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

Feeding lactobacilli impacts lupus progression in (NZBxNZW)F1 lupus-prone mice by enhancing immunoregulation

Although the relationship between autoimmunity and microorganisms is complex, there is evidence that microorganisms can prevent the development of various autoimmune diseases. Lactobacilli are beneficial gut bacteria that play an important role in immune system development. The goals of this study were to assess the ability of three different strains of lactobacilli (L. casei B255, L. reuteri DSM 17509 and L. plantarum LP299v) to control lupus development/progression in (NZBxNZW)F1 (BWF1) lupus-prone mice before and after disease onset, and identify the mechanisms mediating protection. BWF1 mice fed with individual L. casei or L. reuteri before disease onset exhibited delayed lupus onset and increased survival, while feeding L. plantarum had little impact. In vitro treatment of BWF1 dendritic cells with individual lactobacilli strains upregulated IL-10 production to various extents, with L. casei being the most effective. The protection mediated by L. casei was associated with upregulation of B7-1 and B7-2 by antigen presenting cells, two costimulatory molecules important for regulatory T cell (Treg) induction. Moreover, feeding L. casei lead to increased percentages of CD4+Foxp3+ Tregs and IL10-producing T cells in the lymphoid organs of treated mice. More importantly, mice fed L. casei after disease onset remained stable for several months, i.e. exhibited delayed anti-nucleic acid production and kidney disease progression, and increased survival. Therefore, feeding lactobacilli appears to delay lupus progression possibly via mechanisms involving Treg induction and IL-10 production. Altogether, these data support the notion that ingestion of lactobacilli, with immunoregulatory properties, may be a viable strategy for controlling disease development and progression in patients with lupus, i.e. extending remission length and reducing flare frequency

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

APC from CFA-treated NOD mice effectively induce regulatory cell function <i>in vitro</i>.

<p>CD4⁺CD25⁻ responder cells from B6 mice were cultured with irradiated spleen cells (APC) from individual 30 week-old B6, Non-Sick (NS) CFA-treated or Sick NOD mice, anti-CD3 and either with or without CD4⁺CD25⁺ cells from B6 mice (1∶1 regulatory∶responder ratio; A & B). CD4⁺CD25⁻ responder cells from NOD mice were cultured with irradiated spleen cells (APC) from individual 11 week-old NOD mice injected with PBS or CFA three weeks earlier or B6 mice, anti-CD3 and either with or without CD4⁺CD25⁺ cells from NOD mice (1∶1 regulatory∶responder ratio; C & D). Raw cpm data are shown for one representative animal (A & C), and percent inhibition is shown where each point represents an individual animal (B & D). * and ** indicate significant differences from the respective proliferation controls (i.e., responders alone; A & C), or B6 APC (B) at <i>p</i><0.001 and p<0.05, respectively.</p

Disease-free CFA-treated NOD mice possess potent regulatory cells.

<p>Three different doses of CFA containing 50, 100 or 300 µg of <i>Mycobacterium tuberculosis</i> or PBS were administered s.c. to 6 week-old NOD mice. An * indicates a significant difference from the PBS control at <i>p</i><0.03 (A). Pancreata were harvested from 34 week-old Sick (Sick) or CFA-treated Non-Sick (NS) NOD mice and digested with collagenase. Cells from individual mice were labeled with anti-CD4, CD25 antibodies, and Foxp3 antibodies and analyzed by FACS®. Each point represents an individual animal. An ** indicates a significant difference from the Sick NOD group at <i>p</i><0.05 (B & C). Pancreatic LN cells were harvested from 30-week-old CFA-treated Non-Sick NOD mice, and labeled with anti-CD4, CD25 and Foxp3 antibodies, and analyzed by FACS® (D). Pancreatic LN cells from 30-week-old Sick or CFA-treated Non-Sick (NS) were pooled, CD4⁺ cells purified and 0.4×10⁶ cells transferred into 4-wk-old NOD mice. Or purified CD4⁺ cells from pancreatic LN from 30-week-old CFA-treated Non-Sick (NS) were depleted of CD25⁺ cells (NS CD4⁺CD25⁻) and 0.4×10⁶ of these CD4⁺CD25⁻ cells injected into 4-wk-old NOD mice. Blood glucose was monitored weekly and diabetes incidence determined. An *** indicates a significant difference from the untreated controls at <i>p</i><0.005 (E).</p

APC from pre-diabetic and Sick NOD mice are less efficient at maintaining Foxp3 expression <i>in vitro</i>.

<p>CD4⁺CD25⁺ T cells from 12–15 week-old Sick NOD mice were cultured overnight in multiple wells, either alone or with irradiated T-cell depleted spleen cells (APC) from B6, pre-diabetic (11 week-old) NOD or Sick NOD mice and anti-CD3 antibody. Cells were pooled and labeled with anti-CD4, anti-CD25 and anti-Foxp3 antibodies. CD4⁺CD25⁺ T cells and APC were pooled from 3 mice. Cells were analyzed for Foxp3 expression by FACS® after gating on CD4⁺CD25⁺ cells. Representative results from one of two experiments are shown.</p

APC from NOD mice are less efficient at maintaining Foxp3 expression <i>in vitro</i>.

<p>CD4⁺CD25⁺ T cells from 12–15 week-old B6 or Sick NOD mice were cultured overnight in multiple wells, either alone or with B6 or Sick NOD irradiated T-cell depleted spleen cells (APC) and anti-CD3 antibody. Cells were pooled and labeled with anti-CD4, anti-CD25 and anti-Foxp3 antibodies. CD4⁺CD25⁺ T cells and APC were pooled from 3 mice. Cells were analyzed for Foxp3 expression by FACS® after gating on CD4⁺CD25⁺ cells. Representative results from one of four experiments are shown.</p