High-Throughput Screen Identifies Non-Inflammatory Small Molecule Inducers of Trained Immunity

Trained immunity is a form of innate immune memory characterized by epigenetic and metabolic reprogramming in response to specific stimuli. This rewiring can result in increased cytokine and effector responses to pathogenic challenge, providing non-specific protection against disease. It may also improve immune responses to established immunotherapeutics and vaccines. Despite the promise of training for next-generation therapeutic design, most current understanding and experimentation is conducted with complex and heterogeneous biologically derived molecules, such as ß-glucan or the BCG vaccine. This limited collection of training compounds also limits study of the genes most involved in training responses as each molecule has both training and non-training effects. Small molecules with tunable pharmacokinetics and delivery modalities would both assist in the study of trained immunity and its future application for therapeutics. To identify novel small molecule inducers of trained immunity, we screened a library of 2000 drugs and drug-like compounds. Identification of well-defined compounds can improve our understanding of innate immune memory and broaden the scope of its clinical applications. We identified over 2 dozen small molecules in several chemical classes, including the traditionally immunosuppressive glucocorticoids, that induce a training phenotype in the absence of initial immune activation – a current limitation of reported inducers of training. We chose 7 of these top candidates to characterize and establish training activity in vivo. In this work, we expand the number of compounds known to induce trained immunity, creating new avenues for the study and application of innate immune training. Overall design: In order to discover epigentic changes initiated by these novel small molecules, we performed assay for transposase accessible chromatin sequencing (ATAC-seq) on bone marrow derived macrophages (BMDMs) from a single female mouse. In a 96 well plate, we treated 100,000 BMDMs with 10 µM each of seven small molecules found to induce innate immune training in a high throughput screen. As a positive control for training, we also treated BMDMs with 10 µM of Beta Glucan. As negative control for training, we treated BMDMs with phosphate buffered saline (PBS). Three biological replicates each were sequenced for the beta glucan treated BMDMs and for the seven experimental small molecules. We sequenced six biological replicates of the PBS treated BMDMs to increase statistical power. Two sequencing runs were performed on the same samples to increase sequencing depth (see raw files from sequencing run #1 labeled "AEK-RK..." vs from sequencing run #2 "AEK-HRK...").