VEXAS syndrome is characterized by inflammasome activation and monocyte dysregulation.

Acquired mutations in the UBA1 gene were recently identified in patients with severe adult-onset auto-inflammatory syndrome called VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic). However, the precise physiological and clinical impact of these mutations remains poorly defined. Here we study a unique prospective cohort of VEXAS patients. We show that monocytes from VEXAS are quantitatively and qualitatively impaired and display features of exhaustion with aberrant expression of chemokine receptors. In peripheral blood from VEXAS patients, we identify an increase in circulating levels of many proinflammatory cytokines, including IL-1ß and IL-18 which reflect inflammasome activation and markers of myeloid cells dysregulation. Gene expression analysis of whole blood confirms these findings and also reveals a significant enrichment of TNF-α and NFκB signaling pathways that can mediate cell death and inflammation. This study suggests that the control of the nflammasome activation and inflammatory cell death could be therapeutic targets in VEXAS syndrome.

Cell Plasticity in a Mouse Model of Benign Prostate Hyperplasia Drives Amplification of Androgen-Independent Epithelial Cell Populations Sensitive to Antioxidant Therapy.

Benign prostate hyperplasia (BPH) is caused by the nonmalignant enlargement of the transition zone of the prostate gland, leading to lower urinary tract symptoms. Although current medical treatments are unsatisfactory in many patients, the limited understanding of the mechanisms driving disease progression prevents the development of alternative therapeutic strategies. The probasin-prolactin (Pb-PRL) transgenic mouse recapitulates many histopathological features of human BPH. Herein, these alterations parallel urodynamic disturbance reminiscent of lower urinary tract symptoms. Single-cell RNA-sequencing analysis of Pb-PRL mouse prostates revealed that their epithelium mainly includes low-androgen signaling cell populations analogous to Club/Hillock cells enriched in the aged human prostate. These intermediate cells are predicted to result from the reprogramming of androgen-dependent luminal cells. Pb-PRL mouse prostates exhibited increased vulnerability to oxidative stress due to reduction of antioxidant enzyme expression. One-month treatment of Pb-PRL mice with anethole trithione (ATT), a specific inhibitor of mitochondrial ROS production, reduced prostate weight and voiding frequency. In human BPH-1 epithelial cells, ATT decreased mitochondrial metabolism, cell proliferation, and stemness features. ATT prevented the growth of organoids generated by sorted Pb-PRL basal and LSCmed cells, the two major BPH-associated, androgen-independent epithelial cell compartments. Taken together, these results support cell plasticity as a driver of BPH progression and therapeutic resistance to androgen signaling inhibition, and identify antioxidant therapy as a promising treatment of BPH.

Rescuing the cytolytic function of APDS1 patient T cells via TALEN-mediated PIK3CD gene correction.

Gain-of-function mutations in the PIK3CD gene result in activated phosphoinositide 3-kinase δ syndrome type 1 (APDS1). This syndrome is a life-threatening combined immunodeficiency and today there are neither optimal nor long-term therapeutic solutions for APDS1 patients. Thus, new alternative treatments are highly needed. The aim of the present study is to explore one therapeutic avenue that consists of the correction of the PIK3CD gene through gene editing. Our proof-of-concept shows that TALEN-mediated gene correction of the mutated PIK3CD gene in APDS1 T cells results in normalized phospho-AKT levels in basal and activated conditions. Normalization of PI3K signaling was correlated to restored cytotoxic functions of edited CD8+ T cells. At the transcriptomic level, single-cell RNA sequencing revealed corrected signatures of CD8+ effector memory and CD8+ proliferating T cells. This proof-of-concept study paves the way for the future development of a gene therapy candidate to cure activated phosphoinositide 3-kinase δ syndrome type 1.

Single-cell RNA-sequencing of PBMCs from SAVI patients reveals disease-associated monocytes with elevated integrated stress response.

Gain-of-function mutations in stimulator of interferon gene 1 (STING1) result in STING-associated vasculopathy with onset in infancy (SAVI), a severe autoinflammatory disease. Although elevated type I interferon (IFN) production is thought to be the leading cause of the symptoms observed in patients, STING can induce a set of pathways, which have roles in the onset and severity of SAVI and remain to be elucidated. To this end, we performed a multi-omics comparative analysis of peripheral blood mononuclear cells (PBMCs) and plasma from SAVI patients and healthy controls, combined with a dataset of healthy PBMCs treated with IFN-ß. Our data reveal a subset of disease-associated monocyte, expressing elevated CCL3, CCL4, and IL-6, as well as a strong integrated stress response, which we suggest is the result of direct PERK activation by STING. Cell-to-cell communication inference indicates that these monocytes lead to T cell early activation, resulting in their senescence and apoptosis. Last, we propose a transcriptomic signature of STING activation, independent of type I IFN response.

NBEAL2 deficiency in humans leads to low CTLA-4 expression in activated conventional T cells.

Loss of NBEAL2 function leads to grey platelet syndrome (GPS), a bleeding disorder characterized by macro-thrombocytopenia and α-granule-deficient platelets. A proportion of patients with GPS develop autoimmunity through an unknown mechanism, which might be related to the proteins NBEAL2 interacts with, specifically in immune cells. Here we show a comprehensive interactome of NBEAL2 in primary T cells, based on mass spectrometry identification of altogether 74 protein association partners. These include LRBA, a member of the same BEACH domain family as NBEAL2, recessive mutations of which cause autoimmunity and lymphocytic infiltration through defective CTLA-4 trafficking. Investigating the potential association between NBEAL2 and CTLA-4 signalling suggested by the mass spectrometry results, we confirm by co-immunoprecipitation that CTLA-4 and NBEAL2 interact with each other. Interestingly, NBEAL2 deficiency leads to low CTLA-4 expression in patient-derived effector T cells, while their regulatory T cells appear unaffected. Knocking-down NBEAL2 in healthy primary T cells recapitulates the low CTLA-4 expression observed in the T cells of GPS patients. Our results thus show that NBEAL2 is involved in the regulation of CTLA-4 expression in conventional T cells and provide a rationale for considering CTLA-4-immunoglobulin therapy in patients with GPS and autoimmune disease.

Novel repertoire of tau biosensors to monitor pathological tau transformation and seeding activity in living cells.

Aggregates of the tau protein are a well-known hallmark of several neurodegenerative diseases, collectively referred to as tauopathies, including frontal temporal dementia and Alzheimer's disease (AD). Monitoring the transformation process of tau from physiological monomers into pathological oligomers or aggregates in a high-throughput, quantitative manner and in a cellular context is still a major challenge in the field. Identifying molecules able to interfere with those processes is of high therapeutic interest. Here, we developed a series of inter- and intramolecular tau biosensors based on the highly sensitive Nanoluciferase (Nluc) binary technology (NanoBiT) able to monitor the pathological conformational change and self-interaction of tau in living cells. Our repertoire of tau biosensors reliably reports i. molecular proximity of physiological full-length tau at microtubules; ii. changes in tau conformation and self-interaction associated with tau phosphorylation, as well as iii. tau interaction induced by seeds of recombinant tau or from mouse brain lysates of a mouse model of tau pathology. By comparing biosensors comprising different tau forms (i.e. full-length or short fragments, wild-type, or the disease-associated tau(P301L) variant) further insights into the tau transformation process are obtained. Proof-of-concept data for the high-throughput suitability and identification of molecules interfering with the pathological tau transformation processes are presented. This novel repertoire of tau biosensors is aimed to boost the disclosure of molecular mechanisms underlying pathological tau transformation in living cells and to discover new drug candidates for tau-related neurodegenerative diseases.

Prolonged response after TPO-RA discontinuation in primary ITP: results of a prospective multicenter study.

Sustained response off treatment (SROT) after thrombopoietin receptor agonist (TPO-RA) discontinuation has been reported in immune thrombocytopenia (ITP). This prospective multicenter interventional study enrolled adults with persistent or chronic primary ITP and complete response (CR) on TPO-RAs. The primary end point was the proportion of patients achieving SROT (platelet count >30 × 109/L and no bleeding) at week 24 (W24) with no other ITP-specific medications. Secondary end points included the proportion of sustained CR off-treatment (SCROT, platelet count >100 × 109/L and no bleeding) and SROT at W52, bleeding events, and pattern of response to a new course of TPO-RAs. We included 48 patients with a median age of 58.5 years; 30 of 48 had chronic ITP at TPO-RA initiation. In the intention-to-treat analysis, 27 of 48 achieved SROT, 15 of 48 achieved SCROT at W24; 25 of 48 achieved SROT, and 14 of 48 achieved SCROT at W52. No severe bleeding episode occurred in patients who relapsed. Among patients rechallenged with TPO-RA, 11 of 12 achieved CR. We found no significant clinical predictors of SROT at W24. Single-cell RNA sequencing revealed enrichment of a tumor necrosis factor α signaling via NF-κB signature in CD8+ T cells of patients with no sustained response after TPO-RA discontinuation, which was further confirmed by a significant overexpression of CD69 on CD8+ T cells at baseline in these patients as compared with those achieving SCROT/SROT. Our results strongly support a strategy based on progressive tapering and discontinuation of TPO-RAs for patients with chronic ITP who achieved a stable CR on treatment. This trial was registered at www.clinicaltrials.gov as #NCT03119974.

Late-onset enteric virus infection associated with hepatitis (EVAH) in transplanted SCID patients.

BACKGROUND: Allogenic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) are potentially curative treatments for severe combined immunodeficiency (SCID). Late-onset posttreatment manifestations (such as persistent hepatitis) are not uncommon. OBJECTIVE: We sought to characterize the prevalence and pathophysiology of persistent hepatitis in transplanted SCID patients (SCIDH+) and to evaluate risk factors and treatments. METHODS: We used various techniques (including pathology assessments, metagenomics, single-cell transcriptomics, and cytometry by time of flight) to perform an in-depth study of different tissues from patients in the SCIDH+ group and corresponding asymptomatic similarly transplanted SCID patients without hepatitis (SCIDH-). RESULTS: Eleven patients developed persistent hepatitis (median of 6 years after HSCT or GT). This condition was associated with the chronic detection of enteric viruses (human Aichi virus, norovirus, and sapovirus) in liver and/or stools, which were not found in stools from the SCIDH- group (n = 12). Multiomics analysis identified an expansion of effector memory CD8+ T cells with high type I and II interferon signatures. Hepatitis was associated with absence of myeloablation during conditioning, split chimerism, and defective B-cell function, representing 25% of the 44 patients with SCID having these characteristics. Partially myeloablative retransplantation or GT of patients with this condition (which we have named as "enteric virus infection associated with hepatitis") led to the reconstitution of T- and B-cell immunity and remission of hepatitis in 5 patients, concomitantly with viral clearance. CONCLUSIONS: Enteric virus infection associated with hepatitis is related to chronic enteric viral infection and immune dysregulation and is an important risk for transplanted SCID patients with defective B-cell function.

Skeletal Stem/Progenitor Cells in Periosteum and Skeletal Muscle Share a Common Molecular Response to Bone Injury.

Bone regeneration involves skeletal stem/progenitor cells (SSPCs) recruited from bone marrow, periosteum, and adjacent skeletal muscle. To achieve bone reconstitution after injury, a coordinated cellular and molecular response is required from these cell populations. Here, we show that SSPCs from periosteum and skeletal muscle are enriched in osteochondral progenitors, and more efficiently contribute to endochondral ossification during fracture repair as compared to bone-marrow stromal cells. Single-cell RNA sequencing (RNAseq) analyses of periosteal cells reveal the cellular heterogeneity of periosteum at steady state and in response to bone fracture. Upon fracture, both periosteal and skeletal muscle SSPCs transition from a stem/progenitor to a fibrogenic state prior to chondrogenesis. This common activation pattern in periosteum and skeletal muscle SSPCs is mediated by bone morphogenetic protein (BMP) signaling. Functionally, Bmpr1a gene inactivation in platelet-derived growth factor receptor alpha (Pdgfra)-derived SSPCs impairs bone healing and decreases SSPC proliferation, migration, and osteochondral differentiation. These results uncover a coordinated molecular program driving SSPC activation in periosteum and skeletal muscle toward endochondral ossification during bone regeneration. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Antigen receptor-engineered Tregs inhibit CNS autoimmunity in cell therapy using nonredundant immune mechanisms in mice.

CD4+ FOXP3+ Tregs are currently explored to develop cell therapies against immune-mediated disorders, with an increasing focus on antigen receptor-engineered Tregs. Deciphering their mode of action is necessary to identify the strengths and limits of this approach. Here, we addressed this issue in an autoimmune disease of the CNS, EAE. Following disease induction, autoreactive Tregs upregulated LAG-3 and CTLA-4 in LNs, while IL-10 and amphiregulin (AREG) were increased in CNS Tregs. Using genetic approaches, we demonstrated that IL-10, CTLA-4, and LAG-3 were nonredundantly required for the protective function of antigen receptor-engineered Tregs against EAE in cell therapy whereas AREG was dispensable. Treg-derived IL-10 and CTLA-4 were both required to suppress acute autoreactive CD4+ T-cell activation, which correlated with disease control. These molecules also affected the accumulation in the recipients of engineered Tregs themselves, underlying complex roles for these molecules. Noteworthy, despite the persistence of the transferred Tregs and their protective effect, autoreactive T cells eventually accumulated in the spleen of treated mice. In conclusion, this study highlights the remarkable power of antigen receptor-engineered Tregs to appropriately provide multiple suppressive factors nonredundantly necessary to prevent autoimmune attacks.

A monocyte/dendritic cell molecular signature of SARS-CoV-2-related multisystem inflammatory syndrome in children with severe myocarditis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children is generally milder than in adults, but a proportion of cases result in hyperinflammatory conditions often including myocarditis. METHODS: To better understand these cases, we applied a multiparametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. Plasma cytokine and chemokine levels and blood cellular composition were measured, alongside gene expression at the bulk and single-cell levels. FINDINGS: The most severe forms of multisystem inflammatory syndrome in children (MIS-C) related to SARS-CoV-2 that resulted in myocarditis were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomics analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis characterized by sustained nuclear factor κB (NF-κB) activity and tumor necrosis factor alpha (TNF-α) signaling and associated with decreased gene expression of NF-κB inhibitors. We also found a weak response to type I and type II interferons, hyperinflammation, and response to oxidative stress related to increased HIF-1α and Vascular endothelial growth factor (VEGF) signaling. CONCLUSIONS: These results provide potential for a better understanding of disease pathophysiology. FUNDING: Agence National de la Recherche (Institut Hospitalo-Universitaire Imagine, grant ANR-10-IAHU-01; Recherche Hospitalo-Universitaire, grant ANR-18-RHUS-0010; Laboratoire d'Excellence ''Milieu Intérieur," grant ANR-10-LABX-69-01; ANR-flash Covid19 "AIROCovid" and "CoVarImm"), Institut National de la Santé et de la Recherche Médicale (INSERM), and the "URGENCE COVID-19" fundraising campaign of Institut Pasteur.

Direct contribution of skeletal muscle mesenchymal progenitors to bone repair.

Bone regenerates by activation of tissue resident stem/progenitor cells, formation of a fibrous callus followed by deposition of cartilage and bone matrices. Here, we show that mesenchymal progenitors residing in skeletal muscle adjacent to bone mediate the initial fibrotic response to bone injury and also participate in cartilage and bone formation. Combined lineage and single-cell RNA sequencing analyses reveal that skeletal muscle mesenchymal progenitors adopt a fibrogenic fate before they engage in chondrogenesis after fracture. In polytrauma, where bone and skeletal muscle are injured, skeletal muscle mesenchymal progenitors exhibit altered fibrogenesis and chondrogenesis. This leads to impaired bone healing, which is due to accumulation of fibrotic tissue originating from skeletal muscle and can be corrected by the anti-fibrotic agent Imatinib. These results elucidate the central role of skeletal muscle in bone regeneration and provide evidence that skeletal muscle can be targeted to prevent persistent callus fibrosis and improve bone healing after musculoskeletal trauma.

Efficacy of parasternal block to decrease intraoperative opioid use in coronary artery bypass surgery via sternotomy: a randomized controlled trial.

OBJECTIVE: This study aims to assess the effect of a preoperative parasternal plane block (PSB) on opioid consumption required to maintain hemodynamic stability during sternotomy for coronary artery bypass graft surgery. METHODS: This double-blind, randomized, placebo-controlled trial prospectively enrolled 35 patients scheduled for coronary artery bypass graft surgery under general anesthesia with propofol and remifentanil. Patients were randomized to receive preoperative PSB using either ropivacaine (PSB group) or saline solution (placebo group) (1:1 ratio). The primary endpoint was the maximal effect-site concentration of remifentanil required to maintain heart rate and blood pressure within the recommended ranges during sternotomy. RESULTS: Median maximum concentration of remifentanil necessary to maintain adequate hemodynamic status during sternotomy was significantly reduced in PSB group (4.2 (2.5-6.0) ng/mL) compared with placebo group (7.0 (5.2-8.0) ng/mL) (p=0.02). Mean maximum concentration of propofol used to control depth of anesthesia was also reduced (3.9±1.1 µg/mL vs 5.0±1.5 µg/mL, PSB vs placebo, respectively; p=0.02). This reduction in propofol consumption during sternotomy enabled a more adequate level of sedation to be maintained in patients (minimum patient state index was 11.7±8.7 in placebo group and 18.3±6.8 in PSB group; p=0.02). PSB reduced postoperative inflammatory response by limiting concentrations of proinflammatory cytokines IL-8, IL-18, IL-23, IL-33 and MCP-1 measured in the first 7-day after surgery (p<0.05). CONCLUSIONS: Preoperative PSB reduced the maximum concentrations of remifentanil and propofol required to maintain hemodynamic stability and depth of anesthesia during sternotomy. TRIAL REGISTRATION NUMBER: NCT03734159.Sébastien Bloc, M.D.1,2; Brieuc P. Pérot, Ph.D.3; Hadrien Gibert, M.D.1; Jean-Dominique Law Koune, M.D.1; Yannick Burg, M.D.1; Didier Leclerc, M.D.1; Anne-Sophie Vuitton, M.D.1; Christophe De La Jonquière, M.D.1; Marine Luka, L.S.3; Thierry Waldmann, M.D.4; Nicolas Vistarini, M.D.4; Stéphane Aubert, M.D.4; Mickaël M. Ménager, Ph.D.3; Messaouda Merzoug, Ph.D.2; Cécile Naudin, Ph.D.2; Pierre Squara, M.D.2,5.

Maturation and persistence of the anti-SARS-CoV-2 memory B cell response.

Memory B cells play a fundamental role in host defenses against viruses, but to date, their role has been relatively unsettled in the context of SARS-CoV-2. We report here a longitudinal single-cell and repertoire profiling of the B cell response up to 6 months in mild and severe COVID-19 patients. Distinct SARS-CoV-2 spike-specific activated B cell clones fueled an early antibody-secreting cell burst as well as a durable synchronous germinal center response. While highly mutated memory B cells, including pre-existing cross-reactive seasonal Betacoronavirus-specific clones, were recruited early in the response, neutralizing SARS-CoV-2 RBD-specific clones accumulated with time and largely contributed to the late, remarkably stable, memory B cell pool. Highlighting germinal center maturation, these cells displayed clear accumulation of somatic mutations in their variable region genes over time. Overall, these findings demonstrate that an antigen-driven activation persisted and matured up to 6 months after SARS-CoV-2 infection and may provide long-term protection.

Amyloid Beta Peptide Is an Endogenous Negative Allosteric Modulator of Leptin Receptor.

INTRODUCTION: Metabolic dysfunction is now recognized as a pivotal component of Alzheimer's disease (AD), the most common dementia worldwide. However, the precise molecular mechanisms linking metabolic dysfunction to AD remain elusive. OBJECTIVE: Here, we investigated the direct impact of soluble oligomeric amyloid beta (Aß) peptides, the main molecular hallmark of AD, on the leptin system, a major component of central energy metabolism regulation. METHODS: We developed a new time-resolved fluorescence resonance energy transfer-based Aß binding assay for the leptin receptor (LepR) and studied the effect of Aß on LepR function in several in vitro assays. The in vivo effect of Aß on LepR function was studied in an Aß-specific AD mouse model and in pro-opiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus. RESULTS: We revealed specific and high-affinity (Ki = 0.1 nM) binding of Aß to LepR. Pharmacological characterization of this interaction showed that Aß binds allosterically to the extracellular domain of LepR and negatively affects receptor function. Negative allosteric modulation of LepR by Aß was detected at the level of signaling pathways (STAT-3, AKT, and ERK) in vitroand in vivo. Importantly, the leptin-induced response of POMC neurons, key players in the regulation of metabolic function, was completely abolished in the presence of Aß. CONCLUSION: Our data indicate that Aß is a negative allosteric modulator of LepR, resulting in impaired leptin action, and qualify LepR as a new and direct target of Aß oligomers. Preventing the interaction of Aß with LepR might improve both the metabolic and cognitive dysfunctions in AD condition.

Fgfr3 Is a Positive Regulator of Osteoblast Expansion and Differentiation During Zebrafish Skull Vault Development.

Gain or loss-of-function mutations in fibroblast growth factor receptor 3 (FGFR3) result in cranial vault defects highlighting the protein's role in membranous ossification. Zebrafish express high levels of fgfr3 during skull development; in order to study FGFR3's role in cranial vault development, we generated the first fgfr3 loss-of-function zebrafish (fgfr3lof/lof ). The mutant fish exhibited major changes in the craniofacial skeleton, with a lack of sutures, abnormal frontal and parietal bones, and the presence of ectopic bones. Integrated analyses (in vivo imaging and single-cell RNA sequencing of the osteoblast lineage) of zebrafish fgfr3lof/lof revealed a delay in osteoblast expansion and differentiation, together with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis. We conclude that changes in the extracellular matrix within growing bone might impair cell-cell communication, mineralization, and new osteoblast recruitment. © 2020 American Society for Bone and Mineral Research.

Dendritic Cell Maturation Regulates TSPAN7 Function in HIV-1 Transfer to CD4+ T Lymphocytes.

Dendritic cells (DCs) serve a key function in host defense, linking innate detection of microbes to activation of pathogen-specific adaptive immune responses. DCs express cell surface receptors for HIV-1 entry, but are relatively resistant to productive viral replication. They do, however, facilitate infection of co-cultured T-helper cells through a process referred to as trans-infection. We previously showed that tetraspanin 7 (TSPAN7), a transmembrane protein, is involved, through positive regulation of actin nucleation, in the transfer of HIV-1 from the dendrites of immature monocyte-derived DCs (iMDDCs) to activated CD4+ T lymphocytes. Various molecular mechanisms have been described regarding HIV-1 trans-infection and seem to depend on DC maturation status. We sought to investigate the crosstalk between DC maturation status, TSPAN7 expression and trans-infection. We followed trans-infection through co-culture of iMDDCs with CD4+ T lymphocytes, in the presence of CXCR4-tropic replicative-competent HIV-1 expressing GFP. T cell infection, DC maturation status and dendrite morphogenesis were assessed through time both by flow cytometry and confocal microscopy. Our previously described TSPAN7/actin nucleation-dependent mechanism of HIV-1 transfer appeared to be mostly observed during the first 20 h of co-culture experiments and to be independent of HIV replication. In the course of co-culture experiments, we observed a progressive maturation of MDDCs, correlated with a decrease in TSPAN7 expression, a drastic loss of dendrites and a change in the shape of DCs. A TSPAN7 and actin nucleation-independent mechanism of trans-infection, relying on HIV-1 replication, was then at play. We discovered that TSPAN7 expression is downregulated in response to different innate immune stimuli driving DC maturation, explaining the requirement for a TSPAN7/actin nucleation-independent mechanism of HIV transfer from mature MDDCs (mMDDCs) to T lymphocytes. As previously described, this mechanism relies on the capture of HIV-1 by the I-type lectin CD169/Siglec-1 on mMDDCs and the formation of a "big invaginated pocket" at the surface of DCs, both events being tightly regulated by DC maturation. Interestingly, in iMDDCs, although CD169/Siglec-1 can capture HIV-1, this capture does not lead to HIV-1 transfer to T lymphocytes.

A Comprehensive Map of the Monocyte-Derived Dendritic Cell Transcriptional Network Engaged upon Innate Sensing of HIV.

Transcriptional programming of the innate immune response is pivotal for host protection. However, the transcriptional mechanisms that link pathogen sensing with innate activation remain poorly understood. During HIV-1 infection, human dendritic cells (DCs) can detect the virus through an innate sensing pathway, leading to antiviral interferon and DC maturation. Here, we develop an iterative experimental and computational approach to map the HIV-1 innate response circuitry in monocyte-derived DCs (MDDCs). By integrating genome-wide chromatin accessibility with expression kinetics, we infer a gene regulatory network that links 542 transcription factors with 21,862 target genes. We observe that an interferon response is required, yet insufficient, to drive MDDC maturation and identify PRDM1 and RARA as essential regulators of the interferon response and MDDC maturation, respectively. Our work provides a resource for interrogation of regulators of HIV replication and innate immunity, highlighting complexity and cooperativity in the regulatory circuit controlling the response to infection.

GPR50-Ctail cleavage and nuclear translocation: a new signal transduction mode for G protein-coupled receptors.

Transmission of extracellular signals by G protein-coupled receptors typically relies on a cascade of intracellular events initiated by the activation of heterotrimeric G proteins or ß-arrestins followed by effector activation/inhibition. Here, we report an alternative signal transduction mode used by the orphan GPR50 that relies on the nuclear translocation of its carboxyl-terminal domain (CTD). Activation of the calcium-dependent calpain protease cleaves off the CTD from the transmembrane-bound GPR50 core domain between Phe-408 and Ser-409 as determined by MALDI-TOF-mass spectrometry. The cytosolic CTD then translocates into the nucleus assisted by its 'DPD' motif, where it interacts with the general transcription factor TFII-I to regulate c-fos gene transcription. RNA-Seq analysis indicates a broad role of the CTD in modulating gene transcription with ~ 8000 differentially expressed genes. Our study describes a non-canonical, direct signaling mode of GPCRs to the nucleus with similarities to other receptor families such as the NOTCH receptor.

Quantitative assessment of oligomeric amyloid ß peptide binding to α7 nicotinic receptor.

BACKGROUND AND PURPOSE: Progressive dysfunction of cholinergic transmission is a well-known characteristic of Alzheimer's disease (AD). Amyloid ß (Aß) peptide oligomers are known to play a central role in AD and are suggested to impair the function of the cholinergic nicotinic ACh receptor α7 (α7nAChR). However, the mechanism underlying the effect of Aß on α7nAChR function is not fully understood, limiting the therapeutic exploration of this observation in AD. Here, we aimed to detect and characterize Aß binding to α7nAChR, including the possibility of interfering with this interaction for therapeutic purposes. EXPERIMENTAL APPROACH: We developed a specific and quantitative time-resolved FRET (TR-FRET)-based binding assay for Aß to α7nAChR and pharmacologically characterized this interaction. KEY RESULTS: We demonstrated specific and high-affinity (low nanomolar) binding of Aß to the orthosteric binding site of α7nAChR. Aß binding was prevented and reversed by the well-characterized orthosteric ligands of α7nAChR (epibatidine, α-bungarotoxin, methylylcaconitine, PNU-282987, S24795, and EVP6124) and by the type II positive allosteric modulator (PAM) PNU-120596 but not by the type I PAM NS1738. CONCLUSIONS AND IMPLICATIONS: Our TR-FRET Aß binding assay demonstrates for the first time the specific binding of Aß to α7nAChR, which will be a crucial tool for the development, testing, and selection of a novel generation of AD drug candidates targeting Aß/α7nAChR complexes with high specificity and fewer side effects compared to currently approved α7nAChR drugs. LINKED ARTICLES: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc.