Associate Professor University of Florida, United States
Introduction: Dendritic Cells (DCs) are the professional antigen-presenting cells and the bridge between the adaptive and the innate immune systems. By targeting DCs, the adaptive immune system can be “reprogrammed” through tolerogenic interaction between DCs and T-cells. A common polymer, PLGA, was hailed for its tolerogenic abilities, but its performance did not meet expectations. Additional tolerance factors such as IL-10 must be added to create a tolerogenic environment for DCs in novel autoimmune treatments. In contrast, tumor cells have been shown to establish effective tumor-specific tolerance in DCs through unknown methods. However, secreted metabolites such as lactic acid have been observed in high concentrations and have been theorized to induce tumor-specific tolerance. As such, our work seeks to screen, characterize, and harness the tolerogenic properties of TME-derived metabolic byproducts as potential candidates for a novel autoimmune disease therapy.
Materials and
Methods: Generation of Bone Marrow-Derived Dendritic Cells (BMDCs) Dendritic cells were generated from bone marrow from female C57BL6 mice aged 6-12 weeks using a modified 10-day protocol. BMDCs were cultured for 11 days. On day 11, cells were ready for treatment.
Creation of Treatments for DCs Metabolite Molecules were bought from vendors and weighed. Succinic Acid Disodium salt (Succinic Acid), L-Citrulline (Citrulline), ⍺-Ketoglutaric Acid Disodium Salt (⍺-KGA), and Sodium L-Lactate (Lactate), were dissolved at 50mM, and L-Kynurenine (Kynurenine), and L-Tryptophan (Tryptophan), were dissolved at 1mM in fresh BMDC media and filtered to remove contaminants.
Flow Cytometry On day 11, BMDCs were treated with modified media with added metabolites dissolved at desired concentrations. Dendritic cells were stained for CD11c, and the inflammatory surface markers, CD80, CD86, and MHCII using fluorescent antibodies. Cells were then analyzed via flow cytometry.
Results, Conclusions, and Discussions: After Flow cytometry, the data was analyzed by isolating DC populations through the CD11c surface marker and measuring the CD80, CD86, and MHCII surface markers. The effect of Citrulline on DCs in vitro downregulates CD80 when challenged with LPS. Succinic acid and citrulline significantly upregulate the CD86 and expression in DCs and succinic acid upregulates the MHCII expression with and without an LPS challenge. ⍺-KGA, lactate, kynurenine, and tryptophan do not significantly upregulate inflammatory surface markers. This could potentially mean tolerance in DCs. This supports the hypothesis that metabolites can alter the phenotype of dendritic cells in an inflammatory environment. Later research diving into the tolerogenic surface markers of the DCs can expose more effects of metabolites on the DC Phenotype.
