Inhibition of SRC-mediated integrin signaling in bone marrow niche enhances hematopoietic stem cell function.

Interaction with microenvironmental factors is crucial for the regulation of hematopoietic stem cell (HSC) function. Stroma derived factor (SDF)-1α supports HSCs in the quiescent state and is central to the homing of transplanted HSCs. Here, we show that integrin signaling regulates Sdf-1α expression transcriptionally. Systemic deletion of Periostin, an Integrin-αv ligand, showed increased expression of Sdf-1α in bone marrow (BM) niche. Pharmacological inhibition or CRISPR-Cas9-mediated deletion of SRC, resulted in a similar increase in the chemokine expression in vitro. Importantly, systemic SRC-inhibition led to increase in SDF-1α levels in BM plasma. This resulted in a robust increase (14.05 ± 1.22% to 29.11 ± 0.69%) in the homing efficiency of transplanted HSCs. In addition, we observed enhancement in the recovery of blood cell counts following radiation injury, indicating an enhanced hematopoietic function. These results establish a role of SRC-mediated integrin signaling in the transcriptional regulation of Sdf-1α. This mechanism could be harnessed further to improve the hematopoietic function.

Niche-Mediated Integrin Signaling Supports Steady-State Hematopoiesis in the Spleen.

Outside-in integrin signaling regulates cell fate decisions in a variety of cell types, including hematopoietic stem cells (HSCs). Our earlier published studies showed that interruption of periostin (POSTN) and integrin-αv (ITGAV) interaction induces faster proliferation in HSCs with developmental stage-dependent functional effects. In this study, we examined the role of POSTN-ITGAV axis in lymphohematopoietic activity in spleen that hosts a rare population of HSCs, the functional regulation of which is not clearly known. Vav-iCre-mediated deletion of Itgav in the hematopoietic system led to higher proliferation rates, resulting in increased frequency of primitive HSCs in the adult spleen. However, in vitro CFU-C assays demonstrated a poorer differentiation potential following Itgav deletion. This also led to a decrease in the white pulp area with a significant decline in the B cell numbers. Systemic deletion of its ligand, POSTN, phenocopied the effects noted in Vav-Itgav-/- mice. Histological examination of Postn-deficient spleen also showed an increase in the spleen trabecular areas. Importantly, these are the myofibroblasts of the trabecular and capsular areas that expressed high levels of POSTN within the spleen tissue. In addition, vascular smooth muscle cells also expressed POSTN. Through CFU-S12 assays, we showed that hematopoietic support potential of stroma in Postn-deficient splenic hematopoietic niche was defective. Overall, we demonstrate that POSTN-ITGAV interaction plays an important role in spleen lymphohematopoiesis.

The Periostin/Integrin-αv Axis Regulates the Size of Hematopoietic Stem Cell Pool in the Fetal Liver.

We earlier showed that outside-in integrin signaling through POSTN-ITGAV interaction plays an important role in regulating adult hematopoietic stem cell (HSC) quiescence. Here, we show that Itgav deletion results in increased frequency of phenotypic HSCs in fetal liver (FL) due to faster proliferation. Systemic deletion of Postn led to increased proliferation of FL HSCs, albeit without any loss of stemness, unlike Vav-Itgav-/- HSCs. Based on RNA sequencing analysis of FL and bone marrow HSCs, we predicted the involvement of DNA damage response pathways in this dichotomy. Indeed, proliferative HSCs from Postn-deficient FL tissues showed increased levels of DNA repair, resulting in lesser double-strand breaks. Thus POSTN, with its expression majorly localized in the vascular endothelium of FL tissue, acts as a regulator of stem cell pool size during development. Overall, we demonstrate that the duality of response to proliferation in HSCs is developmental stage dependent and can be correlated with DNA damage responses.

Human stem cell-derived monocytes and microglia-like cells reveal impaired amyloid plaque clearance upon heterozygous or homozygous loss of TREM2.

INTRODUCTION: Murine microglia expressing the Alzheimer's disease-linked TREM2R47H mutation display variable decrease in phagocytosis, while impaired phagocytosis is reported following loss of TREM2. However, no data exist on TREM2+/R47H human microglia. Therefore, we created human pluripotent stem cell (hPSC) monocytes and transdifferentiated microglia-like cells (tMGs) to examine the effect of the TREM2+/R47H mutation and loss of TREM2 on phagocytosis. METHODS: We generated isogenic TREM2+/R47H, TREM2+/-, and TREM2-/- hPSCs using CRISPR/Cas9. Following differentiation to monocytes and tMGs, we studied the uptake of Escherichia coli fragments and analyzed amyloid plaque clearance from cryosections of APP/PS1+/- mouse brains. RESULTS: We demonstrated that tMGs resemble cultured human microglia. TREM2+/- and TREM2-/- hPSC monocytes and tMGs phagocytosed significantly less E. coli fragments and cleared less amyloid plaques than wild-type hPSC progeny, with no difference for TREM2+/R47H progeny. DISCUSSION: In vitro phagocytosis of hPSC monocytes and tMGs was not affected by the TREM2+/R47H mutation but was significantly impaired in TREM2+/- and TREM2-/- progeny.

The Impact of Integrin ß2 on Granulocyte/Macrophage Progenitor Proliferation.

Previously, we reported that although the HSPC frequency in bone marrow cells (BMC) was comparable between ß2-/- and ß2+/+ mice, transplantation of ß2-/- BMC into lethally irradiated CD45.1 recipient resulted in more myeloid cell production than ß2+/+ BMC. The objective of this study is to address if integrin ß2 deficiency skews granulocyte/macrophage progenitor (GMP) proliferation. FACS analysis demonstrated that GMP frequency and cell number were higher and megakaryocyte/erythrocyte progenitor frequency and cell number were lower in ß2-/- mice than ß2+/+ mice. However, the common myeloid progenitors (CMP) frequency and cell number were similar between the two groups. The increased GMP number was due to GMP proliferation as evidenced by the percentage of BrdU-incorporating GMP. Whole genome transcriptome analysis identified increased FcεRIα expression in ß2-/- CMP compared to ß2+/+ CMP. FcεRIα expression on ß2-/- GMP was detected increased in ß2-/- mice by qRT-PCR and FACS. Although transplantation of FcεRIαhi GMP or FcεRIαlo GMP into lethally irradiated CD45.1 recipient resulted in comparable myeloid cell production, transplantation of ß2 deficient FcεRIαhi GMP generated more myeloid cells than ß2+/+ FcεRIαhi GMP. GATA2 expression was increased in ß2-/- GMP. Using a luciferase reporter assay, we demonstrated that mutation of the GATA2 binding site in the FcεRIα promoter region diminished FcεRIα transcription. In vitro, the addition of IgE, the ligand of FcεRIα, promoted GMP expansion, which was abrogated by inhibition of JNK phosphorylation. Integrin ß2 deficiency promoted GMP proliferation and myeloid cell production, which was mediated via FcεRIα/IgE-induced JNK phosphorylation in GMP. Stem Cells 2019;37:430-440.

Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis.

Integrins play an important role in haematopoietic stem cell (HSC) maintenance in the bone marrow niche. Here, we demonstrate that Periostin (Postn) via interaction with Integrin-αv (Itgav) regulates HSC proliferation. Systemic deletion of Postn results in peripheral blood (PB) anaemia, myelomonocytosis and lymphopenia, while the number of phenotypic HSCs increases in the bone marrow. Postn-/- mice recover faster from radiation injury with concomitant loss of primitive HSCs. HSCs from Postn-/- mice show accumulation of DNA damage generally associated with aged HSCs. Itgav deletion in the haematopoietic system leads to a similar PB phenotype and HSC-intrinsic repopulation defects. Unaffected by Postn, Vav-Itgav-/- HSCs proliferate faster in vitro, illustrating the importance of Postn-Itgav interaction. Finally, the Postn-Itgav interaction inhibits the FAK/PI3K/AKT pathway in HSCs, leading to increase in p27Kip1 expression resulting in improved maintenance of quiescent HSCs. Together, we demonstrate a role for Itgav-mediated outside-in signalling in regulation of HSC proliferation and stemness.

Hematopoietic stem/progenitor cells directly contribute to arteriosclerotic progression via integrin ß2.

Recent studies described the association between hematopoietic stem/progenitor cell (HSPC) expansion in the bone marrow (BM), leukocytosis in the peripheral blood, and accelerated atherosclerosis. We hypothesized that circulating HSPC may home to inflamed vessels, where they might contribute to inflammation and neointima formation. We demonstrated that Lin(-) Sca-1(+) cKit(+) (LSK cells) in BM and peripheral blood of LDLr(-/-) mice on high fat diet expressed significantly more integrin ß2 , which was responsible for LSK cell adhesion and migration toward ICAM-1 in vitro, and homing to injured arteries in vivo, all of which were blocked with an anti-CD18 blocking antibody. When homed LSK cells were isolated from ligated artery and injected to irradiated recipients, they resulted in BM reconstitution. Injection of CD18(+/+) LSK cells to immunodeficient Balb/C Rag2(-) É£C(-/-) recipients resulted in more severe inflammation and reinforced neointima formation in the ligated carotid artery, compared to mice injected with PBS and CD18(-/-) LSK cells. Hypercholesterolemia stimulated ERK phosphorylation (pERK) in LSK cells of LDLr(-/-) mice in vivo. Blockade of pERK reduced ARF1 expression, leading to decreased integrin ß2 function on HSPC. In addition, integrin ß2 function could be regulated via ERK-independent LRP1 pathway. Integrin ß2 expression on HSPC is regulated by hypercholesterolemia, specifically LDL, in pERK-dependent and -independent manners, leading to increased homing and localization of HSPC to injured arteries, which is highly correlated with arteriosclerosis.

SMAD signaling regulates CXCL12 expression in the bone marrow niche, affecting homing and mobilization of hematopoietic progenitors.

We recently demonstrated that ex vivo activation of SMAD-independent bone morphogenetic protein 4 (BMP4) signaling in hematopoietic stem/progenitor cells (HSPCs) influences their homing into the bone marrow (BM). Here, we assessed whether alterations in BMP signaling in vivo affects adult hematopoiesis by affecting the BM niche. We demonstrate that systemic inhibition of SMAD-dependent BMP signaling by infusion of the BMP antagonist noggin (NGN) significantly increased CXCL12 levels in BM plasma leading to enhanced homing and engraftment of transplanted HSPCs. Conversely, the infusion of BMP7 but not BMP4, resulted in decreased HSPC homing. Using ST2 cells as an in vitro model of BM niche, we found that incubation with neutralizing anti-BMP4 antibodies, NGN, or dorsomorphin (DM) as well as knockdown of Smad1/5 and Bmp4, all enhanced CXCL12 production. Chromatin immunoprecipitation identified the SMAD-binding element in the CXCL12 promoter to which SMAD4 binds. When deleted, increased CXCL12 promoter activity was observed, and NGN or DM no longer affected Cxcl12 expression. Interestingly, BMP7 infusion resulted in mobilization of only short-term HSCs, likely because BMP7 affected CXCL12 expression only in osteoblasts but not in other niche components. Hence, we describe SMAD-dependent BMP signaling as a novel regulator of CXCL12 production in the BM niche, influencing HSPC homing, engraftment, and mobilization.

Regulation of high-density lipoprotein on hematopoietic stem/progenitor cells in atherosclerosis requires scavenger receptor type BI expression.

OBJECTIVE: Recently, we demonstrated that scavenger receptor type BI (SR-BI), a high-density lipoprotein (HDL) receptor, was expressed on murine hematopoietic stem/progenitor cells (HSPC) and infusion of reconstituted HDL and purified human apolipoprotein A-I (apoA-I) suppressed HSPC proliferation. We hypothesized that SR-B1 expression is required for the observed antiproliferative effects of HDL on HSPC. APPROACH AND RESULTS: SR-BI-deficient (SR-BI(-/-)) mice and wild-type controls were fed on chow or high-fat diet (HFD) for 8 to 10 weeks. Under chow diet, a significant increase in Lin(-) Sca1(+) cKit(+) cells (LSK cells, so-called HSPC) was found in the bone marrow of SR-BI(-/-) mice when compared with wild-type mice. HFD induced a further expansion of CD150(+)CD48(-) LSK cells (HSC), HSPC, and granulocyte monocyte progenitors in SR-BI(-/-) mice. Injection of reactive oxygen species inhibitor N-acetylcysteine attenuated HFD-induced HSPC expansion, leukocytosis, and atherosclerosis in SR-BI(-/-) mice. ApoA-I infusion inhibited HSPC cell proliferation, Akt phosphorylation and reactive oxygen species production in HSPC and plaque progression in low-density lipoprotein receptor knockout (LDLr(-/-)) apoA-I(-/-) mice on HFD but had no effect on SR-BI(-/-) mice on HFD. Transplantation of SR-BI(-/-) bone marrow cells into irradiated LDLr(-/-) recipients resulted in enhanced white blood cells reconstitution, inflammatory cell production, and plaque development. In patients with coronary heart disease, HDL levels were negatively correlated with white blood cells count and HSPC frequency in the peripheral blood. By flow cytometry, SR-BI expression was detected on human HSPC. CONCLUSIONS: SR-BI plays a critical role in the HDL-mediated regulation HSPC proliferation and differentiation, which is associated with atherosclerosis progression.

Wnt5a does not support hematopoiesis in stroma-free, serum-free cultures.

Previously we reported that Wnt5a is highly expressed in the murine urogenital ridge-derived UG26-1B6 cells but not embryonic liver-derived EL08-1D2 cells. Mouse long-term repopulating hematopoietic stem cells (LTR-HSC) were maintained in non-contact UG26-1B6 cultures but not EL08-1D2 non-contact cultures, unless Wnt5a was also added to the cultures, suggesting a role for Wnt5a in the in vitro maintenance of LTR-HSC. Here, we investigated if the effect of Wnt5a on adult LTR-HSC activity is HSC-autonomous. To test the effect of Wnt5a on maintenance of LTR-HSC, we performed limiting dilution competitive transplantation assays of murine Lin-Sca1(+) c-kit(+) (LSK) cells cultured for 5 days with TPO and SCF with and without Wnt5a. The effect of Wnt5a on the generation of colony forming units (CFU) and the homing ability of LSK progeny was also tested. No effects were found of Wnt5a on total cell expansion, the number of CFU, or homing ability of day 5 LSK progeny. Furthermore, addition of Wnt5a did not improve, but may have impeded maintenance of LTR-HSC. In conclusion, our data indicate that Wnt5a does not enhance the maintenance and expansion of adult murine LTR-HSCs or committed progenitors cultured in vitro in serum- and stroma-free conditions.

Glypican-3-mediated inhibition of CD26 by TFPI: a novel mechanism in hematopoietic stem cell homing and maintenance.

Directional migration determines hematopoietic stem/progenitor cell (HSPC) homing, which depends upon the interaction between the chemokine CXCL12 and its receptor CXCR4. CD26 is a widely expressed membrane-bound ectopeptidase that cleaves CXCL12 thereby depleting its chemokine activity. We identified tissue-factor pathway inhibitor (TFPI) as a biological inhibitor of CD26 in murine and human HSPCs. We observed low-level TFPI expression in endothelial cells in the bone marrow (BM), which did not increase following radiation injury. Treatment of HSPCs with TFPI in vitro led to enhanced HSPC migration toward CXCL12, as well as homing and engraftment in the BM upon transplantation. We found that Glypican-3 (GPC3), a heparan sulfate proteoglycan expressed on murine as well as human HSPCs, mediated this effect. TFPI did not affect CD26 activity, migration, or homing of GPC3(-/-) HSPCs, while it affected GPC1(-/-) HSPCs similar to wild-type HSPCs. Moreover, proliferation of GPC3(-/-) but not GPC1(-/-) BM HSPCs was significantly increased, which was associated with a decrease in the primitive HSC pool in BM and an increase in proportion of the circulating HSPCs in the peripheral blood. Hence, we present a novel role for TFPI and GPC3 in regulating HSC homing as well as retention in the BM.

A novel role of BMP4 in adult hematopoietic stem and progenitor cell homing via Smad independent regulation of integrin-α4 expression.

UNLABELLED: Although it is well established that BMP4 plays an important role in the development of hematopoietic system, it is less well understood whether BMP4 affects adult hematopoiesis and how. Here, we describe a novel mechanism by which BMP4 regulates homing of murine as well as human hematopoietic stem/progenitor cells (HSPCs). BMP4 treatment of murine BM derived c-kitLin-Sca-1 (KLS) and CD150CD48-KLS cells for up to 5 days in vitro prevented the culture-induced loss of Integrin-α4 (ITGA4) expression as well as homing. The effect on ITGA4 expression in response to BMP4 is mediated via SMAD-independent phosphorylation of p38 MAPK, which activates microphthalmia-associated transcription factor (MITF), known to induce ITGA4 expression. Elevated ITGA4 expression significantly enhanced HSPC attachment to bone marrow stromal cells, homing and long-term engraftment of the BMP4 treated cells compared with the cells cultured without BMP4. BMP4 also induced expression of ITGA4 on human BM derived Lin-CD34 cells in culture, which was associated with improved homing potential. Thus, BMP4 prevents culture-induced loss of ITGA4 expression on HSPCs in a SMAD-independent manner, resulting in improved homing of cultured HSPCs and subsequent hematopoietic reconstitution. KEY POINTS: Cytokine-induced loss of murine as well as human HSPC homing during ex vivo culture can be prevented by addition of BMP4. In HSPCs, BMP4 directly regulates Integrin-α4 expression through SMAD-independent p38 MAPK-mediated signaling.

Hematopoietic stem/progenitor cell proliferation and differentiation is differentially regulated by high-density and low-density lipoproteins in mice.

RATIONALE: Hematopoietic stem/progenitor cells (HSPC) are responsible for maintaining the blood system as a result of their self-renewal and multilineage differentiation capacity. Recently, studies have suggested that HDL cholesterol may inhibit and impaired cholesterol efflux may increase HSPC proliferation and differentiation. OBJECTIVES: We hypothesized that LDL may enhance HSPC proliferation and differentiation while HDL might have the opposing effect which might influence the size of the pool of inflammatory cells. METHODS AND RESULTS: HSPC number and function were studied in hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice on high fat diet. Hypercholesterolemia was associated with increased frequency of HSPC, monocytes and granulocytes in the peripheral blood (PB). In addition, an increased proportion of BM HSPC was in G(2)M of the cell cycle, and the percentage of HSPC and granulocyte-macrophage progenitors (GMP) increased in BM of LDLr(-/-) mice. When BM Lin-Sca-1+cKit+ (i.e. "LSK") cells were cultured in the presence of LDL in vitro we also found enhanced differentiation towards monocytes and granulocytes. Furthermore, LDL promoted lineage negative (Lin-) cells motility. The modulation by LDL on HSPC differentiation into granulocytes and motility was inhibited by inhibiting ERK phosphorylation. By contrast, when mice were infused with human apoA-I (the major apolipoprotein of HDL) or reconstituted HDL (rHDL), the frequency and proliferation of HSPC was reduced in BM in vivo. HDL also reversed the LDL-induced monocyte and granulocyte differentiation in vitro. CONCLUSION: Our data suggest that LDL and HDL have opposing effects on HSPC proliferation and differentiation. It will be of interest to determine if breakdown of HSPC homeostasis by hypercholesterolemia contributes to inflammation and atherosclerosis progression.