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Mir-223 regulates the number and function of myeloid-derived suppressor cells in multiple sclerosis and experimental autoimmune encephalomyelitis

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Abstract

Myeloid-derived cells play important modulatory and effector roles in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells, composed of monocytic (MO) and polymorphonuclear (PMN) fractions, which can suppress T cell activities in EAE. Their role in MS remains poorly characterized. We found decreased numbers of circulating MDSCs, driven by lower frequencies of the MO-MDSCs, and higher MDSC expression of microRNA miR-223 in MS versus healthy subjects. To gain mechanistic insights, we interrogated the EAE model. MiR-223 knock out (miR-223−/−) mice developed less severe EAE with increased MDSC numbers in the spleen and spinal cord compared to littermate controls. MiR-223−/− MO-MDSCs suppressed T cell proliferation and cytokine production in vitro and EAE in vivo more than wild-type MO-MDSCs. They also displayed an increased expression of critical mediators of MDSC suppressive function, Arginase-1(Arg1), and the signal transducer and activator of transcription 3 (Stat3), which herein, we demonstrate being an miR-223 target gene. Consistently, MDSCs from MS patients displayed decreased STAT3 and ARG1 expression compared with healthy controls, suggesting that circulating MDSCs in MS are not only reduced in numbers but also less suppressive. These results support a critical role for miR-223 in modulating MDSC biology in EAE and in MS and suggest potential novel therapeutic applications.

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Abbreviations

MS:

Multiple sclerosis

miRNAs:

MicroRNAs

MDSCs:

Myeloid-derived suppressor cells

EAE:

Experimental autoimmune encephalomyelitis

STAT3:

Signal transducer and activator of transcription 3

ARG1:

Arginase-1

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Acknowledgments

We thank Anne H. Cross, MD for careful reading of the manuscript; Julia Sim and Angela Archambault, PhD for technical assistance and advices; Erin Longbrake, MD PhD for helping with patient enrollment in the study; all MS patients and healthy controls that donated blood for this project as well as the study coordinators that drew the blood: Samantha Lancia, Susan Fox, and Bridgette Clay. LP is a Harry Weaver Neuroscience Scholar of the National Multiple Sclerosis Society (NMSS, JF 2144A2/1) and supported by Fondazione Italiana Sclerosi Multipla (FISM; 2014/R/15). GFW was supported by R01NS083678. LP and GFW were funded by the Dana Foundation “Program in the Neuroimmunology and Brain Infections and Cancer”. CC was supported during the course of this study by a FISM fellowship (2012/B/1) and subsequently by a NMSS fellowship (FG 2010-A1/2). TAF was supported by R01AI102924. Patients were seen for this study in the Neuroclinical Research Unit (NCRU) supported by the National Institute of Health (CO6 RR020092) and Washington University Insitute of Clinical and Translational Sciences-Brain Behavioral and Performance Unit (TR000448).

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Authors and Affiliations

  1. Department of Neurology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8111, St. Louis, MO, 63110, USA

    Claudia Cantoni, Francesca Cignarella, Laura Ghezzi, Bob Mikesell, Bryan Bollman, Gregory F. Wu & Laura Piccio

  2. Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, Fondazione Cà Granda, IRCCS Ospedale Policlinico, Milan, Italy

    Laura Ghezzi

  3. Department of Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, USA

    Melissa M. Berrien-Elliott, Aaron R. Ireland & Todd A. Fehniger

  4. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA

    Gregory F. Wu

  5. Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA

    Gregory F. Wu & Laura Piccio

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  1. Claudia Cantoni
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Electronic supplementary material

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401_2016_1621_MOESM1_ESM.tif

Supplementary Fig. 1. Percentages of MDSCs and MO-MDSCs in the peripheral blood of healthy control subjects (n = 16), untreated and GA-treated RRMS patients (n = 10 and n = 24). a, b Percentages of MDSCs and MO-MDSCs were significantly lower in untreated RRMS patients compared to healthy controls. GA-treated patient showed a modest increase in MDSC and MO-MDSC percentages, but this was not statistically significant. Percentages were calculated relative to a gate which included whole leukocytes. Error bars represent the mean ± SD. P values were calculated by Kruskal–Wallis H test and post hoc analysis (TIFF 9217 kb)

401_2016_1621_MOESM2_ESM.tif

Supplementary Fig. 2. Mir-223 expression in the CNS and lymphoid tissues in C57BL/6 mice during EAE. MiR-223 expression in spleen, brain, lymph nodes, and bone marrow taken from C57BL/6 naïve mice and on days 5, 10 15, and 30 post-EAE immunization (n = 4 mice/time point) analyzed by quantitative RT-PCR. U6 snRNA was used as endogenous controls (TIFF 44468 kb)

401_2016_1621_MOESM3_ESM.tif

Supplementary Fig. 3. Size and cellularity of WT and miR-223−/− lymph node draining the immunization. a At day 6, post-immunization WT and miR-223−/− lymph node draining the site of immunization were isolated and measured in size. b Scatter plot of the percentage of myeloid cells (defined as CD11b+Gr1+ cells) from WT and miR-223−/− lymph nodes. Error bars represent mean ± SEM. A representative experiment from a total of three performed is showed. P values were calculated by Mann–Whitney U test (TIFF 18680 kb)

401_2016_1621_MOESM4_ESM.tif

Supplementary Fig. 4. Absolute number of MO and PMN-MDSCs in naive WT and miR-223−/− mice. Cells were isolated from bone marrow, spleen, blood, and lymph nodes from naive WT and miR-223−/− mice and analyzed by flow cytometry. Gates were set on Zombie−CD45+CD11b+ cells; MO-MDSCs were defined as CD11b+Ly6Chigh cells, PMN-MDSCs as CD11b+Gr1+. Absolute numbers of MO and PMN-MDSCs were calculated from the percentages based on the total number of cells (bone marrow, spleen, and lymph nodes) or on the total number of cells per 100 μl of blood. Error bars represent the mean ± SEM. A representative experiment from a total of two is showed. P values were calculated by Mann–Whitney U test (TIFF 24193 kb)

401_2016_1621_MOESM5_ESM.tif

Supplementary Fig. 5. Characterization of surface markers expression and cytokine profile of MDSCs from immunized WT and miR-223−/− mice. a, b MO and PMN-MDSCs in the spleen and CNS-infiltrating cells from MOG35–55 immunized mice (15 DPI) were examined for the expression of MHC II, CD40, CD86, and PD-L1 by flow cytometry. Expression of individual markers is shown based on gating for MO- (CD11b+Ly6G−Ly6Chigh) and PMN-MDSCs (CD11b+Ly6G+Ly6Clow). Histograms for the different surface markers on MO and PMN-MDSCs in the spleen (a) and in the CNS (b) are referring to representative mice from each group. Red and blue lines indicate mean fluorescence intensity (MFI) in WT and miR-223−/−, respectively. Scatter plots are compiling the data from one experiment (four mice/group) shown as percentages of MO and PMN-MDSCs expressing the specific marker. c Analysis of cytokine (TNF-α, IL-10, and GM-CSF) production in splenic MO and PMN-MDSCs from WT and miR-223−/− mice at 15 DPI. Numbers indicate the percentage of cytokine-producing MO and PMN-MDSCs. Error bars are mean ± SEM. P values were calculated by Mann–Whitney U test (TIFF 153485 kb)

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Cantoni, C., Cignarella, F., Ghezzi, L. et al. Mir-223 regulates the number and function of myeloid-derived suppressor cells in multiple sclerosis and experimental autoimmune encephalomyelitis. Acta Neuropathol 133, 61–77 (2017). https://doi.org/10.1007/s00401-016-1621-6

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