E-selectin-mediated rapid NLRP3 inflammasome activation regulates S100A8/S100A9 release from neutrophils via transient gasdermin D pore formation.

S100A8/S100A9 is a proinflammatory mediator released by myeloid cells during many acute and chronic inflammatory disorders. However, the precise mechanism of its release from the cytosolic compartment of neutrophils is unclear. Here, we show that E-selectin-induced rapid S100A8/S100A9 release during inflammation occurs in an NLRP3 inflammasome-dependent fashion. Mechanistically, E-selectin engagement triggers Bruton's tyrosine kinase-dependent tyrosine phosphorylation of NLRP3. Concomitant potassium efflux via the voltage-gated potassium channel KV1.3 mediates ASC oligomerization. This is followed by caspase 1 cleavage and downstream activation of pore-forming gasdermin D, enabling cytosolic release of S100A8/S100A9. Strikingly, E-selectin-mediated gasdermin D pore formation does not result in cell death but is a transient process involving activation of the ESCRT III membrane repair machinery. These data clarify molecular mechanisms of controlled S100A8/S100A9 release from neutrophils and identify the NLRP3/gasdermin D axis as a rapid and reversible activation system in neutrophils during inflammation.

Vitronectin promotes immunothrombotic dysregulation in the venular microvasculature.

Microvascular immunothrombotic dysregulation is a critical process in the pathogenesis of severe systemic inflammatory diseases. The mechanisms controlling immunothrombosis in inflamed microvessels, however, remain poorly understood. Here, we report that under systemic inflammatory conditions the matricellular glycoproteinvitronectin (VN) establishes an intravascular scaffold, supporting interactions of aggregating platelets with immune cells and the venular endothelium. Blockade of the VN receptor glycoprotein (GP)IIb/IIIa interfered with this multicellular interplay and effectively prevented microvascular clot formation. In line with these experimental data, particularly VN was found to be enriched in the pulmonary microvasculature of patients with non-infectious (pancreatitis-associated) or infectious (coronavirus disease 2019 (COVID-19)-associated) severe systemic inflammatory responses. Targeting the VN-GPIIb/IIIa axis hence appears as a promising, already feasible strategy to counteract microvascular immunothrombotic dysregulation in systemic inflammatory pathologies.

Humanized ß2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

The use of cell-based reporter systems has provided valuable insights into the molecular mechanisms of integrin activation. However, current models have significant drawbacks because their artificially expressed integrins cannot be regulated by either physiological stimuli or endogenous signaling pathways. Here, we report the generation of a Hoxb8 cell line expressing human ß2 integrin that functionally replaced the deleted mouse ortholog. Hoxb8 cells are murine hematopoietic progenitor cells that can be efficiently differentiated into neutrophils and macrophages resembling their primary counterparts. Importantly, these cells can be stimulated by physiological stimuli triggering classical integrin inside-out signaling pathways, ultimately leading to ß2 integrin conformational changes that can be recorded by the conformation-specific antibodies KIM127 and mAb24. Moreover, these cells can be efficiently manipulated via the CRISPR/Cas9 technique or retroviral vector systems. Deletion of the key integrin regulators talin1 and kindlin3 or expression of ß2 integrins with mutations in their binding sites abolished both integrin extension and full activation regardless of whether only one or both activators no longer bind to the integrin. Moreover, humanized ß2 integrin Hoxb8 cells represent a valuable new model for rapidly testing the role of putative integrin regulators in controlling ß2 integrin activity in a physiological context.

Mechanism of integrin activation by talin and its cooperation with kindlin.

Talin-induced integrin binding to extracellular matrix ligands (integrin activation) is the key step to trigger many fundamental cellular processes including cell adhesion, cell migration, and spreading. Talin is widely known to use its N-terminal head domain (talin-H) to bind and activate integrin, but how talin-H operates in the context of full-length talin and its surrounding remains unknown. Here we show that while being capable of inducing integrin activation, talin-H alone exhibits unexpectedly low potency versus a constitutively activated full-length talin. We find that the large C-terminal rod domain of talin (talin-R), which otherwise masks the integrin binding site on talin-H in inactive talin, dramatically enhances the talin-H potency by dimerizing activated talin and bridging it to the integrin co-activator kindlin-2 via the adaptor protein paxillin. These data provide crucial insight into the mechanism of talin and its cooperation with kindlin to promote potent integrin activation, cell adhesion, and signaling.

Low kindlin-3 levels in osteoclasts of kindlin-3 hypomorphic mice result in osteopetrosis due to leaky sealing zones.

Osteoclasts form special integrin-mediated adhesion structures called sealing zones that enable them to adhere to and resorb bone. Sealing zones consist of densely packed podosomes tightly interconnected by actin fibers. Their formation requires the presence of the hematopoietic integrin regulator kindlin-3 (also known as Fermt3). In this study, we investigated osteoclasts and their adhesion structures in kindlin-3 hypomorphic mice expressing only 5-10% of the kindlin-3 level of wild-type mice. Low kindlin-3 expression reduces integrin activity, results in impaired osteoclast adhesion and signaling, and delays cell spreading. Despite these defects, in vitro-generated kindlin-3-hypomorphic osteoclast-like cells arrange their podosomes into adhesion patches and belts, but their podosome and actin organization is abnormal. Remarkably, kindlin-3-hypomorphic osteoclasts form sealing zones when cultured on calcified matrix in vitro and on bone surface in vivo. However, functional assays, immunohistochemical staining and electron micrographs of bone sections showed that they fail to seal the resorption lacunae properly, which is required for secreted proteinases to digest bone matrix. This results in mild osteopetrosis. Our study reveals a new, hitherto understudied function of kindlin-3 as an essential organizer of integrin-mediated adhesion structures, such as sealing zones.

Binding of Rap1 and Riam to Talin1 Fine-Tune ß2 Integrin Activity During Leukocyte Trafficking.

ß2 integrins mediate key processes during leukocyte trafficking. Upon leukocyte activation, the structurally bent ß2 integrins change their conformation towards an extended, intermediate and eventually high affinity conformation, which mediate slow leukocyte rolling and firm arrest, respectively. Translocation of talin1 to integrin adhesion sites by interactions with the small GTPase Rap1 and the Rap1 effector Riam precede these processes. Using Rap1 binding mutant talin1 and Riam deficient mice we show a strong Riam-dependent T cell homing process to lymph nodes in adoptive transfer experiments and by intravital microscopy. Moreover, neutrophils from compound mutant mice exhibit strongly increased rolling velocities to inflamed cremaster muscle venules compared to single mutants. Using Hoxb8 cell derived neutrophils generated from the mutant mouse strains, we show that both pathways regulate leukocyte rolling and adhesion synergistically by inducing conformational changes of the ß2 integrin ectodomain. Importantly, a simultaneous loss of both pathways results in a rolling phenotype similar to talin1 deficient neutrophils suggesting that ß2 integrin regulation primarily occurs via these two pathways.

Rap1 and membrane lipids cooperatively recruit talin to trigger integrin activation.

Recruitment and tethering of talin to the plasma membrane initiate the process of integrin activation. Multiple factors including the Rap1 proteins, RIAM (also known as APBB1IP) and PIP2 bind talin proteins and have been proposed to regulate these processes, but not systematically analyzed. By expressing specific talin mutants into talin-null fibroblasts, we show that binding of the talin F0 domain to Rap1 synergizes with membrane lipid binding of the talin F2 domain during talin membrane targeting and integrin activation, whereas the interaction of the talin rod with RIAM was dispensable. We also characterized a second Rap1-binding site within the talin F1 domain by detailed NMR analysis. Interestingly, while talin F1 exhibited significantly weaker Rap1-binding affinity than talin F0, expression of a talin F1 Rap1-binding mutant inhibited cell adhesion, spreading, talin recruitment and integrin activation similarly to the talin F0 Rap1-binding mutant. Moreover, the defects became significantly stronger when both Rap1-binding sites were mutated. In conclusion, our data suggest a model in which cooperative binding of Rap1 to the talin F0 and F1 domains synergizes with membrane PIP2 binding to spatiotemporally position and activate talins to regulate integrin activity.

A kindlin-3-leupaxin-paxillin signaling pathway regulates podosome stability.

Binding of kindlins to integrins is required for integrin activation, stable ligand binding, and subsequent intracellular signaling. How hematopoietic kindlin-3 contributes to the assembly and stability of the adhesion complex is not known. Here we report that kindlin-3 recruits leupaxin into podosomes and thereby regulates paxillin phosphorylation and podosome turnover. We demonstrate that the activity of the protein tyrosine phosphatase PTP-PEST, which controls paxillin phosphorylation, requires leupaxin. In contrast, despite sharing the same binding mode with leupaxin, paxillin recruitment into podosomes is kindlin-3 independent. Instead, we found paxillin together with talin and vinculin in initial adhesion patches of kindlin-3-null cells. Surprisingly, despite its presence in these early adhesion patches, podosomes can form in the absence of paxillin or any paxillin member. In conclusion, our findings show that kindlin-3 not only activates and clusters integrins into podosomes but also regulates their lifetime by recruiting leupaxin, which controls PTP-PEST activity and thereby paxillin phosphorylation and downstream signaling.

Direct Rap1/Talin1 interaction regulates platelet and neutrophil integrin activity in mice.

Targeting Talin1 to the plasma membrane is a crucial step in integrin activation, which in leukocytes is mediated by a Rap1/RIAM/Talin1 pathway, whereas in platelets, it is RIAM independent. Recent structural, biochemical, and cell biological studies have suggested direct Rap1/Talin1 interaction as an alternative mechanism to recruit Talin1 to the membrane and induce integrin activation. To test whether this pathway is of relevance in vivo, we generated Rap1 binding-deficient Talin1 knockin (Tln13mut) mice. Although Tln13mut mice showed no obvious abnormalities, their platelets exhibited reduced integrin activation, aggregation, adhesion, and spreading, resulting in prolonged tail-bleeding times and delayed thrombus formation and vessel occlusion in vivo. Surprisingly, neutrophil adhesion to different integrin ligands and ß2 integrin-dependent phagocytosis were also significantly impaired, which caused profound leukocyte adhesion and extravasation defects in Tln13mut mice. In contrast, macrophages exhibited no defect in adhesion or spreading despite reduced integrin activation. Taken together, our findings suggest that direct Rap1/Talin1 interaction is of particular importance in regulating the activity of different integrin classes expressed on platelets and neutrophils, which both depend on fast and dynamic integrin-mediated responses.

Structure of Rap1b bound to talin reveals a pathway for triggering integrin activation.

Activation of transmembrane receptor integrin by talin is essential for inducing cell adhesion. However, the pathway that recruits talin to the membrane, which critically controls talin's action, remains elusive. Membrane-anchored mammalian small GTPase Rap1 is known to bind talin-F0 domain but the binding was shown to be weak and thus hardly studied. Here we show structurally that talin-F0 binds to human Rap1b like canonical Rap1 effectors despite little sequence homology, and disruption of the binding strongly impairs integrin activation, cell adhesion, and cell spreading. Furthermore, while being weak in conventional binary binding conditions, the Rap1b/talin interaction becomes strong upon attachment of activated Rap1b to vesicular membranes that mimic the agonist-induced microenvironment. These data identify a crucial Rap1-mediated membrane-targeting mechanism for talin to activate integrin. They further broadly caution the analyses of weak protein-protein interactions that may be pivotal for function but neglected in the absence of specific cellular microenvironments.