- Open Access
Angptl4 is upregulated under inflammatory conditions in the bone marrow of mice, expands myeloid progenitors, and accelerates reconstitution of platelets after myelosuppressive therapy
- Anne Schumacher1,
- Bernd Denecke2,
- Till Braunschweig3,
- Jasmin Stahlschmidt1,
- Susanne Ziegler1,
- Lars-Ove Brandenburg4,
- Matthias B. Stope5,
- Antons Martincuks6,
- Michael Vogt7,
- Dieter Görtz6,
- Annalisa Camporeale8,
- Valeria Poli8,
- Gerhard Müller-Newen6,
- Tim H. Brümmendorf1 and
- Patrick Ziegler1, 9Email author
© Schumacher et al. 2015
- Received: 17 December 2014
- Accepted: 7 May 2015
- Published: 9 June 2015
Upon inflammation, myeloid cell generation in the bone marrow (BM) is broadly enhanced by the action of induced cytokines which are produced locally and at multiple sites throughout the body.
Using microarray studies, we found that Angptl4 is upregulated in the BM during systemic inflammation.
Recombinant murine Angptl4 (rmAngptl4) stimulated the proliferation of myeloid colony-forming units (CFUs) in vitro. Upon repeated in vivo injections, rmAngptl4 increased BM progenitor cell frequency and this was paralleled by a relative increase in phenotypically defined granulocyte-macrophage progenitors (GMPs). Furthermore, in vivo treatment with rmAngptl4 resulted in elevated platelet counts in steady-state mice while allowing a significant acceleration of reconstitution of platelets after myelosuppressive therapy. The administration of rmAngptl4 increased the number of CD61+CD41low-expressing megakaryocytes (MK) in the BM of steady-state and in the spleen of transplanted mice. Furthermore, rmAngptl4 improved the in vitro differentiation of immature MKs from hematopoietic stem and progenitor cells. Mechanistically, using a signal transducer and activator of transcription 3 (STAT3) reporter knockin model, we show that rmAngptl4 induces de novo STAT3 expression in immature MK which could be important for the effective expansion of MKs after myelosuppressive therapy.
Whereas the definitive role of Angptl4 in mediating the effects of lipopolysaccharide (LPS) on the BM has to be demonstrated by further studies involving multiple cytokine knockouts, our data suggest that Angptl4 plays a critical role during hematopoietic, especially megakaryopoietic, reconstitution following stem cell transplantation.
- Inflammatory conditions
- Platelet reconstitution
- Myelosuppressive therapy
Hematopoiesis is a tightly regulated process that leads to the well-balanced production of myeloid, erythroid, and lymphoid cells from a small number of highly proliferative hematopoietic stem and progenitor cells (HSCs and HPCs) . During steady-state conditions, hematopoiesis is controlled by the coordinated action of a complex interplay of supporting growth factors and the signals they deliver through hematopoietic cytokine receptors expressed at the surface of HSPCs [2, 3]. These growth factors are produced in the bone marrow (BM) microenvironment or at multiple sites throughout the body from where they reach their target cells in the BM via the bloodstream [2, 4]. Broadly acting early cytokines, including interleukin (IL)-1, IL2, IL-3, IL-6, and IL-11, enhance the initial stages of hematopoietic development and can be distinguished from more late-acting hematopoietic differentiation-inducing cytokines like granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) . Pattern recognition receptors like Toll-like receptors (especially TLRs 4, 7, and 9) recognize conserved microbial products derived from exogenous pathogens . Inflammatory conditions like bacterial or viral infections increase the production and release of early- and late-acting hematopoietic cytokines, and these cytokines contribute to the rapid replenishment of consumed innate immune effector cells like granulocytes and macrophages . As a result, early hematopoiesis in the BM is significantly skewed towards myeloid cell differentiation and output, leading to an increase of myeloid colony-forming progenitor cells as well as granulocytes in the circulation, a process that is described as emergency myelopoiesis [7, 8]. In response to gram-negative infections, emergency myelopoiesis is mediated by TLR4-expressing non-hematopoietic cells, which sense systemic lipopolysaccharide and by secreting myeloid cytokines such as G-CSF, induce an adequate myelopoietic response within the BM .
The involvement of different cytokines such as G-CSF, GM-CSF, or IL-6 in regulating hematopoiesis during steady states as well as during emergency situations has been shown: respective knockout mice have defects both in production and function of myelopoietic effector cells [10–12]. However, alternative pathways are likely to exist as mice with single or combined deficiencies for G-CSF, GM-CSF, and IL-6 or G-CSF and GM-CSF are still able to mount reactive myelopoietic responses during inflammatory conditions [10, 12–14].
The goal of this study was the identification of novel cytokines with yet unknown function in the hematopoietic system. We therefore analyzed the BM of lipopolysaccharide (LPS)- and vehicle-injected wild-type (WT) mice by gene expression microarray. Among the known candidates, we identified angiopoietin-like 4 (Angptl4) as a predominantly upregulated protein in the BM during inflammatory conditions. Angptl4 has a broad range of activities on hematopoiesis acting both on early hematopoietic progenitors as well as on immature CD61+CD41low-expressing megakaryocytes (MKs). Furthermore, Angptl4 is a potent stimulator of megakaryopoiesis after myelosuppressive therapy.
For the isolation of RNA, tissue samples were homogenized in TRIzol reagent using a bead beater homogenizer (PEQLAB, Erlangen, Germany). All cell samples were lysed in TRIzol reagent, and RNA was purified according to the manufacturer’s instructions (Invitrogen, Carlsbad, USA). All RNA samples were subjected to DNAse I treatment. cDNA was synthesized using random hexamers primer and Superscript III reverse transcriptase according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). For all expression experiments, 100 ng cDNA each was analyzed. Real-time PCR for Angptl4 and G-CSF mRNA expression in the murine BM, liver, spleen, lung, and bone marrow stromal cells, as well as PCR for friend leukemia integration 1 (Fli-1), signal transducer and activator of transcription 3 (STAT3), and nuclear factor erythroid-derived 2 (NF-E2) mRNA in MK cultures was performed using a sequence detector (7500 Fast Real-Time PCR System; Invitrogen) and TaqMan target mixes (Assay-on-Demand Gene expression reagents; Invitrogen).
ELISA cytokine assay
Measurement of mouse G-CSF and Angptl4 was done in serum of LPS vs. control mice according to the manufacturer’s instructions (G-CSF, R&D Systems, Minneapolis, MN, USA; Angptl4, USCN Life Science, USA). BM plasma from the control and LPS-injected mice was prepared by flushing both femurs and tibia with 300 μl of cold PBS into Eppendorf-type centrifuge tubes. Cells/debris were removed by centrifugation at 3000 g for 10 min at 4 °C; BM plasma was stored at −20 °C. Control and LPS-stimulated bone marrow stromal cells (BMSCs) at passage 1 were grown to confluence in a T75 flask and kept for 48 h in 7 ml of Iscove’s Modified Dulbecco’s Medium (IMDM) (GIBCO; Life Technologies, Carlsbad, CA, USA) supplemented with 20 % FCS, 2 mM L-glutamine, 50 nM 2-mercaptoethanol (all reagents from Sigma-Aldrich, St. Louis, MO, USA), antibiotics (GIBCO; Life Technologies, Carlsbad, CA, USA), and with or without LPS (10 μg/ml) stimulation. Supernatants were harvested, cleared by centrifugation, and passed through a 0.45 μm filter. Culture supernatants were analyzed for G-CSF and Angptl4.
Myeloid colony-forming assays
To assess colony-forming unit (CFU) stimulation of murine cytokines, freshly isolated mononuclear BM cells (3 × 104) resuspended in IMDM and supplemented with 20 % FCS, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, stem cell factor (SCF; 10 ng/ml), fms-related tyrosine kinase 3 (FLT3; 10 ng/ml), and thrombopoietin (TPO; 50 ng/ml) were mixed with methylcellulose (Methocult M3231, 2.6 %, StemCell Technologies, Vancouver, Canada) to yield a final concentration of 0.9 % methylcellulose. Additional factors were added in the following concentrations as indicated within the figure: IL-3 (20 ng/ml), GM-CSF (50 ng/ml), G-CSF (50 ng/ml), and Angptl4 (50 ng/ml). For estimation of CFU frequency after Angptl4 stimulation in vivo, 3 × 104 cells were plated in methylcellulose mixed with IMDM (30 % FCS, 2 mM L-glutamine, 50 μM 2-mercaptoethanol) including the following factors: mIL-3 (10 ng/ml), hIL-6 (10 ng/ml), mSCF (10 ng/ml), mGM-CSF (10 ng/ml), mTPO (50 ng/ml), and huEPO (2 U/ml) (all R&D Systems, Minneapolis, MN, USA).
Lethal irradiation and transplantation
Six- to ten-week-old female B6.SJL-PtprcaPep3b/BoyJ mice were lethally irradiated with 2 × 6.5 Gy in a 4-h interval and transplanted with 5 × 105 BM mononuclear cells derived from syngeneic PBS, Angptl4, or non-injected donor mice. All mice were maintained at the animal facility of the university clinic in Aachen, Germany. All animal experiments were approved by the Federal Ministry for Nature, Environment and Consumers’ Protection of the state of North Rhine-Westphalia and were performed in accordance to the respective national, federal, and institutional regulations.
LPS and Angptl4 injection
For microarray and mRNA analysis, the mice were injected once i.p. with 50 μg LPS (1:1 mixture of Escherichia coli K12 and Salmonella minnesota) and analyzed 8 h later. For detection of BM plasma and blood serum G-CSF and Angptl4 levels, the mice were injected twice i.p. with 50 μg LPS in a 48-h interval and analyzed 24 h later. Murine recombinant Angptl4 (250 μg/kg body weight in 100 μl PBS) was injected i.p. for five consecutive days, and the mice were analyzed 48 h later.
In vitro generation of murine megakaryocytes
MKs were developed from lineage-depleted BM cells. Lin+ was depleted from mononuclear cells using a lineage cell depletion kit (Miltenyi, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Lin− cells were seeded at 1 × 105 cells per 1 ml in 48-well plates. Cells were cultured in IMDM containing bovine serum albumin, insulin, transferrin, SCF (25 ng/ml), and antibiotics. Where indicated, Angptl4 (30 ng/ml), TPO (30 ng/ml), or both were added. Cultures were performed at 37 °C in a fully humidified atmosphere of 5 % CO2. After 5 days of culture, cells were subjected to flow cytometry analysis using CD41 (eBioMWReg30) and CD61 (209.G3) antibodies, or smear preparations were prepared using cytospin and stained by the Wright-Giemsa method.
Cells were counted using flow cytometry and Flow-Count fluorospheres (Beckman Coulter, Brea, CA, USA). After washing, harvested cells were resuspended in PBS containing 10 % FCS, 2 mM EDTA, and 7-aminoactinomycin D. Immediately prior to analysis, 50 μl of Flow-Count fluorospheres were added. Absolute cell counts were automatically determined using a Gallios FACS analyzer (Beckman Coulter, Brea, CA, USA). The system software calculated cell numbers using the following formula: cells per microliter = [(viable cells counted)/(fluorospheres counted)] × fluorospheres/microliter (see Additional file 1 for supplementary methods).
Systemic inflammation regulates BM gene clusters associated with immune system process and positive regulation of cytokine production
Angptl4 is upregulated in the BM under inflammatory conditions
As different pathogenic signals evoke different cellular responses, we additionally analyzed mice with (Streptococcus pneumoniae) S.p.-induced experimental meningitis. After S.p. injection through the frontolateral skull, the mice have been shown to rapidly develop gram-positive bacteremia and a systemic inflammatory response , which is dependent on the activity of TLR-2, TLR-4, and TLR-9 [26–28]. We therefore analyzed the BM of the S.p.-injected mice for emergency myelopoiesis characteristic myeloid cell proliferation and differentiation [9, 24, 29]. As in the LPS-injected animals, mature myeloid cells (CD11b+Gr-1high) decreased upon S.p. injection, whereas the frequency of promyelocytes and myelocytes (CD11b+Gr-1low) were found to be increased (Additional file 2: Fig. S2A). Compared to the vehicle-injected animals, the S.p.-injected animals showed an increase of G-CSF and Angptl4 both in BM and in blood plasma, respectively (Additional file 2: Fig. S2B).
To identify the candidate producer cells of Angptl4 during inflammatory conditions, we purified mouse bone marrow stromal cells (BMSCs) from the BM by plastic adherence. Stromal cultures, developed from proliferating mesenchymal precursors, expressed stroma-associated surface markers and could be differentiated into adipocytes and osteoblasts (Additional file 2: Fig. S3A, B). Stimulation of murine BMSC with LPS induced the production of G-CSF and Angptl4 proteins, both of which were barely detectable at the baseline (Fig. 2d). We conclude that systemic inflammatory conditions induce Angptl4 mRNA expression at different sites throughout the body including Angptl4 protein production and release in the BM, the primary site of hematopoiesis.
Angptl4 stimulates the proliferation of myeloid progenitors in vitro and expands myeloid progenitors in vivo
In vivo treatment with Angptl4 results in elevated platelet counts in mice in steady state and after myelosuppressive therapy
Angptl4 increases the number of immature megakaryocytes in vitro
Angptl4 induces de novo STAT3 expression in CD61+CD41low/negative megakaryocytes in vitro
MK maturation is governed by the activity of a group of transcription factors, including GATA-1, ETS family members, NF-E2, and STAT3 [41, 42]. To investigate the relative mRNA expression levels of transcription factors associated with different stages of MK differentiation in response to Angptl4, we chose the ETS family member Fli-1 [43–45], STAT3 [46, 47], NF-E2 , and the GATA binding protein GATA-1. NF-E2 expression is associated with late cytoplasmic maturation stages of MK development ; Fli-1 and STAT3 are believed to be expressed on MK precursors through the promegakaryoblast stage with additional expression of STAT3 in mature megakaryocytes and platelets [46, 47, 50, 51]. GATA-1 has been shown to promote MK differentiation and to restrain abnormal immature MK expansion .
Therefore, the expression levels for Fli-1, STAT3, NF-E2, and GATA-1 mRNA reflect whether the culture contains mainly immature or mature MKs.
It was shown that GATA-1 could bind and inhibit the activity of STAT3 in MKs  and that the transgenic expression of dominant negative STAT3 causes a significant delay in PLT recovery after myelosuppression, suggesting that STAT3 is required for normal regulation of megakaryopoiesis . We therefore focused on STAT3 to determine the effects of Angptl4 on this transcription factor in more detail. To confirm that upregulation of STAT3 mRNA levels by Angptl4 faithfully reflects increased STAT3 protein levels and to identify STAT3 expression in megakaryocytic and non-megakaryocytic lineage cells in culture, we made use of a mouse model with STAT3-yellow fluorescent protein (YFP) knocked into the endogenous STAT3 locus. The mice bearing the STAT3-YFP knockin allele develop normally are fertile and display normal development of different myeloid lineages, such as monocytes, granulocytes, and MKs (data not shown). By measuring YFP, we assessed the expression of STAT3 in MK cultures at day 5 after initiating from Lin−/STAT3-YFP BM cells. In all cytokine-supplemented cultures, STAT3+ cells developed with relative number varying between 15–60 % (Additional file 2: Fig. S4A, B and data not shown) and CD61+CD41low/negative megakaryocytic lineage cells as well as CD61+CD41high megakaryocytic lineage cells could be identified. In non-megakaryocytic lineage cells defined by the lack of CD61 and CD41 surface staining, STAT3+ cells were not detected, suggesting a restriction of STAT3 expression to developing megakaryocytic lineage cells in these cultures (Fig. 7b, c). In line with that, STAT3 expression could be clearly detected in CD61+CD41low/negative as well as CD61+CD41high-expressing cells (Fig. 7c). In CD61+CD41low/negative cells, Angptl4 increased and at the same time TPO decreased the fraction of STAT3+ cells. In CD61+CD41high cells, TPO increased the percentage of STAT3+ cells whereas Angptl4 had no additional effect. The number of STAT3+ cells therefore reflects STAT3 mRNA levels and further supports the impact of Angptl4 action towards immature megakaryocytic lineage cells.
In this study, we identified Angptl4 as an upregulated protein during inflammatory conditions in the BM of mice and determined the effects of recombinant Angptl4 on early and late stages of hematopoieisis in vitro and in vivo. Angptl4 is a secreted glycoprotein with a physiological role in lipid metabolism and a predominant expression in adipose tissue and liver [20, 53, 54]. Angptl4 inhibits the activity of lipoprotein lipase and thereby promotes an increase in circulating triglyceride levels [32, 55, 56]. Further involvement of Angptl4 has been shown to occur in energy homeostasis, wound repair, tumorigenesis, angiogenesis, and redox regulation . As a member of the superfamily of Angptl proteins (including Angptl 1–7), Angptl4 shares sequence homology with angiopoietins and consists of a secretory signal peptide, an N-terminal coiled-coil domain, and a C-terminal fibrinogen-like domain . In close correlation to our data, upregulation of Angptl4 expression has been shown during acute phase responses in the LPS-treated mice in the liver, heart, muscle, and adipose tissue . It was suggested that elevated levels of Angptl4 could contribute to hypertriglyceridemia, which is associated with inflammation; however, a direct effect of Angptl4 on inflammatory pathways has not been demonstrated so far. We here connect inflammation-induced upregulation of Angptl4 with the first hallmark of emergency myelopoiesis: the increase of myeloid progenitors in the BM. However, as LPS induces the production of a variety of different cytokines and multiple cytokines display redundant functions on hematopoiesis, the definitive role of Angptl4 in mediating the effects of LPS on the BM has to be demonstrated by combining multiple cytokine knockouts.
Members of the angiopoietin-like family of proteins, including Angptl4, have been shown to bind to paired immunoglobulin-like receptors, which supports ex vivo expansion of HSCs . Remarkably, Angptl4 did not effectively stimulate expansion of HSCs. When transplanted into conditioned mice, Angptl4 expanded HSCs and supported engraftment for 4 weeks only; however, such effects were not detected 3 or 6 months after transplantation . These findings imply that Angptl4 might have effects on committed progenitor cells rather than on immature self-renewing HSCs. Indeed, our results show that Angptl4 can regulate myeloid cell proliferation at the level of HPCs, and Angptl4 therefore should be added to the growing list of early-acting cytokines such as IL-3 and IL-6. Interestingly, Angptl4, G-CSF, and GM-CSF were equally active in enhancing agents for murine BM colony formation in the presence of SCF, TPO, and Flt3L in vitro. However, when combined, Angptl4 had additive effects with GM-CSF but not with G-CSF to increase colony formation. The mechanism for this action is not known but may involve an increased sensitivity of CFUs to Angptl4 or GM-CSF through receptor upregulation or maturation of progenitors.
In contrast to the action of G-CSF, Angptl4 seems to have more local and limited effects. As the receptor of G-CSF is expressed throughout the granulocytic lineage from granulocytic precursors to mature neutrophils, its increase in the blood has an immediate impact on granulopoiesis . A massive increase in G-CSF upon LPS stimulation is therefore, as a single cytokine, sufficient to translate into leukocytosis and neutrophilia, which is observed during emergency myelopoiesis . Angptl4 has been shown to be regulated by hypoxia and chronic inflammatory responses. Angptl4 functions as a matricellular protein and by inhibiting lipoprotein lipase (LPL), Angptl4 increases serum trigylceride levels . Although Angplt 4 is seen as a physiological mediator of intracellular adipose tissue lipolysis, serum Angptl4 levels do not always correlate with plasma triglyceride levels . It has already been demonstrated that hematopoietic stem cells (HSCs) express PIRB, and PIRB therefore may function as sensor of inflammation through binding to the inflammatory Angptl4 and protecting HSCs from excessive activation and exhaustion .
To see if Angptl4 has any in vivo action, we injected mrAngptl4 into the WT mice for five consecutive days. At the level of progenitor cells, the results obtained showed that in vivo application stimulates the same types of HPCs as are stimulated in vitro by mrAngptl4. However, the effects seen after mrAngptl4 injection were relatively moderate and did not result in a significant increase of white blood cell counts. In addition, we directly assessed the effects on myeloid cells after mrAngptl4 in vivo injection as compared to LPS injection. Whereas LPS treatment clearly induced an increase in promyelocytes and myelocytes in vivo, repeated Angptl4 injection had only a marginal effect on the induction of metamyelocytes and band forms (data not shown). We therefore conclude that Angptl4 might be involved in the fine-tuning of proliferation and differentiation of leukocytes at the level of their progenitors.
In vivo mrAngptl4 applications further lead to an increase of peripheral blood PLTs and an increase of immature MKs in the BM, whereas phenotypically defined MEPs were not affected. In addition, when applied after lethal irradiation and transplantation, mrAngptl4 treatment resulted in a significantly accelerated recovery of PLTs, whereas the transplantation of mrAngptl4 in the in vivo pre-treated BM cells did not, suggesting that Angptl4 exerts its effects mainly on immature MKs downstream of CFU-Meg. In line with that, cytokines, which have been shown to be capable of modifying murine BM cells to accelerate PLT reconstitution after transplantation, such as IL-6 and TPO [36–38], also have been shown to stimulate and/or expand CFU-Meg formation in murine cultures and in vivo. Therefore, it is tempting to speculate that Angptl4 induces platelet production through the interaction with monopotent megakaryocyte-committed progenitors (MKPs) . Common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEPs) can differentiate into MKPs after 72 h in stromal cultures, indicating that MKPs are downstream of these two progenitors. MKPs are cytokine responsive progenitor cells, which do not display self-renewal activity and therefore give rise to platelets for approximately 3 weeks . When we analyzed c-kit+Sca-1−CD150+ CD41+Lin− MKPs, about 50 % of the cells were positive for PIRB (Additional file 2: Fig. S4B). These findings therefore suggest that the increased production of platelets can be mediated by direct interactions between secreted Angptl4 and its receptor, expressed on early MK-committed progenitor cells.
In contrast to MEPs, MKPs do not display any spleen colony-forming activity . The increase of megakaryopoiesis in the spleen after transplantation therefore depends on the number of MEPs, trafficking from the recovering bone marrow to the spleen. The fact that after transplantation, Angplt4 increases relative cell numbers of CD61+CD41low cells in the spleen but not in the BM (Fig. 5) therefore most likely reflects the post-transplant distribution of developing MKPs. In addition, different effects of systemic available Angptl4 on the BM and spleen cannot be excluded at this point.
After LPS injection, the TLR4−/− mice were shown to have a decreased circulating and reticulated platelet count compared to the WT mice. In the WT mice, platelets increased 1 week after a single-dose LPS injection, and this is beyond the time it takes for the circulating platelet pool to turnover in the mice . As platelets have no nuclei, these findings therefore suggest a contribution of TLRs (direct or cytokine mediated) for genomic regulation of platelet production at the level of MKs. In support for this notion are findings which demonstrate that inflammatory processes can increase MK maturation and protein content through TLR2, which may affect platelet function and thrombosis. In line with this, our findings suggest that the increased production of platelets during inflammation can be mediated by direct interactions between secreted Angptl4 and its receptor, expressed on early MK-committed progenitor cells.
The fact that after Angptl4 injection, an increase in immature megakaryocytes and a lack of increase in mature megakaryocytes lead to the production of platelets seems to be counterintuitive at first sight. The in vitro differentiation of CD61+CD41low MKs into platelet-producing CD61+CD41high MKs has been demonstrated, and by following the development of immature CD61+CD41low, mature CD61+CD41high, and platelets for several days, their in vivo dynamics after IL-11 injection suggested that the subsequent increase in CD61+CD41high MKs and platelets was due to de novo maturation from CD61+CD41low MKs . Conversely, we did not detect any increase in CD61+CD41high-expressing MKs after Angptl4 injection.
The accumulation of immature MKs could be either due to an inhibition of the maturation of MKs by Angptl4, the lack of local cytokines which promote terminal differentiation of MKs, or alternatively a proapoptotic effect on mature MKs. For instance, a large number of mature megakaryocytes that appear to undergo cell death as a result of IL-6 administration have been noted previously , and at the same time, IL-6 has a platelet-enhancing effect and promotes megakaryocyte maturation and colony formation [64, 65]. In addition, both intrinsic and extrinsic apoptosis pathways have been described to be implicated in thrombopoiesis. The effects reported after Angptl4 injection therefore may be directly or indirectly related to IL-6 and be part of a homeostatic control mechanism, counterbalancing megakaryocyte hyperplasia. As MK cells have previously been shown to produce IL-6 , we measured IL-6 levels in cytokine-supplemented cultures. After 9 days of culture, the addition of Angptl4 or TPO increased IL-6 levels as compared to culture with SCF alone, with the highest level recovered from combined Angptl4 and TPO cultures (Additional file 2: Fig. S4C).
Furthermore, it has been shown that platelets express PIRB as well as the ortholog of human leukocyte immunoglobulin-like receptor B2 (LILRB2), and Angptl2 has been shown to inhibit platelet activation partially through the PIRB pathway .
In any case, large numbers of CD61+CD41low MKs, induced by Angptl4, lead to their differentiation into CD61+CD41high MKs and an increase in rapid platelet recovery in vivo. This conclusion is strongly supported by our in vitro findings: as shown in Fig. 6b, immature CD61+CD41low/negative MK numbers are increased in Angptl4-supplemented cultures and this leads to an additive increase in mature CD61+CD41high MKs in Angptl4- and TPO-combined cultures.
Angptl4 induces STAT3 protein expression in CD61+CD41low/negative megakaryocytic lineage cells, and STAT3 has been suggested to be important for effective expansion of megakaryocytic progenitor cells in the early stage of megakaryopoiesis, as shown by the delayed recovery of PLTs after myeloablation in mice carrying a dominant negative version of STAT3 . We here establish a functional link between Angptl4 and STAT3 and propose a model in which Angptl4 through regulation of STAT3 expression expands immature MKs, which in the setting of autologous stem cell transplantation represents a potential approach to accelerate the reconstitution of megakaryopoiesis.
Based on the fact that single or combined deficiencies of known hematopoiesis supporting cytokines do not abrogate emergency hematopoiesis, we looked for inflammation-induced cytokines with a yet unknown function in the hematopoietic system. By identifying Angptl4, we provide a new player with effects both on early hematopoietic progenitors as well as on platelet production. We envision further studies aiming at a possible role of Angptl4 as a predictor of platelet engraftment after autologous stem cell transplantation or its use in the acceleration of engraftment in clinical transplantation settings.
This work was funded by a START grant of the Medical faculty of RWTH Aachen University to PZ.
- Kondo M, Wagers AJ, Manz MG, Prohaska SS, Scherer DC, Beilhack GF, et al. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu Rev Immunol. 2003;21:759–806.PubMedView ArticleGoogle Scholar
- Metcalf D. Hematopoietic cytokines. Blood. 2008;111:485–91.PubMed CentralPubMedView ArticleGoogle Scholar
- Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K, Silvennoinen O. Signaling through the hematopoietic cytokine receptors. Annu Rev Immunol. 1995;13:369–98.PubMedView ArticleGoogle Scholar
- Kaushansky K. Lineage-specific hematopoietic growth factors. N Engl J Med. 2006;354:2034–45.PubMedView ArticleGoogle Scholar
- Kumar H, Kawai T, Akira S. Toll-like receptors and innate immunity. Biochem Biophys Res Commun. 2009;388:621–5.PubMedView ArticleGoogle Scholar
- Hamilton JA. Colony-stimulating factors in inflammation and autoimmunity. Nat Rev Immunol. 2008;8:533–44.PubMedView ArticleGoogle Scholar
- Kawakami M, Tsutsumi H, Kumakawa T, Abe H, Hirai M, Kurosawa S, et al. Levels of serum granulocyte colony-stimulating factor in patients with infections. Blood. 1990;76:1962–4.PubMedGoogle Scholar
- Selig C, Nothdurft W. Cytokines and progenitor cells of granulocytopoiesis in peripheral blood of patients with bacterial infections. Infect Immun. 1995;63:104–9.PubMed CentralPubMedGoogle Scholar
- Boettcher S, Ziegler P, Schmid MA, Takizawa H, van Rooijen N, Kopf M, et al. Cutting edge: LPS-induced emergency myelopoiesis depends on TLR4-expressing nonhematopoietic cells. J Immunol. 2012;188:5824–8.PubMedView ArticleGoogle Scholar
- Hibbs ML, Quilici C, Kountouri N, Seymour JF, Armes JE, Burgess AW, et al. Mice lacking three myeloid colony-stimulating factors (G-CSF, GM-CSF, and M-CSF) still produce macrophages and granulocytes and mount an inflammatory response in a sterile model of peritonitis. J Immunol. 2007;178:6435–43.PubMedView ArticleGoogle Scholar
- Lieschke GJ, Grail D, Hodgson G, Metcalf D, Stanley E, Cheers C, et al. Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization. Blood. 1994;84:1737–46.PubMedGoogle Scholar
- Seymour JF, Lieschke GJ, Grail D, Quilici C, Hodgson G, Dunn AR. Mice lacking both granulocyte colony-stimulating factor (CSF) and granulocyte-macrophage CSF have impaired reproductive capacity, perturbed neonatal granulopoiesis, lung disease, amyloidosis, and reduced long-term survival. Blood. 1997;90:3037–49.PubMedGoogle Scholar
- Basu S, Hodgson G, Zhang HH, Katz M, Quilici C, Dunn AR. "Emergency" granulopoiesis in G-CSF-deficient mice in response to Candida albicans infection. Blood. 2000;95:3725–33.PubMedGoogle Scholar
- Walker F, Zhang HH, Matthews V, Weinstock J, Nice EC, Ernst M, et al. IL6/sIL6R complex contributes to emergency granulopoietic responses in G-CSF- and GM-CSF-deficient mice. Blood. 2008;111:3978–85.PubMedView ArticleGoogle Scholar
- Zambon AC, Gaj S, Ho I, Hanspers K, Vranizan K, Evelo CT, et al. GO-Elite: a flexible solution for pathway and ontology over-representation. Bioinformatics (Oxford, England). 2012;28:2209–10.View ArticleGoogle Scholar
- Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium Nature Genetics. 2000;25:25–9.View ArticleGoogle Scholar
- Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011;8:785–6.PubMedView ArticleGoogle Scholar
- Kim I, Kim HG, Kim H, Kim HH, Park SK, Uhm CS, et al. Hepatic expression, synthesis and secretion of a novel fibrinogen/angiopoietin-related protein that prevents endothelial-cell apoptosis. Biochem J. 2000;346(Pt 3):603–10.PubMed CentralPubMedView ArticleGoogle Scholar
- Kersten S, Mandard S, Tan NS, Escher P, Metzger D, Chambon P, et al. Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferator-activated receptor target gene. J Biol Chem. 2000;275:28488–93.PubMedView ArticleGoogle Scholar
- Yoon JC, Chickering TW, Rosen ED, Dussault B, Qin Y, Soukas A, et al. Peroxisome proliferator-activated receptor gamma target gene encoding a novel angiopoietin-related protein associated with adipose differentiation. Mol Cell Biol. 2000;20:5343–9.PubMed CentralPubMedView ArticleGoogle Scholar
- Zhang CC, Kaba M, Iizuka S, Huynh H, Lodish HF. Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells. Nat Med. 2006;12:240–5.PubMed CentralPubMedView ArticleGoogle Scholar
- Zhang CC, Kaba M, Ge G, Xie K, Tong W, Hug C, et al. Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation. Blood. 2008;111:3415–23.PubMed CentralPubMedView ArticleGoogle Scholar
- Quinton LJ, Nelson S, Boé DM, Zhang P, Zhong Q, Kolls JK, et al. The granulocyte colony-stimulating factor response after intrapulmonary and systemic bacterial challenges. J Infect Dis. 2002;185:1476–82.PubMedView ArticleGoogle Scholar
- Ueda Y, Kondo M, Kelsoe G. Inflammation and the reciprocal production of granulocytes and lymphocytes in bone marrow. J Exp Med. 2005;201:1771–80.PubMed CentralPubMedView ArticleGoogle Scholar
- Gerber J, Raivich G, Wellmer A, Noeske C, Kunst T, Werner A, et al. A mouse model of Streptococcus pneumoniae meningitis mimicking several features of human disease. Acta Neuropathol. 2001;101:499–508.PubMedGoogle Scholar
- Malley R, Henneke P, Morse SC, Cieslewicz MJ, Lipsitch M, Thompson CM, et al. Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection. Proc Natl Acad Sci U S A. 2003;100:1966–71.PubMed CentralPubMedView ArticleGoogle Scholar
- Lee KS, Scanga CA, Bachelder EM, Chen Q, Snapper CM. TLR2 synergizes with both TLR4 and TLR9 for induction of the MyD88-dependent splenic cytokine and chemokine response to Streptococcus pneumoniae. Cell Immunol. 2007;245:103–10.PubMed CentralPubMedView ArticleGoogle Scholar
- Albiger B, Dahlberg S, Sandgren A, Wartha F, Beiter K, Katsuragi H, et al. Toll-like receptor 9 acts at an early stage in host defence against pneumococcal infection. Cell Microbiol. 2007;9:633–44.PubMedView ArticleGoogle Scholar
- Lagasse E, Weissman IL. Flow cytometric identification of murine neutrophils and monocytes. J Immunol Methods. 1996;197:139–50.PubMedView ArticleGoogle Scholar
- Jaiswal S, Weissman IL. Hematopoietic stem and progenitor cells and the inflammatory response. Ann N Y Acad Sci. 2009;1174:118–21.PubMedView ArticleGoogle Scholar
- Zhang P, Welsh DA, Siggins 2nd RW, Bagby GJ, Raasch CE, Happel KI, et al. Acute alcohol intoxication inhibits the lineage- c-kit+ Sca-1+ cell response to Escherichia coli bacteremia. J Immunol. 2009;182:1568–76.PubMed CentralPubMedView ArticleGoogle Scholar
- Yoshida K, Shimizugawa T, Ono M, Furukawa H. Angiopoietin-like protein 4 is a potent hyperlipidemia-inducing factor in mice and inhibitor of lipoprotein lipase. J Lipid Res. 2002;43:1770–2.PubMedView ArticleGoogle Scholar
- Matsumura-Takeda K, Sogo S, Isakari Y, Harada Y, Nishioka K, Kawakami T, et al. CD41+/CD45+ cells without acetylcholinesterase activity are immature and a major megakaryocytic population in murine bone marrow. Stem Cells (Dayton, Ohio). 2007;25:862–70.View ArticleGoogle Scholar
- Caen JP, Han ZC, Bellucci S, Alemany M. Regulation of megakaryocytopoiesis. Haemostasis. 1999;29:27–40.PubMedGoogle Scholar
- Hofmann WK, Ottmann OG, Hoelzer D. Memorial lecture. Megakaryocytic growth factors: is there a new approach for management of thrombocytopenia in patients with malignancies? Leukemia. 1999;13(1):S14–8.PubMedView ArticleGoogle Scholar
- MacVittie TJ, Farese AM, Patchen ML, Myers LA. Therapeutic efficacy of recombinant interleukin-6 (IL-6) alone and combined with recombinant human IL-3 in a nonhuman primate model of high-dose, sublethal radiation-induced marrow aplasia. Blood. 1994;84:2515–22.PubMedGoogle Scholar
- Saitoh M, Taguchi K, Yasuda S, Kikumori M, Nishimori T, Suda M, et al. Thrombopoietic activity of recombinant human interleukin-11 in nonhuman primates with ACNU-induced thrombocytopenia. Interferon Cytokine Res. 2000;20:539–45.View ArticleGoogle Scholar
- Wagemaker G, Neelis KJ, Hartong SCC, Wognum AW, Thomas GR, Fielder PJ, et al. The efficacy of recombinant TPO in murine And nonhuman primate models for myelosuppression and stem cell transplantation. Stem Cells (Dayton, Ohio). 1998;16 Suppl 2:127–41.View ArticleGoogle Scholar
- Slayton WB, Georgelas A, Pierce LJ, Elenitoba-Johnson KS, Perry SS, Marx M, et al. The spleen is a major site of megakaryopoiesis following transplantation of murine hematopoietic stem cells. Blood. 2002;100:3975–82.PubMedView ArticleGoogle Scholar
- Zeigler FC, de Sauvage F, Widmer HR, Keller GA, Donahue C, Schreiber RD, et al. In vitro megakaryocytopoietic and thrombopoietic activity of c-mpl ligand (TPO) on purified murine hematopoietic stem cells. Blood. 1994;84:4045–52.PubMedGoogle Scholar
- Kostyak JC, Naik UP. Megakaryopoiesis: transcriptional insights into megakaryocyte maturation. Front Biosci. 2007;12:2050–62.PubMedView ArticleGoogle Scholar
- Goldfarb AN. Transcriptional control of megakaryocyte development. Oncogene. 2007;26:6795–802.PubMedView ArticleGoogle Scholar
- Athanasiou M, Clausen PA, Mavrothalassitis GJ, Zhang XK, Watson DK, Blair DG. Increased expression of the ETS-related transcription factor FLI-1/ERGB correlates with and can induce the megakaryocytic phenotype. Cell Growth Differ. 1996;7:1525–34.PubMedGoogle Scholar
- Wang X, Crispino JD, Letting DL, Nakazawa M, Poncz M, Blobel GA. Control of megakaryocyte-specific gene expression by GATA-1 and FOG-1: role of Ets transcription factors. EMBO J. 2002;21:5225–34.PubMed CentralPubMedView ArticleGoogle Scholar
- Bastian LS, Kwiatkowski BA, Breininger J, Danner S, Roth G. Regulation of the megakaryocytic glycoprotein IX promoter by the oncogenic Ets transcription factor Fli-1. Blood. 1999;93:2637–44.PubMedGoogle Scholar
- Drachman JG, Sabath DF, Fox NE, Kaushansky K. Thrombopoietin signal transduction in purified murine megakaryocytes. Blood. 1997;89:483–92.PubMedGoogle Scholar
- Kirito K, Osawa M, Morita H, Shimizu R, Yamamoto M, Oda A, et al. A functional role of Stat3 in in vivo megakaryopoiesis. Blood. 2002;99:3220–7.PubMedView ArticleGoogle Scholar
- Shivdasani RA, Rosenblatt MF, Zucker-Franklin D, Jackson CW, Hunt P, Saris CJ, et al. Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell. 1995;81:695–704.PubMedView ArticleGoogle Scholar
- Shivdasani RA. The role of transcription factor NF-E2 in megakaryocyte maturation and platelet production. Stem Cells (Dayton, Ohio). 1996;14 Suppl 1:112–5.View ArticleGoogle Scholar
- Ezumi Y, Takayama H, Okuma M. Thrombopoietin, c-Mpl ligand, induces tyrosine phosphorylation of Tyk2, JAK2, and STAT3, and enhances agonists-induced aggregation in platelets in vitro. FEBS Lett. 1995;374:48–52.PubMedView ArticleGoogle Scholar
- Pang L, Xue HH, Szalai G, Wang X, Wang Y, Watson DK, et al. Maturation stage-specific regulation of megakaryopoiesis by pointed-domain Ets proteins. Blood. 2006;108:2198–206.PubMed CentralPubMedView ArticleGoogle Scholar
- Ezoe S, Matsumura I, Gale K, Satoh Y, Ishikawa J, Mizuki M, et al. GATA transcription factors inhibit cytokine-dependent growth and survival of a hematopoietic cell line through the inhibition of STAT3 activity. J Biol Chem. 2005;280:13163–70.PubMedView ArticleGoogle Scholar
- Zhu P, Goh YY, Chin HF, Kersten S, Tan NS. Angiopoietin-like 4: a decade of research. Biosci Rep. 2012;32:211–9.PubMedView ArticleGoogle Scholar
- Mandard S, Zandbergen F, van Straten E, Wahli W, Kuipers F, Müller M, et al. The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity. J Biol Chem. 2006;281:934–44.PubMedView ArticleGoogle Scholar
- Romeo S, Yin W, Kozlitina J, Pennacchio LA, Boerwinkle E, Hobbs HH, et al. Rare loss-of-function mutations in ANGPTL family members contribute to plasma triglyceride levels in humans. J Clin Invest. 2009;119:70–9.PubMed CentralPubMedGoogle Scholar
- Ge H, Yang G, Huang L, Motola DL, Pourbahrami T, Li C. Oligomerization and regulated proteolytic processing of angiopoietin-like protein 4. J Biol Chem. 2004;279:2038–45.PubMedView ArticleGoogle Scholar
- Lu B, Moser A, Shigenaga JK, Grunfeld C, Feingold KR. The acute phase response stimulates the expression of angiopoietin like protein 4. Biochem Biophys Res Commun. 2010;391:1737–41.PubMedView ArticleGoogle Scholar
- Ortega-Senovilla H, Schaefer-Graf U, Meitzner K, Graf K, Abou-Dakn M, Herrera E. Lack of relationship between cord serum angiopoietin-like protein 4 (ANGPTL4) and lipolytic activity in human neonates born by spontaneous delivery. PLoS One. 2013;8, e81201.PubMed CentralPubMedView ArticleGoogle Scholar
- Zheng J, Umikawa M, Cui C, Li J, Chen X, Zhang C, et al. Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development. Nature. 2012;485:656–60.PubMed CentralPubMedView ArticleGoogle Scholar
- Nakorn TN, Miyamoto T, Weissman IL. Characterization of mouse clonogenic megakaryocyte progenitors. Proc Natl Acad Sci U S A. 2003;100:205–10.PubMed CentralPubMedView ArticleGoogle Scholar
- Na Nakorn T, Traver D, Weissman IL, Akashi K. Myeloerythroid-restricted progenitors are sufficient to confer radioprotection and provide the majority of day 8 CFU-S. J Clin Invest. 2002;109:1579–85.PubMed CentralPubMedView ArticleGoogle Scholar
- Jayachandran M, Brunn GJ, Karnicki K, Miller RS, Owen WG, Miller VM. In vivo effects of lipopolysaccharide and TLR4 on platelet production and activity: implications for thrombotic risk. J Appl Physiol (Bethesda, Md : 1985). 2007;102:429–33.View ArticleGoogle Scholar
- Stahl CP, Zucker-Franklin D, Evatt BL, Winton EF. Effects of human interleukin-6 on megakaryocyte development and thrombocytopoiesis in primates. Blood. 1991;78:1467–75.PubMedGoogle Scholar
- Hill RJ, Warren MK, Levin J. Stimulation of thrombopoiesis in mice by human recombinant interleukin 6. J Clin Invest. 1990;85:1242–7.PubMed CentralPubMedView ArticleGoogle Scholar
- Ishibashi T, Kimura H, Uchida T, Kariyone S, Friese P, Burstein SA. Human interleukin 6 is a direct promoter of maturation of megakaryocytes in vitro. Proc Natl Acad Sci U S A. 1989;86:5953–7.PubMed CentralPubMedView ArticleGoogle Scholar
- Wickenhauser C, Lorenzen J, Thiele J, Hillienhof A, Jungheim K, Schmitz B, et al. Secretion of cytokines (interleukins-1 alpha, -3, and -6 and granulocyte-macrophage colony-stimulating factor) by normal human bone marrow megakaryocytes. Blood. 1995;85:685–91.PubMedGoogle Scholar
- Fan X, Shi P, Dai J, Lu Y, Chen X, Liu X, et al. Paired immunoglobulin-like receptor B regulates platelet activation. Blood. 2014;124:2421–30.PubMedView ArticleGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.