Mechanisms of Impaired Neutrophil Migration by Micrornas in Myelodysplastic Syndromes

Mechanisms of Impaired Neutrophil Migration by MicroRNAs in Myelodysplastic Syndromes

This information is current as Meiwan Cao, Yayoi Shikama, Hideo Kimura, Hideyoshi of September 29, 2021. Noji, Kazuhiko Ikeda, Tomoyuki Ono, Kazuei Ogawa, Yasuchika Takeishi and Junko Kimura J Immunol 2017; 198:1887-1899; Prepublished online 27 January 2017;

doi: 10.4049/jimmunol.1600622 Downloaded from http://www.jimmunol.org/content/198/5/1887

Supplementary http://www.jimmunol.org/content/suppl/2017/01/27/jimmunol.160062 Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 74 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/198/5/1887.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Mechanisms of Impaired Neutrophil Migration by MicroRNAs in Myelodysplastic Syndromes

Meiwan Cao,* Yayoi Shikama,*,† Hideo Kimura,‡ Hideyoshi Noji,x,{ Kazuhiko Ikeda,x,‖ Tomoyuki Ono,* Kazuei Ogawa,x Yasuchika Takeishi,x and Junko Kimura*

In myelodysplastic syndromes (MDS), functional defects of neutrophils result in high mortality because of infections; however, the molecular basis remains unclear. We recently found that miR-34a and miR-155 were significantly increased in MDS neutrophils. To clarify the effects of the aberrant microRNA expression on neutrophil functions, we introduced miR-34a, miR-155, or control micro- RNA into neutrophil-like differentiated HL60 cells. Ectopically introduced miR-34a and miR-155 significantly attenuated migration toward chemoattractants fMLF and IL-8, but enhanced degranulation. To clarify the mechanisms for inhibition of migration, we studied the effects of miR-34a and miR-155 on the migration-regulating Rho family members, Cdc42 and Rac1. The introduced miR-34a and miR-155 decreased the fMLF-induced active form of Cdc42 to 29.0 6 15.9 and 39.7 6 4.8% of that in the control cells, Downloaded from respectively, although Cdc42 protein levels were not altered. miR-34a decreased a Cdc42-specific guanine nucleotide exchange factor (GEF), dedicator of cytokinesis (DOCK) 8, whereas miR-155 reduced another Cdc42-specific GEF, FYVE, RhoGEF, and PH domain-containing (FGD) 4. The knockdown of DOCK8 and FGD4 by small interfering RNA suppressed Cdc42 activation and fMLF/IL-8–induced migration. miR-155, but not miR-34a, decreased Rac1 protein, and introduction of Rac1 small interfering RNA attenuated Rac1 activation and migration. Neutrophils from patients showed significant attenuation in migration compared with healthy cells, and protein levels of DOCK8, FGD4, and Rac1 were well correlated with migration toward fMLF (r = 0.642, 0.686, http://www.jimmunol.org/ and 0.436, respectively) and IL-8 (r = 0.778, 0.659, and 0.606, respectively). Our results indicated that reduction of DOCK8, FGD4, and Rac1 contributes to impaired neutrophil migration in MDS. The Journal of Immunology, 2017, 198: 1887–1899.

yelodysplastic syndromes (MDS) are a heterogeneous Neutrophil migration to infection sites is induced by chemo- group of clonal disorders characterized by ineffective attractants, such as fMLF (8, 9) and IL-8 (IL-8/CXCL8) (10, 11). M hematopoiesis resulting in numerical, morphological, Both fMLF- and IL-8/CXCL8–induced migration have been shown and functional abnormalities in blood cells of multiple lineages to be affected in MDS-derived neutrophils (12–14). A previous study

(1–4). Most notably, quantitative and qualitative defects of neu- suggested that disturbed activation of the Rac-ERK pathway and by guest on September 29, 2021 trophilic granulocytes reduce bactericidal and fungicidal activities, PI3K is responsible for the aberrant IL-8/CXCL8–induced migration resulting in life-threatening infections (5–7). MDS-derived neutro- in MDS (14). It has been reported that CD18 plays a critical role phils have demonstrated impairment in migration, production of regarding fMLF-induced migration (15), and that expression of the reactive oxygen species, and phagocytosis. However, the molecular CD11b–CD18 complex is decreased in MDS neutrophils (16). When basis of the neutrophil dysfunction has yet to be clearly defined. stimulated with fMLF, activation of ERK1/2 and protein kinase B (PKB/Akt) was also attenuated in MDS (17). Although fMLF has *Department of Pharmacology, School of Medicine, Fukushima Medical University, been shown to activate various Rho family members that play es- Fukushima 960-1295, Japan; †Center for Medical Education and Career Develop- sential roles in regulating cytoskeletal dynamics (18), there have yet ment, Fukushima Medical University, Fukushima 960-1295, Japan; ‡Department of Hematology, Kita-Fukushima Medical Center, Date 960-0502, Japan; xDepartment of to be studies on whether insufficient activation of Rho proteins is Cardiology and Hematology, School of Medicine, Fukushima Medical University, involved in aberrant fMLF-induced migration of MDS neutrophils. Fukushima 960-1295, Japan; {Department of Medical Oncology, School of Medi- ‖ Among the Rho family members, Cdc42 and Rac1 have been cine, Fukushima Medical University, Fukushima 960-1295, Japan; and Department of Blood Transfusion and Transplantation Immunology, School of Medicine, Fukushima extensively studied as key regulators for cell migration. Cdc42 is Medical University, Fukushima 960-1295, Japan required for actin polymerization and filopodial protrusion to ORCIDs: 0000-0001-5121-5130 (Y.S.); 0000-0002-5269-269X (K.I.). maintain polarity, whereas Rac1 promotes actin assembly to reg- Received for publication April 8, 2016. Accepted for publication December 30, 2016. ulate lamellipodia extension (19–21). Both Rac1 and Cdc42 act as This work was supported by Japanese Society for Promotion of Science Grants-in- molecular switches by cycling between an inactive GDP-bound Aid for Scientific Research (C) MO23591400 (to Y.S.), MO26461409 (to Y.S.), and form and an active GTP-bound form. In response to stimuli, GDP- MO24590325 (to J.K.). bound forms in the cytoplasm are recruited to the membrane, Address correspondence and reprint requests to Dr. Yayoi Shikama, Center for Med- ical Education and Career Development, Fukushima Medical University, 1 Hikarigaoka, where guanine nucleotide exchange factors (GEFs) convert GDP Fukushima 960-1295, Japan. E-mail address: [email protected] to GTP (22, 23). GEFs are categorized into two distinct classes, The online version of this article contains supplemental material. dedicator of cytokinesis (DOCK) proteins and the diffuse B cell Abbreviations used in this article: dbcAMP, dibutyryl cAMP; Dbl, diffuse B cell lymphoma (Dbl) family (23, 24). Of the DOCK proteins, DOCK8 lymphoma; dHL60, differentiated HL60; DOCK, dedicator of cytokinesis; FGD, is a Cdc42-specific GEF that critically regulates migration of FYVE, RhoGEF, and PH domain-containing; FPR, fMLF receptor; GEF, guanine nucleotide exchange factor; MDS, myelodysplastic syndromes; miRNA, microRNA; dendritic cells (25), whereas DOCK2 and DOCK5 are identified as MPO, myeloperoxidase; PKB, protein kinase B; RCMD, refractory cytopenia with potent Rac regulators in neutrophils (26). Like FYVE, RhoGEF, multilineage dysplasia; siRNA, small interfering RNA; TET2, Tet oncogene family and PH domain-containing 2 (FGD2) and FGD3, FGD4, also member 2. known as Frabin (FGD1-related F-actin binding protein), is a Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 Cdc42-specific GEF belonging to the Dbl family (27). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600622 1888 miR-34a AND miR-155 IMPAIRED NEUTROPHIL MIGRATION

We recently reported that miR-34a and miR-155 were signifi- Materials and Methods cantly increased in neutrophils isolated from MDS patients com- Blood donors, ethics, and neutrophil isolation pared with those from healthy cells (28). Aberrant expression of Peripheral blood was obtained from 12 healthy volunteers and 11 MDS microRNAs (miRNAs) affects various cell functions. It has been patients consisting of 9 subjects with refractory cytopenia with multilineage shown that miR-34a, a target of p53 (29), not only inhibits pro- dysplasia (RCMD), 1 with refractory cytopenia with unilineage dysplasia, liferation by inducing apoptosis (30), but also suppresses migra- and 1 with refractory anemia with excess blasts-2, according to the World tion and/or invasion of malignant cell lines via reduction of Health Organization 2008 classification (38). Table I summarizes the metalloproteinases and Fra-1 (31–33). Regarding miR-155, it has clinical data of the patients and the genomic information obtained by target sequencing. Although none of the patients had experienced symptomatic been reported that its overexpression accelerates proliferation of infections for at least 1 y before the blood drawing, patient 8 experienced cancer cells via repression of SHIP-1, a negative regulator of the development of pneumonia several days after blood donation for this study. Akt pathway (34), and inhibits migration of malignant and non- This study and the process of securing written informed consent from the malignant cells (35–37). Because both miR-34a and miR-155 have patients and healthy control subjects were approved by the Ethics Com- mittee of Fukushima Medical University (approval no. 1077), which is been shown to affect migration of various cell types, the aberrantly guided by local policy, national laws, and the World Medical Association increased miR-34a/miR-155 may be a cause of impaired migration Declaration of Helsinki. of MDS neutrophils. Furthermore, a database (www.microRNA. As previously described (39), the granulocyte fraction was obtained by org) predicts that miR-34a and miR-155 target Rac1 and several centrifugation through Lymphoprep (Axis-Shield, Oslo, Norway) followed Cdc42-specific GEFs. Reduction of these molecules by the over- by hypotonic lysis of erythrocytes. More than 92% of cells in the fraction were neutrophilic granulocytes, as confirmed by May-Grunwald€ and expressed miR-34a/miR-155 could impair activation of Cdc42 and Giemsa staining. Rac1, resulting in the inhibition of migration. Downloaded from In this study, we examined whether miR-34a and miR-155 Target sequencing inhibited neutrophil migration by affecting activation of Cdc42 The target sequence was carried out using the Human Myeloid Neoplasms Panel and Rac1. We demonstrated that overexpression of miR-34a and (catalog no. NGHS-003X; Qiagen, Hilden, Germany) for 50 genes (available miR-155 attenuated migration of neutrophil-like differentiated at: https://www.qiagen.com/jp/shop/sample-technologies/dna/dna-preparation/ HL60 (dHL60) cells toward fMLF/IL-8 via targeting different generead-dnaseq-gene-panels-v2?catno=NGHS-0033#geneglobe). Exons of target genes were enriched by multiplex PCR according to the manufacturer’s molecules in Cdc42/Rac-activating pathways. Furthermore, we http://www.jimmunol.org/ instructions. After verifying quality and total amount of the samples by studied the expression levels of the identified target molecules as GeneRead Library Quantification System, next-generation sequencing was well as their relationship with migratory activity in healthy and performed using MiSeq (Illumina, San Diego, CA). Sequence data were an- MDS neutrophils. alyzed with Web-based software, QIAGEN NGS Data Analysis Web Portal by guest on September 29, 2021

FIGURE 1. Differentiation of miRNA-overexpressing cells. (A) Experimental design. miR-34a, miR-155, or control miRNA was introduced into HL60 cells by electroporation. After 24 h, 500 mM dbcAMP was added into culture medium, and the cells were allowed to differentiate toward a neutrophil-like phenotype for 2 d. (B) Cell morphology and CD11b expression. To confirm cell morphology, we stained cells with May-Grunwald€ and Giemsa solutions. To measure cell surface expression of CD11b and CD18 by flow cytometry, we treated cells with mouse monoclonal anti-CD11b conjugated with PE or anti- CD18 with FITC (solid line) or mouse isotype IgG labeled with PE or FITC (broken line). Viable cells formed a single population according to forward and side scatters on flow cytometry, and this population was subjected to single-color analyses. (C) Amounts of MPO stored in dHL60 cells. (D) Amounts of elastase in dHL60 cells. Horizontal bars represent mean values of 9 to 15 experiments. The Journal of Immunology 1889 Downloaded from http://www.jimmunol.org/

FIGURE 2. Effects of miR-34a and miR-155 on degranulation and migration. (A) Effects of miRNA mimic introduction on fMLF-induced migration. (B) Effects of miRNA inhibitors on fMLF-induced migration. The cells treated with miRNA mimics or inhibitors were placed into the upper wells of a polycarbonate membrane chamber system and allowed to migrate through 3-mm pores toward the lower wells containing no chemoattractant (white column) or 10 nM fMLF (black column) at 37˚C for 90 min. The ratios of the cells that migrated to the total cells plated in the upper wells are shown as mean 6 SD of four to seven independent experiments performed in duplicate. (C) Released MPO. (D) Released elastase. Cells were incubated with indicated stimuli (200 nM fMLF, 300 nM C5a, and 5 ng/ml GM-CSF) at 37˚C and centrifuged. The substrate was added to the supernatant to measure enzyme released into the assay buffer. The y-axes indicate the ratios of released enzyme to total enzymes initially stored in granules. *p , 0.05, **p , 0.01. by guest on September 29, 2021

(https://www.qiagen.com/jp/shop/genes-and-pathways/technology-portals/ Quantification of RNA and miRNA browse-ngs/next-generation-sequencing/?workflowstep%3dd7db5450-911c- 44e6-9e30-6fb6d5e11eea). Silent mutations and known germline poly- Total cellular RNA was isolated using Isogen (Nippon Gene, Toyama, morphisms listed in public database (https://www.ncbi.nlm.nih.gov/SNP/) Japan). First-strand cDNA was synthesized as described previously (40). were excluded. Expression of three fMLF receptor (FPR) isoforms (FPR1-3) was quantified by real-time PCR in duplicate using SYBR Premix Ex Taq (Takara Preparation of cells Bio, Otsu, Japan) and normalized by b-actin (40). The primers used for FPR isoforms were FPR1 sense 59-GGCATCATCCGGTTCATCATT-39, A human leukemic cell line HL60 was cultured in RPMI 1640 (Wako antisense 59-AGGGCACTTGTCACATCCACT-39;FPR2sense59-GTCGG- Laboratory Chemicals, Osaka, Japan) supplemented with 10% (v/v) heat- ACCTTGGATTCTTGCT-39,antisense59-CTTTTTGTGGATCTTGGCTG- inactivated FBS (Nichirei Biosciences, Tokyo, Japan). Introduction of CA-39; FPR3 sense 59-CGCACAGTCAACACCATCTG-39,antisense59- 50 nM of the following was carried out by square-pulse electroporation (280 GTCATCACCCTCTTGGCCAGACTC-39. Total cellular RNA was poly- V, 12 ms) using a Gene Pulser (Bio-Rad Laboratories, Hercules, CA): adenylated and reverse transcribed using a Mir-X miRNA First-strand mirVana miRNA mimics (dsRNA oligonucleotides) of miR-34a (has-miR- Synthesis Kit (Clontech Laboratories, Mountain View, CA) to confirm the 34a-5p MC11030), miR-155 (has-miR-155-5p MC12601) and the negative effects of miRNA introduction. The miRNAs were quantified by real-time control (4464058) (Life Technologies, Carlsbad, CA), mirVana miRNA PCR using miR-34a– or miR-155–specific primer and mRQ 39 primer inhibitors (single-stranded chemically modified oligonucleotides) against (Clontech Laboratories) and normalized by U6. miR-34a (has-miR-34a-5p MH11030), miR-155 (has-miR-155-5p MH12601) and the negative control (4464076) (Life Technologies), small interfering Immunofluorescence staining RNAs (siRNAs; DOCK8: sense 59-GAGACUUACUCUUCGAAGAtt-39, antisense 59-UCUUCGAAGAGUAAGUCUCca-39, FGD4: sense 59-GAAG- Cells were stained with mouse monoclonal anti-CD11b labeled with PE GAGACUAAUGAGCAAtt-39,antisense59-UUGCUCAUUAGUCUCCUU- (eBioscience, San Diego, CA), mouse monoclonal CD18 conjugated with Cat-39, Rac1: sense 59-CUACUGUCUUUGACAAUUAtt-39,antisense59- FITC (Sony Biotechnology, San Jose, CA), or isotype match control IgG UAAUUGUCAAAGACAGUAGgg-39), and the negative control (AM4611) labeled with PE or FITC (Beckman Coulter, Marseille, France). The cell- lacking homology for any known gene sequences (Life Technologies). It surface expressions of CD11b and CD18 were analyzed by FACSCanto II was confirmed that introduction of the siRNA control did not affect ex- (BD Biosciences, San Jose, CA). pression levels of DOCK8, FGD4, and Rac1. After 24 h, 500 mMdibutyryl Migration assay cAMP (dbcAMP) (Sigma-Aldrich, St. Louis, MO) was added into the culture medium to induce differentiation toward a neutrophil-like phenotype for For the migration assay, 1 3 105 cells in the upper wells of a polycarbonate a further 48 h (Fig. 1A). For experiments using IL-8, HL60 cells were membrane chamber system with 3-mm pores (Cell Biolabs, San Diego, cultured with 1.25% DMSO for 4 d, and miRNAs or siRNAs were intro- CA) were allowed to migrate into the lower wells containing 10 nM fMLF duced by electroporation 3 d before migration assay. (Sigma-Aldrich) or 100 ng/ml IL-8 (PeproTech, Rocky Hill, NJ) for It was confirmed that the introduction of the controls for miRNA mimics 90 min at 37˚C. After aspirating out the contents remaining in the upper and inhibitors did not alter miR-34a and miR-155 levels. In each experi- wells, the cells were collected from the lower chamber and the reverse side ment, the mimics increased miR-34a/miR-155 levels by .100-fold com- of the membrane, and treated with lysis buffer including CyQuant GR dye pared with those in the control miRNA-treated cells, and the decrease by (Cell Biolabs) for 20 min at room temperature. The fluorescence of the cell inhibitors was more than 80%. lysate was immediately read at 480/520 nm. 1890 miR-34a AND miR-155 IMPAIRED NEUTROPHIL MIGRATION Downloaded from http://www.jimmunol.org/

FIGURE 3. Effects of miR-34a and miR-155 on Cdc42. (A) Cdc42 protein levels in the cells with excessive miRNA. (B) Expression of Cdc42 protein in miRNA inhibitor-treated cells. Cdc42 protein levels were analyzed by immunoblotting. The ratios of Cdc42 band intensities to those of GAPDH are shown by guest on September 29, 2021 as mean 6 SD of three to six independent experiments. (C) Effects of Cdc42 inhibition on fMLF-induced migration. A Cdc42 inhibitor ML141 was added to culture medium at the indicated concentrations, and the cells that migrated were quantiﬁed. The results shown are mean 6 SD of four to ﬁve independent experiments. *p , 0.05. (D) Activation of Cdc42. The cells were incubated with or without 10 mM fMLF for 1 min, and the GTP-bound form of Cdc42 was pulled down by p21-binding domain of human p21-activated protein kinase 1 agarose beads. The bead-bound active Cdc42 and total Cdc42 in cell lysate were visualized by immunoblotting with anti-Cdc42. The ratios of active form to total Cdc42 in fMLF-stimulated cells were calculated. In the graph, the values from the fMLF-stimulated control cells are set as 1.0, and mean 6 SD of three independent experiments are presented.

Degranulation assay tein kinase 1–bound agarose beads (Cell Biolabs) for 45 min at 4˚C. The beads were washed three times with lysis buffer and subjected to immu- The cells that were preincubated with 10 mg/ml cytochalasin B (Sigma- noblotting of Cdc42 or Rac1. Aldrich) in assay buffer (PBS containing 0.9 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES, 10 mM glucose, and 0.1% BSA [pH 7.4]) at 37˚C for Cell lysate preparation 5 min were stimulated with 200 nM fMLF (Sigma-Aldrich), 300 nM human complement C5a (Sigma-Aldrich), or 5 ng/ml human GM-CSF For detection of DOCK8, Rac1, and ERK1/2 in dHL60, the cells were lysed by (PeproTech) for 15 min and centrifuged. To measure myeloperoxidase 1% Nonidet P-40 buffer. For analyses of DOCK8 and Rac1 in the neutrophils, (MPO) activity, we added 0.3 mM H2O2 (Wako Laboratory Chemicals) Cdc42 and DOCK2 in dHL60, and FGD4 in both neutrophils and dHL60, total and 1 mM tetramethylbenzidine (Sigma-Aldrich) to both cell pellets lysed cell lysates were obtained from TCA-precipitated fraction, as previously de- by 0.5% Triton X-100 and supernatant. After incubation for 5 min at room scribed (41). RIPA buffer was used for detection of PKB/Akt in dHL60. temperature, the enzyme reaction was terminated by adding 0.8 M acetic acid and 2 mM sodium azide to immediately read the absorbance at 630 nm. Immunoblotting Elastase activity was analyzed by incubation with 0.45 mM of an elastase Proteins were separated on a 7.5, 10, or 15% NaDodSO4 polyacrylamide gel N L substrate -t-BOC- -alanine-p-nitrophenol ester (Sigma-Aldrich) for 25 min and transferred onto Immobilon-P transfer membrane (Millipore, Billerica, followed by absorbance measurement at 347 nm. MA) for blotting with anti-DOCK8 (ab175208) (Abcam, Cambridge, Pull-down assay of active Cdc42 and Rac1 U.K.), DOCK2 (ab124838; Abcam), FGD4 (ab97785; Abcam), ERK1/2 (ab184699; Abcam), pERK1/2 (Thr202/Thr204) (ab76299; Abcam), PKB/ For Cdc42 and Rac1 activity analyses, 15 3 106 and 5 3 106 cells were Akt (sc1618) (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated incubated in HBSS containing 0.1% BSA with or without fMLF, respec- PKB/Akt (Ser473) (04-736) (Millipore), or Cdc42/Rac1 (BD Biosciences), tively, at 37˚C for 1 min. After immediate centrifugation, the cell pellets respectively. The membranes blotted with the first Abs were treated with a were treated with lysis buffer (25 mM Tris [pH 7.5], 50 mM NaCl, 5 mM HRP-conjugated second Ab (Life Technologies). Anti-a-tubulin (sc-5286) MgCl2, 1 mM EDTA, 1% Nonidet P-40, 10% glycerol, inhibitors for and anti-GAPDH conjugated with HRP (sc-25778) were used as internal proteinase and phosphatase) containing 5 mM diisopropyl fluorophosphates controls. Membranes were soaked in ImmunoStar Zeta (Wako), and sig- (Sigma-Aldrich) for 20 min on ice, and centrifuged at 10,000 3 g for nals were detected using the Molecular Image ChemiDoc XRS Plus Sys- 10 min. While one tenth of the volume of the supernatant was collected for tem (Bio-Rad Laboratories). The reliability of the Abs was tested using quantification of total Cdc42 or Rac1, the remaining volume was incubated positive and negative controls (Supplemental Fig. 1), and the linearity of with 15 mg Rac/Cdc42 (p21)-binding domain of human p21-activated pro- quantification (Supplemental Fig. 2) was confirmed. The Journal of Immunology 1891

Statistical analysis wells migrated through the 3-mm pores regardless of excessive For three-group comparison, one-way ANOVA (IBM SPSS Statistics 17.0) miRNA expression. When fMLF was present in the lower chamber, was used, and the Mann–Whitney U test was used for the two-group 63.4 6 13.4% of the control cells migrated, which was significantly comparison. Correlation coefficients were obtained by bivariate correla- reduced in the miR-34a– and miR-155–overexpressing cells (42.7 6 tion. The p values ,0.05 were considered significant. Regarding expres- 14.6%, p , 0.05, and 40.3 6 9.2%, p , 0.05, respectively) (Fig. 2A). sion of miRNAs and proteins in individuals, the criteria of significant In contrast, the treatment of inhibitors against miR-34a and miR-155 increase and reduction were set as higher and lower expressions than the 6 , two SDs from mean values of healthy control subjects, respectively. increased migration in the presence of fMLF to 72.9 3.5% (p 0.05) and 68.5 6 8.2% (p , 0.05), respectively (Fig. 2B). The mRNA Results levels of three isoforms of the FPR, FPR1, FPR2, and FPR3, were not altered by overexpression of miR-34a or miR-155 (data not shown), Neither miR-34a nor miR-155 affected cell differentiation suggesting that alteration of the FRP expression was not involved in We first examined whether the introduction of miR-34a and miR-155 attenuated migration of miRNA-overexpressing cells. affected dbcAMP-induced differentiation in HL60 cells. As shown in Overexpression of miR-34a and miR-155 enhanced Fig. 1B, dbcAMP treatment induced segmented nuclei and cell-surface degranulation expression of the differentiation marker CD11b, which did not differ regardless of the ectopically introduced miRNAs. CD18 levels were We next examined the effects of miR-34a and miR-155 over- also similar among the three differentiated cells. There were no sig- expression on release of MPO and elastase. In the absence of nificant differences in the amounts of MPO (Fig. 1C) and elastase stimuli, 20–30% of total MPO and elastase were detected in the

(Fig. 1D) stored in the primary granules among the three cell types. assay buffer, which was not altered by overexpression of miRNAs. Downloaded from In contrast, when stimulated with fMLF, C5a, and GM-CSF, the Migration toward fMLF was inhibited by miR-34a and released MPO from the control cells was 81.1 6 7.0, 74.5 6 8.3, miR-155 and 59.3 6 14.1% of total MPO, respectively. The amounts of The effects of miR-34a and miR-155 on fMLF-induced migration were MPO released by these three stimuli were signiﬁcantly greater in analyzed. In the absence of fMLF, 30.0–35.0% of the cells in the upper both cells with ectopic miR-34a (90.8 6 4.6%, p , 0.05; 86.6 6 http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Modiﬁed expression of DOCK8 and FGD4. (A) DOCK8 protein levels in miRNA-introduced cells. (B) Expression of DOCK8 protein in miR-34a inhibitor-treated cells. (C) Effects of miRNA mimic introduction on FGD4 protein. (D) Expression of FGD4 in miR-155 inhibitor-introduced cells. (E) DOCK2 levels in miRNA mimic-induced cells. (F) Expression of DOCK2 in miRNA inhibitor-induced cells. Mean 6 SD of four to six independent experiments are shown. *p , 0.05. 1892 miR-34a AND miR-155 IMPAIRED NEUTROPHIL MIGRATION

4.8%, p , 0.01; 76.4 6 10.1%, p , 0.05) and miR-155 (90.5 6 DOCK8 was a target of miR-34a. Although DOCK8 was not a target 5.5%, p , 0.05; 86.1 6 5.9%, p , 0.01; 77.8 6 8.5%, p , 0.01) of miR-155, miR-155 was predicted to downregulate FGD4, another than in the control cells (Fig. 2C). Elastase released in response to Cdc42-speciﬁc GEF belonging to the Dbl family. In miR-34a–in- these three stimuli was also greater in the cells overexpressing miR- troduced cells, the DOCK8 protein levels were 68.8 6 11.1% of that 34a (59.4 6 14.4%, p , 0.05; 57.8 6 12.9%, p , 0.05; 55.1 6 in the control cells (p , 0.05), whereas miR-155 overexpression did 18.3%, p , 0.05) and miR-155 (59.2 6 11.1%, p , 0.05; 57.1 6 not alter DOCK8 levels (Fig. 4A). When the miR-34a inhibitor was 13.4%, p , 0.05; 55.4 6 11.3%, p , 0.05) than those in the control introduced to dHL60, the DOCK8 protein was increased 2.3 6 0.9- cells (45.6 6 9.8, 44.9 6 11.0, and 41.4 6 10.8%) (Fig. 2D). fold (p , 0.05) (Fig. 4B). In contrast, the FGD4 protein, which was not altered by miR-34a, decreased to 50% of that in the control cells miR-34a and miR-155 inhibited activation, but not expression, (p , 0.05) by introduction of miR-155 (Fig. 4C). The inhibitor of of Cdc42 miR-155 increased FGD4 1.7 6 0.3-fold (p , 0.05) (Fig. 4D). Among migration-regulating Rho proteins, Rac2 and RhoA have been We also measured the protein levels of another GEF decisive for shown to critically regulate degranulation in neutrophils (42–45). neutrophil migration, DOCK2, which is not a target of miR-34a or Therefore, we focused on other Rho proteins, namely Cdc42 and Rac1, miR-155. Neither mimics (Fig. 4E) nor inhibitors (Fig. 4F) of to identify molecules responsible for attenuated migration. Neither miR-34a and miR-155 altered DOCK2 levels. mimics nor inhibitors of miR-34a/miR-155 altered the expression of Cdc42 protein (Fig. 3A, 3B). In the presence of 2.5 mM ML141, a Reduction of DOCK8 and FGD4 attenuated migration and Cdc42 inhibitor, fMLF-induced migration (Fig. 3C) and Cdc42 acti- Cdc42 activation vation (data not shown) were completely blocked, conﬁrming that To clarify whether the reduction of DOCK8 and FGD4 led to the Downloaded from Cdc42 activation was essential for fMLF-induced migration. The GTP- impairment of migration, we introduced siRNAs for DOCK8 and bound active form of Cdc42, which was hardly observed by pull-down FGD4. In DOCK8 siRNA-treated cells (Fig. 5A), fMLF-induced assay without stimulation, became detectable with fMLF stimulation in migration was reduced by 84.6 6 30.7% (Fig. 5B) and correlated the control cells. Introduction of miR-34a and miR-155 resulted in a with DOCK8 protein levels (r = 0.672, p , 0.05). Elevation of decrease of active Cdc42 under stimulation with fMLF to 29.0 6 15.9 GTP-bound Cdc42 was also diminished by 94.3 6 23.5% in

and 39.7 6 4.8% of that in the control cells, respectively (Fig. 3D). DOCK8 siRNA-induced cells compared with that in the control http://www.jimmunol.org/ These data suggest that miR-34a and miR-155 did not target Cdc42 cells (Fig. 5C). When endogenous FGD4 protein was decreased to itself, but targeted some molecules involved in the activation of Cdc42. 51.7 6 24.7% by siRNA (Fig. 5D), fMLF did not increase migration (0.9 6 0.1-fold that in unstimulated control cells) miR-34a and miR-155 downregulated different Cdc42-speciﬁc GEFs (Fig. 5E). Overall, the correlation coefﬁcient between FGD4 levels According to the gene target search program microRNA.org, of the and migration was 0.880 (p , 0.05). Although fMLF upregulated DOCK family members highly expressed in neutrophils, only GTP-bound Cdc42 protein 3.9 6 1.7-fold in the control cells, by guest on September 29, 2021

FIGURE 5. Roles of DOCK8 and FGD4 in fMLF-induced migration. (A) DOCK8 levels after siRNA introduction. DOCK8 siRNA or control siRNA was introduced to HL60 cells by electroporation. The cells were subsequently cultured in the presence of 500 mM dbcAMP, and DOCK8 protein levels were analyzed by immunoblotting 72 h after electroporation. The graph shows the mean 6 SD of seven independent experiments. (B) Effects of DOCK8 siRNA on fMLF-induced migration. The ratios of migrating cells to total cells initially plated into the upper chamber are shown. The value from control siRNAis set as 1.0, and the results from three independent experiments are presented as mean 6 SD. (C) Effects of DOCK8 siRNA on Cdc42 activation. The ratios of GTP-bound Cdc42 to total Cdc42 are presented. The values of unstimulated control cells are set as 1.0, and mean 6 SD of four independent experiments are presented. (D) FGD4 protein levels after siRNA introduction. (E) Effects of FGD4 siRNA on fMLF-induced migration. The ratios of migrating cells to total cells from three independent experiments are shown as mean 6 SD. (F) Modulation of fMLF-induced Cdc42 activation by FGD4 siRNA. The ratios of GTP-bound Cdc42 to total Cdc42 from four independent experiments are shown as mean 6 SD. *p , 0.05. The Journal of Immunology 1893 Downloaded from http://www.jimmunol.org/ FIGURE 6. Roles of Rac1 in fMLF-induced migration. (A) Effects of miR-34a and miR-155 mimics on Rac1 protein. Rac1 was immunoblotted and normalized by GAPDH. The graph shown is from four to six independent experiments. (B) Effects of miR-155 inhibitor on Rac1 expression. (C) Rac1 protein levels after siRNA introduction. The results were obtained from eight independent experiments. (D) Effects of Rac1 siRNA on migration toward fMLF. The ratios of migrating cells to total cells are shown. The value from unstimulated control cells is set as 1.0, and data from five independent experiments are shown as mean 6 SD. (E) Modulation of fMLF-induced Rac1 activation by siRNA introduction. The ratios of GTP-bound Rac1 to total Rac1 in unstimulated control cells are set as 1.0, and data are shown as mean 6 SD of three independent experiments. *p , 0.05. elevation of active Cdc42 was not observed in the FGD4 siRNA- was significantly attenuated by both miR-34a (p , 0.05) and miR- treated cells stimulated with fMLF (0.7 6 0.4-fold of the unsti- 155 (p , 0.05) (Fig. 7A) and enhanced by the inhibitors (Fig. 7B). by guest on September 29, 2021 mulated control) (Fig. 5F). In the presence of ML141, migration of dHL60 toward IL-8 was inhibited, suggesting the involvement of the Cdc42 pathway (Fig. miR-155 downregulated Rac1, resulting in attenuated 7C). When DOCK8 and FGD4 were reduced by siRNA, the migration dHL60 cells barely migrated toward IL-8 (Fig. 7D, 7E). The Rac1 Another Rho protein Rac1 was identified as a target of miR-155 by inhibitor (Fig. 7F) and the introduction of Rac1 siRNA (Fig. 7G) the microRNA.org program. Endogenous Rac1 protein was signifi- also interfered with IL-8–induced migration (p , 0.05 and p , 0. cantly reduced by overexpression of miR-155 (56.2 6 27.9% of that 05, respectively). The migratory response to IL-8 was positively in the controls, p , 0.05), but not by miR-34a (113.6 6 17.8%) correlated with expression levels of DOCK8 (r = 0.650, p , 0.05), (Fig. 6A). The miR-155 inhibitor significantly increased Rac1 FGD4 (r = 0.678, p , 0.05), and Rac1 (r = 0.893, p , 0.05) (161.7 6 17.6% of that in the controls) (p , 0.05) (Fig. 6B). When levels. Thus, DOCK8, FGD4, and Rac1 were involved in not only Rac1 was decreased to 55.0 6 22.1% by siRNA (Fig. 6C), fMLF fMLF- but also IL-8–induced migration. increased migrating cells only by 16.9 6 22.0% (Fig. 6D), which miR-34a and miR-155 attenuated phosphorylation of ERK1/2, was significantly smaller than that in the control siRNA-treated cells but not PKB/Akt (78.0 6 46.5%, p , 0.05). Rac1 levels were highly correlated with fMLF-induced migration (r = 0.840, p , 0.05). As shown in We next investigated the effects of miR-34a/miR-155 on activation Fig. 6E, reduction of Rac1 concealed elevation of GTP-bound Rac1 of ERK1/2 and PKB/Akt by fMLF, which was shown to be disturbed by fMLF (0.7 6 0.3-fold that in unstimulated control cells), whereas in MDS (14, 17, 47). Both miR-34a and miR-155 significantly fMLF increased active Rac1 2.1 6 0.2-fold in the control cells. inhibited the phosphorylation of ERK1/2 by fMLF (5.8 6 3.6-fold, p , 0.05, and 5.6 6 2.8-fold, p , 0.05, respectively, versus con- DOCK8, FGD4, and Rac1 were involved in IL-8–induced trols: 10.6 6 5.3-fold) (Fig. 8A). The fMLF-induced ERK phos- migration phorylation was enhanced by both miR-34a (p , 0.05) and miR-155 The next question was whether reduction of DOCK8, FGD4, and (p , 0.05) inhibitors (Fig. 8B). In contrast, the phosphorylation of Rac1 affects migration toward IL-8, which is known to be regulated PKB/Akt by fMLF was not significantly altered by either mimics by the ERK1/2 and PKB/Akt pathways (14). Because it had been (Fig. 8C) or inhibitors of miR-34a/miR-155 (Fig. 8D). reported that dbcAMP-differentiated cells do not respond che- To examine whether the reduction of the two GEFs and Rac1 motactically to IL-8 (46), we could not detect migration of interfered with activation of ERK1/2 and Akt, we quantified fMLF- dbcAMP-treated HL60 cells to IL-8. Therefore, DMSO was used induced phosphorylation of ERK and Akt under knockdown of as a differentiation inducer. It was confirmed that the cell-surface DOCK8, FGD4, and Rac1. Compared with the control siRNA- expressions of CD11b and CD18 did not differ among the DMSO- treated cells (10.9 6 5.6-fold), ERK phosphorylation was signif- treated three cell types with ectopic miR-34a, miR-155, and icantly attenuated by silencing Rac1 (6.5 6 3.1-fold, p , 0.05), control miRNA (Supplemental Fig. 3). IL-8–induced migration but not DOCK8 (11.2 6 5.9-fold) or FGD4 (9.3 6 4.0-fold) 1894 miR-34a AND miR-155 IMPAIRED NEUTROPHIL MIGRATION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. Effects of DOCK8, FGD4, and Rac1 on IL-8–induced migration. (A) Effects of miR-34a and miR-155 mimics on IL-8–induced migration. (B) IL-8–induced migration under inhibition of miR-34a and miR-155. (C) Migration toward IL-8 in the presence of 2.5 mM ML141, a Cdc42 inhibitor. (D) Effects of DOCK8 siRNA and (E) FGD4 siRNA on migration. (F) IL-8–induced migration in the presence of 50 mM NSC23766, a Rac1 inhibitor. (G) Effects of Rac1 siRNA on migration. Data represent mean 6 SD of three to ﬁve independent experiments. *p , 0.05, **p , 0.01.

(Fig. 8E). In contrast, none of those siRNAs affected PKB/Akt protein levels were not decreased in either patient 3 or 5 with miR- phosphorylation (Fig. 8F). 155 overexpression, whereas a significant reduction of FGD4 protein was observed in patients 1, 2, 4, 8, and 10 (0.47, 0.45, 0.48, Impaired neutrophil migration was correlated with reduced 0.28, and 0.42 versus 1.00 6 0.21 in the controls, respectively). expression of DOCK8, FGD4, and Rac1 Thus, the expression levels of FGD4 protein were not correlated To study whether the reduction of DOCK8, FGD4, and Rac1 was with miR-155 levels. However, there was a strong correlation be- responsible for impaired neutrophil migration in MDS, we isolated tween FGD4 protein levels and fMLF/IL-8–induced migratory ac- peripheral neutrophils from 12 healthy volunteers and 11 MDS tivities (fMLF: r = 0.686, p , 0.01; IL-8: r = 0.659, p , 0.05) patients (Table I), including 7 with high-expression miR-34a (pa- (Fig. 9C). Although significantly low Rac1 expression was ob- tients 1–5, 7, and 10 in Fig. 9B) and 2 (patients 3 and 5) with in- served only in patient 4, who had a normal miR-155 level (0.45 creased miR-155. As shown in Fig. 9A, fMLF-induced increase of versus 1.00 6 0.20 in the controls), Rac1 protein levels positively migrating cells, which was 1.7 6 0.2-fold in the healthy control correlated with fMLF/IL-8–induced migration (fMLF: r = 0.436, subjects, was 1.1 6 0.3-fold in MDS (p , 0.001). IL-8–induced p , 0.05; IL-8: r = 0.606, p , 0.05) (Fig. 9D). Table II summarizes migration was compared between six healthy control subjects and the migratory activities and protein levels in each patient. six patients (patients 6–11), and significant attenuation was found in Taken together, the reductions of DOCK8, FGD4, and Rac1, MDS patients (1.2 6 0.1-fold versus 1.6 6 0.1-fold in the controls, which did not always coincide with miR-34a or miR-155 over- p , 0.01). expression in MDS neutrophils, correlated with attenuation of As depicted in Fig. 9B, MDS patients with high miR-34a levels migratory activity in response to fMLF and IL-8. showed significantly lower DOCK8 protein expression than the healthy volunteers (0.51 6 0.41 versus 1.00 6 0.41, p , 0.01), Discussion and inverse correlation was observed between miR-34a and Using neutrophil-like dHL60 cells, we demonstrated that cell DOCK8 levels (r = 20.464, p , 0.05). The DOCK8 levels pos- migration toward fMLF and IL-8 was attenuated by miR-34a and itively correlated with both fMLF- and IL-8–induced migration miR-155, both of which were aberrantly increased in the neutro- (fMLF: r = 0.642, p , 0.01; IL-8: r = 0.778, p , 0.01). The FGD4 phils from MDS patients. The inhibitory effects of the miRNAs on The Journal of Immunology 1895 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 8. Effects of miR-34a and miR-155 on phosphorylation of ERK1/2 and PKB/Akt. (A) Phosphorylation of ERK1/2 in response to fMLF in the cells with miRNA mimics. (B) ERK1/2 phosphorylation under miRNA inhibition. (C) Effects of miRNA mimics on fMLF-induced PKB/Akt phosphorylation. (D) PKB/Akt phosphorylation under miRNA inhibition. Effects of DOCK8, FGD4, and Rac1 siRNAs on phosphorylation of ERK1/2 (E) and PKB/Akt (F). The cells were stimulated with 1 mM fMLF for 2 min, and the reaction was terminated with ice-cold PBS. The cell lysates were subjected to immunoblotting using the indicated Abs. Mean 6 SD of the ratios of phosphorylated form to total protein from 5 to 10 independent experiments are shown. *p , 0.05. migration were facilitated by reducing two Cdc42-specific GEFs, zones to the plasma membrane, whereas inactivation of Cdc42 DOCK8 and FGD4, and Rac1. Further analyses of healthy and accelerated the production of reactive oxygen species (42, 49), MDS neutrophils indicated that reduction of these molecules was which was dependent on Rac2 activity (50, 51). Therefore, the correlated with the impaired neutrophil migration in MDS. Cdc42 activating pathway was a candidate that caused suppression Overexpression of miR-34a and miR-155 did not seem to affect of migration. In addition to Cdc42, we speculated the involvement the differentiation of HL60 induced by dbcAMP, because no of Rac1. In human neutrophils, although Rac2 is more than 80% of differences were observed in the morphology, expression of the total Rac protein (52, 53), Rac1 is essential for directed mi- CD11b/CD18, and amounts of enzymes in primary granules among gration (54–56). the cells with ectopic miR-34a, miR-155, or control miRNA. TheimpairmentinfMLF–andIL-8–inducedmigrationofneu- Therefore, the three cell types were considered to be in the same trophils, which is essential for pathogen killing at infection sites differentiation stage and available for comparison of functional (57), is a disadvantage to bactericidal functions. In contrast, en- interference by the miRNAs, and the impairment of migration was hanced enzyme release from granules could intensify bactericidal not attributable to CD11b/CD18 levels. efforts and lead to tissue damage (48). We speculate that the coin- In miR-34a– and miR-155–overexpressing cells, inhibition of cidence of attenuated migration and enhanced degranulation in- migration coincided with enhancement of degranulation, sug- creases susceptibility to local infection and deteriorates systemic gesting that Rac2 was unlikely to be involved in the inhibition of inflammation, because MPO stimulates production of ROS and migration. Degranulation is predominantly regulated by Rac2, as chemokines (58). However, according to a previous article by Dang shown by the lack of granule translocation to plasma membrane et al. (59), MPO release from MDS neutrophils was similar to that before docking and exocytosis in Rac2-deficient neutrophils (43, from healthy control subjects. This may be because of neutralization 48). In contrast, previous studies have reported cross talk between of the miR-34a/miR-155–induced enhancement by additional fac- Rac2 and another Rho family member, Cdc42, which is known as tors that have inhibitory effects on degranulation in those patients. a major regulator of neutrophil migration (42, 49). Activation of To our knowledge, it is a novel finding that DOCK8 is involved in Cdc42 inhibited the translocation of Rac2 from the perinuclear miR-34a–mediated inhibition of neutrophil migration induced by 1896 miR-34a AND miR-155 IMPAIRED NEUTROPHIL MIGRATION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 9. Migration and expression of DOCK8, FGD4, and Rac1 in neutrophils from MDS patients. (A) Comparison of neutrophil migration toward fMLF or IL-8 between healthy donors and MDS patients. Data of fMLF-induced migration was obtained from 11 healthy donors and 11 patients including 7 (1–5, 7, and 10) with significantly higher miR-34a expression and 2 (3 and 5) with overexpressed miR-155. Six patients (6–11) were subjected to comparison of IL-8–induced migration with six controls. The ratios of migrating cells to total cells in the absence of a stimulus are set as 1.0. **p , 0.01, ***p , 0.001. (B) Expression of DOCK8. The numbers above the photos indicate expression levels of miR-34a and miR-155, and ratios of band intensities of DOCK8 to those of a-tubulin in immunoblotting. The comparison of DOCK8 protein levels between the healthy control subjects and MDS patients (upper left), correlations between miR-34a and DOCK8 levels (upper right), and fMLF- (lower left) and IL-8–induced migration (lower right) are presented. Closed triangles and open squares represent healthy individuals and MDS patients, respectively. **p , 0.01. (C) Expression of FGD4. (D) Expression of Rac1. The detected FGD4 and Rac1 were normalized by GAPDH. Relationships between FGD4/Rac1 protein levels and miR-155 expression (left) and fMLF- (middle) and IL-8–induced (right) migration are shown. Closed triangles and open squares, respectively, represent healthy controls and MDS patients. NE, not examined. not only fMLF, but also IL-8. A decrease of DOCK8 by miR-34a (60), we speculate that the attenuation of ERK1/2 phosphorylation mimic and an increase of DOCK8 by miR-34a inhibitor confirmed by miR-34a was due to the reduction of MAPK kinase 1. that miR-34a targeted DOCK8, as was suggested by the micro- In MDS, DOCK8 reduction caused by miR-34a overexpression RNA.org program. DOCK2, which lacked modulation by any of seems to be one of the causes of impaired neutrophil migration. the miRNA mimics and inhibitors tested, was used as a negative Both fMLF/IL-8–induced migratory activities and DOCK8 protein control. A previous study showed that CCL21-induced dendritic levels were attenuated in MDS neutrophils, and correlation was cell migration was attenuated via impaired activation of Cdc42 at detected between DOCK8 levels and migration. Overexpression of the leading edge membrane in DOCK8 knockdown mice (25). miR-34a has previously been demonstrated in CD34+ bone mar- This report supports our findings, although we did not distinguish row cells derived from patients with early-stage MDS (61). If between local and global Cdc42 activation. In addition, miR-34a hematopoietic stem cells overexpressing miR-34a survive and inhibited the activation of ERK1/2, which is known to play a differentiate, DOCK8 protein levels could be lowered in various crucial role in regulating cellular migration (14, 17). Because the types of blood cells. DOCK8 deficiency has been shown to affect database microRNA.org indicates that MAPK kinase 1, an up- migration of dendritic cells and CD4+ T cells, B cell activation, stream activator of the ERK1/2 pathway, is a target of miR-34a and CD8+ T cell survival and function (25, 62–64). Thus, miR- The Journal of Immunology 1897

34a–mediated DOCK8 reduction may interfere with acquired and natural immunity in MDS patients. miR-155 inhibited migration via two different mechanisms from miR-34a: reduction of another Cdc42-speciﬁc GEF, FGD4; and a Rho protein Rac1, in dHL60. As the database predicted, ectopic

a miR-155 decreased FGD4 and Rac1, both of which were upregulated by the introduction of the miR-155 inhibitor. Knockdown of FGD4 and Rac1 by siRNA interfered with the activation of Cdc42 NE NE NE NE and Rac1, respectively, resulting in disturbed migration toward TET2(Q810*) TET2(S1107P) TET2(S1290L) fMLF and IL-8. Rac1 was already shown to be required for Genetic Information neutrophil migration (54, 65) However, involvement of FGD4 in

NRAS(V9F) TET2(S1107P) neutrophil migration had not been studied, whereas FGD4 had SF3B1(L1251F) SF3A1(N82I) TET2(S1107P) GATA1 (H71P) been shown to regulate motility of carcinoma cells and motor neurons (66–68). This study demonstrated that FGD4 was indis- NRAS(V9F), TET2(S1107P) NF1(T25651) pensable for regulating migration of blood cells. Overexpression of miR-155 did not affect PKB/Akt. Although attenuated activation of PBK/Akt has been reported in MDS- derived neutrophils and CD34+ cells (17, 47), our ﬁndings sug-

gest that inhibition of PKB/Akt activation is not due to increased Downloaded from CyA None None None PLTs) (RBC) Therapy miR-155. miR-155 targets both SHIP-1, a phosphatase that neg- Cytarabine, PLTs), CyA Azacitidine, atively regulates PKB/Akt (34), and positive regulators of the Akt transfusion (RBC) Transfusion (RBC) aclarubicin, G-CSF

zctdn,transfusion Azacitidine, pathway, PI3Kg subunits p84 and p101 (69). The effects of reduction of these molecules could have countervailed each other. In contrast, ERK1/2 activation was inhibited by miR-155. This effect

could be attributed to silencing Rac1, which activates ERK1/2 (14, http://www.jimmunol.org/ 70), because ERK1/2 itself has no miR-155 binding sites. As Rac1 siRNA suppressed ERK1/2 phosphorylation, miR-155 indirectly 3,del (5)(q?), XY

2 inhibited ERK1/2 activity via Rac1. Thus, miR-155 inhibited 46,XY 46,XY 46,XY 46,XX 46,XX 46,XY 2,

Cytogenetics migration via suppression of FGD4 and Rac1, but not PKB/Akt. 2

48,XY,idem,+21 In neutrophils, the elevation of miR-155 did not seem to be the 16,+mar1,+mar2, +mar3

2 only cause of suppression of FGD4 and Rac1, because expressions

+8, of FGD4 and Rac1 were preserved in the two patients with aberrantly high miR-155 levels, and miR-155–normal patients /l) by guest on September 29, 2021

10 showed a decrease in FGD4 and Rac1. This may be attributed to 10 the fact that the expression of these molecules is regulated by 3 various factors, but not by a single miRNA. For example, FGD4 is also a target of miR-143 and miR-320a, which were recently found to be downregulated and upregulated in CD34+ cells from MDS patients, respectively (61, 71). Downregulation of miR-143 8.4 2.6 9.1 12.2 9.9 24.3 9.4 2.9 47,XY, 8.4 1.2 7.7 5.1 46,XX,del (12)(p?),46,XX Transfusion (RBC, 9.6 5.9 46,XY,del (20)(q11.2g13.3) 7.2 30.5 46,XY,del (5)(q?); 46,XY Transfusion (RBC) 10.3 13.7 46,XX,add (13)(q12); 46,XX Transfusion (RBC, 10.1 11.6 15.0 13.7 46,XY,del (20)(q12q13); 46, may mask the inhibitory effect of miR-155 on FGD4, whereas increased miR-320a could result in the reduction of FGD4 without aberrant expression of miR-155. /l) Hb (g/dl) PLTs ( 9

10 Instead of the ambiguous relationship between the expressions of

3 miR-155 and its target molecules, the attenuated migration of MDS 1.9 1.5 2.2 0.7 1.7 1.1 2.2 3.8 0.4 3.1 1.5 neutrophils seemed to contribute to the suppressed expression of FGD4 and Rac1, because expression levels of FGD4 and Rac1 were signiﬁcantly correlated with fMLF- and IL-8–induced migration. Especially, FGD4, as well as DOCK8, showed a high correlation /l) Neutrophils (

9 coefﬁcient with migratory activity, suggesting that reduction of

10 DOCK8 and FGD4 may be the major regulators of migration in 3 neutrophils. In three patients (patients 3, 5, and 6), however, the reduction in FGD4, Rac1, and DOCK8 was not statistically sig- niﬁcant, whereas migratory activities were signiﬁcantly attenuated. In these patients, accumulation of a subtle decrease of multiple molecules, that is, DOCK8 and Rac1 in patient 6, may result in the impaired migration. Another possibility is that other mechanisms, such as inhibition of PKB/Akt, interfered with migratory response. The clinical relevance of the reduction of the GEFs and Rac1 needs to be further studied. Not only DOCK8 (25, 62–64), but also Rac1 (72), has been shown to affect immune cell functions. Our patients, except patient 8, however, experienced no symptomatic 1 89/M4 RCMD 70/F 2.1 RCMD 3.5 23 74/M5 89/F RCMD RCMD 72/F 3.2 RCMD 2.0 2.1 68 71/F RCMD 82/M RAEB2 3.1 1.9 7 82/M RCMD 3.1 9 64/M RCUD 6.1 No. Age (y)/Sex Subtype WBC ( Asterisk represents stop codon. Patient 1011 75/M 82/F RCMD RCMD 5.3 1.5 a CyA, cyclosporin A; F, female; Hb, hemoglobin concentration; M, male; NE,infections not examined; PLTs, platelets; RAEB2, refractory anemia with excess blasts 2; RCUD, refractory cytopenia with unilineage for dysplasia. at least 1 y. Migratory activities, expression of GEFs

Table I. Hematological and clinical ﬁndings of patients and Rac1, or any blood cell counts did not distinguish patient 8 1898 miR-34a AND miR-155 IMPAIRED NEUTROPHIL MIGRATION

Table II. Protein levels and migration of patients

Patient No. DOCK8/a-Tubulin FGD4/GADPH Rac1/GADPH fMLF-Induced Migration IL-8–Induced Migration 1 0.47 0.47a 0.96 1.25a NE 2 0.41 0.45a 0.85 1.16a NE 3 0.28 0.72 0.71 1.26a NE 4 0.45 0.48a 0.45a 0.90a NE 5 1.41 0.64 0.76 1.22a NE 6 0.44 1.11 0.61 1.07a 1.06a 7 0.13a 0.62 0.74 0.44a 0.98a 8 0.32 0.28a 0.70 1.07a 1.13a 9 0.07a 0.60 1.16 1.21a 1.31a 10 0.41 0.42a 1.00 1.45 1.27a 11 0.32 NE NE 1.33 1.25a Controls 1.00 6 0.41 1.00 6 0.21 1.00 6 0.23 1.7 6 0.2 1.6 6 0.1 aSigniﬁcantly low protein levels or migratory activities, deﬁned as a reduction of .2 SD from the mean values of the healthy controls. NE, not examined.

11. Clark-Lewis, I., B. Moser, A. Walz, M. Baggiolini, G. J. Scott, and R. Aebersold.

from others. Regarding FGD4, of which the mutations are known to Downloaded from 1991. Chemical synthesis, purification, and characterization of two inflammatory result in Charcot-Marie-Tooth disease (66, 73, 74), the effects of proteins, neutrophil activating peptide 1 (interleukin-8) and neutrophil activating FGD4 loss on hematopoiesis are not clear. It is also of interest peptide. Biochemistry 30: 3128–3135. whether the expression of GEFs and Rac1 is connected to any so- 12. Ricevuti, G., A. Mazzone, D. Pasotti, G. Fossati, I. Mazzucchelli, and A. Notario. 1993. The role of integrins in granulocyte dysfunction in myelo- matic mutations frequently observed in MDS. Although Tet dysplastic syndrome. Leuk. Res. 17: 609–619. oncogene family member 2 (TET2) was mutated in at least 6 13. Pasotti, D., A. Mazzone, G. Fossati, I. Mazzucchelli, M. C. Pistone, patients out of 11, more patients need to be studied to determine M. Montagna, N. Parachini, G. Labbate, C. Corti, and G. Ricevuti. 1993. Cor- relations between membrane integrins and granulocyte defects in myelodys- http://www.jimmunol.org/ the roles of TET2 mutations in regulation of GEFs/Rac1 expression. plastic syndromes. Recenti Prog. Med. 84: 742–749. In this study, we identified mechanisms of attenuated migration 14. Fuhler, G. M., G. J. Knol, A. L. Drayer, and E. Vellenga. 2005. Impaired by aberrantly increased miR-34a and miR-155, and demonstrated interleukin-8- and GROalpha-induced phosphorylation of extracellular signal- regulated kinase result in decreased migration of neutrophils from patients that the protein levels of DOCK8, FGD4, and Rac1 are down- with myelodysplasia. J. Leukoc. Biol. 77: 257–266. regulated in MDS neutrophils, which contributed to impaired 15. Kim, G. D., S. E. Lee, H. Yang, H. R. Park, G. W. Son, C. S. Park, and Y. S. Park. migratory activity of MDS-derived neutrophils. 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