Inhibition of LTßR signalling activates WNT-induced regeneration in lung.

Lymphotoxin ß-receptor (LTßR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures1,2, which are associated with severe chronic inflammatory diseases that span several organ systems3-6. How LTßR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTßR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTßR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTßR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTßR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTßR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFß signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/ß-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTßR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures1 and inhibition of apoptosis with tissue-regenerative strategies.

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.

Sfrp1 inhibits lung fibroblast invasion during transition to injury-induced myofibroblasts.

BACKGROUND: Fibroblast-to-myofibroblast conversion is a major driver of tissue remodelling in organ fibrosis. Distinct lineages of fibroblasts support homeostatic tissue niche functions, yet their specific activation states and phenotypic trajectories during injury and repair have remained unclear. METHODS: We combined spatial transcriptomics, multiplexed immunostainings, longitudinal single-cell RNA-sequencing and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. Our findings were validated in idiopathic pulmonary fibrosis patient tissues in situ as well as in cell differentiation and invasion assays using patient lung fibroblasts. Cell differentiation and invasion assays established a function of SFRP1 in regulating human lung fibroblast invasion in response to transforming growth factor (TGF)ß1. MEASUREMENTS AND MAIN RESULTS: We discovered a transitional fibroblast state characterised by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1 + cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage-specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1 + transitional fibroblasts and Cthrc1 + myofibroblasts. TGFß1 downregulated SFRP1 in noninvasive transitional cells and induced their switch to an invasive CTHRC1+ myofibroblast identity. Finally, using loss-of-function studies we showed that SFRP1 modulates TGFß1-induced fibroblast invasion and RHOA pathway activity. CONCLUSIONS: Our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies SFRP1 as a modulator of TGFß1-driven fibroblast phenotypes in fibrogenesis. These findings are relevant in the context of therapeutic interventions that aim at limiting or reversing fibroblast foci formation.

Targeting genomic SARS-CoV-2 RNA with siRNAs allows efficient inhibition of viral replication and spread.

A promising approach to tackle the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. So far it is unclear, which viral replication steps can be efficiently inhibited with siRNAs. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, and thereby terminate replication before start of transcription and prevent virus-induced cell death. Coronaviruses replicate via negative sense RNA intermediates using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5' and 3' end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting common sequences shared by all viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs that targeted open reading frame 1 (ORF1) which only exists in gRNA. In contrast, siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs leading to an impaired antiviral efficacy. Verifying the translational relevance of these findings, we show that a chemically modified siRNA that targets a highly conserved region of ORF1, inhibited SARS-CoV-2 replication ex vivo in explants of the human lung. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with specifically targeting gRNA, might be key for high antiviral efficacy.

Development of a dynamic in vitro stretch model of the alveolar interface with aerosol delivery.

We describe the engineering design, computational modeling, and empirical performance of a moving air-liquid interface (MALI) bioreactor for the study of aerosol deposition on cells cultured on an elastic, porous membrane which mimics both air-liquid interface exposure conditions and mechanoelastic motion of lung tissue during breathing. The device consists of two chambers separated by a cell layer cultured on a porous, flexible membrane. The lower (basolateral) chamber is perfused with cell culture medium simulating blood circulation. The upper (apical) chamber representing the air compartment of the lung is interfaced to an aerosol generator and a pressure actuation system. By cycling the pressure in the apical chamber between 0 and 7 kPa, the membrane can mimic the periodic mechanical strain of the alveolar wall. Focusing on the engineering aspects of the system, we show that membrane strain can be monitored by measuring changes in pressure resulting from the movement of media in the basolateral chamber. Moreover, liquid aerosol deposition at a high dose delivery rate (>1 µl cm-2 min-1 ) is highly efficient (ca. 51.5%) and can be accurately modeled using finite element methods. Finally, we show that lung epithelial cells can be mechanically stimulated under air-liquid interface and stretch-conditions without loss of viability. The MALI bioreactor could be used to study the effects of aerosol on alveolar cells cultured at the air-liquid interface in a biodynamic environment or for toxicological or therapeutic applications.

Cub domain-containing protein 1 negatively regulates TGF-ß signaling and myofibroblast differentiation.

Fibroblasts are thought to be the prime cell type for producing and secreting extracellular matrix (ECM) proteins in the connective tissue. The profibrotic cytokine transforming growth factor-ß1 (TGF-ß1) activates and transdifferentiates fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts, which exhibit increased ECM secretion, in particular collagens. Little information, however, exists about cell-surface molecules on fibroblasts that mediate this transdifferentiation process. We recently identified, using unbiased cell-surface proteome analysis, Cub domain-containing protein 1 (CDCP1) to be strongly downregulated by TGF-ß1. CDCP1 is a transmembrane glycoprotein, the expression and role of which has not been investigated in lung fibroblasts to date. Here, we characterized, in detail, the effect of TGF-ß1 on CDCP1 expression and function, using immunofluorescence, FACS, immunoblotting, and siRNA-mediated knockdown of CDCP1. CDCP1 is present on interstitial fibroblasts, but not myofibroblasts, in the normal and idiopathic pulmonary fibrosis lung. In vitro, TGF-ß1 decreased CDCP1 expression in a time-dependent manner by impacting mRNA and protein levels. Knockdown of CDCP1 enhanced a TGF-ß1-mediated cell adhesion of fibroblasts. Importantly, CDCP1-depleted cells displayed an enhanced expression of profibrotic markers, such as collagen V or α-SMA, which was found to be independent of TGF-ß1. Our data show, for the very first time that loss of CDCP1 contributes to fibroblast to myofibroblast differentiation via a potential negative feedback loop between CDCP1 expression and TGF-ß1 stimulation.

Distinct niches within the extracellular matrix dictate fibroblast function in (cell free) 3D lung tissue cultures.

Cues from the extracellular matrix (ECM) and their functional interplay with cells play pivotal roles for development, tissue repair, and disease. However, the precise nature of this interplay remains elusive. We used an innovative 3D cell culture ECM model by decellularizing 300-µm-thick ex vivo lung tissue scaffolds (d3D-LTCs) derived from diseased and healthy mouse lungs, which widely mimics the native (patho)physiological in vivo ECM microenvironment. We successfully repopulated all d3D-LTCs with primary human and murine fibroblasts, and moreover, we demonstrated that the cells also populated the innermost core regions of the d3D-LTCs in a real 3D fashion. The engrafted fibroblasts revealed a striking functional plasticity, depending on their localization in distinct ECM niches of the d3D-LTCs, affecting the cells' tissue engraftment, cellular migration rates, cell morphologies, and protein expression and phosphorylation levels. Surprisingly, we also observed fibroblasts that were homing to the lung scaffold's interstitium as well as fibroblasts that were invading fibrotic areas. To date, the functional nature and even the existence of 3D cell matrix adhesions in vivo as well as in 3D culture models is still unclear and controversial. Here, we show that attachment of fibroblasts to the d3D-LTCs evidently occurred via focal adhesions, thus advocating for a relevant functional role in vivo. Furthermore, we found that protein levels of talin, paxillin, and zyxin and phosphorylation levels of paxillin Y118, as well as the migration-relevant small GTPases RhoA, Rac, and CDC42, were significantly reduced compared with their attachment to 2D plastic dishes. In summary, our results strikingly indicate that inherent physical or compositional characteristics of the ECM act as instructive cues altering the functional behavior of engrafted cells. Thus, d3D-LTCs might aid to obtain more realistic data in vitro, with a high relevance for drug discovery and mechanistic studies alike.

Pharmacometabolic response to pirfenidone in pulmonary fibrosis detected by MALDI-FTICR-MSI.

Idiopathic pulmonary fibrosis (IPF) is a fatal condition that reduces life expectancy and shows a limited response to available therapies. Pirfenidone has been approved for treatment of IPF, but little is known about the distinct metabolic changes that occur in the lung upon pirfenidone administration.Here, we performed a proof-of-concept study using high-resolution quantitative matrix-assisted laser desorption/ionisation Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI) to simultaneously detect, visualise and quantify in situ endogenous and exogenous metabolites in lungs of mice subjected to experimental fibrosis and human patients with IPF, and to assess the effect of pirfenidone treatment on metabolite levels.Metabolic pathway analysis and endogenous metabolite quantification revealed that pirfenidone treatment restores redox imbalance and glycolysis in IPF tissues, and downregulates ascorbate and aldarate metabolism, thereby likely contributing to in situ modulation of collagen processing. As such, we detected specific alterations in metabolite pathways in fibrosis and, importantly, metabolic recalibration following pirfenidone treatment.Together, these results highlight the suitability of high-resolution MALDI-FTICR-MSI for deciphering the therapeutic effects of pirfenidone and provide a preliminary analysis of the metabolic changes that occur during pirfenidone treatment in vivo These data may therefore contribute to improvement of currently available therapies for IPF.

The instructive extracellular matrix of the lung: basic composition and alterations in chronic lung disease.

The pulmonary extracellular matrix (ECM) determines the tissue architecture of the lung, and provides mechanical stability and elastic recoil, which are essential for physiological lung function. Biochemical and biomechanical signals initiated by the ECM direct cellular function and differentiation, and thus play a decisive role in lung development, tissue remodelling processes and maintenance of adult homeostasis. Recent proteomic studies have demonstrated that at least 150 different ECM proteins, glycosaminoglycans and modifying enzymes are expressed in the lung, and these assemble into intricate composite biomaterials. These highly insoluble assemblies of interacting ECM proteins and their glycan modifications can act as a solid phase-binding interface for hundreds of secreted proteins, which creates an information-rich signalling template for cell function and differentiation. Dynamic changes within the ECM that occur upon injury or with ageing are associated with several chronic lung diseases. In this review, we summarise the available data about the structure and function of the pulmonary ECM, and highlight changes that occur in idiopathic pulmonary fibrosis (IPF), pulmonary arterial hypertension (PAH), chronic obstructive pulmonary disease (COPD), asthma and lung cancer. We discuss potential mechanisms of ECM remodelling and modification, which we believe are relevant for future diagnosis and treatment of chronic lung disease.

Time- and compartment-resolved proteome profiling of the extracellular niche in lung injury and repair.

The extracellular matrix (ECM) is a key regulator of tissue morphogenesis and repair. However, its composition and architecture are not well characterized. Here, we monitor remodeling of the extracellular niche in tissue repair in the bleomycin-induced lung injury mouse model. Mass spectrometry quantified 8,366 proteins from total tissue and bronchoalveolar lavage fluid (BALF) over the course of 8 weeks, surveying tissue composition from the onset of inflammation and fibrosis to its full recovery. Combined analysis of proteome, secretome, and transcriptome highlighted post-transcriptional events during tissue fibrogenesis and defined the composition of airway epithelial lining fluid. To comprehensively characterize the ECM, we developed a quantitative detergent solubility profiling (QDSP) method, which identified Emilin-2 and collagen-XXVIII as novel constituents of the provisional repair matrix. QDSP revealed which secreted proteins interact with the ECM, and showed drastically altered association of morphogens to the insoluble matrix upon injury. Thus, our proteomic systems biology study assigns proteins to tissue compartments and uncovers their dynamic regulation upon lung injury and repair, potentially contributing to the development of anti-fibrotic strategies.

Coactivator-Associated Arginine Methyltransferase-1 Function in Alveolar Epithelial Senescence and Elastase-Induced Emphysema Susceptibility.

Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible loss of lung function and is one of the most prevalent and severe diseases worldwide. A major feature of COPD is emphysema, which is the progressive loss of alveolar tissue. Coactivator-associated arginine methyltransferase-1 (CARM1) regulates histone methylation and the transcription of genes involved in senescence, proliferation, and differentiation. Complete loss of CARM1 leads to disrupted differentiation and maturation of alveolar epithelial type II (ATII) cells. We thus hypothesized that CARM1 regulates the development and progression of emphysema. To address this, we investigated the contribution of CARM1 to alveolar rarefication using the mouse model of elastase-induced emphysema in vivo and small interfering (si)RNA-mediated knockdown in ATII-like LA4 cells in vitro. We demonstrate that emphysema progression in vivo is associated with a time-dependent down-regulation of CARM1. Importantly, elastase-treated CARM1 haploinsufficient mice show significantly increased airspace enlargement (52.5 ± 9.6 µm versus 38.8 ± 5.5 µm; P < 0.01) and lung compliance (2.8 ± 0.32 µl/cm H2O versus 2.4 ± 0.4 µl/cm H2O; P < 0.04) compared with controls. The knockdown of CARM1 in LA4 cells led to decreased sirtuin 1 expression (0.034 ± 0.003 versus 0.022 ± 0.001; P < 0.05) but increased expression of p16 (0.27 ± 0.013 versus 0.31 ± 0.010; P < 0.5) and p21 (0.81 ± 0.088 versus 1.28 ± 0.063; P < 0.01) and higher ß-galactosidase-positive senescent cells (50.57 ± 7.36% versus 2.21 ± 0.34%; P < 0.001) compared with scrambled siRNA. We further demonstrated that CARM1 haploinsufficiency impairs transdifferentiation and wound healing (32.18 ± 0.9512% versus 8.769 ± 1.967%; P < 0.001) of alveolar epithelial cells. Overall, these results reveal a novel function of CARM1 in regulating emphysema development and premature lung aging via alveolar senescence as well as impaired regeneration, repair, and differentiation of ATII cells.

Multidimensional immunolabeling and 4D time-lapse imaging of vital ex vivo lung tissue.

During the last decades, the study of cell behavior was largely accomplished in uncoated or extracellular matrix (ECM)-coated plastic dishes. To date, considerable cell biological efforts have tried to model in vitro the natural microenvironment found in vivo. For the lung, explants cultured ex vivo as lung tissue cultures (LTCs) provide a three-dimensional (3D) tissue model containing all cells in their natural microenvironment. Techniques for assessing the dynamic live interaction between ECM and cellular tissue components, however, are still missing. Here, we describe specific multidimensional immunolabeling of living 3D-LTCs, derived from healthy and fibrotic mouse lungs, as well as patient-derived 3D-LTCs, and concomitant real-time four-dimensional multichannel imaging thereof. This approach allowed the evaluation of dynamic interactions between mesenchymal cells and macrophages with their ECM. Furthermore, fibroblasts transiently expressing focal adhesions markers incorporated into the 3D-LTCs, paving new ways for studying the dynamic interaction between cellular adhesions and their natural-derived ECM. A novel protein transfer technology (FuseIt/Ibidi) shuttled fluorescently labeled α-smooth muscle actin antibodies into the native cells of living 3D-LTCs, enabling live monitoring of α-smooth muscle actin-positive stress fibers in native tissue myofibroblasts residing in fibrotic lesions of 3D-LTCs. Finally, this technique can be applied to healthy and diseased human lung tissue, as well as to adherent cells in conventional two-dimensional cell culture. This novel method will provide valuable new insights into the dynamics of ECM (patho)biology, studying in detail the interaction between ECM and cellular tissue components in their natural microenvironment.

Preclinical validation and imaging of Wnt-induced repair in human 3D lung tissue cultures.

Chronic obstructive pulmonary disease (COPD) is characterised by a progressive loss of lung tissue. Inducing repair processes within the adult diseased lung is of major interest and Wnt/ß-catenin signalling represents a promising target for lung repair. However, the translation of novel therapeutic targets from model systems into clinical use remains a major challenge.We generated murine and patient-derived three-dimensional (3D) ex vivo lung tissue cultures (LTCs), which closely mimic the 3D lung microenvironment in vivo. Using two well-known glycogen synthase kinase-3ß inhibitors, lithium chloride (LiCl) and CHIR 99021 (CT), we determined Wnt/ß-catenin-driven lung repair processes in high spatiotemporal resolution using quantitative PCR, Western blotting, ELISA, (immuno)histological assessment, and four-dimensional confocal live tissue imaging.Viable 3D-LTCs exhibited preserved lung structure and function for up to 5 days. We demonstrate successful Wnt/ß-catenin signal activation in murine and patient-derived 3D-LTCs from COPD patients. Wnt/ß-catenin signalling led to increased alveolar epithelial cell marker expression, decreased matrix metalloproteinase-12 expression, as well as altered macrophage activity and elastin remodelling. Importantly, induction of surfactant protein C significantly correlated with disease stage (per cent predicted forced expiratory volume in 1 s) in patient-derived 3D-LTCs.Patient-derived 3D-LTCs represent a valuable tool to analyse potential targets and drugs for lung repair. Enhanced Wnt/ß-catenin signalling attenuated pathological features of patient-derived COPD 3D-LTCs.

Mechanosensing through focal adhesion-anchored intermediate filaments.

Integrin-based mechanotransduction involves a complex focal adhesion (FA)-associated machinery that is able to detect and respond to forces exerted either through components of the extracellular matrix or the intracellular contractile actomyosin network. Here, we show a hitherto unrecognized regulatory role of vimentin intermediate filaments (IFs) in this process. By studying fibroblasts in which vimentin IFs were decoupled from FAs, either because of vimentin deficiency (V0) or loss of vimentin network anchorage due to deficiency in the cytolinker protein plectin (P0), we demonstrate attenuated activation of the major mechanosensor molecule FAK and its downstream targets Src, ERK1/2, and p38, as well as an up-regulation of the compensatory feedback loop acting on RhoA and myosin light chain. In line with these findings, we show strongly reduced FA turnover rates in P0 fibroblasts combined with impaired directional migration, formation of protrusions, and up-regulation of "stretched" high-affinity integrin complexes. By exploiting tension-independent conditions, we were able to mechanistically link these defects to diminished cytoskeletal tension in both P0 and V0 cells. Our data provide important new insights into molecular mechanisms underlying cytoskeleton-regulated mechanosensing, a feature that is fundamental for controlled cell movement and tumor progression.

Efficient bioactive delivery of aerosolized drugs to human pulmonary epithelial cells cultured in air-liquid interface conditions.

In inhalation therapy, drugs are deposited as aerosols onto the air-facing lung epithelium. The currently used in vitro cell assays for drug testing, however, typically dissolve drugs in the medium, completely covering the cells, which represents an unphysiological drug application scenario. Although physiologically realistic in vitro cell culture models of the pulmonary air-blood barrier are available, reliable, easy-to-handle, and efficient technologies for direct aerosol-to-cell delivery are lacking. Here, we introduce the Air-Liquid Interface (ALI) Cell Exposure-Cloud (ALICE-CLOUD) technology, which uses principles of cloud motion for fast and quantitative delivery of aerosolized liquid drugs to pulmonary cells cultured under realistic ALI conditions. Aerosol-to-cell delivery proved to be highly efficient, reproducible, and rapid when using aerosolized fluorescein as surrogate drug. As a proof-of-concept study for the ALICE-CLOUD, we performed functional efficacy studies with the U.S. Food and Drug Administration-approved proteasome inhibitor, Bortezomib, a novel candidate drug for inhalation therapy. Aerosolized Bortezomib had a pronounced anti-inflammatory effect on human epithelial lung cells (A549), as indicated by a significant reduction of (TNFα-induced) IL-8 promoter activation. Importantly, cell-based therapeutic efficacy of aerosolized Bortezomib under ALI conditions was similar to that under dissolved and nonaerosolized submerged conditions, but with faster uptake kinetics. Our data indicate that the ALICE-CLOUD is a reliable tool for aerosolized drug screening with cells cultured under ALI conditions, which combines ease of handling with rapid, efficient, and dosimetrically accurate drug-to-cell delivery. This may pave the way for screening of inhalable drugs under physiologically more relevant and, hence, potentially more predictive conditions than the currently used submerged cell culture systems.

Cytokine signaling converging on IL11 in ILD fibroblasts provokes aberrant epithelial differentiation signatures.

Introduction: Interstitial lung disease (ILD) is a heterogenous group of lung disorders where destruction and incomplete regeneration of the lung parenchyma often results in persistent architectural distortion of the pulmonary scaffold. Continuous mesenchyme-centered, disease-relevant signaling likely initiates and perpetuates the fibrotic remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area. Methods: With the aim of identifying functional mediators of the lung mesenchymal-epithelial crosstalk with potential as new targets for therapeutic strategies, we developed a 3D organoid co-culture model based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells that form alveolar organoids in presence of lung fibroblasts from fibrotic-ILD patients, in our study referring to cases of pulmonary fibrosis, as well as control cell line (IMR-90). Results: While organoid formation capacity and size was comparable in the presence of fibrotic-ILD or control lung fibroblasts, metabolic activity was significantly increased in fibrotic-ILD co-cultures. Alveolar organoids cultured with fibrotic-ILD fibroblasts further demonstrated reduced stem cell function as reflected by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. To screen for key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used mass spectrometry and characterized the secretome of end stage fibrotic-ILD lung fibroblasts in comparison to non-chronic lung disease (CLD) patient fibroblasts. Out of the over 2000 proteins detected by this experimental approach, 47 proteins were differentially abundant comparing fibrotic-ILD and non-CLD fibroblast secretome. The fibrotic-ILD secretome profile was dominated by chemokines, including CXCL1, CXCL3, and CXCL8, interfering with growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating alveolar organoids with IL11, we recapitulated the co-culture results obtained with primary fibrotic-ILD fibroblasts including changes in metabolic activity. Conclusion: We identified mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD. In our alveolar organoid co-cultures, we were able to highlight the importance of fibroblast-initiated aberrant epithelial differentiation and confirmed IL11 as a key player in fibrotic-ILD pathogenesis by unbiased fibroblast secretome analysis.

Ex vivo tissue perturbations coupled to single-cell RNA-seq reveal multilineage cell circuit dynamics in human lung fibrogenesis.

Pulmonary fibrosis develops as a consequence of failed regeneration after injury. Analyzing mechanisms of regeneration and fibrogenesis directly in human tissue has been hampered by the lack of organotypic models and analytical techniques. In this work, we coupled ex vivo cytokine and drug perturbations of human precision-cut lung slices (hPCLS) with single-cell RNA sequencing and induced a multilineage circuit of fibrogenic cell states in hPCLS. We showed that these cell states were highly similar to the in vivo cell circuit in a multicohort lung cell atlas from patients with pulmonary fibrosis. Using micro-CT-staged patient tissues, we characterized the appearance and interaction of myofibroblasts, an ectopic endothelial cell state, and basaloid epithelial cells in the thickened alveolar septum of early-stage lung fibrosis. Induction of these states in the hPCLS model provided evidence that the basaloid cell state was derived from alveolar type 2 cells, whereas the ectopic endothelial cell state emerged from capillary cell plasticity. Cell-cell communication routes in patients were largely conserved in hPCLS, and antifibrotic drug treatments showed highly cell type-specific effects. Our work provides an experimental framework for perturbational single-cell genomics directly in human lung tissue that enables analysis of tissue homeostasis, regeneration, and pathology. We further demonstrate that hPCLS offer an avenue for scalable, high-resolution drug testing to accelerate antifibrotic drug development and translation.

Spray drying siRNA-lipid nanoparticles for dry powder pulmonary delivery.

While all the siRNA drugs on the market target the liver, the lungs offer a variety of currently undruggable targets which could potentially be treated with RNA therapeutics. Hence, local, pulmonary delivery of RNA nanoparticles could finally enable delivery beyond the liver. The administration of RNA drugs via dry powder inhalers offers many advantages related to physical, chemical and microbial stability of RNA and nanosuspensions. The present study was therefore designed to test the feasibility of engineering spray dried lipid nanoparticle (LNP) powders. Spray drying was performed using 5% lactose solution (m/V), and the targets were set to obtain nanoparticle sizes after redispersion of spray-dried powders around 150 nm, a residual moisture level below 5%, and RNA loss below 15% at maintained RNA bioactivity. The LNPs consisted of an ionizable cationic lipid which is a sulfur-containing analog of DLin-MC3-DMA, a helper lipid, cholesterol, and PEG-DMG encapsulating siRNA. Prior to the spray drying, the latter process was simulated with a novel dual emission fluorescence spectroscopy method to preselect the highest possible drying temperature and excipient solution maintaining LNP integrity and stability. Through characterization of physicochemical and aerodynamic properties of the spray dried powders, administration criteria for delivery to the lower respiratory tract were fulfilled. Spray dried LNPs penetrated the lung mucus layer and maintained bioactivity for >90% protein downregulation with a confirmed safety profile in a lung adenocarcinoma cell line. Additionally, the spray dried LNPs successfully achieved up to 50% gene silencing of the house keeping gene GAPDH in ex vivo human precision-cut lung slices at without increasing cytokine levels. This study verifies the successful spray drying procedure of LNP-siRNA systems maintaining their integrity and mediating strong gene silencing efficiency on mRNA and protein levels both in vitro and ex vivo. The successful spray drying procedure of LNP-siRNA formulations in 5% lactose solution creates a novel siRNA-based therapy option to target respiratory diseases such as lung cancer, asthma, COPD, cystic fibrosis and viral infections.

Inhibition of SARS-CoV-2 replication in the lung with siRNA/VIPER polyplexes.

SARS-CoV-2 has been the cause of a global pandemic since 2019 and remains a medical urgency. siRNA-based therapies are a promising strategy to fight viral infections. By targeting a specific region of the viral genome, siRNAs can efficiently downregulate viral replication and suppress viral infection. However, to achieve the desired therapeutic activity, siRNA requires a suitable delivery system. The VIPER (virus-inspired polymer for endosomal release) block copolymer has been reported as promising delivery system for both plasmid DNA and siRNA in the past years. It is composed of a hydrophilic block for condensation of nucleic acids as well as a hydrophobic, pH-sensitive block that, at acidic pH, exposes the membrane lytic peptide melittin, which enhances endosomal escape. In this study, we aimed at developing a formulation for pulmonary administration of siRNA to suppress SARS-CoV-2 replication in lung epithelial cells. After characterizing siRNA/VIPER polyplexes, the activity and safety profile were confirmed in a lung epithelial cell line. To further investigate the activity of the polyplexes in a more sophisticated cell culture system, an air-liquid interface (ALI) culture was established. siRNA/VIPER polyplexes reached the cell monolayer and penetrated through the mucus layer secreted by the cells. Additionally, the activity against wild-type SARS-CoV-2 in the ALI model was confirmed by qRT-PCR. To investigate translatability of our findings, the activity against SARS-CoV-2 was tested ex vivo in human lung explants. Here, siRNA/VIPER polyplexes efficiently inhibited SARS-CoV-2 replication. Finally, we verified the delivery of siRNA/VIPER polyplexes to lung epithelial cells in vivo, which represent the main cellular target of viral infection in the lung. In conclusion, siRNA/VIPER polyplexes efficiently delivered siRNA to lung epithelial cells and mediated robust downregulation of viral replication both in vitro and ex vivo without toxic or immunogenic side effects in vivo, demonstrating the potential of local siRNA delivery as a promising antiviral therapy in the lung.