Stable lines and clones of long-term proliferating normal, genetically unmodified murine common lymphoid progenitors.

Common lymphoid progenitors (CLPs) differentiate to T and B lymphocytes, dendritic cells, natural killer cells, and innate lymphoid cells. Here, we describe culture conditions that, for the first time, allow the establishment of lymphoid-restricted, but uncommitted, long-term proliferating CLP cell lines and clones from a small pool of these cells from normal mouse bone marrow, without any genetic manipulation. Cells from more than half of the cultured CLP clones could be induced to differentiate to T, B, natural killer, dendritic, and myeloid cells in vitro. Cultured, transplanted CLPs transiently populate the host and differentiate to all lymphoid subsets, and to myeloid cells in vivo. This simple method to obtain robust numbers of cultured noncommitted CLPs will allow studies of cell-intrinsic and environmentally controlled lymphoid differentiation programs. If this method can be applied to human CLPs, it will provide new opportunities for cell therapy of patients in need of myeloid-lymphoid reconstitution

Longitudinal intravital imaging of the femoral bone marrow reveals plasticity within marrow vasculature.

The bone marrow is a central organ of the immune system, which hosts complex interactions of bone and immune compartments critical for hematopoiesis, immunological memory, and bone regeneration. Although these processes take place over months, most existing imaging techniques allow us to follow snapshots of only a few hours, at subcellular resolution. Here, we develop a microendoscopic multi-photon imaging approach called LIMB (longitudinal intravital imaging of the bone marrow) to analyze cellular dynamics within the deep marrow. The approach consists of a biocompatible plate surgically fixated to the mouse femur containing a gradient refractive index lens. This microendoscope allows highly resolved imaging, repeatedly at the same regions within marrow tissue, over months. LIMB reveals extensive vascular plasticity during bone healing and steady-state homeostasis. To our knowledge, this vascular plasticity is unique among mammalian tissues, and we expect this insight will decisively change our understanding of essential phenomena occurring within the bone marrow

Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo.

In T lymphocytes, expression of miR-148a is induced by T-bet and Twist1, and is specific for pro-inflammatory Th1 cells. In these cells, miR-148a inhibits the expression of the pro-apoptotic protein Bim and promotes their survival. Here we use sequence-specific cholesterol-modified oligonucleotides against miR-148a (antagomir-148a) for the selective elimination of pro-inflammatory Th1 cells in vivo. In the murine model of transfer colitis, antagomir-148a treatment reduced the number of pro-inflammatory Th1 cells in the colon of colitic mice by 50% and inhibited miR-148a expression by 71% in the remaining Th1 cells. Expression of Bim protein in colonic Th1 cells was increased. Antagomir-148a-mediated reduction of Th1 cells resulted in a significant amelioration of colitis. The effect of antagomir-148a was selective for chronic inflammation. Antigen-specific memory Th cells that were generated by an acute immune reaction to nitrophenylacetyl-coupled chicken gamma globulin (NP-CGG) were not affected by treatment with antagomir-148a, both during the effector and the memory phase. In addition, antibody titers to NP-CGG were not altered. Thus, antagomir-148a might qualify as an effective drug to selectively deplete pro-inflammatory Th1 cells of chronic inflammation without affecting the protective immunological memory

IL-7 and immobilized Kit-ligand stimulate serum- and stromal cell-free cultures of precursor B-cell lines and clones.

Long-term proliferating, DH JH -rearranged mouse precursor B-cell lines have previously been established in serum- and IL-7-containing media from fetal liver, but not from bone marrow. Serum and stromal cells expose these pre-B cells to undefined factors, hampering accurate analyses of ligand-dependent signaling, which controls pre-B cell proliferation, survival, residence and migration. Here, we describe a novel serum-free, stromal cell-free culture system, which allows us to establish and maintain pre-B cells not only from fetal liver, but also from bone marrow with practically identical efficiencies in proliferation, cloning and differentiation. Surprisingly, recombinant kit-ligand, also called stem cell factor, produced as a kit-ligand-Fc fusion protein, suffices to replace stromal cells and serum, provided that it is presented to cultured pre-B cells in an optimal density in plate-bound, insolubilized, potentially crosslinking form. Additional recombinant CXCL12 and fibronectin have a minor influence on the establishment and maintenance of pre-B cell lines and clones from fetal liver, but are necessary to establish such cell lines from bone marrow

Stable lines and clones of long-term proliferating normal, genetically unmodified murine common lymphoid progenitors.

Common lymphoid progenitors (CLPs) differentiate to T and B lymphocytes, dendritic cells, natural killer cells, and innate lymphoid cells. Here, we describe culture conditions that, for the first time, allow the establishment of lymphoid-restricted, but uncommitted, long-term proliferating CLP cell lines and clones from a small pool of these cells from normal mouse bone marrow, without any genetic manipulation. Cells from more than half of the cultured CLP clones could be induced to differentiate to T, B, natural killer, dendritic, and myeloid cells in vitro. Cultured, transplanted CLPs transiently populate the host and differentiate to all lymphoid subsets, and to myeloid cells in vivo. This simple method to obtain robust numbers of cultured noncommitted CLPs will allow studies of cell-intrinsic and environmentally controlled lymphoid differentiation programs. If this method can be applied to human CLPs, it will provide new opportunities for cell therapy of patients in need of myeloid-lymphoid reconstitution

The miR-221/222 cluster regulates hematopoietic stem cell quiescence and multipotency by suppressing both Fos/AP-1/IEG pathway activation and stress-like differentiation to granulocytes.

Throughout life, hematopoietic stem cells (HSCs), residing in bone marrow (BM), continuously regenerate erythroid/megakaryocytic, myeloid, and lymphoid cell lineages. This steady-state hematopoiesis from HSC and multipotent progenitors (MPPs) in BM can be perturbed by stress. The molecular controls of how stress can impact hematopoietic output remain poorly understood. MicroRNAs (miRNAs) as posttranscriptional regulators of gene expression have been found to control various functions in hematopoiesis. We find that the miR-221/222 cluster, which is expressed in HSC and in MPPs differentiating from them, perturbs steady-state hematopoiesis in ways comparable to stress. We compare pool sizes and single-cell transcriptomes of HSC and MPPs in unperturbed or stress-perturbed, miR-221/222-proficient or miR-221/222-deficient states. MiR-221/222 deficiency in hematopoietic cells was induced in C57BL/6J mice by conditional vav-cre-mediated deletion of the floxed miR-221/222 gene cluster. Social stress as well as miR-221/222 deficiency, alone or in combination, reduced HSC pools 3-fold and increased MPPs 1.5-fold. It also enhanced granulopoisis in the spleen. Furthermore, combined stress and miR-221/222 deficiency increased the erythroid/myeloid/granulocytic precursor pools in BM. Differential expression analyses of single-cell RNAseq transcriptomes of unperturbed and stressed, proficient HSC and MPPs detected more than 80 genes, selectively up-regulated in stressed cells, among them immediate early genes (IEGs). The same differential single-cell transcriptome analyses of unperturbed, miR-221/222-proficient with deficient HSC and MPPs identified Fos, Jun, JunB, Klf6, Nr4a1, Ier2, Zfp36-all IEGs-as well as CD74 and Ly6a as potential miRNA targets. Three of them, Klf6, Nr4a1, and Zfp36, have previously been found to influence myelogranulopoiesis. Together with increased levels of Jun, Fos forms increased amounts of the heterodimeric activator protein-1 (AP-1), which is known to control the expression of the selectively up-regulated expression of the IEGs. The comparisons of single-cell mRNA-deep sequencing analyses of socially stressed with miR-221/222-deficient HSC identify 5 of the 7 Fos/AP-1-controlled IEGs, Ier2, Jun, Junb, Klf6, and Zfp36, as common activators of HSC from quiescence. Combined with stress, miR-221/222 deficiency enhanced the Fos/AP-1/IEG pathway, extended it to MPPs, and increased the number of granulocyte precursors in BM, inducing selective up-regulation of genes encoding heat shock proteins Hspa5 and Hspa8, tubulin-cytoskeleton-organizing proteins Tuba1b, Tubb 4b and 5, and chromatin remodeling proteins H3f3b, H2afx, H2afz, and Hmgb2. Up-regulated in HSC, MPP1, and/or MPP2, they appear as potential regulators of stress-induced, miR-221/222-dependent increased granulocyte differentiation. Finally, stress by serial transplantations of miR-221/222-deficient HSC selectively exhausted their lymphoid differentiation capacities, while retaining their ability to home to BM and to differentiate to granulocytes. Thus, miR-221/222 maintains HSC quiescence and multipotency by suppressing Fos/AP-1/IEG-mediated activation and by suppressing enhanced stress-like differentiation to granulocytes. Since miR-221/222 is also expressed in human HSC, controlled induction of miR-221/222 in HSC should improve BM transplantations

Human and Mouse Hematopoietic Stem Cells Are a Depot for Dormant <i>Mycobacterium tuberculosis</i>

<div><p>An estimated third of the world’s population is latently infected with <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), with no clinical signs of tuberculosis (TB), but lifelong risk of reactivation to active disease. The niches of persisting bacteria during latent TB infection remain unclear. We detect <i>Mtb</i> DNA in peripheral blood selectively in long-term repopulating pluripotent hematopoietic stem cells (LT-pHSCs) as well as in mesenchymal stem cells from latently infected human donors. In mice infected with low numbers of <i>Mtb</i>, that do not develop active disease we, again, find LT-pHSCs selectively infected with <i>Mtb</i>. In human and mouse LT-pHSCs <i>Mtb</i> are stressed or dormant, non-replicating bacteria. Intratracheal injection of <i>Mtb</i>-infected human and mouse LT-pHSCs into immune-deficient mice resuscitates <i>Mtb</i> to replicating bacteria within the lung, accompanied by signs of active infection. We conclude that LT-pHSCs, together with MSCs of <i>Mtb</i>-infected humans and mice serve as a hitherto unappreciated quiescent cellular depot for <i>Mtb</i> during latent TB infection.</p></div

Detection of <i>Mtb</i> infection in different organs and hematopoietic cells of mice day 28 p.i. by <i>Mtb</i> DNA PCR and <i>Mtb</i> CFU.

<p>C57BL/6 mice were infected with 10⁵ CFUs <i>Mtb</i> (H37Rv). <b>(A)</b> Quantification of <i>Mtb</i>-specific DNA by real-time TaqMan PCR using probes targeting <i>MPB64</i> and <i>IS6110</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169119#pone.0169119.s004" target="_blank">S4 Fig</a>) on genomic DNA of 10⁵ lung cells (n = 8), 10⁵ Gr1⁺, CD11c⁺, CD19⁺, Mac1⁺, NK1.1⁺, CD4⁺/8⁺ cells (n = 4; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169119#pone.0169119.s001" target="_blank">S1C Fig</a>), and 10³ LT-pHSCs, ST-pHSCs and MPPs (n = 16; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169119#pone.0169119.s001" target="_blank">S1B Fig</a>). <b>(B)</b> Quantification of <i>Mtb</i>-specific DNA by limiting dilutions using a single-target PCR for <i>IS6110</i> (n = 3; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169119#pone.0169119.s002" target="_blank">S2B Fig</a>). <b>(C)</b> Real-time SYBR green PCR using primers targeting <i>MPB64</i> (n = 4–8; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169119#pone.0169119.s004" target="_blank">S4 Fig</a>). Real-time PCRs were performed in 2 independent runs in technical triplicates and normalized to murine GAPDH. Known <i>Mtb</i> concentrations were used as reference. <b>(D)</b> CFU enumeration on Middlebrook 7H11 agar in cells of lung, spleen and thymus (n = 16). <b>(E)</b> CFU enumeration on Middlebrook 7H11 agar for Lin⁺ cell populations (n = 8). <b>(F)</b> CFU enumeration on Middlebrook 7H11 agar for hematopoietic progenitors (n = 16). Shown are data of 4 independent experiments. Data are shown as median + interquartile. *<i>P</i> ˂ 0.05, **<i>P</i> ˂ 0.005, ***<i>P</i> ˂ 0.0005, ****<i>P</i> ˂ 0.00005 by Mann-Whitney test.</p

A model of a long-term persisting niche for non-replicating <i>Mtb</i> bearing the risk of resuscitation of active <i>Mtb</i>.

<p>Model of LTBI where non-replicating <i>Mtb</i> reside, and perhaps move, between long-lived, resting hematopoietic and non-hematopoietic cells in hypoxic niches in bone marrow and in which actively replicating <i>Mtb</i> can be resuscitated leading to TB.</p

Intratracheal transfer of <i>Mtb</i> infected human and murine pHSCs leads to <i>Mtb</i> growth and increased cellularity into the lungs in transplanted hosts.

<p><b>(A)</b> Injection of Lin^-CD34⁺ and Lin⁺ cells from blood of IGRA⁺ human donors (Donor 12–14) and mouse LT-pHSCs from bone marrow 28 days p.i. into the trachea of <i>Rag2</i>^–/–<i>Il2rg</i>^/–mice (3 mice/population). Transfer of 10² CFUs <i>Mtb</i> was used as positive (n = 3), uninfected pHSCs and Lin⁺ cells of an IGRA⁻donor (Donor 3; n = 1) as negative, control. Recipients were analyzed after 3 weeks. <b>(B)</b> Monitoring of <i>Mtb</i> infection by TaqMan PCR using probes that target <i>MPB64</i> and <i>IS6110</i> together on genomic DNA of 10⁵ lung cells 3 weeks upon transfer. PCRs were performed in technical triplicates and normalized to murine GAPDH. <b>(C)</b> CFU <i>Mtb</i> growth on Middlebrook 7H11 agar in cells of lung, spleen, thymus and non-separated, 10⁵ bone marrow cells 3 weeks upon transfer (n = 3/population). Shown are data from 3 independent experiments. <b>(D)</b> Histopathology of representative lung sections 3 weeks upon transfer. Lungs were stained with hematoxylin/eosin, screened with 5×objectives and verified using a light microscope. Shown are representative data from 3 independent experiments. Data are shown as median + interquartile. Scale bar: 100 μm.</p