Suppression of dendritic cell activation by diabetes autoantigens linked to the cholera toxin B subunit

Abstract

Antigen presenting cells, specifically dendritic cells (DCs) are a focal point in the delicate balance between T cell tolerance and immune responses contributing to the onset of type I diabetes (T1D). Weak adjuvant proteins like the cholera toxin B subunit when linked to autoantigens may sufficiently alter the balance of this initial immune response to suppress the development of autoimmunity. To assess adjuvant enhancement of autoantigen mediated immune suppression of Type 1 diabetes, we examined the cholera toxin B subunit (CTB)-proinsulin fusion protein (CTB-INS) activation of immature dendritic cells (iDC) at the earliest detectable stage of the human immune response. In this study, Incubation of human umbilical cord blood monocyte-derived immature DCs with CTB-INS autoantigen fusion protein increased the surface membrane expression of DC toll-like receptor (TLR-2) while no significant upregulation in TLR-4 expression was detected. Inoculation of iDCs with CTB stimulated the biosynthesis of both CD86 and CD83 co-stimulatory factors demonstrating an immunostimulatory role for CTB in both DC activation and maturation. In contrast, incubation of iDCs with proinsulin partially suppressed CD86 co-stimulatory factor mediated DC activation, while incubation of iDCs with CTB-INS fusion protein completely suppressed iDC biosynthesis of both CD86 and CD83 costimulatory factors. The incubation of iDCs with increasing amounts of insulin did not increase the level of immune suppression but rather activated DC maturation by stimulating increased biosynthesis of both CD86 and CD83 costimulatory factors. Inoculation of iDCs with CTB-INS fusion protein dramatically increased secretion of the immunosuppressive cytokine IL-10 and suppressed synthesis of the pro-inflammatory cytokine IL12/23 p40 subunit protein suggesting that linkage of CTB to insulin (INS) may play an important role in mediating DC guidance of cognate naïve Th0 cell development into immunosuppressive T lymphocytes. Taken together, the experimental data suggests Toll like receptor 2 (TLR-2) plays a dominant role in CTB mediated INS inhibition of DC induced type 1 diabetes onset in human Type 1 diabetes autoimmunity. Further, fusion of CTB to the autoantigen was found to be essential for enhancement of immune suppression as codelivery of CTB and insulin did not significantly inhibit DC costimulatory factor biosynthesis. The experimental data presented supports the hypotheses that adjuvant enhancement of Author to whom correspondence should be addressed: William Langridge; Tel.: +1-909 558-1000 (81362); Fax: +1-909-558-0177 (Center for Health Disparities and Molecular Medicine, Loma Linda University, School of Medicine, Loma Linda, CA. 92354). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its fina...

Mucosal Adjuvants

“…After oral administration, antigens coupled to or in combination with CT-B increase their tolerogenic potential, by a mechanism that is not well discerned but is likely to include regulatory T cells (FoxP3 + ) and/or T-cellderived immunoregulatory cytokines such as TGF-β and IL-10 (Bregenholt et al, 2003;Odumosu et al, 2011;Sun et al, 2010).…”

The Role of Dendritic Cell Maturation in the Induction of Insulin-Dependent Diabetes Mellitus

“…So far, no safe and effective treatment is available to control the onset and progression this life-long debilitating disease (3). The loss of β-cell function in IDDM is mediated principally by CD4 + and CD8 + T cells, and autoreactive effector CD8 + T cells (CTLs) were shown to be responsible for islet β-cell destruction in the non-obese diabetic (NOD) mouse and in human IDDM (4–6). …”

The Role of Dendritic Cells in Tissue-Specific Autoimmunity

“…In a recent finding in our laboratory, incubation of human immature moDCs with CTB-INS autoantigen fusion protein showed an increase in surface expression of TLR2 with no significant upregulation in TLR4 expression [148]. In contrast, inoculation of immature dendritic cells (iDCs) with CTB stimulated the biosynthesis of both CD86 and CD83 costimulatory factors demonstrating an immunostimulatory role for CTB in both DC activation and maturation.…”