Erratum: Piezo1 induces endothelial responses to shear stress via soluble adenylyl Cyclase-IP3R2 circuit.

[This corrects the article DOI: 10.1016/j.isci.2023.106661.].

trans-Endothelial neutrophil migration activates bactericidal function via Piezo1 mechanosensing.

The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.

End binding-3 inhibitor activates regenerative program in age-related macular degeneration.

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

Ubiquitin ligase CHFR mediated degradation of VE-cadherin through ubiquitylation disrupts endothelial adherens junctions.

Vascular endothelial cadherin (VE-cadherin) expressed at endothelial adherens junctions (AJs) is vital for vascular integrity and endothelial homeostasis. Here we identify the requirement of the ubiquitin E3-ligase CHFR as a key mechanism of ubiquitylation-dependent degradation of VE-cadherin. CHFR was essential for disrupting the endothelium through control of the VE-cadherin protein expression at AJs. We observe augmented expression of VE-cadherin in endothelial cell (EC)-restricted Chfr knockout (ChfrΔEC) mice. We also observe abrogation of LPS-induced degradation of VE-cadherin in ChfrΔEC mice, suggesting the pathophysiological relevance of CHFR in regulating the endothelial junctional barrier in inflammation. Lung endothelial barrier breakdown, inflammatory neutrophil extravasation, and mortality induced by LPS were all suppressed in ChfrΔEC mice. We find that the transcription factor FoxO1 is a key upstream regulator of CHFR expression. These findings demonstrate the requisite role of the endothelial cell-expressed E3-ligase CHFR in regulating the expression of VE-cadherin, and thereby endothelial junctional barrier integrity.

Detecting organelle-specific activity of potassium channels with a DNA nanodevice.

Cell surface potassium ion (K+) channels regulate nutrient transport, cell migration and intercellular communication by controlling K+ permeability and are thought to be active only at the plasma membrane. Although these channels transit the trans-Golgi network, early and recycling endosomes, whether they are active in these organelles is unknown. Here we describe a pH-correctable, ratiometric reporter for K+ called pHlicKer, use it to probe the compartment-specific activity of a prototypical voltage-gated K+ channel, Kv11.1, and show that this cell surface channel is active in organelles. Lumenal K+ in organelles increased in cells expressing wild-type Kv11.1 channels but not after treatment with current blockers. Mutant Kv11.1 channels, with impaired transport function, failed to increase K+ levels in recycling endosomes, an effect rescued by pharmacological correction. By providing a way to map the organelle-specific activity of K+ channels, pHlicKer technology could help identify new organellar K+ channels or channel modulators with nuanced functions.

An unexpected role of neutrophils in clearing apoptotic hepatocytes in vivo.

Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here, we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver, and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.

Every day, the immune cells clears the remains of billions of old and damaged cells that have undergone a controlled form of death. Removing them quickly helps to prevent inflammation or the development of autoimmune diseases. While immune cells called neutrophils are generally tasked with removing invading bacteria, macrophages are thought to be responsible for clearing dead cells. However, in healthy tissue, the process occurs so efficiently that it can be difficult to confirm which cells are responsible. To take a closer look, Cao et al. focused on the liver by staining human samples to identify both immune and dead cells. Unexpectedly, there were large numbers of neutrophils visible inside dead liver cells. Further experiments in mice revealed that after entering the dead cells, neutrophils engulfed the contents and digested the dead cell from the inside out. This was a surprising finding because not only are neutrophils not usually associated with dead cells, but immune cells usually engulf cells and bacteria from the outside rather than burrowing inside them. The importance of this neutrophil behaviour was shown when Cao et al. studied samples from patients with an autoimmune disease where immune cells attack the liver. In this case, very few dead liver cells contained neutrophils, and the neutrophils themselves did not seem capable of removing the dead cells, leading to inflammation. This suggests that defective neutrophil function could be a key contributor to this autoimmune disease. The findings identify a new role for neutrophils in maintaining healthy functioning of the liver and reveal a new target in the treatment of autoimmune diseases. In the future, Cao et al. plan to explore whether compounds that enhance clearance of dead cells by neutrophils can be used to treat autoimmune liver disease in mouse models of the disease.

Trained immunity of alveolar macrophages enhances injury resolution via KLF4-MERTK-mediated efferocytosis.

Recent studies suggest that training of innate immune cells such as tissue-resident macrophages by repeated noxious stimuli can heighten host defense responses. However, it remains unclear whether trained immunity of tissue-resident macrophages also enhances injury resolution to counterbalance the heightened inflammatory responses. Here, we studied lung-resident alveolar macrophages (AMs) prechallenged with either the bacterial endotoxin or with Pseudomonas aeruginosa and observed that these trained AMs showed greater resilience to pathogen-induced cell death. Transcriptomic analysis and functional assays showed greater capacity of trained AMs for efferocytosis of cellular debris and injury resolution. Single-cell high-dimensional mass cytometry analysis and lineage tracing demonstrated that training induces an expansion of a MERTKhiMarcohiCD163+F4/80low lung-resident AM subset with a proresolving phenotype. Reprogrammed AMs upregulated expression of the efferocytosis receptor MERTK mediated by the transcription factor KLF4. Adoptive transfer of these trained AMs restricted inflammatory lung injury in recipient mice exposed to lethal P. aeruginosa. Thus, our study has identified a subset of tissue-resident trained macrophages that prevent hyperinflammation and restore tissue homeostasis following repeated pathogen challenges.

Endosomal trafficking of two-pore K+ efflux channel TWIK2 to plasmalemma mediates NLRP3 inflammasome activation and inflammatory injury.

Potassium efflux via the two-pore K+ channel TWIK2 is a requisite step for the activation of NLRP3 inflammasome, however, it remains unclear how K+ efflux is activated in response to select cues. Here, we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in the extrusion of K+. We showed that ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP-ligated purinergic receptor P2X7 each prevented endosomal fusion with the plasmalemma and K+ efflux as well as NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-depleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. We conclude that Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 localization and activity at the cell surface and the downstream activation of the NLRP3 inflammasome. Results show that endosomal trafficking of TWIK2 to the plasmalemma is a potential therapeutic target in acute or chronic inflammatory states.

Piezo1 induces endothelial responses to shear stress via soluble adenylyl Cyclase-IP3R2 circuit.

Endothelial cells (ECs) continuously sense and adapt to changes in shear stress generated by blood flow. Here, we show that the activation of the mechanosensitive channel Piezo1 by defined shear forces induces Ca2+ entry into the endoplasmic reticulum (ER) via the ER Ca2+ ATPase pump. This entry is followed by inositol trisphosphate receptor 2 (IP3R2)-elicited ER Ca2+ release into the cytosol. The mechanism of ER Ca2+ release involves the generation of cAMP by soluble adenylyl cyclase (sAC), leading to IP3R2-evoked Ca2+ gating. Depleting sAC or IP3R2 prevents ER Ca2+ release and blocks EC alignment in the direction of flow. Overexpression of constitutively active Akt1 restores the shear-induced alignment of ECs lacking Piezo1 or IP3R2, as well as the flow-induced vasodilation in endothelial restricted Piezo1 knockout mice. These studies describe an unknown Piezo1-cAMP-IP3R2 circuit as an essential mechanism activating Akt signaling and inducing adaptive changes in ECs to laminar flow.

An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) neutralize infection and are efficacious for the treatment of COVID-19. However, SARS-CoV-2 variants, notably sublineages of B.1.1.529/omicron, have emerged that escape antibodies in clinical use. As an alternative, soluble decoy receptors based on the host entry receptor ACE2 broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE22 .v2.4-IgG1 was previously shown to be effective against SARS-CoV-2 variants when administered intravenously. Here, inhalation of aerosolized sACE22 .v2.4-IgG1 increased survival and ameliorated lung injury in K18-hACE2 mice inoculated with P.1/gamma virus. Loss of catalytic activity reduced the decoy's therapeutic efficacy, which was further confirmed by intravenous administration, supporting dual mechanisms of action: direct blocking of S and turnover of ACE2 substrates associated with lung injury and inflammation. Furthermore, sACE22 .v2.4-IgG1 tightly binds and neutralizes BA.1, BA.2, and BA.4/BA.5 omicron and protects K18-hACE2 mice inoculated with a high dose of BA.1 omicron virus. Overall, the therapeutic potential of sACE22 .v2.4-IgG1 is demonstrated by the inhalation route and broad neutralization potency persists against highly divergent SARS-CoV-2 variants.

Single-cell transcriptomic profiling of lung endothelial cells identifies dynamic inflammatory and regenerative subpopulations.

Studies have demonstrated the phenotypic heterogeneity of vascular endothelial cells (ECs) within a vascular bed; however, little is known about how distinct endothelial subpopulations in a particular organ respond to an inflammatory stimulus. We performed single-cell RNA-Seq of 35,973 lung ECs obtained during baseline as well as postinjury time points after inflammatory lung injury induced by LPS. Seurat clustering and gene expression pathway analysis identified 2 major subpopulations in the lung microvascular endothelium, a subpopulation enriched for expression of immune response genes such as MHC genes (immuneEC) and another defined by increased expression of vascular development genes such as Sox17 (devEC). The presence of immuneEC and devEC subpopulations was also observed in nonhuman primate lungs infected with SARS-CoV-2 and murine lungs infected with H1N1 influenza virus. After the peak of inflammatory injury, we observed the emergence of a proliferative lung EC subpopulation. Overexpression of Sox17 prevented inflammatory activation in ECs. Thus, there appeared to be a "division of labor" within the lung microvascular endothelium in which some ECs showed propensity for inflammatory signaling and others for endothelial regeneration. These results provide underpinnings for the development of targeted therapies to limit inflammatory lung injury and promote regeneration.

Nanoparticle targeting of de novo profibrotic macrophages mitigates lung fibrosis.

The pathogenesis of lung fibrosis involves hyperactivation of innate and adaptive immune pathways that release inflammatory cytokines and growth factors such as tumor growth factor (TGF)ß1 and induce aberrant extracellular matrix protein production. During the genesis of pulmonary fibrosis, resident alveolar macrophages are replaced by a population of newly arrived monocyte-derived interstitial macrophages that subsequently transition into alveolar macrophages (Mo-AMs). These transitioning cells initiate fibrosis by releasing profibrotic cytokines and remodeling the matrix. Here, we describe a strategy for leveraging the up-regulation of the mannose receptor CD206 in interstitial macrophages and Mo-AM to treat lung fibrosis. We engineered mannosylated albumin nanoparticles, which were found to be internalized by fibrogenic CD206+ monocyte derived macrophages (Mo-Macs). Mannosylated albumin nanoparticles incorporating TGFß1 small-interfering RNA (siRNA) targeted the profibrotic subpopulation of CD206+ macrophages and prevented lung fibrosis. The findings point to the potential utility of mannosylated albumin nanoparticles in delivering TGFß-siRNA into CD206+ profibrotic macrophages as an antilung fibrosis strategy.

An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) glycoprotein neutralize infection and are efficacious for the treatment of mild-to-moderate COVID-19. However, SARS-CoV-2 variants have emerged that partially or fully escape monoclonal antibodies in clinical use. Notably, the BA.2 sublineage of B.1.1.529/omicron escapes nearly all monoclonal antibodies currently authorized for therapeutic treatment of COVID-19. Decoy receptors, which are based on soluble forms of the host entry receptor ACE2, are an alternative strategy that broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE2 2 .v2.4-IgG1 was previously shown to be effective in vivo against SARS-CoV-2 variants when administered intravenously. Here, the inhalation of sACE2 2 .v2.4-IgG1 is found to increase survival and ameliorate lung injury in K18-hACE2 transgenic mice inoculated with a lethal dose of the virulent P.1/gamma virus. Loss of catalytic activity reduced the decoy’s therapeutic efficacy supporting dual mechanisms of action: direct blocking of viral S and turnover of ACE2 substrates associated with lung injury and inflammation. Binding of sACE2 2 .v2.4-IgG1 remained tight to S of BA.1 omicron, despite BA.1 omicron having extensive mutations, and binding exceeded that of four monoclonal antibodies approved for clinical use. BA.1 pseudovirus and authentic virus were neutralized at picomolar concentrations. Finally, tight binding was maintained against S from the BA.2 omicron sublineage, which differs from S of BA.1 by 26 mutations. Overall, the therapeutic potential of sACE2 2 .v2.4-IgG1 is further confirmed by inhalation route and broad neutralization potency persists against increasingly divergent SARS-CoV-2 variants.

Albumin Nanoparticle Endocytosing Subset of Neutrophils for Precision Therapeutic Targeting of Inflammatory Tissue Injury.

The complex involvement of neutrophils in inflammatory diseases makes them intriguing but challenging targets for therapeutic intervention. Here, we tested the hypothesis that varying endocytosis capacities would delineate functionally distinct neutrophil subpopulations that could be specifically targeted for therapeutic purposes. By using uniformly sized (∼120 nm in diameter) albumin nanoparticles (ANP) to characterize mouse neutrophils in vivo, we found two subsets of neutrophils, one that readily endocytosed ANP (ANPhigh neutrophils) and another that failed to endocytose ANP (ANPlow population). These ANPhigh and ANPlow subsets existed side by side simultaneously in bone marrow, peripheral blood, spleen, and lungs, both under basal conditions and after inflammatory challenge. Human peripheral blood neutrophils showed a similar duality. ANPhigh and ANPlow neutrophils had distinct cell surface marker expression and transcriptomic profiles, both in naive mice and in mice after endotoxemic challenge. ANPhigh and ANPlow neutrophils were functionally distinct in their capacities to kill bacteria and to produce inflammatory mediators. ANPhigh neutrophils produced inordinate amounts of reactive oxygen species and inflammatory chemokines and cytokines. Targeting this subset with ANP loaded with the drug piceatannol, a spleen tyrosine kinase (Syk) inhibitor, mitigated the effects of polymicrobial sepsis by reducing tissue inflammation while fully preserving neutrophilic host-defense function.

Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants.

Vaccine hesitancy and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) escaping vaccine-induced immune responses highlight the urgency for new COVID-19 therapeutics. Engineered angiotensin-converting enzyme 2 (ACE2) proteins with augmented binding affinities for SARS-CoV-2 spike (S) protein may prove to be especially efficacious against multiple variants. Using molecular dynamics simulations and functional assays, we show that three amino acid substitutions in an engineered soluble ACE2 protein markedly augmented the affinity for the S protein of the SARS-CoV-2 WA-1/2020 isolate and multiple VOCs: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). In humanized K18-hACE2 mice infected with the SARS-CoV-2 WA-1/2020 or P.1 variant, prophylactic and therapeutic injections of soluble ACE22.v2.4-IgG1 prevented lung vascular injury and edema formation, essential features of CoV-2-induced SARS, and above all improved survival. These studies demonstrate broad efficacy in vivo of an engineered ACE2 decoy against SARS-CoV-2 variants in mice and point to its therapeutic potential.

Gasdermin D pores are dynamically regulated by local phosphoinositide circuitry.

Gasdermin D forms large, ~21 nm diameter pores in the plasma membrane to drive the cell death program pyroptosis. These pores are thought to be permanently open, and the resultant osmotic imbalance is thought to be highly damaging. Yet some cells mitigate and survive pore formation, suggesting an undiscovered layer of regulation over the function of these pores. However, no methods exist to directly reveal these mechanistic details. Here, we combine optogenetic tools, live cell fluorescence biosensing, and electrophysiology to demonstrate that gasdermin pores display phosphoinositide-dependent dynamics. We quantify repeated and fast opening-closing of these pores on the tens of seconds timescale, visualize the dynamic pore geometry, and identify the signaling that controls dynamic pore activity. The identification of this circuit allows pharmacological tuning of pyroptosis and control of inflammatory cytokine release by living cells.

Mechanisms of Lung Injury Induced by SARS-CoV-2 Infection.

The lung is the major target organ of SARS-CoV-2 infection, which causes COVID-19. Here, we outline the multistep mechanisms of lung epithelial and endothelial injury induced by SARS-CoV-2: direct viral infection, chemokine/cytokine-mediated damage, and immune cell-mediated lung injury. Finally, we discuss the recent progress in terms of antiviral therapeutics as well as the development of anti-inflammatory or immunomodulatory therapeutic approaches. This review also provides a systematic overview of the models for studying SARS-CoV-2 infection and discusses how an understanding of mechanisms of lung injury will help identify potential targets for future drug development to mitigate lung injury.

CD38-mediated Inhibition of Bruton's Tyrosine Kinase in Macrophages Prevents Endotoxemic Lung Injury.

TLR4 signaling via endotoxemia in macrophages promotes macrophage transition to the inflammatory phenotype through NLRP3 inflammasome activation. This transition event has the potential to trigger acute lung injury (ALI). However, relatively little is known about the regulation of NLRP3 and its role in the pathogenesis of ALI. Here we interrogated the signaling pathway activated by CD38, an ectoenzyme expressed in macrophages, in preventing ALI through suppressing NLRP3 activation. Wild-type and Cd38-knockout (Cd38-/-) mice were used to assess inflammatory lung injury, and isolated macrophages were used to delineate underlying TLR4 signaling pathway. We showed that CD38 suppressed TLR4 signaling in macrophages by inhibiting Bruton's tyrosine kinase (Btk) through the recruitment of Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) and resulting in the dephosphorylation of activated Btk. Cd38-/- mice show enhanced lung polymorphonuclear leukocyte extravasation and severe lung injury. LPS- or polymicrobial sepsis-induced mortality in Cd38-/- mice were markedly augmented compared with wild types. CD38 in macrophages functioned by inhibiting Btk activation through activation of SHP2 and resulting dephosphorylation of Btk, and thereby preventing activation of downstream targets NF-κB and NLRP3. Cd38-/- macrophages displayed markedly increased activation of Btk, NF-κB, and NLRP3, whereas in vivo administration of the Btk inhibitor ibrutinib (a Food and Drug Administration-approved drug) prevented augmented TLR4-induced inflammatory lung injury seen in Cd38-/- mice. Our findings together show upregulation of CD38 activity and inhibition of Btk activation downstream of TLR4 activation as potential strategies to prevent endotoxemic ALI.

KIF13B-mediated VEGFR2 trafficking is essential for vascular leakage and metastasis in vivo.

VEGF-A induces vascular leakage and angiogenesis via activating the cell surface localized receptor VEGF receptor 2 (VEGFR2). The amount of available VEGFR2 at the cell surface is however tightly regulated by trafficking of VEGFR2 by kinesin family 13 B (KIF13B), a plus-end kinesin motor, to the plasma membrane of endothelial cells (ECs). Competitive inhibition of interaction between VEGFR2 and KIF13B by a peptide kinesin-derived angiogenesis inhibitor (KAI) prevented pathological angiogenesis in models of cancer and eye disease associated with defective angiogenesis. Here, we show the protective effects of KAI in VEGF-A-induced vascular leakage and cancer metastasis. Using an EC-specific KIF13B knockout (Kif13b iECKO ) mouse model, we demonstrated the function of EC expressed KIF13B in mediating VEGF-A-induced vascular leakage, angiogenesis, tumor growth, and cancer metastasis. Thus, KIF13B-mediated trafficking of VEGFR2 to the endothelial surface has an essential role in pathological angiogenesis induced by VEGF-A, and is therefore a potential therapeutic target.