Red blood cells present a complex cell plasma membrane architecture with submicrometric organization leading to nanomechanical heterogeneities.

Reovirus infection requires the concerted action of viral and host factors to promote cell entry. After interaction of reovirus attachment protein σ1 with cell-surface carbohydrates and proteinaceous receptors, additional host factors mediate virus internalization. In particular, β1 integrin is required for endocytosis of reovirus virions following junctional adhesion molecule A (JAM-A) binding. While integrin-binding motifs in the surface-exposed region of reovirus capsid protein λ2 are thought to mediate integrin interaction, evidence for direct β1 integrin-reovirus interactions and knowledge of how integrins function to mediate reovirus entry is lacking. Here, we use single-virus force spectroscopy and confocal microscopy to discover a direct interaction between reovirus and β1 integrins. Comparison of interactions between reovirus disassembly intermediates as well as mutants and β1 integrin show that λ2 is the integrin ligand. Finally, using fluidic force microscopy, we demonstrate a functional role for β1 integrin interaction in promoting clathrin recruitment to cell-bound reovirus. Our study demonstrates a direct interaction between reovirus and β1 integrins and offers insights into the mechanism of reovirus cell entry. These results provide new perspectives for the development of efficacious antiviral therapeutics and the engineering of improved viral gene delivery and oncolytic vectors.

Inflammation is a pathophysiological response of innate immunity to infection or tissue damage. This response is among others triggered by factors released by damaged or dying cells, termed damage-associated molecular pattern (DAMP) molecules that act as danger signals. DAMPs interact with pattern recognition receptors (PRRs) to contribute to the induction of inflammation. However, how released peroxiredoxins (PRDXs) are able to activate PRRs, such as Toll-like receptors (TLRs), remains elusive. Here, we used force-distance curve-based atomic force microscopy to investigate the molecular mechanisms by which extracellular human PRDX5 can activate a proinflammatory response. Single-molecule experiments demonstrated that PRDX5 binds to purified TLR4 receptors, on macrophage-differentiated THP-1 cells, and on human TLR4-transfected CHO cells. These findings suggest that extracellular PRDX5 can specifically trigger a proinflammatory response. Moreover, our work also revealed that PRDX5 binding induces a cellular mechanoresponse. Collectively, this study provides insights into the role of extracellular PRDX5 in innate immunity.

Silica nanoparticles (SiNP) trigger a range of innate immune responses in relevant essential organs, such as the liver and the lungs. Inflammatory reactions, including NLRP3 inflammasome activation, have been linked to particulate materials; however, the molecular mechanisms and key actors remain elusive. Although many receptors, including several scavenger receptors, were suggested to participate in SiNP cellular uptake, mechanistic evidence of their role on innate immunity is lacking. Here we present an atomic force microscopy-based approach to physico-mechanically map the specific interaction occurring between nanoparticles and scavenger receptor A1 (SRA1) in vitro on living lung epithelial cells. We find that SiNP recognition by SRA1 on human macrophages plays a key role in mediating NLRP3 inflammasome activation, and we identify cellular mechanical changes as clear indicators of inflammasome activation in human macrophages, greatly advancing our knowledge on the interplay among nanomaterials and innate immunity.

Human peroxiredoxin-5 (PRDX5) is a unique redox-sensitive protein that plays a dual role in brain ischemia-reperfusion injury. While intracellular PRDX5 has been reported to act as a neuroprotective antioxidative enzyme by scavenging peroxides, once released extracellularly from necrotic brain cells, the protein aggravates neural cell death by inducing expression of proinflammatory cytokines in macrophages through activation of Toll-like receptor (TLR) 2 (TLR2) and 4 (TLR4). Although recent evidence showed that PRDX5 was able to interact directly with TLR4, little is known regarding the role of the cysteine redox state of PRDX5 on its DAMP function. To gain insights into the role of PRDX5 redox-active cysteine residues in the TLR4-dependent proinflammatory activity of the protein, we used a recombinant human PRDX5 in the disulfide (oxidized) form and a mutant version lacking the peroxidatic cysteine, as well as chemically reduced and hyperoxidized PRDX5 proteins. We first analyzed the oxidation state and oligomerization profile by Western blot, mass spectrometry, and SEC-MALS. Using ELISA, we demonstrate that the disulfide bridge between the enzymatic cysteines is required to allow improved TLR4-dependent IL-8 secretion. Moreover, single-molecule force spectroscopy experiments revealed that TLR4 alone is not sufficient to discriminate the different PRDX5 redox forms. Finally, flow cytometry binding assays show that disulfide PRDX5 has a higher propensity to bind to the surface of living TLR4-expressing cells than the mutant protein. Taken together, these results demonstrate the importance of the redox state of PRDX5 cysteine residues on TLR4-induced inflammation.