Identification of immunoregulatory peptides from gut microbiota modulating Epstein-Barr virus-induced gene 3 (EBI3)...

Gut microbiota-derived compounds are pivotal in modulating host immunity by regulating the functions of various key innate and adaptive immune cells. Epstein-Barr virus-induced gene 3 (EBI3) serves as the beta subunit shared by the heterodimeric cytokines interleukin (IL)-27 and IL-35. Both these cytokines have been documented to inhibit the development of T helper 2 (Th2) and T helper 17 (Th17) cells, while enhancing the function of regulatory T cells (Tregs). EBI3, itself, has also been shown to regulate cell-mediated immune responses. Despite their critical roles in maintaining immune homeostasis, there is a significant lack of robust, high-throughput-compatible assays to evaluate the secretion of IL-27, IL-35, or EBI3. In this study, we detail the development of a novel amplified luminescent proximity homogeneous assay (AlphaLISA™) to quantify EBI3 secretion by tolerogenic dendritic cells. We utilized this assay to screen a library of 9739 small proteins derived from the human gut microbiota to identify compounds that could stimulate EBI3 secretion. Our findings revealed the immunoregulatory potential of VAC18, an unknown protein from Fusicatenibacter saccharivorans (Clostridiumcluster XIVa) which significantly induces the secretion of both EBI3 and IL-27. This is the first study to demonstrate the effect of gut microbiota derived peptides on the balanced secretion of EBI3 and IL-27.