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Recently, microRNAs (miRNAs) have been shown to be involved in hematopoietic cell development but their role in eosinophilopoeisis has not yet been described. Here we show that miR-223 is up-regulated during eosinophil differentiation in an ex vivo bone marrow derived eosinophil culture system. Targeted ablation of miR-223 leads to an increased proliferation of eosinophil progenitors. We found up-regulation of a miR-223 target gene – IGF1R in the eosinophil progenitor cultures derived from miR-223-/- mice compared to miR-223+/+ littermate controls. The increased proliferation of miR-223-/- eosinophil progenitors was reversed by treatment with the IGF1R inhibitor (picropodophyllin). Whole genome microarray analysis of differentially regulated genes between miR-223+/+ and miR-223-/- eosinophil progenitor cultures identified a specific enrichment in genes that regulate hematologic cell development. Indeed, miR-223-/- eosinophil progenitors had a delay in differentiation. Our results demonstrate that miRNAs regulate the development of eosinophils by influencing eosinophil progenitor growth and differentiation and identify a contributory role for miR-223 in this process.
Eosinophils are multifunctional effector cells implicated in the pathogenesis of a variety of diseases including asthma, eosinophil gastrointestinal disorders and helminth infection (1-3). They differentiate from a common myeloid progenitor in mice through an intermediate granulocyte/macrophage progenitor and then via an eosinophil lineage committed progenitor (4). The cytokine IL-5 is particularly important in eosinophil lineage development as it promotes the selective differentiation of eosinophils and also stimulates the release of mature eosinophils from the bone marrow (5). Indeed, eosinophil progenitors are a subset of granulocyte/macrophage progenitors that are IL5Rα positive and IL-5 induces the growth and maturation of eosinophils (4, 6).
MicoRNAs (miRNA) are short single stranded RNA molecules that silence target genes post-transcriptionally by either inhibiting protein translation or facilitating the degradation of target mRNAs. Different hematopoietic lineages have significant differences in their miRNA expression (7). Various miRNAs have been reported to regulate the differentiation and lineage commitment of hematopoietic progenitor cells (8). However, there is a lack of information on how miRNAs regulate the development of hematopoietic cells after lineage commitment. To the best of our knowledge, regulation of eosinophil progenitor cell proliferation by miRNAs has not been reported. Although multiple cytokines (e.g. IL-3, IL-5 and GM-CSF) and transcription factors (e.g. GATA-1, PU.1) have been shown to regulate the growth of eosinophil progenitors (9), other regulatory mechanisms such as miRNAs likely have a role in regulating or fine-tuning this process. Only one recent report has focused on this, showing that miR-21* could regulate the pro-survival effect of GM-CSF on eosinophils (10).
The expression of miR-223 has been shown to be driven by myeloid transcription factors PU.1 and C/EBP, factors that are important in eosinophilopoiesis (11). Here we show that miR-223 was up-regulated during eosinophil differentiation in an ex vivo bone marrow derived eosinophil culture model. Notably, miR-223 deficient eosinophil progenitor cells showed a hyperproliferative capacity. Mechanistic analysis identified a contributory role for the miR-223 target the IGF1 receptor (IGF1R) in mediating eosinophil progenitor cell proliferation. Gene expression analysis followed by systems biological analysis identified a role for miR-223 in hematopoietic development and cellular growth and function. Consistent with this prediction, miR-223-/- eosinophils had a delay in eosinophil differentiation as assessed by CCR3 expression. Our data suggest that miRNAs can directly regulate the development of eosinophils by influencing the proliferation and differentiation of eosinophil progenitor cells.
The miR-223 gene targeted mice backcrossed for 5 generations into the C57BL/6 background was previously described; the mice were kindly provided to us by Dr. Eran Hornstein (Weizmann Institute of Science, Rehovot, Israel) (12). Littermate controls were used for all experiments. All animals used were housed under specific pathogen-free conditions in accordance with institutional guidelines. The Institutional Animal Care and Use Committee of the Cincinnati Children's Hospital Medical Center approved the use of animals in these experiments.
Bone marrow cells were collected from femur and tibia of the mice and the stem/progenitor cell enriched low-density fraction were isolated by gradient centrifugation using Histopaque 1083 (Sigma) according to manufacturer's protocol. The low-density fraction of bone marrow cells were cultured in IMDM with 10% FBS, 100 U/mL penicillin, and 100 μg/ml streptomycin supplemented with 100 ng/mL stem cell factor and 100 ng/mL FLT-3 ligand (Peprotech) from day 0 to day 4. On day 4, the stem cell factor and FLT-3 ligand were replaced with 10 ng/mL IL-5 and cultured for an additional 10 - 12 days (6). The culture media was changed every other day and cells were counted and concentration was adjusted to 1×106/mL during each media change. Eosinophil maturity was assessed by FACS staining for CCR3 and Siglec-F and/or Diff-Quik staining of cytospin preparations. Eosinophil progenitor proliferation was assessed by counting the cells every 2 days using a hemacytometer.
Total RNA was isolated using miRNeasy mimi Kit according to manufacturer's protocols (Qiagen). Levels of miRNA expression were measured quantitatively by using the TaqMan MicroRNA Assay (Applied Biosystems) following the manufacturer's protocol and assayed on the Applied Biosystems 7900HT Real-Time PCR System. Normalization was performed using U6 small nuclear RNA. Relative expression was calculated using the comparative CT method as previously described (13).
Total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). All primer/probe sets were obtained from Applied Biosytems. Samples were analyzed by TaqMan qRT-PCR for Ccr3 (Assay ID: Mm01216172_m1) and normalized to Hprt1 (Assay ID: Mm00446968_m1). Relative expression was calculated using the comparative CT method.
One million cultured eosinophil progenitor cells were stained with CCR3-FITC (R&D Systems) and SiglecF-PE (BD Bioscience) as CCR3 and Siglec-F are markers for mature eosinophils. Staining was performed on ice for 30 minutes in staining buffer (0.5% BSA, 0.01% NaN3 in 1× HBSS) according to manufacturer's protocol. Data was acquired on a BD FACS Canto I flow cytometer and analyzed using FlowJo.
Cells were rapidly washed in phosphate buffered saline and lysed in M-PER mammalian protein extraction reagent (Pierce) according to manufacturer's protocol. Protease inhibitor cocktails (Pierce) and phosphatase inhibitor cocktails (Pierce) were added to the M-PER protein extraction reagent immediately before lysis. Western blot analysis was performed as previously described (14). The anti-IGF1R antibody was obtained from Cell Signaling Technology. The anti-GAPDH antibody was from Abcam.
Bone marrow derived eosinophils were resuspended at a concentration of 1×106 cells/mL and treated with DMSO or 2 μM of picropodophyllin at day 8 (15). Cell proliferation was determined by cell counting using a hemacytometer on day 10 and day 12. Cell lysates were collected on day 10 and levels of IGF1R expression was determined by western blot. To determine a dose response curve of picropodophyllin, bone marrow derived eosinophils were resuspended at a concentration of 1×106 cells/mL and plated in a 96 well plate at 100 μL per well on day 9. The cells were treated with increasing concentrations of picropodophyllin and the level of cell proliferation was determined using Cell-Titer Glo luminescent cell viability assay (Promega) according to manufacturer's protocol.
The Affymetrix Mouse Gene 1.0ST array was used to compare gene expression profiles between miR-223+/+ and miR-223-/- eosinophil progenitor cultures at day 4, day 8 and day 12. Microarray data were analyzed using the GeneSpring software (Agilent Technologies). Global scaling was performed to compare genes from chip to chip, and a base set of probes was generated by requiring a minimum raw expression level of 20th percentile out of all probes on the microarray. The resulting probe sets were then baseline transformed and filtered on at least 1.5 fold difference between miR-223+/+ and miR-223-/- eosinophil progenitor cultures. Statistical significance was determined at p < 0.05 with Benjamini Hochberg false discovery rate correction. The resulting list of genes was clustered using hierarchical clustering and a heatmap was generated. Biological functional enrichment analysis was carried out using Ingenuity Pathway Analysis (Ingenuity Systems) and ToppGene/ToppCluster (16, 17). The microarray data have been deposited into the Array Express database (www.ebi.ac.uk/arrayexpress) with accession number E-MEXP-3350 in compliance with minimum information about microarray experiment (MIAME) standards.
Statistical analyses were performed with student's t-test or one-way ANOVA with Tukey post-hoc test where appropriate. Statistical significance and the p values were indicated on the figures where appropriate. P values less than 0.05 were considered statistically significant.
Bone marrow derived eosinophils were cultured according to the schematic shown in Figure 1A. Eosinophils were obtained with high purity as determined by FACS staining for CCR3 and Siglec-F double positive cells on day 14 (Fig. 1B). There was up-regulation of miR-223 during the eosinophil differentiation culture from day 4 to day 14 (Fig. 1C), with the most prominent difference seen between days 8 and 14.
To determine the effect of miR-223 on the proliferation of eosinophil progenitor cells, we cultured bone marrow derived eosinophils from miR-223-/- mice. Compared to cultures from the wild-type littermate controls, the miR-223-/- eosinophil progenitor cultures had a prominently increased proliferation with the most prominent effect seen between day 10 and day 14 (Fig. 2A). The miR-223+/+ and miR-223-/- bone marrow derived eosinophils were morphologically indistinguishable from each other at day 8, 10, 12 or 14 (Fig. 2B, Supplemental Fig. 1).
MiR-223 has been shown to target IGF1R (12). The IGF1R is the major physiologic receptor for IGF1 (18). Since IGF1 is a major anabolic hormone that stimulates cell proliferation and is a potent inhibitor of programmed cell death, we decided to investigate whether the levels of IGF1 receptor were differentially regulated between eosinophil cultures derived from miR-223-/- mice and miR-223+/+ littermate controls. It is notable that IGF1 has not been previously examined for its impact on eosinophils or their progenitors. The IGF1R is not expressed at day 4 (Fig.3), indicating that proliferation of progenitor cells under the influence of stem cell factor and FLT-3 ligand is not likely dependent on the levels of IGF1R. However, there were substantial levels of IGF1R expression from day 10 to day 14 of the culture, coinciding with the increased proliferation seen in both the miR-223+/+ and miR-223-/- eosinophil cultures (Fig. 2A, Fig. 3). However, in the miR-223+/+ cultures, the IGF1R level was progressively decreased from day 10 to day 14, reflecting that eosinophil progenitors gradually loose their proliferation capacity during the differentiation process (Fig 2A, Fig. 3). Compared to the miR-223+/+ cultures, the miR-223-/- cultures have significantly increased levels of IGF1R at both day 12 and day 14 (Fig. 3).
To determine whether the up-regulation of IGF1 receptor was responsible for the increased proliferation seen in eosinophil cultures derived from the miR-223-/- mice, we treated eosinophil cultures on day 8 with 2 μM of an IGF1 receptor inhibitor – picropodophyllin or an equivalent volume of DMSO as a control. The DMSO treatment had no effect on eosinophil proliferation. The miR-223-/- cultures treated with DMSO had significant increases in proliferation compared to DMSO treated miR-223+/+ cultures (Fig 4A), confirming our results in Figure 2B. In contrast, treatment with 2 μM of picropodophyllin inhibited the proliferation of both miR-223+/+ and miR-223-/- cultures to a similar extent (Fig. 4A), completely reversing the increased proliferation seen in miR-223-/- cultures. We analyzed the levels of IGF1R expression on day 10 with or without picropodophyllin treatment, and found that picropodophyllin induced a nearly complete down-regulation of IGF1 receptor in both the miR-223+/+ and miR-223-/- cultures (Fig. 4B), in agreement with previous reports that picropodphyllin could inhibit IGF1R expression (19). Dose-response studies demonstrated that picropodophyllin inhibited miR-223+/+ and miR223-/- eosinophil progenitor proliferation with similar IC50 (Fig. 4C).
Interestingly, we identified a delayed up-regulation of CCR3 in miR-223-/- eosinophil progenitor cultures compared to miR-223+/+ eosinophil progenitor cultures. We performed qRT-PCR analysis of the Ccr3 level on day 8, day 10 and day 12 eosinophil progenitor cultures and found that miR-223-/- eosinophil progenitors had decreased up-regulation of Ccr3 compared with miR-223+/+ eosinophil progenitors at all three time points (Fig. 5A), indicative of decreased maturation of the miR-223-/- eosinophil progenitor cells. To determine the surface expression of CCR3 during the eosinophil progenitor culture, we performed FACS analysis of CCR3 and Siglec-F expression from day 8 to day 16 of eosinophil culture. The mature eosinophils are CCR3+ and Siglec-F+. There are less than 2% CCR3+Siglec-F+ cells during the eosinophil culture on day 8, indicating that nearly all cells are in the progenitor stage in both the miR-223+/+ and miR-223-/- cultures (Fig. 5B). While CCR3+SiglecF+ cells begin to appear on day 10, the CCR3+SiglecF+ cells in the miR-223-/- cultures were substantially less than that in the miR-223+/+ cultures. This difference is most pronounced during day 10 and day 12 of the eosinophil progenitor culture (Fig. 5B), concomitant with the onset of the increased proliferation seen in the miR-223-/- cultures (Fig. 2A). This suggests that the increased proliferation of the miR-223-/- eosinophil progenitors is associated with a delay in eosinophil differentiation.
Having identified a decreased differentiation of miR223-/- eosinophils using a targeted approach (focusing on CCR3 and Siglec-F), we were interested in extending this finding at a genome-wide level. Accordingly, a genome wide gene expression microarray analysis was performed on days 4, 8 and 12 of the eosinophil progenitor cultures. There are no differentially regulated genes at day 4 between the miR-223+/+ and miR-223-/- cultures (data not shown), supporting that the progenitor cell growth under the influence of SCF and FLT3L is not dependent on miR-223. At day 8 of the culture, we found 17 down-regulated and 16 up-regulated genes (Fig. 6A). Consistent with our prior findings, Ccr3 was down regulated at day 8 (Fig. 6A, black arrow). The full gene list is in Supplemental Table I. Functional enrichment analysis identified hematological system development and function, cell growth, and regulation of immune response as the top affected biological functions, (Fig. 6B, Supplemental Fig. 2), supporting our observation that miR-223 affects the proliferative responses in eosinophils. Analysis of genes differentially regulated at day 12 of the culture (Fig. 6C) showed similar results, where “Hematological System Development and Function” was identified as the top affected biological pathway (Fig. 6D). The full gene list is in Supplemental Table I.
In this report, we show that miR-223 regulates the proliferation and differentiation of eosinophil progenitors. We found up-regulation of miR-223 in an ex vivo eosinophil differentiation culture model. Targeted ablation of this single miRNA led to a major effect on eosinophil progenitor cells as miR-223-/- cells had a markedly increased proliferation in response to the eosinophil growth factor IL-5. Furthermore, miR-223 deficiency led to a defect in eosinophil maturation as indicated by a delayed up-regulation of surface CCR3 expression.
We found up-regulation of the miR-223 target gene IGF1R in the miR-223-/- eosinophil cultures compared to miR-223+/+ cultures (12). The up-regulation of IGF1R coincides with the onset of the increased proliferation seen in the miR-223-/- eosinophil culture. We subsequently demonstrated that the increased proliferation in the miR-223-/- culture could be reversed by treatment with an IGF1R inhibitor. Similarly, the proliferation of miR-223+/+ eosinophil progenitors could also be blocked by an IGF1R inhibitor. This indicates that the increased proliferation seen in miR-223-/- eosinophil cultures have not bypassed the IGF1R pathway. These data are the first to show that IGF1R is involved in eosinophil development. Several IGF1R inhibitors are currently under development for the treatment of various types of cancer (20). Our data indicate that the IGF1R inhibitors could potentially also be used to treat patients with eosinophilia, such as the hypereosinophilic syndrome (21). Furthermore, we have identified miR-223 as a regulator of eosinophil IGF1R levels. While the up-regulation of IGF1R likely has a contributory role in the increased proliferation seen in the miR-223-/- eosinophil progenitor cultures, we do not preclude the involvement of additional pathways. In particular, our microarray analysis identified multiple additional growth and proliferation related genes differentially regulated between miR-223+/+ and miR-223-/- cultures. These include down-regulation of NAD(P)H:quinone oxidoreductase 1 (NQO1) where NQO1 deficient mice have been found to have a significant increase in blood granulocytes including eosinophils (22). We also noted down-regulation of inhibitor of DNA binding 2 (ID2), whose knockdown has been shown to cause increased eosinophil progenitor growth and delayed eosinophil progenitor differentiation (23).
We found a delayed up-regulation of CCR3 in miR-223-/- eosinophil progenitor cultures, likely representing delayed eosinophil maturation. This is associated with the increased expression of IGF1 receptor, raising the possibility that IGF1 could negatively regulate the expression of CCR3. We did not find significant differences in the mRNA expression of eosinophil granule proteins. We measured the blood eosinophil level in vivo and did not find any difference between the miR-223+/+ and miR-223-/- mice (Supplemental Fig. 3A). This is likely due to in vivo compensation at the stage where multiplipotent progenitors are differentiated into eosinophil lineage committed progenitors. Indeed, when we measured the level of IL5Rα+CCR3+ eosinophil lineage committed progenitors in vivo, we found a decreased level of eosinophil lineage committed progenitors in the miR-223-/- mice (Supplemental Fig. 3B). This likely compensated for the increased proliferative capacity of the miR-223-/- eosinophil progenitor cells.
MiR-223 has been found to be over-expressed in asthma, eosinophilic esophagitis, and atopic dermatitis, where eosinophils are implicated in the disease pathogenesis to varying degrees (24-29). We have recently found that both miR-223 and miR-21 were up-regulated in eosinophilic esophagitis patients (28). They are the top two miRNAs correlated with eosinophil levels in patient esophageal biopsies. Using systems biology analysis, we found that miR-223 and miR-21 co-regulated a set of interacting target genes involved in eosinophil proliferation and differentiation (28). MiR-21 has been known to promote cell proliferation in various cell types by down-regulating a variety of pro-apoptotic genes both directly and indirectly (30). The up-regulation of miR-223 likely provides a check and balance in the system given the ability of miR-223 to promote eosinophil maturation. Furthermore, a recent report showed that miR-21*, a complementary miRNA of miR-21, was up-regulated after GM-CSF treatment and could inhibit the apoptosis of eosinophils (10). This indicates that the minor miRNAs could also have a role in regulating the proliferation of eosinophil progenitors, adding another level of complexity. Future therapies targeting miRNAs, including miR-21 and miR-223 and their minor miR* forms, will likely allow fine-tuing of the eosinophil level in various diseases.
In summary, we have identified miR-223 as a regulator of eosinophil progenitor proliferation. We found that IGF1R is up-regulated during in eosinophil development and miR-223 is a regulator of IGF1R levels. Further elucidating and understanding the roles and regulations of miRNAs during eosinophil development may lead to novel therapeutic targets for eosinophilic disorders.
We would like to thank Dr. Eran Hornstein for providing the miR-223 deficient mice.
This work was supported by the Ruth L. Kirschstein National Research Service Award for individual predoctoral MD/PhD fellows F30HL104892 from the National Heart Lung and Blood Institute (T.X.L), the Ryan Fellowship from Albert J. Ryan Foundation (T.X.L.), and the Organogenesis Training Grant (NIH T32HD046387 supporting T.X.L.). This work was also supported by the University of Cincinnati Medical Scientist Training Program (NIH T32GM063483). Additionally, this work was supported by NIH R01AI083450 (M.E.R), the Campaign Urging Research for Eosinophilic Disease (CURED); the Buckeye Foundation; and the Food Allergy Research Education (FARE, formerly FAI and FAAN).