IL-32 is Elevated in Severe COVID-19 and Drives Expression of Inflammatory Genes in Human Macrophages

IFN-γ-driven M1-like inflammatory macrophage states are shared and abundant across several human immune-mediated inflammatory diseases (IMIDs) including, rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and COVID-19. To identify secreted proteins which drive or suppress M1-like macrophage states we screened >600 human proteins and peptides in cell-based assays using human monocyte-derived macrophages for four phenotypic states (M1-like, M1-like in IFN-γ-primed cells, IFN-γ-like and IL-10-like) based on production of inflammatory cytokines. We identified and validated several hits from our screens including the cytokines IL-32β and IL-32γ but not IL-32α which induced an M1-like inflammatory state in non-primed and IFN-γ-primed macrophages. IL-32β induced macrophage responses such as tolerance, cross-tolerance, activation of inflammatory signalling pathways and transcriptional responses which were overlapping yet distinct to those observed with LPS. Because IL-32 is an orphan cytokine with no rodent orthologue and without an identified receptor or downstream signalling pathway we used genome-wide RNAi screens, small molecule inhibitors and co-localization assays to identify the signalling pathway. We found that IL-32β uses components of the TLR4-MyD88-IRAK1 signalling pathway to mediate inflammatory signalling in human macrophages. Furthermore, by analysing the expression of IL-32β, IFN-γ, their pathway component genes and their specific transcriptional responses in macrophages across multiple human IMID single-cell datasets we identified a potential role for both IL-32β and IFN-γ signalling to macrophages in COVID-19. Analysis of serum from healthy controls and patients with mild and severe COVID-19 revealed elevated levels of IL-32 in severe COVID-19. Collectively, this work identifies IL-32 as a cytokine which synergises with IFN-γ, signals through a MyD88/IRAK-1 pathway, is elevated in severe COVID-19 and induces a transcriptional response which is observed in macrophages in mild and severe COVID-19.