On Top of the Alveolar Epithelium: Surfactant and the Glycocalyx

Gas exchange in the lung takes place via the air-blood barrier in the septal walls of alveoli. The tissue elements that oxygen molecules have to cross are the alveolar epithelium, the interstitium and the capillary endothelium. The epithelium that lines the alveolar surface is covered by a thin and continuous liquid lining layer. Pulmonary surfactant acts at this air-liquid interface. By virtue of its biophysical and immunomodulatory functions, surfactant keeps alveoli open, dry and clean. What needs to be added to this picture is the glycocalyx of the alveolar epithelium. Here, we briefly review what is known about this glycocalyx and how it can be visualized using electron microscopy. The application of colloidal thorium dioxide as a staining agent reveals differences in the staining pattern between type I and type II alveolar epithelial cells and shows close associations of the glycocalyx with intraalveolar surfactant subtypes such as tubular myelin. These morphological findings indicate that specific spatial interactions between components of the surfactant system and those of the alveolar epithelial glycocalyx exist which may contribute to the maintenance of alveolar homeostasis, in particular to alveolar micromechanics, to the functional integrity of the air-blood barrier, to the regulation of the thickness and viscosity of the alveolar lining layer, and to the defence against inhaled pathogens. Exploring the alveolar epithelial glycocalyx in conjunction with the surfactant system opens novel physiological perspectives of potential clinical relevance for future research

Adjunctive therapy with the Tie2 agonist Vasculotide reduces pulmonary permeability in Streptococcus pneumoniae infected and mechanically ventilated mice

Community acquired pneumonia, mainly caused by Streptococcus pneumoniae (S.pn.), is a common cause of death worldwide. Despite adequate antibiotic therapy, pneumococcal pneumonia can induce pulmonary endothelial hyperpermeability leading to acute lung injury, which often requires mechanical ventilation (MV) causing ventilator-induced lung injury (VILI). Endothelial stabilization is mediated by angiopoietin-1 induced Tie2 activation. PEGylated (polyethylene glycol) Tie2-agonist Vasculotide (VT) mimics Angiopietin-1 effects. Recently, VT has been shown to reduce pulmonary hyperpermeability in murine pneumococcal pneumonia. The aim of this study was to determine whether VT reduces lung damage in S.pn. infected and mechanically ventilated mice. Pulmonary hyperpermeability, immune response and bacterial load were quantified in S.pn. infected mice treated with Ampicillin + /-VT and undergoing six hours of MV 24 h post infection. Histopathological lung changes, Tie2-expression and -phosphorylation were evaluated. VT did not alter immune response or bacterial burden, but interestingly combination treatment with ampicillin significantly reduced pulmonary hyperpermeability, histological lung damage and edema formation. Tie2-mRNA expression was reduced by S.pn. infection and/or MV but not restored by VT. Moreover, Tie2 phosphorylation was not affected by VT. These findings indicate that VT may be a promising adjunctive treatment option for prevention of VILI in severe pneumococcal pneumonia

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

Humanized Mouse Model Mimicking Pathology of Human Tuberculosis for in vivo Evaluation of Drug Regimens

Human immune system mice are highly valuable for in vivo dissection of human immune responses. Although they were employed for analyzing tuberculosis (TB) disease, there is little data on the spatial organization and cellular composition of human immune cells in TB granuloma pathology in this model. We demonstrate that human immune system mice, generated by transplanted human fetal liver derived hematopoietic stem cells develop a continuum of pulmonary lesions upon Mycobacterium tuberculosis aerosol infection. In particular, caseous necrotic granulomas, which contribute to prolonged TB treatment time, developed, and had cellular phenotypic spatial-organization similar to TB patients. By comparing two recommended drug regimens, we confirmed observations made in clinical settings: Adding Moxifloxacin to a classical chemotherapy regimen had no beneficial effects on bacterial eradication. We consider this model instrumental for deeper understanding of human specific features of TB pathogenesis and of particular value for the pre-clinical drug development pipeline

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

Single-cell-resolved interspecies comparison shows a shared inflammatory axis and a dominant neutrophil-endothelial program in severe COVID-19

A key issue for research on COVID-19 pathogenesis is the lack of biopsies from patients and of samples at the onset of infection. To overcome these hurdles, hamsters were shown to be useful models for studying this disease. Here, we further leverage the model to molecularly survey the disease progression from time-resolved single-cell RNA sequencing data collected from healthy and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected Syrian and Roborovski hamster lungs. We compare our data to human COVID-19 studies, including bronchoalveolar lavage, nasal swab, and postmortem lung tissue, and identify a shared axis of inflammation dominated by macrophages, neutrophils, and endothelial cells, which we show to be transient in Syrian and terminal in Roborovski hamsters. Our data suggest that, following SARS-CoV-2 infection, commitment to a type 1- or type 3-biased immunity determines moderate versus severe COVID-19 outcomes, respectively

Die Rolle von treml6 in der Entwicklung von B-1a B Zellen Und Der Einfluss von ICOS Kostimulation auf die erworbene Immunantwort gegen M. tuberculosis

TREM Rezeptoren sind aus infektionsimmunologischer Sicht von großem Interesse, da sie TLR Signale inhibieren bzw. verstärken können und somit in der Lage sind, inflammatorische Immunantworten zu regulieren. Diese Doktorarbeit untersucht die immunologische Funktion von murinem TLT-6 (Gen: treml6), einem bislang unerforschten ITIM-tragenden Rezeptor der TREM Rezeptor Familie, mittels Analyse von treml6-WT und eigens generierten treml6-/- Mäusen. Treml6 mRNA wurde vornehmlich von Leukozyten der Peritonealhöhle und lymphoiden Organen exprimiert. Treml6-/- Mäuse waren fortpflanzungsfähig und zeigten normales Wachstum. Die Analyse ihrer Immunzellzusammensetzung zeigte, dass treml6-/- Mäuse eine verringerte Anzahl an B-1 B Vorläuferzellen in der fetalen Leber und im adulten Knochenmark aufwiesen. Möglicherweise bedingt durch diese Reduktion an Vorläuferzellen, hatten sowohl junge als auch erwachsene treml6-/- Mäusen weniger peritoneale B-1a B Zellen als WT Mäuse. Andere B-1 oder B-2 Zellpopulationen in treml6-/- Mäusen waren hingegen nicht beeinträchtigt. Ferner wiesen treml6-/- Mäusen keine Defekte in der Aufrechterhaltung von B-1a B Zellen in der Peripherie, wie zum Beispiel eine erhöhte Apoptosisrate der B Zellen, noch Unterschiede in der Funktionalität der B Zellen, wie z.B. abnormale Antikörpersekretion, auf. Der B-1a B Zelldefekt in treml6-/- Mäusen kann, im Gegensatz zum Großteil der in der Literatur bekannten B-1 B Zelldefekte, ebenfalls nicht auf eine veränderte Signaltransduktion des BCR zurückgeführt werden. Die Ursache für die Reduktion der peritonealen B-1a B Zellen vermuten wir vielmehr in einem Defekt im fetalen Stadium der B-1a B Zellentwicklung in treml6-/- Mäusen. Hinweise hierfür lieferten Zelltransferexperimente. Entsprechend resultierte die Transplantation von adultem treml6-/- KM in Rag1-/- Mäuse in einer normalen Rekonstitution aller B Zellpopulationen des Rezipienten. Transplantierten wir jedoch aus treml6-/- fetalen Leberzellen generierte pre-B I Zellen, so zeigte sich, dass sie im Vergleich zu WT pre-B I B Zellen, eine beschränkte Fähigkeit aufwiesen, das B-1a B Zellkompartiment im Rag1-/- Rezipienten zu füllen. Zusammenfassend schlussfolgern wir, dass treml6 ein positiver Regulator der fetalen B-1a B Zellentwicklung ist. Unsere Daten liefern somit neue Einblicke in die Regulation der B-1a B Zellentwicklung, einem nach wie vor stark diskutierten Forschungsfeld. Ferner haben unsere Untersuchungen die Erkenntnisse über die TREM Rezeptor Familie um eine weitere Funktion, nämlich ihre Beteiligung an der Regulation der Lymphozytenentwicklung, erweitert. Mtb ist weiterhin als globale gesundheitliche Bedrohung anzusehen. T Zellen, insbesondere CD4+ Th1 Zellen und ihr wichtigstes Effektorzytokin IFN-γ, sind für eine schützende Immunantwort gegen Mtb entscheidend. Dessen ungeachtet gelingt es der Immunantwort inklusive der T Zellantwort nicht, die Bakterien vollständig zu eliminieren und Sterilität zu erreichen. Im Zuge vorrausgehender Bemühungen, Faktoren zu identifizieren, die die Th1 Antwort gegen Mtb beeinflussen, stellten wir fest, dass in der murinen Mtb Infektion ein Großteil der IFN-γ sekretierenden CD4+ Th1 Zellen ICOS exprimierten. Dementsprechend lag die Vermutung nahe, dass ICOS eine wichtige Rolle in der Ausbildung der Th1 Antwort gegen Mtb spielt. Wir infizierten WT und ICOS-/- Mäuse mit Mtb, um ein detailiertes Bild der Kinetik und der Art der T Zellantwort in Abwesenheit von ICOS-Kostimulation zu erhalten. ICOS-Defizienz führte zu einer verstärkten polyklonalen CD4+ Th1 Antwort gegen Mtb. Die Ursache hierfür lässt sich in der stark reduzierten Anzahl und Frequenzen der Treg in ICOS-/- Mäusen vermuten. Im Gegensatz zur CD4+ Th1 war die Mtb-spezifische CD8+ T Zellantwort in ICOS-/- Mäusen in der chronischen Phase der Infektion abgeschwächt. Die Reduktion betraf nicht nur Effektor CD8+ T Zellen, sondern auch Effektor-Gedächtnis CD8+ T Zellen in Mtb infizierten ICOS-/- Mäusen. Da nur eine marginale ICOS Expression auf CD8+ T Zellen während der Mtb Infektion festzustellen war, lässt sich die Reduktion der Mtb-spezifischen CD8+ T Zellen nicht auf intrinsische Defekte zurückführen, sondern wurde wahrscheinlich durch beeinträchtigte CD4+ T Zell-Hilfe in ICOS-/- Mäusen verursacht. Insgesamt bestätigten wir unsere Anfangsvermutung, dass ICOS die Th1 T Zellantwort gegen Mtb maßgeblich beeinflusst und konnten zusätzlich zeigen, dass die Stärke der Treg Antwort ICOS-abhängig war. Das Ausmaß, in dem ICOS die einzelnen T Zellpopulationen wie CD4+ Th1, Treg und CD8+ T Zellen und somit auch ihre Interaktion untereinander beeinflusste, unterschied sich zwischen Milz und Lunge. So zeigten ICOS-/- Mäuse in der späten chronischen Phase der Mtb Infektion eine bessere Kontrolle der Erreger in der Milz, nicht aber in der Lunge.TREM receptors are of particular interest in infection immunology since they are able to regulate inflammatory responses by either amplifying or dampening TLR signals. Here we investigated the immunological function of TLT-6 (gene: treml6), a so far undescribed ITIM-containing receptor of the TREM protein family using treml6-WT and in-house generated treml6-/- mice. Treml6 mRNA was broadly expressed among leukocytes of the peritoneal cavity and lymphoid tissues. Treml6-/- mice were fertile and showed normal growth. Analysis of their immune cell composition revealed reduced numbers of B-1 B cell specific precursors in fetal liver and adult BM of treml6-/- mice. Likely as a consequence of this reduction, young and adult treml6-/- mice bared reduced numbers of peritoneal B-1a B cells. Notably, solely peritoneal B-1a B cells, but no other B-1 or B-2 B cell subsets were affected by the lack of treml6. No abnormalities in maintenance of B-1a B cells or in their functionality, such as increased apoptosis rates of B cells or altered antibody responses, respectively, were evident in treml6-/- mice. Moreover, in contrast to most B-1 B cell defects described in the literature, the defect observed in treml6-/- mice was independent of BCR signaling. By means of transplantation experiments it became apparent that the origin of the peritoneal B-1a B cell defect in treml6-/- mice was rooted in a defect during the fetal B cell development. Transplantation of treml6-/- adult BM cells resulted in normal - WT-like - B cell engraftment of Rag1-/- mice. In contrast, transplantation of treml6-/- fetal liver-derived pre-B I cells resulted in poorer B-1a B cell engraftment of recipient Rag1-/- mice when compared to WT pre-B I cells. In sum, we propose that treml6 is a positive regulator of fetal B-1a B cell development. Hence, our data provide new insights into B-1a B cell development; an area of research that is still controversial. Moreover, our study expands the knowledge of the TREM receptor family by ascribing it an additional function, namely regulation of lymphocyte development. Mtb is still a global health threat. T cells, notably CD4+ Th1 cells and their major effector cytokine IFN-γ are crucial for protective immunity against Mtb. However, the immune responses including the T cell response are not sufficient to eliminate the bacilli and to provide sterile immunity. In an initial attempt to identify factors that shape the Th1 response, we observed that CD4+ Th1 cells, notably IFN-γ-secretors, co-expressed ICOS during murine TB. These data pointed to ICOS as an important player in the formation of Th1 responses against Mtb. To gain detailed information about the kinetics and nature of the T cell response in absence of ICOS signaling, we infected WT and ICOS-/- mice with Mtb. ICOS deficiency resulted in an increased polyclonal CD4+ Th1 response against Mtb, most likely caused by robustly reduced numbers and frequencies of Treg in these mice. In contrast to the CD4+ Th1 response, the Mtb-specific CD8+ T cell response was reduced in the absence of ICOS, but only during the later stage of infection. In addition, not only Mtb-specific effector CD8+ T cells, but also effector memory CD8+ T cells were reduced in Mtb infected ICOS-/- mice. The reduction in CD8+ T cells was most likely not CD8+ T cell intrinsic – since CD8+ T cells revealed only marginal ICOS surface expression, but caused by impaired CD4+ T cell help. In sum, we confirmed our initial assumption; ICOS indeed influenced the Th1 cell response against Mtb, and moreover presence of ICOS was mandatory for normal Treg responses. The extent to which ICOS influenced single T cell populations, notably CD4+ Th1, Treg, and CD8+ T cells, and conclusively the interaction among themselves, differed between spleen and lung. As a result, ICOS-/- mice showed improved control of Mtb in the spleens, but not in the lungs during the late chronic phase of infection

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

De Novo-Whole Genome Assembly of the Roborovski Dwarf Hamster (Phodopus roborovskii) Genome: An Animal Model for Severe/Critical COVID-19

The Roborovski dwarf hamster Phodopus roborovskii belongs to the Phodopus genus, one of the seven within Cricetinae subfamily. Like other rodents such as mice, rats, or ferrets, hamsters can be important animal models for a range of diseases. Whereas the Syrian hamster from the genus Mesocricetus is now widely used as a model for mild-to-moderate coronavirus disease 2019, Roborovski dwarf hamster shows a severe-to-lethal course of disease upon infection with the novel human coronavirus severe acute respiratory syndrome coronavirus 2