Inhibiting the immunoproteasome's β5i catalytic activity affects human peripheral blood-derived immune cell viability

Small molecule inhibitors selectively targeting the immunoproteasome subunit β5i are currently being developed for the treatment of autoimmune disorders. However, patients carrying loss-of-function mutations in the gene encoding β5i (Psmb8) suffer from the proteasome-associated autoinflammatory syndromes (PRAAS) emphasizing the need to study pharmacological inhibition of immunoproteasome function in human cells. Here, we characterized the immunomodulatory potential of the selective β5i inhibitor ONX 0914 and Bortezomib, a pan-proteasome inhibitor, in human peripheral blood mononuclear cells (PBMCs). Both compounds efficiently blocked pro-inflammatory cytokine secretion in human whole blood and PBMC cultures stimulated with toll-like receptor (TLR) agonists. Furthermore, the compounds inhibited T cell cytokine production induced by recall antigen CMVpp65 or by polyclonal stimulation. The viability of PBMCs, however, was rapidly decreased in the presence of ONX 0914 and Bortezomib demonstrated by decreased residual cytosolic ATP and increased Annexin V surface binding. Interestingly, HLA-DR ⁺ monocytes were rapidly depleted from the cultures in the presence of ONX 0914 as a β5i-selective inhibitor and Bortezomib. In conclusion, the anti-inflammatory potential of β5i-selective inhibitors is correlating with a cytotoxicity increase in human PBMC subsets ex vivo. Our results provide important insights into the anti-inflammatory mechanism of action of β5i-inhibitors which currently hold the promise as a novel therapy for autoinflammatory diseases.