ABSTRACT
Thymomas and mediastinal lymphomas are the most common mediastinal neoplasms in cats. While mediastinal lymphoma occurs primarily in younger cats (approximately 2 to 4 years of age), thymomas are diagnosed almost exclusively in older cats. However, the following case shows that this is not necessarily always the case.A 3-year- and 11-month-old neutered male European Shorthair was presented with a preliminary report of exercise intolerance and restricted respiration. Both symptoms occurred primarily at high ambient temperatures. Radiographically, a mediastinal mass filled a significant part of the thoracic cavity. A computed tomographic examination and ultrasound-guided TruCut biopsy were performed. Histologic examination suggested a thymoma. A sternal thoracotomy and resection of the circumferential proliferation was conducted. Subsequent histopathologic examination confirmed the suspected diagnosis of a mediastinal thymoma. The cat recovered well postoperatively, and a clinical and radiographic follow-up examination was unremarkable after 8 months.
Subject(s)
Cat Diseases , Thymoma , Thymus Neoplasms , Cats , Animals , Male , Cat Diseases/surgery , Cat Diseases/diagnosis , Cat Diseases/pathology , Cat Diseases/diagnostic imaging , Thymoma/veterinary , Thymoma/surgery , Thymoma/diagnosis , Thymoma/pathology , Thymus Neoplasms/veterinary , Thymus Neoplasms/surgery , Thymus Neoplasms/diagnosis , Thymus Neoplasms/pathology , Mediastinal Neoplasms/veterinary , Mediastinal Neoplasms/surgery , Mediastinal Neoplasms/diagnosis , Mediastinal Neoplasms/pathology , Tomography, X-Ray Computed/veterinaryABSTRACT
The pulmonary epithelial glycocalyx is rich in glycosaminoglycans such as hyaluronan and heparan sulfate. Despite their presence, the importance of these glycosaminoglycans in bacterial lung infections remains elusive. To address this, we intranasally inoculated mice with Streptococcus pneumoniae in the presence or absence of enzymes targeting pulmonary hyaluronan and heparan sulfate, followed by characterization of subsequent disease pathology, pulmonary inflammation, and lung barrier dysfunction. Enzymatic degradation of hyaluronan and heparan sulfate exacerbated pneumonia in mice, as evidenced by increased disease scores and alveolar neutrophil recruitment. However, targeting epithelial hyaluronan in combination with Streptococcus pneumoniae infection further exacerbated systemic disease, indicated by elevated splenic bacterial load and plasma levels of pro-inflammatory cytokines. In contrast, enzymatic cleavage of heparan sulfate resulted in increased bronchoalveolar bacterial burden, lung damage and pulmonary inflammation in mice infected with Streptococcus pneumoniae. Accordingly, heparinase-treated mice also exhibited disrupted lung barrier integrity as evidenced by higher alveolar edema scores and vascular protein leakage into the airways. This finding was corroborated in a human alveolus-on-a-chip platform, confirming that heparinase treatment also disrupts the human lung barrier during Streptococcus pneumoniae infection. Notably, enzymatic pre-treatment with either hyaluronidase or heparinase also rendered human epithelial cells more sensitive to pneumococcal-induced barrier disruption, as determined by transepithelial electrical resistance measurements, consistent with our findings in murine pneumonia. Taken together, these findings demonstrate the importance of intact hyaluronan and heparan sulfate in limiting pneumococci-induced damage, pulmonary inflammation, and epithelial barrier function and integrity. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
ABSTRACT
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.
Subject(s)
COVID-19 , Endothelial Cells , Lung , Neutrophils , SARS-CoV-2 , Single-Cell Analysis , COVID-19/immunology , COVID-19/virology , COVID-19/pathology , Animals , Humans , Neutrophils/immunology , SARS-CoV-2/immunology , Lung/pathology , Lung/virology , Lung/immunology , Cricetinae , Endothelial Cells/virology , Endothelial Cells/pathology , Inflammation/pathology , Mesocricetus , Disease Models, Animal , Male , Species SpecificityABSTRACT
The development of effective SARS-CoV-2 vaccines has been essential to control COVID-19, but significant challenges remain. One problem is intramuscular administration, which does not induce robust mucosal immune responses in the upper airways-the primary site of infection and virus shedding. Here we compare the efficacy of a mucosal, replication-competent yet fully attenuated virus vaccine, sCPD9-ΔFCS, and the monovalent mRNA vaccine BNT162b2 in preventing transmission of SARS-CoV-2 variants B.1 and Omicron BA.5 in two scenarios. Firstly, we assessed the protective efficacy of the vaccines by exposing vaccinated male Syrian hamsters to infected counterparts. Secondly, we evaluated transmission of the challenge virus from vaccinated and subsequently challenged male hamsters to naïve contacts. Our findings demonstrate that the live-attenuated vaccine (LAV) sCPD9-ΔFCS significantly outperformed the mRNA vaccine in preventing virus transmission in both scenarios. Our results provide evidence for the advantages of locally administered LAVs over intramuscularly administered mRNA vaccines in preventing infection and reducing virus transmission.
Subject(s)
COVID-19 Vaccines , COVID-19 , Animals , Cricetinae , Male , Humans , BNT162 Vaccine , COVID-19/prevention & control , mRNA Vaccines , SARS-CoV-2 , Mesocricetus , Antibodies, Viral , Antibodies, NeutralizingABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0276115.].
ABSTRACT
Influenza A virus (IAV) defective interfering particles (DIPs) are considered as new promising antiviral agents. Conventional DIPs (cDIPs) contain a deletion in the genome and can only replicate upon co-infection with infectious standard virus (STV), during which they suppress STV replication. We previously discovered a new type of IAV DIP "OP7" that entails genomic point mutations and displays higher antiviral efficacy than cDIPs. To avoid safety concerns for the medical use of OP7 preparations, we developed a production system that does not depend on infectious IAV. We reconstituted a mixture of DIPs consisting of cDIPs and OP7 chimera DIPs, in which both harbor a deletion in their genome. To complement the defect, the deleted viral protein is expressed by the suspension cell line used for production in shake flasks. Here, DIP preparations harvested are not contaminated with infectious virions, and the fraction of OP7 chimera DIPs depended on the multiplicity of infection. Intranasal administration of OP7 chimera DIP material was well tolerated in mice. A rescue from an otherwise lethal IAV infection and no signs of disease upon OP7 chimera DIP co-infection demonstrated the remarkable antiviral efficacy. The clinical development of this new class of broad-spectrum antiviral may contribute to pandemic preparedness.
Subject(s)
Coinfection , Influenza A virus , Influenza, Human , Animals , Mice , Humans , Defective Viruses/genetics , Influenza A virus/genetics , Virus Replication , Antiviral Agents/pharmacologyABSTRACT
The development of atopic dermatitis in infancy, and subsequent allergies, such as asthma in later childhood, is known as the atopic march. The mechanism is largely unknown, however the course of disease indicates an inter-epithelial crosstalk, through the onset of inflammation in the skin and progression to other mucosal epithelia. In this study, we investigated if and how skin-lung epithelial crosstalk contributes to the development of the atopic march. First, we emulated inter-epithelial crosstalk through indirect coculture of bioengineered atopic-like skin disease models and three-dimensional bronchial epithelial models triggering an asthma-like phenotype in the latter. A subsequent secretome analysis identified thrombospondin-1, CD44, complement factor C3, fibronectin, and syndecan-4 as potentially relevant skin-derived mediators. Because these mediators are extracellular matrix-related proteins, we then studied the involvement of the extracellular matrix, unveiling distinct proteomic, transcriptomic, and ultrastructural differences in atopic samples. The latter indicated extracellular matrix remodeling triggering the release of the above-mentioned mediators. In vivo mouse data showed that exposure to these mediators dysregulated activated circadian clock genes which are increasingly discussed in the context of atopic diseases and asthma development. Our data point toward the existence of a skin-lung axis that could contribute to the atopic march driven by skin extracellular matrix remodeling.
ABSTRACT
Introduction: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the danger posed by human coronaviruses. Rapid emergence of immunoevasive variants and waning antiviral immunity decrease the effect of the currently available vaccines, which aim at induction of neutralizing antibodies. In contrast, T cells are marginally affected by antigen evolution although they represent the major mediators of virus control and vaccine protection against virus-induced disease. Materials and methods: We generated a multi-epitope vaccine (PanCoVac) that encodes the conserved T cell epitopes from all structural proteins of coronaviruses. PanCoVac contains elements that facilitate efficient processing and presentation of PanCoVac-encoded T cell epitopes and can be uploaded to any available vaccine platform. For proof of principle, we cloned PanCoVac into a non-integrating lentivirus vector (NILV-PanCoVac). We chose Roborovski dwarf hamsters for a first step in evaluating PanCoVac in vivo. Unlike mice, they are naturally susceptible to SARS-CoV-2 infection. Moreover, Roborovski dwarf hamsters develop COVID-19-like disease after infection with SARS-CoV-2 enabling us to look at pathology and clinical symptoms. Results: Using HLA-A*0201-restricted reporter T cells and U251 cells expressing a tagged version of PanCoVac, we confirmed in vitro that PanCoVac is processed and presented by HLA-A*0201. As mucosal immunity in the respiratory tract is crucial for protection against respiratory viruses such as SARS-CoV-2, we tested the protective effect of single-low dose of NILV-PanCoVac administered via the intranasal (i.n.) route in the Roborovski dwarf hamster model of COVID-19. After infection with ancestral SARS-CoV-2, animals immunized with a single-low dose of NILV-PanCoVac i.n. did not show symptoms and had significantly decreased viral loads in the lung tissue. This protective effect was observed in the early phase (2 days post infection) after challenge and was not dependent on neutralizing antibodies. Conclusion: PanCoVac, a multi-epitope vaccine covering conserved T cell epitopes from all structural proteins of coronaviruses, might protect from severe disease caused by SARS-CoV-2 variants and future pathogenic coronaviruses. The use of (HLA-) humanized animal models will allow for further efficacy studies of PanCoVac-based vaccines in vivo.
Subject(s)
COVID-19 , Viral Vaccines , Cricetinae , Humans , Animals , Mice , COVID-19/prevention & control , SARS-CoV-2 , COVID-19 Vaccines , Epitopes, T-Lymphocyte , Administration, Intranasal , Antibodies, Neutralizing , HLA-A AntigensABSTRACT
Live attenuated vaccines (LAVs) administered via the mucosal route may offer better control of the COVID-19 pandemic than non-replicating vaccines injected intramuscularly. Conceptionally, LAVs have several advantages, including presentation of the entire antigenic repertoire of the virus, and the induction of strong mucosal immunity. Thus, immunity induced by LAV could offer superior protection against future surges of COVID-19 cases caused by emerging SARS-CoV-2 variants. However, LAVs carry the risk of unintentional transmission. To address this issue, we investigated whether transmission of a SARS-CoV-2 LAV candidate can be blocked by removing the furin cleavage site (FCS) from the spike protein. The level of protection and immunity induced by the attenuated virus with the intact FCS was virtually identical to the one induced by the attenuated virus lacking the FCS. Most importantly, removal of the FCS completely abolished horizontal transmission of vaccine virus between cohoused hamsters. Furthermore, the vaccine was safe in immunosuppressed animals and showed no tendency to recombine in vitro or in vivo with a SARS-CoV-2 field strain. These results indicate that removal of the FCS from SARS-CoV-2 LAV is a promising strategy to increase vaccine safety and prevent vaccine transmission without compromising vaccine efficacy.
Subject(s)
COVID-19 Vaccines , COVID-19 , Animals , Cricetinae , Humans , COVID-19/prevention & control , Pandemics , SARS-CoV-2 , Vaccines, Attenuated , Antibodies, Viral , Antibodies, NeutralizingABSTRACT
Vaccines play a critical role in combating the COVID-19 pandemic. Future control of the pandemic requires improved vaccines with high efficacy against newly emerging SARS-CoV-2 variants and the ability to reduce virus transmission. Here we compare immune responses and preclinical efficacy of the mRNA vaccine BNT162b2, the adenovirus-vectored spike vaccine Ad2-spike and the live-attenuated virus vaccine candidate sCPD9 in Syrian hamsters, using both homogeneous and heterologous vaccination regimens. Comparative vaccine efficacy was assessed by employing readouts from virus titrations to single-cell RNA sequencing. Our results show that sCPD9 vaccination elicited the most robust immunity, including rapid viral clearance, reduced tissue damage, fast differentiation of pre-plasmablasts, strong systemic and mucosal humoral responses, and rapid recall of memory T cells from lung tissue after challenge with heterologous SARS-CoV-2. Overall, our results demonstrate that live-attenuated vaccines offer advantages over currently available COVID-19 vaccines.
Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Cricetinae , Humans , Vaccines, Attenuated , COVID-19/prevention & control , COVID-19 Vaccines , BNT162 Vaccine , Pandemics , MesocricetusABSTRACT
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.
Subject(s)
MicroRNAs , Pneumonia, Pneumococcal , Animals , Humans , Mice , Inflammation/genetics , Inflammation/microbiology , Inflammation/pathology , Lung/pathology , Mice, Knockout , MicroRNAs/genetics , Pneumonia, Pneumococcal/genetics , Pneumonia, Pneumococcal/microbiology , Pneumonia, Pneumococcal/pathology , Streptococcus pneumoniaeABSTRACT
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.
Subject(s)
Bacteriophages , Immunity, Humoral , Animals , Mice , Mice, Inbred C57BL , Pseudomonas aeruginosa , Escherichia coliABSTRACT
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.
Subject(s)
Circadian Clocks , Ventilator-Induced Lung Injury , Mice , Animals , Circadian Clocks/genetics , ARNTL Transcription Factors/genetics , ARNTL Transcription Factors/metabolism , Lung , Ventilator-Induced Lung Injury/genetics , Ventilator-Induced Lung Injury/metabolism , Circadian Rhythm/genetics , Mice, Inbred C57BLABSTRACT
Pneumonia is the most common cause of the acute respiratory distress syndrome (ARDS). Here, we identified loss of endothelial cystic fibrosis transmembrane conductance regulator (CFTR) as an important pathomechanism leading to lung barrier failure in pneumonia-induced ARDS. CFTR was down-regulated after Streptococcus pneumoniae infection ex vivo or in vivo in human or murine lung tissue, respectively. Analysis of isolated perfused rat lungs revealed that CFTR inhibition increased endothelial permeability in parallel with intracellular chloride ion and calcium ion concentrations ([Cl-]i and [Ca2+]i). Inhibition of the chloride ion-sensitive with-no-lysine kinase 1 (WNK1) protein with tyrphostin 47 or WNK463 replicated the effect of CFTR inhibition on endothelial permeability and endothelial [Ca2+]i, whereas WNK1 activation by temozolomide attenuated it. Endothelial [Ca2+]i transients and permeability in response to inhibition of either CFTR or WNK1 were prevented by inhibition of the cation channel transient receptor potential vanilloid 4 (TRPV4). Mice deficient in Trpv4 (Trpv4-/-) developed less lung edema and protein leak than their wild-type littermates after infection with S. pneumoniae. The CFTR potentiator ivacaftor prevented lung CFTR loss, edema, and protein leak after S. pneumoniae infection in wild-type mice. In conclusion, lung infection caused loss of CFTR that promoted lung edema formation through intracellular chloride ion accumulation, inhibition of WNK1, and subsequent disinhibition of TRPV4, resulting in endothelial calcium ion influx and vascular barrier failure. Ivacaftor prevented CFTR loss in the lungs of mice with pneumonia and may, therefore, represent a possible therapeutic strategy in people suffering from ARDS due to severe pneumonia.
Subject(s)
Chlorides , Pneumonia , Humans , Mice , Animals , Calcium , Lung , Cystic Fibrosis Transmembrane Conductance Regulator , TRPV Cation ChannelsABSTRACT
Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Currently, such models have received widespread attention in the study of SARS-CoV-2 causing the COVID-19 pandemic. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human alveolar-like organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human alveolar-like organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and FURIN as exemplary host factors of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human alveolar-like organoids and the subsequent qPCR to quantify the expression level of ACE2, TMPRSS2, and FURIN as host factors of SARS-CoV-2 on the mRNA level. Protocol set 3 contains an in-depth explanation on how to infect human alveolar-like organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human alveolar-like organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human alveolar-like organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human alveolar-like organoids based on material from different donors.
Subject(s)
COVID-19 , Humans , COVID-19/metabolism , SARS-CoV-2 , Furin/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Pandemics , Lung/metabolism , OrganoidsABSTRACT
Recent studies have revealed the dynamic and complex evolution of CLCA1 gene homologues in and between mammals and birds with a particularly high diversity in mammals. In contrast, CLCA2 has only been found as a single copy gene in mammals, to date. Furthermore, CLCA2 has only been investigated in few mammalian species but not in birds. Here, we established core genomic, protein biochemical and expressional properties of CLCA2 in several bird species and compared them with mammalian CLCA2. Chicken, turkey, quail and ostrich CLCA2 were compared to their mammalian orthologues using in silico, biochemical and expressional analyses. CLCA2 was found highly conserved not only at the level of genomic and exon architecture but also in terms of the canonical CLCA2 protein domain organization. The putatively prototypical galline CLCA2 (gCLCA2) was cloned and immunoblotting as well as immunofluorescence analyses of heterologously expressed gCLCA2 revealed protein cleavage, glycosylation patterns and anchoring in the plasma membrane similar to those of most mammalian CLCA2 orthologues. Immunohistochemistry found highly conserved CLCA2 expression in epidermal keratinocytes in all birds and mammals investigated. Our results suggest a highly conserved and likely evolutionarily indispensable role of CLCA2 in keratinocyte function. Its high degree of conservation on the genomic, biochemical and expressional levels stands in contrast to the dynamic structural complexities and proposed functional diversifications between mammalian and avian CLCA1 homologues, insinuating a significant degree of negative selection of CLCA2 orthologues among birds and mammals. Finally, and again in contrast to CLCA1, the high conservation of CLCA2 makes it a strong candidate for studying basic properties of the functionally still widely unresolved CLCA gene family.
Subject(s)
Chickens , Mammals , Animals , Mammals/genetics , Chickens/genetics , Quail/genetics , Genomics , Turkeys/geneticsABSTRACT
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.
Subject(s)
Pneumonia, Pneumococcal , Receptor, TIE-2/agonists , Ventilator-Induced Lung Injury , Ampicillin/pharmacology , Angiopoietin-1/pharmacology , Animals , Anti-Bacterial Agents/pharmacology , Lung/pathology , Mice , Mice, Inbred C57BL , Peptide Fragments , Permeability , Pneumonia, Pneumococcal/drug therapy , Polyethylene Glycols/pharmacology , RNA, Messenger/pharmacology , Respiration, Artificial , Streptococcus pneumoniae , Ventilator-Induced Lung Injury/prevention & controlABSTRACT
Mechanisms of epithelial renewal in the alveolar compartment remain incompletely understood. To this end, we aimed to characterize alveolar progenitors. Single-cell RNA-sequencing (scRNA-seq) analysis of the HTII-280+/EpCAM+ population from adult human lung revealed subclusters enriched for adult stem cell signature (ASCS) genes. We found that alveolar progenitors in organoid culture in vitro show phenotypic lineage plasticity as they can yield alveolar or bronchial cell-type progeny. The direction of the differentiation is dependent on the presence of the GSK-3ß inhibitor, CHIR99021. By RNA-seq profiling of GSK-3ß knockdown organoids we identified additional candidate target genes of the inhibitor, among others FOXM1 and EGF. This gives evidence of Wnt pathway independent regulatory mechanisms of alveolar specification. Following influenza A virus (IAV) infection organoids showed a similar response as lung tissue explants which confirms their suitability for studies of sequelae of pathogen-host interaction.
Subject(s)
Lung , Organoids , Cell Differentiation/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Lung/metabolism , Organoids/metabolism , Wnt Signaling PathwayABSTRACT
The systemic immune response to viral infection is shaped by master transcription factors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA sequencing approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alarmin transcription) as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling, characterized PIRAT as a nuclear decoy RNA, keeping PU.1 from binding to alarmin promoters and promoting its binding to pseudogenes in naïve monocytes. NF-κB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Alarmin expression is additionally enhanced by the up-regulation of the lncRNA LUCAT1, which promotes NF-κB-dependent gene expression at the expense of targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncoding RNA networks in systemic antiviral responses to SARS-CoV-2 in humans.