Background: Epidemiological studies have identified pneumonia to be closely linked to an increased risk for cardiovascular events. Potential causality of this association, however, remains to be established, and underlying mechanisms are yet to be resolved. Here, we tested the hypothesis that prior pulmonary infection with Streptococcus pneumoniae accelerates the development of an atherosclerotic phenotype in mice. Such a phenotype would explain the increased cardiovascular risk in the epidemiological data. Methods: Atheroprone ApoE−/− mice were fed a high fat diet, and after 6 weeks nasally infected with either 2.5 x 107 S. pneumoniae or vehicle control. Antibiotic therapy was initiated 60h post infection (p.i.) by s.c. injections of ampicillin on 5 consecutive days followed by oral administration of amoxicillin for 10 days. At days 35 or 90 post-infection, aortas were harvested and assessed for vasoreactivity and arterial stiffness by wire myography. Morphological changes of the aortic vessel wall and eNOS expression levels were quantified by western blot and histological stainings. Results: Unexpectedly, 35 days p.i. aortas of infected mice presented with decreased aortic stiffness, increased endothelial-independent vasorelaxation and decreased wall thickness due to lower collagen-1 expression and reduced elastin fragmentation when compared to sham animals. After 90 days, however, these effects had reversed and animals with prior pneumonia showed increased aortic stiffness, attenuated endothelial dependent vasorelaxation that was associated with reduced eNOS protein expression and phosphorylation, and increased atherosclerotic plaque burden relative to vehicle control. Conclusion: Single episodes of pneumonia promote a delayed pro-atherogenic phenotype that manifests as endothelial dysfunction, aortic stiffening and enhanced plaque burden. Underlying mechanisms are currently under investigation to identify risk predictors for patient stratification and clinical targets for prevention of cardiovascular disease following pneumonia. Funding: German Ministry of Education and Research (BMBF) in the framework of SYMPATH (01ZX1906A). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background Recent studies have demonstrated a link between respiratory infections and increased short-term risk of cardiovascular disease (CVD). However, the molecular mechanisms underlying the increased cardiovascular risk after respiratory infections are only poorly understood. Here, we aimed to decipher pathophysiological circuits of pneumonia associated CVD in experimental models of bacterial pneumonia and vascular injury. Methods C57BL/6J mice were exposed to intranasal inoculation with either Streptococcus pneumoniae ( S. pneumoniae ) serotype 4 (pneumonia group) or phosphate buffered saline (PBS) (control group). 24 hours post infectionem (p.i.) mice were treated with antibiotics until the end of the study. On day 7 p.i. carotid artery injury (CI) was induced by electric stimulation and vascular repair was analyzed 3 days after injury. Plasma proteomic analyses were performed by Olink Bioscience. Primary human aortic endothelial cells (HAECs) were used to study alterations of the endothelial functional properties, bioenergetic state and thrombogenic potential in vitro . Intravital fluorescence microscopy equipped with video recording was applied to measure thrombus formation in real-time. Results Bacterial pneumonia impaired repair capacity of the endothelium after vascular injury. Proteomic analyses revealed significantly higher plasma levels of glucagon in mice after recovery from pneumonia relative to controls, which was further confirmed by ELISA detecting glucagon. Mechanistically, we found that glucagon impaired mitochondrial bioenergetics and migratory potential in HAECs and induced an inflammatory response. Moreover, glucagon fostered vascular thrombogenicity as demonstrated by increased thrombocyte adhesion to HAECs and accelerated carotid artery thrombus formation in vivo . Acute application of the glucagon-like peptide-1 receptor (GLP1-R) agonist liraglutide to lower blood glucagon levels, restored vascular repair potential and attenuated vascular thrombogenicity in mice with pneumonia. Conclusions Our findings reveal a novel mechanism that associates elevated circulatory glucagon levels to dysfunctional endothelium and increased vascular thrombogenicity, suggesting glucagon signaling as a potential therapeutic target to prevent pneumonia-induced cardiovascular events. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl - channel and ABC transporter; its mutations cause the clinical picture of cystic fibrosis (CF). Of late, CFTR has emerged as an important regulator of platelet function, as CFTR dysfunction causes agonist-induced platelet hyperactivation. These findings are reminiscent of platelets from SARS-CoV-2 infected patients since thromboembolic complications represent hallmarks of severe COVID-19 that may critically contribute to morbidity and mortality. CFTR modulators have recently been introduced as a treatment for patients with various CFTR mutations, but have also been reported by us and others to enhance channel function of wild type CFTR. We therefore postulated that CFTR modulators may exert anti-coagulant effects on platelets of healthy donors (HD) and COVID-19 patients.We recruited 36 COVID-19 patients with moderate, and 34 COVID-19 patients with severe disease course (all w/o anti-platelet drugs), and 38 HDs. In line with our hypothesis, we observed significant reductions in platelet agonists adenosine diphosphate (ADP)- or thrombin receptor activating protein-6 (TRAP6)-induced CD62p/CD63 expression, Ca 2+ -mobilization, aggregation, and adhesion of platelets from HDs by pre-treatment with ivacaftor. In blood from COVID-19 patients, platelet activation correlates with disease severity, as demonstrated by a 5-fold and 8-fold increase in the proportion of CD62p + platelets from patients with moderate and severe disease, respectively, relative to HDs. Similarly, the proportion of CD63 + platelets in patients with severe COVID-19 was 2-fold higher than in HDs. Retrospective analysis of clinical data from a total of 4,050 CF patients with COVID-19 receiving single or combination therapy of ivacaftor, lumacaftor, tezacaftor, or elexacaftor in comparison to an untreated cohort revealed that CF therapy reduced the relative risk to suffer thromboembolism-associated cardiovascular events such as heart attack or deep vein thrombosis by 50.0% or 61.1%, respectively, suggesting an anti-thrombotic effect of CFTR modulators in CF COVID-19 patients. In line with this observation, ex vivo pre-treatment of platelets from acute COVID-19 patients with ivacaftor reduced Ca 2+ mobilization, adhesion, and aggregation of platelets .Our results demonstrate an anticoagulant effect of CFTR potentiators on platelets from HDs and severe COVID-19 patients and thus, suggest CFTR potentiators as a promising strategy to reduce the risk of thrombotic events in the clinical management of COVID-19 and similar pro-thrombotic disease states. F. Behrens received funding from the Berlin Institute of Health (BIH). L. Michalick reports grants from the BIH and the German Centre for Cardiovascular Research (DZHK). A. Haghikia is participant in the BIH-Charité Advanced Clinician Scientist Pilotprogram funded by the Charité - Universitätsmedizin Berlin and the BIH and reports a research grant within the BIH & MDC Focus Area Translational Vascular Biomedicine. R. Preissner reports partial funding of this work by the German Research Foundation (KFO339, TRR295). M. Witzenrath reports grants from the German Research Foundation (SFB-TR84 C06 and C09, SFB 1449 B02), and from the German Ministry of Education and Research (BMBF) in the framework of CAPSyS (01ZX1604B, 01ZX1304B), SYMPATH (01ZX1906A), PROVID (01KI20160A), Phage4Cure (16GW0141), MAPVAP (16GW0247) and NUM-NAPKON. W. M. Kuebler reports grants from the German Research Foundation (SFB-TR84 A2 and C9, SFB 1449 B1, SFB 1470 A4, KU1218/9-1, KU1218/11-1, and KU1218/12-1), the BMBF in the framework of SYMPATH (01ZX1906A) and PROVID (01KI20160A), and the DZHK. S. Simmons reports grants from the DZHK and the German Foundation for Heart Research (F-09-19). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Respiratory infections caused by multidrug-resistant Acinetobacter baumannii are difficult to treat and associated with high mortality among critically ill hospitalized patients. Bacteriophages (phages) eliminate pathogens with high host specificity and efficacy. However, the lack of appropriate preclinical experimental models hampers the progress of clinical development of phages as therapeutic agents. Therefore, we tested the efficacy of a purified lytic phage, vB_AbaM_Acibel004, against multidrug-resistant A. baumannii clinical isolate RUH 2037 infection in immunocompetent mice and a human lung tissue model. Sham- and A. baumannii-infected mice received a single-dose of phage or buffer via intratracheal aerosolization. Group-specific differences in bacterial burden, immune and clinical responses were compared. Phage-treated mice not only recovered faster from infection-associated hypothermia but also had lower pulmonary bacterial burden, lower lung permeability, and cytokine release. Histopathological examination revealed less inflammation with unaffected inflammatory cellular recruitment. No phage-specific adverse events were noted. Additionally, the bactericidal effect of the purified phage on A. baumannii was confirmed after single-dose treatment in an ex vivo human lung infection model. Taken together, our data suggest that the investigated phage has significant potential to treat multidrug-resistant A. baumannii infections and further support the development of appropriate methods for preclinical evaluation of antibacterial efficacy of phages.

The high incidence of thrombotic events suggests a possible role of the contact system pathway in COVID-19 pathology. In this study, we determined the altered levels of factor XII (FXII) and its activation products in critically ill patients with COVID-19 in comparison with patients with severe acute respiratory distress syndrome related to the influenza virus (acute respiratory distress syndrome [ARDS]-influenza). Compatible with those data, we found rapid consumption of FXII in COVID-19 but not in ARDS-influenza plasma. Interestingly, the lag phase in fibrin formation, triggered by the FXII activator kaolin, was not prolonged in COVID-19, as opposed to that in ARDS-influenza. Confocal and electron microscopy showed that increased FXII activation rate, in conjunction with elevated fibrinogen levels, triggered formation of fibrinolysis-resistant, compact clots with thin fibers and small pores in COVID-19. Accordingly, clot lysis was markedly impaired in COVID-19 as opposed to that in ARDS-influenza. Dysregulated fibrinolytic system, as evidenced by elevated levels of thrombin-activatable fibrinolysis inhibitor, tissue-plasminogen activator, and plasminogen activator inhibitor-1 in COVID-19 potentiated this effect. Analysis of lung tissue sections revealed widespread extra- and intravascular compact fibrin deposits in patients with COVID-19. A compact fibrin network structure and dysregulated fibrinolysis may collectively contribute to a high incidence of thrombotic events in COVID-19.

Atherosclerosis is one of the leading causes of death worldwide. Biomathematical modelling of the underlying disease and therapy processes might be a useful aid to develop and improve preventive and treatment concepts of atherosclerosis. We here propose a biomathematical model of murine atherosclerosis under different diet and treatment conditions including lipid modulating compound and antibiotics. The model is derived by translating known biological mechanisms into ordinary differential equations and by assuming appropriate response kinetics to the applied interventions. We explicitly describe the dynamics of relevant immune cells and lipid species in atherosclerotic lesions including the degree of blood vessel occlusion due to growing plaques. Unknown model parameters were determined by fitting the predictions of model simulations to time series data derived from mice experiments. Parameter fittings resulted in a good agreement of model and data for all 13 experimental scenarios considered. The model can be used to predict the outcome of alternative treatment schedules of combined antibiotic, immune modulating, and lipid lowering agents under high fat or normal diet. We conclude that we established a comprehensive biomathematical model of atherosclerosis in mice. We aim to validate the model on the basis of further experimental data.

Endothelial dysfunction and increased microvascular permeability are hallmarks of severe COVID‐19. At present, the underlying mechanisms of endothelial barrier failure in COVID‑19 remain elusive. Here, we show that increased thrombin activity in plasma from severe COVID‐19 patients activates endothelial protease‐activated receptor (PAR1), which mediates barrier failure by triggering TRPV4‐mediated Ca2+ influx in lung microvascular endothelial cells.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic lung condition characterized by progressively deteriorating respiratory function. Exacerbations due to lung infections are thought to promote disease progression, and the presence of Streptococcus in the lung microbiome has been linked to progression of IPF. The aim of this study was to evaluate the impact of pulmonary fibrosis on susceptibility to pneumococcal pneumonia and subsequent bacteremia. Methods: The effect of subclinical low-dose infection with Streptococcus pneumoniae via the nasal route was studied using fos-related antigen-2 (Fra-2) transgenic (TG) mice, which develop spontaneous progressive pulmonary fibrosis. Two days after infection bacterial load was assessed in lung tissue, bronchoalveolar lavage (BAL), blood and spleen. Leukocyte subsets and cytokine levels were analyzed in BAL and blood. Additionally, lung compliance and arterial blood gases were assessed. Results: Low dose lung infection with Streptococcus pneumoniae in Fra-2 TG mice resulted in substantial pneumonia including weight loss, increased lung bacterial load and bacteremia, whereas WT mice remained mostly unaffected. Compared to WT mice BAL alveolar macrophages were reduced in Fra-2 TG mice. Proinflammatory cytokines and chemokines were elevated upon infection in BAL supernatant and plasma of Fra-2 TG mice. Following infection lung compliance was decreased in Fra-2 TG mice. Conclusion: Pulmonary fibrosis increases susceptibility to pneumococcal pneumonia and bacteremia possibly via impaired alveolar bacterial clearance.

Approximately 20% of symptomatic patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection progress to severe coronavirus disease 2019 (COVID-19) with critical hypoxaemia fulfilling the criteria of acute respiratory distress syndrome (ARDS). Consistent with the classic features of ARDS, severe COVID-19 is characterised by ground glass opacities on computed tomography imaging and diffuse alveolar damage post mortem [1], suggesting permeability-type lung oedema as driver of respiratory failure. Consistent with this concept, autopsy findings show severe lung endothelial injury in patients who succumbed to COVID-19 [2]. The plasma of COVID-19 patients induces pulmonary microvascular barrier failure, which increases with disease severity. Here, a screening platform to test for plasma mediators and the therapeutic potential of barrier stabilising compounds is reported. https://bit.ly/3k4C0tB

Idiopathic pulmonary fibrosis (IPF) is a deadly condition characterized by progressive respiratory dysfunction. Exacerbations due to airway infections are believed to promote disease progression, and presence of Streptococcus in the lung microbiome has been associated with the progression of IPF and mortality. The aim of this study was to analyze the effect of lung fibrosis on susceptibility to pneumococcal pneumonia and bacteremia. The effects of subclinical (low dose) infection with Streptococcus pneumoniae were studied in a well characterized fos-related antigen-2 (Fra-2) transgenic (TG) mouse model of spontaneous, progressive pulmonary fibrosis. Forty-eight hours after transnasal infection with S. pneumoniae, bacterial load was assessed in lung tissue, bronchoalveolar lavage (BAL), blood, and spleen. Leukocyte subsets and cytokine levels were analyzed in BAL and blood. Lung compliance and arterial blood gases were assessed. In contrast to wildtype mice, low dose lung infection with S. pneumoniae in Fra-2 TG mice resulted in substantial pneumonia including weight loss, increased lung bacterial load, and bacteremia. BAL alveolar macrophages were reduced in Fra-2 TG mice compared to the corresponding WT mice. Proinflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, and CXCL1) were elevated upon infection in BAL supernatant and plasma of Fra-2 TG mice. Lung compliance was decreased in Fra-2 TG mice following low dose infection with S. pneumoniae. Pulmonary fibrosis increases susceptibility to pneumococcal pneumonia and bacteremia possibly via impaired alveolar bacterial clearance.

The high incidence of thrombotic events suggests a possible role of the contact system pathway in COVID-19 pathology. Here, we demonstrate altered levels of factor XII (FXII) and its activation products in two independent cohorts of critically ill COVID-19 patients in comparison to patients suffering from severe acute respiratory distress syndrome due to influenza virus (ARDS-influenza). Compatible with this data, we report rapid consumption of FXII in COVID-19, but not in ARDS-influenza, plasma. Interestingly, the kaolin clotting time was not prolonged in COVID-19 as compared to ARDS-influenza. Using confocal and electron microscopy, we show that increased FXII activation rate, in conjunction with elevated fibrinogen levels, triggers formation of fibrinolysis-resistant, compact clots with thin fibers and small pores in COVID-19. Accordingly, we observed clot lysis in 30% of COVID-19 patients and 84% of ARDS-influenza subjects. Analysis of lung tissue sections revealed wide-spread extra- and intra-vascular compact fibrin deposits in COVID-19. Together, our results indicate that elevated fibrinogen levels and increased FXII activation rate promote thrombosis and thrombolysis resistance via enhanced thrombus formation and stability in COVID-19.

Study objective Endothelial dysfunction and increased microvascular permeability are hallmarks of severe COVID-19. At present, the extent of endothelial barrier failure and its underlying mechanisms in COVID‑19 remain unclear. We hypothesized that endothelial leak results from bioactive mediators released in COVID-19 rather than direct endothelial infection and can thus be recapitulated ex vivo by treating endothelial cells with patient plasma, thus providing a personalized screening platform for barrier-protective interventions in COVID-19. Methods Citrate plasma was sampled as part of the Pa-COVID-19 cohort study (ethics approval EA2/066/20) in patients with moderate (hospitalized, no invasive ventilation; WHO severity score: 3-4) and severe (high flow O2 or intubated and mechanically ventilated; WHO severity score: 5-7) COVID‑19. Plasma samples were diluted to 10% (v/v) in cell culture medium without FCS and tested for their ability to disrupt barrier integrity of primary human pulmonary microvascular endothelial cells (HPMEC) monolayers by electrical cell-substrate impedance sensing (ECIS), immunofluorescence for endothelial VE-cadherin and F-actin, and real-time Ca2+ imaging. Plasma from healthy donors served as control. Results COVID-19 plasma was virus-free but caused endothelial barrier disruption as measured by ECIS and gap formation in HPMEC monolayers. The extent of barrier disruption increased with disease severity but varied considerably between endothelial cells from different microvascular beds (lung/heart >> skin). The TRPV4-antagonist HC-067047 prevented the endothelial Ca2+ response to COVID-19 plasma and protected endothelial barrier integrity in lung microvascular cells. Conclusion Here, we identify TRPV4 as critical regulator of microvascular permeability in COVID‑19. Targeting TRPV4-mediated endothelial barrier failure may present a promising adjunctive therapy in COVID-19.

Introduction: In 2014, the WHO proclaimed the beginning of a post-antibiotic era [1]. As alternatives to antibiotics, bacteriophages are increasingly coming into focus to combat the increasing number of resistant bacteria. In order to reach infected sites in the lungs and reduce unwanted systemic effects, nebulization of drugs is a favorable way of application. The infectivity of phages with different tail lengths after nebulization was tested under various conditions. This project is part of Phage4Cure, a BMBF-funded study examining the use of phages against lung infections. Methods: Long-tailed (Siphoviridae), short-tailed (Podoviridae) and medium long tailed (Myoviridae) phages from the order Caudovirales were analyzed. Four different phages per morphotype were nebulized with a JET nebulizer. Additionally, three phages (two Myoviridae, one Podovirus) were purified according to laboratory standards. By using the double-agar overlay method, titers after nebulization were compared to the stock solution. Results: Phages of the type Siphoviridae showed the highest titer losses during nebulization and Podoviridae the lowest. Among the Myoviridae variable and phage-dependent titer loss was observed. In the mesh nebulization system, all three phages showed a loss of titer after nebulization independent of the purification degree. Only one of the purified phages showed a higher titer loss after nebulization compared to lysate. Conclusion: For successful phage inhalation therapy, the effects of nebulizer technology, the size of the phage tail and purity must be considered. [1] www.who.int/mediacentre/news/releases/2014/amr-report/en/

Purpose of review In this brief review, we discuss the current epidemiological data and latest results from basic research on the cardiovascular sequelae after lower respiratory tract infection. Recent findings Novel epidemiological evidence substantiates the association between pneumonia and subsequent cardiovascular events (CVEs) in the short- and long-term after viral or bacterial acute infection. Biomarkers such as cardiac troponin or coronary artery calcium may represent useful predictive tools for the detection of cardiac involvement during and after pneumonia. Particularly, Streptococcus pneumoniae directly cause cardiac damage by invasion into the myocardium and formation of microscopic lesions finally leading to the development of cardiac scarring in rodents and nonhuman primates. In addition, a causal relationship between pulmonary inflammation and atherosclerotic plaque formation in systemic arteries has emerged that appears to involve a mechanistic role for neutrophil granulocytes. However, many key pathomechanisms by which pneumonia may trigger or promote subsequent CVEs still remain unclear. Summary Pneumonia may deleteriously impact cardiovascular function. Direct cardiomyocyte destruction by pathogens as well as host inflammatory response associated effects including atherosclerotic plaque development and/or rupture have been observed. Details of underlying mechanisms need to be further investigated to deliver future perspectives for the prevention of CVEs subsequent to pneumonia.

Chronic lung diseases are frequently complicated by lung and airway infections. The rise of new resistance mechanisms is alarming, and MDR bacteria, including Acinetobacter baumannii, are spreading globally. Rediscovery of phage-therapy could be a solution to overcome this problem. High specificity of bacteriophages and their effectivity in lysis make phages attractive as antibiotics are failing more and more. However, phages need to be properly chosen and characterized according to quality standards, and preparations must be highly purified.

Pneumonia may be caused by a wide range of pathogens and is considered the most common infectious cause of death in humans. Murine acute lung infection models mirror human pathologies in many aspects and contribute to our understanding of the disease and the development of novel treatment strategies. Despite progress in other fields of tissue imaging, histopathology remains the most conclusive and practical read out tool for the descriptive and semiquantitative evaluation of mouse pneumonia and therapeutic interventions. Here, we systematically describe and compare the distinctive histopathological features of established models of acute pneumonia in mice induced by Streptococcus (S.) pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Legionella pneumophila, Escherichia coli, Middle East respiratory syndrome (MERS) coronavirus, influenza A virus (IAV) and superinfection of IAV-incuced pneumonia with S. pneumoniae. Systematic comparisons of the models revealed striking differences in the distribution of lesions, the characteristics of pneumonia induced, principal inflammatory cell types, lesions in adjacent tissues, and the detectability of the pathogens in histological sections. We therefore identified core criteria for each model suitable for practical semiquantitative scoring systems that take into account the pathogen- and model-specific patterns of pneumonia. Other critical factors that affect experimental pathologies are discussed, including infectious dose, time kinetics, and the genetic background of the mouse strain. The substantial differences between the model-specific pathologies underscore the necessity of pathogen- and model-adapted criteria for the comparative quantification of experimental outcomes. These criteria also allow for the standardized validation and comparison of treatment strategies in preclinical models.