An increase in LSK ⁻ CD150⁻ cells reflects apoptosis of HSCs.

<p>(<b>A</b>) The LSK⁻ cell population is basically CD150-negative. LSK⁺ (Lin⁻Sca1⁺c-Kit⁺) cells and LSK⁻ (Lin⁻Sca1⁺c-Kit⁻) cells from bone marrow of B6 mice were stained with CD150, and analyzed by FACS. (<b>B</b>) High apoptotic rates in bone marrow LSK⁻CD150⁻ cells. LSK⁺ and LSK⁻ cells from bone marrow of B6 mice were stained with CD150, further labeled with 7AAD and Annexin V, and analyzed by FACS. Apoptotic rate for each cell population was indicated. (<b>C</b>) An increase in LSK⁻CD150⁻ cells reflects apoptosis of HSCs. B6 mice were treated with irradiation or 5-FU for different days as indicated. LSK⁺ and LSK⁻ cells from bone marrow of B6 mice were stained with CD150, were further labeled with 7AAD and Annexin V, and analyzed by FACS.</p

Irradiation and 5-FU treatment promote cellular transition of LSK to LSK⁻ cells.

<p>(<b>A</b>) Lethal irradiation causes an increase in the percentage of apoptotic LSK⁻ cells. Mice were treated by two split doses of 550-cGy gamma irradiation (separated by 3 hours), and the apoptotic cells (7AAD⁺Annexin V⁺) in the LSK⁻ population were analyzed by FACS at different time points (n = 4). **: <i>p</i><0.01. (<b>B</b>) Total number and percentages of LSK and LSK⁻ cells in bone marrow of lethally irradiated WT mice were determined at different time points (n = 4). *: <i>p</i><0.05; **: <i>p</i><0.01. (<b>C</b>) Total number and percentages of LSK and LSK⁻ in bone marrow of 5-FU treated WT mice at different time points. The mice were treated with 5-FU (200 mg/kg) by intravenous injection, and the percentages of LSK and LSK⁻ cells were monitored at different time points (n = 4). *: <i>p</i><0.05; **: <i>p</i><0.01. (<b>D</b>) The apoptotic rate of LSK⁻ cells in 5-FU treated mice. WT mice were treated with 5-FU, and apoptotic rate of LSK⁻ cells were monitored at different time points (n = 4). *: <i>p</i><0.05; **: <i>p</i><0.01.</p

Long-term treatment of CML mice with imatinib fails to promote the transition of LSCs to BCR-ABL expressing LSK⁻ cells.

<p>(<b>A</b>) Apoptotic rates between LSCs and BCR-ABL expressing LSK⁻ cells in CML mice were compared. At day 14 after induction of CML, apoptotic rate in GFP⁺LSK⁻ was slightly higher than that of LSCs. (<b>B</b>) The percentage of GFP⁺LSK⁻ cells was increased after imatinib treatment for 12 days, beginning at day 8 after induction of CML (100 mg/kg). (<b>C</b>) The percentages of LSCs and GFP⁺LSK⁻ cells in bone marrow of CML mice were compared after imatinib treatment for 55 days. *: <i>p</i><0.05; **: <i>p</i><0.01. (<b>D</b>) <i>Icsbp</i> expression was increased in bone marrow cells of mice treated with lethal irradiation. Bone marrow cells were collected from lethally irradiated mice at day 0, 2 and 4 after irradiation. Total RNA was extracted and <i>Icsbp</i> expression was determined by real-time PCR. *: <i>p</i><0.05. (<b>E</b>) <i>Icsbp</i> expression in bone marrow cells was determined in 5-FU treated mice. Bone marrow cells were collected from 5-FU treated mice at day 0, 2, 4 and 6 after the treatment, and total RNA was extracted and <i>Icsbp</i> expression was determined by real-time PCR. *: <i>p</i><0.05; **: <i>p</i><0.01. (<b>F</b>) Proposed molecular model for the control of the transition of LSK/LSCs and LSK⁻ cells. The balance between LSK and LSK⁻ cells is controlled by the <i>Icsbp</i>/<i>Lyn</i> pathway in normal hematopoiesis and by <i>Alox5</i> in BCR-ABL induced CML.</p

The <i>Icsbp</i>-<i>Lyn</i> pathway controls the transition of LSK to LSK⁻ cells.

<p>(<b>A</b>) The percentages and numbers of LSK, LSK⁻ and Lin⁻Sca1⁻c-Kit⁺ were compared between WT and <i>Icsbp^−/−</i> mice (n = 4 for each group). *: <i>p</i><0.05; **: <i>p</i><0.01. (<b>B</b>) The percentages of LSK, LSK⁻ and Lin⁻Sca1⁻c-Kit⁺ were compared between WT and BXH2 mice. (<b>C</b>) Cell cycle analysis of progenitor and stem cells in <i>Icsbp^−/−</i> mice. Bone marrow cells were stained with Hoechst blue, and DNA contents, represented by the percentages of progenitor and stem cells in the S+G2M phase of the cell cycle, were examined by FACS (n = 4). **: <i>p</i><0.01. (<b>D</b>) Apoptotic rates of progenitor and stem cells in <i>Icsbp^−/−</i> mice. The cells were labeled with 7AAD and Annexin V, and the percentages of progenitor and stem cells were determined by FACS (n = 4). **: <i>p</i><0.01. (<b>E</b>) <i>Lyn</i> expression in bone marrow cells was compared between WT and <i>Icsbp^−/−</i> mice. Bone marrow cells were collected from WT and <i>Icsbp</i>^−/− mice, respectively, and <i>Lyn</i> expression was detected by real-time PCR (n = 3). *: <i>p</i><0.05. (<b>F</b>) The percentages of LSK and LSK⁻ were compared between WT and <i>Lyn^−/−</i> mice. <i>Lyn</i> deletion caused an increase in the percentage of LSK and decrease in the percentage of LSK⁻ cells. (<b>G</b>) Overexpression of <i>Lyn</i> caused an increase in the LSK⁻ population. <i>Lyn</i>^−/− bone marrow cells were transduced with <i>MSCV-GFP</i> or <i>MSCV-Lyn-GFP</i> retrovirus, followed by transplantation of the transduced cells into lethally irradiated WT recipient mice. At day 60 after the transplantation, the percentages of LSK and LSK⁻ cells were determined by FACS.</p

Suppression of LSCs through disturbing the <i>Alox5</i> or <i>Hsp90</i> pathways is associated with enhanced transition of LSCs to BCR-ABL-expressing LSK⁻ cells.

<p>(<b>A</b>) BCR-ABL⁺LSK cells give rise to LSK⁻ cells in CML mice. 1×10⁶ bone marrow cells (containing BCR-ABL⁺LSK cells) from primary CML mice were transplanted into each secondary recipient mouse. 15 days later, bone marrow cells were analyzed by FACS for the presence of BCR-ABL⁺LSK and LSK⁻ cells. (<b>B</b>) <i>Alox5</i> deletion enhanced the transition of LSCs (GFP⁺LSK) to GFP⁺ LSK⁻ cells. At day 20 after induction of CML, the percentage of LSCs and GFP⁺ LSK⁻ cells in bone marrow of recipients of BCR-ABL transduced WT donor marrow cells were determined (top panel), and the percentage of GFP⁺LSK⁻ cells was lower than that of LSCs. In recipients of BCR-ABL transduced <i>Alox5^−/−</i> donor marrow cells, the percentage of GFP⁺ LSK⁻ cells became higher than that of LSCs (middle panel). At day 90, the percentage of GFP⁺LSK⁻ cells reached as high as 50% (bottom panel) (n = 4 for each group). **: <i>p</i><0.01. (<b>C</b>) Inhibition of <i>Alox5</i> function by Zileuton enhanced the transition of LSCs to BCR-ABL expressing Lin⁻Sca1⁺c-Kit⁻ cells. CML mice were treated with Zileuton (300 mg/kg body weight, twice a day) for 90 days and the percentages of LSCs and GFP⁺LSK⁻ were monitored by FACS (n = 4 for each group). **: <i>p</i><0.01. (<b>D</b>) The percentages of LSCs and BCR-ABL-T315I expressing Lin⁻Sca1⁺c-Kit⁻ cells were measured by FACS after IPI-504 treatment. Bone marrow cells from CML mice were cultured under a stem cell culture condition in the presence of IPI-504 (100 nM) for 6 days, and the percentages of LSCs and GFP⁺LSK⁻ were determined by FACS. *: <i>p</i><0.05.</p

The LSK⁻ cell population is derived from LSK cells and provides an apoptotic cellular pathway for LSK cells.

<p>(<b>A</b>) LSK (Lin⁻Sca1⁺c-Kit⁺) cells (1×10³) and LSK⁻ (Lin⁻Sca1⁺c-Kit⁻) cells (1×10⁴) were sorted from bone marrow cells of CD45.1⁺ wild type (WT) mice by FACS, and transferred into lethal irradiated CD45.2⁺ WT recipient mice. 12 weeks later, bone marrow cells were collected and stained with antibodies for CD45.1, lineage markers, Sca−1 and c-Kit. Peripheral blood cells were also collected and stained with antibodies for CD45.1, Gr-1, Mac−1 and B220. (<b>B</b>) LSK cells were sorted from bone marrow of C56BL/6 (B6) mice by FACS and were irradiated (2000 cGy, once) or treated with 5-FU <i>in vitro</i>. The cells were cultured for 24 hours and then analyzed by FACS. (<b>C</b>) Apoptotic rates of progenitor and stem cells in vivo. Progenitor and stem cells were labeled with 7AAD and Annexin V, and analyzed by FACS (n = 5). **: <i>p</i><0.01. (<b>D</b>) Cell cycle analysis of progenitor and stem cells in vivo. The cells were stained with Hoechst Blue, and the cells in the S+G2M phase were analyzed by FACS (n = 5). **: <i>p</i><0.01.</p

Longitudinal changes in the serum levels of (a) tumor necrosis factor-α (TNF-α), (b) interleukin-1β (IL-1β), and (c) IL-6 in 176 healthy infants over the first year of life.

<p>Symbols and error bars are mean±SEM plotted at the mean ages of the study visits.</p

An early islet transcriptional signature is associated with local inflammation in autoimmune diabetes

Identifying the early islet cellular processes of autoimmune type 1 diabetes (T1D) in humans is challenging given the absence of symptoms during this period and the inaccessibility of the pancreas for sampling. Here, we study temporal events in pancreatic islets in LEW.1WR1 rats, in which autoimmune diabetes can be induced with virus infection, by performing transcriptional analysis of islets harvested during the pre-diabetic period. Single-cell RNA-Seq and differential expression analyses of islets from pre-diabetic rats reveal subsets of β and a cells under stress as evidenced by heightened expression, over time, of a transcriptional signature characterized by interferon-stimulated genes, chemokines including Cxcl10, major histocompatibility class I, and genes for the ubiquitin-proteasome system. Mononuclear phagocytes show increased expression of inflammatory markers. RNA-in situ hybridization of rat pancreatic tissue defines the spatial distribution of Cxcl10+ β and a cells and their association with CD8+ T cell infiltration, a hallmark of insulitis and islet destruction. Our studies define early islet transcriptional events during immune cell recruitment to islets and reveal spatial associations between stressed β and a cells and immune cells. Insights into such early processes can assist in the development of therapeutic and prevention strategies for T1D.</p