Human macrophages activate a coordinated program linking inflammation and cell death in response to intracellular Poly(I:C).

Background: Macrophages sense viral double-stranded RNA (dsRNA) via the innate immune receptors TLR3 and MDA5 whose activation drives a robust inflammatory program, often coupled to cell death. TLR3/TRIF-dependent apoptosis has been described in response to dsRNA. Growing evidence suggests that dsRNA may trigger multiple forms of cell death with mechanisms yet to be elucidated. In addition to TLR/TRIF axis, the necroptosis regulator RIPK3 has been implicated in both inflammatory response and cell death during viral infections in several cell types. However, its role in regulating these two events in human macrophages remains unclear. Methods: Here, we investigated in human monocyte-derived macrophages (hMDMs) the mechanisms of dsRNA induced cell death and its temporal and molecular linkage to inflammatory signaling, with a focus on the interplay between RIPK3 and the TLR3/TRIF pathways. Results: We found that intracellular Poly(I:C), a synthetic analogue of dsRNA, induced cell death with apoptotic and necroptotic features. TRIF-mediated signaling cascade and RIPK3 jointly contributed to caspase-8 activation leading to apoptosis. RIPK3 also promoted necroptosis by inducing MLKL phosphorylation. Downstream of caspases, Poly(I:C) induced cleavage of Gasdermin E (GSDME), which remained intracellular and localized in mitochondria contributing to the amplification of caspase-3 activation. RIPK3-dependent signaling drove the release of IFN-β and TNF-α and promoted the upregulation of the sensors TLR3 and MDA5, thereby amplifying the inflammatory axis. The inflammatory response preceded cell death, with RIPK3 being the key regulator of the early signaling and TLR3/TRIF becoming involved at later timepoints. Conclusions: Collectively, these findings identify RIPK3 as key mediator coordinating dsRNA-induced inflammatory signaling and regulated cell death in human macrophages.