Mendelian randomization studies revealed a causal role for remnant cholesterol in cardiovascular disease. Remnant particles accumulate in the arterial wall, potentially propagating local and systemic ...inflammation. We evaluated the impact of remnant cholesterol on arterial wall inflammation, circulating monocytes, and bone marrow in patients with familial dysbetalipoproteinemia (FD).
Arterial wall inflammation and bone marrow activity were measured using
F-FDG PET/CT. Monocyte phenotype was assessed with flow cytometry. The correlation between remnant levels and hematopoietic activity was validated in the CGPS (Copenhagen General Population Study). We found a 1.2-fold increase of
F-FDG uptake in the arterial wall in patients with FD (n=17, age 60±8 years, remnant cholesterol: 3.26 2.07-5.71) compared with controls (n=17, age 61±8 years, remnant cholesterol 0.29 0.27-0.40;
<0.001). Monocytes from patients with FD showed increased lipid accumulation (lipid-positive monocytes: Patients with FD 92% 86-95, controls 76% 66-81,
=0.001, with an increase in lipid droplets per monocyte), and a higher expression of surface integrins (CD11b, CD11c, and CD18). Patients with FD also exhibited monocytosis and leukocytosis, accompanied by a 1.2-fold increase of
F-FDG uptake in bone marrow. In addition, we found a strong correlation between remnant levels and leukocyte counts in the CGPS (n=103 953,
for trend 5×10-276). In vitro experiments substantiated that remnant cholesterol accumulates in human hematopoietic stem and progenitor cells coinciding with myeloid skewing.
Patients with FD have increased arterial wall and cellular inflammation. These findings imply an important inflammatory component to the atherogenicity of remnant cholesterol, contributing to the increased cardiovascular disease risk in patients with FD.
Bone marrow (BM) mesenchymal stromal cells (MSCs) provide microenvironmental support to hematopoietic stem and progenitor cells (HSPCs). Culture-expanded MSCs are interesting candidates for cellular ...therapies due to their immunosuppressive and regenerative potential which can be further enhanced by pretreatment with interferon-gamma (IFN-γ). However, it remains unknown whether IFN-γ can also influence hematopoietic support by BM-MSCs. In this study, we elucidate the impact of IFN-γ on the hematopoietic support of BM-MSCs. We found that IFN-γ increases expression of interleukin (IL)-6 and stem cell factor by human BM-MSCs. IFN-γ-treated BM-MSCs drive HSPCs toward myeloid commitment in vitro, but impair subsequent differentiation of HSPC. Moreover, IFN-γ-ARE-Del mice with increased IFN-γ production specifically lose their BM-MSCs, which correlates with a loss of hematopoietic stem cells' quiescence. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, we conclude that IFN-γ negatively affects maintenance of BM-MSCs and their hematopoietic support in vitro and in vivo.
: The bone marrow (BM) is the main site of metastases and relapse in patients with neuroblastoma (NB). BM-residing mesenchymal stromal cells (MSCs) were shown to promote tumor cell survival and ...chemoresistance. Here we characterize the MSC compartment of the metastatic NB BM niche.
: Fresh BM of 62 NB patients (all stages), and control fetal and adult BM were studied by flow cytometry using well-established MSC-markers (CD34-, CD45-, CD90+, CD105+), and CD146 and CD271 subtype-markers. FACS-sorted BM MSCs and tumor cells were validated by qPCR. Moreover, isolated MSCs were tested for multilineage differentiation and Colony-forming-unit-fibroblasts (CFU-Fs) capacity.
: Metastatic BM contains a higher number of MSCs (
< 0.05) with increased differentiation capacity towards the osteoblast lineage. Diagnostic BM contains a MSC-subtype (CD146+CD271-), only detected in BM of patients with metastatic-NB, determined by flow cytometry. FACS-sorting clearly discriminated MSC(-subtypes) and NB fractions, validated by mRNA and DNA qPCR. Overall, the CD146+CD271- subtype decreased during therapy and was detected again in the majority of patients at relapse.
: We demonstrate that the neuroblastoma BM-MSC compartment is different in quantity and functionality and contains a metastatic-niche-specific MSC-subtype. Ultimately, the MSCs contribution to tumor progression could provide targets with potential for eradicating resistant metastatic disease.
Cell adhesion and migration are important determinants of homing and development of hematopoietic stem and progenitor cells (HSPCs) in bone marrow (BM) niches. The extracellular matrix protein ...transforming growth factor-β (TGF-β) inducible gene H3 (BIGH3) is involved in adhesion and migration, although the effect of BIGH3 is highly cell type-dependent. BIGH3 is abundantly expressed by mesenchymal stromal cells, while its expression in HSPCs is relatively low unless induced by certain BM stressors. Here, we set out to determine how BIGH3 modulates HSPC adhesion and migration. We show that primary HSPCs adhere to BIGH3-coated substrates, which is, in part, integrin-dependent. Overexpression of BIGH3 in HSPCs and HL60 cells reduced the adhesion to the substrate fibronectin in adhesion assays, which was even more profound in electrical cell-substrate impedance sensing (ECIS) assays. Accordingly, the CXCL12 induced migration over fibronectin-coated surface was reduced in BIGH3-expressing HSPCs. The integrin expression profile of HSPCs was not altered upon BIGH3 expression. Although expression of BIGH3 did not alter actin polymerization in response to CXCL12, it inhibited the PMA-induced activation of the small GTPase RAC1 as well as the phosphorylation and activation of extracellular-regulated kinases (ERKs). Reduced activation of ERK and RAC1 may be responsible for the inhibition of cell adhesion and migration by BIGH3 in HSPCs. Induced BIGH3 expression upon BM stress may contribute to the regulation of BM homeostasis.
Abstract
Aim
Preclinical work indicates that low-density lipoprotein cholesterol (LDL-C) not only drives atherosclerosis by directing the innate immune response at plaque level but also augments ...proinflammatory monocyte production in the bone marrow (BM) compartment. In this study, we aim to unravel the impact of LDL-C on monocyte production in the BM compartment in human subjects.
Methods and results
A multivariable linear regression analysis in 12 304 individuals of the EPIC-Norfolk prospective population study showed that LDL-C is associated with monocyte percentage (β = 0.131 95% CI: 0.036–0.225; P = 0.007), at the expense of granulocytes (β = −0.876 95% CI: −1.046 to −0.705; P < 0.001). Next, we investigated whether altered haematopoiesis could explain this monocytic skewing by characterizing CD34+ BM haematopoietic stem and progenitor cells (HSPCs) of patients with familial hypercholesterolaemia (FH) and healthy normocholesterolaemic controls. The HSPC transcriptomic profile of untreated FH patients showed increased gene expression in pathways involved in HSPC migration and, in agreement with our epidemiological findings, myelomonocytic skewing. Twelve weeks of cholesterol-lowering treatment reverted the myelomonocytic skewing, but transcriptomic enrichment of monocyte-associated inflammatory and migratory pathways persisted in HSPCs post-treatment. Lastly, we link hypercholesterolaemia to perturbed lipid homeostasis in HSPCs, characterized by lipid droplet formation and transcriptomic changes compatible with increased intracellular cholesterol availability.
Conclusions
Collectively, these data highlight that LDL-C impacts haematopoiesis, promoting both the number and the proinflammatory activation of circulating monocytes. Furthermore, this study reveals a potential contributory role of HSPC transcriptomic reprogramming to residual inflammatory risk in FH patients despite cholesterol-lowering therapy.
Graphical Abstract
LDL-C impacts hematopoiesis, promoting both the number and the proinflammatory activation of circulating monocytes.
Experimental work posits that acute ischaemic events trigger haematopoietic activity, driving monocytosis, and atherogenesis. Considering the chronic low-grade inflammatory state in atherosclerosis, ...we hypothesized that haematopoietic hyperactivity is a persistent feature in cardiovascular disease (CVD). Therefore, we aimed to assess the activity of haematopoietic organs and haematopoietic stem and progenitor cells (HSPCs) in humans.
First, we performed 18F-fluorodeoxyglucose positron emission tomographic (18F-FDG PET) imaging in 26 patients with stable atherosclerotic CVD (ischaemic event >12 months ago), and 25 matched controls. In splenic tissue, 18F-FDG uptake was 2.68 ± 0.65 in CVD patients vs. 1.75 ± 0.54 in controls (1.6-fold higher; P< 0.001), and in bone marrow 3.20 ± 0.76 vs. 2.72 ± 0.46 (1.2-fold higher; P = 0.003), closely related to LDL cholesterol levels (LDLc, r = 0.72). Subsequently, we determined progenitor potential of HSPCs harvested from 18 patients with known atherosclerotic CVD and 30 matched controls; both groups were selected from a cohort of cancer patients undergoing autologous stem cell transplantation. In CVD patients, the normalized progenitor potential, expressed as the number of colony-forming units-granulocyte/monocyte (CFU-GM) colonies/CD34+ cell, was 1.6-fold higher compared with matched controls (P < 0.001). Finally, we assessed the effects of native and oxidized lipoproteins on HSPCs harvested from healthy donors in vitro. Haematopoietic stem and progenitor cells displayed a 1.5-fold increased CFU-GM capacity in co-culture with oxidized LDL in vitro (P = 0.002), which was inhibited by blocking oxidized phospholipids via E06 (P = 0.001).
Collectively, these findings strengthen the case for a chronically affected haematopoietic system, potentially driving the low-grade inflammatory state in patients with atherosclerosis.
Culture expanded mesenchymal stromal cells (MSCs) are being extensively studied for therapeutic applications, including treatment of graft-versus-host disease, osteogenesis imperfecta and for ...enhancing engraftment of hematopoietic stem cells after transplantation. Thus far, clinical trials have shown that the therapeutic efficiency of MSCs is variable, which may in part be due to inefficient cell migration. Here we demonstrate that human MSCs display remarkable low migratory behaviour compared to other mesodermal-derived primary human cell types. We reveal that specifically in MSCs the nucleus is irregularly shaped and nuclear lamina are prone to wrinkling. In addition, we show that expression of Lamin A/C is relatively high in MSCs. We further demonstrate that in vitro MSC migration through confined pores is limited by their nuclei, a property that might correlate to the therapeutic inefficiency of administered MSC in vivo. Silencing expression of Lamin A/C in MSCs improves nuclear envelope morphology, promotes the protrusive activity of MSCs through confined pores and enhances their retention in the lung after intravenous administration in vivo. Our findings suggest that the intrinsic nuclear lamina properties of MSCs underlie their limited capacity to migrate, and that strategies that target the nuclear lamina might alter MSC-based cellular therapies.
Type 3 innate lymphoid cells (ILC3) are important in tissue homeostasis. In the gut, ILC3 repair damaged epithelium and suppress inflammation. In allogeneic hematopoietic cell transplantation (HCT), ...ILC3 protect against graft-versus-host disease (GvHD), most likely by restoring tissue damage and preventing inflammation. We hypothesize that supplementing HCT grafts with interleukin-22 (IL-22)-producing ILC3 may prevent acute GvHD. We therefore explored ex vivo generation of human IL-22-producing ILC3 from hematopoietic stem and progenitor cells (HSPC) obtained from adult, neonatal and fetal sources. We established a stroma-free system culturing human cord blood-derived CD34
HSPC with successive cytokine mixes for 5 weeks. We analyzed the presence of phenotypically defined ILC, their viability, proliferation and IL-22 production (after stimulation) by flow cytometry and enzyme-linked immunosorbent assay (ELISA). We found that the addition of recombinant human IL-15 and the enhancer of zeste homolog 1/2 inhibitor UNC1999 promoted ILC3 generation. Similar results were demonstrated when UNC1999 was added to CD34
HSPC derived from healthy adult granulocyte colony-stimulating factor mobilized peripheral blood and bone marrow, but not fetal liver. UNC1999 did not negatively impact IL-22 production in any of the HSPC sources. Finally, we observed that autologous HSPC mobilized from the blood of adults with hematological malignancies also developed into ILC3, albeit with a significantly lower capacity. Together, we developed a stroma-free protocol to generate large quantities of IL-22-producing ILC3 from healthy adult human HSPC that can be applied for adoptive transfer to prevent GvHD after allogeneic HCT.
It has been proposed that differences may exist between umbilical cord blood (CB) platelets and adult peripheral blood (APB) platelets, including altered protein levels of the main platelet ...integrins. We have now compared the protein expression profiles of CB and APB platelets employing a label-free comparative proteomics approach. Aggregation studies showed that CB platelets effectively aggregate in the presence of thromboxane A2 analogue, collagen, and peptide agonists of the proteinase-activated receptors 1 and 4. In agreement with previous studies, higher concentrations of the agonists were required to initiate aggregation in the CB platelets. Mass spectrometry analysis revealed no significant difference in the expression levels of critical platelet receptors like glycoprotein (GP)Ib, GPV, GPIX, and integrin αIIbβ3. This was confirmed using flow cytometry-based approaches. Gene ontology enrichment analysis revealed that elevated proteins in CB platelets were in particular enriched in proteins contributing to mitochondrial energy metabolism processes. The reduced proteins were enriched in proteins involved in, among others, platelet degranulation and activation. In conclusion, this study reveals that the CB and APB platelets are distinct. In particular, changes were observed for proteins that belong to metabolic and energy generation processes and not for the critical adhesive platelet integrins and glycoproteins.
Maintenance of hematopoietic stem cells (HSCs) and regulation of their quiescence and self-renewal is critical for maintaining a lifelong supply of blood cells. The ability of HSCs to stay quiescent ...is thought to depend on their specific niche in the bone marrow (BM). Mesenchymal stromal cells (MSC) in the BM are multipotent stem cells that form part of the vascular HSC niche and provide micro-environmental support to HSCs both in vivo and upon expansion ex vivo. Culture-expanded MSCs also exhibit immunomodulatory properties that can be enhanced by pre-treatment with interferon-gamma (IFN-γ). BM MSC are thus attractive candidates for cellular therapy after hematopoietic stem cell transplantation, for promoting rapid hematopoietic recovery and reducing the incidence or severity of graft versus host disease. Although IFN-γ pre-treatment can improve the immunomodulatory properties of MSCs, elevated IFN-γ levels have also been associated with anemia and BM failure in multiple chronic inflammatory diseases. While the impact of IFN-γ on HSC has been elucidated in recent years, it remains largely unknown whether IFN-γ can also influence hematopoietic support by BM stromal cells.
In this study, we aim to elucidate the impact of IFN-γ on hematopoietic support of BM MSC.
We show that in vitro expansion of primary BM MSC cultures from healthy donors was significantly reduced in the presence of IFN-γ, and this effect could be reproduced in the BM stromal cell line MS-5. Concurrently, IFN-γ diminished the clonal capacity of BM MSC, as measured by CFU-F assays. In addition, BM MSC that were pre-stimulated with IFN-γ produced significantly lower levels of CXCL12, suggesting a loss of hematopoietic support potential. Indeed, support of CD34+ hematopoietic stem and progenitor cells (HSPC) in a co-culture assay was greatly reduced in when MSC were pre-treated with IFN-γ.
To determine the impact of IFN-γ on BM MSC in vivo, we investigated the BM stromal compartment of IFN-γ AU-rich element deleted (ARE-Del) mice, which constitutively express IFN-γ in steady state conditions. FACS analysis revealed a remodeling of the BM stromal compartment in ARE-Del mice compared to littermate controls, with significantly fewer MSCs, identified as CD45-Ter119-CD31-CD51+PDGFRa+ cells. Numbers of other stromal cell subsets, such as osteoblasts and fibroblasts, were not altered.
The reduction of BM MSC in ARE-Del mice coincided with a loss of quiescence in HSCs; only 35% of long term HSC (LT-HSC) in ARE-Del mice were quiescent, compared to 70% in WT mice, as determined by Ki-67 staining. Loss of quiescence in LT-HSC did not lead to increased self-renewal, but rather induced increased differentiation towards short-term HSC and multi-potent progenitors.
We then sorted LT-HSC from WT and ARE-Del mice and performed in vitro HSC culture assays in the absence of IFN-γ. Absolute numbers of LT-HSC were rapidly decreased in ARE-Del compared to WT cultures after 3 and 7 days of HSC culture, while numbers of more differentiated progenitors were increased. These data indicate that an IFN-γ-mediated loss of BM MSC in ARE-Del mice leads to loss of quiescent LT-HSCs and induces a tendency towards HSC differentiation over self-renewal.
In conclusion, we have shown that IFN-γ has a negative impact on expansion and hematopoietic support of BM MSC in vitro and in vivo across species. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, it is obvious from our data that IFN-γ impairs their HSC supporting function. These data also provide more insight in the underlying mechanism by which IFN-γ contributes to the pathogenesis of anemia and BM failure.
No relevant conflicts of interest to declare.
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