Infection Mobilizes Hematopoietic Stem Cells through Cooperative NOD-like Receptor and Toll-like Receptor Signaling

Abstract Abstract 556 We have documented that deregulation of the Toll-like Receptor 2 (TLR2) centered innate immunity signals contribute to the pathogenesis of myelodysplastic syndromes (MDS). Of relevance, oncogenically active mutations of MYD88, a signal adaptor protein for TLR signal, have recently been identified as recurrent genetic lesions in both B-cell lymphoma and in chronic lymphocytic leukemia (CLL) (Vu N et al. Nature 2010 and Puente et al. Nature 2011). This information further supports the importance of innate immunity deregulation in leukemogenesis. To further characterize this pathway in MDS, we analyzed potential genetic alteration and expression level of MYD88 in patients of MDS. In a cohort of 40 MDS whole bone marrow mononuclear cell DNA, we first performed pyrosequencing analysis focusing on a list of previously reported MYD88 mutations (V217, W218, S219, I220, S222, M232, S243, L265, and T294). We did not detect mutation of any these hotspots on MYD88 in MDS. We then expanded the sequencing efforts to the entire coding region of MYD88 using an approach that combines PCR amplification and massive parallel sequencing. Still, no mutation of MYD88 was detected using this technique. We then examined the expression of MYD88 in CD34+ hematopoietic stem cells from 65 patients with MDS. In comparison to healthy donors, 26% of MDS patients (N=17) presented a more than 2 fold increase of MYD88 RNA, and 15% (N=10) had a 30%–90% increase. In average, MYD88 RNA level was 1.7 fold increased compared to control. Of potential clinical relevance, patients with higher MYD88 RNA expression in bone marrow CD34+ cells (above median value) (N=33) had a propensity of shorter period (24.4 mo) of overall survival (OS) in comparison to patients with lower levels of MYD88 expression (N=32) (32 mo)(P=0.05). We also found that patients with higher levels of MYD88 expression (split at 0.8 fold to controls) tended to have higher WBC (P=0.02). We have previously shown that blockade of the TLR2 mediated innate immunity signaling in MDS CD34+ cells could positively regulate erythroid lineage differentiation. To evaluate the potential of MYD88 blockade, we applied a 26 AA MYD88 inhibitory peptide that blocks its homodimerization (Invivogen, San Diego, CA) on primary CD34+ cells isolated from patients with lower-risk MDS (N=5). Methylcellulose medium supported colony formation assays indicated that the presence of MYD88 inhibitor led to an average of 60% increase for the numbers of erythroid colonies and a 30% increase for the numbers of total colonies. At the same time, we did not observe these effects of MYD88 blockade on the CD34+ cells isolated from the patients of higher-risk MDS (N=3). IL-8 is one of the key downstream transcriptional targets of the TLR-MYD88-NFkB innate immunity signaling that was documented to be elevated in bone marrow plasma of MDS. ELISA assays also indicated that blockade MYD88 in cultured MDS CD34+ cells led to a decrease of IL-8 concentration in medium. Taken together, these results indicated that MYD88 is overexpressed in hematopoietic stem cells of MDS and that blockade of MYD88 mediated innate immunity signaling may have therapeutic potential in treating patients with MDS. Disclosures: Kantarjian: Genzyme: Research Funding.

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CD34+ hematopoietic stem cells are mobilized with exercise independently of beta-2 adrenergic receptor signaling

Brain Behavior and Immunity ◽

10.1016/j.bbi.2017.07.087 ◽

2017 ◽

Vol 66 ◽

pp. e22 ◽

Cited By ~ 1

Author(s):

N.H. Agha ◽

H.E. Kunz ◽

R. Graff ◽

R. Azadan ◽

F.L. Baker ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Adrenergic Receptor ◽

Receptor Signaling ◽

Hematopoietic Stem ◽

Beta 2

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Toll-like receptor 2 expression on c-kit+ cells tracks the emergence of embryonic definitive hematopoietic progenitors

Nature Communications ◽

10.1038/s41467-019-13150-0 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 2

Author(s):

Jana Balounová ◽

Iva Šplíchalová ◽

Martina Dobešová ◽

Michal Kolář ◽

Karel Fišer ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Toll Like Receptor ◽

Toll Like Receptors ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Mammalian Embryos ◽

Toll Like Receptor 2 ◽

Early Mouse

AbstractHematopoiesis in mammalian embryos proceeds through three successive waves of hematopoietic progenitors. Since their emergence spatially and temporally overlap and phenotypic markers are often shared, the specifics regarding their origin, development, lineage restriction and mutual relationships have not been fully determined. The identification of wave-specific markers would aid to resolve these uncertainties. Here, we show that toll-like receptors (TLRs) are expressed during early mouse embryogenesis. We provide phenotypic and functional evidence that the expression of TLR2 on E7.5 c-kit+ cells marks the emergence of precursors of erythro-myeloid progenitors (EMPs) and provides resolution for separate tracking of EMPs from primitive progenitors. Using in vivo fate mapping, we show that at E8.5 the Tlr2 locus is already active in emerging EMPs and in progenitors of adult hematopoietic stem cells (HSC). Together, this data demonstrates that the activation of the Tlr2 locus tracks the earliest events in the process of EMP and HSC specification.

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IL3-Receptor Signaling Is Dispensable For The Generation and Maintenance Of Jak2V617F-Induced Myeloproliferative Neoplasm (MPN

Blood ◽

10.1182/blood.v122.21.2841.2841 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2841-2841

Author(s):

Therese Vu ◽

Claudia Bruedigam ◽

Rebecca Austin ◽

Catherine Paine-Kuhn ◽

Hayley S. Ramshaw ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Cell Count ◽

White Cell Count ◽

White Cell ◽

Receptor Signaling ◽

Short Term ◽

Hematopoietic Stem

Abstract Myeloproliferative neoplasms (MPN) such as polycythemia vera, essential thrombocythemia and primary myelofibrosis are clonal diseases driven by acquired somatic mutations in hematopoietic stem cells (HSCs), the most common of which is JAK2V617F. JAK2V617F leads to cytokine hypersensitivity and activation of JAK-STAT signaling in the presence of an erythropoietin (EPO), thrombopoietin (TPO) or interleukin-3 (IL3) cytokine receptor scaffold (Nature 2005; 434:1144-8, Leukemia 2008; 22:1828-40). Physiological Jak2V617F expression in long-term hematopoietic stem cells (LT-HSCs; lineagelowcKit+Sca1+CD150+CD48-) is necessary and sufficient to generate MPN, and these LT-HSCs contain the sole reservoir of Jak2V617F MPN-initiating stem cells (Cancer Cell 2010; 17:584-96; Blood 2012; 120:166-72). Although Jak2V617F-mediated EPO hypersensitivity drives erythroid expansion and in vitro EPO-independent colony formation, the EPO receptor is not expressed on Jak2V617F HSC populations (Blood 2012; 120:166-72). This suggests that hypersensitivity to IL3 and/or TPO signaling is responsible for driving LT-HSC proliferation and survival in vivo. To determine the respective contributions of IL3 and TPO signaling to Jak2V617F-induced MPN, pStat5 was measured in HSC and progenitor populations from E2ACre+Jak2V617F+/- knockin mice (hereafter Jak2VF) after stimulation with rmIL3 or rmTPO. Committed myeloid progenitors showed robust pStat5 stimulation with rmIL3, but only low level stimulation with rmTPO (TPO 460.25 ± 25.02 MFI vs. IL3 598.25 ± 69.05 MFI, p<0.05, Vehicle 362.25 ± 76.66 MFI). In contrast, LT-HSCs showed strong induction of pStat5 signaling with rmTPO stimulation but less so with rmIL3 stimulation (TPO 571 ± 47.36 MFI vs. IL3 487.5 ± 53.69 MFI, p=0.05, Vehicle 249.5 ± 47.63 MFI). Concordant with these results, TPO signaling appears essential for the generation of Jak2V617F-induced MPN (ASH 2012 Abstract 427). To determine the contribution of IL3 receptor signaling in Jak2V617F-induced MPN, we crossed Jak2VF knockin mice with mice lacking the common beta subunit of the IL3 receptor that is responsible for signal transduction of IL3, IL5 and GM-CSF (Jak2VFIL3Rb-/-). Jak2VFIL3Rb-/- mice developed MPN with similar latency and mortality to Jak2VF controls. There were no differences in peripheral blood white cell count (16.51 ± 1.5×109/L vs. 14.89 ± 2.4×109/L, p=0.39, n=3), haematocrit (67.97 ± 7.85×109/L vs. 61.97 ± 5.73×109/L, p=0.35, n=3) or extramedullary erythropoiesis (spleen weight, 665 ± 107mg vs. 541 ± 106mg, p=0.22, n=3) between Jak2VFIL3Rb-/- and Jak2VF mice respectively. In competitive bone marrow transplantation assays, all recipients of Jak2VFIL3Rb-/- or Jak2VF bone marrow developed MPN with similar diagnostic parameters such as elevated white cell count (14.29 ± 3.41×109/L vs. 18.38 ± 5.78×109/L, p=0.11, n=8), hematocrit (63.4 ± 16.5% vs. 50.5 ± 13%, p=0.16, n=8) and splenomegaly (529 ± 86mg vs. 465 ± 119mg, p=0.23, n=8) in Jak2VFIL3Rb-/- and Jak2VF mice respectively. Jak2VFIL3Rb-/- bone marrow cells initially showed reduced short-term (4 weeks) engraftment and white cell count in recipients compared to the Jak2V617F group (p<0.0005). However after 16 weeks post-transplant there was no difference in chimerism between recipients of Jak2VFIL3Rb-/- or JakVF cells. IL3 was unable to stimulate pStat5 in Jak2VFIL3Rb-/- LT-HSCs or progenitors, but was preserved in Jak2VF LT-HSCs and progenitors. These data show that IL3Rb signaling is dispensable for Jak2V617F-induced MPN and LT-HSC function, however may regulate short-term myeloid progenitor cell expansion. TPO signaling appears preferentially important for Jak2VF LT-HSC pStat5 induction, whereas IL3 is more important for pStat5 induction in progenitor cells. These findings will help to inform strategies aimed at targeting long term Jak2V617F-initiating HSC populations. Disclosures: No relevant conflicts of interest to declare.

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