DNA sensing via the cGAS/STING pathway activates the immunoproteasome and adaptive T-cell immunity.

The immunoproteasome is a specialized type of proteasome involved in MHC class I antigen presentation, antiviral adaptive immunity, autoimmunity, and is also part of a broader response to stress. Whether the immunoproteasome is regulated by DNA stress, however, is not known. We here demonstrate that mitochondrial DNA stress upregulates the immunoproteasome and MHC class I antigen presentation pathway via cGAS/STING/type I interferon signaling resulting in cell autonomous activation of CD8+ T cells. The cGAS/STING-induced adaptive immune response is also observed in response to genomic DNA and is conserved in epithelial and mesenchymal cells of mice and men. In patients with idiopathic pulmonary fibrosis, chronic activation of the cGAS/STING-induced adaptive immune response in aberrant lung epithelial cells concurs with CD8+ T-cell activation in diseased lungs. Genetic depletion of the immunoproteasome and specific immunoproteasome inhibitors counteract DNA stress induced cytotoxic CD8+ T-cell activation. Our data thus unravel cytoplasmic DNA sensing via the cGAS/STING pathway as an activator of the immunoproteasome and CD8+ T cells. This represents a novel potential pathomechanism for pulmonary fibrosis that opens new therapeutic perspectives.

Loss of ANT1 Increases Fibrosis and Epithelial Cell Senescence in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by progressive lung scarring and remodeling. Although treatments exist that slow disease progression, IPF is irreversible, and there is no cure. Cellular senescence, a major hallmark of aging, has been implicated in IPF pathogenesis, and mitochondrial dysfunction is increasingly recognized as a driver of senescence. Adenine nucleotide translocases (ANTs) are abundant mitochondrial ATP-ADP transporters critical for regulating cell fate and maintaining mitochondrial function. We sought to determine how alterations in ANTs influence cellular senescence in pulmonary fibrosis. We found that SLC25A4 (solute carrier family 25 member 4) (ANT1) and SLC25A5 (ANT2) expression is reduced in the lungs of patients with IPF, particularly within alveolar type II (AT2) cells, by single-cell RNA sequencing and tissue staining. Loss of ANT1 by siRNA in lung epithelial cells resulted in increased senescence markers such as ß-galactosidase and p21, with a reduction in the ratio of nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide. Bleomycin-treated ANT1 knockdown cells also had increased senescence markers compared with bleomycin-treated control cells. Loss of ANT1 in AT2 cells resulted in a reduction in alveolar organoid growth, with an increase in p21 by staining. Global loss of ANT1 resulted in worse lung fibrosis and increased senescence in the bleomycin- and asbestos-induced mouse models of pulmonary fibrosis. In summary, loss of ANT1 contributes to IPF pathogenesis through mitochondrial dysfunction, increased senescence, and decreased regenerative capacity of AT2 cells, resulting in enhanced lung fibrosis. Modulation of ANTs presents a new therapeutic avenue that may alter cellular senescence pathways and limit pulmonary fibrosis.

WNT Signalling in Lung Physiology and Pathology.

The main physiological function of the lung is gas exchange, mediated at the interface between the alveoli and the pulmonary microcapillary network and facilitated by conducting airway structures that regulate the transport of these gases from and to the alveoli. Exposure to microbial and environmental factors such as allergens, viruses, air pollution, and smoke contributes to the development of chronic lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and lung cancer. Respiratory diseases as a cluster are the commonest cause of chronic disease and of hospitalization in children and are among the three most common causes of morbidity and mortality in the adult population worldwide. Many of these chronic respiratory diseases are associated with inflammation and structural remodelling of the airways and/or alveolar tissues. They can often only be treated symptomatically with no disease-modifying therapies that normalize the pathological tissue destruction driven by inflammation and remodelling. In search for novel therapeutic strategies for these diseases, several lines of evidence revealed the WNT pathway as an emerging target for regenerative strategies in the lung. WNT proteins, their receptors, and signalling effectors have central regulatory roles under (patho)physiological conditions underpinning lung function and (chronic) lung diseases and we summarize these roles and discuss how pharmacological targeting of the WNT pathway may be utilized for the treatment of chronic lung diseases.

Microfluidics for secretome analysis under enhanced endogenous signaling.

Cell secretome, the complex set of proteins that are secreted by the cells, is a fundamental mechanism of cell-cell communication both in vitro and in vivo. In vivo, the analysis of proteins secreted into body fluids can bring to the identification of biomarkers for important physiopathological conditions. However, due to the complexity of the protein content of body fluids, a better understanding of the secreted proteins by different cell types is highly desirable and can be performed in vitro for dissection. To this aim, microfluidic culture systems could be particularly relevant because of the accumulation of extrinsic endogenous signals at microliter scale, which better preserves the self-regulation occurring in the small interstitial spaces in vivo. In this work, we perform a quantitative study to compare the secretome in microfluidics and in a standard well plate. Human foreskin fibroblasts are used as a case study. This work also represents an important technological advance in terms of feasibility of high-throughput quantitative protein analyses in microfluidics.

Cell Adhesion Molecules in Fibrotic Diseases.

Mechanisms underlying the pathogenesis of tissue fibrosis remain incompletely understood. Emerging evidence suggests that cell adhesion molecules (CAMs) are critical in fibrotic progression in many organs, including lung, kidney, skin, and liver. CAMs promote cell-cell and cell-extracellular matrix (ECM) interactions to maintain tissue architecture and normal function in homeostasis. However, dysregulated expression and function of CAMs can lead to chronic inflammation and tissue fibrosis. The major families of CAMs include integrins, cadherins, selectins, and immunoglobulins. Here, we review the role of the CAMs in fibrosis development across various organs with a focus on integrins and cadherins, and discuss their respective roles in the development of pulmonary fibrosis.

Spatial mapping of cellular senescence: emerging challenges and opportunities.

Cellular senescence is a well-established driver of aging and age-related diseases. There are many challenges to mapping senescent cells in tissues such as the absence of specific markers and their relatively low abundance and vast heterogeneity. Single-cell technologies have allowed unprecedented characterization of senescence; however, many methodologies fail to provide spatial insights. The spatial component is essential, as senescent cells communicate with neighboring cells, impacting their function and the composition of extracellular space. The Cellular Senescence Network (SenNet), a National Institutes of Health (NIH) Common Fund initiative, aims to map senescent cells across the lifespan of humans and mice. Here, we provide a comprehensive review of the existing and emerging methodologies for spatial imaging and their application toward mapping senescent cells. Moreover, we discuss the limitations and challenges inherent to each technology. We argue that the development of spatially resolved methods is essential toward the goal of attaining an atlas of senescent cells.

Powering the formation of alveoli.

Two cell types in the lung need specific numbers and distributions of mitochondria for alveoli to form correctly.

Dynamic Point Cloud Denoising via Manifold-to-Manifold Distance.

3D dynamic point clouds provide a natural discrete representation of real-world objects or scenes in motion, with a wide range of applications in immersive telepresence, autonomous driving, surveillance, etc. Nevertheless, dynamic point clouds are often perturbed by noise due to hardware, software or other causes. While a plethora of methods have been proposed for static point cloud denoising, few efforts are made for the denoising of dynamic point clouds, which is quite challenging due to the irregular sampling patterns both spatially and temporally. In this paper, we represent dynamic point clouds naturally on spatial-temporal graphs, and exploit the temporal consistency with respect to the underlying surface (manifold). In particular, we define a manifold-to-manifold distance and its discrete counterpart on graphs to measure the variation-based intrinsic distance between surface patches in the temporal domain, provided that graph operators are discrete counterparts of functionals on Riemannian manifolds. Then, we construct the spatial-temporal graph connectivity between corresponding surface patches based on the temporal distance and between points in adjacent patches in the spatial domain. Leveraging the initial graph representation, we formulate dynamic point cloud denoising as the joint optimization of the desired point cloud and underlying graph representation, regularized by both spatial smoothness and temporal consistency. We reformulate the optimization and present an efficient algorithm. Experimental results show that the proposed method significantly outperforms independent denoising of each frame from state-of-the-art static point cloud denoising approaches, on both Gaussian noise and simulated LiDAR noise.

Chronic WNT/ß-catenin signaling induces cellular senescence in lung epithelial cells.

The rapid expansion of the elderly population has led to the recent epidemic of age-related diseases, including increased incidence and mortality of chronic lung diseases, such as Idiopathic Pulmonary Fibrosis (IPF). Cellular senescence is a major hallmark of aging and has a higher occurrence in IPF. The lung epithelium represents a major site of tissue injury, cellular senescence and aberrant activity of developmental pathways such as the WNT/ß-catenin pathway in IPF. The potential impact of WNT/ß-catenin signaling on alveolar epithelial senescence in general as well as in IPF, however, remains elusive. Here, we characterized alveolar epithelial cells of aged mice and assessed the contribution of chronic WNT/ß-catenin signaling on alveolar epithelial type (AT) II cell senescence. Whole lungs from old (16-24 months) versus young (3 months) mice had relatively less epithelial (EpCAM+) but more inflammatory (CD45+) cells, as assessed by flow cytometry. Compared to young ATII cells, old ATII cells showed decreased expression of the ATII cell marker Surfactant Protein C along with increased expression of the ATI cell marker Hopx, accompanied by increased WNT/ß-catenin activity. Notably, when placed in an organoid assay, old ATII cells exhibited decreased progenitor cell potential. Chronic canonical WNT/ß-catenin activation for up to 7 days in primary ATII cells as well as alveolar epithelial cell lines induced a robust cellular senescence, whereas the non-canonical ligand WNT5A was not able to induce cellular senescence. Moreover, chronic WNT3A treatment of precision-cut lung slices (PCLS) further confirmed ATII cell senescence. Simultaneously, chronic but not acute WNT/ß-catenin activation induced a profibrotic state with increased expression of the impaired ATII cell marker Keratin 8. These results suggest that chronic WNT/ß-catenin activity in the IPF lung contributes to increased ATII cell senescence and reprogramming. In the fibrotic environment, WNT/ß-catenin signaling thus might lead to further progenitor cell dysfunction and impaired lung repair.

The Microfluidic Environment Reveals a Hidden Role of Self-Organizing Extracellular Matrix in Hepatic Commitment and Organoid Formation of hiPSCs.

The specification of the hepatic identity during human liver development is strictly controlled by extrinsic signals, yet it is still not clear how cells respond to these exogenous signals by activating secretory cascades, which are extremely relevant, especially in 3D self-organizing systems. Here, we investigate how the proteins secreted by human pluripotent stem cells (hPSCs) in response to developmental exogenous signals affect the progression from endoderm to the hepatic lineage, including their competence to generate nascent hepatic organoids. By using microfluidic confined environment and stable isotope labeling with amino acids in cell culture-coupled mass spectrometry (SILAC-MS) quantitative proteomic analysis, we find high abundancy of extracellular matrix (ECM)-associated proteins. Hepatic progenitor cells either derived in microfluidics or exposed to exogenous ECM stimuli show a significantly higher potential of forming hepatic organoids that can be rapidly expanded for several passages and further differentiated into functional hepatocytes. These results prove an additional control over the efficiency of hepatic organoid formation and differentiation for downstream applications.

Extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture.

Organoids have extensive therapeutic potential and are increasingly opening up new avenues within regenerative medicine. However, their clinical application is greatly limited by the lack of effective GMP-compliant systems for organoid expansion in culture. Here, we envisage that the use of extracellular matrix (ECM) hydrogels derived from decellularized tissues (DT) can provide an environment capable of directing cell growth. These gels possess the biochemical signature of tissue-specific ECM and have the potential for clinical translation. Gels from decellularized porcine small intestine (SI) mucosa/submucosa enable formation and growth of endoderm-derived human organoids, such as gastric, hepatic, pancreatic, and SI. ECM gels can be used as a tool for direct human organoid derivation, for cell growth with a stable transcriptomic signature, and for in vivo organoid delivery. The development of these ECM-derived hydrogels opens up the potential for human organoids to be used clinically.

Prevalence and plasmid characterization of the qnrD determinant in Enterobacteriaceae isolated from animals, retail meat products, and humans.

qnrD, unlike other qnr genes, is mainly located on small nonconjugative plasmids. We investigated the presence of qnrD among 1,373 Enterobacteriaceae isolates in China. Twelve qnrD-positive strains were detected, and all were nonsusceptible to fluoroquinolones. The complete sequence of plasmids showed that the qnrD determinants were located on two plasmids with a respective size of ~4.2 and 2.7 k-bp. Interestingly, the identification of qnrD in this study revealed the highest prevalence of Proteeae among Enterobacteriaceae identified.