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Busch K.,German Cancer Research Center | Klapproth K.,German Cancer Research Center | Barile M.,German Cancer Research Center | Flossdorf M.,German Cancer Research Center | And 7 more authors.
Nature | Year: 2015

Haematopoietic stem cells (HSCs) are widely studied by HSC transplantation into immune- and blood-cell-depleted recipients. Single HSCs can rebuild the system after transplantation. Chromosomal marking, viral integration and barcoding of transplanted HSCs suggest that very low numbers of HSCs perpetuate a continuous stream of differentiating cells. However, the numbers of productive HSCs during normal haematopoiesis, and the flux of differentiating progeny remain unknown. Here we devise a mouse model allowing inducible genetic labelling of the most primitive Tie2+ HSCs in bone marrow, and quantify label progression along haematopoietic development by limiting dilution analysis and data-driven modelling. During maintenance of the haematopoietic system, at least 30% or ∼5,000 HSCs are productive in the adult mouse after label induction. However, the time to approach equilibrium between labelled HSCs and their progeny is surprisingly long, a time scale that would exceed the mouse's life. Indeed, we find that adult haematopoiesis is largely sustained by previously designated "short-term" stem cells downstream of HSCs that nearly fully self-renew, and receive rare but polyclonal HSC input. By contrast, in fetal and early postnatal life, HSCs are rapidly used to establish the immune and blood system. In the adult mouse, 5-fluoruracil-induced leukopenia enhances the output of HSCs and of downstream compartments, thus accelerating haematopoietic flux. Label tracing also identifies a strong lineage bias in adult mice, with several-hundred-fold larger myeloid than lymphoid output, which is only marginally accentuated with age. Finally, we show that transplantation imposes severe constraints on HSC engraftment, consistent with the previously observed oligoclonal HSC activity under these conditions. Thus, we uncover fundamental differences between the normal maintenance of the haematopoietic system, its regulation by challenge, and its re-establishment after transplantation. HSC fate mapping and its linked modelling provide a quantitative framework for studying in situ the regulation of haematopoiesis in health and disease. © 2015 Macmillan Publishers Limited. All rights reserved.

Gomez-Pinilla P.J.,Catholic University of Leuven | Farro G.,Catholic University of Leuven | Di Giovangiulio M.,Catholic University of Leuven | Stakenborg N.,Catholic University of Leuven | And 8 more authors.
PLoS ONE | Year: 2014

Introduction: Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3Cre/+, devoid of mast cells but with intact Kit signaling. Design: The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. KitW-sh/W-sh and Cpa3Cre/+ mice, and by use of the mast cell stabilizer cromolyn. Results: KitW-sh/W-sh mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, Cpa3Cre/+ mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3Cre/+ mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (Cpa3+/+). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI. Conclusions: Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI. © 2014 Gomez-Pinilla et al.

Dhaeze T.,transnational University of Limburg | Stinissen P.,transnational University of Limburg | Liston A.,Autoimmune Genetics Laboratory | Liston A.,Catholic University of Leuven | Hellings N.,transnational University of Limburg
Autoimmunity Reviews | Year: 2015

Regulatory T cells (Tregs) are essential in maintaining tolerance to self. Several lines of evidence indicate that Tregs are functionally impaired in a variety of autoimmune diseases, leading to inefficient regulation of autoimmune T cells. Recent findings also suggest that Tregs are essential in controlling autoreactive B cells. The recently identified follicular regulatory T cell subset (TFR) is thought to regulate the production of autoantibodies in the germinal center (GC) response. Here we provide an update on the role of Tregs in controlling the GC response, and whether defective control over B cell tolerance contributes to autoimmunity. © 2015 Elsevier B.V.

Dooley J.,Autoimmune Genetics Laboratory | Dooley J.,Catholic University of Leuven | Linterman M.A.,University of Cambridge | Liston A.,Autoimmune Genetics Laboratory | Liston A.,Catholic University of Leuven
Immunological Reviews | Year: 2013

MicroRNAs are short, 19-24 nucleotide long, RNA molecules capable of regulating the longevity and, to a lesser extent, translation of messenger RNA (mRNA) species. The function of the microRNA network, and indeed, even that of individual microRNA species, can have profoundly different roles in even a single cell type as the microRNA/mRNA composition evolves. As the role of microRNA within T cells has come under increasing scrutiny, several distinct checkpoints have been demonstrated to have a particular reliance on microRNA regulation. MicroRNAs are arguably most important in T cells during the earliest and last stages in T-cell biology. The first stages of early thymic differentiation have a crucial reliance on the microRNA network, while later stages and peripheral homeostasis are largely, although not completely, microRNA-independent. The most profound effects on T cells are in the activation of effector and regulatory functions of conventional and regulatory T cells, where microRNA deficiency results in a near-complete loss of function. In this review, we focus on integrating the research on individual microRNA into a more global understanding of the function of the microRNA regulatory network in T cells. © 2013 John Wiley & Sons A/S.

Ucar O.,German Cancer Research Center | Tykocinski L.-O.,German Cancer Research Center | Tykocinski L.-O.,University of Heidelberg | Dooley J.,Autoimmune Genetics Laboratory | And 4 more authors.
European Journal of Immunology | Year: 2013

The establishment and maintenance of central tolerance depends to a large extent on the ability of medullary thymic epithelial cells to express a variety of tissue-restricted antigens, the so-called promiscuous gene expression (pGE). Autoimmune regulator (Aire) is to date the best characterised transcriptional regulator known to at least partially coordinate pGE. There is accruing evidence that the expression of Aire-dependent and -independent genes is modulated by higher order chromatin configuration, epigenetic modifications and post-transcriptional control. Given the involvement of microRNAs (miRNAs) as potent post-transcriptional modulators of gene expression, we investigated their role in the regulation of pGE in purified mouse and human thymic epithelial cells (TECs). Microarray profiling of TEC subpopulations revealed evolutionarily conserved cell type and differentiation-specific miRNA signatures with a subset of miRNAs being significantly upregulated during terminal medullary thymic epithelial cell differentiation. The differential regulation of this subset of miRNAs was correlated with Aire expression and some of these miRNAs were misexpressed in the Aire knockout thymus. In turn, the specific absence of miRNAs in TECs resulted in a progressive reduction of Aire expression and pGE, affecting both Aire-dependent and -independent genes. In contrast, the absence of miR-29a only affected the Aire-dependent gene pool. These findings reveal a mutual interdependence of miRNA and Aire. © 2013 The Authors. European Journal of Immunology published byWiley-VCH Verlag GmbH & Co. KGaA Weinheim.

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