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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.

Blood platelets are best known for their roles in hemostasis and thrombosis, but platelets also make important contributions to inflammation, immunity, and inflammatory resolution. Experiments involving depletion, genetic modification, and live imaging of platelets in animal models have increased our mechanistic understanding of platelet contributions to inflammation. In this minireview, we provide a critical overview of experimental techniques for manipulating and imaging platelets in inflammation models. We then highlight studies using innovative approaches to elucidate molecular mechanisms through which platelet subsets, platelet Fc gamma receptors, and pro-resolution platelet functions influence inflammatory responses. We also propose future technologies and research directions which might move us closer to harnessing of platelet functions for improved therapeutic modulation of inflammatory diseases.

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.

Progressive fibrosing interstitial lung diseases (PFILDs) cause substantial morbidity and mortality. Antifibrotic agents slow progression, but most of the clinical need remains unmet. The archetypal PFILD is idiopathic pulmonary fibrosis (IPF). Chronic progression is driven by transforming growth factor (TGF-)β1 signalling. It is punctuated by inflammatory flares known as acute exacerbations (AE-IPF), which are associated with accelerated decline and high mortality. We hypothesized that acute injury responses underlying exacerbations and the mechanisms of chronic fibrosis overlap at the molecular level, via a cell surface assembly nucleated by galectin-3 that we term the ‘gal-3-fibrosome’. We focused upon a putative pro-inflammatory galectin-3 ligand, the CD98:integrin complex. Our data indicate CD98 and β1-integrin co-localise with galectin-3 within epithelial cells in IPF lung tissue, and within 40 nm in human lung tissue treated with TGF-β1 compared to controls. CD98 is required for interleukin (IL-)6 and IL-8 responses to biochemical and biophysical conditions mimicking stimuli of AE-IPF in vivo, ex vivo and in cells, and for an interstitial neutrophilic response in a mouse model. We demonstrate this pathway progresses via intracellular influx of Ca2+ mediated by TRPV4, and NF-κB activation, operating in positive feedback. Lastly we show the CD98- and galectin-3-dependence of IL-6 and IL-8 responses to the SARS-CoV-2 spike protein receptor binding domain and the conservation of this response pattern between lung epithelial cells and monocyte-derived macrophages. Taken together our findings identify CD98 as a key mediator of both pro-fibrotic and acute inflammatory responses in the lung with relevance to AE- and chronic progression of IPF, and the priming of fibrotic lungs for acute inflammatory responses. They similarly implicate CD98 and galectin-3 as mediators of COVID pneumonitis and worse outcomes in ILD patients with COVID.

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.

Streptococcal pneumonia is a worldwide health problem that kills ∼2 million people each year, particularly young children, the elderly, and immunosuppressed individuals. Alveolar macrophages and neutrophils provide the early innate immune response to clear pneumococcus from infected lungs. However, the level of neutrophil involvement is context dependent, both in humans and in mouse models of the disease, influenced by factors such as bacterial load, age, and coinfections. Here, we show that the G protein–coupled receptor (GPCR) adaptor protein norbin (neurochondrin, NCDN), which was hitherto known as a regulator of neuronal function, is a suppressor of neutrophil-mediated innate immunity. Myeloid norbin deficiency improved the immunity of mice to pneumococcal infection by increasing the involvement of neutrophils in clearing the bacteria, without affecting neutrophil recruitment or causing autoinflammation. It also improved immunity during Escherichia coli–induced septic peritonitis. It increased the responsiveness of neutrophils to a range of stimuli, promoting their ability to kill bacteria in a reactive oxygen species–dependent manner, enhancing degranulation, phagocytosis, and the production of reactive oxygen species and neutrophil extracellular traps, raising the cell surface levels of selected GPCRs, and increasing GPCR-dependent Rac and Erk signaling. The Rac guanine-nucleotide exchange factor Prex1, a known effector of norbin, was dispensable for most of these effects, which suggested that norbin controls additional downstream targets. We identified the Rac guanine-nucleotide exchange factor Vav as one of these effectors. In summary, our study presents the GPCR adaptor protein norbin as an immune suppressor that limits the ability of neutrophils to clear bacterial infections.

Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b 1 platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti–mouse CD49b-PE (phycoerythrin) antibody. Platelet–endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b 1 platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism.

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.

Haemolysis that occurs in intravascular haemolytic disorders, such as sickle cell disease and malaria, is associated with inflammation and platelet activation. Alveolar haemorrhage, for example following primary blast lung injury (PBLI) or acute respiratory distress syndrome (ARDS), results in the escape of erythrocytes (RBCs) into alveolar spaces, where they subsequently lyse and release their intracellular contents. However, the inflammatory effects of RBCs in the airways are not fully understood. We hypothesized that RBCs in the airway induce an inflammatory response, associated with platelet activation. By instilling whole RBCs or lysed RBCs into the airways of mice, we have demonstrated that whole RBCs elicit macrophage accumulation in the lung. However, lysed RBCs induce significant inflammatory cell recruitment, particularly neutrophils and this was associated with a 50% increase in circulating platelet neutrophil complexes (PNCs). Platelet depletion prior to lysed RBC exposure in the lung resulted in reduced neutrophil recruitment, suggesting that the presence of intracellular RBC components in the airways can elicit inflammation that is platelet dependent. To identify specific platelet dependent signalling pathways involved in neutrophil recruitment, anti-P-selectin ligand and anti-PSGL1 blocking antibodies were tested, however neither affected neutrophil recruitment. These findings implicate an involvement for other, as yet unidentified platelet-dependent signalling and adhesion mechanisms. Further understanding of how platelets contribute to lung inflammation induced by the presence of RBCs could offer novel therapeutic approaches to attenuate inflammation that occurs in conditions associated with alveolar haemorrhage.

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.

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.

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.

Platelet activation occurs in patients with allergic inflammation, and platelets can be activated directly by allergen via an IgE-dependent process. Platelets have been shown to activate APCs such as CD11c+ dendritic cells in vitro. Although CD11c+ dendritic cells are a requisite for allergen sensitization, the role of platelets in this process is unknown. In this study, we investigated whether platelets were necessary for allergen sensitization. Balb/c mice sensitized to ovalbumin were exposed to subsequent aerosolized allergen (ovalbumin challenge). We analyzed lung CD11c+ cell activation, colocalization with platelets, and some other indices of inflammation. The role of platelets at the time of allergen sensitization was assessed through platelet depletion experiments restricted to the period of sensitization. Platelets colocalized with airway CD11c+ cells, and this association increased after allergen sensitization as well as after subsequent allergen exposure. Temporary platelet depletion (>95%) at the time of allergen sensitization led to a suppression of IgE and IL-4 synthesis and to a decrease in the pulmonary recruitment of eosinophils, macrophages, and lymphocytes after subsequent allergen exposure. Furthermore, in mice previously depleted of platelets at the time of sensitization, the recovered platelet population was shown to have reduced expression of FcεRI. Pulmonary CD11c+ cell recruitment was suppressed in these mice after allergen challenge, suggesting that the migration of CD11c+ cells in vivo may be dependent on direct platelet recognition of allergen. We conclude that platelets are necessary for efficient host sensitization to allergen. This propagates the subsequent inflammatory response during secondary allergen exposure and increases platelet association with airway CD11c+ cells.

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.

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.

The activation of platelets during host defence and inflammatory disorders has become increasingly documented. Clinical studies of patients with asthma reveal heightened platelet activation and accumulation into lung tissue. Accompanying studies in animal models of allergic lung inflammation, using protocols of experimentally induced thrombocytopenia proclaim an important role for platelets during the leukocyte recruitment cascade, tissue integrity, and lung function. The functions of platelets during these inflammatory events are clearly distinct to platelet functions during haemostasis and clot formation, and have led to the concept that a dichotomy (or polytomy, depending on what else platelets do) in platelet activation exists. The platelet, therefore, presents us with novel opportunities for modulating these inflammatory responses. This review discusses the rationale and effectiveness of current anti-platelet drugs in their use to supress inflammation with regard to asthma, and the need to consider novel possibilities for pharmacological modulation of platelet function associated with inflammation that are pharmacologically distinct to current anti-platelet therapies.

Summary Introduction Platelets are under investigation for their role in host defence and inflammatory lung diseases and have been demonstrated to be recruited to the lung. However, the mechanisms and consequences of platelet recruitment into lungs are poorly understood. We have utilised a murine model to investigate the mechanisms of platelet involvement in lung inflammation induced by intranasal administration of LPS. Objectives Our aim was to characterise lung platelet recruitment following LPS inhalation in mice using immunohistochemistry, and non-invasive and invasive radiolabelled platelet tracking techniques. Results Intranasal administration of LPS caused an increase in lung platelet staining in lung tissue and elicited the recruitment of radiolabelled platelets into the lung. Prior to these responses in the lung, we observed an earlier decrease in blood platelet counts, temporally associated with platelet recruitment to the liver and spleen. Non-invasive measurements of thoracic radioactivity reflected changes in blood counts rather than extravascular lung platelet recruitment. However, both in situ counting of radiolabelled platelets and immunostaining for platelet surface markers showed LPS-induced increases in extravascular platelets into lung airspaces suggesting that some of the platelets recruited to the lung enter air spaces. Conclusions Intranasal administration of LPS activates the innate immune response which includes a fall in peripheral blood platelet counts with subsequent platelet recruitment to the lung, spleen and liver, measured by immunohistochemistry and radiolabelling techniques.

Gustav Born achieved scientific fame for his application of light transmission aggregometry to the study of platelet function, but also led interdisciplinary research teams in pioneering quantitative in vivo imaging studies of platelet aggregation and leukocyte adhesion, and in conducting the first research into the biomechanical factors underlying atherosclerotic plaque rupture. Gus Born also communicated both current research findings and an integrated understanding of cardiovascular biology to a wide audience through acting as scientific advisor on several television productions. Using footage from two of these films, we discuss Gustav Born's scientific achievements and legacy.

Platelets have been implicated in pulmonary inflammatory cell recruitment following exposure to allergic and non-allergic stimuli, but little is known about the role of platelets in response to pulmonary infection with Pseudomonas aeruginosa (P. aeruginosa). In this study, we have investigated the impact of the experimental depletion of circulating platelets on a range of inflammatory and bacterial parameters, and their subsequent impact on mortality in a murine model of pulmonary infection with P. aeruginosa. P. aeruginosa infection in mice induced a mild but significant state of peripheral thrombocytopenia in addition to pulmonary platelet accumulation. Increased platelet activation was detected in infected mice through increased levels of the platelet derived mediators PF-4 and β-TG in bronchoalveolar lavage fluid (BALF) and blood plasma. In mice depleted of circulating platelets, pulmonary neutrophil recruitment was significantly reduced 24 hours’ post infection, whilst the incidence of systemic dissemination of bacteria was significantly increased compared to non-platelet depleted control mice. Furthermore, mortality rates were increased in bacterial infected mice depleted of circulating platelets. This work demonstrates a role for platelets in the host response towards a gram-negative bacterial respiratory infection.

Platelet activation occurs during host defence and in various inflammatory disorders. In animal models of infection and inflammation, experimental depletion of platelets leads to significantly reduced leukocyte recruitment and impaired clearance of pathogens from the lung. It is now appreciated that purinergic receptor activation is required for leukocyte activation, motility and adhesion, and platelet interactions with leukocytes can be modulated by purinergic stimulation of platelets. Here, we have investigated the role of platelet P2Y1, P2Y12, P2Y14, and P2X1 receptors on leukocyte recruitment and chemotaxis. Mice were administered either vehicle controls or selective P2Y1, P2Y12, P2Y14, or P2X1 antagonists intravenously before intranasal administration of lipopolysaccharide (LPS) to investigate the effect of these drugs on pulmonary leukocyte recruitment, peripheral platelet counts, bleeding times, and ex vivo platelet aggregation. Separately, platelets were incubated with P2Y1, P2Y12, P2X1 antagonists, or P2Y14 agonists to assess effects on platelet-induced neutrophil chemotaxis in vitro. Pulmonary neutrophil recruitment induced by intranasal LPS administration was inhibited in mice administered either with P2Y1 or P2Y14 antagonists, but not with P2Y12 or P2X1 antagonists. Furthermore, the administration of either a P2Y1 or a P2Y14 antagonist reversed the incidence of peripheral thrombocytopaenia associated with LPS exposure. Bleeding times were significantly increased in mice administered P2Y1, P2Y12, or P2X1 antagonists, whilst ex vivo platelet aggregation to ADP was significantly reduced. These haemostatic responses remained unaltered following antagonism of P2Y14. In vitro chemotaxis assays revealed direct antagonism of platelet P2Y1, but not P2Y12 or P2X1 receptors suppressed platelet-dependent neutrophil motility towards Macrophage derived chemokine (MDC, CCL22). Furthermore, the stimulation of platelets with selective P2Y14 agonists (UDP-glucose, MRS2690) resulted in significant platelet-dependent neutrophil chemotaxis. These results reveal a role for P2Y1 and P2Y14 activation of platelets following exposure to LPS, whilst haemostatic indices were unaffected by inhibition of platelet function with the P2Y14 antagonist in response to LPS.

The small GTPase Rac is required for neutrophil recruitment during inflammation, but its guanine-nucleotide exchange factor (GEF) activators seem dispensable for this process, which led us to investigate the possibility of cooperation between Rac-GEF families. Thioglycollate-induced neutrophil recruitment into the peritoneum was more severely impaired in P-Rex1(-/-) Vav1(-/-) (P1V1) or P-Rex1(-/-) Vav3(-/-) (P1V3) mice than in P-Rex null or Vav null mice, suggesting cooperation between P-Rex and Vav Rac-GEFs in this process. Neutrophil transmigration and airway infiltration were all but lost in P1V1 and P1V3 mice during lipopolysaccharide (LPS)-induced pulmonary inflammation, with altered intercellular adhesion molecule 1-dependent slow neutrophil rolling and strongly reduced L- and E-selectin-dependent adhesion in airway postcapillary venules. Analysis of adhesion molecule expression, neutrophil adhesion, spreading, and migration suggested that these defects were only partially neutrophil-intrinsic and were not obviously involving vascular endothelial cells. Instead, P1V1 and P1V3 platelets recapitulated the impairment of LPS-induced intravascular neutrophil adhesion and recruitment, showing P-Rex and Vav expression in platelets to be crucial. Similarly, during ovalbumin-induced allergic inflammation, pulmonary recruitment of P1V1 and P1V3 eosinophils, monocytes, and lymphocytes was compromised in a platelet-dependent manner, and airway inflammation was essentially abolished, resulting in improved airway responsiveness. Therefore, platelet P-Rex and Vav family Rac-GEFs play important proinflammatory roles in leukocyte recruitment.