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

NVP-LDE225, a Potent and Selective SMOOTHENED Antagonist Reduces Melanoma Growth <i>In Vitro</i> and <i>In Vivo</i>

<div><p>Melanoma is one of the most aggressive cancers and its incidence is increasing worldwide. So far there are no curable therapies especially after metastasis. Due to frequent mutations in members of the mitogen-activated protein kinase (MAPK) signaling pathway, this pathway is constitutively active in melanoma. It has been shown that the SONIC HEDGEHOG (SHH)-GLI and MAPK signaling pathway regulate cell growth in many tumors including melanoma and interact with each other in the regulation of cell proliferation and survival.</p><p>Here we show that the SHH-GLI pathway is active in human melanoma cell lines as they express downstream target of this pathway <i>GLI1</i>. Expression of <i>GLI1</i> was significantly higher in human primary melanoma tissues harboring BRAF^V600E mutation than those with wild type BRAF. Pharmacologic inhibition of BRAF^V600E in human melanoma cell lines resulted in decreased expression of GLI1 thus demonstrating interaction of SHH-GLI and MAPK pathways. Inhibition of SHH-GLI pathway by the novel small molecule inhibitor of smoothened NVP-LDE225 was followed by inhibition of cell growth and induction of apoptosis in human melanoma cell lines, interestingly with both BRAF^V600E and BRAF^{Wild Type} status. NVP-LDE225 was potent in reducing cell proliferation and inducing tumor growth arrest <i>in vitro</i> and <i>in vivo</i>, respectively and these effects were superior to the natural compound cyclopamine.</p><p>Finally, we conclude that inhibition of SHH-GLI signaling pathway in human melanoma by the specific smoothened inhibitor NVP-LDE225 could have potential therapeutic application in human melanoma even in the absence of BRAF^V600E mutation and warrants further investigations.</p></div

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

The effect of NVP-LDE225 on human melanoma cell viability.

<p>Human melanoma cell lines LOX IMVI, MEWO, SK-MEL-2, UACC 257, WM 115 and MEL FH were treated with different concentrations of NVP-LDE225, cyclopamine or the vehicle (DMSO). Cell viability at specific time points was measured by MTT assay. Experiments were performed in triplicates. One representative experiment is shown. Mean values with SD are shown.</p

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

<i>In vivo</i> GLI1 expression after intratumoral administration of NVP-LDE225.

<p>LOX OMVI human melanoma cells were injected s.c into both flanks as mentioned above. Tumors were treated intratumorally on daily basis with vehicle (<b>A & B</b>) or NVP-LDE225 (<b>C & D</b>). Immunofluorescent microscopy of GLI1was performed on isolated tumor tissues. GLI1 staining was performed by overnight incubation of sections at 4°C with rabbit anti-human polyclonal Ab (<b>B & D</b>, NBP1-78259, Novus Biologicals, Littleton, CO) or isotype control (<b>A & C</b>) followed by an 1 hr-incubation with Alexa Fluor® 488 Donkey IgG, anti-rabbit (A21206, Invitrogen, Carlsbad, CA) at RT (green). Counterstaining of nuclei was performed with propidium iodide (red). Pictures were taken on a confocal laser-scanning microscope system (LSM 410; Zeiss). Yellow color corresponds to double positive (anti-GLI1 and propidium iodide) nuclear staining.</p

Numbers of <i>Mtb</i> DNA copies (<i>MPB64</i> qPCR) and CFUs in 10⁴ cells of different cell populations.

<p>Data are shown as median + interquartile (n = 4–8).</p

Human peripheral Lin^–CD34⁺, Lin^–CD34⁺CD38^lowCD90⁺, SP⁺ pHSCs as well as CD271⁺CD45^- MSCs of IGRA⁺ donors harbour <i>Mtb</i> DNA.

<p>Lin⁺, Lin^–CD34⁺, Lin^–CD34⁺CD38^lowCD90⁺, Lin^–CD34⁺CD38⁺CD90^–, Lin^–SP⁺ and Lin⁺ SP⁻cells (Donors 1, 6, 8, 9) were purified from blood of IGRA⁺ (n = 8) and IGRA⁻donors (n = 7; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169119#pone.0169119.s001" target="_blank">S1A Fig</a>). CD271⁺CD45^- MSCs (Donors 10 and 13) were purified from blood of IGRA⁺ donors (n = 2; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169119#pone.0169119.s001" target="_blank">S1B Fig</a>). CD1c⁺, CD14⁺, CD16⁺, CD4⁺/8⁺, CD15⁺, CD19⁺, and CD56⁺ cells were prepared from blood of IGRA⁺ donors (n = 3). Genomic DNA prepared from 10³ hematopoietic progenitors and MSCs as well as 10⁵ Lin⁺ cells from IGRA⁺ and IGRA^- donors were tested for the presence of <i>Mtb</i> DNA by PCR. <b>(A, E)</b> Quantification of <i>Mtb</i>-specific DNA by real-time TaqMan PCR using probes that target <i>MPB64</i> and <i>IS6110</i> together (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169119#pone.0169119.s004" target="_blank">S4 Fig</a>). <b>(B)</b> Genomic DNA of 10³ hematopoietic progenitors from IGRA⁺ and IGRA^- donors were tested by PCR for a DNA fragment present in <i>Mtb</i>, but not in BCG. <b>(C)</b> Quantification of <i>Mtb</i>-specific DNA by limiting dilutions using a single-target PCR for <i>IS6110</i> (Donor 13, 14; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169119#pone.0169119.s002" target="_blank">S2A Fig</a>). <b>(D, E)</b> Quantification of <i>Mtb</i>-specific DNA by real-time SYBR green PCR using primers that target <i>MPB64</i> alone (<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 human GAPDH. Known <i>Mtb</i> concentrations were used as reference. <b>(F)</b> CFU <i>Mtb</i> growth on Middlebrook 7H11 agar plates (n = 2–3). Data are shown as median + interquartile.</p

Murine and human pHSCs are infected with <i>Mtb</i> expressing dormancy genes.

<p><b>(A)</b> Expression analyses on RNA isolated from <i>Mtb</i>-infected mouse lung cells and purified LT-pHSCs (n = 8). <b>(B)</b> Expression analyses on RNA isolated from Lin^–CD34⁺ pHSCs from IGRA⁺ donors (n = 6) and <i>Mtb</i>-infected human monocytic leukemia cell line 96 h p.i. (n = 3). Expression analyses were done by real-time TaqMan PCR for <i>SigA</i>, <i>DosR</i>, <i>c-lat</i> and <i>hspX</i>. <i>SigA</i> was used as reference for <i>Mtb</i>. Real-time TaqMan PCRs were performed in 3 independent runs in technical triplicates. Data are shown as median + interquartile. *<i>P</i> ˂ 0.05, **<i>P</i> ˂ 0.005, ***<i>P</i> ˂ 0.005 by Mann-Whitney test.</p