COPD basal cells are primed towards secretory to multiciliated cell imbalance driving increased resilience to environmental stressors.

INTRODUCTION: Environmental pollutants injure the mucociliary elevator, thereby provoking disease progression in chronic obstructive pulmonary disease (COPD). Epithelial resilience mechanisms to environmental nanoparticles in health and disease are poorly characterised. METHODS: We delineated the impact of prevalent pollutants such as carbon and zinc oxide nanoparticles, on cellular function and progeny in primary human bronchial epithelial cells (pHBECs) from end-stage COPD (COPD-IV, n=4), early disease (COPD-II, n=3) and pulmonary healthy individuals (n=4). After nanoparticle exposure of pHBECs at air-liquid interface, cell cultures were characterised by functional assays, transcriptome and protein analysis, complemented by single-cell analysis in serial samples of pHBEC cultures focusing on basal cell differentiation. RESULTS: COPD-IV was characterised by a prosecretory phenotype (twofold increase in MUC5AC+) at the expense of the multiciliated epithelium (threefold reduction in Ac-Tub+), resulting in an increased resilience towards particle-induced cell damage (fivefold reduction in transepithelial electrical resistance), as exemplified by environmentally abundant doses of zinc oxide nanoparticles. Exposure of COPD-II cultures to cigarette smoke extract provoked the COPD-IV characteristic, prosecretory phenotype. Time-resolved single-cell transcriptomics revealed an underlying COPD-IV unique basal cell state characterised by a twofold increase in KRT5+ (P=0.018) and LAMB3+ (P=0.050) expression, as well as a significant activation of Wnt-specific (P=0.014) and Notch-specific (P=0.021) genes, especially in precursors of suprabasal and secretory cells. CONCLUSION: We identified COPD stage-specific gene alterations in basal cells that affect the cellular composition of the bronchial elevator and may control disease-specific epithelial resilience mechanisms in response to environmental nanoparticles. The identified phenomena likely inform treatment and prevention strategies.

Granulocyte Colony-Stimulating Factor is a Determinant of Severe Bronchopulmonary Dysplasia and Coincident Retinopathy.

Bronchopulmonary dysplasia (BPD), also called chronic lung disease of immaturity, afflicts approximately one third of all extremely premature infants, causing lifelong lung damage. There is no effective treatment other than supportive care. Retinopathy of prematurity (ROP), which impairs vision irreversibly, is common in BPD, suggesting a related pathogenesis. However, specific mechanisms of BPD and ROP are not known. Herein, a neonatal mouse hyperoxic model of coincident BPD and retinopathy was used to screen for candidate mediators, which revealed that granulocyte colony-stimulating factor (G-CSF), also known as colony-stimulating factor 3, was up-regulated significantly in mouse lung lavage fluid and plasma at postnatal day 14 in response to hyperoxia. Preterm infants with more severe BPD had increased plasma G-CSF. G-CSF-deficient neonatal pups showed significantly reduced alveolar simplification, normalized alveolar and airway resistance, and normalized weight gain compared with wild-type pups after hyperoxic lung injury. This was associated with a marked reduction in the intensity, and activation state, of neutrophilic and monocytic inflammation and its attendant oxidative stress response, and protection of lung endothelial cells. G-CSF deficiency also provided partial protection against ROP. The findings in this study implicate G-CSF as a pathogenic mediator of BPD and ROP, and suggest the therapeutic utility of targeting G-CSF biology to treat these conditions.

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.

Delineating morbidity patterns in preterm infants at near-term age using a data-driven approach.

BACKGROUND: Long-term survival after premature birth is significantly determined by development of morbidities, primarily affecting the cardio-respiratory or central nervous system. Existing studies are limited to pairwise morbidity associations, thereby lacking a holistic understanding of morbidity co-occurrence and respective risk profiles. METHODS: Our study, for the first time, aimed at delineating and characterizing morbidity profiles at near-term age and investigated the most prevalent morbidities in preterm infants: bronchopulmonary dysplasia (BPD), pulmonary hypertension (PH), mild cardiac defects, perinatal brain pathology and retinopathy of prematurity (ROP). For analysis, we employed two independent, prospective cohorts, comprising a total of 530 very preterm infants: AIRR ("Attention to Infants at Respiratory Risks") and NEuroSIS ("Neonatal European Study of Inhaled Steroids"). Using a data-driven strategy, we successfully characterized morbidity profiles of preterm infants in a stepwise approach and (1) quantified pairwise morbidity correlations, (2) assessed the discriminatory power of BPD (complemented by imaging-based structural and functional lung phenotyping) in relation to these morbidities, (3) investigated collective co-occurrence patterns, and (4) identified infant subgroups who share similar morbidity profiles using machine learning techniques. RESULTS: First, we showed that, in line with pathophysiologic understanding, BPD and ROP have the highest pairwise correlation, followed by BPD and PH as well as BPD and mild cardiac defects. Second, we revealed that BPD exhibits only limited capacity in discriminating morbidity occurrence, despite its prevalence and clinical indication as a driver of comorbidities. Further, we demonstrated that structural and functional lung phenotyping did not exhibit higher association with morbidity severity than BPD. Lastly, we identified patient clusters that share similar morbidity patterns using machine learning in AIRR (n=6 clusters) and NEuroSIS (n=8 clusters). CONCLUSIONS: By capturing correlations as well as more complex morbidity relations, we provided a comprehensive characterization of morbidity profiles at discharge, linked to shared disease pathophysiology. Future studies could benefit from identifying risk profiles to thereby develop personalized monitoring strategies. TRIAL REGISTRATION: AIRR: DRKS.de, DRKS00004600, 28/01/2013. NEuroSIS: ClinicalTrials.gov, NCT01035190, 18/12/2009.

Hypoxia-Inducible Factor-1α in SM22α-Expressing Cells Modulates Alveolarization.

Worldwide, the incidence of both preterm births and chronic lung disease of infancy, or bronchopulmonary dysplasia, remains high. Infants with bronchopulmonary dysplasia have larger and fewer alveoli, a lung pathology that can persist into adulthood. Although recent data point to a role for hypoxia-inducible factor-1α (HIF-1α) in mediating pulmonary angiogenesis and alveolarization, the cell-specific role of HIF-1α remains incompletely understood. Thus, we hypothesized that HIF-1α, in a distinct subset of mesenchymal cells, mediates postnatal alveolarization. To test the hypothesis, we generated mice with a cell-specific deletion of HIF-1α by crossing SM22α promoter-driven Cre mice with HIF-1αflox/flox mice (SM22α-HIF-1α-/-), determined SM-22α-expressing cell identity using single-cell RNA sequencing, and interrogated samples from preterm infants. Deletion of HIF-1α in SM22α-expressing cells had no effect on lung structure at day 3 of life. However, at 8 days, there were fewer and larger alveoli, a difference that persisted into adulthood. Microvascular density, elastin organization, and peripheral branching of the lung vasculature were decreased in SM22α-HIF-1α-/- mice, compared with control mice. Single-cell RNA sequencing demonstrated that three mesenchymal cell subtypes express SM22α: myofibroblasts, airway smooth muscle cells, and vascular smooth muscle cells. Pulmonary vascular smooth muscle cells from SM22α-HIF-1α-/- mice had decreased angiopoietin-2 expression and, in coculture experiments, a diminished capacity to promote angiogenesis that was rescued by angiopoietin-2. Angiopoietin-2 expression in tracheal aspirates of preterm infants was inversely correlated with overall mechanical ventilation time, a marker of disease severity. We conclude that SM22α-specific HIF-1α expression drives peripheral angiogenesis and alveolarization in the lung, perhaps by promoting angiopoietin-2 expression.

Prenatal vitamin D supplementation mitigates inflammation-related alveolar remodeling in neonatal mice.

The development of chronic lung disease in the neonate, also known as bronchopulmonary dysplasia (BPD), is the most common long-term complication in prematurely born infants. In BPD, the disease-characteristic inflammatory response culminates in nonreversible remodeling of the developing gas exchange area, provoked by the impact of postnatal treatments such as mechanical ventilation (MV) and oxygen treatment. To evaluate the potential of prenatal treatment regimens to modulate this inflammatory response and thereby impact the vulnerability of the lung toward postnatal injury, we designed a multilayered preclinical mouse model. After administration of either prenatal vitamin D-enriched (VitD+; 1,500 IU/g food) or -deprived (VitD-; <10 IU/kg) food during gestation in C57B6 mice (the onset of mating until birth), neonatal mice were exposed to hyperoxia (FiO2 = 0.4) with or without MV for 8 h at days 5-7 of life, whereas controls spontaneously breathed room air. Prenatal vitamin D supplementation resulted in a decreased number of monocytes/macrophages in the neonatal lung undergoing postnatal injury together with reduced TGF-ß pathway activation. In consequence, neonatal mice that received a VitD+ diet during gestation demonstrated less extracellular matrix (ECM) remodeling upon lung injury, reflected by the reduction of pulmonary α-smooth muscle actin-positive fibroblasts, decreased collagen and elastin deposition, and lower amounts of interstitial tissue in the lung periphery. In conclusion, our findings support strategies that attempt to prevent vitamin D insufficiency during pregnancy as they could impact lung health in the offspring by mitigating inflammatory changes in neonatal lung injury and ameliorating subsequent remodeling of the developing gas exchange area.NEW & NOTEWORTHY Vitamin D-enriched diet during gestation resulted in reduced lung inflammation and matrix remodeling in neonatal mice exposed to clinically relevant, postnatal injury. The results underscore the need to monitor the subclinical effects of vitamin D insufficiency that impact health in the offspring when other risk factors come into play.

Magnetic resonance imaging-based scoring of the diseased lung in the preterm infant with bronchopulmonary dysplasia: UNiforme Scoring of the disEAsed Lung in BPD (UNSEAL BPD).

Neonatal chronic lung disease lacks standardized assessment of lung structural changes. We addressed this clinical need by the development of a novel scoring system [UNSEAL BPD (UNiforme Scoring of the disEAsed Lung in BPD)] using T2-weighted single-shot fast-spin-echo sequences from 3 T MRI in very premature infants with and without bronchopulmonary dysplasia (BPD). Quantification of interstitial and airway remodeling, emphysematous changes, and ventilation inhomogeneity was achieved by consensus scoring on a five-point Likert scale. We successfully identified moderate and severe disease by logistic regression [area under the curve (AUC), 0.89] complemented by classification tree analysis revealing gestational age-specific structural changes. We demonstrated substantial interreader reproducibility (weighted Cohen's κ 0.69) and disease specificity (AUC = 0.91). Our novel MRI score enables the standardized assessment of disease-characteristic structural changes in the preterm lung exhibiting significant potential as a quantifiable endpoint in early intervention clinical trials and long-term disease monitoring.

MRI pulmonary artery flow detects lung vascular pathology in preterms with lung disease.

BACKGROUND: Pulmonary vascular disease (PVD) affects the majority of preterm neonates with bronchopulmonary dysplasia (BPD) and significantly determines long-term mortality through undetected progression into pulmonary hypertension. Our objectives were to associate characteristics of pulmonary artery (PA) flow and cardiac function with BPD-associated PVD near term using advanced magnetic resonance imaging (MRI) for improved risk stratification. METHODS: Preterms <32 weeks postmenstrual age (PMA) with/without BPD were clinically monitored including standard echocardiography and prospectively enrolled for 3 T MRI in spontaneous sleep near term (AIRR (Attention to Infants at Respiratory Risks) study). Semi-manual PA flow quantification (phase-contrast MRI; no BPD n=28, mild BPD n=35 and moderate/severe BPD n=25) was complemented by cardiac function assessment (cine MRI). RESULTS: We identified abnormalities in PA flow and cardiac function, i.e. increased net forward volume right/left ratio, decreased mean relative area change and pathological right end-diastolic volume, to sensitively detect BPD-associated PVD while correcting for PMA (leave-one-out area under the curve 0.88, sensitivity 0.80 and specificity 0.81). We linked these changes to increased right ventricular (RV) afterload (RV-arterial coupling (p=0.02), PA mid-systolic notching (t2; p=0.015) and cardiac index (p=1.67×10-8)) and correlated echocardiographic findings. Identified in moderate/severe BPD, we successfully applied the PA flow model in heterogeneous mild BPD cases, demonstrating strong correlation of PVD probability with indicators of BPD severity, i.e. duration of mechanical ventilation (rs=0.63, p=2.20×10-4) and oxygen supplementation (rs=0.60, p=6.00×10-4). CONCLUSIONS: Abnormalities in MRI PA flow and cardiac function exhibit significant, synergistic potential to detect BPD-associated PVD, advancing the possibilities of risk-adapted monitoring.

Impaired function and delayed regeneration of dendritic cells in COVID-19.

Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DCs) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute illness to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage-HLADR+ cells lacking DC markers. Increased frequency of CD163+ CD14+ cells within the recently discovered DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of programmed death-ligand 1 (PD-L1) in conventional DCs (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naïve CD4+ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.

Validation of disease-specific biomarkers for the early detection of bronchopulmonary dysplasia.

OBJECTIVE: To demonstrate and validate the improvement of current risk stratification for bronchopulmonary dysplasia (BPD) early after birth by plasma protein markers (sialic acid-binding Ig-like lectin 14 (SIGLEC-14), basal cell adhesion molecule (BCAM), angiopoietin-like 3 protein (ANGPTL-3)) in extremely premature infants. METHODS AND RESULTS: Proteome screening in first-week-of-life plasma samples of n = 52 preterm infants <32 weeks gestational age (GA) on two proteomic platforms (SomaLogic®, Olink-Proteomics®) confirmed three biomarkers with significant predictive power: BCAM, SIGLEC-14, and ANGPTL-3. We demonstrate high sensitivity (0.92) and specificity (0.86) under consideration of GA, show the proteins' critical contribution to the predictive power of known clinical risk factors, e.g., birth weight and GA, and predicted the duration of mechanical ventilation, oxygen supplementation, as well as neonatal intensive care stay. We confirmed significant predictive power for BPD cases when switching to a clinically applicable method (enzyme-linked immunosorbent assay) in an independent sample set (n = 25, p < 0.001) and demonstrated disease specificity in different cohorts of neonatal and adult lung disease. CONCLUSION: While successfully addressing typical challenges of clinical biomarker studies, we demonstrated the potential of BCAM, SIGLEC-14, and ANGPTL-3 to inform future clinical decision making in the preterm infant at risk for BPD. TRIAL REGISTRATION: Deutsches Register Klinische Studien (DRKS) No. 00004600; https://www.drks.de . IMPACT: The urgent need for biomarkers that enable early decision making and personalized monitoring strategies in preterm infants with BPD is challenged by targeted marker analyses, cohort size, and disease heterogeneity. We demonstrate the potential of the plasma proteins BCAM, SIGLEC-14, and ANGPTL-3 to identify infants with BPD early after birth while improving the predictive power of clinical variables, confirming the robustness toward proteome assays and proving disease specificity. Our comprehensive analysis enables a phase-III clinical trial that allows full implementation of the biomarkers into clinical routine to enable early risk stratification in preterms with BPD.

Variable functional connectivity architecture of the preterm human brain: Impact of developmental cortical expansion and maturation.

Functional connectivity (FC) is known to be individually unique and to reflect cognitive variability. Although FC can serve as a valuable correlate and potential predictor of (patho-) physiological nervous function in high-risk constellations, such as preterm birth, templates for individualized FC analysis are lacking, and knowledge about the capacity of the premature brain to develop FC variability is limited. In a cohort of prospectively recruited, preterm-born infants undergoing magnetic resonance imaging close to term-equivalent age, we show that the overall pattern could be reliably detected with a broad range of interindividual FC variability in regions of higher-order cognitive functions (e.g., association cortices) and less interindividual variability in unimodal regions (e.g., visual and motor cortices). However, when comparing the preterm and adult brains, some brain regions showed a marked shift in variability toward adulthood. This shift toward greater variability was strongest in cognitive networks like the attention and frontoparietal networks and could be partially predicted by developmental cortical expansion. Furthermore, FC variability was reflected by brain tissue characteristics indicating cortical maturation. Brain regions with high functional variability (e.g., the inferior frontal gyrus and temporoparietal junction) displayed lower cortical maturation at birth compared with somatosensory cortices. In conclusion, the overall pattern of interindividual variability in FC is already present preterm; however, some brain regions show increased variability toward adulthood, identifying characteristic patterns, such as in cognitive networks. These changes are related to postnatal cortical expansion and maturation, allowing for environmental and developmental factors to translate into marked individual differences in FC.

Validation of in vitro models for smoke exposure of primary human bronchial epithelial cells.

The bronchial epithelium is constantly challenged by inhalative insults including cigarette smoke (CS), a key risk factor for lung disease. In vitro exposure of bronchial epithelial cells using CS extract (CSE) is a widespread alternative to whole CS (wCS) exposure. However, CSE exposure protocols vary considerably between studies, precluding direct comparison of applied doses. Moreover, they are rarely validated in terms of physiological response in vivo and the relevance of the findings is often unclear. We tested six different exposure settings in primary human bronchial epithelial cells (phBECs), including five CSE protocols compared with wCS exposure. We quantified cell-delivered dose and directly compared all exposures using expression analysis of 10 well-established smoke-induced genes in bronchial epithelial cells. CSE exposure of phBECs was varied in terms of differentiation state, exposure route, duration of exposure, and dose. Gene expression was assessed by quantitative real-time PCR (qPCR) and Western Blot analysis. Cell type-specific expression of smoke-induced genes was analyzed by immunofluorescent analysis. Three surprisingly dissimilar exposure types, namely, chronic CSE treatment of differentiating phBECs, acute CSE treatment of submerged basal phBECs, and wCS exposure of differentiated phBECs performed best, resulting in significant upregulation of seven (chronic CSE) and six (acute wCS, acute submerged CSE exposure) out of 10 genes. Acute apical or basolateral exposure of differentiated phBECs with CSE was much less effective despite similar doses used. Our findings provide guidance for the design of human in vitro CS exposure models in experimental and translational lung research.

Relationship between impaired BMP signalling and clinical risk factors at early-stage vascular injury in the preterm infant.

INTRODUCTION: Chronic lung disease, that is, bronchopulmonary dysplasia (BPD) is the most common complication in preterm infants and develops as a consequence of the misguided formation of the gas-exchange area undergoing prenatal and postnatal injury. Subsequent vascular disease and its progression into pulmonary arterial hypertension critically determines long-term outcome in the BPD infant but lacks identification of early, disease-defining changes. METHODS: We link impaired bone morphogenetic protein (BMP) signalling to the earliest onset of vascular pathology in the human preterm lung and delineate the specific effects of the most prevalent prenatal and postnatal clinical risk factors for lung injury mimicking clinically relevant conditions in a multilayered animal model using wild-type and transgenic neonatal mice. RESULTS: We demonstrate (1) the significant reduction in BMP receptor 2 (BMPR2) expression at the onset of vascular pathology in the lung of preterm infants, later mirrored by reduced plasma BMP protein levels in infants with developing BPD, (2) the rapid impairment (and persistent change) of BMPR2 signalling on postnatal exposure to hyperoxia and mechanical ventilation, aggravated by prenatal cigarette smoke in a preclinical mouse model and (3) a link to defective alveolar septation and matrix remodelling through platelet derived growth factor-receptor alpha deficiency. In a treatment approach, we partially reversed vascular pathology by BMPR2-targeted treatment with FK506 in vitro and in vivo. CONCLUSION: We identified impaired BMP signalling as a hallmark of early vascular disease in the injured neonatal lung while outlining its promising potential as a future biomarker or therapeutic target in this growing, high-risk patient population.

TRPM7 is the central gatekeeper of intestinal mineral absorption essential for postnatal survival.

Zn2+, Mg2+, and Ca2+ are essential minerals required for a plethora of metabolic processes and signaling pathways. Different categories of cation-selective channels and transporters are therefore required to tightly control the cellular levels of individual metals in a cell-specific manner. However, the mechanisms responsible for the organismal balance of these essential minerals are poorly understood. Herein, we identify a central and indispensable role of the channel-kinase TRPM7 for organismal mineral homeostasis. The function of TRPM7 was assessed by single-channel analysis of TRPM7, phenotyping of TRPM7-deficient cells in conjunction with metabolic profiling of mice carrying kidney- and intestine-restricted null mutations in Trpm7 and animals with a global "kinase-dead" point mutation in the gene. The TRPM7 channel reconstituted in lipid bilayers displayed a similar permeability to Zn2+ and Mg2+ Consistently, we found that endogenous TRPM7 regulates the total content of Zn2+ and Mg2+ in cultured cells. Unexpectedly, genetic inactivation of intestinal rather than kidney TRPM7 caused profound deficiencies specifically of Zn2+, Mg2+, and Ca2+ at the organismal level, a scenario incompatible with early postnatal growth and survival. In contrast, global ablation of TRPM7 kinase activity did not affect mineral homeostasis, reinforcing the importance of the channel activity of TRPM7. Finally, dietary Zn2+ and Mg2+ fortifications significantly extended the survival of offspring lacking intestinal TRPM7. Hence, the organismal balance of divalent cations critically relies on one common gatekeeper, the intestinal TRPM7 channel.

Multiplatform Approach for Plasma Proteomics: Complementarity of Olink Proximity Extension Assay Technology to Mass Spectrometry-Based Protein Profiling.

The plasma proteome is the ultimate target for biomarker discovery. It stores an endless amount of information on the pathophysiological status of a living organism, which is, however, still difficult to comprehensively access. The high complexity of the plasma proteome can be addressed by either a system-wide and unbiased tool such as mass spectrometry (LC-MS/MS) or a highly sensitive targeted immunoassay such as the proximity extension assay (PEA). To address relevant differences and important shared characteristics, we tested the performance of LC-MS/MS in the data-dependent and data-independent acquisition modes and Olink PEA to measure circulating plasma proteins in 173 human plasma samples from a Southern German population-based cohort. We demonstrated the measurement of more than 300 proteins with both LC-MS/MS approaches applied, mainly including high-abundance plasma proteins. By the use of the PEA technology, we measured 728 plasma proteins, covering a broad dynamic range with high sensitivity down to pg/mL concentrations. Then, we quantified 35 overlapping proteins with all three analytical platforms, verifying the reproducibility of data distributions, measurement correlation, and gender-based differential expression. Our work highlights the limitations and the advantages of both targeted and untargeted approaches and proves their complementary strengths. We demonstrated a significant gain in proteome coverage depth and subsequent biological insight by a combination of platforms-a promising approach for future biomarker and mechanistic studies.

Altered relaxation times in MRI indicate bronchopulmonary dysplasia.

We developed a MRI protocol using transverse (T2) and longitudinal (T1) mapping sequences to characterise lung structural changes in preterm infants with bronchopulmonary dysplasia (BPD). We prospectively enrolled 61 infants to perform 3-Tesla MRI of the lung in quiet sleep. Statistical analysis was performed using logistic Group Lasso regression and logistic regression. Increased lung T2 relaxation time and decreased lung T1 relaxation time indicated BPD yielding an area under the curve (AUC) of 0.80. Results were confirmed in an independent study cohort (AUC 0.75) and mirrored by lung function testing, indicating the high potential for MRI in future BPD diagnostics. TRIAL REGISTRATION: DRKS00004600.

CX3CR1-fractalkine axis drives kinetic changes of monocytes in fibrotic interstitial lung diseases.

Circulating immune cell populations have been shown to contribute to interstitial lung disease (ILD). In this study, we analysed circulating and lung resident monocyte populations, and assessed their phenotype and recruitment from the blood to the lung in ILD. Flow cytometry analysis of blood samples for quantifying circulating monocytes was performed in 105 subjects: 83 with ILD (n=36, n=28 and n=19 for nonspecific interstitial pneumonia, hypersensitivity pneumonitis and connective-tissue disease-associated ILD, respectively), as well as 22 controls. Monocyte localisation and abundance were assessed using immunofluorescence and flow cytometry of lung tissue. Monocyte populations were cultured either alone or with endothelial cells to assess fractalkine-dependent transmigration pattern. We show that circulating classical monocytes (CM) were increased in ILD compared with controls, while nonclassical monocytes (NCM) were decreased. CM abundance correlated inversely with lung function, while NCM abundance correlated positively. Both CCL2 and CX3CL1 concentrations were increased in plasma and lungs of ILD patients. Fractalkine co-localised with ciliated bronchial epithelial cells, thereby creating a chemoattractant gradient towards the lung. Fractalkine enhanced endothelial transmigration of NCM in ILD samples only. Immunofluorescence, as well as flow cytometry, showed an increased presence of NCM in fibrotic niches in ILD lungs. Moreover, NCM in the ILD lungs expressed increased CX3CR1, M2-like and phagocytic markers. Taken together, our data support that in ILD, fractalkine drives the migration of CX3CR1+ NCM to the lungs, thereby perpetuating the local fibrotic process.

Cell-surface phenotyping identifies CD36 and CD97 as novel markers of fibroblast quiescence in lung fibrosis.

Fibroblasts play an important role in lung homeostasis and disease. In lung fibrosis, fibroblasts adopt a proliferative and migratory phenotype, with increased expression of α-smooth muscle actin (αSMA) and enhanced secretion of extracellular matrix components. Comprehensive profiling of fibroblast heterogeneity is limited because of a lack of specific cell-surface markers. We have previously profiled the surface proteome of primary human lung fibroblasts. Here, we sought to define and quantify a panel of cluster of differentiation (CD) markers in primary human lung fibroblasts and idiopathic pulmonary fibrosis (IPF) lung tissue, using immunofluorescence and FACS analysis. Fibroblast function was assessed by analysis of replicative senescence. We observed the presence of distinct fibroblast phenotypes in vivo, characterized by various combinations of Desmin, αSMA, CD36, or CD97 expression. Most markers demonstrated stable expression over passages in vitro, but significant changes were observed for CD36, CD54, CD82, CD106, and CD140a. Replicative senescence of fibroblasts was observed from passage 10 onward. CD36- and CD97-positive but αSMA-negative cells were present in remodeled areas of IPF lungs. Transforming growth factor (TGF)-ß treatment induced αSMA and collagen I expression but repressed CD36 and CD97 expression. We identified a panel of stable surface markers in human lung fibroblasts, applicable for positive-cell isolation directly from lung tissue. TGF-ß exposure represses CD36 and CD97 expression, despite increasing αSMA expression; we therefore identified complex surface protein changes during fibroblast-myofibroblast activation. Coexistence of quiescence and activated fibroblast subtypes in the IPF lung suggests dynamic remodeling of fibroblast activation upon subtle changes to growth factor exposure in local microenvironmental niches.

Activation of the NF-κB pathway alters the phenotype of MSCs in the tracheal aspirates of preterm infants with severe BPD.

Mesenchymal stromal cells (MSCs) are released into the airways of preterm infants following lung injury. These cells display a proinflammatory phenotype and are associated with development of severe bronchopulmonary dysplasia (BPD). We aimed to characterize the functional properties of MSCs obtained from tracheal aspirates of 50 preterm infants who required invasive ventilation. Samples were separated by disease severity. The increased proliferative capacity of MSCs was associated with longer duration of mechanical ventilation and higher severity of BPD. Augmented growth depended on nuclear accumulation of NFκBp65 and was accompanied by reduced expression of cytosolic α-smooth muscle actin (α-SMA). The central role of NF-κB signaling was confirmed by inhibition of IκBα phosphorylation. The combined score of proliferative capacity, accumulation of NFκBp65, and expression of α-SMA was used to predict the development of severe BPD with an area under the curve (AUC) of 0.847. We mimicked the clinical situation in vitro, and stimulated MSCs with IL-1ß and TNF-α. Both cytokines induced similar and persistent changes as was observed in MSCs obtained from preterm infants with severe BPD. RNA interference was employed to investigate the mechanistic link between NFκBp65 accumulation and alterations in phenotype. Our data indicate that determining the phenotype of resident pulmonary MSCs represents a promising biomarker-based approach. The persistent alterations in phenotype, observed in MSCs from preterm infants with severe BPD, were induced by the pulmonary inflammatory response. NFκBp65 accumulation was identified as a central regulatory mechanism. Future preclinical and clinical studies, aimed to prevent BPD, should focus on phenotype changes in pulmonary MSCs.

Differential effects of Nintedanib and Pirfenidone on lung alveolar epithelial cell function in ex vivo murine and human lung tissue cultures of pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease. Repetitive injury and reprogramming of the lung epithelium are thought to be critical drivers of disease progression, contributing to fibroblast activation, extracellular matrix remodeling, and subsequently loss of lung architecture and function. To date, Pirfenidone and Nintedanib are the only approved drugs known to decelerate disease progression, however, if and how these drugs affect lung epithelial cell function, remains largely unexplored. METHODS: We treated murine and human 3D ex vivo lung tissue cultures (3D-LTCs; generated from precision cut lung slices (PCLS)) as well as primary murine alveolar epithelial type II (pmATII) cells with Pirfenidone or Nintedanib. Murine 3D-LTCs or pmATII cells were derived from the bleomycin model of fibrosis. Early fibrotic changes were induced in human 3D-LTCs by a mixture of profibrotic factors. Epithelial and mesenchymal cell function was determined by qPCR, Western blotting, Immunofluorescent staining, and ELISA. RESULTS: Low µM concentrations of Nintedanib (1 µM) and mM concentrations of Pirfenidone (2.5 mM) reduced fibrotic gene expression including Collagen 1a1 and Fibronectin in murine and human 3D-LTCs as well as pmATII cells. Notably, Nintedanib stabilized expression of distal lung epithelial cell markers, especially Surfactant Protein C in pmATII cells as well as in murine and human 3D-LTCs. CONCLUSIONS: Pirfenidone and Nintedanib exhibit distinct effects on murine and human epithelial cells, which might contribute to their anti-fibrotic action. Human 3D-LTCs represent a valuable tool to assess anti-fibrotic mechanisms of potential drugs for the treatment of IPF patients.