NOD dendritic cells stimulated with Lactobacilli preferentially produce IL-10 versus IL-12 and decrease diabetes incidence.

Dendritic cells (DCs) from NOD mice produced high levels of IL-12 that induce IFNγ-producing T cells involved in diabetes development. We propose to utilize the microorganism ability to induce tolerogenic DCs to abrogate the proinflammatory process and prevent diabetes development. NOD DCs were stimulated with Lactobacilli (nonpathogenic bacteria targeting TLR2) or lipoteichoic acid (LTA) from Staphylococcus aureus (TLR2 agonist). LTA-treated DCs produced much more IL-12 than IL-10 and accelerated diabetes development when transferred into NOD mice. In contrast, stimulation of NOD DCs with L. casei favored the production of IL-10 over IL-12, and their transfer decreased disease incidence which anti-IL-10R antibodies restored. These data indicated that L. casei can induce NOD DCs to develop a more tolerogenic phenotype via production of the anti-inflammatory cytokine, IL-10. Evaluation of the relative production of IL-10 and IL-12 by DCs may be a very useful means of identifying agents that have therapeutic potential.

Decreased frequencies of CD4+CD25+Foxp3+ cells and the potent CD103+ subset in peripheral lymph nodes correlate with autoimmune disease predisposition in some strains of mice.

The CD4(+)CD25(+)Foxp3(+) cells are essential for regulation of the immune response, and the integrin, CD103 (α(E)ß(7)), identifies a potent subset of these cells. Defects in CD4(+)CD25(+)Foxp3(+) cells are thought to contribute to susceptibility to autoimmune disease in predisposed individuals. Studies evaluating the quality and quantity of CD4(+)CD25(+)Foxp3(+) regulatory cell populations in the context of autoimmune disease susceptibility have been inconclusive, and few if any, have analyzed the CD103 subset. In this study, we analyzed regulatory T cells (Tregs) from different strains of mice with varying degrees of susceptibility to autoimmune disease. We found no differences in the ability of CD4(+)CD25(+) or the CD103(+) subset of Tregs from young female (NZB × NZW)F1 (BWF1), SJL, C57BL/6, or BALB/c mice to suppress CD4(+)CD25(- ) responders in vitro. Analysis of CD4(+)Foxp3(+) and CD4(+)CD25(+)CD103(+) cell frequencies in lymphoid organs revealed that BWF1 mice had dramatically lower percentages of both populations in the lymph node (LN) than the other strains, and lower percentages in the spleen in all but the C57BL/6 strain. We next determined whether these findings extended to another autoimmune-prone strain. Similar to BWF1 mice, percentages of CD4(+)Foxp3(+) and CD4(+)CD25(+)CD103(+) cells were significantly lower in predisease NOD mice. The low frequencies of CD4(+)Foxp3(+) and CD4(+)CD25(+)CD103(+) cells in BWF1 and NOD mice were not due to deficiencies in either thymic production or homeostatic proliferation. These data indicate that decreased percentages of CD4(+)Foxp3(+) cells and particularly, CD4(+)CD25(+)CD103(+) cells in LN correlate with the predisposition to spontaneous development of autoimmune disease.

APC activation restores functional CD4(+)CD25(+) regulatory T cells in NOD mice that can prevent diabetes development.

BACKGROUND: Defects in APC and regulatory cells are associated with diabetes development in NOD mice. We have shown previously that NOD APC are not effective at stimulating CD4(+)CD25(+) regulatory cell function in vitro. We hypothesize that failure of NOD APC to properly activate CD4(+)CD25(+) regulatory cells in vivo could compromise their ability to control pathogenic cells, and activation of NOD APC could restore this defect, thereby preventing disease. METHODOLOGY/PRINCIPAL FINDINGS: To test these hypotheses, we used the well-documented ability of complete Freund's adjuvant (CFA), an APC activator, to prevent disease in NOD mice. Phenotype and function of CD4(+)CD25(+) regulatory cells from untreated and CFA-treated NOD mice were determined by FACS, and in vitro and in vivo assays. APC from these mice were also evaluated for their ability to activate regulatory cells in vitro. We have found that sick NOD CD4(+)CD25(+) cells expressed Foxp3 at the same percentages, but decreased levels per cell, compared to young NOD or non-NOD controls. Treatment with CFA increased Foxp3 expression in NOD cells, and also increased the percentages of CD4(+)CD25(+)Foxp3(+) cells infiltrating the pancreas compared to untreated NOD mice. Moreover, CD4(+)CD25(+) cells from pancreatic LN of CFA-treated, but not untreated, NOD mice transferred protection from diabetes. Finally, APC isolated from CFA-treated mice increased Foxp3 and granzyme B expression as well as regulatory function by NOD CD4(+)CD25(+) cells in vitro compared to APC from untreated NOD mice. CONCLUSIONS/SIGNIFICANCE: These data suggest that regulatory T cell function and ability to control pathogenic cells can be enhanced in NOD mice by activating NOD APC.

Deficiency in NOD antigen-presenting cell function may be responsible for suboptimal CD4+CD25+ T-cell-mediated regulation and type 1 diabetes development in NOD mice.

Various defects in antigen-presenting cells (APCs) and T-cells, including regulatory cells, have been associated with type 1 diabetes development in NOD mice. CD4(+)CD25(+) regulatory cells play a crucial role in controlling various autoimmune diseases, and a deficiency in their number or function could be involved in disease development. The current study shows that NOD mice had fewer CD4(+)CD25(+) regulatory cells, which expressed normal levels of glucocorticoid-induced tumor necrosis factor receptor and cytotoxic T-lymphocyte-associated antigen-4. We have also found that NOD CD4(+)CD25(+) cells regulate poorly in vitro after stimulation with anti-CD3 and NOD APCs in comparison with B6 CD4(+)CD25(+) cells stimulated with B6 APCs. Surprisingly, stimulation of NOD CD4(+)CD25(+) cells with B6 APCs restored regulation, whereas with the reciprocal combination, NOD APCs failed to activate B6 CD4(+)CD25(+) cells properly. Interestingly, APCs from disease-free (>30 weeks of age), but not diabetic, NOD mice were able to activate CD4(+)CD25(+) regulatory function in vitro and apparently in vivo because only spleens of disease-free NOD mice contained potent CD4(+)CD25(+) regulatory cells that prevented disease development when transferred into young NOD recipients. These data suggest that the failure of NOD APCs to activate CD4(+)CD25(+) regulatory cells may play an important role in controlling type 1 diabetes development in NOD mice.