A Therapeutic Non-self-reactive SARS-CoV-2 Antibody Protects from Lung Pathology in a COVID-19 Hamster Model.

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from 10 COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb, CV07-209, neutralized authentic SARS-CoV-2 with an IC50 value of 3.1 ng/mL. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2-neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss, and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.

Recognition of microbial viability via TLR8 drives TFH cell differentiation and vaccine responses.

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (TFH cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting TFH cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust TFH cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of TFH cell differentiation and a promising target for TFH cell-skewing vaccine adjuvants.

Virus-induced senescence is a driver and therapeutic target in COVID-19.

Derailed cytokine and immune cell networks account for the organ damage and the clinical severity of COVID-19 (refs. 1-4). Here we show that SARS-CoV-2, like other viruses, evokes cellular senescence as a primary stress response in infected cells. Virus-induced senescence (VIS) is indistinguishable from other forms of cellular senescence and is accompanied by a senescence-associated secretory phenotype (SASP), which comprises pro-inflammatory cytokines, extracellular-matrix-active factors and pro-coagulatory mediators5-7. Patients with COVID-19 displayed markers of senescence in their airway mucosa in situ and increased serum levels of SASP factors. In vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, which mirrored hallmark features of COVID-19 such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue1,8,9. Moreover, supernatant from VIS cells, including SARS-CoV-2-induced senescence, induced neutrophil extracellular trap formation and activation of platelets and the clotting cascade. Senolytics such as navitoclax and a combination of dasatinib plus quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-infected hamsters and mice. Our findings mark VIS as a pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest that senolytic targeting of virus-infected cells is a treatment option against SARS-CoV-2 and perhaps other viral infections.

Ventilator-induced Lung Injury Is Modulated by the Circadian Clock.

Rationale: Mechanical ventilation (MV) is life-saving but may evoke ventilator-induced lung injury (VILI). Objectives: To explore how the circadian clock modulates severity of murine VILI via the core clock component BMAL1 (basic helix-loop-helix ARNT like 1) in myeloid cells. Methods: Myeloid cell BMAL1-deficient (LysM (lysozyme 2 promoter/enhancer driving cre recombinase expression)Bmal1-/-) or wild-type control (LysMBmal1+/+) mice were subjected to 4 hours MV (34 ml/kg body weight) to induce lung injury. Ventilation was initiated at dawn or dusk or in complete darkness (circadian time [CT] 0 or CT12) to determine diurnal and circadian effects. Lung injury was quantified by lung function, pulmonary permeability, blood gas analysis, neutrophil recruitment, inflammatory markers, and histology. Neutrophil activation and oxidative burst were analyzed ex vivo. Measurements and Main Results: In diurnal experiments, mice ventilated at dawn exhibited higher permeability and neutrophil recruitment compared with dusk. Experiments at CT showed deterioration of pulmonary function, worsening of oxygenation, and increased mortality at CT0 compared with CT12. Wild-type neutrophils isolated at dawn showed higher activation and reactive oxygen species production compared with dusk, whereas these day-night differences were dampened in LysMBmal1-/- neutrophils. In LysMBmal1-/- mice, circadian variations in VILI severity were dampened and VILI-induced mortality at CT0 was reduced compared with LysMBmal1+/+ mice. Conclusions: Inflammatory response and lung barrier dysfunction upon MV exhibit diurnal variations, regulated by the circadian clock. LysMBmal1-/- mice are less susceptible to ventilation-induced pathology and lack circadian variation of severity compared with LysMBmal1+/+ mice. Our data suggest that the internal clock in myeloid cells is an important modulator of VILI.

Hamster models of COVID-19 pneumonia reviewed: How human can they be?

The dramatic global consequences of the coronavirus disease 2019 (COVID-19) pandemic soon fueled quests for a suitable model that would facilitate the development and testing of therapies and vaccines. In contrast to other rodents, hamsters are naturally susceptible to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the Syrian hamster (Mesocricetus auratus) rapidly developed into a popular model. It recapitulates many characteristic features as seen in patients with a moderate, self-limiting course of the disease such as specific patterns of respiratory tract inflammation, vascular endothelialitis, and age dependence. Among 4 other hamster species examined, the Roborovski dwarf hamster (Phodopus roborovskii) more closely mimics the disease in highly susceptible patients with frequent lethal outcome, including devastating diffuse alveolar damage and coagulopathy. Thus, different hamster species are available to mimic different courses of the wide spectrum of COVID-19 manifestations in humans. On the other hand, fewer diagnostic tools and information on immune functions and molecular pathways are available than in mice, which limits mechanistic studies and inference to humans in several aspects. Still, under pandemic conditions with high pressure on progress in both basic and clinically oriented research, the Syrian hamster has turned into the leading non-transgenic model at an unprecedented pace, currently used in innumerable studies that all aim to combat the impact of the virus with its new variants of concern. As in other models, its strength rests upon a solid understanding of its similarities to and differences from the human disease, which we review here.

ADAR1 Is Required for Dendritic Cell Subset Homeostasis and Alveolar Macrophage Function.

RNA editing by adenosine deaminases acting on dsRNA (ADAR) has become of increasing medical relevance, particularly because aberrant ADAR1 activity has been associated with autoimmunity and malignancies. However, the role of ADAR1 in dendritic cells (DC), representing critical professional APCs, is unknown. We have established conditional murine CD11c Cre-mediated ADAR1 gene ablation, which did not induce general apoptosis in CD11c+ cells but instead manifests in cell type-specific effects in DC subpopulations. Bone marrow-derived DC subset analysis revealed an incapacity to differentiate CD103 DC+ in both bulk bone marrow and purified pre-DC lineage progenitor assays. ADAR1 deficiency further resulted in a preferential systemic loss of CD8+/CD103+ DCs, revealing critical dependency on ADAR1, whereas other DC subpopulations were moderately affected or unaffected. Additionally, alveolar macrophages were depleted and dysfunctional, resembling pulmonary alveolar proteinosis. These results reveal an unrecognized role of ADAR1 in DC subset homeostasis and unveils the cell type-specific effects of RNA editing.

Digital Image Analyses on Whole-Lung Slides in Mouse Models of Acute Pneumonia.

Descriptive histopathology of mouse models of pneumonia is essential in assessing the outcome of infections, molecular manipulations, or therapies in the context of whole lungs. Quantitative comparisons between experimental groups, however, have been limited to laborious stereology or ill-defined scoring systems that depend on the subjectivity of a more or less experienced observer. Here, we introduce self-learning digital image analyses that allow us to transform optical information from whole mouse lung sections into statistically testable data. A pattern-recognition-based software and a nuclear count algorithm were adopted to quantify user-defined pathologies from whole slide scans of lungs infected with Streptococcus pneumoniae or influenza A virus compared with PBS-challenged lungs. The readout parameters "relative area affected" and "nuclear counts per area" are proposed as relevant criteria for the quantification of lesions from hematoxylin and eosin-stained sections, also allowing for the generation of a heat map of, for example, immune cell infiltrates with anatomical assignments across entire lung sections. Moreover, when combined with immunohistochemical labeling of marker proteins, both approaches are useful for the identification and counting of, for example, immune cell populations, as validated here by direct comparisons with flow cytometry data. The solutions can easily and flexibly be adjusted to specificities of different models or pathogens. Automated digital analyses of whole mouse lung sections may set a new standard for the user-defined, high-throughput comparative quantification of histological and immunohistochemical images. Still, our algorithms established here are only a start, and need to be tested in additional studies and other applications in the future.

Sphingosine Kinase 1 Regulates Inflammation and Contributes to Acute Lung Injury in Pneumococcal Pneumonia via the Sphingosine-1-Phosphate Receptor 2.

OBJECTIVES: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia. DESIGN: Controlled, in vitro, ex vivo, and in vivo laboratory study. SUBJECTS: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells. INTERVENTIONS: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin. MEASUREMENTS AND MAIN RESULTS: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1 mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase. CONCLUSIONS: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.

Soluble mucus component CLCA1 modulates expression of leukotactic cytokines and BPIFA1 in murine alveolar macrophages but not in bone marrow-derived macrophages.

The secreted airway mucus cell protein chloride channel regulator, calcium-activated 1, CLCA1, plays a role in inflammatory respiratory diseases via as yet unidentified pathways. For example, deficiency of CLCA1 in a mouse model of acute pneumonia resulted in reduced cytokine expression with less leukocyte recruitment and the human CLCA1 was shown to be capable of activating macrophages in vitro. Translation of experimental data between human and mouse models has proven problematic due to several CLCA species-specific differences. We therefore characterized activation of macrophages by CLCA1 in detail in solely murine ex vivo and in vitro models. Only alveolar but not bone marrow-derived macrophages freshly isolated from C57BL6/J mice increased their expression levels of several pro-inflammatory and leukotactic cytokines upon CLCA1 stimulation. Among the most strongly regulated genes, we identified the host-protective and immunomodulatory airway mucus component BPIFA1, previously unknown to be expressed by airway macrophages. Furthermore, evidence from an in vivo Staphylococcus aureus pneumonia mouse model suggests that CLCA1 may also modify BPIFA1 expression in airway epithelial cells. Our data underscore and specify the role of mouse CLCA1 in inflammatory airway disease to activate airway macrophages. In addition to its ability to upregulate cytokine expression which explains previous observations in the Clca1-deficient S. aureus pneumonia mouse model, modulation of BPIFA1 expression expands the role of CLCA1 in airway disease to involvement in more complex downstream pathways, possibly including liquid homeostasis, airway protection, and antimicrobial defense.

Delay in antibiotic therapy results in fatal disease outcome in murine pneumococcal pneumonia.

BACKGROUND: Community-acquired pneumonia (CAP) remains a major cause of death worldwide. Mechanisms underlying the detrimental outcome despite adequate antibiotic therapy and comorbidity management are still not fully understood. METHODS: To model timely versus delayed antibiotic therapy in patients, mice with pneumococcal pneumonia received ampicillin twice a day starting early (24 h) or late (48 h) after infection. Clinical readouts and local and systemic inflammatory mediators after early and late antibiotic intervention were examined. RESULTS: Early antibiotic intervention rescued mice, limited clinical symptoms and restored fitness, whereas delayed therapy resulted in high mortality rates. Recruitment of innate immune cells remained unaffected by antibiotic therapy. However, both early and late antibiotic intervention dampened local levels of inflammatory mediators in the alveolar spaces. Early treatment protected from barrier breakdown, and reduced levels of vascular endothelial growth factor (VEGF) and perivascular and alveolar edema formation. In contrast, at 48 h post infection, increased pulmonary leakage was apparent and not reversed by late antibiotic treatment. Concurrently, levels of VEGF remained high and no beneficial effect on edema formation was evident despite therapy. Moreover, early but not late treatment protected mice from a vast systemic inflammatory response. CONCLUSIONS: Our data show that only early antibiotic therapy, administered prior to breakdown of the alveolar-capillary barrier and systemic inflammation, led to restored fitness and rescued mice from fatal streptococcal pneumonia. The findings highlight the importance of identifying CAP patients prior to lung barrier failure and systemic inflammation and of handling CAP as a medical emergency.

Ventilator-induced lung injury is aggravated by antibiotic mediated microbiota depletion in mice.

BACKGROUND: Antibiotic exposure alters the microbiota, which can impact the inflammatory immune responses. Critically ill patients frequently receive antibiotic treatment and are often subjected to mechanical ventilation, which may induce local and systemic inflammatory responses and development of ventilator-induced lung injury (VILI). The aim of this study was to investigate whether disruption of the microbiota by antibiotic therapy prior to mechanical ventilation affects pulmonary inflammatory responses and thereby the development of VILI. METHODS: Mice underwent 6-8 weeks of enteral antibiotic combination treatment until absence of cultivable bacteria in fecal samples was confirmed. Control mice were housed equally throughout this period. VILI was induced 3 days after completing the antibiotic treatment protocol, by high tidal volume (HTV) ventilation (34 ml/kg; positive end-expiratory pressure = 2 cmH2O) for 4 h. Differences in lung function, oxygenation index, pulmonary vascular leakage, macroscopic assessment of lung injury, and leukocyte and lymphocyte differentiation were assessed. Control groups of mice ventilated with low tidal volume and non-ventilated mice were analyzed accordingly. RESULTS: Antibiotic-induced microbiota depletion prior to HTV ventilation led to aggravation of VILI, as shown by increased pulmonary permeability, increased oxygenation index, decreased pulmonary compliance, enhanced macroscopic lung injury, and increased cytokine/chemokine levels in lung homogenates. CONCLUSIONS: Depletion of the microbiota by broad-spectrum antibiotics prior to HTV ventilation renders mice more susceptible to developing VILI, which could be clinically relevant for critically ill patients frequently receiving broad-spectrum antibiotics.

Validation of Digital Microscopy Compared With Light Microscopy for the Diagnosis of Canine Cutaneous Tumors.

Integration of new technologies, such as digital microscopy, into a highly standardized laboratory routine requires the validation of its performance in terms of reliability, specificity, and sensitivity. However, a validation study of digital microscopy is currently lacking in veterinary pathology. The aim of the current study was to validate the usability of digital microscopy in terms of diagnostic accuracy, speed, and confidence for diagnosing and differentiating common canine cutaneous tumor types and to compare it to classical light microscopy. Therefore, 80 histologic sections including 17 different skin tumor types were examined twice as glass slides and twice as digital whole-slide images by 6 pathologists with different levels of experience at 4 time points. Comparison of both methods found digital microscopy to be noninferior for differentiating individual tumor types within the category epithelial and mesenchymal tumors, but diagnostic concordance was slightly lower for differentiating individual round cell tumor types by digital microscopy. In addition, digital microscopy was associated with significantly shorter diagnostic time, but diagnostic confidence was lower and technical quality was considered inferior for whole-slide images compared with glass slides. Of note, diagnostic performance for whole-slide images scanned at 200× magnification was noninferior in diagnostic performance for slides scanned at 400×. In conclusion, digital microscopy differs only minimally from light microscopy in few aspects of diagnostic performance and overall appears adequate for the diagnosis of individual canine cutaneous tumors with minor limitations for differentiating individual round cell tumor types and grading of mast cell tumors.

Vasculotide reduces pulmonary hyperpermeability in experimental pneumococcal pneumonia.

BACKGROUND: Community-acquired pneumonia (CAP) is a significant cause of morbidity and mortality worldwide. Despite effective antimicrobial therapy, CAP can induce pulmonary endothelial hyperpermeability resulting in life-threatening lung failure due to an exaggerated host-pathogen interaction. Treatment of acute lung injury is mainly supportive because key elements of inflammation-induced barrier disruption remain undetermined. Angiopoietin-1 (Ang-1)-mediated Tie2 activation reduces, and the Ang-1 antagonist Ang-2 increases, inflammation and endothelial permeability in sepsis. Vasculotide (VT) is a polyethylene glycol-clustered Tie2-binding peptide that mimics the actions of Ang-1. The aim of our study was to experimentally test whether VT is capable of diminishing pneumonia-induced lung injury. METHODS: VT binding and phosphorylation of Tie2 were analyzed using tryptophan fluorescence spectroscopy and phospho-Tie-2 enzyme-linked immunosorbent assay. Human and murine lung endothelial cells were investigated by immunofluorescence staining and electric cell-substrate impedance sensing. Pulmonary hyperpermeability was quantified in VT-pretreated, isolated, perfused, and ventilated mouse lungs stimulated with the pneumococcal exotoxin pneumolysin (PLY). Furthermore, Streptococcus pneumoniae-infected mice were therapeutically treated with VT. RESULTS: VT showed dose-dependent binding and phosphorylation of Tie2. Pretreatment with VT protected lung endothelial cell monolayers from PLY-induced disruption. In isolated mouse lungs, VT decreased PLY-induced pulmonary permeability. Likewise, therapeutic treatment with VT of S. pneumoniae-infected mice significantly reduced pneumonia-induced hyperpermeability. However, effects by VT on the pulmonary or systemic inflammatory response were not observed. CONCLUSIONS: VT promoted pulmonary endothelial stability and reduced lung permeability in different models of pneumococcal pneumonia. Thus, VT may provide a novel therapeutic perspective for reduction of permeability in pneumococcal pneumonia-induced lung injury.

Pulmonary immunostimulation with MALP-2 in influenza virus-infected mice increases survival after pneumococcal superinfection.

Pulmonary infection with influenza virus is frequently complicated by bacterial superinfection, with Streptococcus pneumoniae being the most prevalent causal pathogen and hence often associated with high morbidity and mortality rates. Local immunosuppression due to pulmonary influenza virus infection has been identified as a major cause of the pathogenesis of secondary bacterial lung infection. Thus, specific local stimulation of the pulmonary innate immune system in subjects with influenza virus infection might improve the host defense against secondary bacterial pathogens. In the present study, we examined the effect of pulmonary immunostimulation with Toll-like receptor 2 (TLR-2)-stimulating macrophage-activating lipopeptide 2 (MALP-2) in influenza A virus (IAV)-infected mice on the course of subsequent pneumococcal superinfection. Female C57BL/6N mice infected with IAV were treated with MALP-2 on day 5 and challenged with S. pneumoniae on day 6. Intratracheal MALP-2 application increased proinflammatory cytokine and chemokine release and enhanced the recruitment of leukocytes, mainly neutrophils, into the alveolar space of IAV-infected mice, without detectable systemic side effects. Local pulmonary instillation of MALP-2 in IAV-infected mice 24 h before transnasal pneumococcal infection considerably reduced the bacterial number in the lung tissue without inducing exaggerated inflammation. The pulmonary viral load was not altered by MALP-2. Clinically, MALP-2 treatment of IAV-infected mice increased survival rates and reduced hypothermia and body weight loss after pneumococcal superinfection compared to those of untreated coinfected mice. In conclusion, local immunostimulation with MALP-2 in influenza virus-infected mice improved pulmonary bacterial elimination and increased survival after subsequent pneumococcal superinfection.

Murine CLCA5 is uniquely expressed in distinct niches of airway epithelial cells.

The murine mCLCA5 protein is a member of the chloride channel regulators, calcium-activated (CLCA) family and is suspected to play a role in airway mucus cell differentiation. Although mCLCA5 mRNA was previously found in total lung extracts, the expressing cells and functions in the naive murine respiratory tract are unknown. Therefore, mCLCA5 protein expression was identified by immunohistochemistry and confocal laser scanning microscopy using entire lung sections of naive mice. Moreover, we determined mRNA levels of functionally related genes (mClca3, mClca5, Muc5ac and Muc5b) and quantified mCLCA5-, mCLCA3- and CC10-positive cells and periodic acid-Schiff-positive mucus cells in naive, PBS-treated or Staphylococcus aureus-infected mice. We also investigated mCLCA5 protein expression in Streptococcus pneumoniae and influenza virus lung infection models. Finally, we determined species-specific differences in the expression patterns of the murine mCLCA5 and its human and porcine orthologs, hCLCA2 and pCLCA2. The mCLCA5 protein is uniquely expressed in highly select bronchial epithelial cells and submucosal glands in naive mice, consistent with anatomical locations of progenitor cell niches. Under conditions of challenge (PBS, S. aureus, S. pneumoniae, influenza virus), mRNA and protein expression strongly declined with protein recovery only in models retaining intact epithelial cells. In contrast to mice, human and porcine bronchial epithelial cells do not express their respective mCLCA5 orthologs and submucosal glands had fewer expressing cells, indicative of fundamental differences in mice versus humans and pigs.

Prospectively defined murine mesenchymal stem cells inhibit Klebsiella pneumoniae-induced acute lung injury and improve pneumonia survival.

BACKGROUND: Numerous studies have described the immunosuppressive capacity of mesenchymal stem cells (MSC) but these studies use mixtures of heterogeneous progenitor cells for in vitro expansion. Recently, multipotent MSC have been prospectively identified in murine bone marrow (BM) on the basis of PDFGRa(+) SCA1(+) CD45(-) TER119(-) (PαS) expression but the immunomodulatory capacity of these MSC is unknown. METHODS: We isolated PαS MSC by high-purity FACS sorting of murine BM and after in vitro expansion we analyzed the in vivo immunomodulatory activity during acute pneumonia. PαS MSC (1 × 10(6)) were applied intratracheally 4 h after acute respiratory Klebsiella pneumoniae induced infection. RESULTS: PαS MSC treatment resulted in significantly reduced alveolitis and protein leakage in comparison to mock-treated controls. PαS MSC-treated mice exhibited significantly reduced alveolar TNF-α and IL-12p70 expression, while IL-10 expression was unaffected. Dissection of respiratory dendritic cell (DC) subsets by multiparameter flow cytometry revealed significantly reduced lung DC infiltration and significantly reduced CD86 costimulatory expression on lung CD103(+) DC in PαS MSC-treated mice. In the post-acute phase of pneumonia, PαS MSC-treated animals exhibited significantly reduced respiratory IL-17(+) CD4(+) T cells and IFN-γ(+) CD4(+) T cells. Moreover, PαS MSC treatment significantly improved overall pneumonia survival and did not increase bacterial load. CONCLUSION: In this study we demonstrated for the first time the feasibility and in vivo immunomodulatory capacity of prospectively defined MSC in pneumonia.

Miniaturized bronchoscopy enables unilateral investigation, application, and sampling in mice.

Lung diseases, including pneumonia and asthma, are among the most prevalent human disorders, and murine models have been established to investigate their pathobiology and develop novel treatment approaches. Whereas bronchoscopy is valuable for diagnostic and therapeutic procedures in patients, no equivalent for small rodents has been established. Here, we introduce a miniaturized video-bronchoscopy system offering new opportunities in experimental lung research. With an outer diameter of 0.75 mm, it is possible to advance the optics into the main bronchi of mice. An irrigation channel allows bronchoalveolar lavage and unilateral application of substances to one lung. Even a unilateral infection is possible, enabling researchers to use the contralateral lung as internal control.

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.

Dependence of the Ki67 Labelling Index of Selected Canine Tumours on Patient Age, Sex and Tumour Size.

Cell proliferation is a fundamental criterion in the assessment of malignant progression of many tumours and an essential parameter in several grading schemes. However, proliferation may be dependent on patient age and other variables, as shown in normal tissues, cultured cells and human neoplasms. We thus hypothesized that age or other patient or tumour-related parameters might generally affect proliferation in canine neoplasms, which might be of value for optimizing prognostic algorithms. We performed linear regression analyses to associate age, sex and tumour size with digitally quantified immunohistochemical Ki67 labelling indices (Ki67-LIs) of 495 canine tumours, including cutaneous mast cell tumours (MCTs, n = 70), soft tissue sarcomas (n = 61), plasmacytomas (n = 86), trichoblastomas (n = 62) and perianal gland adenomas (PGAs, n = 95) as well as testicular interstitial (n = 65) and Sertoli cell tumours (n = 56). In MCTs, the Ki67-LI increased 1.13-fold per year of age (P <0.05) in bitches but not in males. Conversely, in PGAs it rose 1.10-fold per year in males (P <0.05) while it decreased 0.95-fold in bitches (P = 0.37). Only in MCTs and PGAs was the Ki67-LI associated with tumour size, albeit in oppositional directions (MCTs: 1.26-fold per cm diameter, P <0.01; PGAs: 0.76-fold, P <0.01). No correlations were found in the other tumour types. The few sex-dependent correlations with patient age and tumour size established here indicate highly tumour-type specific mechanisms, but the diagnostic consequences are uncertain.