Towards Inhaled Phage Therapy in Western Europe

The emergence of multidrug-resistant bacteria constitutes a great challenge for modern medicine, recognized by leading medical experts and politicians worldwide. Rediscovery and implementation of bacteriophage therapy by Western medicine might be one solution to the problem of increasing antibiotic failure. In some Eastern European countries phage therapy is used for treating infectious diseases. However, while the European Medicines Agency (EMA) advised that the development of bacteriophage-based therapies should be expedited due to its significant potential, EMA emphasized that phages cannot be recommended for approval before efficacy and safety have been proven by appropriately designed preclinical and clinical trials. More evidence-based data is required, particularly in the areas of pharmacokinetics, repeat applications, immunological reactions to the application of phages as well as the interactions and effects on bacterial biofilms and organ-specific environments. In this brief review we summarize advantages and disadvantages of phage therapy and discuss challenges to the establishment of phage therapy as approved treatment for multidrug-resistant bacteria

Repetitive Exposure to Bacteriophage Cocktails against Pseudomonas aeruginosa or Escherichia coli Provokes Marginal Humoral Immunity in Naïve Mice

Phage therapy of ventilator-associated pneumonia (VAP) is of great interest due to the rising incidence of multidrug-resistant bacterial pathogens. However, natural or therapy-induced immunity against therapeutic phages remains a potential concern. In this study, we investigated the innate and adaptive immune responses to two different phage cocktails targeting either Pseudomonas aeruginosa or Escherichia coli—two VAP-associated pathogens—in naïve mice without the confounding effects of a bacterial infection. Active or UV-inactivated phage cocktails or buffers were injected intraperitoneally daily for 7 days in C57BL/6J wild-type mice. Blood cell analysis, flow cytometry analysis, assessment of phage distribution and histopathological analysis of spleens were performed at 6 h, 10 days and 21 days after treatment start. Phages reached the lungs and although the phage cocktails were slightly immunogenic, phage injections were well tolerated without obvious adverse effects. No signs of activation of innate or adaptive immune cells were observed; however, both active phage cocktails elicited a minimal humoral response with secretion of phage-specific antibodies. Our findings show that even repetitive injections lead only to a minimal innate and adaptive immune response in naïve mice and suggest that systemic phage treatment is thus potentially suitable for treating bacterial lung infections

MicroRNA-223 Dampens Pulmonary Inflammation during Pneumococcal Pneumonia

Community-acquired pneumonia remains a major contributor to global communicable disease-mediated mortality. Neutrophils play a leading role in trying to contain bacterial lung infection, but they also drive detrimental pulmonary inflammation, when dysregulated. Here we aimed at understanding the role of microRNA-223 in orchestrating pulmonary inflammation during pneumococcal pneumonia. Serum microRNA-223 was measured in patients with pneumococcal pneumonia and in healthy subjects. Pulmonary inflammation in wild-type and microRNA-223-knockout mice was assessed in terms of disease course, histopathology, cellular recruitment and evaluation of inflammatory protein and gene signatures following pneumococcal infection. Low levels of serum microRNA-223 correlated with increased disease severity in pneumococcal pneumonia patients. Prolonged neutrophilic influx into the lungs and alveolar spaces was detected in pneumococci-infected microRNA-223-knockout mice, possibly accounting for aggravated histopathology and acute lung injury. Expression of microRNA-223 in wild-type mice was induced by pneumococcal infection in a time-dependent manner in whole lungs and lung neutrophils. Single-cell transcriptome analyses of murine lungs revealed a unique profile of antimicrobial and cellular maturation genes that are dysregulated in neutrophils lacking microRNA-223. Taken together, low levels of microRNA-223 in human pneumonia patient serum were associated with increased disease severity, whilst its absence provoked dysregulation of the neutrophil transcriptome in murine pneumococcal pneumonia

Preclinical Assessment of Bacteriophage Therapy against Experimental Acinetobacter baumannii Lung Infection

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

Bitter taste signaling in tracheal epithelial brush cells elicits innate immune responses to bacterial infection

Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into firstline defense mechanisms against bacterial infections of the lung

Bitter taste signaling in tracheal epithelial brush cells elicits innate immune responses to bacterial infection.

peer reviewedConstant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first-line defense mechanisms against bacterial infections of the lung

Investigation of potential immunomodulatory effects of moxifloxacin in severe murine pneumococcal pneumonia

Die ambulant erworbene Pneumonie ist die häufigste tödlich verlaufende Infektionskrankheit weltweit. Streptococcus pneumoniae (S. pneumoniae) ist der mit Abstand häufigste Erreger der ambulant erworbenen Pneumonie. Trotz spezifischer antimikrobieller Therapien und stetiger Bemühungen, die Behandlung von Risikopatienten zu verbessern, wird die Mortalität bei immunkompetenten, antibiotisch adäquat therapierten Patienten mit schwerer Pneumonie auf Intensivstationen weiterhin mit über 20 % angegeben. Die unkontrollierte Aktivierung des Immunsystems scheint bei der schweren Pneumonie eine zentrale Rolle in der Entstehung eines akuten Lungenversagens sowie von Sepsis und Multiorganversagen zu spielen. Daher könnte die gezielte Limitierung überschießender Entzündungsreaktionen kombiniert mit einer effektiven antimikrobiellen Therapie den Verlauf der schweren Pneumonie begünstigen. Chinolone könnten neben ihren gut untersuchten antimikrobiellen Eigenschaften auch antiinflammatorisch wirksam sein. Besonders Moxifloxacin, das unter anderem in der Behandlung der Pneumonie eingesetzt wird, hemmte in vitro die Freisetzung von proinflammatorischen Zytokinen aus humanen Monozyten und humanen Alveolarepithelzellen nach Stimulation mit hitzeinaktivierten Pneumokokken oder Lipopolysacchariden, jedoch gibt es wenig Evidenz aus Untersuchungen mit lebenden Bakterien. Klinische Untersuchungen zeichnen ein weniger eindeutiges Bild. Während in einer retrospektiven klinischen Studie ein besseres Überleben von Patienten mit schwerer ambulant erworbener Pneumonie unter Moxifloxacin-Therapie, verglichen mit Laktam-Monotherapie, beobachtet wurde, konnte prospektiv kein klinischer Nutzen für Patienten mit Sepsis nachgewiesen werden, die zusätzlich zu einer Carbapenem-Therapie Moxifloxacin erhielten. Ziel der vorliegenden Dissertationsarbeit war es daher experimentell zu prüfen, ob für Moxifloxacin immunmodulatorische Eigenschaften in Modellen der Pneumokokkenpneumonie nachweisbar sind. Hierfür wurden in einem ex vivo Modell vitale humane Lungenproben mit TNF- oder S. pneumoniae stimuliert und mit Ampicillin, Moxifloxacin oder Lösungsmittel behandelt und der Einfluss von Moxifloxacin im Vergleich zu Ampicillin, einer der Standardtherapien der ambulant erworbenen Pneumonie, auf die ausgelöste Entzündungsreaktion bestimmt. Für Ampicillin sind keine auf das Immunsystem wirkenden Eigenschaften bekannt. Nach der Stimulation mit TNF- wurde vermehrt IL-6 und IL-8 produziert. Die Behandlung mit Moxifloxacin resultierte in einer verminderten Bildung von IL-6 im Gegensatz zur Behandlung mit Ampicillin. Die Infektion mit S. pneumoniae führte ebenfalls zu einer gesteigerten Produktion der untersuchten Zytokine, die jedoch durch Behandlung mit Moxifloxacin oder Ampicillin nicht reduziert wurde. Darüber hinaus wurde die Moxifloxacin- Therapie in der murinen Pneumokokkenpneumonie im Vergleich zu einer Behandlung mit Ampicillin hinsichtlich Parametern der Entzündungsreaktion, der Erregerelimination, pulmonalvaskulärer Schrankenstörung, histopathologischer Lungenveränderungen und klinischem Verlauf der Infektion untersucht. Mäuse wurden mit S. pneumoniae infiziert und beginnend 24 Stunden nach der Infektion in 12 Stunden-Intervallen mit Ampicillin, Moxifloxacin oder Lösungsmittel behandelt. Zusätzlich dienten Mäuse als Kontrolltiere, die mit Lösungsmittel scheininfiziert und behandelt wurden. Die infizierten Mäuse entwickelten innerhalb von 24 Stunden eine schwere Pneumonie. Alle antibiotisch behandelten Tiere erholten sich im Beobachtungszeitraum fast vollständig von klinischen Zeichen der Pneumonie. Die Behandlung mit Moxifloxacin führte zu einer effektiveren pulmonalen Erregerelimination ab 36 Stunden nach der Infektion sowie zu einer besseren Reduktion von Bakteriämien im Gegensatz zu Ampicillin. Mit Ampicillin behandelte Mäuse zeigten eine erhöhte pulmonalvaskuläre Permeabilität 36 Stunden nach der Infektion im Gegensatz zu Moxifloxacin behandelten Mäusen. Dabei war die verminderte Erregerlast in den mit Moxifloxacin behandelten Tieren nicht ursächlich für die niedrigere pulmonalvaskuläre Permeabilität, was durch Untersuchungen an einer zusätzlichen Versuchsgruppe gezeigt werden konnte, die gleichzeitig mit Ampicillin und Moxifloxacin behandelt wurde. Die kombinierte Behandlung führte zu einer mit der Ampicillin-Monotherapie vergleichbaren Permeabilität. Auch die Effekte beider Antibiotika bezüglich aller weiteren untersuchten Parameter waren vergleichbar. Insbesondere war die pulmonale und systemische Inflammation trotz geringerer Erregerdichte unter Moxifloxacin-Therapie im Gegensatz zur Ampicillin-Therapie nicht reduziert. Zusätzlich wurde der direkte Einfluss von Moxifloxacin und Ampicillin auf die Integrität eines Endothelzellmonolayers mit oder ohne Thrombin-Stimulation durch Messung des transzellulären elektrischen Widerstands getestet. Moxifloxacin erhöhte in vitro weder die basale Integrität des Monolayers, noch reduzierte es die durch Thrombin verursachte Störung der interzellulären Kontakte. Auch Ampicillin hatte keinen Einfluss auf die Stabilität der Barriere. Die Ergebnisse der vorliegenden Studie zeigten, dass Moxifloxacin die TNF-induzierte Inflammation in humanem Lungengewebe im Gegensatz zu Ampicillin reduzierte. Jedoch ließen sich weder in humanem Lungengewebe, noch in der schweren murinen Pneumonie nach Infektion mit S. pneumoniae antiinflammatorische Eigenschaften von Moxifloxacin im Vergleich zu Ampicillin nachweisen. Moxifloxacin war Ampicillin bezüglich der Eliminierung pulmonaler Erregerlast und Bakteriämien überlegen. Ampicillin-behandelte Tiere wiesen allerdings eine erhöhte pulmonalvaskuläre Permeabilität in der akuten Phase der Pneumonie auf und diese Beobachtung war unabhängig von Effekten des Moxifloxacin.With regard to causes of morbidity and mortality worldwide, community-acquired pneumonia holds the highest significance and Streptococcus pneumoniae (S. pneumoniae) is the most prevalent causative pathogen of community-acquired pneumonia. Despite adequate antibiotic therapy and constant endeavour to improve the clinical outcome of high risk patients, existing treatment and drug regimens are still insufficient, considering the high mortality (above 20 %) for immunocompetent patients in intensive care units hospitalized for severe community-acquired pneumonia. The main contributing factor to an adverse outcome of patients suffering from severe pneumonia is an excessive activation of the innate immune system, which leads to the development of acute respiratory failure, sepsis and multiple organ failure. Thus, limitation of exaggerated inflammatory reactions, combined with an effective antibiotic treatment might improve the outcome of affected patients. Efficient therapeutic protocols of various respiratory infections utilize Quinolones. Apart from their well described antimicrobial characteristics, Quinolones were suggested to exert anti-inflammatory effects. Particularly moxifloxacin has been shown to inhibit the release of proin-flammatory cytokines in in vitro experiments, using human monocytes and human alveolar epithelial cells stimulated with heat-inactivated pneumococci or lipopolysaccharides. However, clinical studies demonstrated a less clear picture. In a retrospective study, patients with severe community-acquired pneumonia were treated with moxifloxacin, which resulted in improved outcome as compared to monotherapy with -lactam antibiotics. It was hypothesized that potential immunomodulatory features of moxifloxacin were responsible for this improvement of patient survival. In contrast, a prospective study showed no clinical benefit for patients with sepsis, who were treated with moxifloxacin in addition to carbapenem antibiotics. This study aimed at analysing the impact of moxifloxacin on the inflammatory host response in ex vivo cultured intact human lung tissue and in a murine model of severe pneumococcal pneumonia. Therefore, ex vivo cultured human lung tissue was stimulated with TNF- or infected with S. pneumoniae, treated with ampicillin, moxifloxacin or solvent and proinflammatory cytokines were quantified in the supernatant. For ampicillin which is one standard treatment in community-acquired pneumonia, anti-inflammatory effects have not been described so far. Therefore, it was used as reference antibiotic in this study. Upon stimulation with TNF- human lung tissue produced IL-6 and IL-8, which was reduced by moxifloxacin but not by ampicillin. Infection with S. pneumoniae also induced IL-6 and IL-8 production in human lung tissue which was not influenced by treatment with moxifloxacin or ampicillin. Additionally, a murine model was established, in which antibiotic treatment with ampicillin or moxifloxacin was applied in severe pneumococcal pneumonia to mimic the clinical treatment initiated in the emergency department. Mice infected with a lethal dosage of S. pneumoniae were treated in 12-hour intervals with ampicillin, moxifloxacin or solvent, starting 24 hours post infection. In addition, sham infected and solvent treated animals were investigated. At specified time points post infection, the bacterial burden in lung, blood and spleen was analysed. Cytokine levels were measured in bronchoalveolar lavage fluid and blood plasma. Furthermore, proinflammatory gene regulation in murine lung tissue was investigated by real-time polymerase chain reaction. Bronchoalveolar lavage fluid and blood leukocytes were differentiated and pulmonary vascular permeability was determined. Additionally, lung tissue from infected mice was histologically analysed. Upon infection with S. pneumoniae, mice developed severe pneumonia within 24 hours post infection. Antibiotic treated mice were rescued from developing acute respiratory distress syndrome and sepsis, and recovered from clinical signs of pneumonia within the observation period. Pulmonary bacterial burden was lower in moxifloxacin treated mice in contrast to ampicillin from 36 hours post infection on, and fewer cases with bacteraemia were observed when compared with ampicillin treated mice. At 36 hours post infection, ampicillin treated mice showed increased pulmonary vascular permeability, which could not be attributed to the lower bacterial burden in moxifloxacin treated mice. This was shown by an additionally investigated group of mice treated with moxifloxacin and ampicillin simultaneously. Animals in this group displayed equally high permeability as mice with ampicillin monotherapy. Bronchoalveolar lavage fluid and blood cytokine levels, proinflammatory gene expression, blood leukocytes and morphologic lung injury scores were similar in mice treated with moxifloxacin or ampicillin. To estimate a potential effect of moxifloxacin on the integrity of the cellular monolayer, transcellular electrical resistance of human umbilical vein endothelial cell monolayers was measured in the presence of moxifloxacin or ampicillin with or without thrombin stimulation. Moxifloxacin exerted no direct stabilising effects on the endothelial barrier, and it also did not reduce thrombin- induced disruption of integrity. The same applied to ampicillin. In summary, in human lung tissue moxifloxacin treatment reduced TNF-α induced inflammation in contrast to ampicillin. However, ampicillin and moxifloxacin showed similar effects in pneumococci-induced inflammation of human lung tissue and in severe murine pneumococcal pneumonia. Nevertheless, treatment with moxifloxacin reduced pulmonary bacterial load and bacteraemia in contrast to ampicillin. Pulmonary vascular permeability in the acute phase of murine pneumococcal pneumonia was enhanced by ampicillin in contrast to moxifloxacin treatment, which was independent from effects of moxifloxacin

A biomathematical model of immune response and barrier function in mice with pneumococcal lung infection

Pneumonia is one of the leading causes of death worldwide. The course of the disease is often highly dynamic with unforeseen critical deterioration within hours in a relevant proportion of patients. Besides antibiotic treatment, novel adjunctive therapies are under development. Their additive value needs to be explored in preclinical and clinical studies and corresponding therapy schedules require optimization prior to introduction into clinical practice. Biomathematical modeling of the underlying disease and therapy processes might be a useful aid to support these processes. We here propose a biomathematical model of murine immune response during infection with Streptococcus pneumoniae aiming at predicting the outcome of different treatment schedules. The model consists of a number of non-linear ordinary differential equations describing the dynamics and interactions of the pulmonal pneumococcal population and relevant cells of the innate immune response, namely alveolar- and inflammatory macrophages and neutrophils. The cytokines IL-6 and IL-10 and the chemokines CCL2, CXCL1 and CXCL5 are considered as major mediators of the immune response. We also model the invasion of peripheral blood monocytes, their differentiation into macrophages and bacterial penetration through the epithelial barrier causing blood stream infections. We impose therapy effects on this system by modelling antibiotic therapy and treatment with the novel C5a-inactivator NOX-D19. All equations are derived by translating known biological mechanisms into equations and assuming appropriate response kinetics. Unknown model parameters were determined by fitting the predictions of the model to time series data derived from mice experiments with close-meshed time series of state parameters. Parameter fittings resulted in a good agreement of model and data for the experimental scenarios. The model can be used to predict the performance of alternative schedules of combined antibiotic and NOX-D19 treatment. We conclude that we established a comprehensive biomathematical model of pneumococcal lung infection, immune response and barrier function in mice allowing simulations of new treatment schedules. We aim to validate the model on the basis of further experimental data. We also plan the inclusion of further novel therapy principles and the translation of the model to the human situation in the near future