Actin Dynamics Regulates Dendritic Cell-Mediated Transfer of HIV-1 to T Cells.

Whereas human dendritic cells (DCs) are largely resistant to productive infection with HIV-1, they have a unique ability to take up the virus and transmit it efficiently to T lymphocytes through a process of trans-infection or trans-enhancement. To elucidate the molecular and cell biological mechanism for trans-enhancement, we performed an shRNA screen of several hundred genes involved in organelle and membrane trafficking in immature human monocyte-derived dendritic cells (MDDCs). We identified TSPAN7 and DNM2, which control actin nucleation and stabilization, as having important and distinct roles in limiting HIV-1 endocytosis and in maintaining virus particles on dendrites, which is required for efficient transfer to T lymphocytes. Further characterization of this process may provide insights not only into the role of DCs in transmission and dissemination of HIV-1 but also more broadly into mechanisms controlling capture and internalization of pathogens.

Tetraspanin 7 and its closest paralog tetraspanin 6: membrane organizers with key functions in brain development, viral infection, innate immunity, diabetes and cancer.

Tetraspanin (TSPAN) protein family forms a family of transmembrane proteins that act as organizers/scaffold for other proteins. TSPANs are primarily present on plasma membranes although they are also found in other biological membranes. They are organized in tetraspanin-enriched microdomains (TEMs), which allow spatiotemporal tuning of protein functions through the control of their membrane localization. TSPAN6 and TSPAN7 are close paralogs expressed in different tissues, TSPAN7 being highly expressed in the brain. Their functions only started to be unveiled in the late 2000's and are still poorly understood. Here, we introduce how TSPAN7 was first highlighted has a protein mutated in some forms of X-linked mental retardation, which was later proposed to be caused by defects in neuronal morphogenesis and synaptic transmission. We then discuss the impacts TSPAN7 has on cell morphology of dendritic cells and osteoclasts, through rearrangement of actin cytoskeleton and how TSPAN7 was shown to be a target of autoantibody in patients suffering from type 1 diabetes. Finally, we are addressing the double edge sword that is TSPAN7 in cancer. In the second part of this review, we address the known roles of TSPAN6 and how this protein was shown to participate in synaptic transmission and in amyloid precursor protein secretion, which may contribute to Alzheimer's disease pathology. We conclude this review by discussing the anti-inflammatory effect of TSPAN6.

LYST controls the biogenesis of the endosomal compartment required for secretory lysosome function.

Chediak-Higashi syndrome (CHS) is caused by mutations in the gene encoding LYST protein, the function of which remains poorly understood. Prominent features of CHS include defective secretory lysosome exocytosis and the presence of enlarged, lysosome-like organelles in several cell types. In order to get further insight into the role of LYST in the biogenesis and exocytosis of cytotoxic granules, we analyzed cytotoxic T lymphocytes (CTLs) from patients with CHS. Using confocal microscopy and correlative light electron microscopy, we showed that the enlarged organelle in CTLs is a hybrid compartment that contains proteins components from recycling-late endosomes and lysosomes. Enlargement of cytotoxic granules results from the progressive clustering and then fusion of normal-sized endolysosomal organelles. At the immunological synapse (IS) in CHS CTLs, cytotoxic granules have limited motility and appear docked while nevertheless unable to degranulate. By increasing the expression of effectors of lytic granule exocytosis, such as Munc13-4, Rab27a and Slp3, in CHS CTLs, we were able to restore the dynamics and the secretory ability of cytotoxic granules at the IS. Our results indicate that LYST is involved in the trafficking of the effectors involved in exocytosis required for the terminal maturation of perforin-containing vesicles into secretory cytotoxic granules.

TSPAN7, effector of actin nucleation required for dendritic cell-mediated transfer of HIV-1 to T cells.

Dendritic cells (DCs) have essential roles in early detection of pathogens and activation of both innate and adaptive immune responses. Whereas human DCs are resistant to productive HIV-1 replication, they have a unique ability to take up virus and transmit it efficiently to T lymphocytes. By doing that, HIV-1 may evade, at least in part, the first line of defense of the immune system, exploiting DCs instead to facilitate rapid infection of a large pool of immune cells. While performing an shRNA screen in human primary monocyte-derived DCs, to gain insights into this cell biological process, we discovered the role played by tetraspanin-7 (TSPAN7). This member of the tetraspanin family appears to be a positive regulator of actin nucleation and stabilization, through the ARP2/3 complex. By doing so, TSPAN7 limits HIV-1 endocytosis and maintains viral particles on actin-rich dendrites for an efficient transfer toward T lymphocytes. While studying the function of TSPAN7 in the control of actin nucleation, we also discovered the existence in DCs of two opposing forces at the plasma membrane: actin nucleation, a protrusive force which seems to counterbalance actomyosin contraction.

Inherited defects in lymphocyte cytotoxic activity.

The granule-dependent cytotoxic activity of lymphocytes plays a critical role in the defense against virally infected cells and tumor cells. The importance of this cytotoxic pathway in immune regulation is evidenced by the severe and often fatal condition, known as hemophagocytic lymphohistiocytic syndrome (HLH) that occurs in mice and humans with genetically determined impaired lymphocyte cytotoxic function. HLH manifests as the occurrence of uncontrolled activation of T lymphocytes and macrophages infiltrating multiple organs. In this review, we focus on recent advances in the characterization of effectors regulating the release of cytotoxic granules, and on the role of this cytotoxic pathway in lymphocyte homeostasis and immune surveillance. Analysis of the mechanisms leading to the occurrence of hemophagocytic syndrome designates gamma-interferon as an attractive therapeutic target to downregulate uncontrolled macrophage activation, which sustains clinical and biological features of HLH.

PIK3CA inhibition in models of proliferative glomerulonephritis and lupus nephritis.

Proliferative glomerulonephritis is a severe condition often leading to kidney failure. There is a significant lack of effective treatment for these disorders. Here, following the identification of a somatic PIK3CA gain-of-function mutation in podocytes of a patient, we demonstrate using multiple genetically engineered mouse models, single-cell RNA sequencing and spatial transcriptomics the crucial role played by this pathway for proliferative glomerulonephritis development by promoting podocyte proliferation, dedifferentiation and inflammation. Additionally, we show that alpelisib, a PI3Kα inhibitor, improves glomerular lesions and kidney function in different mouse models of proliferative glomerulonephritis and lupus nephritis by targeting podocytes. Surprisingly, we determined that pharmacological inhibition of PI3Kα affects B and T lymphocyte population in lupus nephritis mouse models with decrease in the production of proinflammatory cytokines, autoantibodies and glomerular complement deposition, which are all characteristic features of PI3K delta (PI3Kδ) inhibition, the primary PI3K isoform expressed in lymphocytes. Importantly, PI3Kα inhibition does not impact lymphocyte function under normal conditions. These findings were then confirmed in human lymphocytes isolated from patients with active lupus nephritis. In conclusion, we demonstrate the major role played by PI3Kα in proliferative glomerulonephritis and show that in this condition, alpelisib acts on both podocytes and the immune system.

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.

Trained immunity as a possible newcomer in autoinflammatory and autoimmune diseases pathophysiology.

Autoimmune disorders have been well characterized over the years and many pathways-but not all of them-have been found to explain their pathophysiology. Autoinflammatory disorders, on the other hand, are still hiding most of their molecular and cellular mechanisms. During the past few years, a newcomer has challenged the idea that only adaptive immunity could display memory response. Trained immunity is defined by innate immune responses that are faster and stronger to a second stimulus than to the first one, being the same or not. In response to the trained immunity inducer, and through metabolic and epigenetic changes of hematopoietic stem and progenitor cells in the bone marrow that are transmitted to their cellular progeny (peripheral trained immunity), or directly of tissue-resident cells (local innate immunity), innate cells responsiveness and functions upon stimulation are improved in the long-term. Innate immunity can be beneficial, but it could also be detrimental when maladaptive. Here, we discuss how trained immunity could contribute to the physiopathology of autoimmune and autoinflammatory diseases.

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.

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.

A newly identified isoform of Slp2a associates with Rab27a in cytotoxic T cells and participates to cytotoxic granule secretion.

Cytotoxic T lymphocytes (CTLs) and natural killer cells help control infections and tumors via a killing activity that is mediated by the release of cytotoxic granules. Granule secretion at the synapse formed between the CTL and the target cell leads to apoptosis of the latter. This process involves polarization of the CTL's secretory machinery and cytotoxic granules. The small GTPase Rab27a and the hMunc13-4 protein have been shown to be required for both granule maturation and granule docking and priming at the immunologic synapse. Using a tandem affinity purification technique, we identified a previously unknown hematopoietic form of Slp2a (Slp2a-hem) and determined that it is a specific effector of the active form of Rab27a. This interaction occurs in vivo in primary CTLs. We have shown that (1) Rab27a recruits Slp2a-hem on vesicular structures in peripheral CTLs and (2) following CTL-target cell conjugate formation, the Slp2a-hem/Rab27a complex colocalizes with perforin-containing granules at the immunologic synapse, where it binds to the plasma membrane through its C2 domains. The overexpression of a dominant-negative form of Slp2a-hem markedly impaired exocytosis of cytotoxic granules-indicating that Slp2a is required for cytotoxic granule docking at the immunologic synapse.

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.

Staphylococcus aureus Impairs the Function of and Kills Human Dendritic Cells via the LukAB Toxin.

Staphylococcus aureus is a human pathogen responsible for high morbidity and mortality worldwide. Recurrent infections with this bacterium are common, suggesting that S. aureus thwarts the development of sterilizing immunity. S. aureus strains that cause disease in humans produce up to five different bicomponent toxins (leukocidins) that target and lyse neutrophils, innate immune cells that represent the first line of defense against S. aureus infections. However, little is known about the role of leukocidins in blunting adaptive immunity. Here, we explored the effects of leukocidins on human dendritic cells (DCs), antigen-presenting cells required for the development of adaptive immunity. Using an ex vivo infection model of primary human monocyte-derived dendritic cells, we found that S. aureus, including strains from different clonal complexes and drug resistance profiles, effectively kills DCs despite efficient phagocytosis. Although all purified leukocidins could kill DCs, infections with live bacteria revealed that S. aureus targets and kills DCs primarily via the activity of leukocidin LukAB. Moreover, using coculture experiments performed with DCs and autologous CD4+ T lymphocytes, we found that LukAB inhibits DC-mediated activation and proliferation of primary human T cells. Taken together, the data determined in the study reveal a novel immunosuppressive strategy of S. aureus whereby the bacterium blunts the development of adaptive immunity via LukAB-mediated injury of DCs.IMPORTANCE Antigen-presenting cells such as dendritic cells (DCs) fulfill an indispensable role in the development of adaptive immunity by producing proinflammatory cytokines and presenting microbial antigens to lymphocytes to trigger a faster, specific, and long-lasting immune response. Here, we studied the effect of Staphylococcus aureus toxins on human DCs. We discovered that the leukocidin LukAB hinders the development of adaptive immunity by targeting human DCs. The ability of S. aureus to blunt the function of DCs could help explain the high frequency of recurrent S. aureus infections. Taken together, the results from this study suggest that therapeutically targeting the S. aureus leukocidins may boost effective innate and adaptive immune responses by protecting innate leukocytes, enabling proper antigen presentation and T cell activation.

Quantitative Measurements of HIV-1 and Dextran Capture by Human Monocyte-derived Dendritic Cells (MDDCs).

The aim of this protocol is to describe how to measure and quantify the amount of HIV-1 particles and dextran molecules internalized in human monocyte derived dendritic cells (MDDCs), using three different techniques: flow cytometry, quantitative PCR and confocal microscopy. BACKGROUND: This protocol was developed in order to assess the changes of HIV-1 internalization upon disruption of actin nucleation in human monocyte derived dendritic cells. Following a shRNA screen to identify genes important for HIV-1 transfer from dendritic cells to T cells, we observed that a disruption of actin nucleation leads to a switch from actin rich dendrites to blebs, due to an excess of actomyosin contraction. As a consequence, a decrease of HIV-1 transfer and an increase of HIV-1 internalization due to bleb retraction-driven macropinocytosis were observed. We concluded that effectors of actin nucleation and stabilization were key to maintain HIV-1 on actin-rich dendrites and to limit its endocytosis, for efficient transfer to T lymphocytes (Menager and Littman, 2016).

Munc18-2 deficiency causes familial hemophagocytic lymphohistiocytosis type 5 and impairs cytotoxic granule exocytosis in patient NK cells.

Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous autosomal recessive immune disorder characterized by the occurrence of uncontrolled activation of lymphocytes and macrophages infiltrating multiple organs. Disease-causing mutations in the perforin (PRF1; also known as FHL2), Munc13-4 (UNC13D; also known as FHL3), and syntaxin-11 (STX11; also known as FHL4) genes have been identified in individuals with FHL. These genes all encode proteins involved in the cytotoxic activity of lymphocytes. Here, we show that the gene encoding syntaxin-binding protein 2 (Munc18-2; official gene symbol STXBP2) is mutated in another subset of patients with FHL (designated by us as "FHL5"). Lymphoblasts isolated from these patients had strongly decreased STXBP2 protein expression, and NK cells exhibited impaired cytotoxic granule exocytosis, a defect that could be overcome by ectopic expression of wild-type STXBP2. Furthermore, we provide evidence that syntaxin-11 is the main partner of STXBP2 in lymphocytes, as its expression required the presence of STXBP2. Our work shows that STXBP2 deficiency causes FHL5. These data indicate that STXBP2 is required at a late step of the secretory pathway for the release of cytotoxic granules by binding syntaxin 11, another component of the intracellular membrane fusion machinery.

Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4.

Cytotoxic T lymphocytes and natural killer cells exert their cytotoxic activity through the polarized secretion of cytotoxic granules at the immunological synapse. Rab27a and hMunc13-4 are critical effectors of the exocytosis of cytotoxic granules. Here we show that the cytotoxic function of lymphocytes requires the cooperation of two types of organelles: the lysosomal cytotoxic granule and the endosomal 'exocytic vesicle'. Independently of Rab27a, hMunc13-4 mediated the assembly of Rab11(+) recycling and Rab27(+) late endosomal vesicles, constituting a pool of vesicles destined for regulated exocytosis. It also primed cytotoxic granule fusion, possibly through interaction with active Rab27a. Cytotoxic T lymphocyte-target cell recognition induced rapid polarization of both types of organelles, which coalesced near the cell-cell contact area. Our data provide insight into the regulation of the generation and release of cytotoxic granules by effector cytotoxic T lymphocytes and natural killer cells.