FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3041120131> ?p ?o ?g. }

• W3041120131 endingPage "55" @default.
• W3041120131 startingPage "42" @default.
• W3041120131 abstract "•Chronic stimulation of TLR2/6, but not TLR1/2, accelerates leukemic transformation in the NHD13 mouse model of MDS.•Conversely, loss of TLR6, but not TLR1, extends survival in NHD13 mice.•TLR2/6 agonist treatment leads to cell-autonomous expansion of premalignant HSPCs.•TLR2/6 agonist treatment is associated with activation of Myc and mTORC1 in HPSCs. Toll-like receptor 2 (TLR2) expression is increased on hematopoietic stem and progenitor cells (HSPCs) of patients with myelodysplastic syndromes (MDS), and enhanced TLR2 signaling is thought to contribute to MDS pathogenesis. Notably, TLR2 heterodimerizes with TLR1 or TLR6, and while high TLR2 is associated with lower-risk disease, high TLR6, but not TLR1, correlates with higher-risk disease. This raises the possibility of heterodimer-specific effects of TLR2 signaling in MDS, and in the work described here, we tested the effects of specific modulation of TLR1/2 versus TLR2/6 signaling on premalignant HSPCs. Indeed, chronic stimulation of TLR2/6, but not TLR1/2, accelerates leukemic transformation in the NHD13 mouse model of MDS, and conversely, loss of TLR6, but not TLR1, slows this process. TLR2/6 stimulation expands premalignant HSPCs, and chimeric mouse studies revealed that cell-autonomous signaling contributes to this expansion. Finally, TLR2/6 stimulation is associated with an enrichment of Myc and mTORC1 activities. While Myc inhibition partially suppressed the TLR2/6 agonist-mediated expansion of premalignant HSPCs, inhibition of mTORC1 exacerbated it, suggesting that these pathways play opposite roles in regulating the effects of TLR2/6 ligation on HSPCs. Together, these data reveal heterodimer-specific effects of TLR2 signaling on premalignant HSPCs, with TLR2/6 signaling promoting their expansion and leukemic transformation. Toll-like receptor 2 (TLR2) expression is increased on hematopoietic stem and progenitor cells (HSPCs) of patients with myelodysplastic syndromes (MDS), and enhanced TLR2 signaling is thought to contribute to MDS pathogenesis. Notably, TLR2 heterodimerizes with TLR1 or TLR6, and while high TLR2 is associated with lower-risk disease, high TLR6, but not TLR1, correlates with higher-risk disease. This raises the possibility of heterodimer-specific effects of TLR2 signaling in MDS, and in the work described here, we tested the effects of specific modulation of TLR1/2 versus TLR2/6 signaling on premalignant HSPCs. Indeed, chronic stimulation of TLR2/6, but not TLR1/2, accelerates leukemic transformation in the NHD13 mouse model of MDS, and conversely, loss of TLR6, but not TLR1, slows this process. TLR2/6 stimulation expands premalignant HSPCs, and chimeric mouse studies revealed that cell-autonomous signaling contributes to this expansion. Finally, TLR2/6 stimulation is associated with an enrichment of Myc and mTORC1 activities. While Myc inhibition partially suppressed the TLR2/6 agonist-mediated expansion of premalignant HSPCs, inhibition of mTORC1 exacerbated it, suggesting that these pathways play opposite roles in regulating the effects of TLR2/6 ligation on HSPCs. Together, these data reveal heterodimer-specific effects of TLR2 signaling on premalignant HSPCs, with TLR2/6 signaling promoting their expansion and leukemic transformation. The myelodysplastic syndromes are a group of hematopoietic stem and progenitor cell (HSPC) disorders characterized by abnormal hematopoiesis and a high risk of transformation to acute leukemia [1Jacobs A Myelodysplastic syndromes: pathogenesis, functional abnormalities, and clinical implications.J Clin Pathol. 1985; 38: 1201-1217Crossref PubMed Scopus (102) Google Scholar,2Galton DA The myelodysplastic syndromes.Clin Lab Haematol. 1984; 6: 99-112Crossref PubMed Scopus (40) Google Scholar]. Numerous prior studies have described enhanced innate immune signaling and, in particular, increased toll-like receptor (TLR) signaling in the CD34+ stem and progenitor cells of patients with MDS [3Wei Y Dimicoli S Bueso-Ramos C et al.Toll-like receptor alterations in myelodysplastic syndrome.Leukemia. 2013; 27: 1832-1840Crossref PubMed Scopus (123) Google Scholar, 4Starczynowski DT Kuchenbauer F Argiropoulos B et al.Identification of miR-145 and miR-146a as mediators of the 5q– syndrome phenotype.Nat Med. 2010; 16: 49-58Crossref PubMed Scopus (535) Google Scholar, 5Dimicoli S Wei Y Bueso-Ramos C et al.Overexpression of the toll-like receptor (TLR) signaling adaptor MYD88, but lack of genetic mutation, in myelodysplastic syndromes.PLoS One. 2013; 8: e71120Crossref PubMed Scopus (56) Google Scholar, 6Maratheftis CI Andreakos E Moutsopoulos HM Voulgarelis M Toll-like receptor-4 is up-regulated in hematopoietic progenitor cells and contributes to increased apoptosis in myelodysplastic syndromes.Clin Cancer Res. 2007; 13: 1154-1160Crossref PubMed Scopus (112) Google Scholar, 7Velegraki M Papakonstanti E Mavroudi I et al.Impaired clearance of apoptotic cells leads to HMGB1 release in the bone marrow of patients with myelodysplastic syndromes and induces TLR4-mediated cytokine production.Haematologica. 2013; 98: 1206-1215Crossref PubMed Scopus (49) Google Scholar, 8Varney ME Niederkorn M Konno H et al.Loss of Tifab, a del(5q) MDS gene, alters hematopoiesis through derepression of Toll-like receptor-TRAF6 signaling.J Exp Med. 2015; 212: 1967-1985Crossref PubMed Scopus (75) Google Scholar, 9Varney ME Melgar K Niederkorn M Smith MA Barreyro L Starczynowski DT Deconstructing innate immune signaling in myelodysplastic syndromes.Exp Hematol. 2015; 43: 587-598Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Monlish DA Bhatt ST Schuettpelz LG The role of toll-like receptors in hematopoietic malignancies.Front Immunol. 2016; 7: 390Crossref PubMed Scopus (66) Google Scholar, 11Zeng Q Shu J Hu Q et al.Apoptosis in human myelodysplastic syndrome CD34+ cells is modulated by the upregulation of TLRs and histone H4 acetylation via a beta-arrestin 1 dependent mechanism.Exp Cell Res. 2016; 340: 22-31Crossref PubMed Scopus (17) Google Scholar]. This enhanced TLR signaling is thought to contribute to the ineffective hematopoiesis and cell death that are prominent features of lower-risk disease [12Sallman DA Cluzeau T Basiorka AA List A Unraveling the pathogenesis of MDS: the NLRP3 inflammasome and pyroptosis drive the MDS phenotype.Front Oncol. 2016; 6: 151Crossref PubMed Scopus (66) Google Scholar]. Among the TLR family members, RNA levels of TLR2 have been reported to be increased in the majority of patients with MDS, with the highest expression in patients with low-risk disease, by International Prognostic Scoring System [3Wei Y Dimicoli S Bueso-Ramos C et al.Toll-like receptor alterations in myelodysplastic syndrome.Leukemia. 2013; 27: 1832-1840Crossref PubMed Scopus (123) Google Scholar,11Zeng Q Shu J Hu Q et al.Apoptosis in human myelodysplastic syndrome CD34+ cells is modulated by the upregulation of TLRs and histone H4 acetylation via a beta-arrestin 1 dependent mechanism.Exp Cell Res. 2016; 340: 22-31Crossref PubMed Scopus (17) Google Scholar]. Given its pervasive overexpression and the fact that chronic TLR stimulation is known to impair normal hematopoietic stem cell function [13Esplin BL Shimazu T Welner RS et al.Chronic exposure to a TLR ligand injures hematopoietic stem cells.J Immunol. 2011; 186: 5367-5375Crossref PubMed Scopus (231) Google Scholar, 14Herman AC Monlish DA Romine MP Bhatt ST Zippel S Schuettpelz LG Systemic TLR2 agonist exposure regulates hematopoietic stem cells via cell-autonomous and cell-non-autonomous mechanisms.Blood Cancer J. 2016; 6: e437Crossref PubMed Scopus (34) Google Scholar, 15Takizawa H Fritsch K Kovtonyuk LV et al.Pathogen-induced TLR4-TRIF innate immune signaling in hematopoietic stem cells promotes proliferation but reduces competitive fitness.Cell Stem Cell. 2017; 21 (e225): 225-240Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 16Zhang H Rodriguez S Wang L et al.Sepsis induces hematopoietic stem cell exhaustion and myelosuppression through distinct contributions of TRIF and MYD88.Stem Cell Rep. 2016; 6: 940-956Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar], TLR2 is an attractive therapeutic target for patient with MDS [17Gil-Perez A Montalban-Bravo G Management of myelodysplastic syndromes after failure of response to hypomethylating agents.Ther Adv Hematol. 2019; 102040620719847059Crossref Scopus (24) Google Scholar]; however, its role in the disease remains unclear. To study the role of TLR2 signaling in MDS, we used the NUP98–HOXD13 (NHD13) mouse model. This well-characterized transgenic MDS model expresses the NUP98–HOXD13 fusion from the hematopoietic Vav-1 promoter and exhibits many of the salient features of human MDS including cytopenias, bone marrow dysplasia, and transformation to acute leukemia [18Lin YW Slape C Zhang Z Aplan PD NUP98–HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia.Blood. 2005; 106: 287-295Crossref PubMed Scopus (165) Google Scholar]. Notably, the HSPCs of the NHD13 mice, like those of humans with MDS, have increased expression of TLR2, and we recently reported that complete loss of TLR2 or the TLR signaling adaptor MyD88 is associated with earlier leukemic transformation in the NHD13 mice [19Monlish DA Bhatt ST Duncavage EJ et al.Loss of Toll-like receptor 2 results in accelerated leukemogenesis in the NUP98–HOXD13 mouse model of MDS.Blood. 2018; 131: 1032-1035Crossref PubMed Scopus (10) Google Scholar]. Mechanistic studies revealed that loss of TLR2 resulted in a reduction of activated caspase-1 and lower rates of cell death in preleukemic HSPCs [19Monlish DA Bhatt ST Duncavage EJ et al.Loss of Toll-like receptor 2 results in accelerated leukemogenesis in the NUP98–HOXD13 mouse model of MDS.Blood. 2018; 131: 1032-1035Crossref PubMed Scopus (10) Google Scholar]. Together, these data suggest that some TLR signaling may be protective against the progression of MDS to acute leukemia, and are consistent with patient data indicating that high TLR2 expression correlates with lower-risk disease and better overall survival [3Wei Y Dimicoli S Bueso-Ramos C et al.Toll-like receptor alterations in myelodysplastic syndrome.Leukemia. 2013; 27: 1832-1840Crossref PubMed Scopus (123) Google Scholar,11Zeng Q Shu J Hu Q et al.Apoptosis in human myelodysplastic syndrome CD34+ cells is modulated by the upregulation of TLRs and histone H4 acetylation via a beta-arrestin 1 dependent mechanism.Exp Cell Res. 2016; 340: 22-31Crossref PubMed Scopus (17) Google Scholar]. TLR2 functions largely as a heterodimer with TLR1 or TLR6, and notably, while high TLR2 is associated with lower-risk disease, high TLR6 correlates with higher-risk disease and a trend toward poorer survival [3Wei Y Dimicoli S Bueso-Ramos C et al.Toll-like receptor alterations in myelodysplastic syndrome.Leukemia. 2013; 27: 1832-1840Crossref PubMed Scopus (123) Google Scholar]. This raises the intriguing possibility that there are heterodimer-specific effects of TLR2 signaling in MDS pathogenesis, and herein we used the NHD13 mouse model to test the effects of specific modulation of TLR1/2 versus TLR2/6 signaling on premalignant HSPCs. Indeed, we find that chronic treatment of NHD13 mice with a TLR2/6 agonist, but not a TLR1/2 agonist, leads to more robust cycling and expansion of HSPCs, and accelerates the development of leukemia and death. Conversely, loss of TLR6, but not TLR1, extends survival of the NHD13 mice. TLR2/6 agonist treatment is associated with a marked increase in serum interleukin (IL)-6; however, loss of IL-6 did not mitigate the TLR2/6 agonist-specific expansion of HSPCs. Rather, chimeric mouse studies suggested that the TLR2/6 agonist-mediated effects on premalignant HSPCs are, at least in part, cell autonomous. Finally, RNA profiling studies found that TLR2/6 stimulation is associated with enrichment of Myc and mTORC1 targets. Myc inhibition partially mitigated the TLR2/6 agonist-mediated expansion of premalignant HSPCs. Conversely, mTORC1 inhibition resulted in a dramatic exacerbation of the TLR2/6 agonist-mediated HSPC expansion in NHD13 mice, suggesting that Myc and mTORC1 have opposite roles in mediating the effects of TLR2/6 stimulation on premalignant HSPCs. Collectively, these data reveal that there are indeed heterodimer-specific effects of TLR2 signaling on premalignant HSPCs, with TLR2/6 stimulation, but not TLR1/2 stimulation, promoting HSPC expansion and leukemogenesis in the NHD13 mouse model. C57BL/6J, C57BL/6 (B6.SJL-Ptprca Pepcb/BoyJ; CD45.1), NUP98–HOXD13 (C57BL/6-Tg(Vav1-NUP98/HOXD13)G2Apla/J), Il-6–/– (B6.129S2-Il6tm1Kopf/J), and Tlr1–/– (B6.129S1-Tlr1tm1Flv/J) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Tlr6–/– mice were obtained from Oriental BioService, Inc. (Kyoto, Japan). All mice were maintained on a C57BL/6J background. Sex- and age-matched mice were used in accordance with the guidelines of the Washington University Animal Studies Committee. PAM3CSK4 and PAM2CSK4 (InvivoGen, San Diego, CA)were reconstituted in sterile water and delivered by intraperitoneal (IP) injection. Unless otherwise indicated, mice were administered 1 µg PAM2CSK4, 25 µg PAM3CSK4, or water (vehicle) per dose, 3 doses/week, for the duration of the study. For short-term treatment regimens, the final dose was administered 24 hours before euthanization. For JQ1 treatments, mice were administered 1 mg JQ1 via IP injection 2 hours before TLR2 agonist treatment [20Belkina AC Nikolajczyk BS Denis GV BET protein function is required for inflammation: Brd2 genetic disruption and BET inhibitor JQ1 impair mouse macrophage inflammatory responses.J Immunol. 2013; 190: 3670-3678Crossref PubMed Scopus (302) Google Scholar]. JQ1 (Adooq Bioscience, Irvine, CA)was prepared as a 50 mg/mL stock in DMSO, then diluted to 5 mg/mL with 10% 2-hydroxypropyl-β-cyclodextrin. For rapamycin treatments, mice were administered 0.15 mg rapamycin IP daily (dissolved in 2% dimethyl sulfoxide [DMSO], 30% polyethylene glycol [PEG] 300, and 5% Tween 80 in double-distilled water; Selleck Chemicals, Houston, TX). Thirty thousand KSL cells were sorted into 10% trichloroacetic acid, pelleted at 14,000 rpm for 10 min at 4°C, and washed twice with acetone. Dried pellets were resuspended in solubilization buffer (9 mol/L urea, 2% Triton X-100, and 1% dithiothreitol [DTT]), and incubated with loading dye at 70°C for 10 min. Samples were run on 4%–12% Bis–Tris Plus Gels (Invitrogen, Carlsbad, CA) and transferred to polyvinyl difluoride (PVDF) membranes. Membranes were probed for phospho-STAT1 (No. 9167, Cell Signaling, Danvers), STAT1 (No. 9172, Cell Signaling), pErk1/2 (No. 4370, Cell Signaling), Erk1/2 (No. 4696, Cell Signaling), and rabbit anti-mouse HRP-conjugated secondary antibody (No. 7074, Cell Signaling). Proteins were detected using Pierce ECL Western Blotting Substrate (ThermoFisher, Kalamazoo, MI) on chemiluminescence film. Moribund mice were euthanized, and 1 × 106 bone marrow or spleen cells and 100 μL of blood cells were prepared in 200 µL of fluorescence-activated cell sorting (FACS) buffer supplemented with bovine serum albumin (A10008-01, Gibco, Gaithersburg, MD). The cells were spun onto glass slides using a Shandon Cytospin 3 (8 min at 500 rpm) and stained using Protocol Hema 3 Wright–Giemsa stain (ThermoFisher). Images were captured with an Axioskop 2 microscope equipped with an Axiocam camera and Zen software (Carl Zeiss Microscopy, LLC, Thornwood, NY). Cells were also prepared and stained for flow cytometry using the antibodies under Supplementary Data (online only, available at www.exphem.org). Blood was obtained by retro-orbital sampling. Bone marrow cells were isolated by centrifugation of femurs at 6,000 rpm for 3 min [21Amend SR Valkenburg KC Pienta KJ Murine hind limb long bone dissection and bone marrow isolation.J Vis Exp. 2016; 110: 53936Google Scholar]. Spleen cells were harvested by being crushed through a 100 µmol/L strainer. Cells were processed for staining as previously described [22Schuettpelz LG Gopalan PK Giuste FO Romine MP van Os R Link DC Kruppel-like factor 7 overexpression suppresses hematopoietic stem and progenitor cell function.Blood. 2012; 120: 2981-2989Crossref PubMed Scopus (31) Google Scholar] and stained using the antibodies listed in the Supplementary Data. Cell counts were determined using the Hemavet HV950 (Drew Scientific, Miami Lake, FL). Stained cells were analyzed on a Gallios flow cytometer (Beckman Coulter, Indianapolis, IN). Data were analyzed with FlowJo software (version 10.5.3, TreeStar, Ashland, OR). Bone marrow cells were stained for surface markers (see Supplementary Data), fixed using the BD Cytofix/Cytoperm Kit (BD Biosciences, Franklin Lakes, NJ), blocked with 5% goat serum, stained with mouse anti-human Ki-67 (clone B56, BD Pharmingen) per the manufacturer's instructions, washed, and resuspended in 4′,6-diamidino-2-phenylindone (DAPI)-containing FACS buffer. Bone marrow cells were stained for surface markers (Supplementary Data) and then washed in 1 × binding buffer, and 2 × 106 cells were stained with the Annexin V Apoptosis Detection Kit APC (ThermoFisher) before flow cytometry analysis. Bone marrow cells were obtained by crushing bones in phosphate-buffered saline supplemented with 2% bovine serum albumin. Single-cell suspensions were filtered using a 40-μm filter before staining with antibodies (see Supplementary Data) on ice for 20 min. c-Kit+ cells were pre-enriched before sorting on a BD FACSAria Fusion (BD Biosciences) by selection with biotin-conjugated paramagnetic beads using the MACS Separator (Miltenyi Biotec, Auburn, CA). Nonviable cells were excluded by DAPI staining. One thousand KSL cells were sorted into 300 µL of FACS buffer supplemented with 2% bovine serum albumin, then transferred to 3 mL of MethoCult GF M3434 (Stem Cell Technologies, Vancouver, BC, Canada) and plated onto 35 × 10-mm Petri dishes in duplicate. Colonies were scored after 7 days of growth at 37°C in a humidified chamber with 5% CO2. Cells were then collected, pooled, and replated at a density of 1 × 103 cells per well. For transplantation studies of cells from treated NHD13 mice, 1 × 103 KSL cells were sorted and transplanted into sublethally irradiated (250 cGy) wild-type (WT; CD45.1) mice. Mice were treated with a single dose of TLR agonist. After 4 hours, blood was obtained via cardiac puncture, allowed to clot at room temperature for 1–2 hours, and then centrifuged for 10 min at 6,000 rpm. Serum was assessed using the Proteome Profiler Mouse Cytokine Array Kit (R&D Systems, Minneapolis, MN), according to the manufacturer's instructions. Densitometry was performed using ImageJ (National Institutes of Health, Bethesda, MD). For IL-6 measurements, serum was assessed using the mouse IL-6 Quantikine ELISA kit (R&D Systems). Chimeric mice were generated by transplanting a 1:1 mixture of whole bone marrow cells from NHD13 mice (CD45.1/CD45.2) with bone marrow from NHD13;Tlr2–/– or NHD13;Tlr6–/– mice (CD45.2) into lethally irradiated WT (CD45.1) recipients. CD45.1/CD45.2 NHD13 mice were generated by crossing NHD13 (CD45.2) mice with C57BL/6 (B6.SJL-Ptprc*Pep3bBoyJ) mice to generate CD45.1/CD45.2 NHD13 mice. A total of 2 × 106 cells was transplanted via retro-orbital injection into lethally irradiated (two doses of 550 cGy) WT CD45.1 mice. Cells were allowed to engraft for at least 12 weeks before further analysis. RNA was prepared from sorted KSL cells using NucleoSpin RNA XS (Macherey–Nagel, Bethlehem, PA), amplified using the Sigma Complete Whole Transcriptome Amplification Kit (WTA2, Millipore Sigma, Darmstadt, Germany), and analyzed using the Agilent Mouse Gene Expression v2 8 × 60K Microarray (Agilent). Gene set enrichment analysis (GSEA) was performed using the GSEA software (Broad Institute). Data are presented as mean ± SEM, unless otherwise stated. Statistical significance was assessed using an unpaired, two-tailed Student t test or the Log-rank (Mantel–Cox) test. GraphPad Prism (version 8.0.2, GraphPad Software, San Diego, CA) was used for all statistical analyses. In all cases, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. We previously reported that young adult NHD13 mice have increased surface TLR2 on their hematopoietic stem and progenitor (c-Kit+Sca-1+Lineage- [KSL]) cells compared with WT littermates [19Monlish DA Bhatt ST Duncavage EJ et al.Loss of Toll-like receptor 2 results in accelerated leukemogenesis in the NUP98–HOXD13 mouse model of MDS.Blood. 2018; 131: 1032-1035Crossref PubMed Scopus (10) Google Scholar]. Using flow cytometry, we determined that surface expression of the TLR2 binding partners, TLR1 and TLR6, is also increased on KSL cells of the NHD13 mice compared with WT littermates (Figure 1A,B; Supplementary Figure E1, online only, available at www.exphem.org). We then asked how chronic exposure to TLR1/2 versus TLR2/6 ligands influences hematopoiesis and disease progression in the NHD13 mice. To this end, NHD13 mice and WT littermate controls were treated chronically (three times weekly) with the TLR1/2 agonist PAM3CSK4, the TLR2/6 agonist PAM2CSK4, or vehicle (water) alone. We used doses of these agonists that had similar effects on the HSPCs of WT mice (Figure 2A–G) and that also had similar effects on the activation of known downstream targets of TLR2 signaling, including phosphorylation of STAT1 and ERK1/2 (Figure 1C; Supplementary Figure E2, online only, available at www.exphem.org). Long-term treatment with these agonists had minimal effects on WT mice, who exhibited only a modest reduction in platelets with the TLR1/2 agonist (PAM3CSK4, Figure 1D–F) and, with the exception of one death in the TLR2/6 agonist (PAM2CSK4) cohort from an unknown cause, lived >500 days (at which time they were sacrificed/censored from the study, Figure 1G). Consistent with prior reports [18Lin YW Slape C Zhang Z Aplan PD NUP98–HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia.Blood. 2005; 106: 287-295Crossref PubMed Scopus (165) Google Scholar,19Monlish DA Bhatt ST Duncavage EJ et al.Loss of Toll-like receptor 2 results in accelerated leukemogenesis in the NUP98–HOXD13 mouse model of MDS.Blood. 2018; 131: 1032-1035Crossref PubMed Scopus (10) Google Scholar,23Slape CI Saw J Jowett JB et al.Inhibition of apoptosis by BCL2 prevents leukemic transformation of a murine myelodysplastic syndrome.Blood. 2012; 120: 2475-2483Crossref PubMed Scopus (35) Google Scholar], we found reduced white blood cell (WBC) and platelet counts in NHD13 mice compared with WT controls at baseline. In contrast to WT mice, treatment of NHD13 mice with the TLR2/6 agonist increased WBC counts in preleukemic mice (Figure 1D), and both agonists further decreased platelet counts (Figure 1F). Notably, treatment of the NHD13 mice with the TLR2/6 agonist (PAM2CSK4), but not the TLR1/2 agonist (PAM3CSK4), was associated with earlier death than water treatment alone (Figure 1G). The cause of death for each mouse was determined using flow cytometry and histopathologic evaluation by a hematopathologist (EJD) following the Bethesda criteria [24Kogan SC Ward JM Anver MR et al.Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice.Blood. 2002; 100: 238-245Crossref PubMed Scopus (345) Google Scholar]. The most common determined cause of death in the NHD13 mice, including those treated with the TLR2/6 agonist, was leukemia (Figure 1H,I; Supplementary Table E1, online only, available at www.exphem.org). Supporting a role for TLR6 in promoting leukemogenesis, we found that loss of TLR6, but not TLR1, is associated with significantly longer survival in the NHD13 mice (Supplementary Figure E3 and Supplementary Tables E2 and E3, online only, available at www.exphem.org). In addition, compared with NHD13 mice, NHD13;Tlr6–/– mice more often died of MDS rather than leukemia (Supplementary Figure E3 and Supplementary Table E2). Of note, neither TLR1 nor TLR6 loss affected the peripheral blood counts of NHD13 mice (Supplementary Figure E3).Figure 2TLR2/6 agonist treatment expands premalignant HSPCs. (A) Young adult (6- to 8-week) NHD13 mice or WT littermates were treated with a 2-week course (six doses total) of TLR2/6 agonist (PAM2CSK4, 1 µg/dose) or TLR1/2 agonist (PAM3CSK4, 25 µg/dose), and bone marrow was analyzed by flow cytometry. Shown are representative flow plots of the c-Kit+Sca-1+Lineage– (KSL) cells from the different treatment groups. The frequency among whole bone marrow cells (B) and total numbers (C) of KSL cells in the bone marrow of the different groups, as well as the frequency (D) and total numbers (E) of long-term HSCs (Lineage-c–Kit+Sca-1+CD150+CD48– [KSL SLAM] cells) are shown (see Supplementary Figure E3 for representative gating). Percentages of KSL cells in G0 (F) and S/G2/M (G) of the cell cycle as determined by Ki-67 and DAPI staining. (H) Representative Ki-67 and DAPI staining of KSL cells from water-, PAM2CSK4-, and PAM3CSK4-treated NHD13 mice. (I) Annexin V staining of KSL cells from the indicated groups. (J) NHD13 mice were crossed to Tlr1–/–, Tlr2–/–, or Tlr6–/– mice, as indicated, and treated with PAM2CSK4 or PAM3CSK4 as indicated for 6 doses as above. Shown are the frequencies of KSL cells in the indicated groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, nd = no statistical difference by unpaired Student t test. Error bars represent the mean ± EM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To gain insight into the differential effects of TLR1/2 versus TLR2/6 signaling on survival of the NHD13 mice, we looked at the effects of the different agonist treatments on the stem and progenitor cell populations (Figure 2A). Specifically, young adult NHD13 mice and WT littermate controls were treated for 2 weeks with either the TLR1/2 or TLR2/6 agonist (using the same doses and schedule as described for chronic treatment), and HSPC numbers were assessed by flow cytometry. While the different agonists had similar effects on WT mice, increasing both KSL cells and KSL CD150+CD48– (KSL SLAM, or long-term hematopoietic stem cells), the TLR2/6 agonist (PAM2CSK4) expanded KSL and KSL SLAM cells in the NHD13 mice to a significantly greater extent than the TLR1/2 agonist PAM3CSK4 (Figure 2B–E; Supplementary Figure E4A, online only, available at www.exphem.org). Of note, myeloid progenitors were expanded in the WT mice in response to the TLR1/2 agonist, but not the TLR2/6 agonist (Supplementary Figure E4B,C). Consistent with the greater expansion of the KSL cells in the NHD13 mice in response to TLR2/6 agonist treatment, we found a higher percentage of these cells were actively cycling (Figure 2F–H) compared with those of the TLR1/2-agonist treated mice or those from mice treated with water alone. The HSPCs of NHD13 mice have high rates of cell death [23Slape CI Saw J Jowett JB et al.Inhibition of apoptosis by BCL2 prevents leukemic transformation of a murine myelodysplastic syndrome.Blood. 2012; 120: 2475-2483Crossref PubMed Scopus (35) Google Scholar,25Choi CW Chung YJ Slape C Aplan PD Impaired differentiation and apoptosis of hematopoietic precursors in a mouse model of myelodysplastic syndrome.Haematologica. 2008; 93: 1394-1397Crossref PubMed Scopus (22) Google Scholar], and this cell death (as measured by Annexin V staining, Figure 2I) is modestly reduced in the TLR2/6 agonist-treated animals compared with the TLR1/2 agonist-treated ones (with a trending reduction in death compared with water-treated controls). The KSL population from NHD13 mice has previously been reported to be capable of serial replating in methylcellulose and to contain the transplantable, disease-initiating cells [26Cheng G Liu F Asai T et al.Loss of p300 accelerates MDS-associated leukemogenesis.Leukemia. 2017; 31: 1382-1390Crossref PubMed Scopus (25) Google Scholar, 27Chung YJ Choi CW Slape C Fry T Aplan PD Transplantation of a myelodysplastic syndrome by a long-term repopulating hematopoietic cell.Proc Natl Acad Sci USA. 2008; 105: 14088-14093Crossref PubMed Scopus (32) Google Scholar, 28Guirguis AA Slape CI Failla LM et al.PUMA promotes apoptosis of hematopoietic progenitors driving leukemic progression in a mouse model of myelodysplasia.Cell Death Differ. 2016; 23: 1049-1059Crossref PubMed Scopus (11) Google Scholar]. To confirm that the expanded KSL population in the PAM2CSK4-treated mice retains these properties, we sorted KSL cells from WT and NHD13 mice treated for 2 weeks with the TLR1/2 (PAM3CSK4) or TLR2/6 (PAM2CSK4) agonist, or water alone, and performed serial replatings in MethoCult. Indeed, the KSL cells from TLR2 agonist-treated NHD13 mice were capable of generating colonies through serial replatings, while the WT KSL cells failed after the third replating (Supplementary Figure E5A, online only, available at www.exphem.org). In addition, sorted KSL cells from TLR2 agonist-treated or control NHD13 mice were capable of long-term engraftment in sublethally irradiated WT recipient mice, albeit with a predominately Gr-1+ population (Supplementary Figure E5B,C). These findings are similar to those for the cells from water control-treated NHD13 mice. Thus, TLR2/6 agonist exposure expands premalignant HSPCs in the NHD13 mice to a greater extent than TLR1/2 agonist treatment, and these expanded cells remain capable of serial replating and long-term repopulation. Finally, to rule out the possibility that off-target effects of the TLR agonists are responsible for HSPC expansion, we treated TLR-deficient mice with PAM2CSK4 or PAM3CSK4. As expected, the TLR2/6 agonist-mediated expansion" @default.
• W3041120131 created "2020-07-16" @default.
• W3041120131 creator A5004783856 @default.
• W3041120131 creator A5023805918 @default.
• W3041120131 creator A5026084358 @default.
• W3041120131 creator A5044476970 @default.
• W3041120131 creator A5050851135 @default.
• W3041120131 creator A5053436400 @default.
• W3041120131 creator A5055739074 @default.
• W3041120131 creator A5057334251 @default.
• W3041120131 creator A5071450878 @default.
• W3041120131 creator A5072599021 @default.
• W3041120131 date "2020-08-01" @default.
• W3041120131 modified "2023-09-23" @default.
• W3041120131 title "TLR2/6 signaling promotes the expansion of premalignant hematopoietic stem and progenitor cells in the NUP98–HOXD13 mouse model of MDS" @default.
• W3041120131 cites W1961505479 @default.
• W3041120131 cites W1973465167 @default.
• W3041120131 cites W1979345599 @default.
• W3041120131 cites W1987445150 @default.
• W3041120131 cites W1995969368 @default.
• W3041120131 cites W2005759082 @default.
• W3041120131 cites W2009949436 @default.
• W3041120131 cites W2014357007 @default.
• W3041120131 cites W2019608583 @default.
• W3041120131 cites W2020920210 @default.
• W3041120131 cites W2063013559 @default.
• W3041120131 cites W2069089745 @default.
• W3041120131 cites W2069737466 @default.
• W3041120131 cites W2080403219 @default.
• W3041120131 cites W2080796596 @default.
• W3041120131 cites W2092213185 @default.
• W3041120131 cites W2096258302 @default.
• W3041120131 cites W2100295977 @default.
• W3041120131 cites W2106229108 @default.
• W3041120131 cites W2124789340 @default.
• W3041120131 cites W2128877614 @default.
• W3041120131 cites W2131095909 @default.
• W3041120131 cites W2132327973 @default.
• W3041120131 cites W2136072463 @default.
• W3041120131 cites W2144770839 @default.
• W3041120131 cites W2144896888 @default.
• W3041120131 cites W2150469988 @default.
• W3041120131 cites W2152600551 @default.
• W3041120131 cites W2210729715 @default.
• W3041120131 cites W2214429730 @default.
• W3041120131 cites W2225951295 @default.
• W3041120131 cites W2231320725 @default.
• W3041120131 cites W2298584763 @default.
• W3041120131 cites W2419253007 @default.
• W3041120131 cites W2423491126 @default.
• W3041120131 cites W2442550790 @default.
• W3041120131 cites W2526134968 @default.
• W3041120131 cites W2529711900 @default.
• W3041120131 cites W2556255420 @default.
• W3041120131 cites W2597817312 @default.
• W3041120131 cites W2739047154 @default.
• W3041120131 cites W2791765802 @default.
• W3041120131 cites W2898144712 @default.
• W3041120131 cites W2943855331 @default.
• W3041120131 doi "https://doi.org/10.1016/j.exphem.2020.07.001" @default.
• W3041120131 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7673652" @default.
• W3041120131 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/32652111" @default.
• W3041120131 hasPublicationYear "2020" @default.
• W3041120131 type Work @default.
• W3041120131 sameAs 3041120131 @default.
• W3041120131 citedByCount "8" @default.
• W3041120131 countsByYear W30411201312021 @default.
• W3041120131 countsByYear W30411201312022 @default.
• W3041120131 countsByYear W30411201312023 @default.
• W3041120131 crossrefType "journal-article" @default.
• W3041120131 hasAuthorship W3041120131A5004783856 @default.
• W3041120131 hasAuthorship W3041120131A5023805918 @default.
• W3041120131 hasAuthorship W3041120131A5026084358 @default.
• W3041120131 hasAuthorship W3041120131A5044476970 @default.
• W3041120131 hasAuthorship W3041120131A5050851135 @default.
• W3041120131 hasAuthorship W3041120131A5053436400 @default.
• W3041120131 hasAuthorship W3041120131A5055739074 @default.
• W3041120131 hasAuthorship W3041120131A5057334251 @default.
• W3041120131 hasAuthorship W3041120131A5071450878 @default.
• W3041120131 hasAuthorship W3041120131A5072599021 @default.
• W3041120131 hasBestOaLocation W30411201311 @default.
• W3041120131 hasConcept C109159458 @default.
• W3041120131 hasConcept C15729860 @default.
• W3041120131 hasConcept C201750760 @default.
• W3041120131 hasConcept C28328180 @default.
• W3041120131 hasConcept C502942594 @default.
• W3041120131 hasConcept C86803240 @default.
• W3041120131 hasConcept C95444343 @default.
• W3041120131 hasConceptScore W3041120131C109159458 @default.
• W3041120131 hasConceptScore W3041120131C15729860 @default.
• W3041120131 hasConceptScore W3041120131C201750760 @default.
• W3041120131 hasConceptScore W3041120131C28328180 @default.
• W3041120131 hasConceptScore W3041120131C502942594 @default.
• W3041120131 hasConceptScore W3041120131C86803240 @default.
• W3041120131 hasConceptScore W3041120131C95444343 @default.
• W3041120131 hasLocation W30411201311 @default.
• W3041120131 hasLocation W30411201312 @default.
• W3041120131 hasLocation W30411201313 @default.

• next