FOXK2 targeting by the SCF-E3 ligase subunit FBXO24 for ubiquitin mediated degradation modulates mitochondrial respiration.

FOXK2 is a crucial transcription factor implicated in a wide array of biological activities and yet understanding of its molecular regulation at the level of protein turnover is limited. Here we identify that FOXK2 undergoes degradation in lung epithelia in the presence of the virulent pathogens P. aeruginosa and K. pneumoniae through ubiquitin-proteasomal processing. FOXK2 through its carboxyl-terminus (aa 428-478) binds the Skp-Cullin-F-box ubiquitin E3 ligase subunit FBXO24 that mediates multisite polyubiquitylation of the transcription factor resulting in its nuclear degradation. FOXK2 was detected within mitochondria and targeted depletion of the transcription factor or cellular expression of FOXK2 mutants devoid of key carboxyl-terminal domains significantly impaired mitochondrial function. In experimental bacterial pneumonia, Fbxo24 heterozygous mice exhibited preserved mitochondrial function and Foxk2 protein levels compared to wild-type littermates. The results suggest a new mode of regulatory control of mitochondrial energetics through modulation of FOXK2 cellular abundance.

Single Cell MALDI-MS Imaging of Lipids and Proteins in Senescent Fibroblasts.

In this study, we evaluate the lipidomic and proteomic profiles of human lung fibroblasts (hLFs) to interrogate changes occurring due to senescence. To study single cell populations, a comparison of cell adhered onto slides utilizing poly-D-lysine versus centrifugal force deposition was first analyzed to determine whether specific alterations were observed between preparations. The poly-D-lysine approach was than utilized to interrogate the lipidome of the cell populations and further evaluate potential applications of the MALDI-IHC platform for single-cell level analyses. Altogether, our results show the ability to detect lipids implicated in senescence and alterations to protein expression between normal and senescent fibroblast populations. This report is the first time that the MALDI-IHC system has been utilized at a single-cell level for analyzing the expression of proteins.

RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in the development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health, and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from patients with PAH. Endothelial haploinsufficiency of RAB7 caused spontaneous pulmonary hypertension (PH) in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA-Seq, and RAB7-silenced ECs showed impaired angiogenesis and expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, suggesting inhibition of autophagy at the predegradation level. Furthermore, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in rats with chronic hypoxia/SU5416. In conclusion, we demonstrate for the first time to our knowledge the fundamental impairment of EC function by loss of RAB7, causing PH, and show RAB7 activation to be a potential therapeutic strategy in a preclinical model of PH.

LEF1 isoforms regulate cellular senescence and aging.

The study of aging and its mechanisms, such as cellular senescence, has provided valuable insights into age-related pathologies, thus contributing to their prevention and treatment. The current abundance of high-throughput data combined with the surge of robust analysis algorithms has facilitated novel ways of identifying underlying pathways that may drive these pathologies. For the purpose of identifying key regulators of lung aging, we performed comparative analyses of transcriptional profiles of aged versus young human subjects and mice, focusing on the common age-related changes in the transcriptional regulation in lung macrophages, T cells, and B immune cells. Importantly, we validated our findings in cell culture assays and human lung samples. Our analysis identified lymphoid enhancer binding factor 1 (LEF1) as an important age-associated regulator of gene expression in all three cell types across different tissues and species. Follow-up experiments showed that the differential expression of long and short LEF1 isoforms is a key regulatory mechanism of cellular senescence. Further examination of lung tissue from patients with idiopathic pulmonary fibrosis, an age-related disease with strong ties to cellular senescence, revealed a stark dysregulation of LEF1. Collectively, our results suggest that LEF1 is a key factor of aging, and its differential regulation is associated with human and murine cellular senescence.

Transcriptional changes of the aging lung.

Aging is a natural process associated with declined organ function and higher susceptibility to developing chronic diseases. A systemic single-cell type-based study provides a unique opportunity to understand the mechanisms behind age-related pathologies. Here, we use single-cell gene expression analysis comparing healthy young and aged human lungs from nonsmoker donors to investigate age-related transcriptional changes. Our data suggest that aging has a heterogenous effect on lung cells, as some populations are more transcriptionally dynamic while others remain stable in aged individuals. We found that monocytes and alveolar macrophages were the most transcriptionally affected populations. These changes were related to inflammation and regulation of the immune response. Additionally, we calculated the LungAge score, which reveals the diversity of lung cell types during aging. Changes in DNA damage repair, fatty acid metabolism, and inflammation are essential for age prediction. Finally, we quantified the senescence score in aged lungs and found that the more biased cells toward senescence are immune and progenitor cells. Our study provides a comprehensive and systemic analysis of the molecular signatures of lung aging. Our LungAge signature can be used to predict molecular signatures of physiological aging and to detect common signatures of age-related lung diseases.

Intact mitochondrial function in the setting of telomere-induced senescence.

Mitochondria play essential roles in metabolic support and signaling within all cells. Congenital and acquired defects in mitochondria are responsible for several pathologies, including premature entrance to cellar senescence. Conversely, we examined the consequences of dysfunctional telomere-driven cellular senescence on mitochondrial biogenesis and function. We drove senescence in vitro and in vivo by deleting the telomere-binding protein TRF2 in fibroblasts and hepatocytes, respectively. Deletion of TRF2 led to a robust DNA damage response, global changes in transcription, and induction of cellular senescence. In vitro, senescent cells had significant increases in mitochondrial respiratory capacity driven by increased cellular and mitochondrial volume. Hepatocytes with dysfunctional telomeres maintained their mitochondrial respiratory capacity in vivo, whether measured in intact cells or purified mitochondria. Induction of senescence led to the upregulation of overlapping and distinct genes in fibroblasts and hepatocytes, but transcripts related to mitochondria were preserved. Our results support that mitochondrial function and activity are preserved in telomere dysfunction-induced senescence, which may facilitate continued cellular functions.

End-Stage Idiopathic Pulmonary Fibrosis Lung Microenvironment Promotes Impaired NK Activity.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic age-related chronic lung disease characterized by the accumulation of senescent cells. Whether impaired immune response is responsible for the accumulation of senescent cells in the IPF lung remains unknown. In this study, we characterized the NK phenotype in IPF lungs via flow cytometry using 5-dodecanoylaminofluorescein di-ß-d-galactopyranoside, markers of tissue residence, and chemokine receptors. The effect of the lung microenvironment was evaluated using lung fibroblast (LF) conditioned media (CM), and the bleomycin-induced pulmonary fibrosis mouse model was used to assess the in vivo relationship between NK cells and the accumulation of senescent cells. We found that NK cells from the lower lobe of IPF patients exhibited immune-senescent and impaired CD57-NKG2A+ phenotype. We also observed that culture of NK cells from healthy donors in CM from IPF lower lobe lung fibroblasts induced a senescent-like phenotype and impaired cytotoxic capacity. There is an impaired NK recruitment by LF, and NKs presented decreased migration toward their CM. In addition, NK cell-depleted mice treated with bleomycin showed increased collagen deposition and accumulation of different populations of senescent cells compared with controls. The IPF lung microenvironment induces a dysfunctional NK phenotype limiting the clearance of lung senescent cells and the resolution of lung fibrosis. We propose that impaired NK activity could be one of the mechanisms responsible for perpetuating the accumulation of senescent cells in IPF lungs.

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.

LEF1 isoforms regulate cellular senescence and aging.

Background: The study of aging and its mechanisms, such as cellular senescence, has provided valuable insights into age-related pathologies, thus contributing to their prevention and treatment. The current abundance of high throughput data combined with the surge of robust analysis algorithms has facilitated novel ways of identifying underlying pathways that may drive these pathologies. Methods: With the focus on identifying key regulators of lung aging, we performed comparative analyses of transcriptional profiles of aged versus young human subjects and mice, focusing on the common age-related changes in the transcriptional regulation in lung macrophages, T cells, and B immune cells. Importantly, we validated our findings in cell culture assays and human lung samples. Results: We identified Lymphoid Enhancer Binding Factor 1 (LEF1) as an important age-associated regulator of gene expression in all three cell types across different tissues and species. Follow-up experiments showed that the differential expression of long and short LEF1 isoforms is a key regulatory mechanism of cellular senescence. Further examination of lung tissue from patients with Idiopathic Pulmonary Fibrosis (IPF), an age-related disease with strong ties to cellular senescence, we demonstrated a stark dysregulation of LEF1. Conclusions: Collectively, our results suggest that the LEF1 is a key factor of aging, and its differential regulation is associated with human and murine cellular senescence.

Antiviral CD8+ T-cell immune responses are impaired by cigarette smoke and in COPD.

BACKGROUND: Virus infections drive COPD exacerbations and progression. Antiviral immunity centres on the activation of virus-specific CD8+ T-cells by viral epitopes presented on major histocompatibility complex (MHC) class I molecules of infected cells. These epitopes are generated by the immunoproteasome, a specialised intracellular protein degradation machine, which is induced by antiviral cytokines in infected cells. METHODS: We analysed the effects of cigarette smoke on cytokine- and virus-mediated induction of the immunoproteasome in vitro, ex vivo and in vivo using RNA and Western blot analyses. CD8+ T-cell activation was determined in co-culture assays with cigarette smoke-exposed influenza A virus (IAV)-infected cells. Mass-spectrometry-based analysis of MHC class I-bound peptides uncovered the effects of cigarette smoke on inflammatory antigen presentation in lung cells. IAV-specific CD8+ T-cell numbers were determined in patients' peripheral blood using tetramer technology. RESULTS: Cigarette smoke impaired the induction of the immunoproteasome by cytokine signalling and viral infection in lung cells in vitro, ex vivo and in vivo. In addition, cigarette smoke altered the peptide repertoire of antigens presented on MHC class I molecules under inflammatory conditions. Importantly, MHC class I-mediated activation of IAV-specific CD8+ T-cells was dampened by cigarette smoke. COPD patients exhibited reduced numbers of circulating IAV-specific CD8+ T-cells compared to healthy controls and asthmatics. CONCLUSION: Our data indicate that cigarette smoke interferes with MHC class I antigen generation and presentation and thereby contributes to impaired activation of CD8+ T-cells upon virus infection. This adds important mechanistic insight on how cigarette smoke mediates increased susceptibility of smokers and COPD patients to viral infections.

Early events marking lung fibroblast transition to profibrotic state in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is an age-associated progressive lung disease with accumulation of scar tissue impairing gas exchange. Previous high-throughput studies elucidated the role of cellular heterogeneity and molecular pathways in advanced disease. However, critical pathogenic pathways occurring in the transition of fibroblasts from normal to profibrotic have been largely overlooked. METHODS: We used single cell transcriptomics (scRNA-seq) from lungs of healthy controls and IPF patients (lower and upper lobes). We identified fibroblast subclusters, genes and pathways associated with early disease. Immunofluorescence assays validated the role of MOXD1 early in fibrosis. RESULTS: We identified four distinct fibroblast subgroups, including one marking the normal-to-profibrotic state transition. Our results show for the first time that global downregulation of ribosomal proteins and significant upregulation of the majority of copper-binding proteins, including MOXD1, mark the IPF transition. We find no significant differences in gene expression in IPF upper and lower lobe samples, which were selected to have low and high degree of fibrosis, respectively. CONCLUSIONS: Early events during IPF onset in fibroblasts include dysregulation of ribosomal and copper-binding proteins. Fibroblasts in early stage IPF may have already acquired a profibrotic phenotype while hallmarks of advanced disease, including fibroblast foci and honeycomb formation, are still not evident. The new transitional fibroblasts we discover could prove very important for studying the role of fibroblast plasticity in disease progression and help develop early diagnosis tools and therapeutic interventions targeting earlier disease states.

RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from PAH patients. Endothelial haploinsufficiency of RAB7 caused spontaneous PH in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA sequencing and RAB7 silenced ECs showed impaired angiogenesis, expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, which suggests inhibition of autophagy at the pre-degradation level. Further, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in chronic hypoxia/SU5416 rats. In conclusion, we demonstrate here for the first time the fundamental impairment of EC function by loss of RAB7 that leads to PH and show RAB7 activation as a potential therapeutic strategy in a preclinical model of PH.

CYB5R3 in type II alveolar epithelial cells protects against lung fibrosis by suppressing TGF-ß1 signaling.

Type II alveolar epithelial cell (AECII) redox imbalance contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a deadly disease with limited treatment options. Here, we show that expression of membrane-bound cytochrome B5 reductase 3 (CYB5R3), an enzyme critical for maintaining cellular redox homeostasis and soluble guanylate cyclase (sGC) heme iron redox state, is diminished in IPF AECIIs. Deficiency of CYB5R3 in AECIIs led to sustained activation of the pro-fibrotic factor TGF-ß1 and increased susceptibility to lung fibrosis. We further show that CYB5R3 is a critical regulator of ERK1/2 phosphorylation and the sGC/cGMP/protein kinase G axis that modulates activation of the TGF-ß1 signaling pathway. We demonstrate that sGC agonists (BAY 41-8543 and BAY 54-6544) are effective in reducing the pulmonary fibrotic outcomes of in vivo deficiency of CYB5R3 in AECIIs. Taken together, these results show that CYB5R3 in AECIIs is required to maintain resilience after lung injury and fibrosis and that therapeutic manipulation of the sGC redox state could provide a basis for treating fibrotic conditions in the lung and beyond.

Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy.

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1-/D mice). Ckmm-Cre+/- ;Ercc1-/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre+/- ;Ercc1-/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre+/- ;Ercc1-/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre+/- ;Ercc1-/fl and Ercc1-/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.

scResolve: Recovering single cell expression profiles from multi-cellular spatial transcriptomics.

Many popular spatial transcriptomics techniques lack single-cell resolution. Instead, these methods measure the collective gene expression for each location from a mixture of cells, potentially containing multiple cell types. Here, we developed scResolve, a method for recovering single-cell expression profiles from spatial transcriptomics measurements at multi-cellular resolution. scResolve accurately restores expression profiles of individual cells at their locations, which is unattainable from cell type deconvolution. Applications of scResolve on human breast cancer data and human lung disease data demonstrate that scResolve enables cell type-specific differential gene expression analysis between different tissue contexts and accurate identification of rare cell populations. The spatially resolved cellular-level expression profiles obtained through scResolve facilitate more flexible and precise spatial analysis that complements raw multi-cellular level analysis.

Cross-talk between TSC2 and the extracellular matrix controls pulmonary vascular proliferation and pulmonary hypertension.

Increased proliferation and survival of cells in small pulmonary arteries (PAs) drive pulmonary arterial hypertension (PAH). Because cell growth mediated by the mTOR-containing mTORC1 complex is inhibited by tuberous sclerosis complex 2 (TSC2), we investigated the role of this GTPase-activating protein in PAH pathology. TSC2 abundance was decreased in remodeled small PAs and PA vascular smooth muscle cells (PAVSMCs) from patients with PAH or from rodent pulmonary hypertension (PH) models, as well as PAVSMCs maintained on substrates that reproduced pathology-induced stiffness. Accordingly, mice with smooth muscle-specific reduction in TSC2 developed PH. At the molecular level, decreased TSC2 abundance led to stiffness-induced PAVSMC proliferation, increased abundance of the mechanosensitive transcriptional coactivators YAP/TAZ, and enhanced mTOR kinase activity. Moreover, extracellular matrix (ECM) produced by TSC2-deficient PAVSMCs stimulated the proliferation of nondiseased PA adventitial fibroblasts and PAVSMCs through fibronectin and its receptor, the α5ß1 integrin. Reconstituting TSC2 in PAVSMCs from patients with PAH through overexpression or treatment with the SIRT1 activator SRT2104 decreased YAP/TAZ abundance, mTOR activity, and ECM production, as well as inhibited proliferation and induced apoptosis. In two rodent models of PH, SRT2104 treatment restored TSC2 abundance, attenuated pulmonary vascular remodeling, and ameliorated PH. Thus, TSC2 in PAVSMCs integrates ECM composition and stiffness with pro-proliferative and survival signaling, and restoring TSC2 abundance could be an attractive therapeutic option to treat PH.

Noncanonical HIPPO/MST Signaling via BUB3 and FOXO Drives Pulmonary Vascular Cell Growth and Survival.

RATIONALE: The MSTs (mammalian Ste20-like kinases) 1/2 are members of the HIPPO pathway that act as growth suppressors in adult proliferative diseases. Pulmonary arterial hypertension (PAH) manifests by increased proliferation and survival of pulmonary vascular cells in small PAs, pulmonary vascular remodeling, and the rise of pulmonary arterial pressure. The role of MST1/2 in PAH is currently unknown. OBJECTIVE: To investigate the roles and mechanisms of the action of MST1 and MST2 in PAH. METHODS AND RESULTS: Using early-passage pulmonary vascular cells from PAH and nondiseased lungs and mice with smooth muscle-specific tamoxifen-inducible Mst1/2 knockdown, we found that, in contrast to canonical antiproliferative/proapoptotic roles, MST1/2 act as proproliferative/prosurvival molecules in human PAH pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts and support established pulmonary vascular remodeling and pulmonary hypertension in mice with SU5416/hypoxia-induced pulmonary hypertension. By using unbiased proteomic analysis, gain- and loss-of function approaches, and pharmacological inhibition of MST1/2 kinase activity by XMU-MP-1, we next evaluated mechanisms of regulation and function of MST1/2 in PAH pulmonary vascular cells. We found that, in PAH pulmonary arterial adventitial fibroblasts, the proproliferative function of MST1/2 is caused by IL-6-dependent MST1/2 overexpression, which induces PSMC6-dependent downregulation of forkhead homeobox type O 3 and hyperproliferation. In PAH pulmonary arterial vascular smooth muscle cells, MST1/2 acted via forming a disease-specific interaction with BUB3 and supported ECM (extracellular matrix)- and USP10-dependent BUB3 accumulation, upregulation of Akt-mTORC1, cell proliferation, and survival. Supporting our in vitro observations, smooth muscle-specific Mst1/2 knockdown halted upregulation of Akt-mTORC1 in small muscular PAs of mice with SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Together, this study describes a novel proproliferative/prosurvival role of MST1/2 in PAH pulmonary vasculature, provides a novel mechanistic link from MST1/2 via BUB3 and forkhead homeobox type O to the abnormal proliferation and survival of pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts, remodeling and pulmonary hypertension, and suggests new target pathways for therapeutic intervention.

Interferon Lambda Signaling in Macrophages Is Necessary for the Antiviral Response to Influenza.

Interferon lambda (IFNλ) signaling is a promising therapeutic target against viral infection in murine models, yet little is known about its molecular regulation and its cognate receptor, interferon lambda receptor 1 (IFNLR1) in human lung. We hypothesized that the IFNλ signaling axis was active in human lung macrophages. In human alveolar macrophages (HAMs), we observed increased IFNLR1 expression and robust increase in interferon-stimulated gene (ISG) expression in response to IFNλ ligand. While human monocytes express minimal IFNLR1, differentiation of monocytes into macrophages with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) increased IFNLR1 mRNA, IFNLR1 protein expression, and cellular response to IFNλ ligation. Conversely, in mice, M-CSF or GM-CSF stimulated macrophages failed to produce ISGs in response to related ligands, IFNL2 or IFNL3, suggesting that IFNLR1 signaling in macrophages is species-specific. We next hypothesized that IFNλ signaling was critical in influenza antiviral responses. In primary human airway epithelial cells and precision-cut human lung slices, influenza infection substantially increased IFNλ levels. Pretreatment of both HAMs and differentiated human monocytes with IFNL1 significantly inhibited influenza infection. IFNLR1 knockout in the myeloid cell line, THP-1, exhibited reduced interferon responses to either direct or indirect exposure to influenza infection suggesting the indispensability of IFNLR1 for antiviral responses. These data demonstrate the presence of IFNλ - IFNLR1 signaling axis in human lung macrophages and a critical role of IFNλ signaling in combating influenza infection.