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 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021 • No Triage! 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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