Purified Polyclonal Naturally-Occuring Immunoglobulin M (nlgM) Inhibits Pro-Inflammatory Cytokine Secretion as Well as Prevents the Onset and Progression of Autoimmune Type 1 Diabetes Mellitus

Abstract

Introduction: Type 1 diabetes (T1DM) is a multistage, T-cell-mediated autoimmune disease that involves the progressive destruction of insulin-producing β-cells, resulting in the complete loss of insulin secretion. Studies indicate that innate immunity and non-specific inflammation might contribute to both, the early induction and amplification of the immune assault against pancreatic β-cells as well as the stabilization and maintenance of insulitis. Cytokines derived from the Th1 subset of CD4 T cells, such as INF-gamma, TNF-alpha, IL-2, IL-12 are actively involved in β-cell destruction, while those derived from Th2/Th3 cells, such as IL-4, IL-10 and TGF-β are thought to inhibit β-cell destruction. Th17 cells too are potent inducers of tissue inflammation and autoimmunity and may have a role in T1DM. Tregs are ineffective in controlling TH-17-mediated inflammation as seen in auto-immune insulitis and in allograft rejection. nIgM deficiency is associated with an increased tendency towards autoimmune disease development, while elevated levels of nIgM anti-leucocyte autoantibodies are associated with fewer graft rejections. Objective: The objective is to determine if purified polyclonal nIgM therapy could a) mitigate inflammation by inhibiting proinflammatory cytokine secretion and b) prevent the onset and progression of T1DM in diabetes-prone non-obese diabetic (NOD) mice. Methods: IgM and IgG were purified by size-exclusion column chromatography (Sephacryl S-300 HR) from heat-inactivated WT-BL6 murine sera. Freshly isolated splenocytes (5×106 cells in 0.5ml RPMI1640 culture media), obtained from 3-6 week old NOD mice (n=4), were activated with LPS (0.35μg/ml) and soluble anti-CD3 (1.0μg/ml) and cultured for 4 days at 37oC, 5% CO2, with or without purified 3-5mg nIgM. TNF-alpha, IFN-gamma and IL-17A in supernatants were quantitated using ELISA kits. 4-7 wk-old female NOD litter-mates, divided into control and test groups, received either saline/BSA (n=30, controls) or 100ug nIgM intraperitoneal (n=23, test) followed by 50ug nIgM biweekly for 18-22wks. Results: nIgM significantly inhibited TNF-alpha secretion (activated splenoctyes cultured without nIgM vs. with nIgM; 4443.1±210.5 vs.101.1±52.5pg/ml; p< 0.001); IFN-gamma secretion (3244.2±160.6 vs. 0 pg/ml; p< 0.001) and IL-17A (732.2±66.4 vs. 5.3±9.2pg/ml, p< 0.001) from LPS+anti-CD3 activated splenocytes. Incubation of islets with 2-8ug IgM did not affect insulin secretion in response to glucose challenge compared to controls, in glucose-stimulated insulin secretion assay. 80% of the control mice became diabetic by 18-20wks of age with severe insulitis and periductal/perivascular inflammation, while 0/23 of nIgM-treated mice became diabetic (p< 0.001). The islets in nIgM-treated group revealed normal islet appearance, mild peri-insulitis as well as β-cell neogenesis. nIgM therapy initiated at 11wks of age resulted in diabetes in only 15% of nIgM-treated animals (n=10) compared to 80% of controls (p< 0.001). Mildly diabetic mice were restored to normoglycemia with tri-weekly nIgM therapy alone (n=5), whilst severely diabetic mice required minimal dose islet transplant (75 islets equivalents) additionally to restore normoglycemia (n=3). Islet allotransplantation studies are underway. Conclusion: nIgM therapy has tremendous clinical potential in mitigating inflammation by inhibiting proinflammatory cytokine secretion as well as in preventing the onset and progression of autoimmune T1DM. Its role as a therapeutic interventional adjunct in islet transplantation is currently under evaluation.