The guardians of pulmonary harmony: alveolar macrophages orchestrating the symphony of lung inflammation and tissue homeostasis.

Recent breakthroughs in single-cell sequencing, advancements in cellular and tissue imaging techniques, innovations in cell lineage tracing, and insights into the epigenome collectively illuminate the enigmatic landscape of alveolar macrophages in the lung under homeostasis and disease conditions. Our current knowledge reveals the cellular and functional diversity of alveolar macrophages within the respiratory system, emphasising their remarkable adaptability. By synthesising insights from classical cell and developmental biology studies, we provide a comprehensive perspective on alveolar macrophage functional plasticity. This includes an examination of their ontology-related features, their role in maintaining tissue homeostasis under steady-state conditions and the distinct contribution of bone marrow-derived macrophages (BMDMs) in promoting tissue regeneration and restoring respiratory system homeostasis in response to injuries. Elucidating the signalling pathways within inflammatory conditions, the impact of various triggers on tissue-resident alveolar macrophages (TR-AMs), as well as the recruitment and polarisation of macrophages originating from the bone marrow, presents an opportunity to propose innovative therapeutic approaches aimed at modulating the equilibrium between phenotypes to induce programmes associated with a pro-regenerative or homeostasis phenotype of BMDMs or TR-AMs. This, in turn, can lead to the amelioration of disease outcomes and the attenuation of detrimental inflammation. This review comprehensively addresses the pivotal role of macrophages in the orchestration of inflammation and resolution phases after lung injury, as well as ageing-related shifts and the influence of clonal haematopoiesis of indeterminate potential mutations on alveolar macrophages, exploring altered signalling pathways and transcriptional profiles, with implications for respiratory homeostasis.

Impact of different tissue dissociation protocols on endothelial cell recovery from developing mouse lungs.

Flow cytometry and fluorescence-activated cell sorting are widely used to study endothelial cells, for which the generation of viable single-cell suspensions is an essential first step. Two enzymatic approaches, collagenase A and dispase, are widely employed for endothelial cell isolation. In this study, the utility of both enzymatic approaches, alone and in combination, for endothelial cell isolation from juvenile and adult mouse lungs was assessed, considering the number, viability, and subtype composition of recovered endothelial cell pools. Collagenase A yielded an 8-12-fold superior recovery of viable endothelial cells from lung tissue from developing mouse pups, compared to dispase, although dispase proved superior in efficiency for epithelial cell recovery. Single-cell RNA-Seq revealed that the collagenase A approach yielded a diverse endothelial cell subtype composition of recovered endothelial cell pools, with broad representation of arterial, capillary, venous, and lymphatic lung endothelial cells; while the dispase approach yielded a recovered endothelial cell pool highly enriched for one subset of general capillary endothelial cells, but poor representation of other endothelial cells subtypes. These data indicate that tissue dissociation markedly influences the recovery of endothelial cells, and the endothelial subtype composition of recovered endothelial cell pools, as assessed by single-cell RNA-Seq.

GLI1+ Cells Contribute to Vascular Remodeling in Pulmonary Hypertension.

BACKGROUND: The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs). METHODS: Gli1Cre-ERT2; tdTomatoflox mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature. RESULTS: The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an Acta2high fraction of cells with pathways in cancer and MAPK (mitogen-activated protein kinase) signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation. CONCLUSIONS: Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies.

Adoptive transfer of donor B lymphocytes: a phase 1/2a study for patients after allogeneic stem cell transplantation.

ABSTRACT: Immune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is slow and patients carry a high and prolonged risk of opportunistic infections. We hypothesized that the adoptive transfer of donor B cells can foster after HSCT immuno-reconstitution. Here, we report, to our knowledge, the results of a first-in-human phase 1/2a study aimed to evaluate the feasibility and safety of adoptively transferred donor B cells and to test their activity upon recall vaccination. Good manufactoring practice (GMP) B-cell products were generated from donor apheresis products using 2-step magnetic cell separation. Fifteen patients who had undergone allo-HSCT were enrolled and treated after taper of immunosuppression (median, day +148; range, 130-160). Patients received 4 different doses of B cells (0.5 × 106 to 4.0 × 106 B cells per kg body weight). To test the activity of infused donor memory B cells in vivo, patients were vaccinated with a pentavalent vaccine 7 days after B-cell transfer. We observed the mobilization of plasmablasts and an increase in serum titers against vaccine antigens, with a stronger response in patients receiving higher B-cell numbers. Analysis of immunoglobulin VH-sequences by next-generation sequencing revealed that plasmablasts responding to vaccination originated from memory B-cell clones from the donor. Donor B-cell transfer was safe, as no Epstein-Barr virus (EBV) reactivation was observed, and only low-grade graft-versus-host disease (GVHD) occurred in 4 out of 15 patients. This pilot trial may pave the way for further studies exploring the adoptive transfer of memory B cells to reduce the frequency of infections after allo-HSCT. This trial was registered at ClinicalTrial.gov as #NCT02007811.

Alveolar macrophage-expressed Plet1 is a driver of lung epithelial repair after viral pneumonia.

Influenza A virus (IAV) infection mobilizes bone marrow-derived macrophages (BMDM) that gradually undergo transition to tissue-resident alveolar macrophages (TR-AM) in the inflamed lung. Combining high-dimensional single-cell transcriptomics with complex lung organoid modeling, in vivo adoptive cell transfer, and BMDM-specific gene targeting, we found that transitioning ("regenerative") BMDM and TR-AM highly express Placenta-expressed transcript 1 (Plet1). We reveal that Plet1 is released from alveolar macrophages, and acts as important mediator of macrophage-epithelial cross-talk during lung repair by inducing proliferation of alveolar epithelial cells and re-sealing of the epithelial barrier. Intratracheal administration of recombinant Plet1 early in the disease course attenuated viral lung injury and rescued mice from otherwise fatal disease, highlighting its therapeutic potential.

Analysis of acute COVID-19 including chronic morbidity: protocol for the deep phenotyping National Pandemic Cohort Network in Germany (NAPKON-HAP).

BACKGROUND: The severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) pandemic causes a high burden of acute and long-term morbidity and mortality worldwide despite global efforts in containment, prophylaxis, and therapy. With unprecedented speed, the global scientific community has generated pivotal insights into the pathogen and the host response evoked by the infection. However, deeper characterization of the pathophysiology and pathology remains a high priority to reduce morbidity and mortality of coronavirus disease 2019 (COVID-19). METHODS: NAPKON-HAP is a multi-centered prospective observational study with a long-term follow-up phase of up to 36 months post-SARS-CoV-2 infection. It constitutes a central platform for harmonized data and biospecimen for interdisciplinary characterization of acute SARS-CoV-2 infection and long-term outcomes of diverging disease severities of hospitalized patients. RESULTS: Primary outcome measures include clinical scores and quality of life assessment captured during hospitalization and at outpatient follow-up visits to assess acute and chronic morbidity. Secondary measures include results of biomolecular and immunological investigations and assessment of organ-specific involvement during and post-COVID-19 infection. NAPKON-HAP constitutes a national platform to provide accessibility and usability of the comprehensive data and biospecimen collection to global research. CONCLUSION: NAPKON-HAP establishes a platform with standardized high-resolution data and biospecimen collection of hospitalized COVID-19 patients of different disease severities in Germany. With this study, we will add significant scientific insights and provide high-quality data to aid researchers to investigate COVID-19 pathophysiology, pathology, and chronic morbidity.

RSV vaccination strategies for high-risk patients 2023: a collaborative position paper by leading German medical societies and organizations.

Respiratory syncytial virus (RSV) inflicts severe illness and courses of infections not only in neonates, infants, and young children, but also causes significant morbidity and mortality in older adults and in people with immunosuppression, hemato-oncologic disease, chronic lung disease, or cardiovascular disease. In June and August 2023, effective vaccines against RSV were approved for the first time by the European Medicines Agency (EMA) for the EU. The respective pivotal studies showed a very high efficacy of the vaccine in preventing severe RSV-associated respiratory infections. At this point, use of the respective vaccines is restricted to persons aged 60 years or older, according to the registration studies. We therefore recommend use of the vaccination in persons aged 60 years or older. In addition, we recommend use of the vaccination in adults of any age with severe pulmonary or cardiovascular pre-existing conditions, as well as in adults with significant immune compromise, after individual consultation with the treating physician. Cost coverage can be applied for individually with the responsible health insurance company.

Prevention of CMV/EBV reactivation by double-specific T cells in patients after allogeneic stem cell transplantation: results from the randomized phase I/IIa MULTIVIR-01 study.

Introduction: Allogeneic stem cell transplantation is used to cure hematologic malignancies or deficiencies of the hematopoietic system. It is associated with severe immunodeficiency of the host early after transplant and therefore early reactivation of latent herpesviruses such as CMV and EBV within the first 100 days are frequent. Small studies and case series indicated that application of herpes virus specific T cells can control and prevent disease in this patient population. Methods: We report the results of a randomized controlled multi centre phase I/IIa study (MULTIVIR-01) using a newly developed T cell product with specificity for CMV and EBV derived from the allogeneic stem cell grafts used for transplantation. The study aimed at prevention and preemptive treatment of both viruses in patients after allogeneic stem cell transplantation targeting first infusion on day +30. Primary endpoints were acute transfusion reaction and acute-graft versus-host-disease after infusion of activated T cells. Results: Thirty-three patients were screened and 9 patients were treated with a total of 25 doses of the T cell product. We show that central manufacturing can be achieved successfully under study conditions and the product can be applied without major side effects. Overall survival, transplant related mortality, cumulative incidence of graft versus host disease and number of severe adverse events were not different between treatment and control groups. Expansion of CMV/EBV specific T cells was observed in a fraction of patients, but overall there was no difference in virus reactivation. Discussion: Our study results indicate peptide stimulated epitope specific T cells derived from stem cell grafts can be administered safely for prevention and preemptive treatment of reactivation without evidence for induction of acute graft versus host disease. Clinical trial registration: https://clinicaltrials.gov, identifier NCT02227641.

Alveolar macrophages in tissue homeostasis, inflammation, and infection: evolving concepts of therapeutic targeting.

Alveolar macrophages (AMs) are the sentinel cells of the alveolar space, maintaining homeostasis, fending off pathogens, and controlling lung inflammation. During acute lung injury, AMs orchestrate the initiation and resolution of inflammation in order to ultimately restore homeostasis. This central role in acute lung inflammation makes AMs attractive targets for therapeutic interventions. Single-cell RNA-Seq and spatial omics approaches, together with methodological advances such as the generation of human macrophages from pluripotent stem cells, have increased understanding of the ontogeny, function, and plasticity of AMs during infectious and sterile lung inflammation, which could move the field closer to clinical application. However, proresolution phenotypes might conflict with proinflammatory and antibacterial responses. Therefore, therapeutic targeting of AMs at vulnerable time points over the course of infectious lung injury might harbor the risk of serious side effects, such as loss of antibacterial host defense capacity. Thus, the identification of key signaling hubs that determine functional fate decisions in AMs is of the utmost importance to harness their therapeutic potential.

Blind spot in endocarditis guidelines: Mycobacterium chimaera prosthetic valve endocarditis after cardiac surgery-a case series.

Background: The recently published 2023 Duke-ISCVID Criteria for Infective Endocarditis for the first time consider mycobacteria (esp. Mycobacterium chimaera) as 'typical' microorganisms for prosthetic valve endocarditis (major criteria). This reflects the ongoing worldwide outbreak of M. chimaera prosthetic valve endocarditis. Case summary: Our case series demonstrates a diagnostic pathway for mycobacterial endocarditis. Symptoms are unspecific, and standard microbiological testing does not result in identification of the causative agent (see Graphical Abstract); therefore patients require special microbiological and imaging diagnostics. One patient with early diagnosis and stringent antibiotic and surgical therapy survived. Two patients with disseminated infection at the time point of diagnosis had fatal outcomes. Discussion: The diagnostic approach in our small retrospective case series is in line with the new modified Duke criteria and underlines the diagnostic gap in the previous definitions. Outcome of M. chimaera prosthetic valve endocarditis is related to timely diagnosis and anti-mycobacterial as well as surgical treatment. Non-tuberculous mycobacteria should be given more attention in future endocarditis guidelines.

Influenza virus decreases albumin uptake and megalin expression in alveolar epithelial cells.

Introduction: Acute respiratory distress syndrome (ARDS) is a common complication of influenza virus (IV) infection. During ARDS, alveolar protein concentrations often reach 40-90% of plasma levels, causing severe impairment of gas exchange and promoting deleterious alveolar remodeling. Protein clearance from the alveolar space is at least in part facilitated by the multi-ligand receptor megalin through clathrin-mediated endocytosis. Methods: To investigate whether IV infection impairs alveolar protein clearance, we examined albumin uptake and megalin expression in MLE-12 cells and alveolar epithelial cells (AEC) from murine precision-cut lung slices (PCLS) and in vivo, under IV infection conditions by flow cytometry and western blot. Transcriptional levels from AEC and broncho-alveolar lavage (BAL) cells were analyzed in an in-vivo mouse model by RNAseq. Results: IV significantly downregulated albumin uptake, independently of activation of the TGF-ß1/GSK3ß axis that has been previously implicated in the regulation of megalin function. Decreased plasma membrane abundance, total protein levels, and mRNA expression of megalin were associated with this phenotype. In IV-infected mice, we identified a significant upregulation of matrix metalloproteinase (MMP)-14 in BAL fluid cells. Furthermore, the inhibition of this protease partially recovered total megalin levels and albumin uptake. Discussion: Our results suggest that the previously described MMP-driven shedding mechanisms are potentially involved in downregulation of megalin cell surface abundance and clearance of excess alveolar protein. As lower alveolar edema protein concentrations are associated with better outcomes in respiratory failure, our findings highlight the therapeutic potential of a timely MMP inhibition in the treatment of IV-induced ARDS.

Association of ward-level antibiotic consumption with healthcare-associated Clostridioides difficile infections: an ecological study in five German university hospitals, 2017-2019.

OBJECTIVES: To analyse the influence of antibiotic consumption on healthcare-associated healthcare onset (HAHO) Clostridioides difficile infection (CDI) in a German university hospital setting. METHODS: Monthly ward-level antibiotic consumption measured in DDD/100 patient days (pd) and CDI surveillance data from five university hospitals in the period 2017 through 2019 were analysed. Uni- and multivariable analyses were performed with generalized estimating equation models. RESULTS: A total of 225 wards with 7347 surveillance months and 4 036 602 pd participated. With 1184 HAHO-CDI cases, there was a median incidence density of 0.17/1000 pd (IQR 0.03-0.43) across all specialties, with substantial differences among specialties. Haematology-oncology wards showed the highest median incidence density (0.67/1000 pd, IQR 0.44-1.01), followed by medical ICUs (0.45/1000 pd, IQR 0.27-0.73) and medical general wards (0.32/1000 pd, IQR 0.18-0.53). Multivariable analysis revealed carbapenem (mostly meropenem) consumption to be the only antibiotic class associated with increased HAHO-CDI incidence density. Each carbapenem DDD/100 pd administered increased the HAHO-CDI incidence density by 1.3% [incidence rate ratio (IRR) 1.013; 95% CI 1.006-1.019]. Specialty-specific analyses showed this influence only to be valid for haematological-oncological wards. Overall, factors like ward specialty (e.g. haematology-oncology ward IRR 2.961, 95% CI 2.203-3.980) or other CDI cases on ward had a stronger influence on HAHO-CDI incidence density (e.g. community-associated CDI or unknown association case in same month IRR 1.476, 95% CI 1.242-1.755) than antibiotic consumption. CONCLUSIONS: In the German university hospital setting, monthly ward-level carbapenem consumption seems to increase the HAHO-CDI incidence density predominantly on haematological-oncological wards. Furthermore, other patient-specific factors seem to be equally important to control HAHO-CDI.

Nicotine promotes e-cigarette vapour-induced lung inflammation and structural alterations.

BACKGROUND: Electronic cigarette (e-cigarette) vapour is gaining popularity as an alternative to tobacco smoking and can induce acute lung injury. However, the specific role of nicotine in e-cigarette vapour and its long-term effects on the airways, lung parenchyma and vasculature remain unclear. RESULTS: In vitro exposure to nicotine-containing e-cigarette vapour extract (ECVE) or to nicotine-free e-cigarette vapour extract (NF ECVE) induced changes in gene expression of epithelial cells and pulmonary arterial smooth muscle cells (PASMCs), but ECVE in particular caused functional alterations (e.g. a decrease in human and mouse PASMC proliferation by 29.3±5.3% and 44.3±8.4%, respectively). Additionally, acute inhalation of nicotine-containing e-cigarette vapour (ECV) but not nicotine-free e-cigarette vapour (NF ECV) increased pulmonary endothelial permeability in isolated lungs. Long-term in vivo exposure of mice to ECV for 8 months significantly increased the number of inflammatory cells, in particular lymphocytes, compared to control and NF ECV in the bronchoalveolar fluid (BALF) (ECV: 853.4±150.8 cells·mL-1; control: 37.0±21.1 cells·mL-1; NF ECV: 198.6±94.9 cells·mL-1) and in lung tissue (ECV: 25.7±3.3 cells·mm-3; control: 4.8±1.1 cells·mm-3; NF ECV: 14.1±2.2 cells·mm-3). BALF cytokines were predominantly increased by ECV. Moreover, ECV caused significant changes in lung structure and function (e.g. increase in airspace by 17.5±1.4% compared to control), similar to mild tobacco smoke-induced alterations, which also could be detected in the NF ECV group, albeit to a lesser degree. In contrast, the pulmonary vasculature was not significantly affected by ECV or NF ECV. CONCLUSIONS: NF ECV components induce cell type-specific effects and mild pulmonary alterations, while inclusion of nicotine induces significant endothelial damage, inflammation and parenchymal alterations.

Autoantibodies are highly prevalent in non-SARS-CoV-2 respiratory infections and critical illness.

The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non-SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non-SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

Clathrin-Mediated Albumin Clearance in Alveolar Epithelial Cells of Murine Precision-Cut Lung Slices.

A hallmark of acute respiratory distress syndrome (ARDS) is an accumulation of protein-rich alveolar edema that impairs gas exchange and leads to worse outcomes. Thus, understanding the mechanisms of alveolar albumin clearance is of high clinical relevance. Here, we investigated the mechanisms of the cellular albumin uptake in a three-dimensional culture of precision-cut lung slices (PCLS). We found that up to 60% of PCLS cells incorporated labeled albumin in a time- and concentration-dependent manner, whereas virtually no uptake of labeled dextran was observed. Of note, at a low temperature (4 °C), saturating albumin receptors with unlabeled albumin and an inhibition of clathrin-mediated endocytosis markedly decreased the endocytic uptake of the labeled protein, implicating a receptor-driven internalization process. Importantly, uptake rates of albumin were comparable in alveolar epithelial type I (ATI) and type II (ATII) cells, as assessed in PCLS from a SftpcCreERT2/+: tdTomatoflox/flox mouse strain (defined as EpCAM+CD31-CD45-tdTomatoSPC-T1α+ for ATI and EpCAM+CD31-CD45-tdTomatoSPC+T1α- for ATII cells). Once internalized, albumin was found in the early and recycling endosomes of the alveolar epithelium as well as in endothelial, mesenchymal, and hematopoietic cell populations, which might indicate transcytosis of the protein. In summary, we characterize albumin uptake in alveolar epithelial cells in the complex setting of PCLS. These findings may open new possibilities for pulmonary drug delivery that may improve the outcomes for patients with respiratory failure.

GLI1+ cells are a source of repair-supportive mesenchymal cells (RSMCs) during airway epithelial regeneration.

Repair-supportive mesenchymal cells (RSMCs) have been recently reported in the context of naphthalene (NA)-induced airway injury and regeneration. These cells transiently express smooth muscle actin (Acta2) and are enriched with platelet-derived growth factor receptor alpha (Pdgfra) and fibroblast growth factor 10 (Fgf10) expression. Genetic deletion of Ctnnb1 (gene coding for beta catenin) or Fgf10 in these cells using the Acta2-Cre-ERT2 driver line after injury (defined as NA-Tam condition; Tam refers to tamoxifen) led to impaired repair of the airway epithelium. In this study, we demonstrate that RSMCs are mostly captured using the Acta2-Cre-ERT2 driver when labeling occurs after (NA-Tam condition) rather than before injury (Tam-NA condition), and that their expansion occurs mostly between days 3 and 7 following NA treatment. Previous studies have shown that lineage-traced peribronchial GLI1+ cells are transiently amplified after NA injury. Here, we report that Gli1 expression is enriched in RSMCs. Using lineage tracing with Gli1Cre-ERT2 mice combined with genetic inactivation of Fgf10, we show that GLI1+ cells with Fgf10 deletion fail to amplify around the injured airways, thus resulting in impaired airway epithelial repair. Interestingly, Fgf10 expression is not upregulated in GLI1+ cells following NA treatment, suggesting that epithelial repair is mostly due to the increased number of Fgf10-expressing GLI1+ cells. Co-culture of SCGB1A1+ cells with GLI1+ cells isolated from non-injured or injured lungs showed that GLI1+ cells from these two conditions are similarly capable of supporting bronchiolar organoid (or bronchiolosphere) formation. Single-cell RNA sequencing on sorted lineage-labeled cells showed that the RSMC signature resembles that of alveolar fibroblasts. Altogether, our study provides strong evidence for the involvement of mesenchymal progenitors in airway epithelial regeneration and highlights the critical role played by Fgf10-expressing GLI1+ cells in this context.

FGFR2b signalling restricts lineage-flexible alveolar progenitors during mouse lung development and converges in mature alveolar type 2 cells.

The specification, characterization, and fate of alveolar type 1 and type 2 (AT1 and AT2) progenitors during embryonic lung development are poorly defined. Current models of distal epithelial lineage formation fail to capture the heterogeneity and dynamic contribution of progenitor pools present during early development. Furthermore, few studies explore the pathways involved in alveolar progenitor specification and fate. In this paper, we build upon our previously published work on the regulation of airway epithelial progenitors by fibroblast growth factor receptor 2b (FGFR2b) signalling during early (E12.5) and mid (E14.5) pseudoglandular stage lung development. Our results suggest that a significant proportion of AT2 and AT1 progenitors are lineage-flexible during late pseudoglandular stage development, and that lineage commitment is regulated in part by FGFR2b signalling. We have characterized a set of direct FGFR2b targets at E16.5 which are likely involved in alveolar lineage formation. These signature genes converge on a subpopulation of AT2 cells later in development and are downregulated in AT2 cells transitioning to the AT1 lineage during repair after injury in adults. Our findings highlight the extensive heterogeneity of pneumocytes by elucidating the role of FGFR2b signalling in these cells during early airway epithelial lineage formation, as well as during repair after injury.

Corrigendum: Neospora caninum infection triggers S-phase arrest and alters nuclear characteristics in primary bovine endothelial host cells.

[This corrects the article DOI: 10.3389/fcell.2022.946335.].

Neospora caninum Infection Triggers S-phase Arrest and Alters Nuclear Characteristics in Primary Bovine Endothelial Host Cells.

Neospora caninum represents a major cause of abortive disease in bovines and small ruminants worldwide. As a typical obligate intracellular apicomplexan parasite, N. caninum needs to modulate its host cell for successful replication. In the current study, we focused on parasite-driven interference with host cell cycle progression. By performing DNA content-based cell cycle phase analyses in N. caninum-infected primary bovine umbilical vein endothelial cells (BUVEC), a parasite-driven S-phase arrest was detected at both 24 and 32 h p. i., being paralleled by fewer host cells experiencing the G0/G1 cell cycle phase. When analyzing S-subphases, proliferation cell nuclear antigen (per PCNA)-based experiments showed a reduced population of BUVEC in the late S-phase. Analyses on key molecules of cell cycle regulation documented a significant alteration of cyclin A2 and cyclin B1 abundance in N. caninum-infected host endothelial cells, thereby confirming irregularities in the S-phase and S-to-G2/M-phase transition. In line with cell cycle alterations, general nuclear parameters revealed smaller nuclear sizes and morphological abnormalities of BUVEC nuclei within the N. caninum-infected host cell layer. The latter observations were also confirmed by transmission electron microscopy (TEM) and by analyses of lamin B1 as a marker of nuclear lamina, which illustrated an inhomogeneous nuclear lamin B1 distribution, nuclear foldings, and invaginations, thereby reflecting nuclear misshaping. Interestingly, the latter finding applied to both non-infected and infected host cells within parasitized BUVEC layer. Additionally, actin detection indicated alterations in the perinuclear actin cap formation since typical nucleo-transversal filaments were consistently lacking in N. caninum-infected BUVEC, as also documented by significantly decreased actin-related intensities in the perinuclear region. These data indicate that N. caninum indeed alters host cell cycle progression and severely affects the host cell nuclear phenotype in primary bovine endothelial host cells. In summary, these findings add novel data on the complex N. caninum-specific modulation of host cell and nucleus, thereby demonstrating clear differences in cell cycle progression modulation driven by other closely related apicomplexans like Toxoplasma gondii and Besnotia besnoiti.