First line of defense: viral RNA-induced cell death mechanisms in macrophages and neutrophil responses to live bacteria.

Inflammation is a fundamental protective response that relies on the coordinated action of innate immune cells to eliminate pathogens, remove damaged tissues, and restore homeostasis. This work addresses two complementary aspects of inflammatory regulation, focusing on (i) the mechanisms of macrophage cell death induced by viral RNA mimetics, and (ii) the ability of human neutrophils to discriminate live from dead bacteria, with particular attention to Group B Streptococcus (GBS). First, we investigated how human macrophages respond to cytosolic Poly(I:C), a synthetic analogue of viral dsRNA. Our findings demonstrate that Poly(I:C) triggers a mixed lytic cell death program involving pyroptosis secondary to apoptosis and necroptosis. This response is mediated by TLR3 and requires TRIF-dependent activation of caspase-8, caspase-3, and GSDME, while GSDMD remains inactive. Importantly, Poly(I:C)-induced macrophage death is also caused by RIPK3, which leads to necroptosis, and occurs independently of NLRP3 inflammasome activation. Although Poly(I:C) did not prime or activate inflammasome pathways in unprimed macrophages, it acted as a secondary signal in LPS-primed cells; however, transfection reagents influenced these outcomes, prompting ongoing optimization. Overall, our data identify a TLR3–TRIF–caspase-8/3–GSDME axis as the central driver of dsRNA-induced lytic death in human macrophages. Second, we examined how neutrophils detect and respond to GBS. We show that human neutrophils preferentially sense live GBS through the combined activity of formyl peptide receptors (FPR1 and FPR2) and TLR8. TLR8-mediated recognition of bacterial RNA emerged as the dominant pathway for cytokines and ROS production, while FPR1 and FPR2 selectively engaged distinct formylated GBS peptides, acting as crucial determinants of bacterial viability sensing. Neutrophil activation was markedly reduced when either TLR8 or FPR signaling was inhibited, indicating that robust inflammatory responses require concurrent engagement of both receptor systems. This multisensory mechanism likely functions as a safeguard to ensure that potent neutrophil effector functions are deployed only in the presence of live, potentially dangerous bacteria. Together, these findings highlight key innate immune mechanisms governing inflammatory responses to viral and bacterial stimuli, revealing how macrophages integrate dsRNA-driven death pathways and how neutrophils combine FPRs and TLR8 signaling to discriminate live from dead microbes and calibrate inflammation accordingly.