Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus

Abstract

Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2'-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans.

Keywords: Type 1 diabetes; antibody; beta cell (B-cell); pancreatic islet; serpin.

FIGURE 1.

Increase in the number of pancreatic islets in mice exposed to serpin B13 antibodies. A, 3-week-old NOD female mice with distinct levels of endogenous serpin B13 Ab were sacrificed, and their pancreata were subjected to pancreatic islet isolation. B, scheme of Ab injection in Balb/c mice depicted in C and D. 2-Week-old Balb/c male mice injected with serpin B13 mAb or IgG control were sacrificed at 8 weeks of age and their pancreata were subjected to pancreatic islet isolation (C) or examined by IF for insulin-positive cell cluster sections measuring >58 μm in diameter (D). E, scheme of Ab injection in Balb/c mice depicted in F and G. Balb/c male mice injected at 2 weeks, 8 weeks, and 4 months of age with serpin B13 mAb or IgG control were sacrificed at 9 months of age and their pancreata were examined by IF exactly as in D (F) or analyzed for beta cell mass as described under ”Experimental Procedures“ (G). Data are presented as mean ± S.E.

FIGURE 2.

Increased pancreatic islet cellularity in serpin B13 mAb-treated mice. A and B, Balb/c male mice were injected with a low (4 × 2.5 μg) or high (4 × 25 μg) dose of serpin B13 mAb or IgG control, as described in the legend to Fig. 1B. The animals were sacrificed at 8 weeks old, and their pancreata were prepared for insulin staining and FACS. The average total cellularity (A) and beta cell number per islet (B) are shown. The islets from experiments depicted in Fig. 1C were used for the analysis. C and D, Balb/c male mice were injected with serpin B13 mAb or IgG control (4 × 25 μg), as described in the legend to Fig. 1, B (2-month follow up), and E (9-month follow up), respectively. The data depicted in C and D are based on mice that were also used for the analysis depicted in Fig. 1, D and F, respectively. Data are presented as mean ± S.E.

FIGURE 3.

Linear regression analysis of beta cellularity according to islet section size in IgG- and serpin B13 mAb-treated mice. Pancreatic tissue blocks were obtained from the same Balb/c male mice that were used in the experiments depicted in Fig. 1. Tissue sections were stained with an anti-insulin Ab and DAPI, and nuclei in small and larger beta cell cluster sections were quantified. A, analysis of small islet sections. A total of 20,429 nuclei in the IgG-treated group and 23,497 nuclei in the serpin B13 mAb-treated group were examined to generate the formulae. B, analysis of larger islet sections. A total of 36,598 nuclei in the IgG-treated group and 41,589 nuclei in the serpin B13 mAb-treated group were examined to generate the formulae.

FIGURE 4.

Increased beta cell 5-EdU incorporation in serpin B13 mAb-treated mice. A, strategy for Ab injection and 5-EdU incorporation. B, representative staining of pancreatic islets for Edu. Red, insulin; green, EdU; blue, DAPI. C, the percentages of 5-EdU beta cells in islet sections per animal in small and larger islets. D, percent increase in the number of 5-EdU⁺ beta cells and E, pancreatic islet sections with at least one 5-EdU⁺ beta cell following serpin B13 mAb injection. Data are presented as mean ± S.E.

FIGURE 5.

Effect of serpin antibodies on Reg gene expression in pancreatic islets. Female NOD mice (A) with or without endogenous serpin autoantibodies, as well as healthy (B) or STZ-treated (C) male Balb/c mice injected with serpin mAb or control IgG (exactly as depicted in Figs. 1B and 5C, inset, respectively) were subjected to quantitative PCR to assess in their islets expression of Reg and other genes (a, Arx; b, Brn4; c, Hlxb9; d, Insm1; e, Irx1; f, lrx2; g, Isl1; h, MafA; i, Nkx2.2; j, Pax4; k, Pax6; l, Neurod1; m, Ins2; n, Ngn3; o, Nkx6.1; p, Pdx1; q, Reg1; r, Reg2; s, Reg3a; t, Reg3b; u, Reg3d; v, Reg3g; and w, Reg4). Analysis depicted in B is confined to islets with <200 μm diameter. D, Reg gene expression in human islets incubated with serpin B13 mAb (or control IgG) alone, or an extract of pancreatic ductal (or nonductal) epithelium containing these antibodies. Data are presented as mean ± S.E.

FIGURE 6.

Amelioration of STZ-induced diabetes in Balb/c mice treated with serpin vaccine. Balb/c male pups were intraperitoneal injected on two occasions (days +5 and +10) with 10 μg of mouse serpin B13 diluted in PBS (open, n = 8) or PBS alone (black, n = 7) and injected with a single dose of 100 mg/kg of STZ on day +10 to induce diabetes. Mice were then monitored for blood glucose levels at 1-week intervals, starting on day +17, as indicated.

FIGURE 7.

Glucose control in Balb/c mice subjected to serpin B13 mAb treatment. A, strategy for Ab and STZ injection. B, plasma insulin levels at fasting and 1 h after glucose intraperitoneal injection (postprandial). C, blood glucose levels at baseline and every 30 min for 2 h after glucose injection (top). The data also are expressed as the area under the curve (AUC) for all time points combined (bottom). IgG group: n = 10; serpin Ab group: n = 8.

FIGURE 8.

Impact of antibodies to serpin B13 on IDAA1C in recent-onset T1D patients. Fifty-four placebo subjects previously recruited to Type 1 Diabetes TrialNet protocols were examined for baseline secretion of serpin B13 antibodies and analyzed for IDAA1C at baseline and follow-up at 3, 6, and 12 months. The negative samples were compared with samples that showed weak (+) (A) or strong (++) (B) binding to serpin B13. Linear regression was used for the analysis. Data are presented as mean ± S.E.

FIGURE 9.

Impact of antibodies to serpin B13 on C-peptide response in recent-onset T1D patients. Serpin B13 Ab was measured at baseline and its impact examined with regard to C-peptide concentration, either fasting (A) or stimulated at 90 min during mixed-meal tolerance test (MMTT) (B). Two-way analyses of variance with Dunnett's was used to compare C-peptide at baseline versus the 3-, 6-, and 12-month follow up.