Thromboxane-Prostanoid Receptor Signaling Drives Persistent Fibroblast Activation in Pulmonary Fibrosis.

Rationale: Although persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF), mechanisms regulating persistent fibroblast activation in the lungs have not been fully elucidated. Objectives: On the basis of our observation that lung fibroblasts express TBXA2R (thromboxane-prostanoid receptor) during fibrosis, we investigated the role of TBXA2R signaling in fibrotic remodeling. Methods: We identified TBXA2R expression in lungs of patients with IPF and mice and studied primary mouse and human lung fibroblasts to determine the impact of TBXA2R signaling on fibroblast activation. We used TBXA2R-deficient mice and small-molecule inhibitors to investigate TBXA2R signaling in preclinical lung fibrosis models. Measurements and Main Results: TBXA2R expression was upregulated in fibroblasts in the lungs of patients with IPF and in mouse lungs during experimental lung fibrosis. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, thereby suggesting that an alternative ligand activates profibrotic TBXA2R signaling. In contrast to thromboxane, F2-isoprostanes, which are nonenzymatic products of arachidonic acid induced by reactive oxygen species, were persistently elevated during fibrosis. F2-isoprostanes induced TBXA2R signaling in fibroblasts and mediated a myofibroblast activation profile due, at least in part, to potentiation of TGF-ß (transforming growth factor-ß) signaling. In vivo treatment with the TBXA2R antagonist ifetroban reduced profibrotic signaling in the lungs, protected mice from lung fibrosis in three preclinical models (bleomycin, Hermansky-Pudlak mice, and radiation-induced fibrosis), and markedly enhanced fibrotic resolution after bleomycin treatment. Conclusions: TBXA2R links oxidative stress to fibroblast activation during lung fibrosis. TBXA2R antagonists could have utility in treating pulmonary fibrosis.

Chronic lung diseases are associated with gene expression programs favoring SARS-CoV-2 entry and severity.

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyze the transcriptomes of 611,398 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observe a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD AT2 cells express higher levels of genes linked directly to the efficiency of viral replication and the innate immune response. Additionally, we identify basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection.

Chronic lung diseases are associated with gene expression programs favoring SARS-CoV-2 entry and severity.

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyzed the transcriptomes of 605,904 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observed a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD epithelial cells expressed higher levels of genes linked directly to the efficiency of viral replication and innate immune response. Additionally, we identified basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection.

Single-cell RNA sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis.

Pulmonary fibrosis (PF) is a form of chronic lung disease characterized by pathologic epithelial remodeling and accumulation of extracellular matrix (ECM). To comprehensively define the cell types, mechanisms, and mediators driving fibrotic remodeling in lungs with PF, we performed single-cell RNA sequencing of single-cell suspensions from 10 nonfibrotic control and 20 PF lungs. Analysis of 114,396 cells identified 31 distinct cell subsets/states. We report that a remarkable shift in epithelial cell phenotypes occurs in the peripheral lung in PF and identify several previously unrecognized epithelial cell phenotypes, including a KRT5- /KRT17 + pathologic, ECM-producing epithelial cell population that was highly enriched in PF lungs. Multiple fibroblast subtypes were observed to contribute to ECM expansion in a spatially discrete manner. Together, these data provide high-resolution insights into the complexity and plasticity of the distal lung epithelium in human disease and indicate a diversity of epithelial and mesenchymal cells contribute to pathologic lung fibrosis.

Epithelial Injury and Dysfunction in the Pathogenesis of Idiopathic PulmonaryFibrosis.

Idiopathic pulmonary fibrosis is a disease of older adults leading to progressive dyspnea and reduced exercise capacity, typically resulting in death within 3-5years of diagnosis. Underlying genetic susceptibility combined with environmental insults is proposed to trigger a chronic wound repair response, leading to activation of the fibrotic cascade. Perturbations in several molecular pathways mediate vulnerability of the alveolar epithelium to injurious agents, including the unfolded protein response, autophagy, mitophagy, and cellular senescence. These cellular responses are intricately intertwined and link genetic susceptibility to the progressive fibrotic phenotype. Ongoing studies investigating these pathways in type II alveolar epithelial cells show promise for identifying new targeted interventions that could prevent or halt the progression of IPF.

Tenosynovial chondromatosis of the flexor hallucis longus in a 17-year-old girl.

Tenosynovial chondromatosis is a benign chondrogenic metaplasia of extra-articular synovial tissue. The most common locations for tenosynovial chondromatosis to develop are the hands and feet. The condition has rarely been reported in children. We present a case of tenosynovial chondromatosis of the flexor hallucis longus in a 17-year-old girl. The presentation was unusual not only due to the location and young age of the patient but also the absence of any palpable mass on physical exam and complete lack of calcification of the cartilage bodies. Initial diagnosis was made by MRI. The patient underwent tenosynovectomy with an excellent postoperative recovery at 6-month follow-up. Histopathology confirmed the diagnosis of tenosynovial chondromatosis.

Resident progenitors, not exogenous migratory cells, generate the majority of visceral mesothelium in organogenesis.

Historically, analyses of mesothelial differentiation have focused on the heart where a highly migratory population of progenitors originating from a localized "extrinsic" source moves to and over the developing organ. This model long stood alone as the paradigm for generation of this cell type. Here, using chick/quail chimeric grafting and subsequent identification of mesothelial cell populations, we demonstrate that a different mechanism for the generation of mesothelia exists in vertebrate organogenesis. In this newly discovered model, mesothelial progenitors are intrinsic to organs of the developing digestive and respiratory systems. Additionally, we demonstrate that the early heart stands alone in its ability to recruit an entirely exogenous mesothelial cell layer during development. Thus, the newly identified "organ intrinsic" model of mesotheliogenesis appears to predominate while the long-studied cardiac model of mesothelial development may be the outlier.

Comprehensive timeline of mesodermal development in the quail small intestine.

BACKGROUND: To generate the mature intestine, splanchnic mesoderm diversifies into six different tissue layers each with multiple cell types through concurrent and complex morphogenetic events. Hindering the progress of research in the field is the lack of a detailed description of the fundamental morphological changes that constitute development of the intestinal mesoderm. RESULTS: We used immunofluorescence and morphometric analyses of wild-type and Tg(tie1:H2B-eYFP) quail embryos to establish a comprehensive timeline of mesodermal development in the avian intestine. The following landmark features were analyzed from appearance of the intestinal primordium through generation of the definitive structure: radial compartment formation, basement membrane dynamics, mesothelial differentiation, mesenchymal expansion and growth patterns, smooth muscle differentiation, and maturation of the vasculature. In this way, structural relationships between mesodermal components were identified over time. CONCLUSIONS: This integrated analysis presents a roadmap for investigators and clinicians to evaluate diverse experimental data obtained at individual stages of intestinal development within the longitudinal context of intestinal morphogenesis.

Identification of a novel developmental mechanism in the generation of mesothelia.

Mesothelium is the surface layer of all coelomic organs and is crucial for the generation of their vasculature. Still, our understanding of the genesis of this essential cell type is restricted to the heart where a localized exogenous population of cells, the proepicardium, migrates to and envelops the myocardium supplying mesothelial, vascular and stromal cell lineages. Currently it is not known whether this pattern of development is specific to the heart or applies broadly to other coelomic organs. Using two independent long-term lineage-tracing studies, we demonstrate that mesothelial progenitors of the intestine are intrinsic to the gut tube anlage. Furthermore, a novel chick-quail chimera model of gut morphogenesis reveals these mesothelial progenitors are broadly distributed throughout the gut primordium and are not derived from a localized and exogenous proepicardium-like source of cells. These data demonstrate an intrinsic origin of mesothelial cells to a coelomic organ and provide a novel mechanism for the generation of mesothelial cells.

Canonical Wnt signaling is required for ophthalmic trigeminal placode cell fate determination and maintenance.

Cranial placodes are ectodermal regions that contribute extensively to the vertebrate peripheral sensory nervous system. The development of the ophthalmic trigeminal (opV) placode, which gives rise only to sensory neurons of the ophthalmic lobe of the trigeminal ganglion, is a useful model of sensory neuron development. While key differentiation processes have been characterized at the tissue and cellular levels, the signaling pathways governing opV placode development have not. Here we tested in chick whether the canonical Wnt signaling pathway regulates opV placode development. By introducing a Wnt reporter into embryonic chick head ectoderm, we show that the canonical pathway is active in Pax3+ opV placode cells as, or shortly after, they are induced to express Pax3. Blocking the canonical Wnt pathway resulted in the failure of targeted cells to adopt or maintain an opV fate, as assayed by the expression of various markers including Pax3, FGFR4, Eya2, and the neuronal differentiation markers Islet1, neurofilament, and NeuN, although, surprisingly, it led to upregulation of Neurogenin2, both in the opV placode and elsewhere in the ectoderm. Activating the canonical Wnt signaling pathway, however, was not sufficient to induce Pax3, the earliest specific marker of the opV placode. We conclude that canonical Wnt signaling is necessary for normal opV placode development, and propose that other molecular cues are required in addition to Wnt signaling to promote cells toward an opV placode fate.