Viral diseases are consistently among the most pervasive healthcare burdens in the world. A key feature of viral diseases like COVID-19 and influenza A that contributes their mortality is their propensity to trigger hyperinflammatory immune responses in infected individuals. While much is known about the antiviral response, there remain considerable deficits in our understanding of the processes that lead to excessive inflammation. For example, while much is understood about the NLRP3 inflammasome, there is far less understood about how antiviral Toll-like receptors like TLR3 can potentially prime its activation. In this RTF proposal, we describe a research strategy that departs from the status quo and aims to establish a concrete linkage between TLR3 signaling and NLRP3 inflammasome activation, with IRE1 as its linchpin. We propose to block IRE1 activity concomitant with poly I:C exposure in human and mouse macrophage-like cell lines to determine if the NLRP3 inflammasome can still be activated, as measured by mitochondrial DNA release, caspase-1 activation, and interleukin-1 beta release. We also propose to compare the transcriptomes of poly I:C-stimulated, IRE1-inhibited macrophages with poly I:C-stimulated macrophages. These will be undergraduate-driven experiments completed and analyzed in our lab at the University of Maine at Augusta (UMA). We will consult with the Maine INBRE Bioinformatics Core for support in analyzing these data so that we may better understand the multiple layers of regulation that control the inflammatory response. These pilot studies will yield critical data that will inform future proposals aimed at understanding the processes that drive hyperinflammatory responses seen in viral diseases like COVID-19 and influenza A.