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. 2013 Jul 1;210(7):1311-29.
doi: 10.1084/jem.20112615. Epub 2013 Jun 3.

Therapeutic targeting of NOTCH signaling ameliorates immune-mediated bone marrow failure of aplastic anemia

Justine E Roderick  1 , Gabriela Gonzalez-Perez, Christina Arieta Kuksin, Anushka Dongre, Emily R Roberts, Janani Srinivasan, Chester Andrzejewski Jr, Abdul H Fauq, Todd E Golde, Lucio Miele, Lisa M Minter
Affiliations

Therapeutic targeting of NOTCH signaling ameliorates immune-mediated bone marrow failure of aplastic anemia

Justine E Roderick et al. J Exp Med. .

Abstract

Severe aplastic anemia (AA) is a bone marrow (BM) failure (BMF) disease frequently caused by aberrant immune destruction of blood progenitors. Although a Th1-mediated pathology is well described for AA, molecular mechanisms driving disease progression remain ill defined. The NOTCH signaling pathway mediates Th1 cell differentiation in the presence of polarizing cytokines, an action requiring enzymatic processing of NOTCH receptors by γ-secretase. Using a mouse model of AA, we demonstrate that expression of both intracellular NOTCH1(IC) and T-BET, a key transcription factor regulating Th1 cell differentiation, was increased in spleen and BM-infiltrating T cells during active disease. Conditionally deleting Notch1 or administering γ-secretase inhibitors (GSIs) in vivo attenuated disease and rescued mice from lethal BMF. In peripheral T cells from patients with untreated AA, NOTCH1(IC) was significantly elevated and bound to the TBX21 promoter, showing NOTCH1 directly regulates the gene encoding T-BET. Treating patient cells with GSIs in vitro lowered NOTCH1(IC) levels, decreased NOTCH1 detectable at the TBX21 promoter, and decreased T-BET expression, indicating that NOTCH1 signaling is responsive to GSIs during active disease. Collectively, these results identify NOTCH signaling as a primary driver of Th1-mediated pathogenesis in AA and may represent a novel target for therapeutic intervention.

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Figures

Figure 1.
Figure 1.
Induction of BMF in AA mice recapitulates human disease. (A) BM cellularity (top left), weight loss (top right), and peripheral pancytopenia (bottom) at day 17 after disease induction for control mice (open boxes; n = 9) or AA mice (closed boxes; n = 11). (B) Representative hematoxylin and eosin staining of sternum from one AA mouse (bottom) compared with one irradiation control (top). Bars, 200 µm. (C) The relative expression of Ifng, Tnf, Tbx21, Gzmb, Prf1, and Pf4 in T cells isolated from spleens and BM of control and AA mice was determined by real-time PCR and normalized to naive T cells isolated from irradiation controls (n = 3–5 samples of pooled T cells, with each sample generated from four to eight mice). Data represent the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired Student’s t test.
Figure 2.
Figure 2.
NOTCH1 is increased during immune-mediated BMF. Flow cytometric detection of NOTCH1IC was validated using conventional immunoblotting methods. (A) CD4+ and CD8+ T cells from C57BL/6 mice were left unstimulated (first lane) or stimulated for 48 h with anti-CD3ε plus anti-CD28 in the absence (second lane) or presence (third lane) of GSIs. Whole cell lysates were generated from one half of the sample, separated by SDS-PAGE, and probed with clone N1A (top), stripped, and reprobed with Val1744 (middle), both of which recognize NOTCH1IC. Blots were stripped again then reprobed with anti–β-actin to verify equal loading. Data are representative of three independent replicates. (B) The other half of the sample was used to assess NOTCH1IC by flow cytometry using anti-NOTCH1IC, clone N1A, in total T cells (left), CD4+ T cells (middle), and CD8+ T cells (right). Data are representative of three independent replicates. (C) Percentages of NOTCH1IC-expressing cells in spleens and BM of control and AA mice were determined by flow cytometry; a representative histogram shows NOTCH1IC expression in CD4+ and CD8+ T cells isolated from naive spleens and from spleens and BM of mice with BMF (n = 9–11 mice/group). (D) Expression of Notch1, Notch2, Notch3, and Hes1 in T cells isolated from control spleens and from spleens and BM of AA mice was determined by real-time PCR (n = 3–5 samples, each pooled from four to eight mice). (E and F) Percent positive (E) and MFI (F) of NOTCH1IC (n = 9–11)-, NOTCH2 (n = 4)-, and NOTCH3 (n = 4)-expressing CD4+ and CD8+ T cells from spleens and BM of control and AA mice. (G) Representative histograms show NOTCH1IC, NOTCH2, and NOTCH3 expression in CD4+ and CD8+ T cells isolated from naive spleens and from spleens and BM of mice with BMF (n = 4–11 mice/group, as noted in F). Data represent the mean ± SEM. *, P < 0.05; ***, P < 0.001.
Figure 3.
Figure 3.
Abrogating NOTCH1 signaling reduces expression of signature proinflammatory proteins. (A) Schematic of experimental approach for generating and evaluating N1−/− mice. (B) Relative expression of Notch1 in T cells isolated from the spleens of N1−/− mice was determined by real-time PCR and normalized to naive T cells isolated from control littermates (data are the mean ± SEM of at least three replicates). (C–F) CD4+ and CD8+ T cells from C57BL/6 mice were treated with vehicle only (DMSO) or with the GSI IL-CHO (GSI) before being stimulated for 72 h with anti-CD3ε plus anti-CD28. CD4+ and CD8+ T cells from N1−/− mice were stimulated for 72 h with anti-CD3ε plus anti-CD28. After 72 h, the level of T-BET (C) and GRANZYME B (D) was determined. (E and F) After a further 5-h restimulation with anti-CD3ε, in the presence of GolgiPlug, IFN-γ was determined by intracellular staining and flow cytometric methods (for C–F, data represent the mean ± SEM of at least three independent replicates). (G–J) Human CD4+ and CD8+ T cells were treated with vehicle only (DMSO), with the NS-GSI JLK-6 (NS-GSI), or with the NOTCH-inhibiting GSI IL-CHO (GSI) before being stimulated for 72 h with anti-CD3ε plus anti-CD28, under Th1-polarizing conditions. After 72 h, expression of NOTCH1IC (G and H), T-BET (I), and GRANZYME B (J) were determined by flow cytometry (for G–J, data are the mean ± SEM of at least three replicates). *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired Student’s t test.
Figure 4.
Figure 4.
Conditionally deleting Notch1 ameliorates disease in AA mice. (A) Representative hematoxylin and eosin staining of BM from one AA mouse each whose BMF was induced with cre control, polyIC control, cre + polyIC control, or Notch1 conditional KO (N1−/−) splenocytes. Bars, 200 µm. (B and C) BM cellularity (B) and percentages of BM-infiltrating T cells (C) were determined in AA mice induced with N1−/− splenocytes (n = 10) and compared with AA mice induced with cre control (n = 4), polyIC control (n = 6), or cre + polyIC control (n = 4) splenocytes. (D) Flow cytometric analysis of NOTCH1IC in T cells isolated from the BM of cre control (n = 4), polyIC control (n = 6), cre + polyIC control (n = 4), or N1−/− mice after treatment with polyIC. (E) Kaplan–Meier survival estimates of AA mice induced with N1−/− splenocytes (n = 12) compared with animals induced with cre control (n = 6), polyIC control (n = 6), or cre + polyIC control (n = 7) splenocytes. Data represent the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired Student’s t test; log-rank test for survival estimates.
Figure 5.
Figure 5.
GSI treatment attenuates lethal BMF in AA mice. (A) Representative hematoxylin and eosin staining of BM from one control chow–fed AA mouse (top right) compared with BM from one irradiation control mouse (top left) or from one each, GSI-treated AA mouse (bottom). Bars, 200 µm. (B) BM cellularity, weight change, and peripheral white and red blood cell counts were assessed in AA mice left untreated (n = 11–14) or treated with vehicle alone (DMSO; n = 4) or with GSIs administered in rodent chow, beginning 14 d before BMF induction (n = 12–22), or by i.p. injection, beginning 3 d before BMF induction (n = 5). (C) Percentages of BM-infiltrating CD4+ and CD8+ T cells were quantified in untreated (n = 9), vehicle-treated (DMSO; n = 4), and GSI-treated animals (n = 5–22). (D) IFN-γ (left) and TNF (right) were measured in the plasma of control (n = 9), AA mice (n = 11–14), and AA mice receiving GSI in rodent chow (n = 12–22). (E) NOTCH1IC in BM-infiltrating T cells (n = 11–22 mice/condition) was determined by flow cytometry; representative histograms of NOTCH1IC staining within BM-infiltrating CD4+ and CD8+ T cells from one control and one GSI-treated AA mouse. (F) Kaplan–Meier survival estimates for AA mice fed control chow or GSI chow (P < 0.001, as determined by the log-rank test). Data represent the mean ± SEM (n = 4–22 animals). *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired Student’s t test.
Figure 6.
Figure 6.
Therapeutic administration of GSIs prolongs survival of AA mice. (A) Representative hematoxylin and eosin staining of BM from one AA mouse each whose treatment with vehicle only (DMSO; top) or GSIs (bottom) was begun 5 d after BMF induction. Bars, 200 µm. (B–E) BM cellularity (B), weight change (C), and circulating white (D) and red cells (E) were determined in vehicle-only–treated AA mice (n = 5) and GSI-treated (i.p.) AA mice (n = 5) beginning 5 d after disease induction. (F) Percentages of BM-infiltrating T cells were determined for control (γIR + DMSO [n = 4] and γIR + GSI [n = 4]), vehicle-treated (BMF + DMSO; n = 5), and GSI-treated (BMF + GSI; n = 5) AA mice. (G) Kaplan–Meier survival estimates for AA mice fed control chow or GSI chow beginning 5 d after disease induction (P = 0.002, log-rank test). Data represent the mean ± SEM (n = 4 to 8 mice/group). **, P < 0.01; ***, P < 0.001; unpaired Student’s t test.
Figure 7.
Figure 7.
NOTCH1IC is increased in PBMCs of patients with untreated AA and is functionally active. (A and B) Flow cytometric analyses of NOTCH1IC in CD4+ (A) and CD8+ (B) T cells from healthy controls (n = 6) and patients with AA (n = 9) who had not received prior IST. Percentages of NOTCH1IC-positive cells are indicated on the left-hand y axes, whereas NOTCH1IC protein expression is indicated by MFI on the right-hand y axes. (C) Expression of NOTCH1 and NOTCH-regulated genes HES1, TBX21, CDKN1A, NRARP, IFNG, TNF, DTX1, and PTCRA in PBMCs from healthy controls (n = 4–5) and AA patients (n = 6) was determined by real-time PCR. Data represent the mean ± SEM. *, P < 0.05; unpaired Student’s t test with Welch’s correction applied when variances were significantly different.
Figure 8.
Figure 8.
NOTCH1 regulates Th1-associated molecules through its direct regulation of the TBX21 promoter. Peripheral T cells from AA patients (n = 3–6) were treated with vehicle only (DMSO) or with 40 µM GSIs and then stimulated in vitro for 72 h. (A–F) CD4+ T cells were analyzed by intracellular staining and flow cytometry for expression of NOTCH1IC (A), T-BET (C), and IFN-γ (E); CD8+ T cells were analyzed for expression of NOTCH1IC (B), GRANZYME B (D), and IFN-γ (F); representative histograms from one patient each are shown to the right of collated data for each protein evaluated. (G) Schematic representation of the TBX21 promoter showing relative location of CSL-binding sites and regions amplified by primer sets #1 and #2 (not shown to scale). (H) Representative negative image of agarose gel showing two amplified regions of the TBX21 promoter immunoprecipitated using antibodies specific for NOTCH1 and CSL from PBMCs of one healthy control (left) and one AA patient who had not received prior IST (right). (I) Quantification of band intensities of three healthy control samples and two AA patient samples subjected to ChIP. (J) Representative negative image of agarose gel showing two amplified regions of the TBX21 promoter immunoprecipitated using antibodies specific for NOTCH1 and CSL from PBMCs of one Th1-polarized healthy control (left) and one stimulated AA patient (right) after 48 h of treatment with vehicle only (DMSO) or with GSIs. (K) Quantification of band intensities of three Th1-polarized healthy control samples and three stimulated AA patient samples treated with vehicle only (DMSO) or with GSIs for 48 h before ChIP. Data represent the mean ± SEM of three independent replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired Student’s t test.
Figure 9.
Figure 9.
Extended GSI treatment does not compromise hematopoietic stem cell engraftment or long-term hematopoiesis. (A–D) Donor mice were fed control (open bars) or GSI chow (gray bars) for 6 mo before harvesting and transplanting total BM cells into lethally irradiated recipients (first reconstitution; n = 1–8 mice/group). Some mice were fed GSI chow for an additional 4 mo (diagonal stripe bars) before all mice (n = 4) were harvested and total BM cells from each group were transplanted into a second group of lethally irradiated recipients (second reconstitution; n = 1–8 mice/group). Mice were followed for an additional 2 mo. At each harvest, BM cellularity (A), distribution of stem and progenitor cells (B), lineage markers (C), and clonogenic potential (D) were assessed. Data represent the mean ± SEM.
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References

    1. Adler S.H., Chiffoleau E., Xu L., Dalton N.M., Burg J.M., Wells A.D., Wolfe M.S., Turka L.A., Pear W.S. 2003. Notch signaling augments T cell responsiveness by enhancing CD25 expression. J. Immunol. 171:2896–2903 - PubMed
    1. Auderset F., Schuster S., Coutaz M., Koch U., Desgranges F., Merck E., MacDonald H.R., Radtke F., Tacchini-Cottier F. 2012. Redundant Notch1 and Notch2 signaling is necessary for IFNγ secretion by T helper 1 cells during infection with Leishmania major. PLoS Pathog. 8:e1002560 10.1371/journal.ppat.1002560 - DOI - PMC - PubMed
    1. Bagby G.C., Meyers G. 2009. Myelodysplasia and acute leukemia as late complications of marrow failure: future prospects for leukemia prevention. Hematol. Oncol. Clin. North Am. 23:361–376 10.1016/j.hoc.2009.01.006 - DOI - PMC - PubMed
    1. Bloom M.L., Wolk A.G., Simon-Stoos K.L., Bard J.S., Chen J., Young N.S. 2004. A mouse model of lymphocyte infusion-induced bone marrow failure. Exp. Hematol. 32:1163–1172 10.1016/j.exphem.2004.08.006 - DOI - PubMed
    1. Chen J. 2005. Animal models for acquired bone marrow failure syndromes. Clin. Med. Res. 3:102–108 10.3121/cmr.3.2.102 - DOI - PMC - PubMed

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