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Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy.

Despite the availability of effective vaccines against COVID-19, booster vaccinations are needed to maintain vaccine-induced protection against variant strains and breakthrough infections. This study aimed to investigate the efficacy, safety, and immunogenicity of the Ad26.COV2.S vaccine (Janssen) as primary vaccination plus a booster dose.ENSEMBLE2 is a randomised, double-blind, placebo-controlled, phase 3 trial including crossover vaccination after emergency authorisation of COVID-19 vaccines. Adults aged at least 18 years without previous COVID-19 vaccination at public and private medical practices and hospitals in Belgium, Brazil, Colombia, France, Germany, the Philippines, South Africa, Spain, the UK, and the USA were randomly assigned 1:1 via a computer algorithm to receive intramuscularly administered Ad26.COV2.S as a primary dose plus a booster dose at 2 months or two placebo injections 2 months apart. The primary endpoint was vaccine efficacy against the first occurrence of molecularly confirmed moderate to severe-critical COVID-19 with onset at least 14 days after booster vaccination, which was assessed in participants who received two doses of vaccine or placebo, were negative for SARS-CoV-2 by PCR at baseline and on serology at baseline and day 71, had no major protocol deviations, and were at risk of COVID-19 (ie, had no PCR-positive result or discontinued the study before day 71). Safety was assessed in all participants; reactogenicity, in terms of solicited local and systemic adverse events, was assessed as a secondary endpoint in a safety subset (approximately 6000 randomly selected participants). The trial is registered with ClinicalTrials.gov, NCT04614948, and is ongoing.Enrolment began on Nov 16, 2020, and the primary analysis data cutoff was June 25, 2021. From 34 571 participants screened, the double-blind phase enrolled 31 300 participants, 14 492 of whom received two doses (7484 in the Ad26.COV2.S group and 7008 in the placebo group) and 11 639 of whom were eligible for inclusion in the assessment of the primary endpoint (6024 in the Ad26.COV2.S group and 5615 in the placebo group). The median (IQR) follow-up post-booster vaccination was 36·0 (15·0-62·0) days. Vaccine efficacy was 75·2% (adjusted 95% CI 54·6-87·3) against moderate to severe-critical COVID-19 (14 cases in the Ad26.COV2.S group and 52 cases in the placebo group). Most cases were due to the variants alpha (B.1.1.7) and mu (B.1.621); endpoints for the primary analysis accrued from Nov 16, 2020, to June 25, 2021, before the global dominance of delta (B.1.617.2) or omicron (B.1.1.529). The booster vaccine exhibited an acceptable safety profile. The overall frequencies of solicited local and systemic adverse events (evaluated in the safety subset, n=6067) were higher among vaccine recipients than placebo recipients after the primary and booster doses. The frequency of solicited adverse events in the Ad26.COV2.S group were similar following the primary and booster vaccinations (local adverse events, 1676 [55·6%] of 3015 vs 896 [57·5%] of 1559, respectively; systemic adverse events, 1764 [58·5%] of 3015 vs 821 [52·7%] of 1559, respectively). Solicited adverse events were transient and mostly grade 1-2 in severity.A homologous Ad26.COV2.S booster administered 2 months after primary single-dose vaccination in adults had an acceptable safety profile and was efficacious against moderate to severe-critical COVID-19. Studies assessing efficacy against newer variants and with longer follow-up are needed.Janssen ResearchDevelopment.

Microbes, toxins, therapeutics and cells are often instilled into lungs of mice to model diseases and test experimental interventions. Consistent pulmonary delivery is critical for experimental power and reproducibility, but we observed variation in outcomes between handlers using different anesthetic approaches for intranasal dosing into mice. We therefore used a radiotracer to quantify lung delivery after intranasal dosing under inhalational (isoflurane) versus injectable (ketamine/xylazine) anesthesia in C57BL/6 mice. We found that ketamine/xylazine anesthesia resulted in delivery of a greater proportion (52±9%) of an intranasal dose to lungs relative to isoflurane anesthesia (30±15%). This difference in pulmonary dose delivery altered key outcomes in a model of viral pneumonia, with mice anesthetized with ketamine/xylazine for intranasal infection with influenza A virus developing worse lung pathology and more consistently losing body weight relative to control animals randomized to isoflurane anesthesia. Pulmonary dosing efficiency through oropharyngeal aspiration was not affected by anesthetic method and resulted in delivery of 63±8% of dose to lungs, and a non-surgical intratracheal dosing approach further increased lung delivery to 92±6% of dose. We conclude that anesthetic approach and dosing route can impact pulmonary dosing efficiency. These factors should be considered when planning and reporting studies involving delivery of fluids to lungs of mice.

The biology of human granzymes remains enigmatic in part due to our inability to probe their functions outside of in vitro assays or animal models with divergent granzyme species. We hypothesize that the biology of human granzymes could be better elaborated with a translational imaging technology to reveal the contexts in which granzymes are secreted and biochemically active in vivo. Here, we advance toward this goal by engineering a Granzyme targeting Restricted Interaction Peptide specific to family member B (GRIP B) to measure secreted granzyme B (GZMB) biochemistry with positron emission tomography. A proteolytic cleavage of 64Cu-labeled GRIP B liberates a radiolabeled form of Temporin L, which sequesters the radioisotope by binding to adjacent phospholipid bilayers. Thus, at extended time points postinjection (i.e., hours, not seconds), tissue biodistribution of the radioisotope in vivo reflects relative units of the GZMB activity. As a proof of concept, we show in three syngeneic mouse cancer models that 64Cu-GRIP B detects GZMB from T cells activated with immune checkpoint inhibitors (CPI). Remarkably, the radiotracer detects the proteolysis within tumors but also in lymphoid tissue, where immune cells are activated by a systemic CPI. Control experiments with an uncleavable analogue of 64Cu-GRIP B and tumor imaging studies in germline GZMB knockout mice were applied to show that 64Cu-GRIP B is specific for GZMB proteolysis. Furthermore, we explored a potential noncytotoxic function for GZMB by applying 64Cu-GRIP B to a model of pulmonary inflammation. In summary, we demonstrate that granzyme biochemistry can be assessed in vivo using an imaging modality that can be scaled vertically into human subjects., A protease-activated chemosensor for in vivo molecular imaging enables the detection of activated immune cells by specifically harnessing granzyme B catalysis.

Clinical and molecular heterogeneity are common features of human disease. Understanding the basis for heterogeneity has led to major advances in therapy for many cancers and pulmonary diseases such as cystic fibrosis and asthma. Although heterogeneity of risk factors, disease severity, and outcomes in survivors are common features of the acute respiratory distress syndrome (ARDS), many challenges exist in understanding the clinical and molecular basis for disease heterogeneity and using heterogeneity to tailor therapy for individual patients. This report summarizes the proceedings of the 2021 Aspen Lung Conference, which was organized to review key issues related to understanding clinical and molecular heterogeneity in ARDS. The goals were to review new information about ARDS phenotypes, to explore multicellular and multisystem mechanisms responsible for heterogeneity, and to review how best to account for clinical and molecular heterogeneity in clinical trial design and assessment of outcomes. The report concludes with recommendations for future research to understand the clinical and basic mechanisms underlying heterogeneity in ARDS to advance the development of new treatments for this life-threatening critical illness.

The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 infections has led to a substantial unmet need for treatments, many of which will require testing in appropriate animal models of this disease. Vaccine trials are already underway, but there remains an urgent need to find other therapeutic approaches to either target SARS-CoV-2 or the complications arising from viral infection, particularly the dysregulated immune response and systemic complications which have been associated with progression to severe COVID-19. At the time of writing, in vivo studies of SARS-CoV-2 infection have been described using macaques, cats, ferrets, hamsters, and transgenic mice expressing human angiotensin I converting enzyme 2 (ACE2). These infection models have already been useful for studies of transmission and immunity, but to date only partly model the mechanisms involved in human severe COVID-19. There is therefore an urgent need for development of animal models for improved evaluation of efficacy of drugs identified as having potential in the treatment of severe COVID-19. These models need to reproduce the key mechanisms of COVID-19 severe acute respiratory distress syndrome and the immunopathology and systemic sequelae associated with this disease. Here, we review the current models of SARS-CoV-2 infection and COVID-19-related disease mechanisms and suggest ways in which animal models can be adapted to increase their usefulness in research into COVID-19 pathogenesis and for assessing potential treatments. LINKED ARTICLES: This article is part of a themed issue on The Pharmacology of COVID-19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc.

Dendritic cells (DCs) are crucial for initiating adaptive immune responses. However, the factors that control DC positioning and homeostasis are incompletely understood. We found that type-2 conventional DCs (cDC2s) in the spleen depend on Gα 13 and adhesion G protein–coupled receptor family member-E5 (Adgre5, or CD97) for positioning in blood-exposed locations. CD97 function required its autoproteolytic cleavage. CD55 is a CD97 ligand, and cDC2 interaction with CD55-expressing red blood cells (RBCs) under shear stress conditions caused extraction of the regulatory CD97 N-terminal fragment. Deficiency in CD55-CD97 signaling led to loss of splenic cDC2s into the circulation and defective lymphocyte responses to blood-borne antigens. Thus, CD97 mechanosensing of RBCs establishes a migration and gene expression program that optimizes the antigen capture and presentation functions of splenic cDC2s.

Significance Precise gene editing allows engineering of immune receptors and ligands to reduce the immunogenicity of cells, and strategies for the generation of immune-evasive stem cell sources are currently being developed. This article describes the translational aspect of generating universally transplantable, disease-specific, therapeutic cell products. We provide proof of concept that immune-engineered cells can treat major cardiovascular and pulmonary diseases in fully allogeneic subjects without utilizing any immunosuppression. The translational aspect is emphasized by showing improvements in clinically relevant outcome measures, which are widely used in human trials. Depending on the feasibility of large-scale manufacturing of universal cell therapeutics, this approach could enable cost-effective cell therapy., The emerging field of regenerative cell therapy is still limited by the few cell types that can reliably be differentiated from pluripotent stem cells and by the immune hurdle of commercially scalable allogeneic cell therapeutics. Here, we show that gene-edited, immune-evasive cell grafts can survive and successfully treat diseases in immunocompetent, fully allogeneic recipients. Transplanted endothelial cells improved perfusion and increased the likelihood of limb preservation in mice with critical limb ischemia. Endothelial cell grafts transduced to express a transgene for alpha1-antitrypsin (A1AT) successfully restored physiologic A1AT serum levels in mice with genetic A1AT deficiency. This cell therapy prevented both structural and functional changes of emphysematous lung disease. A mixture of endothelial cells and cardiomyocytes was injected into infarcted mouse hearts, and both cell types orthotopically engrafted in the ischemic areas. Cell therapy led to an improvement in invasive hemodynamic heart failure parameters. Our study supports the development of hypoimmune, universal regenerative cell therapeutics for cost-effective treatments of major diseases.

Megakaryocytes are normal cellular components of the blood returning to the heart and entering the lungs, and historical data has pointed to a role of the lungs in platelet production. Recent studies using intravital microscopy have demonstrated that platelet release occurs in the lung from bone marrow megakaryocytes that embolize into the lung circulation.

Pulmonary ischemia-reperfusion injury (IRI) is a clinical syndrome of acute lung injury that occurs after lung transplantation or remote organ ischemia. IRI causes early mortality and has no effective therapies. While NK cells are innate lymphocytes capable of recognizing injured cells, their roles in acute lung injury are incompletely understood. Here, we demonstrated that NK cells were increased in frequency and cytotoxicity in 2 different IRI mouse models. We showed that NK cells trafficked to the lung tissue from peripheral reservoirs and were more mature within lung tissue. Acute lung ischemia-reperfusion injury was blunted in a NK cell-deficient mouse strain but restored with adoptive transfer of NK cells. Mechanistically, NK cell NKG2D receptor ligands were induced on lung endothelial and epithelial cells following IRI, and antibody-mediated NK cell depletion or NKG2D stress receptor blockade abrogated acute lung injury. In human lung tissue, NK cells were increased at sites of ischemia-reperfusion injury and activated NK cells were increased in prospectively collected human bronchoalveolar lavage in subjects with severe IRI. These data support a causal role for recipient peripheral NK cells in pulmonary IRI via NK cell NKG2D receptor ligation. Therapies targeting NK cells may hold promise in acute lung injury.

Rationale: Circulating monocytes can have proinflammatory or proreparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet-derived β2M (β-2 microglobulin) and TGF-β (transforming growth factor β) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes, respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. Objective: To determine the molecular mechanisms and signal transduction pathways by which β2M and TGF-β regulate monocyte responses both in vitro and in vivo. Methods and Results: Wild-type– (WT) and platelet-specific β2M knockout mice were treated intravenously with either β2M or TGF-β to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma β2M increased proinflammatory monocytes, while increased plasma TGFβ increased proreparative monocytes. TGF-βR (TGF-β receptor) inhibition blunted monocyte responses to both β2M and TGF-β in vivo. Using imaging flow cytometry, we found that β2M decreased monocyte SMAD2/3 nuclear localization, while TGF-β promoted SMAD nuclear translocation but decreased noncanonical/inflammatory (JNK [jun kinase] and NF-κB [nuclear factor-κB] nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. β2M, but not TGF-β, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked noncanonical SMAD-independent monocyte signaling and skewed monocytes towards a proreparative monocyte response. Conclusions: Our findings indicate that elevated plasma β2M and TGF-β dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor but induce SMAD-dependent canonical signaling (TGF-β) versus noncanonical SMAD-independent signaling (β2M) in a ubiquitin ligase dependent manner. This work has broad implications as β2M is increased in several inflammatory conditions, while TGF-β is increased in fibrotic diseases. Graphic Abstract: A graphic abstract is available for this article.

Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals1-3, many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs)3 across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.

Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow-resident (BM-resident) cells. However, platelet-producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared with BM Mks. We therefore sought to define the immune functions of lung Mks. Using single-cell RNA sequencing of BM and lung myeloid-enriched cells, we found that lung Mks, which we term MkL, had gene expression patterns that are similar to antigen-presenting cells. This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment, as evidenced by BM Mks having an MkL-like phenotype under the influence of pathogen receptor challenge and lung-associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4+ T cell activation in an MHC II-dependent manner both in vitro and in vivo. These data indicated that MkL had key immune regulatory roles dictated in part by the tissue environment.

Rapid neutrophil recruitment to sites of inflammation is crucial for innate immune responses. Here, we reveal that the G-protein-coupled receptor GPR35 is upregulated in activated neutrophils, and it promotes their migration. GPR35-deficient neutrophils are less recruited from blood vessels into inflamed tissue, and the mice are less efficient in clearing peritoneal bacteria. Using a bioassay, we find that serum and activated platelet supernatant stimulate GPR35, and we identify the platelet-derived serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) as a GPR35 ligand. GPR35 function in neutrophil recruitment is strongly dependent on platelets, with the receptor promoting transmigration across platelet-coated endothelium. Mast cells also attract GPR35+ cells via 5-HIAA. Mice deficient in 5-HIAA show a loss of GPR35-mediated neutrophil recruitment to inflamed tissue. These findings identify 5-HIAA as a GPR35 ligand and neutrophil chemoattractant and establish a role for platelet- and mast cell-produced 5-HIAA in cell recruitment to the sites of inflammation and bacterial clearance.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense., One Sentence Summary: In severe COVID-19 patients, the immune system fails to generate cells that define mild disease; antibodies in their serum actively prevents the successful production of those cells.

Formaldehyde (FA), an economically important and ubiquitous chemical, has been classified as a human carcinogen and myeloid leukemogen. However, the underlying mechanisms of leukemogenesis remain unclear. Unlike many classical leukemogens that damage hematopoietic stem/progenitor cells (HSC/HPC) directly in the bone marrow, FA-as the smallest, most reactive aldehyde-is thought to be incapable of reaching the bone marrow through inhalation exposure. A recent breakthrough study discovered that mouse lung contains functional HSC/HPC that can produce blood cells and travel bi-directionally between the lung and bone marrow, while another early study reported the presence of HSC/HPC in rat nose. Based on these findings, we hypothesized that FA inhalation could induce toxicity in HSC/HPC present in mouse lung and/or nose rather than in the bone marrow. To test this hypothesis, we adapted a commercially available protocol for culturing burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte, macrophage (CFU-GM) colonies from bone marrow and spleen to also enable culture of these colonies from mouse lung and nose, a novel application of this assay. We reported that in vivo exposure to FA at 3mg/m3 or ex vivo exposure up to 400µM FA decreased the formation of both colony types from mouse lung and nose as well as from bone marrow and spleen. These findings, to the best of our knowledge, are the first empiricallyto show that FA exposure can damage mouse pulmonary and olfactory HSC/HPC and provide potential biological plausibility for the induction of leukemia at the sites of entry rather than the bone marrow.

BACKGROUND Consensus definitions for transfusion-related acute lung injury (TRALI) and transfusion-associated circulatory overload (TACO) have recently been revised; however, pulmonary transfusion reactions remain difficult to diagnose. We hypothesized that N-terminal pro-brain natriuretic peptide (NT-proBNP) levels could have utility in the identification and classification of pulmonary transfusion reactions. STUDY DESIGN AND METHODS We performed a secondary analysis of a case-control study of pulmonary transfusion reactions at four academic hospitals. We evaluated clinical data and measured NT-proBNP levels prior to and following transfusion in patients with TACO (n = 160), transfused acute respiratory distress syndrome (ARDS) [n = 51], TRALI [n = 12], TACO/TRALI [n = 7], and controls [n = 335]. We used Wilcoxon Rank-Sum tests to compare NT-proBNP levels, and classification and regression tree (CART) algorithms to produce a ranking of covariates in order of relative importance for differentiating TACO from transfused controls. RESULTS Pre-transfusion NT-proBNP levels were elevated in cases of transfused ARDS and TACO (both P

The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 infections has led to substantial unmet need for treatments, many of which will require testing in appropriate animal models of this disease. Vaccine trials are already underway, but there remains an urgent need to find other therapeutic approaches to either target SARS-CoV-2 or the complications arising from viral infection, particularly the dysregulated immune response and systemic complications which have been associated with progression to severe COVID-19. At the time of writing, in vivo studies of SARS-CoV-2 infection have been described using macaques, cats, ferrets, hamsters, and transgenic mice expressing human angiotensin I converting enzyme 2 (ACE2). These infection models have already been useful for studies of transmission and immunity, but to date only partially model the mechanisms implicated in human severe COVID-19. There is therefore an urgent need for development of animal models for improved evaluation of efficacy of drugs identified as having potential in the treatment of severe COVID-19. These models need to recapitulate key mechanisms of COVID-19 severe acute respiratory distress syndrome and reproduce the immunopathology and systemic sequelae associated with this disease. Here, we review the current models of SARS-CoV-2 infection and COVID-19-related disease mechanisms and suggest ways in which animal models can be adapted to increase their usefulness in research into COVID-19 pathogenesis and for assessing potential treatments.

In this Perspective, autopsy results and literature are presented supporting the hypothesis that neutrophil extracellular traps (NETs) may contribute to organ damage and mortality in COVID-19. If correct, existing drugs that target NETs, although unspecific, may benefit COVID-19 patients., Coronavirus disease 2019 (COVID-19) is a novel, viral-induced respiratory disease that in ∼10–15% of patients progresses to acute respiratory distress syndrome (ARDS) triggered by a cytokine storm. In this Perspective, autopsy results and literature are presented supporting the hypothesis that a little known yet powerful function of neutrophils—the ability to form neutrophil extracellular traps (NETs)—may contribute to organ damage and mortality in COVID-19. We show lung infiltration of neutrophils in an autopsy specimen from a patient who succumbed to COVID-19. We discuss prior reports linking aberrant NET formation to pulmonary diseases, thrombosis, mucous secretions in the airways, and cytokine production. If our hypothesis is correct, targeting NETs directly and/or indirectly with existing drugs may reduce the clinical severity of COVID-19.

Cystic fibrosis (CF) lung disease is characterized by an inflammatory response that can lead to terminal respiratory failure. The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in CF, and we hypothesized that dysfunctional CFTR in platelets, which are key participants in immune responses, is a central determinant of CF inflammation. We found that deletion of CFTR in platelets produced exaggerated acute lung inflammation and platelet activation after intratracheal LPS or Pseudomonas aeruginosa challenge. CFTR loss of function in mouse or human platelets resulted in agonist-induced hyperactivation and increased calcium entry into platelets. Inhibition of the transient receptor potential cation channel 6 (TRPC6) reduced platelet activation and calcium flux, and reduced lung injury in CF mice after intratracheal LPS or Pseudomonas aeruginosa challenge. CF subjects receiving CFTR modulator therapy showed partial restoration of CFTR function in platelets, which may be a convenient approach to monitoring biological responses to CFTR modulators. We conclude that CFTR dysfunction in platelets produces aberrant TRPC6-dependent platelet activation, which is a major driver of CF lung inflammation and impaired bacterial clearance. Platelets and TRPC6 are what we believe to be novel therapeutic targets in the treatment of CF lung disease.

Antibodies targeting human leukocyte antigen (HLA)/major histocompatibility complex (MHC) proteins limit successful transplantation and transfusion, and their presence in blood products can cause lethal transfusion-related acute lung injury (TRALI). It is unclear which cell types are bound by these anti-leukocyte antibodies to initiate an immunologic cascade resulting in lung injury. We therefore conditionally removed MHC class I (MHC I) from likely cellular targets in antibody-mediated lung injury. Only the removal of endothelial MHC I reduced lung injury and mortality, related mechanistically to absent endothelial complement fixation and lung platelet retention. Restoration of endothelial MHC I rendered MHC I-deficient mice susceptible to lung injury. Neutrophil responses, including neutrophil extracellular trap (NET) release, were intact in endothelial MHC I-deficient mice, whereas complement depletion reduced both lung injury and NETs. Human pulmonary endothelial cells showed high HLA class I expression, and posttransfusion complement activation was increased in clinical TRALI. These results indicate that the critical source of antigen for anti-leukocyte antibodies is in fact the endothelium, which reframes our understanding of TRALI as a rapid-onset vasculitis. Inhibition of complement activation may have multiple beneficial effects of reducing endothelial injury, platelet retention, and NET release in conditions where antibodies trigger these pathogenic responses.

The immune system is designed to robustly respond to pathogenic stimuli but to be tolerant to endogenous ligands to not trigger autoimmunity. Here, we studied an endogenous damage-associated molecular pattern, mitochondrial DNA (mtDNA), during primary graft dysfunction (PGD) after lung transplantation. We hypothesized that cell-free mtDNA released during lung ischemia-reperfusion triggers neutrophil extracellular trap (NET) formation via TLR9 signaling. We found that mtDNA increases in the BAL fluid of experimental PGD (prolonged cold ischemia followed by orthotopic lung transplantation) and not in control transplants with minimal warm ischemia. The adoptive transfer of mtDNA into the minimal warm ischemia graft immediately before lung anastomosis induces NET formation and lung injury. TLR9 deficiency in neutrophils prevents mtDNA-induced NETs, and TLR9 deficiency in either the lung donor or recipient decreases NET formation and lung injury in the PGD model. Compared with human lung transplant recipients without PGD, severe PGD was associated with high levels of BAL mtDNA and NETs, with evidence of relative deficiency in DNaseI. We conclude that mtDNA released during lung ischemia-reperfusion triggers TLR9-dependent NET formation and drives lung injury. In PGD, DNaseI therapy has a potential dual benefit of neutralizing a major NET trigger (mtDNA) in addition to dismantling pathogenic NETs.

Transfusion clinique et biologique - In Press.Proof corrected by the author Available online since mardi 18 juin 2019

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BACKGROUND It is increasingly recognized that recipient risk factors play a prominent role in possible transfusion-related acute lung injury (pTRALI) and transfusion-associated circulatory overload (TACO). We hypothesized that both transfusion and recipient factors including natriuretic peptides could be used to distinguish TRALI from TACO and pTRALI. STUDY DESIGN AND METHODS We performed a post hoc analysis of a case-control study of pulmonary transfusion reactions conducted at the University of California at San Francisco and Mayo Clinic, Rochester. We evaluated clinical data and brain natriuretic peptides (BNP) levels drawn after transfusion in patients with TRALI (n = 21), pTRALI (n = 26), TACO (n = 22), and controls (n = 24). Logistic regression and receiver operating characteristics curve analyses were used to determine the accuracy of clinical and biomarker predictors in differentiating TRALI from TACO and pTRALI. RESULTS We found that pTRALI and TACO were associated with older age, higher fluid balance, and elevated BNP levels relative to those of controls and TRALI. The following variables were useful in distinguishing cases of pTRALI and TACO from TRALI: age more than 70 years, BNP levels more than 1000 pg/mL, 24-hour fluid balance of more than 3 L, and a lower number of transfused blood components. Using the above variables, our logistic model had a 91% negative predictive value in the differential diagnosis of TRALI. CONCLUSIONS Models incorporating readily available clinical and biomarker data can be used to differentiate transfusion-related respiratory complications. Additional studies examining recipient risk factors and the likelihood of TRALI may be useful in decision making regarding donor white blood cell antibody testing.

Background: The acute respiratory distress syndrome (ARDS) is a life-threating disorder that contributes significantly to critical illness. No specific pharmacological interventions directed at lung injury itself have proven effective in improving outcome of patients with ARDS. Platelet activation was identified as a key component in ARDS pathophysiology and may provide an opportunity for preventive and therapeutic strategies. We hypothesize that use of acetyl salicylic acid (ASA) may prevent and/or attenuate lung injury. Methods: We conducted a systematic review of preclinical studies and meta-analysis of clinical studies investigating the efficacy of ASA in the setting of lung injury. Medline, embase, and cochrane databases were searched. Results: The literature search yielded 1,314 unique articles. Fifteen preclinical studies and eight clinical studies fulfilled the in-and exclusion criteria. In the animal studies, the overall effect of ASA was positive, e.g., ASA improved survival and attenuated inflammation and pulmonary edema. Mechanisms of actions involved, among others, are interference with the neutrophil-platelets interaction, reduction of leukotrienes, neutrophil extracellular traps, and prostaglandins. High-dose ASA may be the drug of choice. A meta-analysis of three clinical studies showed an association between ASA use and a reduced incidence of ARDS (OR 0.59, 95% CI 0.36-0.98), albeit with substantial between-study heterogeneity. All studies had their own shortcomings in methodological quality. Conclusion: This systematic review of preclinical studies and meta-analysis of clinical studies suggests a beneficial role for ASA in ARDS prevention and treatment. However, the currently available data is insufficient to justify an indication for ASA in ARDS. The body of literature does support further studies in humans. We suggest clinical trials in which the mechanisms of action of ASA in lung injury models are being evaluated to guide optimal timing and dose, before prospective randomized trials.

A decade ago, definitions of “transfusionߚrelated acute lung injury (TRALI)” and “possible TRALI” were standardized for research and clinical diagnosis. Since then, evidence has confirmed that TRALI is often due to transfusion of white blood cell antibodies to at-risk patients, and the term “TRALI, antibody mediated” is appropriate for such cases. Other TRALI cases are non–antibody mediated. Because specific, nonantibody transfusion factors have not yet been confirmed to cause TRALI in humans, the general term “TRALI, non–antibody mediated” is appropriate for such cases. In contrast, evidence is against possible TRALI being due to transfusion with the more likely cause of the acute respiratory distress syndrome (ARDS) being the alternative ARDS risk factor present in these patients. We propose to drop the misleading term “possible TRALI” and to rename this category of cases as “transfused ARDS.” These nomenclature updates will more accurately categorize ARDS cases that develop after transfusion.

Platelets are recruited to inflammatory foci and contribute to host defence and inflammatory responses. Compared to platelet recruitment in hemostasis and thrombosis, the mechanisms of platelet recruitment in inflammation and host defence are poorly understood. Neutrophil recruitment to lung airspaces following inhalation of bacterial LPS requires platelets and PSGL-1 in mice. Given this association between platelets and neutrophils, we investigated whether recruitment of platelets to lungs of mice following LPS inhalation was dependent on PSGL-1, P-selectin, or interaction with neutrophils. BALB/c mice were administered intranasal LPS (O55:B5, 5 mg/kg) and 48 hours later lungs were collected, and platelets and neutrophils were quantified in tissue sections by immunohistochemistry. The effects of functional blocking antibody treatments targeting the platelet-neutrophil adhesion molecules P-selectin, or PSGL-1, or treatment with a neutrophil depleting antibody targeting Ly6G, were tested on the extent of LPS-induced lung platelet recruitment. Separately in Pf4-Cre×mTmG mice, two-photon intravital microscopy was used to image platelet adhesion in live lungs. Inhalation of LPS caused both platelet and neutrophil recruitment to the lung vasculature. However, decreasing lung neutrophil recruitment by blocking PSGL-1, P-selectin, or depleting blood neutrophils had no effect on lung platelet recruitment. Lung intravital imaging revealed increased adhesion of platelets in the lung microvasculature which was not associated with thrombus formation. In conclusion, platelet recruitment to lungs in response to LPS occurs through mechanisms distinct from those mediating neutrophil recruitment, or the occurrence of pulmonary emboli.

Author(s): Vlaar, Alexander PJ; Toy, Pearl; Fung, Mark; Looney, Mark R; Juffermans, Nicole P; Bux, Juergen; Bolton-Maggs, Paula; Peters, Anna L; Silliman, Christopher C; Kor, Daryl J; Kleinman, Steve | Abstract: BackgroundTransfusion-related acute lung injury (TRALI) is a serious complication of blood transfusion and is among the leading causes of transfusion-related morbidity and mortality in most developed countries. In the past decade, the pathophysiology of this potentially life-threatening syndrome has been increasingly elucidated, large cohort studies have identified associated patient conditions and transfusion risk factors, and preventive strategies have been successfully implemented. These new insights provide a rationale for updating the 2004 consensus definition of TRALI.Study design and methodsAn international expert panel used the Delphi methodology to develop a redefinition of TRALI by modifying and updating the 2004 definition. Additionally, the panel reviewed issues related to TRALI nomenclature, patient conditions associated with acute respiratory distress syndrome (ARDS) and TRALI, TRALI pathophysiology, and standardization of reporting of TRALI cases.ResultsIn the redefinition, the term "possible TRALI" has been dropped. The terminology of TRALI Type I (without an ARDS risk factor) and TRALI Type II (with an ARDS risk factor or with mild existing ARDS) is proposed. Cases with an ARDS risk factor that meet ARDS diagnostic criteria and where respiratory deterioration over the 12 hours before transfusion implicates the risk factor as causative should be classified as ARDS. TRALI remains a clinical diagnosis and does not require detection of cognate white blood cell antibodies.ConclusionsClinicians should report all cases of posttransfusion pulmonary edema to the transfusion service so that further investigation can allow for classification of such cases as TRALI (Type I or Type II), ARDS, transfusion-associated circulatory overload (TACO), or TRALI or TACO cannot distinguish or an alternate diagnosis.

Rationale: Extracellular DNA (eDNA) and neutrophil extracellular traps (NETs) are implicated in multiple inflammatory diseases. NETs mediate inflammasome activation and IL-1β secretion from monocytes and cause airway epithelial cell injury, but the role of eDNA, NETs, and IL-1β in asthma is uncertain. Objectives: To characterize the role of activated neutrophils in severe asthma through measurement of NETs and inflammasome activation. Methods: We measured sputum eDNA in induced sputum from 399 patients with asthma in the Severe Asthma Research Program-3 and in 94 healthy control subjects. We subdivided subjects with asthma into eDNA-low and -high subgroups to compare outcomes of asthma severity and of neutrophil and inflammasome activation. We also examined if NETs cause airway epithelial cell damage that can be prevented by DNase. Measurements and Main Results: We found that 13% of the Severe Asthma Research Program-3 cohort is "eDNA-high," as defined by sputum eDNA concentrations above the upper 95th percentile value in health. Compared with eDNA-low patients with asthma, eDNA-high patients had lower Asthma Control Test scores, frequent history of chronic mucus hypersecretion, and frequent use of oral corticosteroids for maintenance of asthma control (all P values

Pathogens express a set of proteins required for establishing and maintaining an infection, termed virulence life-style genes (VLGs). Due to their outsized importance in pathogenesis, VLG products are attractive targets for the next generation of antimicrobials. However, precise manipulation of VLG expression in the context of infection is technically challenging, limiting our ability to understand the roles of VLGs in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of gene knockdown tools that are transferred by conjugation and stably integrate into pathogen genomes that we call “Mobile-CRISPRi”. Here we show the efficacy of Mobile-CRISPRi in controlling VLG expression in a murine infection model. We optimize Mobile-CRISPRi inPseudomonas aeruginosafor use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid non-specific toxicity. As a proof of principle, we demonstrate that knockdown of a VLG encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of VLGs across many bacterial species and pathogenesis models.ImportanceAntibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically these genetic drivers have been difficult to manipulate, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study virulence life-style genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

Purpose Primary graft dysfunction (PGD) occurs in 1/3 of all lung transplants. It is defined by epithelial dysfunction and innate immune cell infiltration. Natural killer (NK) cells are innate lymphocytes that are activated by NKG2D receptor binding of specific stress molecules. We hypothesized that NK cells mediate PGD by recognizing stress molecules induced on lung epithelial cells and causing injury via direct cytotoxicity. Methods In an established experimental PGD model, left hilar clamp (HC) was compared to sham (S) surgery in C57BL/6 mice. Stress molecules (RAE-1, MULT1) and NK cell receptors were measured by flow cytometry median fluorescent intensity (MFI) on dissociated lung cells. Mice were given blocking anti-NKG2D or isotype control antibodies preceding HC. Human stress molecules (MICA, MICB, ULBP1, 3, and 2/5/6) were measured by flow cytometry on bronchial epithelial cells incubated for 4 hours in hypoxia (1% O2) versus normoxia conditions. The Mann-Whitney U test was applied for pairwise comparisons. Results NK cells were increased as a percent of lymphocytes (p = 0.008) and by count (p = 0.04) in HC (n = 5) versus S (n = 5) lungs and infiltrated airways (Figure 1A). The NKG2D receptor was increased on NK cells following HC (p = 0.005). The stress molecules MULT1 (p = 0.0002) and RAE-1 (Figure 1B, p = 0.002) were increased on epithelial cells in HC compared to S lungs. NKG2D blockade (n = 9) resulted in less pulmonary edema following HC compared to isotype control antibody (Figure 1C, n = 9, p = 0.04). Hypoxic human bronchial epithelial cells (n = 6) had increased MICB (p = 0.008), ULBP-1 (p = 0.05), and ULBP-2,5,6 (Figure 1 D, p = 0.004) compared to normoxic cells (n = 6). Conclusion PGD induces NK cell stress ligands leading to NK cell recruitment that contributes to PGD. NKG2D blockade, under investigation in clinical trials of other diseases, resulted in decreased lung injury. The induction of human NKG2D stress molecules during hypoxia suggests that this NK cell receptor-ligand interaction may mediate PGD.

Transfusion-related acute lung injury (TRALI) is the leading cause of mortality from blood product transfusions. Clinical and experimental studies support a multievent model of injury in which a variety of risk factors (‘first hit’) render the transfusion recipient susceptible to lung injury when transfused with a blood product containing antibody and non-antibody mediators that provide the ‘second hit’ in the model. Our understanding of the mechanisms of antibody-based TRALI has been advanced by the major histocompatibility complex (MHC) Class I monoclonal antibody model of TRALI in mice. Challenge of lipopolysaccharide (LPS)-primed BALB/c mice with cognate MHC Class I antibody produces severe protein-rich pulmonary oedema, and the lung injury response is critically dependent on the involvement of neutrophils and platelets. In the lung microcirculation, neutrophils and platelets spatially interact forming heterotypic aggregates that can be blocked with aspirin, which also reduces lung injury and mortality in a lipoxin-dependent pathway. Activated platelets also trigger the formation of neutrophil extracellular traps (NETs), which can be found in the lungs and blood of experimental and clinical cases of TRALI. Several lines of evidence point to the pathogenicity of NETs in TRALI. Inhibiting the formation of NETs by targeting platelet activation (aspirin) ameliorates lung injury. Dismantling the NET structure using deoxyribonuclease 1 (DNase1) or inhibiting extracellular histones, a key component of NETs, also robustly protects from lung injury. In summary, experimental modelling of TRALI has identified a novel pathophysiologic cascade involving neutrophils, platelets and NETs and has in turn illuminated rational therapeutic approaches for the prevention or treatment of TRALI.

The lung represents a unique immune environment. The primary function of the lung is to enable gas exchange by facilitating the transfer of oxygen into and carbon dioxide out of the blood. However, as a direct byproduct of this process the lung is also constantly exposed to particles, allergens, and pathogens alongside air itself. Due to this, the pulmonary immune system exists in a fine balance between quiescence and inflammation, deviations from which can lead to a failure in respiratory function. A rich history exists attempting to define the critical features of lung immunity, and most recently advances in intravital microscopy have enabled the visualization of intercellular immune dynamics in both steady-state and a variety of disease conditions. In this review, we will summarize a variety of approaches to intravital lung imaging as well as how its application has advanced our understanding of normal lung function as well as disease states such as pulmonary metastasis, asthma, and lung injury.

OBJECTIVES:Transfusion-associated circulatory overload is characterized by hydrostatic pulmonary edema following blood transfusion. Restrictive transfusion practice may affect the occurrence and severity of transfusion-associated circulatory overload in critically ill patients. We sought to examine contemporary risk factors and outcomes for transfusion-associated circulatory overload. DESIGN:Case-control study. SETTING:Four tertiary care hospitals. PATIENTS:We prospectively enrolled 200 patients with transfusion-associated circulatory overload identified by active surveillance and 405 controls matched by transfusion intensity. INTERVENTIONS:None. MEASUREMENTS AND MAIN RESULTS:Among 20,845 transfused patients who received 128,263 blood components from May 2015 until July 2016, transfusion-associated circulatory overload incidence was one case per 100 transfused patients. In addition to cardiovascular comorbidities, multivariable analysis identified the following independent predictors of transfusion-associated circulatory overload: acute kidney injury, emergency surgery, pretransfusion diuretic use, and plasma transfusion-the latter especially in females. Compared with matched controls, transfusion-associated circulatory overload cases were more likely to require mechanical ventilation (71% vs 49%; p < 0.001), experienced longer intensive care and hospital lengths of stay following transfusion, and had higher mortality (21% vs 11%; p = 0.02) even after adjustment for other potentially confounding variables. CONCLUSIONS:Despite restrictive transfusion practice, transfusion-associated circulatory overload remains a frequent complication of transfusion and is an independent risk factor for in-hospital morbidity and mortality. In addition to cardiovascular and renal risk factors, plasma transfusion was associated with transfusion-associated circulatory overload after controlling for other covariates. Additional research is needed to examine the benefit of reduced erythrocyte or plasma exposure in patients at high risk for transfusion-associated circulatory overload.

Neutrophils dominate the early immune response in pathogen-induced acute lung injury, but efforts to harness their responses have not led to therapeutic advancements. Neutrophil extracellular traps (NETs) have been proposed as an innate defense mechanism responsible for pathogen clearance, but there are concerns that NETs may induce collateral damage to host tissues. Here, we detected NETs in abundance in mouse models of severe bacterial pneumonia/acute lung injury and in human subjects with acute respiratory distress syndrome (ARDS) from pneumonia or sepsis. Decreasing NETs reduced lung injury and improved survival after DNase I treatment or with partial protein arginine deiminase 4 deficiency (PAD4+/–). Complete PAD4 deficiency (PAD4–/–) reduced NETs and lung injury but was counterbalanced by increased bacterial load and inflammation. Importantly, we discovered that the lipoxin pathway could be a potent modulator of NET formation, and that mice deficient in the lipoxin receptor (Fpr2–/–) produced excess NETs leading to increased lung injury and mortality. Lastly, we observed in humans that increased plasma NETs were associated with ARDS severity and mortality, and lower plasma DNase I levels were associated with the development of sepsis-induced ARDS. We conclude that a critical balance of NETs is necessary to prevent lung injury and to maintain microbial control, which has important therapeutic implications.

Author(s): Roubinian, Nareg H; Looney, Mark R; Kor, Daryl J; Lowell, Clifford A; Gajic, Ognjen; Hubmayr, Rolf D; Gropper, Michael A; Koenigsberg, Monique; Wilson, Gregory A; Matthay, Michael A; Toy, Pearl; Murphy, Edward L; TRALI Study Group | Abstract: BACKGROUND:Pulmonary transfusion reactions are important complications of blood transfusion, yet differentiating these clinical syndromes is diagnostically challenging. We hypothesized that biologic markers of inflammation could be used in conjunction with clinical predictors to distinguish transfusion-related acute lung injury (TRALI), transfusion-associated circulatory overload (TACO), and possible TRALI. STUDY DESIGN AND METHODS:In a nested case-control study performed at the University of California at San Francisco and Mayo Clinic, Rochester, we evaluated clinical data and blood samples drawn before and after transfusion in patients with TRALI (n = 70), possible TRALI (n = 48), TACO (n = 29), and controls (n = 147). Cytokines measured included granulocyte-macrophage-colony-stimulating factor, interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor-α. Logistic regression and receiver operating characteristics curve analyses were used to determine the accuracy of clinical predictors and laboratory markers in differentiating TACO, TRALI, and possible TRALI. RESULTS:Before and after transfusion, IL-6 and IL-8 were elevated in patients with TRALI and possible TRALI relative to controls, and IL-10 was elevated in patients with TACO and possible TRALI relative to that of TRALI and controls. For all pulmonary transfusion reactions, the combination of clinical variables and cytokine measurements displayed optimal diagnostic performance, and the model comparing TACO and TRALI correctly classified 92% of cases relative to expert panel diagnoses. CONCLUSIONS:Before transfusion, there is evidence of systemic inflammation in patients who develop TRALI and possible TRALI but not TACO. A predictive model incorporating readily available clinical and cytokine data effectively differentiated transfusion-related respiratory complications such as TRALI and TACO.