Deletion of Carboxypeptidase E in ß-Cells Disrupts Proinsulin Processing but Does Not Lead to Spontaneous Development of Diabetes in Mice.

Carboxypeptidase E (CPE) facilitates the conversion of prohormones into mature hormones and is highly expressed in multiple neuroendocrine tissues. Carriers of CPE mutations have elevated plasma proinsulin and develop severe obesity and hyperglycemia. We aimed to determine whether loss of Cpe in pancreatic ß-cells disrupts proinsulin processing and accelerates development of diabetes and obesity in mice. Pancreatic ß-cell-specific Cpe knockout mice (ßCpeKO; Cpefl/fl x Ins1Cre/+) lack mature insulin granules and have elevated proinsulin in plasma; however, glucose-and KCl-stimulated insulin secretion in ßCpeKO islets remained intact. High-fat diet-fed ßCpeKO mice showed weight gain and glucose tolerance comparable with those of Wt littermates. Notably, ß-cell area was increased in chow-fed ßCpeKO mice and ß-cell replication was elevated in ßCpeKO islets. Transcriptomic analysis of ßCpeKO ß-cells revealed elevated glycolysis and Hif1α-target gene expression. On high glucose challenge, ß-cells from ßCpeKO mice showed reduced mitochondrial membrane potential, increased reactive oxygen species, reduced MafA, and elevated Aldh1a3 transcript levels. Following multiple low-dose streptozotocin injections, ßCpeKO mice had accelerated development of hyperglycemia with reduced ß-cell insulin and Glut2 expression. These findings suggest that Cpe and proper proinsulin processing are critical in maintaining ß-cell function during the development of hyperglycemia. ARTICLE HIGHLIGHTS: Carboxypeptidase E (Cpe) is an enzyme that removes the carboxy-terminal arginine and lysine residues from peptide precursors. Mutations in CPE lead to obesity and type 2 diabetes in humans, and whole-body Cpe knockout or mutant mice are obese and hyperglycemic and fail to convert proinsulin to insulin. We show that ß-cell-specific Cpe deletion in mice (ßCpeKO) does not lead to the development of obesity or hyperglycemia, even after prolonged high-fat diet treatment. However, ß-cell proliferation rate and ß-cell area are increased, and the development of hyperglycemia induced by multiple low-dose streptozotocin injections is accelerated in ßCpeKO mice.

Topical Adjuvant Application during Subcutaneous Vaccination Promotes Resident Memory T Cell Generation.

Skin tissue resident memory T cells (TRM) provide superior protection to a second infection. In this study, we evaluated the use of topical CpG oligodeoxynucleotide (ODN) as adjuvant to generate skin TRM in mice. Topical or s.c. CpG ODN adjuvant administration at the time of a s.c. Ag injection led to an accumulation of CD103- CD8 T cells in the epidermis. However, only mice with CpG ODN administered topically had significant numbers of CD103+ Ag-specific CD8 T cells persisting in the local epidermal skin, enhanced circulating memory cells in the blood, and showed protection from intradermal challenge with melanoma cells. Generation of Ag-specific CD8 T cells was dependent on TLR9 expression on hematopoietic cells and partially dependent on receptor expression on stromal cells. Topical challenge of immunized mice at a distal site led to significant expansion of Ag-specific T cells in the blood and accumulation in the challenged skin. We demonstrate that local and systemic T cell memory can be generated with topical CpG ODN at the time of s.c. immunization, suggesting a new method of enhancing current vaccine formulations to generate tissue TRM.

Expansion of antigen-specific regulatory T cells with the topical vitamin d analog calcipotriol.

1,25-Dihydroxyvitamin D(3) is immunosuppressive both in vivo and in vitro. Topical vitamin D analogs such as calcipotriol alter keratinocyte function, but their effects on cutaneous immune responses are less well understood. We demonstrate that exposure of the skin to calcipotriol before transcutaneous immunization with OVA protein and CpG adjuvant prevents Ag-specific CD8(+) T cell priming coincident with Langerhans cell depletion in the skin. Immunization through calcipotriol-treated skin induces CD4(+)CD25(+) regulatory T cells (Treg) that prevent subsequent Ag-specific CD8(+) T cell proliferation and IFN-gamma production. Treg induced by calcipotriol are able to inhibit the induction and the elicitation of protein contact hypersensitivity. Topical calcipotriol treatment also induces RANKL (receptor activator of NF-kappaB ligand) expression by keratinocytes, a TNF family member involved in modulation of skin dendritic cells. UV light B induces Ag-specific tolerance when it is applied before transcutaneous immunization. We suggest that UV light B-induced tolerance is induced via a vitamin D receptor-dependent mechanism as vitamin D receptor (VDR) knockout mice fail to increase FoxP3(+) Treg in their peripheral draining lymph node following irradiation. Additionally, keratinocytes of VDR(-/-) mice fail to induce RANKL upon UV irradiation or calcipotriol treatment. The in vivo expansion of Ag-specific Treg with the topical application of the vitamin D analog calcipotriol followed by transcutaneous immunization is a simple method to augment functional Ag-specific CD4(+)CD25(+)Foxp3(+) Treg populations and mimics Ag-specific UV-induced tolerance.

A quantitative comparison of rates of phagocytosis and digestion of apoptotic cells by macrophages from normal (BALB/c) and diabetes-prone (NOD) mice.

Macrophages play an important role in clearing apoptotic debris from tissue. Defective or reduced clearance, seen, for instance, in non-obese diabetic (NOD) mice, has been correlated with initiation of autoimmune (Type 1) diabetes (T1D) (O'Brien BA, Huang Y, Geng X, Dutz JP, Finegood DT. Diabetes 51: 2481-2488, 2002). To validate such a link, it is essential to quantify the reduced clearance (for example, by comparison to BALB/c control mice) and to determine which elements of that clearance are impaired. Recently, we fit data for the time course of in vitro macrophage feeding experiments to basic models of macrophage clearance dynamics, thus quantifying kinetics of uptake and digestion of apoptotic cells in both mouse strains (Marée AFM, Komba M, Dyck C, Labeçki M, Finegood DT, Edelstein-Keshet L. J Theor Biol 233: 533-551, 2005). In the cycle of modeling and experimental investigation, we identified the importance of 1) measuring short-, intermediate-, and long-time data (to increase the accuracy of parameter fits), and 2) designing experiments with distinct observable regimes, including engulfment-only and digestion-only phases. Here, we report on new results from experiments so designed. In comparing macrophages from the two strains, we find that NOD macrophage engulfment of apoptotic cells is 5.5 times slower than BALB/c controls. Significantly, our new data demonstrate that digestion is at least two times slower in NOD, in contrast with previous conclusions. Moreover, new data enable us to detect an acceleration in engulfment (after the first engulfment) in both strains, but much smaller in NOD macrophages.

Quantifying macrophage defects in type 1 diabetes.

Macrophages from animals prone to autoimmune (type 1) diabetes differ from those of diabetes-resistant animals in processing and clearing apoptotic cells. Using in vitro time-course assays of the number of engulfed apoptotic cells observed within macrophages, we quantified these differences in non-obese diabetic (NOD) versus Balb/c mice. Simple models lead to several elementary parameter estimation techniques. We used these to compute approximate rates of macrophage engulfment and digestion of apoptotic cells from basic features of the data (such as initial rise-times, phagocytic index and percent phagocytosis). Combining these estimates with full fitting of a sequence of model variants to the data, we find that macrophages from normal (Balb/c) mice engulf apoptotic cells up to four times faster than macrophages from the diabetes-prone (NOD) mice. Further, Balb/c macrophages appear to undergo an activation step before achieving their high engulfment rate. In NOD macrophages, we did not see evidence for this activation step. Rates of digestion of engulfed apoptotic cells by macrophages are similar in both types. Since macrophage clearance is an important mechanism of disposal of self-antigen, these macrophage defects could potentially be a factor in predisposition to type 1 diabetes.