Modification of adverse inflammation is required to cure new-onset type 1 diabetic hosts

Abstract

In nonobese diabetic (NOD) mice with overt new-onset type 1 diabetes mellitus (T1DM), short-term treatment with a "triple-therapy" regimen [rapamycin plus agonist IL-2-related and antagonist-type, mutant IL-15-related Ig fusion proteins (IL-2.Ig and mutIL-15.Ig)] halts autoimmune destruction of insulin-producing beta cells and restores both euglycemia and immune tolerance to beta cells. Increases in the mass of insulin-producing beta cells or circulating insulin levels were not linked to the restoration of euglycemia. Instead, the restoration of euglycemia was linked to relief from an inflammatory state that impaired the host's response to insulin. Both restoration of immune tolerance to beta cells and relief from the adverse metabolic effects of an inflammatory state in insulin-sensitive tissues appear essential for permanent restoration of normoglycemia in this T1DM model. Thus, this triple-therapy regimen, possessing both tolerance-inducing and select antiinflammatory properties, may represent a prototype for therapies able to restore euglycemia and self-tolerance in T1DM.

Fig. 1.

Expression of cytotoxic T lymphocyte, Th1, and proinflammatory cytokine genes were grossly reduced in RPM + IL-2.Ig + mutIL-15.Ig triple-therapy-treated hosts. To quantitatively analyze gene-expression profiles, pancreatic-draining lymph nodes were harvested from newly diabetic (n = 4, onset of T1DM within 1 week), old-diabetic (n = 4, diabetic >30 days), and RPM + IL-2.Ig + mutIL-15.Ig-treated new-onset diabetic mice (n = 4, at day 50 after initiation of treatment). Expression of granzyme B, IFNγ, and the IL-1β and TNFα proinflammatory cytokine genes were analyzed on a linear scale and expressed relative to GAPDH expression.

Fig. 2.

Islet histology of spontaneous diabetic NOD mice at recent onset of disease and 52 days after ending treatment with RPM + IL-2.Ig + mutIL-15.Ig triple-therapy treatment (at least 70 days after onset). In recent onset, most islets are atrophic (A), with mainly glucagon-positive cells remaining after destruction of beta cells; a few residual islets (same islet in B and C) with many beta cells are present but manifest invasive insulitis. In these hyperglycemic mice, beta cells are partially degranulated, as seen in C. After treatment and restoration of a euglycemic state, here seen as 237 days after onset, atrophic islets are most prevalent (D); the islets with significant numbers of beta cells (same islet E and F) are surrounded by, but no longer invaded by, mononuclear leukocytes, have a more defined boundary of endocrine cells, and are no longer degranulated. Glucagon immunostaining was used in A, B, D, and E; insulin immunostaining was used in C and F.

Fig. 3.

Insulin tolerance test in NOD mice. The RPM + IL-2.Ig + mutIL-15.Ig triple therapy ablates insulin resistance in diabetic NOD mice. Insulin tolerance tests were performed in (i) age-matched spontaneous new-onset diabetic NOD mice, (ii) the RPM + IL-2.Ig + mutIL-15.Ig-treated spontaneous new-inset NOD mice, and (iii) nondiabetic NOD mice. Food was withheld 3 h before testing. Animals were weighed and blood samples were collected at 0 min; animals were injected i.p. with 0.75 units/kg of regular human insulin (Novolin; Novo Nordisk). Blood samples were then collected at 15, 30, and 60 min. The results were expressed as the percentage of the initial blood-glucose concentration. NOD-sp triple therapy, triple-therapy-treated NOD-sp mice.

Fig. 4.

Triple-therapy (RPM + IL-2.Ig + mutIL-15.Ig) treatment restores insulin signaling in new-onset diabetic NOD mice. Mice were fasted overnight and injected with human insulin (20 units per kg of body weight i.p.) to acutely stimulate insulin signaling. Skeletal muscle (gastrocnemius) was dissected and frozen in liquid nitrogen for immunoblotting analysis of insulin-signaling proteins. Each blot represents a different mouse.