Profibrotic monocyte-derived alveolar macrophages are expanded in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19.

Monocyte-derived alveolar macrophages drive lung injury and fibrosis in murine models and are associated with pulmonary fibrosis in humans. Monocyte-derived alveolar macrophages have been suggested to develop a phenotype that promotes lung repair as injury resolves. We compared single-cell and cytokine profiling of the alveolar space in a cohort of 35 patients with post-acute sequelae of COVID-19 who had persistent respiratory symptoms and abnormalities on a computed tomography scan of the chest that subsequently improved or progressed. The abundance of monocyte-derived alveolar macrophages, their gene expression programs, and the level of the monocyte chemokine CCL2 in bronchoalveolar lavage fluid positively associated with the severity of radiographic fibrosis. Monocyte-derived alveolar macrophages from patients with resolving or progressive fibrosis expressed the same set of profibrotic genes. Our findings argue against a distinct reparative phenotype in monocyte-derived alveolar macrophages, highlighting their utility as a biomarker of failed lung repair and a potential target for therapy.

Roles of vimentin in health and disease.

More than 27 yr ago, the vimentin knockout (Vim-/- ) mouse was reported to develop and reproduce without an obvious phenotype, implying that this major cytoskeletal protein was nonessential. Subsequently, comprehensive and careful analyses have revealed numerous phenotypes in Vim-/- mice and their organs, tissues, and cells, frequently reflecting altered responses in the recovery of tissues following various insults or injuries. These findings have been supported by cell-based experiments demonstrating that vimentin intermediate filaments (IFs) play a critical role in regulating cell mechanics and are required to coordinate mechanosensing, transduction, signaling pathways, motility, and inflammatory responses. This review highlights the essential functions of vimentin IFs revealed from studies of Vim-/- mice and cells derived from them.

The kinase Jnk2 promotes stress-induced mitophagy by targeting the small mitochondrial form of the tumor suppressor ARF for degradation.

Mitophagy is essential for cellular homeostasis, but how mitophagy is regulated is largely unknown. Here we found that the kinase Jnk2 was required for stress-induced mitophagy. Jnk2 promoted ubiquitination and proteasomal degradation of the small mitochondrial form of the tumor suppressor ARF (smARF). Loss of Jnk2 led to the accumulation of smARF, which induced excessive autophagy that resulted in lysosomal degradation of the mitophagy adaptor p62 at steady state. Depletion of p62 prevented Jnk2-deficient cells from mounting mitophagy upon stress. Jnk2-deficient mice displayed defective mitophagy, which resulted in tissue damage under hypoxic stress, as well as hyperactivation of inflammasomes and increased mortality in sepsis. Our findings define a unique mechanism of maintaining immunological homeostasis that protects the host from tissue damage and mortality.

Suppression of inflammation and acute lung injury by Miz1 via repression of C/EBP-δ.

Inflammation is essential for host defense but can cause tissue damage and organ failure if unchecked. How the inflammation is resolved remains elusive. Here we report that the transcription factor Miz1 was required for terminating lipopolysaccharide (LPS)-induced inflammation. Genetic disruption of the Miz1 POZ domain, which is essential for the transactivation or repression activity of Miz1, resulted in hyperinflammation, lung injury and greater mortality in LPS-treated mice but a lower bacterial load and mortality in mice with Pseudomonas aeruginosa pneumonia. Loss of the Miz1 POZ domain prolonged the expression of proinflammatory cytokines. After stimulation, Miz1 was phosphorylated at Ser178, which was required for recruitment of the histone deacetylase HDAC1 to repress transcription of the gene encoding C/EBP-δ, an amplifier of inflammation. Our data provide a long-sought mechanism underlying the resolution of LPS-induced inflammation.

Defining a timeline of colon pathologies after keratin 8 loss: rapid crypt elongation and diarrhea are followed by epithelial erosion and cell exfoliation.

Keratins are epithelial intermediate filament proteins that play a crucial role in cellular stress protection, with K8 being the most abundant in the colon. The intestinal epithelial-specific K8-deficient mouse model (K8flox/flox;Villin-Cre) exhibits characteristics of inflammatory bowel disease, including diarrhea, crypt erosion, hyperproliferation, and decreased barrier function. Nevertheless, the order in which these events occur and whether they are a direct cause of K8 loss or a consequence of one event inducing another remains unexplored. Increased knowledge about early events in the disruption of colon epithelial integrity would help to understand the early pathology of inflammatory and functional colon disorders and develop preclinical models and diagnostics of colonic diseases. Here, we aimed to characterize the order of physiological events after Krt8 loss by utilizing K8flox/flox;Villin-CreERt2 mice with tamoxifen-inducible Krt8 deletion in intestinal epithelial cells, and assess stool analysis as a noninvasive method to monitor real-time gene expression changes following Krt8 loss. K8 protein was significantly decreased within a day after induction, followed by its binding partners, K18 and K19 from day 4 onward. The sequential colonic K8 downregulation in adult mice leads to immediate diarrhea and crypt elongation with activation of proliferation signaling, followed by crypt loss and increased neutrophil activity within 6-8 days, highlighting impaired water balance and crypt elongation as the earliest colonic changes upon Krt8 loss. Furthermore, epithelial gene expression patterns were comparable between colon tissue and stool samples, demonstrating the feasibility of noninvasive monitoring of gut epithelia in preclinical research utilizing Cre-LoxP-based intestinal disease models.NEW & NOTEWORTHY Understanding the order in which physiological and molecular events occur helps to recognize the onset of diseases and improve their preclinical models. We utilized Cre-Lox-based inducible keratin 8 deletion in mouse intestinal epithelium to characterize the earliest events after keratin 8 loss leading to colitis. These include diarrhea and crypt elongation, followed by erosion and neutrophil activity. Our results also support noninvasive methodology for monitoring colon diseases in preclinical models.

Gigaxonin is required for intermediate filament transport.

Gigaxonin is an adaptor protein for E3 ubiquitin ligase substrates. It is necessary for ubiquitination and degradation of intermediate filament (IF) proteins. Giant axonal neuropathy is a pathological condition caused by mutations in the GAN gene that encodes gigaxonin. This condition is characterized by abnormal accumulation of IFs in both neuronal and non-neuronal cells; however, it is unclear what causes IF aggregation. In this work, we studied the dynamics of IFs using their subunits tagged with a photoconvertible protein mEOS 3.2. We have demonstrated that the loss of gigaxonin dramatically inhibited transport of IFs along microtubules by the microtubule motor kinesin-1. This inhibition was specific for IFs, as other kinesin-1 cargoes, with the exception of mitochondria, were transported normally. Abnormal distribution of IFs in the cytoplasm can be rescued by direct binding of kinesin-1 to IFs, demonstrating that transport inhibition is the primary cause for the abnormal IF distribution. Another effect of gigaxonin loss was a more than 20-fold increase in the amount of soluble vimentin oligomers in the cytosol of gigaxonin knock-out cells. We speculate that these oligomers saturate a yet unidentified adapter that is required for kinesin-1 binding to IFs, which might inhibit IF transport along microtubules causing their abnormal accumulation.

Transcriptional Evaluation of the Ductus Arteriosus at the Single-Cell Level Uncovers a Requirement for Vim (Vimentin) for Complete Closure.

OBJECTIVE: Failure to close the ductus arteriosus, patent ductus arteriosus, accounts for 10% of all congenital heart defects. Despite significant advances in patent ductus arteriosus management, including pharmacological treatment targeting the prostaglandin pathway, a proportion of patients fail to respond and must undergo surgical intervention. Thus, further refinement of the cellular and molecular mechanisms that govern vascular remodeling of this vessel is required. METHODS: We performed single-cell RNA-sequencing of the ductus arteriosus in mouse embryos at E18.5 (embryonic day 18.5), and P0.5 (postnatal day 0.5), and P5 to identify transcriptional alterations that might be associated with remodeling. We further confirmed our findings using transgenic mouse models coupled with immunohistochemistry analysis. RESULTS: The intermediate filament vimentin emerged as a candidate that might contribute to closure of the ductus arteriosus. Indeed, mice with genetic deletion of vimentin fail to complete vascular remodeling of the ductus arteriosus. To seek mechanisms, we turned to the RNA-sequencing data that indicated changes in Jagged1 with similar profile to vimentin and pointed to potential links with Notch. In fact, Notch3 signaling was impaired in vimentin null mice and vimentin null mice phenocopies patent ductus arteriosus in Jagged1 endothelial and smooth muscle deleted mice. CONCLUSIONS: Through single-cell RNA-sequencing and by tracking closure of the ductus arteriosus in mice, we uncovered the unexpected contribution of vimentin in driving complete closure of the ductus arteriosus through a mechanism that includes deregulation of the Notch signaling pathway.

Age-related Differences in the Nasal Mucosal Immune Response to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 180 million people since the onset of the pandemic. Despite similar viral load and infectivity rates between children and adults, children rarely develop severe illness. Differences in the host response to the virus at the primary infection site are among the mechanisms proposed to account for this disparity. Our objective was to investigate the host response to SARS-CoV-2 in the nasal mucosa in children and adults and compare it with the host response to respiratory syncytial virus (RSV) and influenza virus. We analyzed clinical outcomes and gene expression in the nasal mucosa of 36 children with SARS-CoV-2, 24 children with RSV, 9 children with influenza virus, 16 adults with SARS-CoV-2, and 7 healthy pediatric and 13 healthy adult controls. In both children and adults, infection with SARS-CoV-2 led to an IFN response in the nasal mucosa. The magnitude of the IFN response correlated with the abundance of viral reads, not the severity of illness, and was comparable between children and adults infected with SARS-CoV-2 and children with severe RSV infection. Expression of ACE2 and TMPRSS2 did not correlate with age or presence of viral infection. SARS-CoV-2-infected adults had increased expression of genes involved in neutrophil activation and T-cell receptor signaling pathways compared with SARS-CoV-2-infected children, despite similar severity of illness and viral reads. Age-related differences in the immune response to SARS-CoV-2 may place adults at increased risk of developing severe illness.

Targeted deletion of keratin 8 in intestinal epithelial cells disrupts tissue integrity and predisposes to tumorigenesis in the colon.

Keratin 8 (K8) is the main intestinal epithelial intermediate filament protein with proposed roles for colonic epithelial cell integrity. Here, we used mice lacking K8 in intestinal epithelial cells (floxed K8 and Villin-Cre1000 and Villin-CreERt2) to investigate the cell-specific roles of intestinal epithelial K8 for colonocyte function and pathologies. Intestinal epithelial K8 deletion decreased K8 partner proteins, K18-K20, 75-95%, and the remaining keratin filaments were located at the colonocyte apical regions with type II K7, which decreased 30%. 2-Deoxy-2-[18F]-fluoroglucose positron emission tomography in vivo imaging identified a metabolic phenotype in the lower gut of the conditional K8 knockouts. These mice developed intestinal barrier leakiness, mild diarrhea, and epithelial damage, especially in the proximal colon. Mice exhibited shifted differentiation from enterocytes to goblet cells, displayed longer crypts and an increased number of Ki67 + transit-amplifying cells in the colon. Significant proproliferative and regenerative signaling occurred in the IL-22, STAT3, and pRb pathways, with minor effects on inflammatory parameters, which, however, increased in aging mice. Importantly, colonocyte K8 deletion induced a dramatically increased sensitivity to azoxymethane-induced tumorigenesis. In conclusion, intestinal epithelial K8 plays a significant role in colonocyte epithelial integrity maintenance, proliferation regulation and tumor suppression.

The cGAS-STING pathway: The role of self-DNA sensing in inflammatory lung disease.

The presence of DNA in the cytosol is usually a sign of microbial infections, which alerts the host innate immune system to mount a defense response. Cyclic GMP-AMP synthase (cGAS) is a critical cytosolic DNA sensor that elicits robust innate immune responses through the production of the second messenger, cyclic GMP-AMP (cGAMP), which binds and activates stimulator of interferon genes (STING). However, cGAS binds to DNA irrespective of DNA sequence, therefore, self-DNA leaked from the nucleus or mitochondria can also serve as a cGAS ligand to activate this pathway and trigger extensive inflammatory responses. Dysregulation of the cGAS-STING pathway is responsible for a broad array of inflammatory and autoimmune diseases. Recently, evidence has shown that self-DNA release and cGAS-STING pathway over-activation can drive lung disease, making this pathway a promising therapeutic target for inflammatory lung disease. Here, we review recent advances on the cGAS-STING pathway governing self-DNA sensing, highlighting its role in pulmonary disease.

Age-Dependent Differences in T-Cell Responses to Influenza A Virus.

Respiratory infections from influenza A virus (IAV) cause substantial morbidity and mortality in children relative to adults. T cells play a critical role in the host response to IAV by supporting the innate and humoral responses, mediating cytotoxic activity, and promoting recovery. There are age-dependent differences in the number, subsets, and localization of T cells, which impact the host response to pathogens. In this article, we first review how T cells recognize IAV and examine differences in the resting T-cell populations between juveniles and adults. Next, we describe how the juvenile CD4+, CD8+, and regulatory T-cell responses compare with those in adults and discuss the potential physiologic and clinical consequences of the differences. Finally, we explore the roles of two unconventional T-cell types in the juvenile response to influenza, natural-killer T cells and γδ T cells. A clear understanding of age-dependent differences in the T-cell response is essential to developing therapies to prevent or reverse the deleterious effects of IAV in children.

Mitochondrial 8-oxoguanine DNA glycosylase mitigates alveolar epithelial cell PINK1 deficiency, mitochondrial DNA damage, apoptosis, and lung fibrosis.

Alveolar epithelial cell (AEC) apoptosis, arising from mitochondrial dysfunction and mitophagy defects, is important in mediating idiopathic pulmonary fibrosis (IPF). Our group established a role for the mitochondrial (mt) DNA base excision repair enzyme, 8-oxoguanine-DNA glycosylase 1 (mtOGG1), in preventing oxidant-induced AEC mtDNA damage and apoptosis and showed that OGG1-deficient mice have increased lung fibrosis. Herein, we determined whether mice overexpressing the mtOGG1 transgene (mtOgg1tg) are protected against lung fibrosis and whether AEC mtOGG1 preservation of mtDNA integrity mitigates phosphatase and tensin homolog-induced putative kinase 1 (PINK1) deficiency and apoptosis. Compared with wild type (WT), mtOgg1tg mice have diminished asbestos- and bleomycin-induced pulmonary fibrosis that was accompanied by reduced lung and AEC mtDNA damage and apoptosis. Asbestos and H2O2 promote the MLE-12 cell PINK1 deficiency, as assessed by reductions in the expression of PINK1 mRNA and mitochondrial protein expression. Compared with WT, Pink1-knockout (Pink1-KO) mice are more susceptible to asbestos-induced lung fibrosis and have increased lung and alveolar type II (AT2) cell mtDNA damage and apoptosis. AT2 cells from Pink1-KO mice and PINK1-silenced (siRNA) MLE-12 cells have increased mtDNA damage that is augmented by oxidative stress. Interestingly, mtOGG1 overexpression attenuates oxidant-induced MLE-12 cell mtDNA damage and apoptosis despite PINK1 silencing. mtDNA damage is increased in the lungs of patients with IPF as compared with controls. Collectively, these findings suggest that mtOGG1 maintenance of AEC mtDNA is crucial for preventing PINK1 deficiency that promotes apoptosis and lung fibrosis. Given the key role of AEC apoptosis in pulmonary fibrosis, strategies aimed at preserving AT2 cell mtDNA integrity may be an innovative target.

Inflammatory Monocytes Drive Influenza A Virus-Mediated Lung Injury in Juvenile Mice.

Healthy children are more likely to die of influenza A virus (IAV) infection than healthy adults. However, little is known about the mechanisms underlying the impact of young age on the development of life-threatening IAV infection. We report increased mortality in juvenile mice compared with adult mice at each infectious dose of IAV. Juvenile mice had sustained elevation of type I IFNs and persistent NLRP3 inflammasome activation in the lungs, both of which were independent of viral titer. Juvenile mice, but not adult mice, had increased MCP-1 levels that remained high even after viral clearance. Importantly, continued production of MCP-1 was associated with persistent recruitment of monocytes to the lungs and prolonged elevation of inflammatory cytokines. Transcriptional signatures of recruited monocytes to the juvenile and adult IAV-infected lungs were assessed by RNA-seq. Genes associated with a proinflammatory signature were upregulated in the juvenile monocytes compared with adult monocytes. Depletion of monocytes with anti-CCR2 Ab decreased type I IFN secretion, NLRP3 inflammasome activation, and lung injury in juvenile mice. This suggests an exaggerated inflammatory response mediated by increased recruitment of monocytes to the lung, and not an inability to control viral replication, is responsible for severe IAV infection in juvenile mice. This study provides insight into severe IAV infection in juveniles and identifies key inflammatory monocytes that may be central to pediatric acute lung injury secondary to IAV.

Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis.

Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

Future Research Directions in Pneumonia. NHLBI Working Group Report.

Pneumonia is a complex pulmonary disease in need of new clinical approaches. Although triggered by a pathogen, pneumonia often results from dysregulations of host defense that likely precede infection. The coordinated activities of immune resistance and tissue resilience then dictate whether and how pneumonia progresses or resolves. Inadequate or inappropriate host responses lead to more severe outcomes such as acute respiratory distress syndrome and to organ dysfunction beyond the lungs and over extended time frames after pathogen clearance, some of which increase the risk for subsequent pneumonia. Improved understanding of such host responses will guide the development of novel approaches for preventing and curing pneumonia and for mitigating the subsequent pulmonary and extrapulmonary complications of pneumonia. The NHLBI assembled a working group of extramural investigators to prioritize avenues of host-directed pneumonia research that should yield novel approaches for interrupting the cycle of unhealthy decline caused by pneumonia. This report summarizes the working group's specific recommendations in the areas of pneumonia susceptibility, host response, and consequences. Overarching goals include the development of more host-focused clinical approaches for preventing and treating pneumonia, the generation of predictive tools (for pneumonia occurrence, severity, and outcome), and the elucidation of mechanisms mediating immune resistance and tissue resilience in the lung. Specific areas of research are highlighted as especially promising for making advances against pneumonia.

A Beginner's Guide to Analysis of RNA Sequencing Data.

Since the first publications coining the term RNA-seq (RNA sequencing) appeared in 2008, the number of publications containing RNA-seq data has grown exponentially, hitting an all-time high of 2,808 publications in 2016 (PubMed). With this wealth of RNA-seq data being generated, it is a challenge to extract maximal meaning from these datasets, and without the appropriate skills and background, there is risk of misinterpretation of these data. However, a general understanding of the principles underlying each step of RNA-seq data analysis allows investigators without a background in programming and bioinformatics to critically analyze their own datasets as well as published data. Our goals in the present review are to break down the steps of a typical RNA-seq analysis and to highlight the pitfalls and checkpoints along the way that are vital for bench scientists and biomedical researchers performing experiments that use RNA-seq.

Vimentin expression is required for the development of EMT-related renal fibrosis following unilateral ureteral obstruction in mice.

Most renal transplants ultimately fail secondary to chronic allograft nephropathy (CAN). Vimentin (vim) is a member of the intermediate filament family of proteins and has been shown to be important in the development of CAN. One of the pathways leading to chronic renal fibrosis after transplant is thought to be epithelial to mesenchymal transition (EMT). Even though vim expression is one of the main steps of EMT, it is unknown whether vim expression is required for EMT leading to renal fibrosis and allograft loss. To this end, the role of vim in renal fibrosis was determined via unilateral ureteral obstruction (UUO) in vim knockout mice (129 svs6 vim -/-). Following UUO, kidneys were recovered and analyzed via Western blotting, immunofluorescence, and transcriptomics. Cultured human proximal renal tubular (HK-2) cells were subjected to lentiviral-driven inhibition of vim expression and then treated with transforming growth factor (TGF)-ß to undergo EMT. Immunoblotting as well as wound healing assays were used to determine development of EMT. Western blotting analyses of mice undergoing UUO reveal increased levels of vim soon after UUO. As expected, interstitial collagen deposition increased in control mice following UUO but decreased in vim -/- kidneys. Immunofluorescence analyses also revealed altered localization of ß-catenin in vim -/- mice undergoing UUO without significant changes in mRNA levels. However, RNA sequencing revealed a decrease in ß-catenin-dependent genes in vim -/- kidneys. Finally, vim-silenced HK-2 cell lines undergoing EMT were shown to have decreased cellular migration during wound healing. We conclude that vim inhibition decreases fibrosis following UUO by possibly altering ß-catenin localization and downstream signaling.

The O-glycosylated ectodomain of FXYD5 impairs adhesion by disrupting cell-cell trans-dimerization of Na,K-ATPase ß1 subunits.

FXYD5 (also known as dysadherin), a regulatory subunit of the Na,K-ATPase, impairs intercellular adhesion by a poorly understood mechanism. Here, we determined whether FXYD5 disrupts the trans-dimerization of Na,K-ATPase molecules located in neighboring cells. Mutagenesis of the Na,K-ATPase ß1 subunit identified four conserved residues, including Y199, that are crucial for the intercellular Na,K-ATPase trans-dimerization and adhesion. Modulation of expression of FXYD5 or of the ß1 subunit with intact or mutated ß1-ß1 binding sites demonstrated that the anti-adhesive effect of FXYD5 depends on the presence of Y199 in the ß1 subunit. Immunodetection of the plasma membrane FXYD5 was prevented by the presence of O-glycans. Partial FXYD5 deglycosylation enabled antibody binding and showed that the protein level and the degree of O-glycosylation were greater in cancer than in normal cells. FXYD5-induced impairment of adhesion was abolished by both genetic and pharmacological inhibition of FXYD5 O-glycosylation. Therefore, the extracellular O-glycosylated domain of FXYD5 impairs adhesion by interfering with intercellular ß1-ß1 interactions, suggesting that the ratio between FXYD5 and α1-ß1 heterodimer determines whether the Na,K-ATPase acts as a positive or negative regulator of intercellular adhesion.