Cell-specific Manner Research Articles

Nox, Nox, Are You There? The Role of NADPH Oxidases in the Peripheral Nervous System.

Significance: Reactive oxygen species (ROS) contribute to multiple aspects of peripheral nervous system (PNS) biology ranging from physiological processes (e.g., axonal outgrowth and regeneration) to pathophysiology (e.g., nerve degeneration). Although ROS are derived from multiple sources, NADPH oxidase (Nox) family members are dedicated to ROS generation. Noxs are expressed in the PNS, and their overexpression is associated with detrimental effects on nerve function and contributes, at least in part, to peripheral neuropathies. Recent Advances: Of the seven members, studies mostly focused on Nox1, Nox2, and Nox4, which are expressed in the PNS in a cell-specific manner. We have also recently identified human Nox5 in sural nerve biopsies. When maintained at homeostatic levels, Noxs regulate several aspects of peripheral nerve health, most notably neurite outgrowth and axonal regeneration following nerve lesion. While Nox2 and Nox4 dysregulation is a major source of oxidative stress in PNS disorders, including neuropathic pain and diabetic peripheral neuropathy, recent evidence also implicates Nox1 and Nox5. Critical Issues: Although there is compelling evidence for a direct role of Noxs on nerve function, little is known about their subcellular localization, intercellular regulation, and interaction. These, together with redox signaling, are considered crucial components of nerve redox status. In addition, the lack of isoform-specific inhibitors limits conclusions about the physiological role of Noxs in the PNS and their therapeutic potential in peripheral neuropathies. Future Directions: Future research using isoform-specific genetic and pharmacological approaches are therefore needed to better understand the significance of Nox enzymes in PNS (patho) physiology. Antioxid. Redox Signal. 37, 613-630.

Hem-1 regulates protective humoral immunity and limits autoantibody production in a B cell-specific manner.

Hematopoietic protein-1 (Hem-1) is a member of the actin-regulatory WASp family verprolin homolog (WAVE) complex. Loss-of-function variants in the NCKAP1L gene encoding Hem-1 were recently discovered to result in primary immunodeficiency disease (PID) in children, characterized by poor specific Ab responses, increased autoantibodies, and high mortality. However, the mechanisms of how Hem-1 deficiency results in PID are unclear. In this study, we utilized constitutive and B cell–specific Nckap1l-KO mice to dissect the importance of Hem-1 in B cell development and functions. B cell–specific disruption of Hem-1 resulted in reduced numbers of recirculating follicular (FO), marginal zone (MZ), and B1 B cells. B cell migration in response to CXCL12 and -13 were reduced. T-independent Ab responses were nearly abolished, resulting in failed protective immunity to Streptococcus pneumoniae challenge. In contrast, T-dependent IgM and IgG2c, memory B cell, and plasma cell responses were more robust relative to WT control mice. B cell–specific Hem-1–deficient mice had increased autoantibodies against multiple autoantigens, and this correlated with hyperresponsive BCR signaling and increased representation of CD11c+T-bet+ age-associated B cell (ABC cells) — alterations associated with autoimmune diseases. These results suggest that dysfunctional B cells may be part of a mechanism explaining why loss-of-function Hem-1 variants result in recurring infections and autoimmunity.

Characterization of cellular subpopulations and their gene expression by single-cell RNA sequencing in canine atopic dermatitis

Abstract Atopic Dermatitis (AD) is the most common, chronic, and inflammatory allergic skin disease worldwide, with a high prevalence both in children and adults, particularly in developed countries. There are limited effective treatments for severe allergic skin diseases available, thus, there is an unmet need of better understanding the molecular mechanisms and cellular populations involved in AD. Since AD in canines closely resembles the disease in humans, they are an attractive animal model for further studies. Leveraging single-cell RNA sequencing, we identified the cellular composition and shifts in cell populations in canine epidermis of atopic dogs compared to the epidermis from healthy controls. Fibroblasts and keratinocytes represented the largest cell populations, with fibroblasts decreasing in the atopic epidermis. Other populations such as endothelial vascular cells showed differences as well. Shifts in immune cell distribution included changes in subpopulations of dendritic cells and monocytes in the atopic skin. NK and T cells, particularly CD8 T cells were increased. Subclustering revealed specific cellular changes in AD dogs. We found signifiant changes in gene expression in most cell populations, with some genes differentially expressed across cells, while others were expressed in a cell specific manner. We also observed subtle differences between samples from animals with different disease progression, identifying gene expression markers changing with AD severity. Here we show that single-cell sequencing is a useful tool to define the cellular composition in canine atopic dermatitis, resolving changes in gene expression at the cellular level to advance our understanding of disease development and progression. Supported by intramural funds from Western University of Health Sciences

Characterization of a Novel Anti-inflammatory Biogenic Amine

Abstract Tryptophan (Trp) is an essential amino acid whose catabolism generates a cascade of over fifty molecules in a cell-specific manner. Three major Trp metabolic pathways have been described: synthesis of serotonin, synthesis of melatonin and generation of kynurenines. In mammalian cells, 90% of Trp is processed through the kynurenine pathway and kynurenine metabolites are regarded as one of the most powerful mechanism for immune regulation. We recently reported a novel biogenic amine, 3HKA, generated through a still uncharacterized lateral pathway of Trp catabolism. This novel biogenic amine is generated by both professional (Dendritic Cells) and non-professional (Lymphatic Endothelial Cells) antigen presenting cells and, differently from all others Trp metabolites, display unique “in vivo” immunosuppressive capabilities. In vitro, 3-HKA exhibited an anti-inflammatory profile by inhibiting the IFN-g-mediated STAT1/NF-kB pathway in both mouse and human dendritic cells (DCs) with a consequent decrease in the release of pro-inflammatory chemokines and cytokines; most notably, TNFa, IL-6, and IL12p70. In vivo, 3-HKA exerted protective effects in an experimental mouse model of Psoriasis by decreasing skin thickness, erythema, scaling and fissuring, reducing TNFa, IL-1b, IFN-g, and IL-17 production, and inhibiting generation of effector CD8+ T cells. Similarly, in a mouse model of nephrotoxic nephritis, besides reducing inflammatory cytokines, 3-HKA improved proteinuria and serum urea nitrogen, overall ameliorating the immune-mediated glomerulonephritis and renal dysfunction. Overall we propose that this novel biological amine is a crucial component of Trp-mediated immune tolerance.

Circadian control of macrophages in the tumor microenvironment.

Abstract Introduction All leukocytes tested to date have functional circadian clocks, and nearly every arm of the immune response is subject to circadian regulation. Circadian clocks instruct the time-of-day-dependent, rhythmic expression of genes in a tissue- and cell-specific manner. In macrophages (mΦs), the circadian clock regulates several factors that are critical to executing effective immune responses. Tumor-associated mΦs are major contributors to immune suppression in the tumor microenvironment (TME). Evidence suggests that metabolically stressful factors in the TME such as acidic pH and nutrient limitation promote mΦ-mediated immune suppression, and recent data point to dysregulation of the circadian clock downstream of metabolic stress. Methods We study the effect of TME-associated metabolic stress on the circadian clock of mΦs in vitro by culturing bone marrow-derived mΦs in conditions mimicking acidic pH and nutrient limitations that have been observed in the TME. To study the impact of mΦ-intrinsic circadian rhythms on tumorigenesis in vivo, we use mice genetically engineered to have a myeloid cell-specific disruption of the circadian clock via deletion of the key clock protein BMAL1. Results Oscillation of core clock proteins is altered in mΦs subjected to TME-associated metabolic stress. Additionally, we observe increased tumor growth in mice co-injected with mΦs whose circadian clocks were disrupted compared to mice co-injected with mΦs whose circadian clocks were functional. Conclusion Our data suggests that stressful conditions associated with the TME can alter the mΦ circadian clock, and that a functional circadian clock in mΦs can suppress tumor growth in a syngeneic murine tumor model of pancreatic cancer. This research has been supported by the following fellowships and grants: 2021-Current: Wilmot Predoctoral Cancer Research Fellowship, Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY 2020-2021: NIH T32 Training Grant in Cellular, Biochemical & Molecular Sciences, University of Rochester Medical Center, Rochester, NY

One genome, many cell states: epigenetic control of innate immunity.

A hallmark of the innate immune system is its ability to rapidly initiate short-lived or sustained transcriptional programs in a cell-specific and pathogen-specific manner that is dependent on dynamic chromatin states. Much of the epigenetic landscape is set during cellular differentiation; however, pathogens and other environmental cues also induce changes in chromatin that can either promote tolerance or 'train' innate immune cells for amplified secondary responses. We review chromatin processes that enable innate immune cell differentiation and functional transcriptional responses in naive or experienced cells, in concert with signal transduction and cellular metabolic shifts. We discuss how immune chromatin mechanisms are maladapted in disease and novel therapeutic approaches for cellular reprogramming.

HIPK2 directs cell type–specific regulation of STAT3 transcriptional activity in Th17 cell differentiation

SignificanceSTAT3 (signal transducer and activator of transcription 3) is a master transcription factor that organizes cellular responses to cytokines and growth factors and is implicated in inflammatory disorders. STAT3 is a well-recognized therapeutic target for human cancer and inflammatory disorders, but how its function is regulated in a cell type-specific manner has been a major outstanding question. We discovered that Stat3 imposes self-directed regulation through controlling transcription of its own regulator homeodomain-interacting protein kinase 2 (Hipk2) in a T helper 17 (Th17) cell-specific manner. Our validation of the functional importance of the Stat3-Hipk2 axis in Th17 cell development in the pathogenesis of T cell-induced colitis in mice suggests an approach to therapeutically treat inflammatory bowel diseases that currently lack a safe and effective therapy.

Activation of 20-HETE Synthase Triggers Oxidative Injury and Peripheral Nerve Damage in Type 2 Diabetic Mice

Diabetic Peripheral Neuropathy (DPN), highly prevalent among patients with diabetes, is characterized by peripheral nerve dysfunction. Reactive Oxygen Species (ROS) overproduction has been suggested to orchestrate diabetic complications including DPN. Untargeted antioxidant therapy has exhibited limited efficacy, highlighting a critical need to explore ROS sources altered in a cell-specific manner in DPN. Cytochromes P450 (CYP) enzymes are prominent sources of ROS. Particularly, the 20-HETE synthase, CYP4A, is reported to mediate diabetes-induced renal, retinal, and cardiovascular injuries. This work investigates the role of CYP4A/20-HETE in DPN and their mechanisms of action. Non-obese type 2 Diabetic mice (MKR) were used and treated with a CYP4A-inhibitor (HET0016) or AMPK-activator (Metformin). Peripheral nerves of MKR mice reflect increased CYP4A and 20-HETE levels, concurrent with altered myelin proteins and sensorimotor deficits. This was associated with increased ROS production and altered Beclin-1 and LC3 protein levels, indicative of disrupted autophagic responses in tandem with AMPK inactivation. AMPK activation via Metformin restored nerve integrity, reduced ROS production, and regulated autophagy. Interestingly, similar outcomes were revealed upon HET0016 treatment whereby ROS production, autophagic responses, and AMPK signaling were normalized in diabetic mice. Altogether, the results highlight hyperglycemia-mediated oxidative injury in DPN through a novel CYP4A/20-HETE/AMPK pathological axis. PerspectiveTo our knowledge, this is the first study to highlight the role of CYPs/20-HETE-induced oxidative injury in the pathogenesis of diabetic peripheral neuropathy. Targeting the identified pathological axis CYP4A/20-HETE/AMPK may be of clinical potential in predicting and alleviating peripheral nerve injury in patients with Type 2 Diabetes Mellitus.

LncRNA EDAL restricts rabies lyssavirus replication in a cell-specific and infection route-dependent manner.

Rabies, caused by rabies lyssavirus (RABV), is a fatal disease among humans and almost all warm-blooded animals. Our previous study showed that the long non-coding RNA (lncRNA) EZH2 degradation-associated lncRNA (EDAL) effectively inhibits RABV infection both in vitro and in vivo by degrading EZH2 and promoting the transcription of an antiviral gene, Pcp4l1. Herein, we found that recombinant RABV expressing EDAL (rRABV-EDAL) restricts RABV replication in primary granule neurons but not in primary cortical neurons or astrocytes. Further study revealed that EDAL induced EZH2 protein degradation and thereby decreased trimethylation of lysine 27 on the histone 3 (H3K27me3) level in granule neuron cells but not in cortical neurons or astrocytes. Furthermore, rRABV-EDAL infection induces more Pcp4l1 mRNA transcription in granule neurons, while there are almost no obvious changes in cortical neurons or astrocytes. Consistently, compared with the parent virus RABV, reduced pathogenicity of rRABV-EDAL was observed in mice post-intranasal infection but not intramuscular infection. These results suggest that the lncRNA EDAL restricts RABV replication in a cell-specific and infection route-dependent manner.

An Essential Role for Alzheimer's-Linked Amyloid Beta Oligomers in Neurodevelopment: Transient Expression of Multiple Proteoforms during Retina Histogenesis.

Human amyloid beta peptide (Aβ) is a brain catabolite that at nanomolar concentrations can form neurotoxic oligomers (AβOs), which are known to accumulate in Alzheimer’s disease. Because a predisposition to form neurotoxins seems surprising, we have investigated whether circumstances might exist where AβO accumulation may in fact be beneficial. Our investigation focused on the embryonic chick retina, which expresses the same Aβ as humans. Using conformation-selective antibodies, immunoblots, mass spectrometry, and fluorescence microscopy, we discovered that AβOs are indeed present in the developing retina, where multiple proteoforms are expressed in a highly regulated cell-specific manner. The expression of the AβO proteoforms was selectively associated with transiently expressed phosphorylated Tau (pTau) proteoforms that, like AβOs, are linked to Alzheimer’s disease (AD). To test whether the AβOs were functional in development, embryos were cultured ex ovo and then injected intravitreally with either a beta-site APP-cleaving enzyme 1 (BACE-1) inhibitor or an AβO-selective antibody to prematurely lower the levels of AβOs. The consequence was disrupted histogenesis resulting in dysplasia resembling that seen in various retina pathologies. We suggest the hypothesis that embryonic AβOs are a new type of short-lived peptidergic hormone with a role in neural development. Such a role could help explain why a peptide that manifests deleterious gain-of-function activity when it oligomerizes in the aging brain has been evolutionarily conserved.

The transcriptional corepressor CTBP-1 acts with the SOX family transcription factor EGL-13 to maintain AIA interneuron cell identity in Caenorhabditis elegans.

Cell identity is characterized by a distinct combination of gene expression, cell morphology, and cellular function established as progenitor cells divide and differentiate. Following establishment, cell identities can be unstable and require active and continuous maintenance throughout the remaining life of a cell. Mechanisms underlying the maintenance of cell identities are incompletely understood. Here, we show that the gene ctbp-1, which encodes the transcriptional corepressor C-terminal binding protein-1 (CTBP-1), is essential for the maintenance of the identities of the two AIA interneurons in the nematode Caenorhabditis elegans. ctbp-1 is not required for the establishment of the AIA cell fate but rather functions cell-autonomously and can act in later larval stage and adult worms to maintain proper AIA gene expression, morphology and function. From a screen for suppressors of the ctbp-1 mutant phenotype, we identified the gene egl-13, which encodes a SOX family transcription factor. We found that egl-13 regulates AIA function and aspects of AIA gene expression, but not AIA morphology. We conclude that the CTBP-1 protein maintains AIA cell identity in part by utilizing EGL-13 to repress transcriptional activity in the AIAs. More generally, we propose that transcriptional corepressors like CTBP-1 might be critical factors in the maintenance of cell identities, harnessing the DNA-binding specificity of transcription factors like EGL-13 to selectively regulate gene expression in a cell-specific manner.

Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia

During gram-negative septicemia, interactions between platelets and neutrophils initiate a detrimental feedback loop that sustains neutrophil extracellular trap (NET) induction, disseminated intravascular coagulation, and inflammation. Understanding intracellular pathways that control platelet-neutrophil interactions is essential for identifying new therapeutic targets. Here, we found that thrombin signaling induced activation of the transcription factor NFAT in platelets. Using genetic and pharmacologic approaches, as well as iNFATuation, a newly developed mouse model in which NFAT activation can be abrogated in a cell-specific manner, we demonstrated that NFAT inhibition in activated murine and human platelets enhanced their activation and aggregation, as well as their interactions with neutrophils and NET induction. During gram-negative septicemia, NFAT inhibition in platelets promoted disease severity by increasing disseminated coagulation and NETosis. NFAT inhibition also partially restored coagulation exvivo in patients with hypoactive platelets. Our results define non-transcriptional roles for NFAT that could be harnessed to address pressing clinical needs.

Mice transgenic for human CTLA4-CD28 fusion gene show proliferation and transformation of ATLL-like and AITL-like T cells

ABSTRACT CTLA4-CD28 gene fusion has been reported to occur in diverse types of T cell lymphoma. The fusion event is expected to convert inhibitory signals to activating signals and promote proliferation and potentially transformation of T cells. To test the function of the CTLA4-CD28 fusion gene in vivo, we generated a murine model that expresses the gene in a T cell-specific manner. The transgenic mice have shorter life spans and display inflammatory responses including lymphadenopathy and splenomegaly. T cells in turn show higher levels of activation and infiltrate various organs including the lung and skin. T cells, in particular CD4+ helper T cells, were also readily transplantable to immunocompromised mice. Transcriptomic profiling revealed that the gene expression pattern in CD4 + T cells closely resembles that of adult T cell leukemia/lymphoma (ATLL) and that of angioimmunoblastic T cell lymphoma (AITL) tissues. Consistently, we detected supernumerary FOXP3+ cells and PD-1+ cells in transgenic and transplanted mice. This is the first report demonstrating the transforming activity of the CTLA4-CD28 fusion gene in vivo, and this murine model should be useful in dissecting the molecular events downstream to this mutation.

3110 – MULTIPARTITE SUPER-ENHANCERS MAY ACT IN AN ORIENTATION-DEPENDENT MANNER

Transcriptional enhancers regulate gene expression in a developmental-stage and cell-specific manner. They were originally defined as individual regulatory elements that activate expression regardless of distance and orientation to their cognate genes. Genome-wide studies have shown that the mammalian enhancer landscape is much more complex, with different classes of individual enhancers and clusters of enhancer-like elements combining in additive, synergistic and redundant manners, possibly acting as single, integrated regulatory elements. These so-called super-enhancers are largely defined as clusters of enhancer-like elements which recruit particularly high levels of Mediator and often drive high levels of expression of key lineage-specific genes. Here, we analysed 78 erythroid-specific super-enhancers and showed that, as units, they preferentially interact in a directional manner, to drive expression of their cognate genes. Using the well characterised α-globin super-enhancer, we show that inverting this entire structure severely downregulates α-globin expression and activates flanking genes 5’ of the super-enhancer. Our detailed genetic dissection of the α-globin locus clearly attributes the cluster's functional directionality to its sequence orientation, demonstrating that, unlike regular enhancers, super-enhancer act in an orientation-dependent manner. Together, these findings identify a novel emergent property of super-enhancers and revise current models by which enhancers are thought to contact and activate their cognate genes. Transcriptional enhancers regulate gene expression in a developmental-stage and cell-specific manner. They were originally defined as individual regulatory elements that activate expression regardless of distance and orientation to their cognate genes. Genome-wide studies have shown that the mammalian enhancer landscape is much more complex, with different classes of individual enhancers and clusters of enhancer-like elements combining in additive, synergistic and redundant manners, possibly acting as single, integrated regulatory elements. These so-called super-enhancers are largely defined as clusters of enhancer-like elements which recruit particularly high levels of Mediator and often drive high levels of expression of key lineage-specific genes. Here, we analysed 78 erythroid-specific super-enhancers and showed that, as units, they preferentially interact in a directional manner, to drive expression of their cognate genes. Using the well characterised α-globin super-enhancer, we show that inverting this entire structure severely downregulates α-globin expression and activates flanking genes 5’ of the super-enhancer. Our detailed genetic dissection of the α-globin locus clearly attributes the cluster's functional directionality to its sequence orientation, demonstrating that, unlike regular enhancers, super-enhancer act in an orientation-dependent manner. Together, these findings identify a novel emergent property of super-enhancers and revise current models by which enhancers are thought to contact and activate their cognate genes.

Chronic Daily House Dust Mite Exposure in Mice is an Effective Model to Quantify the Effect of Pharmacologic Agents on Discrete Stages of Artery Remodeling in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a heterogenous and incurable disease marked by varying degrees of pulmonary vascular remodeling. This vascular remodeling, which includes thickening of the smooth muscle layer (an early finding) and formation of occlusive neointimal lesions (a late finding) in the pulmonary arteries, is a major driver of morbidity and mortality in PH. Available PH therapies consist of vasodilators that do not specifically target lesion formation or expansion and neither prevent progression nor reverse disease. This paucity of curative treatments highlights the need for new drug discovery targeting crucial steps of artery remodeling in PH. The cell dynamics and molecular signals driving neointimal lesion formation have been difficult to elucidate as classic mouse models of PH do not develop neointima. Here, we detail the methods to generate a robust and non-genetic mouse model of PH with medial thickening and neointimal lesion formation in the pulmonary arteries, through chronic exposure to an inflammatory stimulus-house dust mite (HDM). This model rapidly generates human-like pulmonary arterial lesions following a reproducible time course, allowing scrutiny of the cellular and molecular mechanisms controlling each stage of artery remodeling. Further, we outline optimal tissue handling, sectioning, and staining methodologies for detailed quantitative analysis of artery medial thickening and neointimal lesion formation and expansion. Finally, we present a method for staged pharmacologic intervention to identify molecules and pathways required at each step of the pulmonary arterial remodeling process. The advantages of this mouse model of PH over currently available animal models are five-fold. (i) It allows the use of the full range of genetic and single cell tools available in mice to manipulate and study the process of vascular remodeling seen in human disease, including the formation of neointimal lesions in a controlled and cell specific manner. (ii) The vascular lesions develop in a stereotyped manner with predictable timing, allowing for pharmacologic manipulation at discrete stages of vessel remodeling. (iii) It is rapid, with development of PH and vascular remodeling in a timeframe of two to eight weeks. (iv) It uses simple techniques and requires neither surgery, unusual equipment, or extensive personnel training. (v) The staining and quantitation methodologies we present are a significant improvement over those currently in use in the field. We hope that dissemination of this model and the associated detailed methods will speed up the development of novel and more effective PH therapeutics. Graphic abstract: Chronic perivascular inflammation induces medial thickening and neointima formation in pulmonary arteries, following a stereotyped time course, and allowing staged pharmacologic intervention during specific remodeling events, as well as quantitative assessment of vascular changes.

Molecular and cellular functions of long non-coding RNAs in prostate and breast cancer.

Long noncoding RNAs (lncRNAs) are defined as noncoding RNA transcripts with a length greater than 200 nucleotides. Research over the last decade has made great strides in our understanding of lncRNAs, especially in the biology of their role in cancer. In this article, we will briefly discuss the biogenesis and characteristics of lncRNAs, then review their molecular and cellular functions in cancer by using prostate and breast cancer as examples. LncRNAs are abundant, diverse, and evolutionarily, less conserved than protein-coding genes. They are often expressed in a tumor and cell-specific manner. As a key epigenetic factor, lncRNAs can use a wide variety of molecular mechanisms to regulate gene expression at each step of the genetic information flow pathway. LncRNAs display widespread effects on cell behavior, tumor growth, and metastasis. They act intracellularly and extracellularly in an autocrine, paracrine and endocrine fashion. Increased understanding of lncRNA's role in cancer has facilitated the development of novel biomarkers for cancer diagnosis, led to greater understanding of cancer prognosis, enabled better prediction of therapeutic responses, and promoted identification of potential targets for cancer therapy.

Translating Ribosome Affinity Purification (TRAP) of Cell Type-specific mRNA from Mouse Brain Lysates.

Mammalian tissues are highly heterogenous and complex, posing a challenge in understanding the molecular mechanisms regulating protein expression within various tissues. Recent studies have shown that translation at the level of the ribosome is highly regulated, and can vary independently of gene expression observed at a transcriptome level, as well as between cell populations, contributing to the diversity of mammalian tissues. Earlier methods that analyzed gene expression at the level of translation, such as polysomal- or ribosomal-profiling, required large amounts of starting material to isolate enough RNA for analysis by microarray or RNA-sequencing. Thus, rare or less abundant cell types within tissues were not able to be properly studied with these methods. Translating ribosome affinity purification (TRAP) utilizes the incorporation of an eGFP-affinity tag on the large ribosome subunit, driven by expression of cell-type specific Cre-lox promoters, to allow for identification and capture of transcripts from actively translating ribosomes in a cell-specific manner. As a result, TRAP offers a unique opportunity to evaluate the entire mRNA translation profile within a specific cell type, and increase our understanding regarding the cellular complexity of mammalian tissues. Graphical abstract: Schematic demonstrating TRAP protocol for identifying ribosome-bound transcripts specifically within cerebellar Purkinje cells.

Chemogenetics of cell surface receptors: beyond genetic and pharmacological approaches.

Cell surface receptors transmit extracellular information into cells. Spatiotemporal regulation of receptor signaling is crucial for cellular functions, and dysregulation of signaling causes various diseases. Thus, it is highly desired to control receptor functions with high spatial and/or temporal resolution. Conventionally, genetic engineering or chemical ligands have been used to control receptor functions in cells. As the alternative, chemogenetics has been proposed, in which target proteins are genetically engineered to interact with a designed chemical partner with high selectivity. The engineered receptor dissects the function of one receptor member among a highly homologous receptor family in a cell-specific manner. Notably, some chemogenetic strategies have been used to reveal the receptor signaling of target cells in living animals. In this review, we summarize the developing chemogenetic methods of transmembrane receptors for cell-specific regulation of receptor signaling. We also discuss the prospects of chemogenetics for clinical applications.

Interferon-Induced Transmembrane Proteins Inhibit Infection by the Kaposi's Sarcoma-Associated Herpesvirus and the Related Rhesus Monkey Rhadinovirus in a Cell-Specific Manner.

ABSTRACTThe interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral proteins that inhibit the entry of enveloped viruses. We analyzed the effect of IFITMs on the gamma-2 herpesviruses Kaposi’s sarcoma-associated herpesvirus (KSHV) and the closely related rhesus monkey rhadinovirus (RRV). We used CRISPR/Cas9-mediated gene knockout to generate A549 cells, human foreskin fibroblasts (HFF), and human umbilical vein endothelial cells (HUVEC) with combined IFITM1/2/3 knockout and identified IFITMs as cell-dependent inhibitors of KSHV and RRV infection in A549 cells and HFF but not HUVEC. IFITM overexpression revealed IFITM1 as the relevant IFITM that inhibits KSHV and RRV infection. Fluorescent KSHV particles did not pronouncedly colocalize with IFITM-positive compartments. However, we found that KSHV and RRV glycoprotein-mediated cell-cell fusion is enhanced upon IFITM1/2/3 knockout. Taken together, we identified IFITM1 as a cell-dependent restriction factor of KSHV and RRV that acts at the level of membrane fusion. Of note, our results indicate that recombinant IFITM overexpression may lead to results that are not representative for the situation at endogenous levels. Strikingly, we observed that the endotheliotropic KSHV circumvents IFITM-mediated restriction in HUVEC despite high IFITM expression, while influenza A virus (IAV) glycoprotein-driven entry into HUVEC is potently restricted by IFITMs even in the absence of interferon. Mechanistically, we found that KSHV colocalizes less with IFITM1 and IFITM2 in HUVEC than in A549 cells immediately after attachment, potentially contributing to the observed difference in restriction.

The Sympathetic Nervous System Modulates Cancer Vaccine Activity through Monocyte-Derived Cells.

The sympathetic nervous system (SNS) is an important regulator of immune cell function during homeostasis and states of inflammation. Recently, the SNS has been found to bolster tumor growth and impair the development of antitumor immunity. However, it is unclear whether the SNS can modulate APC function. Here, we investigated the effects of SNS signaling in murine monocyte-derived macrophages (moMФ) and dendritic cells (DCs) and further combined the nonspecific β-blocker propranolol with a peptide cancer vaccine for the treatment of melanoma in mice. We report that norepinephrine treatment dramatically altered moMФ cytokine production, whereas DCs were unresponsive to norepinephrine and critically lack β2-adrenergic receptor expression. In addition, we show that propranolol plus cancer vaccine enhanced peripheral DC maturation, increased the intratumor proportion of effector CD8+ T cells, and decreased the presence of intratumor PD-L1+ myeloid-derived suppressor cells. Furthermore, this combination dramatically reduced tumor growth compared with vaccination alone. Taken together, these results offer insights into the cell-specific manner by which the SNS regulates the APC immune compartment and provide strong support for the use of propranolol in combination with cancer vaccines to improve patient response rates and survival.