Investigating the importance of B cells and antibodies during Trichuris muris infection using the IgMi mouse

From Springer Nature via Jisc Publications RouterHistory: received 2020-04-24, rev-recd 2020-06-24, registration 2020-07-17, accepted 2020-07-17, pub-electronic 2020-08-10, online 2020-08-10, pub-print 2020-09Publication status: PublishedFunder: Indonesian endowment fund for education, Phd StudentshipFunder: Biotechnology and Biological Sciences Research Council UK, PhD StudenshipFunder: Medical Research Council UK; Grant(s): MR/NO22661/1 to KJEFunder: Biotechnology and Biological Sciences Research Council UK; Grant(s): BB/P018157/1 to KJEAbstract: The IgMi mouse has normal B cell development; its B cells express an IgM B cell receptor but cannot class switch or secrete antibody. Thus, the IgMi mouse offers a model system by which to dissect out antibody-dependent and antibody-independent B cell function. Here, we provide the first detailed characterisation of the IgMi mouse post-Trichuris muris (T. muris) infection, describing expulsion phenotype, cytokine production, gut pathology and changes in T regulatory cells, T follicular helper cells and germinal centre B cells, in addition to RNA sequencing (RNA seq) analyses of wild-type littermates (WT) and mutant B cells prior to and post infection. IgMi mice were susceptible to a high-dose infection, with reduced Th2 cytokines and elevated B cell-derived IL-10 in mesenteric lymph nodes (MLN) compared to controls. A low-dose infection regime revealed IgMi mice to have significantly more apoptotic cells in the gut compared to WT mice, but no change in intestinal inflammation. IL-10 levels were again elevated. Collectively, this study showcases the potential of the IgMi mouse as a tool for understanding B cell biology and suggests that the B cell plays both antibody-dependent and antibody-independent roles post high- and low-dose T. muris infection. Key messages: During a high-dose T. muris infection, B cells are important in maintaining the Th1/Th2 balance in the MLN through an antibody-independent mechanism. High levels of IL-10 in the MLN early post-infection, and the presence of IL-10-producing B cells, correlates with susceptibility to T. muris infection. B cells maintain gut homeostasis during chronic T. muris infection via an antibody-dependent mechanism

Deep Learning Application in Security and Privacy - Theory and Practice:A Position Paper

Technology is shaping our lives in a multitude of ways. This is fuelled by a technology infrastructure, both legacy and state of the art, composed of a heterogeneous group of hardware, software, services and organisations. Such infrastructure faces a diverse range of challenges to its operations that include security, privacy, resilience, and quality of services. Among these, cybersecurity and privacy are taking the centre-stage, especially since the General Data Protection Regulation (GDPR) came into effect. Traditional security and privacy techniques are overstretched and adversarial actors have evolved to design exploitation techniques that circumvent protection. With the ever-increasing complexity of technology infrastructure, security and privacy-preservation specialists have started to look for adaptable and flexible protection methods that can evolve (potentially autonomously) as the adversarial actor changes its techniques. For this, Artificial Intelligence (AI), Machine Learning (ML) and Deep Learning (DL) were put forward as saviours. In this paper, we look at the promises of AI, ML, and DL stated in academic and industrial literature and evaluate how realistic they are. We also put forward potential challenges a DL based security and privacy protection technique has to overcome. Finally, we conclude the paper with a discussion on what steps the DL and the security and privacy-preservation community have to take to ensure that DL is not just going to be hype, but an opportunity to build a secure, reliable, and trusted technology infrastructure on which we can rely on for so much in our lives

Ezh2 is essential for the generation of functional yolk sac derived erythro-myeloid progenitors

From Springer Nature via Jisc Publications RouterHistory: received 2020-05-19, accepted 2021-10-27, registration 2021-11-08, collection 2021-12, pub-electronic 2021-12-02, online 2021-12-02Publication status: PublishedFunder: Kay Kendall Leukaemia Fund (KKLF); doi: https://doi.org/10.13039/501100000402; Grant(s): KKL811Funder: RCUK | Medical Research Council (MRC); doi: https://doi.org/10.13039/501100000265; Grant(s): MR/L006340/1, MC_UU_12009/5Funder: Cancer Research UK (CRUK); doi: https://doi.org/10.13039/501100000289; Grant(s): C42639/A26988, C5759/A27412Funder: Bloodwise; doi: https://doi.org/10.13039/501100007903; Grant(s): 19014Abstract: Yolk sac (YS) hematopoiesis is critical for the survival of the embryo and a major source of tissue-resident macrophages that persist into adulthood. Yet, the transcriptional and epigenetic regulation of YS hematopoiesis remains poorly characterized. Here we report that the epigenetic regulator Ezh2 is essential for YS hematopoiesis but dispensable for subsequent aorta–gonad–mesonephros (AGM) blood development. Loss of EZH2 activity in hemogenic endothelium (HE) leads to the generation of phenotypically intact but functionally deficient erythro-myeloid progenitors (EMPs), while the generation of primitive erythroid cells is not affected. EZH2 activity is critical for the generation of functional EMPs at the onset of the endothelial-to-hematopoietic transition but subsequently dispensable. We identify a lack of Wnt signaling downregulation as the primary reason for the production of non-functional EMPs. Together, our findings demonstrate a critical and stage-specific role of Ezh2 in modulating Wnt signaling during the generation of EMPs from YS HE

RUNX1B Expression is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis

The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into "pro-erythroid" and "pro-megakaryocyte" populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to establish a pre-leukemic environment

HDAC1 and HDAC2 Modulate TGF-β Signaling during Endothelial-to-Hematopoietic Transition

The first hematopoietic stem and progenitor cells are generated during development from hemogenic endothelium (HE) through trans-differentiation. The molecular mechanisms underlying this endothelial-to-hematopoietic transition (EHT) remain poorly understood. Here, we explored the role of the epigenetic regulators HDAC1 and HDAC2 in the emergence of these first blood cells in vitro and in vivo. Loss of either of these epigenetic silencers through conditional genetic deletion reduced hematopoietic transition from HE, while combined deletion was incompatible with blood generation. We investigated the molecular basis of HDAC1 and HDAC2 requirement and identified TGF-β signaling as one of the pathways controlled by HDAC1 and HDAC2. Accordingly, we experimentally demonstrated that activation of this pathway in HE cells reinforces hematopoietic development. Altogether, our results establish that HDAC1 and HDAC2 modulate TGF-β signaling and suggest that stimulation of this pathway in HE cells would be beneficial for production of hematopoietic cells for regenerative therapies

<i>Runx1-P2-hCD4</i>^<i>+</i> CMPs have enriched multilineage output.

<p><b>A-B.</b> Expression of <i>Runx1-P1-GFP</i> and <i>P2-hCD4</i> in <i>P1-GFP</i>::<i>P2-hCD4/+</i> E12.5, E13.5 and E14.5 FL CD41^- CD150⁺ CMPs. A. Representative FACS plots. B. Quantitation of the proportions of <i>P1</i>^<i>-</i> <i>P2</i>^<i>-</i>, <i>P1</i>^<i>+</i> <i>P2</i>^<i>-</i>, <i>P1</i>^<i>+</i> <i>P2</i>^<i>+</i> and <i>P1</i>^<i>-</i> <i>P2</i>^<i>+</i> CMPs as a percentage of total live red blood cell lysed fetal liver cells. N = 3 <b>C.</b> Differential CFU-C activity of wild type, <i>P2-hCD4</i>^<i>-</i> and <i>P2-hCD4</i>^<i>+</i> E14.5 FL CD41^- CD150⁺ CMPs. N = 7 <b>D-E.</b> Lineage output of day 7 OP9 co-cultured wild type, <i>P2-hCD4</i>^<i>-</i> and <i>P2-hCD4</i>^<i>+</i> CD41^- CD150⁺ CMPs. D. Representative FACS plots of CD11b, GR1, TER119 and CD41. E. Proportion of granulocyte/monocyte, erythroid and megakaryocyte cells. N = 7 <b>F-G.</b> Lineage output of single cultured <i>P2</i>^<i>-</i> and <i>P2</i>^<i>+</i> CMPs in OP9 co-cultures. F. Table of plating efficiency. G. Ternary plots displaying proportions of granulocyte/monocyte, erythroid and megakaryocyte cells in each positive well for wild type, <i>P2-hCD4</i>^<i>-</i> and <i>P2-hCD4</i>^<i>+</i> CD41^- CD150⁺ CMPs. <b>H-I.</b> Short-term (14 hours) differentiation of <i>P2</i>^<i>-</i> and <i>P2</i>^<i>+</i> CD41^- CD150⁺ CMPs in pro-myeloid liquid culture. H. Representative FACS plots of CD16/32/CD34 (top) and CD150/CD41 (middle and bottom) expression in the LK gate of cultured CMPs. I. Proportions of immunophenotypic MkPs, CD41^- CD150^- MEPs, CD41^- CD150⁺ MEPs, GMPs, CD41^- CD150^- CMPs and CD41^- CD150⁺ CMPs in short-term cultures. N = 3.</p

Distinct subsets within the classically defined CMP and MEP compartments can be segregated on the basis of CD55, CD150 and CD31 expression.

<p><b>A-B.</b> Proposed models of fetal liver myeloid progenitor hierarchy. A. In wild type fetal liver, commitment to the erythroid lineage correlates with downregulation of the <i>Runx1b</i> and <i>Gfi1</i> TFs, plus the cell surface markers CD150, CD31, CD45 and CD48. B. RUNX1-null fetal liver hematopoietic progenitors have impaired Mk and erythroid differentiation (dark blue bars), whereas RUNX1C-null (<i>P1-MRIPV)</i> hematopoietic progenitors have impaired megakaryocyte specification, displaying enhanced erythroid and granulocyte/monocyte commitment (light blue arrows).</p