Enhancing human islet xenotransplant survival and function in diabetic immunocompetent mice through LRH-1/NR5A2 pharmacological activation

Abstract

The intricate etiology of type 1 diabetes mellitus (T1D), characterized by harmful interactions between the immune system and insulin-producing beta cells, has hindered the development of effective therapies including human islet transplantation, which requires strong immunosuppressants that impair beta cell survival and function. As such alternative immunomodulating therapies are required for successful transplantation. The discovery that pharmacological activation of the nuclear receptor LRH-1/NR5A2 can reverse hyperglycemia in mouse models of T1D by altering, and not suppressing the autoimmune attack, prompted us to investigate whether LRH-1/NR5A2 activation could improve human islet function/survival after xenotransplantation in immunocompetent mice. Human islets were transplanted under the kidney capsule of streptozotocin (STZ)-induced diabetic mice, and treatment with BL001 (LRH-1/NR5A2 agonist) or vehicle was administered one week post-transplant. Our study, encompassing 3 independent experiments with 3 different islet donors, revealed that mice treated for 8 weeks with BL001 exhibited lower blood glucose levels correlating with improved mouse survival rates as compared to vehicle-treated controls. Human C-peptide was detectable in BL001-treated mice at both 4 and 8 weeks indicating functional islet beta cells. Accordingly, in mice treated with BL001 for 8 weeks, the beta cell mass was preserved, while a significant decrease in alpha cells was observed compared to mice treated with BL001 for only 4 weeks. In contrast, vehicle-treated mice exhibited a reduction in insulin-expressing cells at 8 weeks compared to those at 4 weeks. These results suggest that BL001 significantly enhances the survival, engraftment, and functionality of human islets in a STZ-induced diabetic mouse model.

Keywords: LRH-1; NR5A2; human islet; nuclear receptor; pharmacological treatment; type 1 diabetes; xenotranplantation.

Figure 1

BL001 improves human islet graft survival and function in STZ-treated immunocompetent C57BL/6j mice. (A) Experimental design of the 4-week BL001 treatment post-xenotransplantation experiment. (B) Weekly measurement of non-fasting blood glucose and (C) Kaplan Meier survival curve. (D) OGTT performed at 4 weeks post-BL001/Vehicle treatment. Mice were fasted for 6 hours before the OGTT. (E) Area under the curve (AUC) corresponding to the OGTT. Student t-test, ^# p=0003 and ^& p=0.0037 as compared to control non-STZ mice. (F) Human C-peptide plasma levels at 4 weeks post-BL001/Vehicle treatment. Data are presented as means ± SEM. *p<0.05 student t-test. (G) Mouse C-peptide plasma levels at 4 weeks post-BL001/Vehicle treatment. Data are presented as means ± SEM. Student t-test, ^# p=002 and ^& p=0.0003 as ompared to control non-STZ mice. (H) Representative immunofluorescence images of kidney sections from mice euthanized at 4 weeks post-BL001/Vehicle treatment, displaying staining for insulin (INS), glucagon (GCG) staining along with nuclear DAPI staining. Quantitative analysis of (I) insulin (INS), (J) glucagon (GCG), and (K) CD4⁺ areas, normalized to the DAPI-positive area per graft, at 4 weeks post BL001 treatment. (L) Experimental design of the 8-week BL001 treatment post-xenotransplantation experiment. (M) Weekly measurement of non-fasting glycemia and (N) Kaplan Meier survival curve (O). Human C-peptide plasma levels at 6-week vehicle-treated mice and 8-week BL001-treated mice. Data are presented as means ± SEM. **p<0.01 student t-test. (P) Mouse C-peptide plasma levels at 6-week vehicle-treated mice and 8-week BL001-treated mice. Data are presented as means ± SEM. ^# p=0001 and ^& p=0.0002 Student t-test as compared to control non-STZ mice. (Q) Representative immunofluorescence images of kidney sections from mice at 6 weeks post-vehicle treatment and at 8 weeks post-BL001 treatment, displaying staining for insulin (INS), glucagon (GCG) staining along with nuclear DAPI staining. Quantitative analysis of (R) insulin (INS), (S) glucagon (GCG), and (T) CD4⁺ areas, normalized to the DAPI-positive area per graft, at 4 weeks post BL001 treatment. Data are presented as means ± SEM. *p<0.05 and ****p<0.0001 student t-test. ns, non-significant. (U) Representative immunofluorescence images of pancreas sections from mice at 6 weeks post-vehicle treatment and at 8 weeks post-BL001 treatment. displaying staining for insulin (INS), glucagon (GCG) staining along with nuclear DAPI staining.