Studies have demonstrated the phenotypic heterogeneity of vascular endothelial cells (ECs) within a vascular bed; however, little is known about how distinct endothelial subpopulations in a particular organ respond to an inflammatory stimulus. We performed single cell RNA-sequencing of 35,973 lung ECs obtained during the baseline state as well as post-injury time points following inflammatory lung injury induced by lipopolysaccharide. Seurat clustering and gene expression pathway analysis identified two major subpopulations in the lung microvascular endothelium, a subpopulation enriched for expression of immune response genes such as major histocompatibility complex genes (immuneEC) and another defined by increased expression of vascular development genes such as Sox17 (devEC). The presence of immuneEC and devEC subpopulations was also observed in non-human primate lungs infected with SARS-CoV-2 and murine lungs infected with H1N1 influenza virus. Following the peak of inflammatory injury, we observed the emergence of a proliferative lung EC subpopulation. Overexpression of Sox17 prevented inflammatory activation in ECs. Thus, there appears to be a" division of labor" within the lung microvascular endothelium with some ECs showing propensity for inflammatory signaling and others for endothelial regeneration. These results provide underpinnings for the development of targeted therapies to limit inflammatory lung injury and promote regeneration.

Breathing disorders (BD) (apnoeas/hypopneas, periodic breathing) are highly prevalent in chronic heart failure (CHF) and are associated with altered central respiratory control. Ample evidence identifies the retrotrapezoid nucleus (RTN) as an important chemosensitivity region for ventilatory control and generation of BD in CHF, however little is known about the cellular mechanisms underlying the RTN/BD relationship. Within the RTN, astrocyte-mediated purinergic signalling modulates respiration, but the potential contribution of RTN astrocytes to BD in CHF has not been explored.Selective neuron and/or astrocyte-targeted interventions using either optogenetic and chemogenetic manipulations in the RTN of CHF rats were used to unveil the contribution of the RTN on the development/maintenance of BD, the role played by astrocytes in BD and the molecular mechanism underpinning these alterations.We showed that episodic photo-stimulation of RTN neurons triggered BD in healthy rats, and that RTN neurons ablation in CHF animals eliminates BD. Also, we found a reduction in astrocytes activity and ATP bioavailability within the RTN of CHF rats, and that chemogenetic restoration of normal RTN astrocyte activity and ATP levels improved breathing regularity in CHF. Importantly, P"X/ P2X7 receptor (P2X7r) expression was reduced in RTN astrocytes from CHF rats and viral vector-mediated delivery of human P2X7 P2X7r into astrocytes increases ATP bioavailability and abolished BD.Our results support that RTN astrocytes play a pivotal role on BD generation and maintenance in the setting CHF by a mechanism encompassing P2X7r signalling.This study was funded by the National Research and Development Agency of Chile (ANID).

Human papillomaviruses are DNA viruses that ubiquitously infect humans and have been associated with hyperproliferative lesions. The recently discovered mouse specific papillomavirus (MmuPV1) provides the opportunity to study papillomavirus infections in vivo in the context of a common laboratory mouse model (Mus musculus). To date, a major challenge in the field has been the lack of tools to identify, observe, and characterize individually the papillomavirus hosting cells and also trace the progeny of these cells over time. Here, we present the successful generation of an in vivo lineage-tracing model of MmuPV1-harboring cells and their progeny by means of genetic reporter activation. Following the validation of the system both in vitro and in vivo, we used it to provide a proof-of-concept of its utility. Using flow-cytometry analysis, we observed increased proliferation dynamics and decreased MHC-I cell surface expression in MmuPV1-treated tissues which could have implications in tissue regenerative capacity and ability to clear the virus. This model is a novel tool to study the biology of the MmuPV1 host-pathogen interactions.

Corticotropin-releasing factor type-1 (CRF1) receptors are critical to stress responses because they allow neurons to respond to CRF released in response to stress. Our understanding of the role of CRF1-expressing neurons in CRF-mediated behaviors has been largely limited to mouse experiments due to the lack of genetic tools available to selectively visualize and manipulate CRF1+ cells in rats. Here, we describe the generation and validation of a transgenic CRF1-Cre-tdTomato rat. We report that Crhr1 and Cre mRNA expression are highly colocalized in both the central amygdala (CeA), composed of mostly GABAergic neurons, and in the basolateral amygdala (BLA), composed of mostly glutamatergic neurons. In the CeA, membrane properties, inhibitory synaptic transmission, and responses to CRF bath application in tdTomato+ neurons are similar to those previously reported in GFP+ cells in CRFR1-GFP mice. We show that stimulatory DREADD receptors can be targeted to CeA CRF1+ cells via virally delivered Cre-dependent transgenes, that transfected Cre/tdTomato+ cells are activated by clozapine-n-oxide in vitro and in vivo, and that activation of these cells in vivo increases anxiety-like and nocifensive behaviors. Outside the amygdala, we show that Cre-tdTomato is expressed in several brain areas across the brain, and that the expression pattern of Cre-tdTomato cells is similar to the known expression pattern of CRF1 cells. Given the accuracy of expression in the CRF1-Cre rat, modern genetic techniques used to investigate the anatomy, physiology, and behavioral function of CRF1+ neurons can now be performed in assays that require the use of rats as the model organism.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes a number of strategies to modulate viral and host mRNA translation. Here, we used ribosome profiling in SARS-CoV-2-infected model cell lines and primary airway cells grown at an air-liquid interface to gain a deeper understanding of the translationally regulated events in response to virus replication. We found that SARS-CoV-2 mRNAs dominate the cellular mRNA pool but are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy despite notable accumulation of ribosomes within the slippery sequence on the frameshifting element. In a highly permissive cell line model, although SARS-CoV-2 infection induced the transcriptional upregulation of numerous chemokine, cytokine, and interferon-stimulated genes, many of these mRNAs were not translated efficiently. The impact of SARS-CoV-2 on host mRNA translation was more subtle in primary cells, with marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data reveal the key role of mRNA translation in SARS-CoV-2 replication and highlight unique mechanisms for therapeutic development. IMPORTANCE SARS-CoV-2 utilizes a number of strategies to modulate host responses to ensure efficient propagation. Here, we used ribosome profiling in SARS-CoV-2-infected cells to gain a deeper understanding of the translationally regulated events in infected cells. We found that although viral mRNAs are abundantly expressed, they are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy and alternative translation initiation sites that help increase the coding potential of its RNAs. In permissive cells, SARS-CoV-2 infection induced the translational repression of numerous innate immune mediators. Though the impact of SARS-CoV-2 on host mRNA translation was more subtle in primary airway cell cultures, we noted marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data provide new insight into how SARS-CoV-2 modulates innate host responses and highlight unique mechanisms for therapeutic intervention.

Mechanisms regulating oligodendrocyte differentiation, developmental myelination and myelin maintenance in adulthood are complex and still not completely described. Their understanding is crucial for the development of new protective or therapeutic strategies in demyelinating pathologies such as multiple sclerosis. In this perspective, we have investigated the role of Cyclin-dependent kinase 7 (Cdk7), a kinase involved in cell-cycle progression and transcription regulation, in the oligodendroglial lineage. We generated a conditional knock-out mouse model in which Cdk7 is invalidated in post-mitotic oligodendrocytes. At the end of developmental myelination, the number and diameter of myelinated axons, as well as the myelin structure, thickness and protein composition, were normal. However, in young adult and in aged mice, there was a higher number of small caliber myelinated axons associated with a decreased mean axonal diameter, myelin sheaths of large caliber axons were thinner, and the level of some major myelin-associated proteins was reduced. These defects were accompanied by the appearance of an abnormal clasping phenotype. We also used an in vitro oligodendroglial model and showed that Cdk7 pharmacological inhibition led to an altered myelination-associated morphological modification combined with a decreased expression of myelin-specific genes. Altogether, we identified novel functions for Cdk7 in CNS myelination.

Oligodendrocytes are glial cells that support and insulate axons in the central nervous system through the production of myelin. Oligodendrocytes arise throughout embryonic and early postnatal development from oligodendrocyte precursor cells (OPCs), and recent work demonstrated that they are a transcriptional heterogeneous cell population, but the regional and functional implications of this heterogeneity are less clear. Here, we apply in situ sequencing (ISS) to simultaneously probe the expression of 124 marker genes of distinct oligodendrocyte populations, providing comprehensive maps of the corpus callosum, cingulate, motor, and somatosensory cortex in the brain, as well as gray matter (GM) and white matter (WM) regions in the spinal cord, at postnatal (P10), juvenile (P20), and young adult (P60) stages. We systematically compare the abundances of these populations and investigate the neighboring preference of distinct oligodendrocyte populations.We observed that oligodendrocyte lineage progression is more advanced in the juvenile spinal cord compared to the brain, corroborating with previous studies. We found myelination still ongoing in the adult corpus callosum while it was more advanced in the cortex. Interestingly, we also observed a lateral-to-medial gradient of oligodendrocyte lineage progression in the juvenile cortex, which could be linked to arealization, as well as a deep-to-superficial gradient with mature oligodendrocytes preferentially accumulating in the deeper layers of the cortex. The ISS experiments also exposed differences in abundances and population dynamics over time between GM and WM regions in the brain and spinal cord, indicating regional differences within GM and WM, and we found that neighboring preferences of some oligodendroglia populations are altered from the juvenile to the adult CNS.Overall, our ISS experiments reveal spatial heterogeneity of oligodendrocyte lineage progression in the brain and spinal cord and uncover differences in the timing of oligodendrocyte differentiation and myelination, which could be relevant to further investigate functional heterogeneity of oligodendroglia, especially in the context of injury or disease.

Secreted signals in patterning systems often induce repressive signals that shape their distributions in space and time. In developing growth plates (GPs) of endochondral long bones, Parathyroid hormone-like hormone (Pthlh) inhibits Indian hedgehog (Ihh) to form a negative-feedback loop that controls GP progression and bone size. Whether similar systems operate in other bones and how they arise during embryogenesis remain unclear. We show that Pthlha expression in the zebrafish craniofacial skeleton precedes chondrocyte differentiation and restricts where cells undergo hypertrophy, thereby initiating a future GP. Loss of Pthlha leads to an expansion of cells expressing a novel early marker of the hypertrophic zone (HZ), entpd5a, and later HZ markers, such as ihha, whereas local Pthlha misexpression induces ectopic entpd5a expression. Formation of this early pre-HZ correlates with onset of muscle contraction and requires mechanical force; paralysis leads to loss of entpd5a and ihha expression in the pre-HZ, mislocalized pthlha expression and no subsequent ossification. These results suggest that local Pthlh sources combined with force determine HZ locations, establishing the negative-feedback loop that later maintains GPs.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen. In cell culture, CCHFV is sensed by the cytoplasmic RNA sensor retinoic acid-inducible gene I (RIG-I) molecule and its adaptor molecule mitochondrial antiviral signaling (MAVS) protein. MAVS initiates both type I interferon (IFN-I) and proinflammatory responses. Here, we studied the role MAVS plays in CCHFV infection in mice in both the presence and absence of IFN-I activity. MAVS-deficient mice were not susceptible to CCHFV infection when IFN-I signaling was active and showed no signs of disease. When IFN-I signaling was blocked by antibody, MAVS-deficient mice lost significant weight, but were uniformly protected from lethal disease, whereas all control mice succumbed to infection. Cytokine activity in the infected MAVS-deficient mice was markedly blunted. Subsequent investigation revealed that CCHFV infected mice lacking TNF-α receptor signaling (TNFA-R-deficient), but not IL-6 or IL-1 activity, had more limited liver injury and were largely protected from lethal outcomes. Treatment of mice with an anti-TNF-α neutralizing antibody also conferred partial protection in a post-virus exposure setting. Additionally, we found that a disease causing, but non-lethal strain of CCHFV produced more blunted inflammatory cytokine responses compared to a lethal strain in mice. Our work reveals that MAVS activation and cytokine production both contribute to CCHFV pathogenesis, potentially identifying new therapeutic targets to treat this disease.

02 May 2022: This Accepted Article published in error. The article is under embargo and will publish in Early View in July 2022.This article is protected by

The pathogenesis of squamous cell neoplasms arising in the lacrimal drainage system is poorly understood, and the underlying genomic drivers for disease development remain unexplored. We aimed to investigate the genomic aberrations in carcinomas arising in the LDS and correlate the findings to human papillomavirus (HPV) status. The HPV analysis was performed using HPV DNA PCR, HPV E6/E7 mRNA in-situ hybridization, and p16 immunohistochemistry. The genomic characterization was performed by targeted DNA sequencing of 523 cancer-relevant genes. Patients with LDS papilloma (n = 17) and LDS carcinoma (n = 15) were included. There was a male predominance (68%) and a median age at diagnosis of 46.0 years (range 27.5-65.5 years) in patients with papilloma and 63.8 years (range 34.0-87.2 years) in patients with carcinoma. Transcriptional activity of the HPV E6/E7 oncogenes was detected in the whole tumor thickness in 12/15 (80%) papillomas (HPV6, 11, 16) and 10/15 (67%) squamous cell carcinomas (SCC) (HPV11: 3/15 (20%) and HPV16: 7/15 (47%)). Pathogenic variants in PIK3CA, FGFR3, AKT1, and PIK3R1, wildtype TP53, p16 overexpression, and deregulated high-risk E6/E7 transcription characterized the HPV16-positive SCC. The deregulated pattern of HPV E6/E7 expression, correlating with HPV DNA presence and p16 positivity, supports a causal role of HPV in a subset of LDS papillomas and carcinomas. The viral and molecular profile of LDS SCC resembles that of other HPV-driven SCC.

Genomic and epigenomic studies revealed dysregulation of long non-coding RNAs in many cancer entities, including liver cancer. We identified an epigenetic mechanism leading to upregulation of the long intergenic non-coding RNA 152 (LINC00152) expression in human hepatocellular carcinoma (HCC). Here, we aimed to characterize a potential competing endogenous RNA (ceRNA) network, in which LINC00152 exerts oncogenic functions by sponging miRNAs, thereby affecting their target gene expression. Database and gene expression data of human HCC were integrated to develop a potential LINC00152-driven ceRNA in silico. RNA immunoprecipitation and luciferase assay were used to identify miRNA binding to LINC00152 in human HCC cells. Functionally active players in the ceRNA network were analyzed using gene editing, siRNA or miRNA mimic transfection, and expression vectors in vitro. RNA expression in human HCC in vivo was validated by RNA in situ hybridization. Let-7c-5p, miR-23a-3p, miR-125a-5p, miR-125b-5p, miR-143a-3p, miR-193-3p, and miR-195-5p were detected as new components of the potential LINC00152 ceRNA network in human HCC. LINC00152 was confirmed to sponge miR143a-3p in human HCC cell lines, thereby limiting its binding to their respective target genes, like KLC2. KLC2 was identified as a central mediator promoting pro-tumorigenic effects of LINC00152 overexpression in HCC cells. Furthermore, co-expression of LINC00152 and KLC2 was observed in human HCC cohorts and high KLC2 expression was associated with shorter patient survival. Functional assays demonstrated that KLC2 promoted cell proliferation, clonogenicity and migration in vitro. The LINC00152-miR-143a-3p-KLC2 axis may represent a therapeutic target in human HCC.