Role of syntaxin-11 and munc18-2 in lymphocyte cytotoxic granule exocytosis

Mutations in genes required for the exocytosis of cytotoxic granules by NK cells and cytotoxic T lymphocytes are associated with early-onset familial hemophagocytic lymphohistiocytosis (FHL). In this project, we examined how certain missense mutations in genes encoding syntaxin-11 and Munc18-2 abolish exocytosis and cause disease. Furthermore, we dissected the role of another protein suspected to play a role in cytotoxic granule exocytosis, VAMP8. In the first study, we characterized an FHL5 patient (Munc18-2 p.Q432X and p.S545L) who developed Hodgkin lymphoma with underlying Epstein-Barr virus (EBV) infection. The tissue displayed high infiltrates of cytotoxic T lymphocytes responsive to EBV-derived peptides, yet the cells had dramatically reduced Munc18- 2 protein levels and were unable to undergo cytotoxic granule exocytosis. The patient is an important example of how Munc18-2 missense mutations impairing exocytosis can lead to late-onset HLH and, due to decreased immunosurveillance, might result in cancer. In the second study, we examined the pathophysiological mechanism underlying an N-terminal syntaxin-11 mutation (syntaxin-11 p.L58P) associated with FHL4 in three unrelated patients. These patients displayed defective cytotoxic granule exocytosis despite the functionally important SNARE domain of syntaxin-11 being intact. As the p.L58 is an evolutionary conserved amino acid, we hypothesized that the mutation interrupts interactions with Munc18-2. Further, we determined whether other conserved N-terminal syntaxin-11 residues also contribute to Munc18-2 binding. We found that the interaction is dependent on both an intact syntaxin-11 N-terminal peptide as well as Habc domain because mutations in either completely abolished binding to Munc18-2. It is thus plausible that syntaxin-11, analogous to the syntaxin- 1 / Munc18-1 interaction, employs distinct binding modes to different domains of Munc18-2. The interruption of the syntaxin-11 / Munc18-2 interaction could explain the reduced syntaxin-11 expression levels. Lastly, cytotoxic granule exocytosis is dependent on several proteins being part of the fusion complex between a cytotoxic granule and plasma membrane, yet the v-SNARE mediating granule fusion remains elusive. In the third study, we suspected VAMP8 to play a key role in this fusion process. However, VAMP8 did not localize to cytotoxic granules but instead to recycling endosomes. Upon T cell receptor stimulation, recycling endosomes were recruited to the immune synapse and fused with the plasma membrane. As VAMP8 knockdown blocked cytotoxic granule release, we hypothesized that VAMP8+ recycling endosomes might deliver an important component for the cytotoxic fusion machinery to the plasma membrane. Indeed, recycling endosomes transported syntaxin-11 to the plasma membrane, with VAMP8 knockdown hampering syntaxin-11 delivery. In summary, these data provide a deeper understanding of FHL and the molecular mechanisms of cytotoxic granule exocytosis. We describe a possible link between FHL and cancer which may have diagnostic, prognostic and treatment implications for other FHL patients. Further, we show how N-terminal syntaxin-11 mutations can cause disease, with data hinting towards a meticulous series of interaction modes necessary for syntaxin-11 maintenance and cytotoxic granule secretion. Finally, we find that VAMP8 delivers syntaxin-11 to the immunological synapse in human cytotoxic lymphocytes, demonstrating an unexpected role of recycling endosomes in cytotoxic granule exocytosis

Development of classical Hodgkin’s lymphoma in an adult with biallelic STXBP2 mutations

Experimental model systems have delineated an important role for cytotoxic lymphocytes in the immunosurveillance of cancer. In humans, perforin-deficiency has been associated with occurrence of hematologic malignancies. Here, we describe an Epstein-Barr virus-positive classical Hodgkin's lymphoma in a patient harboring biallelic mutations in STXBP2, a gene required for exocytosis of perforin-containing lytic granules and associated with familial hemophagocytic lymphohistocytosis. Cytotoxic T lymphocytes were found infiltrating the tumor, and a high frequency of Epstein-Barr virus-specific cytotoxic T lymphocytes were detected in peripheral blood. However, lytic granule exocytosis and cytotoxicity by cytotoxic T lymphocytes, as well as natural killer cells, were severely impaired in the patient. Thus, the data suggest a link between defective lymphocyte exocytosis and development of lymphoma in STXBP2-deficient patients. Therefore, with regards to treatment of familial hemophagocytic lymphohistocytosis patients with mutations in genes required for lymphocyte exocytosis, it is important to consider both the risks of hemophagocytic lymphohistocytosis and malignancy.Swedish Research CouncilSwedish Cancer FoundationSwedish Children’s Cancer FoundationHistiocytosis AssociationClas Groschinsky’s Memorial FundJeanssons FoundationÅke Olsson Foundation for Hematological ResearchÅke Wiberg FoundationKarolinska Institute Research FoundationStockholm County Council (ALF project)Publishe

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Isolation of murine bone marrow by centrifugation or flushing for the analysis of hematopoietic cells – a comparative study

Investigation of the bone marrow as the main compartment of hematopoiesis is critical in many research fields. Here, we adapted a centrifugation-based method for the isolation of murine bone marrow and compared it to the traditional flushing method. Analysis of primary hematopoietic stem cells, immune cells, and megakaryocytes revealed a comparable distribution of cellular (sub)populations. Furthermore, in vitro differentiated megakaryocytes displayed unaltered proplatelet formation. Strikingly, bone marrow isolation by centrifugation was considerably faster than the flushing method and significantly increased the cell yield. Thus, the centrifugation-based isolation method is highly suitable for the study of murine bone marrow cells

Knockout of LASP1 in CXCR4 expressing CML cells promotes cell persistence, proliferation and TKI resistance

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the constitutively active BCR‐ABL tyrosine kinase. The LIM and SH3 domain protein 1 (LASP1) has recently been identified as a novel BCR‐ABL substrate and is associated with proliferation, migration, tumorigenesis and chemoresistance in several cancers. Furthermore, LASP1 was shown to bind to the chemokine receptor 4 (CXCR4), thought to be involved in mechanisms of relapse. In order to identify potential LASP1‐mediated pathways and related factors that may help to further eradicate minimal residual disease (MRD), the effect of LASP1 on processes involved in progression and maintenance of CML was investigated. The present data indicate that not only overexpression of CXCR4, but also knockout of LASP1 contributes to proliferation, reduced apoptosis and migration as well as increased adhesive potential of K562 CML cells. Furthermore, LASP1 depletion in K562 CML cells leads to decreased cytokine release and reduced NK cell‐mediated cytotoxicity towards CML cells. Taken together, these results indicate that in CML, reduced levels of LASP1 alone and in combination with high CXCR4 expression may contribute to TKI resistance

Surgical site infection after gastrointestinal surgery in children : an international, multicentre, prospective cohort study

Introduction Surgical site infection (SSI) is one of the most common healthcare-associated infections (HAIs). However, there is a lack of data available about SSI in children worldwide, especially from low-income and middle-income countries. This study aimed to estimate the incidence of SSI in children and associations between SSI and morbidity across human development settings. Methods A multicentre, international, prospective, validated cohort study of children aged under 16 years undergoing clean-contaminated, contaminated or dirty gastrointestinal surgery. Any hospital in the world providing paediatric surgery was eligible to contribute data between January and July 2016. The primary outcome was the incidence of SSI by 30 days. Relationships between explanatory variables and SSI were examined using multilevel logistic regression. Countries were stratified into high development, middle development and low development groups using the United Nations Human Development Index (HDI). Results Of 1159 children across 181 hospitals in 51 countries, 523 (45 center dot 1%) children were from high HDI, 397 (34 center dot 2%) from middle HDI and 239 (20 center dot 6%) from low HDI countries. The 30-day SSI rate was 6.3% (33/523) in high HDI, 12 center dot 8% (51/397) in middle HDI and 24 center dot 7% (59/239) in low HDI countries. SSI was associated with higher incidence of 30-day mortality, intervention, organ-space infection and other HAIs, with the highest rates seen in low HDI countries. Median length of stay in patients who had an SSI was longer (7.0 days), compared with 3.0 days in patients who did not have an SSI. Use of laparoscopy was associated with significantly lower SSI rates, even after accounting for HDI. Conclusion The odds of SSI in children is nearly four times greater in low HDI compared with high HDI countries. Policies to reduce SSI should be prioritised as part of the wider global agenda.Peer reviewe