis-(R,R)-tartarate ditellurane] could preserve β cells function and mass. These beneficial effects were reflected in decreased incidence of diabetes, improved glucose clearance, preservation of body weight, and increased survival. The normal glucose levels were associated with increased insulin levels, preservation of β cell mass and increased islet size. Importantly, this protective activity could be demonstrated when the compounds were administered either at the early pre-diabetic phase with no or initial insulitis, at the pre-diabetic stage with advanced insulitis, or even at the advanced, overtly diabetic stage. We further demonstrate that both tellurium compounds prevent migration of autoimmune lymphocytes to the pancreas, via inhibition of the α4β7 integrin activity. Indeed, the decreased migration resulted in diminished pancreatic islets damage both with respect to their size, β cell function, and caspase-3 activity, the hallmark of apoptosis. Most importantly, AS101 and SAS significantly elevated the number of T regulatory cells in the pancreas, thus potentially controlling the autoimmune process. We show that the compounds inhibit pancreatic caspase-1 activity followed by decreased levels of the inflammatory cytokines IL-1β and IL-17 in the pancreas. These properties enable the compounds to increase the proportion of Tregs in the pancreatic lymph nodes. AS101 and SAS have been previously shown to regulate specific integrins through a unique redox mechanism. Our current results suggest that amelioration of disease in NOD mice by this unique mechanism is due to decreased infiltration of pancreatic islets combined with increased immune regulation, leading to decreased inflammation within the islets. As these tellurium compounds show remarkable lack of toxicity in clinical trials (AS101) and pre-clinical studies (SAS), they may be suitable for the treatment of type-1 diabetes.

Keywords: IL-1β; Tregs; diabetes; inflammation; integrin; tellurium.

Figure 1

Expression of the β7 integrin subunit on T and B cells in various organs of NOD mice. Single cell suspensions from spleens (A), peripheral LN (B), and PLN (C) from 8-week-old NOD mice were stained with FITC-conjugated anti CD3, PE-conjugated anti β7 and APC-conjugated anti B220. CD3⁺β7⁺ positive cells represent T cells expressing the β7 integrin subunit. B220⁺β7⁺ positive cells represent B cells expressing the β7 integrin subunit. The results represent one experiment representative of three performed.

Figure 2

AS101 and SAS inhibit in vitro the activity of the α4β7 integrin in various organs of NOD mice. 5 × 10⁵ single cells from spleens (A), peripheral LN (B), and PLN (C) from 10 week old NOD mice were cultured on BSA or MAdCAM-1-coated micro wells for 2 h in the presence or absence of different concentrations of SAS or AS101The cells were washed twice and then subjected to XTT reagent according to the manufacturer's instructions. The results represent means±SE of three different experiments. ^*p < 0.001 decrease vs. MAdCAM-1 without AS101 or SAS (SAS 0).

Figure 3

AS101 and SAS inhibit in vitro the activity of the a4β7 integrin on both T and B spleen cells of NOD mice. T and B spleen cells from 10 week old mice were sorted by FACS, using anti CD3 or anti B220 antibodies. Single cells (5 × 10⁵) of each lineage (A = T cells; B = B cells) were cultured on BSA or MAdCAM-1-coated microwells for 2 h in the presence or absence of different concentrations of SAS or 1 μg/ml AS101. The cells were washed twice and then subjected to the XTT reagents according to the manufacturer's instructions. The results represent means ± SE from three different experiments. ^*p < 0.01; ^**p < 0.001 decrease vs. MAdCAM-1 without AS101 or SAS (SAS 0).

Figure 4

SAS inhibits in vitro attachment of T and B lymphocytes from NOD mice to the high endothelial venular ('HEV') cells (SVEC-4). Cells were washed twice. Eighty percent confluent endothelial SVEC cells in 6 well plates were stimulated with TNFα to induce MAdCAM-1 expression. After 10 h, 3 × 10⁶ splenocytes from 10 week old NOD mice were added in the presence or absence of various concentrations of SAS o.n. Culture plates were washed and the attached cells were removed and stained with FITC-conjugated anti CD3, and APC-conjugated anti B220 antibodies for the detection of attached T (A) or B (B) cells. The results represent one experiment, representative of three performed.

Figure 5

Treatment with AS101 starting at 5–6 weeks of age prevents the development of T1D in NOD mice. AS101 was administered i.p. every other day at different doses to female NOD mice, starting at 5–6 weeks of age, until 40 weeks. The incidence of diabetes (glucose levels at least 200 mg/dL (A), body weight (B), and percent survival (C) were monitored. n = 10 animals/group. ^**p < 0.05 decrease vs. PBS; ^*p < 0.05 increase vs. PBS; #p < 0.05 increase vs. PBS.

Figure 6

Treatment with AS101 starting at 12 weeks of age prevents the development of T1D in NOD mice. PBS or AS101 at 0.5 mg/kg/injection was administered i.p. every other day at different doses to female NOD mice, starting at 12 weeks of age, until 40 weeks. The incidence of diabetes (A), body weight (B), and percent survival (C) were monitored, n = 10/group. (D,E) AS101 at 0.5 mg/kg/injection was administered i.p. every other day at different doses to female NOD mice, starting at 14 weeks of age. Survival (D) and body weight (E) of mice was monitored. n = 5 mice/group. ##p < 0.005 increase vs. PBS. #p < 0.01 decrease vs. PBS; ^*p < 0.05 increase vs. PBS.

Figure 7

Treatment of NOD mice with SAS or AS101 decreases clinical symptoms of T1D in NOD mice. SAS at different concentrations or AS101 at 1 mg/kg/injection were administered i.p. every other day to female NOD mice, starting at either 5–6 weeks of age (A–D), or at 3 weeks of age (E-H) until 30 weeks. Blood glucose levels (^*p < 0.005 decrease vs PBS) (A,E), body weight (^**p < 0,001; ^*p<p < 0.005 increase vs. PBS) (B,F), percent incidence of diabetes (^*p < 0.005 decrease vs. PBS; ^**p < 0.001) (C,G), and survival (^*p < 0.005 increase vs. PBS) (D,H) were monitored. N = 10 mice/group.

Figure 8

Effect of the compounds on islet morphology and insulitis. AS101 or SAS at 1mg/kg/injection were administered i.p. every other day to female NOD mice, starting at 3 weeks of age. At 12 weeks (A) (beginning of insulitis) or at 20 weeks (B) (severe diabetes)., mice were starved O.N. and injected i.p. with 2gr/kg glucose. Blood glucose levels were monitored at different time points. ^**p < 0.01 vs. PBS; ^*p < 0.05 vs. PBS. N = 10/group. (C–F) At 20 weeks, pancreatic heads were fixed and stained with H&E. (C) Pre-diabetic mice islets at 5w. (D) PBS. (E) AS101 (1 mg/kg/injection). (F) SAS (1 mg/kg/injection). Results show representative data of five mice/group. X200. (G) Percent insulitis of 5 mice/group at 20 weeks in comparison to islets from 5 weeks old mice (pre-diabetic). 20 islets/mouse were scored. ^*p < 0.01 vs. PBS. (H) Percentage of infiltrating cells according to the severity of insulitis. Grade 0, No insulitis (no infiltration); Grade 1, Pre-insulitis (<25% of the islet area infiltrated); Grade 2, Mild insulitis (<50% of the islet area infiltrated); Grade 3, Severe insulitis (>50% of the islet area infiltrated); Grade 4, Massive insulitis (total loss of β cell mass).

Figure 9

AS101 and SAS prevent islet damage and preserve islet function. (A)AS101 or SAS at 1 mg/kg/injection were administered i.p. every other day to female NOD mice, starting at 3 weeks of age. At 20 weeks, pancreatic heads were fixed and stained for insulin (red), glucagon (green), and nuclear staining (blue). Pancreata from 5 week old mice (pre-diabetic) served as a control. (a) Pre-diabetic; (b) PBS; (c) AS101; (d) SAS. The results are representative of 3 mice/group. 10 slides/mouse were visualized. X400. (B) Microscopic determination of islet radius. Results represent means ± SE of 5 mice/group. ^*p < 0.01 vs PBS. (C) Pancreas cell lysates from 20w old mice were analyzed for caspase-3 activity by colorimetric assay. Results represent means ± SE of 5 mice/group. ^*p < 0.01 vs. all groups.

Figure 10

Treatment with AS101 or SAS attenuates α4β7 integrin activity and suppresses inflammatory cytokines in NOD mice. (A) AS101 or SAS at 1 mg/kg/injection were administered i.p. every other day to female NOD mice, starting at 3 weeks of age until 20 weeks. Pre-diabetic mice at 5 weeks of age served as controls. Lymphocytes from PAN LN at 5 × 10⁵/well were isolated and incubated in 96 well plates coated with BSA or MAdCAM-1 for 2 h. Nonattached cells were then washed twice, and the adherent cells were subjected to XTT. The results represent means ± SE of 8 mice/group. ^*p < 0.01 vs. MadCAM-1 alone. (B) IL-1β protein levels from 20 w old mice. Pancreatic cell lysates were evaluated for IL-1β by ELISA. The results represent means ± SE of 5 mice/group. ^*p < 0.01 vs. PBS. (C) At 20 weeks pancreas cell lysates were analyzed for caspase-1 activity by a colorimetric assay, IL-17 (D) and IFNγ (E) protein levels by ELISA. The results represent means ± SE of 3 mice/group. ^*p < 0.001 vs. all.

Figure 11

Treatment with AS101 or SAS increases the proportion of T regulatory cells in PLN of NOD mice. AS101 or SAS were administered i.p. at 1 mg/kg/injection every other day to female NOD mice, starting at 3 weeks of age. At 20 weeks lymphocytes from PLN were isolated and stained for CD4, CD25 and Foxp3. (A) PBS; (B) SAS (1 mg/kg/injection); (C) AS101 1mg/kg/injection. Each curve represents cells isolated from different animal N = 5/group. (D) Evaluation of the proportion of regulatory cells in treated mice. The results represent means ± SE of 5 mice/group.^*p < 0.001 vs. PBS.

Figure 12

AS101 and SAS inhibit α4β7 integrin activity and α4β7-depenfent migration of T effector cells and Tregs. (A) T effector cells and (B) T regulatory cells were isolated from spleens of 20w old NOD female mice. Cells at 5 × 10⁵/well were incubated in 96 well plates coated with BSA or MAdCAM-1 for 2 hours. Nonattached cells were then washed twice, and the adherent cells were subjected to XTT. The results represent means ± SE of 4 mice/group. ^*p < 0.001 vs. BSA; ^**p < 0.005 vs. MAdCAM-1 alone (SAS 0), (C) T effector cells and T regulatory cells were isolated from spleens of 20w old NOD female mice. Cells (2.5 × 10⁵) were incubated for 1 h in the presence of mobilized MadCAM-1 and various doses of SAS or AS101 and then loaded onto 8 mm polycarbonate membrane inserts. The bottom chambers were filled with FBS (20%) and MadCAM-1 (100 μg/ml) serving as a chemo attractant. Migrated cells were quantified after 24 h. The results represent means±SE of 4 mice/group. ^*p < 0.001 vs control (SAS 0 of the relevant cell subset). ^**p < 0.05 (comparison between controls of both cell subsets). (D) Tregs and T effector cells were isolated from spleens of 20w old female NOD mice and stained for the expression of α4β7. The results represent one experiment representative of three performed with 3 mice/group.