Mertk Is a Functional Regulator of Myelin Phagocytosis by Human Myeloid Cells Luke M

MerTK Is a Functional Regulator of Myelin Phagocytosis by Human Myeloid Cells Luke M. Healy, Gabrielle Perron, So-Yoon Won, Mackenzie A. Michell-Robinson, Ayman Rezk, Samuel K. Ludwin, This information is current as Craig S. Moore, Jeffery A. Hall, Amit Bar-Or and Jack P. of October 6, 2021. Antel J Immunol published online 9 March 2016 http://www.jimmunol.org/content/early/2016/03/08/jimmun ol.1502562 Downloaded from

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 9, 2016, doi:10.4049/jimmunol.1502562 The Journal of Immunology

MerTK Is a Functional Regulator of Myelin Phagocytosis by Human Myeloid Cells

Luke M. Healy,* Gabrielle Perron,* So-Yoon Won,* Mackenzie A. Michell-Robinson,* Ayman Rezk,* Samuel K. Ludwin,*,† Craig S. Moore,‡ Jeffery A. Hall,x Amit Bar-Or,* and Jack P. Antel*

Multifocal inflammatory lesions featuring destruction of lipid-rich myelin are pathologic hallmarks of multiple sclerosis. Lesion activity is assessed by the extent and composition of myelin uptake by myeloid cells present in such lesions. In the inflamed CNS, myeloid cells are comprised of brain-resident microglia, an endogenous cell population, and monocyte-derived macrophages, which infiltrate from the systemic compartment. Using microglia isolated from the adult human brain, we demonstrate that myelin phagocytosis is dependent on the polarization state of the cells. Myelin ingestion is significantly enhanced in cells exposed to b TGF- compared with resting basal conditions and markedly reduced in classically activated polarized cells. Transcriptional Downloaded from analysis indicated that TGF-b–treated microglia closely resembled M0 cells. The tyrosine kinase phagocytic receptor MerTK was one of the most upregulated among a select number of differentially expressed genes in TGF-b–treated microglia. In contrast, MerTK and its known ligands, growth arrest-specific 6 and Protein S, were downregulated in classically activated cells. MerTK expression and myelin phagocytosis were higher in CNS-derived microglia than observed in monocyte-derived macrophages, both basally and under all tested polarization conditions. Specific MerTK inhibitors reduced myelin phagocytosis and the resultant

anti-inflammatory biased cytokine responses for both cell types. Defining and modulating the mechanisms that regulate myelin http://www.jimmunol.org/ phagocytosis has the potential to impact lesion and disease evolution in multiple sclerosis. Relevant effects would include enhancing myelin clearance, increasing anti-inflammatory molecule production by myeloid cells, and thereby permitting subsequent tissue repair. The Journal of Immunology, 2016, 196: 000–000.

yelin destruction and uptake by myeloid cells in mul- and others have observed that human adult brain-derived micro- tiple sclerosis (MS) lesions is used as an index of glia in vitro display an increased ability to phagocytose myelin as M lesion activity (1). In MS, the CNS myeloid population compared with MDMs (6, 7). Phagocytosis is more robust in al- is comprised of endogenous microglia and monocyte-derived ternatively activated (M2) microglia and MDMs and reduced in by guest on October 6, 2021 macrophages (MDMs), derived from peripheral blood circulation classically activated (M1) cells (6). The mechanisms regulating (2, 3). Previous studies have referred to myelin-containing mye- this differential activity remain to be defined and are the subject of loid cells as foamy macrophages. Ingestion of myelin by MDMs the current study. in vitro induces an anti-inflammatory cytokine response, a phe- Efficient myelin clearance is an essential requirement for tissue notype also observed in human MS lesions (4, 5). Previously, we repair and remyelination (8, 9). Myelin debris is a major obstacle to the repair process both as a physical impediment and through *Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Mon- the expression of axon growth inhibitory molecules, including treal, Quebec H3A 2B4, Canada; †Department of Pathology and Molecular Medicine, CD47, which mediates the activation of SIRPa expressed on ‡ Queen’s University, Kingston, Ontario K7L 3N6, Canada; Division of BioMedical myeloid cells (10, 11). The specific molecules regulating myelin Sciences, Faculty of Medicine, Memorial University, St. John’s, Newfoundland A1B 3V6, Canada; and xDepartment of Neurosurgery, McGill University, Montreal, Que- phagocytosis are incompletely defined (for review, see Ref. 12). bec H3A 2B4, Canada Studies on phagocytosis of myelin fragments opsonized with ORCIDs: 0000-0001-9496-2216 (L.M.H.); 0000-0003-1150-7483 (G.P.); 0000-0001- components of the complement system or anti-myelin autoanti- 6633-1206 (S.-Y.W.); 0000-0001-8712-2665 (M.A.M.-R.); 0000-0003-3333-435X bodies have indicated roles for both complement receptor 3 and (C.S.M.); 0000-0001-8403-8986 (J.A.H.). FcRs in the uptake of myelin (13, 14). However, significant Received for publication December 9, 2015. Accepted for publication January 28, 2016. phagocytosis is observed upon blocking complement and remov- This work was supported by a grant from the Multiple Sclerosis Society of Canada. ing autoantibodies indicating roles for additional receptors. The scavenger receptor family has also been implicated with increased The sequences presented in this article have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus (https://www.ncbi.nlm. expression of SR-AI/II, LOX-1, and LRP-1 in the rim and the nih.gov/geo/query/acc.cgi?acc=GSE76737) under accession number GSE76737. perilesional normal-appearing white matter of chronic active and Address correspondence and reprint requests to Dr. Jack P. Antel, Montreal Neuro- inactive MS lesions (15). logical Institute, McGill University, 3801 Rue University, Room 111, Montreal, QC In a previous study profiling gene expression of mouse and H3A 2B4, Canada. E-mail address: [email protected] human microglia, expression of the tyrosine kinase receptor The online version of this article contains supplemental material. MerTK and its natural ligands, growth arrest-specific 6 (Gas6) Abbreviations used in this article: FSC, forward light scatter; Gas6, growth arrest- specific 6; KO, knockout; M0, maintained in culture under basal conditions; M1, and Protein S (ProS), were identified as part of a molecular classically activated; M2, alternatively activated; MDM, monocyte-derived macro- signature uniquely or highly enriched in microglia relative to phage; MS, multiple sclerosis; ProS, protein S; PS, phosphatidylserine; qPCR, quan- other myeloid cell populations (16). MerTK is a member of the titative PCR; TAC, Transcriptome Analysis Console; TAM, Tyro3, Axl, and MerTK. Tyro3, Axl, and MerTK (TAM) family of receptors and has Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 an important role in the homeostatic, phagocytic clearance of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502562 2 THE ROLE OF MerTK IN MYELIN PHAGOCYTOSIS apoptotic cells. Its natural ligands, Gas6 and ProS, bind both to Materials and Methods phosphatidylserine (PS) expressed on the surface of apoptotic Cell culture (human monocyte-derived macrophages and cells and to TAM receptors on the surface of phagocytes (17, 18). microglia) Myelin is a lipid-rich membrane that contains PS (4.8% of total PBMCs were isolated from whole blood using Ficoll–Paque density myelin) as part of its structural makeup (19). Studies of the systemic gradient centrifugation (GE Healthcare). CD11b+ cell isolation was immune response have also shown that activation of TAM receptors done using immunomagnetic bead selection according to manufac- results in the dampening of proinflammatory signaling pathways turer’s instructions to achieve 95–99% purity (Miltenyi Biotec). (20). Recently, polymorphisms in the MerTK gene have been linked Monocytes were cultured (5 3 105 cells/ml) in RPMI 1640 media to an increased susceptibility of developing MS (21, 22). supplemented with 10% FBS, 0.1% penicillin/streptomycin, and 0.1% To define the molecular mechanisms underlying myelin glutamine. MDMs were differentiated in vitro by using 25 ng/ml phagocytosis by myeloid cells, we combined gene expression M-CSF for 6 d. Human microglia were isolated from adult brain tissue using previously described protocols (23). Briefly, adult microglia analyses with functional assays using microglia isolated from were derived from surgical resections of brain tissue from pharmaco- the adult human CNS and macrophages generated from pe- logically intractable nonmalignant cases of temporal lobe epilepsy. ripheral blood monocytes of healthy individuals. Both cell types Brain tissue was mechanically dissociated and enzymatically digested were examined after being maintained in culture under basal using trypsin and DNAse prior to mechanical separation through a conditions (M0), traditional M1/M2 polarization conditions, or nylon mesh filter. Tissues then underwent ultracentrifugation to remove in the presence of TGF-b. The latter is implicated in mouse myelin. Dissociated cells were centrifuged, counted, and plated at 2 3 6 studies to reproduce the homeostatic state of the cells in vitro 10 cells/ml in MEM supplemented with 5% FBS and 0.1% penicillin/ basedoncomparisonwithfreshly isolated microglia (16). TAM streptomycin, and 0.1% glutamine. Microglia were grown for 3 d, collected, plated at 1 3 105 cells/ml, and maintained in culture for 6 d, Downloaded from receptor gene expression was further evaluated in microglia during which time cells were polarized to an M1 (IFN-g and LPS) and immediatelyexvivo.Ourresults implicate MerTK as a sig- M2a (IL-4 and IL-13) phenotype as previously described (6). Mtgf cells nificant regulator of myelin phagocytosis and the resultant in- received recombinant human TGF-b (50ng/ml)ondays1and5.M2c flammatory response in human myeloid cell populations. cells were generated with the addition of IL-10 (20 ng/ml) to the http://www.jimmunol.org/ by guest on October 6, 2021

FIGURE 1. Polarization-driven modulation of human adult microglial genomic profiles. (A) Three-dimensional principal component analysis (PCA) plot of robust multichip average–normalized data for all probe sets. (B) Unsupervised hierarchical clustering and heat map of 4827 genes differentially expressed between polarization conditions (ANOVA, p , 0.01, n = 3). Data are log transformed and normalized. Upregulated genes are displayed in red and downregulated genes in green. Dendrogram provides a measure of the relatedness of gene expression in each sample (at top) and for each gene (at right). (C) Volcano plots: comparison of gene expression between M0 microglia and Mtgf, M1, M2a, or M2c. Upregulated genes are displayed in red and downregulated genes in green. The size of the data points is inversely proportional to the p value (ANOVA, p , 0.05, fold change .2or,22). (D) Gene expression of microglia M1 (microRNA-155 [MiR-155]), M2a (CLEC10A), and M2c (CD163) polarization markers. Data are expressed in biweight average signal (log2) 6 SD. Data are also presented as fold change of signal intensity compared with M0 (n = 3). One-way ANOVA and a Bonferroni multiple-comparison test. The Journal of Immunology 3 standard M2a protocol on days 1 and 6. All studies were approved by with either control isotype Ab or appropriate Ab test Ab. Cells were the institutional review board of McGill University. washed and fixed in 1% formaldehyde. Flow cytometry was performed using the FACS Fortessa flow cytometer (BD Biosciences). Myeloid cells Quantification of mRNA expression by quantitative real-time were gated based on side scatter-area and forward light scatter (FSC)-area. PCR Doublets were excluded using FSC-area and FSC-height. Live cells were gated based on live-dead staining (Aqua; Life Technologies). Cells were lysed in TRIzol reagent (Invitrogen) and stored at 280˚C. Total RNA extraction was performed using standard protocols followed by Microarray analysis DNAse treatment according to the manufacturer’s instructions (Qiagen). For gene expression analysis, random hexaprimers and Moloney murine Gene expression profile of polarized cells was measured using a Human Gene leukemia virus reverse transcriptase were used to perform standard reverse ST 2.0 Microarray Chip (Affymetrix). Raw intensity data files were nor- transcription. Analysis of individual gene expression was conducted using malized using the robust multichip average algorithm, averaged with Tukey specific TaqMan probes to assess expression relative to Gapdh (house- biweight average algorithm, and transformed to the log2 scale using the keeping gene). Fold change calculations were performed using the 2DD Affymetrix Expression Console. Principal component analysis plots were threshold cycle method. generated with FlexArray. Data was exported from Expression Console into Affymetrix Transcriptome Analysis Console (TAC) software. Statistical Quantification of cytokine secretion by ELISA analysis of data was performed using one-way ANOVA (unpaired). Volcano plots and heat maps with unsupervised hierarchical clustering (p , 0.01) were Cell-culture supernatants were collected following in vitro experiments and also generated using TAC. A significance threshold of p , 0.05 and fold 2 stored at 80˚C. ELISA for TNF-a was performed in duplicate following change .2or,22 was applied to volcano plots. TAC used the Euclidean the manufacturer’s instructions (BD Biosciences). distance as the distance metric between objects and the complete linkage Phagocytosis assay method to compute the distances between clusters of objects in the generation of the heat maps. Gene expression upregulated or downregulated by at , Human myelin was isolated as previously described (24). Briefly, white least 2-fold with p 0.05 was considered to be statistically significant. Downloaded from matter obtained from surgical resections of the CNS was mechanically GraphPad Prism 5 (GraphPad) was used to perform statistical analyses homogenized in 0.32 mol sucrose and subjected to repeated sucrose den- (ANOVA and Bonferroni multiple comparison test) and plot individual gene sity centrifugation and osmotic shocks to separate myelin from other expression. Data are expressed as Tukey biweight average 6 SD (n =3). cellular components. The myelin was found to be endotoxin-free using Heat maps of specific genes were generated using GENE-E. Data are pre- the Limulus amebocyte lysate test (Sigma-Aldrich). To evaluate myelin sented as relative expression for each row, and the color gradient is set be- phagocytosis by myeloid cells, cells were plated in 12-well plates and tween the row maximum and row minimum. Protein interaction networks polarized as previously described. Myelin was incubated with a pH- were visualized with Cytoscape. A color gradient was used to represent gene sensitive dye (pH-Rodamine; Invitrogen) for 1 h in PBS (pH 8). Dyed (nodes) expression levels. Blue indicates downregulated expression and red http://www.jimmunol.org/ myelin was pelleted, resuspended in PBS (pH8), and added to myeloid indicates upregulated expression. A bold node outline represents a fold cells to a final concentration of 20 mg/ml. Live -cell recording of myelin change .2or,22, and a black outline indicates p , 0.05. uptake was performed on cells plated on Labtek glass-bottom dishes (see Supplemental Videos 1–3) using an inverted, incubated microscopy Results chamber (VivaView; Olympus Life Sciences). Following 3-h incubation with dyed myelin, cells were washed, detached, pelleted, and passed Polarization-driven modulation of human adult microglial through a flow cytometer (FACS Fortessa; BD Biosciences), in which genomic profiles internalized myelin was quantified. To explore the molecular mechanisms that govern the observed Flow cytometry functional diversity in microglial phagocytosis of myelin, we by guest on October 6, 2021 Human myeloid cells were detached gently using 2 mmol EDTA/PBS and conducted a comprehensive measurement of protein coding and blocked in FACS buffer supplemented with 10% normal human serum and long intergenic noncoding RNA transcripts by Affymetrix-based normal mouse IgG (3 mg/ml). Cells were then incubated at 4˚C for 30 min transcriptomal microarray (Human Gene 2.0 ST Array; Affyme-

FIGURE 2. Polarized human myeloid cells display differential abilities to phagocytosis human myelin. (A) Polarized microglia were exposed to dye-labeled human myelin for 3 h. Ingestion was recorded in an incubated microscopy chamber. (B) Basal phagocytic ability of human microglia (n = 6) and MDMs (n = 17) was compared after 3-h exposure to myelin. Data represented as d mean fluorescence intensity (DMFI) compared with no added myelin. Unpaired Student t test. Following a 3-h exposure to dye-labeled myelin, cells were detached, and internalized myelin was quantified by flow cytometry. The phagocytic ability of differentially polarized microglia (n =6)(C) and human MDMs (n = 17) (D) were determined. Data in (B), (C), and (D) are presented as a percentage of M0 phagocytosis levels. One-way ANOVA and a Newman– Keuls multiple-comparison post hoc analysis. 4 THE ROLE OF MerTK IN MYELIN PHAGOCYTOSIS trix). Microglia from different donors, subjected to the same MDMs were incubated with human myelin conjugated to a polarization conditions, clustered together (Fig. 1A, 1B). M0 pH-sensitive dye. Microglia exposed to fluorescently labeled microglia (basal culture condition) were most closely related myelin were incubated in a microscopy chamber in which my- to Mtgf microglia, with only 33 genes being differentially elin uptake could be visualized overtime(seeSupplemental expressed between these cell types—21 upregulated in Mtgf Videos 1–3). Internalized, fluorescing myelin is illustrated in and 12 downregulated in Mtgf (Fig. 1C). The transcriptional Fig. 2A. Cells incubated with myelin for 3 h were detached, profile of M0 microglia was most distinct from M1 cells, with and internalized myelin was quantified by flow cytometry. 837 genes upregulated and 618 genes downregulated in the M1 Basal myelin phagocytosis by MDMs, as measured by flow cells including the expected upregulation of microRNA-155 cytometry at 3 h, was significantly less as compared with (Fig.1D).CellsexposedtoM-CSF/IL-4/IL-13 (M2a) and microglia (Fig. 2B). Following a 3-h incubation with myelin, those with added IL-10 (M2c) had 302 and 415 differentially total myelin uptake by TGF-b–treated microglia (Mtgf) expressed genes, respectively, as compared with M0 (Fig. 1C). exceeded all other cells, with M2c cells also showing an en- Examples of differentially expressed M2-associated genes hanced ability to phagocytose compared with M1-treated such as CLEC10A in M2a and CD163 in M2c are shown in cells, which displayed a depressed capacity to phagocytose Fig. 1D. (Fig. 2C). MDM ability to phagocytose myelin was assessed in an identical manner. MDMs showed a similar pattern of Polarized human myeloid cells display differential abilities to phagocytosis observed between activation states, with en- phagocytose human myelin hanced uptake by Mtgf cells and significantly decreased up- To investigate the phagocytic capacities of differently polar- take by M1 activated cells (Mtgf . M2c . M2a = M0 . M1) ized CNS- and peripherally derived myeloid cells, microglia and (Fig. 2D). Downloaded from http://www.jimmunol.org/ by guest on October 6, 2021

FIGURE 3. TGF-b induces microglial expression of phagocytosis-related genes. (A) Heat map of 33 genes differentially expressed in Mtgf versus M0 microglia (ANOVA, p , 0.05, fold change .2or,22, n = 3). Upregulated gene expression is displayed in red and downregulated gene expression in blue. Genes are in decreasing order of fold change comparing Mtgf to M0. (B) Heat map of genes involved in phagocytosis in microglia. Genes are in decreasing order of fold change. MerTK interaction networks including differential gene expression for Mtgf compared with M0 (C) and M1 (D) as compared with M0 cells. Thick black outline represents a fold change .2or,22. Black outline represents p , 0.05, gray outline represents p . 0.05. The Journal of Immunology 5

TGF-b induces microglial expression of phagocytosis-related genes Differential modulation of human MDM genomic profiles and When comparing gene expression in Mtgf to M0 microglia, functional response to phagocytosis MerTK, a phagocytic tyrosine kinase receptor belonging to the Having completed analysis of the differentially polarized micro- TAM family was among a select number of genes most upreg- glial transcriptome, we carried out a microarray analysis of MDMs ulated in TGF-b–treated cells (Fig. 3A). MerTK was the most activated under the same conditions as the microglia. Results in- upregulated gene when we considered expression of genes indicated Mtgf MDMs to be considerably more distinct from their volved in phagocytosis (Fig. 3B) (Supplemental Fig. 1A). In- M0 counterparts than found in microglia, with 242 (58 upregu- teraction network maps indicated that a number of molecules lated) differentially expressed genes (Fig. 5A). MerTK was again interacting with MerTK were upregulated by TGF-b, including among the most upregulated genes when comparing overall (Fig. 5A) known natural ligands for MerTK, ProS and Gas6 (Fig. 3C). or phagocytic gene (Fig. 5B, Supplemental Fig. 1B) expression. Conversely, these same molecules were downregulated under qPCR validation showed a similar pattern of MerTK gene ex- proinflammatory, M1 conditions (Fig. 3D). pression as found in microglia, under corresponding activation conditions (Mtgf . M2c = M2a = M0 . M1) (Fig. 5C). Tyro3 TGF-b enhances expression of the TAM receptor family expression was also enhanced by TGF-b but not to the degree of members MerTK or Axl. Axl expression, as with microglia, was increased Next, we performed quantitative PCR (qPCR) analysis of dif- in both the TGF-b and M1 conditions (Fig. 5C). TAM receptor ferentially polarized human adult microglia to validate our expression was similar in MDMs as compared with microglia, microarray findings. qPCR confirmed that MerTK was upregu- with both MerTK and Axl being more highly expressed than lated in Mtgf and downregulated in M1 cells as compared with M0 Tyro3 (Fig. 5D). In TGF-b–treated MDMs, myelin ingestion Downloaded from microglia (Fig. 4A). Little or no change was seen between M2 significantly enhanced expression of mRNA for both MerTK and andM0cells(Fig.4A).BothTyro3 and Axl were upregulated in Axl, suggesting a possible positive-feedback signaling loop, by Mtgf cells, with Axl expression also being enhanced in proin- which the process of phagocytosis drives TAM receptor expres- flammatory M1 cells (Fig. 4A). Analysis of immediately ex vivo sion (Fig. 5E). This phagocytosis-driven upregulation of MerTK + (CD11b bead–selected) microglia showed higher expression of and Axl expression was not observed in M1 MDMs (Fig. 5F). MerTK and Axl compared with Tyro3 (Fig. 4B). TGF-b time course studies performed by adding TGF-b to basal microglia TAM receptor protein expression in polarized human myeloid http://www.jimmunol.org/ showed a time-dependent increase in expression of all three cells receptors, with peak expression at 24 h and MerTK showing the To determine whether the pattern of MerTK mRNA expression is most significant upregulation (Fig. 4C). translated into changes at the cell surface, receptor levels in both by guest on October 6, 2021

FIGURE 4. TGF-b enhances expression of TAM receptor family members in human adult microglia. (A) qPCR validation of TAM receptor expression in human adult microglia. Expression of all three TAM receptors is significantly increased in Mtgf cells as compared with M0 cells, with MerTK showing the most significant upregulation. Axl mRNA levels are also increased in M1 cells. (B) Relative TAM receptor mRNA expression levels in freshly isolated CD11b+ bead–selected cells from the noninflamed human CNS show similar expression of both MerTK and Axl with significantly less expression of Tyro3 mRNA. Data presented as fold change relative to MerTK levels. Expression levels normalized to Gapdh (n = 3). One-way ANOVAwith a Newman–Keuls multiple-comparison post hoc analysis. (C) TGF-b treatment time-course experiment shows significant increase in TAM receptor mRNA levels, peaking at 24 h and decreasing thereafter. Data presented as fold change relative to the 0-h time point. Expression levels normalized to Gapdh (n = 4). One-way ANOVA and a Newman–Keuls multiple comparison post hoc analysis. 6 THE ROLE OF MerTK IN MYELIN PHAGOCYTOSIS Downloaded from

FIGURE 5. Differential modulation of human MDM genomic proﬁles and functional response to phagocytosis. (A) Heat map of genes differentially expressed in Mtgf compared with M0 MDMs (ANOVA, p , 0.05, fold change .2 and ,22, n = 3). Genes are in decreasing order of fold change. (B) Heat map of genes involved in phagocytosis in MDMs. Genes are in decreasing order of fold change comparing Mtgf to M0. (C) qPCR validation of TAM

receptor expression in MDMs. Expression levels normalized to Gapdh (n = 7). Data presented as fold change relative to M0 levels. One-way ANOVAwith http://www.jimmunol.org/ Bonferroni post hoc analysis. (D) Expression of TAM receptor mRNA in MDM is similar to what was seen previously in freshly isolated myeloid cells from the CNS, with comparable expression of MerTK and Axl mRNA and significantly less expression of Tyro3. Relative expression levels of MerTK and Axl mRNA were measured by qPCR following MDM ingestion of human myelin. Expression levels normalized to Gapdh (n = 3). Data presented as fold change relative to MerTK levels. One-way ANOVAwith Bonferroni post hoc analysis. (E) TGF-b–treated MDMs significantly upregulate MerTK and Axl mRNA levels following a 3-h exposure to human myelin. (F) This effect is not observed in M1 cells. Expression levels normalized to Gapdh (n = 7). Unpaired Student t test. microglia and MDMs were investigated by flow cytometry. As of myelin by myeloid cells is regulated by MerTK (Fig. 7B). by guest on October 6, 2021 compared with M0 cells, MerTK was increased in Mtgf microglia UNC1062 (MerTK IC50 = 1.1 nmol) is a first-generation MerTK and MDMs (Fig. 6Ai). MerTK expression was decreased in M1 antagonist; UNC2025 (MerTK IC50 = 0.74 nmol) is a second- microglia and MDMs, with expression higher in M2c than M2a in generation antagonist with improved drug metabolism and phar- both cell types (Fig. 6Ai). Tyro3 protein expression was induced macokinetic properties (25, 26). A 1-h pretreatment with both in TGF-b–treated microglia relative to all other polarization types. MerTK antagonists (1 mmol) significantly decreased phagocy- Tyro3 expression was increased in M2c MDMs as compared with tosis of human myelin in M0, Mtgf, and M2c MDMs (Fig. 7B). M1-treated cells (Fig. 6Aii), which correlates with previously This inhibition was most pronounced in microglia treated with observed changes in mRNA levels (Figs. 4A, 5C). Axl protein UNC2025, in which phagocytosis of myelin was brought down expression was not enhanced by TGF-b treatment, and a decrease below M0 levels (Fig. 7C). UNC2025 also inhibited M2c in protein expression was observed under M1 conditions in MDMs phagocytosis, most likely through antagonism of basal MerTK (Fig. 6Aiii). In both microglia and MDMs, the expression levels of receptors in these cells (Fig. 6A). However, the greatest degree all three TAM receptors were as follows: MerTK . Tyro3 . Axl of phagocytic inhibition was observed in TGF-b–treated cells (Fig. 6B). Overall, basal Axl expression was significantly lower in (Fig. 7B, 7C) due to an increased expression of MerTK by these both microglia and MDMs (Fig. 6B), as compared with MerTK cells (Fig. 6A). and Tyro3, contrasting the enhanced Axl mRNA levels observed in TGF-b–treated or M1 activated myeloid cells (Figs. 4A, 5C). The effect of MerTK antagonism on cytokine expression and The low expression of Axl observed by flow cytometry is likely production by myeloid cells due to increased cleavage of the ligand-bound receptor. Basal Incubation of MDMs with human myelin (40 mg/ml for 3 h) caused MerTK expression levels were significantly higher in microglia as an increase in mRNA expression of the anti-inflammatory cyto- compared with MDMs (Fig. 6C). kine, IL-10, as tested in TGF-b–treated MDMs (Fig. 8A). This effect was inhibited using MerTK-specific antagonist UNC2025 Phagocytosis correlates with MerTK expression and can be (Fig. 8A). Addition of myelin to M1 cells, shown previously to inhibited by pharmacological antagonism of MerTK have a reduced phagocytic capacity, did not induce changes in In TGF-b–treated MDMs, a significant correlation was observed IL-10 (Fig. 8A). Pretreatment of M0 microglia with 300 nmol between cell-surface MerTK expression and the degree of myelin UNC1062resultedinanincreaseinLPS-inducedTNF-a re- phagocytosis (r2 = 0.761; p = 0.0003) (Fig. 7A). This correlation is lease, as measured by ELISA (Fig. 8B). The effects of a second- lost in M1-treated cells (r2 = 0.0061) (Fig. 7A). Cell numbers generation MerTK inhibitor UNC2025 were investigated in limited similar experiments from being performed in microglia. MDMs (insufficient microglia available). We used two known MerTK receptor antagonists (UNC1062 and TGF-b–treated MDMs were incubated with 1 mmol UNC2025, UNC2025) to directly determine the extent to which phagocytosis and the expression of TAM receptor and pro/anti-inflammatory The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ FIGURE 6. TAM receptor protein expression in polarized human myeloid cells. TAM receptor expression levels in differently polarized microglia and MDMs were analyzed by flow cytometry. (Ai) Expression of MerTK is significantly enhanced in TGF-b–treated microglia and MDMs with a significant downregulation of the receptor in both M1 cell types. (Aii) Tyro3 is significantly upregulated in TGF-b–treated microglia and M2c MDMs as compared with M1 MDMs. (Aiii) Axl shows no significant difference across all microglial treatment groups with a downregulation observed in M1 MDMs. Data from individual experiments presented as a percentage of M0 expression levels. One-way ANOVA and a Newman–Keuls multiple-comparison post hoc analysis. (B) Comparison of basal MerTK expression between microglia (n = 6) and MDMs (n = 8) shows significantly higher basal expression in microglia than in MDMs. (C) Comparison of protein expression levels of all three TAM receptors in both microglia (n = 6) and (D) MDMs (n = 8) reveals MerTK to be the most highly expressed receptor, followed by Tyro3 with significantly less Axl being expressed by both myeloid cell types. One-way ANOVA and a Newman–Keuls multiple-comparison post hoc analysis. by guest on October 6, 2021 cytokine mRNA were analyzed by qPCR. Significant upregula- of proliferation both in vivo and in vitro, and capacity to survive tion of proinflammatory cytokine mRNA levels (IL-6, IL-1b,and in vitro in the absence of specific growth factor supplementation TNF-a) was observed in Mtgf MDMs treated with UNC2025 (27, 28). In rodents, TGF-b supplementation in vitro sustains the alone (Fig. 8C). In addition, treatment of Mtgf MDMs with basal or homeostatic status of these cells, and deletion of TGF-b UNC2025 alone resulted in a significant inhibition of TGF-b– results in failure of microglia development and survival (16). Our induced MerTK and Axl mRNA expression (Fig. 8D). Therefore, transcriptional analysis of human microglia showed that microglia activation of MerTK and Axl may result in an increased ex- under basal culture conditions closely resemble the profile of pression of these receptors through a positive-feedback mecha- TGF-b–treated cells and are markedly different from M1- and nism, which can be inhibited by pharmacological antagonism of M2-activated cells. There were significantly more differences in the MerTK receptor. No change in IL-10 expression was ob- gene expression between M0 and Mtgf MDMs than seen in the served (Fig. 8D). corresponding microglial profiles. This is likely due to MDMs requiring the presence of M-CSF for maintenance in vitro, which is not the case for microglia. Discussion MerTK was among the most upregulated of the select number The current study demonstrates the contribution of the TAM of genes differentially expressed between microglia and MDMs receptor MerTK to the phagocytosis of myelin by human adult under M0 and Mtgf conditions and the most upregulated among microglia and MDMs. TAM receptor expression levels cor- genes with known phagocytic roles. TAM receptors are known related with phagocytic function in differentially polarized cells to participate in the phagocytic clearance of cells undergoing and with enhanced activity of microglia as compared with apoptosis (29, 30). PS on the outer plasma membrane leaflet of MDMs. Phagocytic activity was highest in myeloid cells exposed apoptotic cells serves as a recognition or eat-me signal de- to TGF-b; transcriptome analyses identified MerTK as being tected by TAM receptor ligands (31). As mentioned previ- the phagocytic receptor most upregulated by TGF-b. Specific ously, PS is a component of myelin, a lipid-rich membrane MerTK inhibitors reduced myelin phagocytosis and the resultant that becomes exposed to the extracellular environment dur- anti-inflammatory cytokine responses for both cell types. An- ing myelin disruption (19). Molecules known to interact with tagonism of MerTK directly promoted a proinflammatory cyto- MerTK were also upregulated in TGF-b–treated microglia, kine response profile in these myeloid cells. including its natural ligands, Gas6 and ProS. Similar to their The adult human CNS-derived microglia used in this study have cognate receptor, these molecules were downregulated in M1 biological properties distinct from murine microglia, including lack cells, indicating a complete inhibition of the MerTK pathway 8 THE ROLE OF MerTK IN MYELIN PHAGOCYTOSIS Downloaded from

FIGURE 7. Phagocytosis correlates with MerTK expression and can be inhibited by pharmacological antagonism of MerTK. A correlation analysis was performed by comparing MerTK expression levels in MDMs with the cells capacity to phagocytose human myelin. (A) The strongest correlation between http://www.jimmunol.org/ MerTK expression levels and phagocytosis was observed in Mtgf MDMs (r2 = 0.761, p = 0.0003, n = 13). This correlation is abolished in M1 MDMs (r2 = 0.0061, n = 13). MerTK receptor antagonists (UNC1062 and UNC2025) were used to investigate the contribution of MerTK to TGF-b–induced increase in phagocytosis of myelin by microglia and MDMs. (B and C) Both antagonists (1-h pretreatment, 1 mmol), although to a greater extent UNC2025, caused an inhibition of phagocytosis in both Mtgf MDMs (n = 13) and microglia (n = 6). Phagocytosis was also inhibited in M2c cells, although this was not statistically significant. Data presented as a percentage of M0 phagocytosis levels. One-way ANOVA and a Newman–Keuls multiple-comparison post hoc analysis. under proinflammatory conditions leading to an almost com- cuprizone toxin demyelination model. In this model, Gas62/2 plete loss in phagocytic ability. Overall expression of MerTK mice show increased microglial number and greater demyelin- by guest on October 6, 2021 was lower in MDMs as compared with CNS-derived microglia. ation and inflammatory activity compared with wild-type ani- Tyro3 protein expression was induced in M2c MDMs, corre- mals, consistent with the ability of Gas6 to modulate microglial sponding to changes at the mRNA level. In contrast to MerTK, activation (35, 36). In addition, direct administration of Axl mRNA expression was increased under proinflammatory M1 recombinant human Gas6 into the CNS in this model resulted in conditions, as recently reported in mouse bone marrow–derived enhanced debris clearance, remyelination, and axonal survival macrophages and dendritic cells following LPS stimulation (32). (37). High levels of soluble MerTK and Axl have been detected These observations indicate the differential regulation of these in MS lesions correlating with low levels of Gas6, suggesting TAM receptors in myeloid cells, suggesting roles for MerTK and that these soluble TAM receptors may sequester Gas6 and thereby Axl under specific inflammatory conditions. Unlike MerTK, we inhibit the phagocytic capacity of lesion-associated myeloid observed dissociation between Axl mRNA and protein levels, and cells (38). this is likely due to cleavage of Gas6-bound Axl as recently de- M2c-polarized cells showed increased phagocytic activity scribed (32). It has been suggested that TAM receptor expression compared with M2a and M0 cells, without differences in overall and function varies depending on the cell type and organ in which MerTK gene or protein expression, implicating involvement of it is expressed. Dendritic cells rely on Axl/Tyro3 to clear apoptotic other receptors in myelin phagocytosis. M2c cells showed up- cells, whereas macrophages in the thymus require MerTK to regulation of FcgR1a, an FcR known to enhance myelin uptake by phagocytose apoptotic thymocytes and maintain tissue homeo- microglia, and downregulation of SIRPa, a molecule that inhibits stasis (33). Our data suggest that myeloid-expressed MerTK is the phagocytosis through its interaction with CD47 (10, 39, 40). most prominent TAM receptor involved in the phagocytic clear- However, phagocytic activity by M2c cells was reduced by ance of myelin debris. MerTK antagonism, albeit to a much lesser extent than what was A strong correlation exists between the expression of MerTK and seen in Mtgf cells, likely reflecting antagonism of the basal phagocytic ability of myeloid cells in TGF-b–treated cells. Specific MerTK expressed in these cells. MerTK antagonists (UNC1062 and UNC2025; Meryx Pharma) Our current in vitro studies further demonstrate that myelin significantly reduced myelin uptake in both microglia and MDMs, ingestion has functional consequences on the inflammation-related with the effect being most evident in the TGF-b–treated cells, properties of myeloid cells. We show that myelin uptake in myeloid likely due to the enhanced MerTK expression in these cells (25, cells is sufficient to induce expression of the anti-inflammatory 34). MerTK mRNA expression levels were further increased in cytokine IL-10. The effect of the small-molecule MerTK antag- TGF-b–treated cells following myelin ingestion, potentially con- onists on reducing this response is likely secondary to their capacity tributing to enhanced phagocytic activity. Conversely, this effect to block myelin uptake, as incubation with the antagonist alone did was not seen with the poorly phagocytosing M1 MDMs. The not alter IL-10 production. Conversely, application of the antag- MerTK pathway has been implicated in myelin clearance in the onist induced a proinflammatory cytokine response in MDMs with The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 8. The effect of MerTK antagonism on cytokine expression and production by myeloid cells. Relative expression levels of IL-10 mRNA in MDMs were measured by qPCR following ingestion of human myelin, in the presence and absence of the MerTK antagonist UNC2025. (A) TGF-b–treated

MDMs upregulate IL-10 mRNA levels following a 3-h exposure to human myelin. This effect is significantly inhibited by a 1-h pretreatment with 1 mmol by guest on October 6, 2021 UNC2025. These effects are not observed in M1 cells (n = 7). (B) Human adult microglia (n = 4) were incubated with 10 nmol and 300 nmol UNC1062 1 h prior to addition of LPS (100 ng/ml for 3 h). Release of TNF-a was measured by ELISA. LPS-alone treatment represented by dotted line. One-way ANOVA and Bonferroni post hoc analysis. (C) One-hour treatment with 1 mmol UNC2025 resulted in significant upregulation proinflammatory cytokine mRNA levels (IL-6, IL-1b, and TNF-a) in Mtgf MDMs. (D) TGF-b–induced upregulation of MerTK and Axl mRNA was significantly inhibited by UNC2025. No change in IL-10 levels was observed. Expression levels normalized to Gapdh (n = 13). One-way ANOVA and Bonferroni post hoc analysis. significant upregulation of IL-6, TNF-a, and IL-1b, consistent its associated anti-inflammatory response and illustrate that this with previous studies using dendritic cells (20). The role of TAM pathway is amenable to pharmacological manipulation. Develop- receptors in inhibiting systemic inflammatory responses has been ment of specific MerTK agonists will be required to determine demonstrated in TAM receptor knockout (KO) animals. Triple whether enhancing MerTK-dependent myelin phagocytosis can KO (MerTK2/2Axl2/2Tyro32/2) mice develop severe immune promote tissue protection and repair in MS. dysregulation including splenomegaly, high titers of circulating autoantibodies, and clinical features of broad-spectrum autoim- Acknowledgments mune disease (41, 42). Single KO, MerTK2/2 mice also dis- We thank Manon Blain for technical assistance, Dr. S. Aljarallah for proof- played hypersensitivity to LPS, leading to endotoxic shock in reading the manuscript, and Dr. A. Greenhalgh for support and critical dis- response to low-dose LPS (43). Deletion of Gas6 resulted cussion throughout the project. in increased severity of experimental autoimmune encephalopa- thy with significantly increased numbers of Iba1+ microglia/ Disclosures macrophages (44). The authors have no financial conflicts of interest. Myelin phagocytosis is an important requirement to permit tissue repair and remyelination following inflammation-mediated injury (9, 11). This is achieved by removing myelin debris, thus per- References mitting entry of oligodendrocyte precursor cells. Phagocytosing 1. Popescu, B. F., I. Pirko, and C. F. Lucchinetti. 2013. Pathology of multiple sclerosis: where do we stand? Continuum (Minneap. Minn.) 19(4 Multiple myeloid cells are also implicated as sources of molecular signals Sclerosis): 901–921. that attract and support the differentiation of the oligodendrocyte 2. Lawson, L. J., V. H. Perry, P. Dri, and S. Gordon. 1990. Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. precursor cells (45, 46). Understanding and potentially manipu- Neuroscience 39: 151–170. lating the phagocytic clearance of myelin has clear consequences 3. Katsumoto, A., H. Lu, A. S. Miranda, and R. M. Ransohoff. 2014. Ontogeny and functions of central nervous system macrophages. J. Immunol. 193: 2615–2621. for MS. Using small-molecule antagonists, we demonstrate 4. Boven, L. A., M. Van Meurs, M. Van Zwam, A. Wierenga-Wolf, R. Q. Hintzen, MerTK’s involvement in the process of myelin phagocytosis and R. G. Boot, J. M. Aerts, S. Amor, E. E. Nieuwenhuis, and J. D. Laman. 2006. 10 THE ROLE OF MerTK IN MYELIN PHAGOCYTOSIS

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