Macrophage regulatory mechanisms in atherosclerosis: The interplay of lipids and inflammation - Chapter 6: Myeloid Kdm6b disruption results in advanced atherosclerosis

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Macrophage regulatory mechanisms in atherosclerosis

The interplay of lipids and inflammation

Neele, A.E.

Publication date

2018

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Citation for published version (APA):

Neele, A. E. (2018). Macrophage regulatory mechanisms in atherosclerosis: The interplay of

lipids and inflammation.

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Chapter 6

Myeloid Kdm6b disruption results in

advanced atherosclerosis

Annette E. Neele, Marion J.J. Gijbels, Saskia van der Velden, Marten A. Hoeksema,

Marieke C.S. Boshuizen, Koen H.M. Prange, Jan Van den Bossche,Annelie Shami, Tina Lucas, Stefanie Dimmeler, Esther Lutgens, Menno P.J. de Winther

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108

Abstract

Background and Aims: Atherosclerosis is lipid-driven chronic inflammatory disorder of

the arteries and monocytes and macrophages play a central role in this process. Within the atherosclerotic lesion macrophages can scavenge modified lipids and become so-called foam cells. We previously reported that the pro-fibrotic transcriptional profile of peritoneal foam cells is controlled by the epigenetic enzyme Kdm6b (also known as Jmjd3). Therefore, we studied the involvement of myeloid Kdm6b in atherosclerosis.

Material and Methods: Bone marrow of myeloid Kdm6b deficient (Kdm6bdel_{) mice or}

wild type littermates (Kdm6bwt_{) was transplanted to lethally irradiated Ldlr}-/- _mice

which were fed a high fat diet for 9 weeks to induce atherosclerosis.

Results: Lesion size was similar between Kdm6bwt _{and Kdm6b}del_{transplanted mice.}

However, lesions of Kdm6bdel mice contained more collagen and necrosis. Pathway analysis on peritoneal foam cells showed that the pathway involved in leukocyte chemotaxis was most significantly upregulated. Although macrophage and neutrophil content were similar after 9 weeks of high fat diet feeding, the relative increase in collagen content and necrosis revealed that atherosclerotic lesions in Kdm6bdel _mice

progress faster.

Conclusion: Myeloid Kdm6b deficiency results in more advanced atherosclerosis.

109

Introduction

Atherosclerosis is a lipid-driven chronic inflammatory disorder of the arteries (1). Monocytes and macrophages play a key role in the initiation and progression of atherosclerotic lesions (1-3). Once monocytes enter the arterial wall and mature into macrophages, they can scavenge modified lipoproteins and thereby become foam cells (1). Besides having foam cell characteristics, macrophages can adopt different activation states based on their environmental triggers. Skewing macrophages to cells with anti-inflammatory features would be beneficial in chronic inflammatory diseases like atherosclerosis (4-6). This can be accomplished by targeting epigenetic processes in macrophages since these are critical for monocyte and macrophage activation (7, 8). Histone modifications are one of the central epigenetic mechanisms of gene regulation referring to posttranslational modifications at histone tails. Histone H3K27 trimethylation is a repressive histone mark catalyzed by the polycomb repressive complex 2 (PRC2) (9) and can be removed by the demethylases Jumonji-C domain 3 (Kdm6b; Jmjd3), Ubiquitously-Transcribed X Chromosome Tetratricopeptide Repeat Protein (Utx) and Ubiquitously-Transcribed Y Chromosome Tetratricopeptide Repeat Protein (Uty) (10). The role of these demethylases in macrophage polarization has been extensively studied and Kdm6b is regulated in response to numeral triggers regulating both inflammatory and anti-inflammatory responses (11-18). We previously reported that peritoneal foam cells from Kdm6b deficient mice, have reduced expression of pro-fibrotic genes and pathways (19). Fibrosis is also important in atherosclerosis. Advanced atherosclerotic lesions are characterized by a fibrous cap which is mainly built of smooth muscle cells and collagen (20). Giving the fact that atherosclerosis is driven by immune cells like macrophages, and upon lesion progression a fibrous cap is built, we studied the involvement of myeloid Kdm6b in atherosclerosis. We found that atherosclerotic lesions of Kdm6b deficient mice were more advanced as they contained more collagen and exhibited more necrosis.

Materials and Methods

Atherosclerosis experiment

For our experiments we made use of C57BL/6 low density lipoprotein receptor knock out mice (Ldlr-/-_{) since these mice are prone to develop atherosclerosis. Ldlr}−/−_mice

were obtained from Jackson laboratories. A bone marrow transplantation (BMT) was performed with either LysM-cre+ Kdm6bfl/fl_{mice (Kdm6b}del_{) or LysM-cre- Kdm6b}fl/fl

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108

Abstract

Background and Aims: Atherosclerosis is lipid-driven chronic inflammatory disorder of

the arteries and monocytes and macrophages play a central role in this process. Within the atherosclerotic lesion macrophages can scavenge modified lipids and become so-called foam cells. We previously reported that the pro-fibrotic transcriptional profile of peritoneal foam cells is controlled by the epigenetic enzyme Kdm6b (also known as Jmjd3). Therefore, we studied the involvement of myeloid Kdm6b in atherosclerosis.

Material and Methods: Bone marrow of myeloid Kdm6b deficient (Kdm6bdel_{) mice or}

wild type littermates (Kdm6bwt_{) was transplanted to lethally irradiated Ldlr}-/- _mice

which were fed a high fat diet for 9 weeks to induce atherosclerosis.

Results: Lesion size was similar between Kdm6bwt _{and Kdm6b}del_{transplanted mice.}

However, lesions of Kdm6bdel mice contained more collagen and necrosis. Pathway analysis on peritoneal foam cells showed that the pathway involved in leukocyte chemotaxis was most significantly upregulated. Although macrophage and neutrophil content were similar after 9 weeks of high fat diet feeding, the relative increase in collagen content and necrosis revealed that atherosclerotic lesions in Kdm6bdel _mice

progress faster.

Conclusion: Myeloid Kdm6b deficiency results in more advanced atherosclerosis.

109

Introduction

Atherosclerosis is a lipid-driven chronic inflammatory disorder of the arteries (1). Monocytes and macrophages play a key role in the initiation and progression of atherosclerotic lesions (1-3). Once monocytes enter the arterial wall and mature into macrophages, they can scavenge modified lipoproteins and thereby become foam cells (1). Besides having foam cell characteristics, macrophages can adopt different activation states based on their environmental triggers. Skewing macrophages to cells with anti-inflammatory features would be beneficial in chronic inflammatory diseases like atherosclerosis (4-6). This can be accomplished by targeting epigenetic processes in macrophages since these are critical for monocyte and macrophage activation (7, 8). Histone modifications are one of the central epigenetic mechanisms of gene regulation referring to posttranslational modifications at histone tails. Histone H3K27 trimethylation is a repressive histone mark catalyzed by the polycomb repressive complex 2 (PRC2) (9) and can be removed by the demethylases Jumonji-C domain 3 (Kdm6b; Jmjd3), Ubiquitously-Transcribed X Chromosome Tetratricopeptide Repeat Protein (Utx) and Ubiquitously-Transcribed Y Chromosome Tetratricopeptide Repeat Protein (Uty) (10). The role of these demethylases in macrophage polarization has been extensively studied and Kdm6b is regulated in response to numeral triggers regulating both inflammatory and anti-inflammatory responses (11-18). We previously reported that peritoneal foam cells from Kdm6b deficient mice, have reduced expression of pro-fibrotic genes and pathways (19). Fibrosis is also important in atherosclerosis. Advanced atherosclerotic lesions are characterized by a fibrous cap which is mainly built of smooth muscle cells and collagen (20). Giving the fact that atherosclerosis is driven by immune cells like macrophages, and upon lesion progression a fibrous cap is built, we studied the involvement of myeloid Kdm6b in atherosclerosis. We found that atherosclerotic lesions of Kdm6b deficient mice were more advanced as they contained more collagen and exhibited more necrosis.

Materials and Methods

Atherosclerosis experiment

For our experiments we made use of C57BL/6 low density lipoprotein receptor knock out mice (Ldlr-/-_{) since these mice are prone to develop atherosclerosis. Ldlr}−/−_mice

were obtained from Jackson laboratories. A bone marrow transplantation (BMT) was performed with either LysM-cre+ Kdm6bfl/fl_{mice (Kdm6b}del_{) or LysM-cre- Kdm6b}fl/fl

littermates (Kdm6bwt_{). Kdm6b}fl/fl _{mice were kindly provided by the laboratory of}

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110

Stefanie Dimmeler (21). Crossbreeding with LysM-cre was performed in our mice facility. Briefly, 40 (20 per group), 10-week old female Ldlr-/-_{mice were divided over}

filter-top cages and provided antibiotics water (neomycin (100 mg/L, Sigma, Zwijndrecht, the Netherlands) and polymyxin B sulfate (60,000 U/L, Invitrogen, Bleiswijk, The Netherlands)) from 1 week pre-BMT until 5 weeks post-BMT. The animals received 2 x 6 Gy total body irradiation on two consecutive days. Bone marrow was isolated from two Kdm6bdel_{and two Kdm6b}wt_{mice, resuspended in}

RPMI1640 (Gibco, Breda, The Netherlands) with 5 U/ml heparin and 2 % iFCS (Gibco, Breda, the Netherlands), and 5*106_{cells were injected intravenously per irradiated}

mouse. Bone marrow transplantation efficiency was determined with qPCR for the Ldlr on DNA isolated from blood (GE Healthcare, Eindhoven, the Netherlands). Three mice were excluded from the analysis due to inefficient bone marrow transplantation (≤80 %). Five weeks after the BMT, the mice were put on a high fat diet (0.15 % cholesterol, 16 % fat, Arie Blok Diets, The Netherlands) for 9 weeks. After sacrifice, hearts were taken out and frozen in Tissue-Tek (DAKO, Eindhoven, The Netherlands) for histology. Blood samples were taken before the start of the diet and before sacrifice for lipid profiling and immune cell flow cytometry. Two mice were sacrificed before the end of experiment since they reached the human endpoints. One additional mice was excluded from the analysis due to insufficient tissue quality. A final of 16 Kdm6bdel

mice were compared to 18 Kdm6bwt _{mice for the statistical analysis. All animal}

experiments were conducted at the University of Amsterdam and approved (permit: DBC10AD) by the Committee for Animal Welfare of the Academic Medical Center, University of Amsterdam.

Histochemistry

Atherosclerotic lesions from the heart were cut in sections of 7 μm on a Leica 3050 cryostat at −25 °C. Cross sections of every 42 μm were stained with Toluidin Blue (0.2 % in PBS, Sigma-Aldrich, Gillingham, UK) to determine lesion size. Lesion size was measured by use of adobe photoshop CS4 and the sum of the three valves is presented. Lesion severity was scored by an experienced pathologist as early (intimal xanthoma), moderate (pathological intimal thickening) and advanced (fibrous cap atheroma) as described elsewhere (22). Sirius red staining was performed for 30 minutes to measure collagen content (0.05 % direct Red in saturated picric acid, Sigma, Zwijndrecht, the Nederlands). Images were obtained using a Leica DM3000 microscope and quantified with photoshop CS4 where collagen quantified as the percentage of total lesion size. Cap thickness was defined as the thinnest part of where cap is visible. For immunohistochemistry slides were fixed in aceton and blocked with Avidin/Biotin Blocking Kit (Vector Laboratories, Burlingame, USA).

111

Herafter cells were incubated with MOMA-2 (1:4000, AbD serotec, Uden, the Netherlands) to stain for macrophages. Biotin-labeled rabbit anti–rat antibody (1:300, Dako, Eindhoven, the Netherlands) was used as a secondary antibody. The signal was amplified using ABC kit (Vector Laboratories, Burlingame, USA) and visualized with the AEC kit (Vector Laboratories, Burlingame, USA). Necrosis area was measured based on toluidin Blue staining by our pathologist and corrected for total plaque size. The EnzChek gelatinase assay kit was used to measure gelatinase activity (Molecular Probes, ThermoFisher scientific, Waltham, MA, USA). Briefly, the slides were fixed with 100 % ethanol for 20 minutes. Next, the slides were incubated with the gelatin solution in a final concentration of 20 µg/ml covered with parafilm and incubated in a moister box for 2 hours at 37 °C. To stop the reaction, samples were fixed in 3.7 % formaldehyde for 10 minutes and stained with DAPI for 5 minutes. Fluorescence images were obtained using a Leica DM3000 microscope and quantified with photoshop CS4. Gelatinase activity (green fluorescence) was corrected for total plaque size.

Bone marrow-derived macrophage culture and collagen synthesis by VSMCs

Bone marrow was isolated from femurs and tibias of Kdm6bwt_{and Kdm6b}del_{mice by}

flushing. The cells were cultured in RPMI-1640 with 25mM HEPES and 2mM L-glutamine which was supplemented with 10 % FCS, penicillin (100 U/ml), streptomycin (100 µg/ml) and 15 % L929-conditioned medium as a source of M-CSF for 8 days. On day 8, cells were stimulated with 50 µg/ml oxLDL, 50 µg/ml acLDL (Alfa Aesar, Karlsruhe, Germany) or left unstimulated for 24 hours. Supernatants were collected and used for the collagen production by vascular smooth muscle cells (VSMCs) assays. Primary mouse VSMCs were isolated and cultured in DMEM/F12 with 20 % FCS (Gibco) on 0.1 % gelatin coated plates. 5*104_{VSMCs were plated per well in a gelatin-coated}

24-wells plate. After overnight adherence, cells were starved (0 % FCS) for 48 hours and next incubated with bone marrow-derived macrophage (BMDM) supernatants for 24 hours. Hereafter the supernatant was removed and VSMCs were fixed in 3.7% formaldehyde and stained with 1 % Sirius Red in 0.01 M HCl. Cells were lysed with 0.01 M NaOH and absorption was measured on a plate reader at 544nm. Gelatin was used as a standard for quantification.

Peritoneal macrophages

Four days prior to the sacrifice five mice per group (Kdm6bwt_{or Kdm6b}del_{) were}

injected intraperitoneally with thioglycollate medium (3%, Fisher, Bleiswijk, The Netherlands) as published (19).

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110

Stefanie Dimmeler (21). Crossbreeding with LysM-cre was performed in our mice facility. Briefly, 40 (20 per group), 10-week old female Ldlr-/-_{mice were divided over}

filter-top cages and provided antibiotics water (neomycin (100 mg/L, Sigma, Zwijndrecht, the Netherlands) and polymyxin B sulfate (60,000 U/L, Invitrogen, Bleiswijk, The Netherlands)) from 1 week pre-BMT until 5 weeks post-BMT. The animals received 2 x 6 Gy total body irradiation on two consecutive days. Bone marrow was isolated from two Kdm6bdel_{and two Kdm6b}wt_{mice, resuspended in}

RPMI1640 (Gibco, Breda, The Netherlands) with 5 U/ml heparin and 2 % iFCS (Gibco, Breda, the Netherlands), and 5*106_{cells were injected intravenously per irradiated}

mouse. Bone marrow transplantation efficiency was determined with qPCR for the Ldlr on DNA isolated from blood (GE Healthcare, Eindhoven, the Netherlands). Three mice were excluded from the analysis due to inefficient bone marrow transplantation (≤80 %). Five weeks after the BMT, the mice were put on a high fat diet (0.15 % cholesterol, 16 % fat, Arie Blok Diets, The Netherlands) for 9 weeks. After sacrifice, hearts were taken out and frozen in Tissue-Tek (DAKO, Eindhoven, The Netherlands) for histology. Blood samples were taken before the start of the diet and before sacrifice for lipid profiling and immune cell flow cytometry. Two mice were sacrificed before the end of experiment since they reached the human endpoints. One additional mice was excluded from the analysis due to insufficient tissue quality. A final of 16 Kdm6bdel

mice were compared to 18 Kdm6bwt _{mice for the statistical analysis. All animal}

experiments were conducted at the University of Amsterdam and approved (permit: DBC10AD) by the Committee for Animal Welfare of the Academic Medical Center, University of Amsterdam.

Histochemistry

Atherosclerotic lesions from the heart were cut in sections of 7 μm on a Leica 3050 cryostat at −25 °C. Cross sections of every 42 μm were stained with Toluidin Blue (0.2 % in PBS, Sigma-Aldrich, Gillingham, UK) to determine lesion size. Lesion size was measured by use of adobe photoshop CS4 and the sum of the three valves is presented. Lesion severity was scored by an experienced pathologist as early (intimal xanthoma), moderate (pathological intimal thickening) and advanced (fibrous cap atheroma) as described elsewhere (22). Sirius red staining was performed for 30 minutes to measure collagen content (0.05 % direct Red in saturated picric acid, Sigma, Zwijndrecht, the Nederlands). Images were obtained using a Leica DM3000 microscope and quantified with photoshop CS4 where collagen quantified as the percentage of total lesion size. Cap thickness was defined as the thinnest part of where cap is visible. For immunohistochemistry slides were fixed in aceton and blocked with Avidin/Biotin Blocking Kit (Vector Laboratories, Burlingame, USA).

111

Herafter cells were incubated with MOMA-2 (1:4000, AbD serotec, Uden, the Netherlands) to stain for macrophages. Biotin-labeled rabbit anti–rat antibody (1:300, Dako, Eindhoven, the Netherlands) was used as a secondary antibody. The signal was amplified using ABC kit (Vector Laboratories, Burlingame, USA) and visualized with the AEC kit (Vector Laboratories, Burlingame, USA). Necrosis area was measured based on toluidin Blue staining by our pathologist and corrected for total plaque size. The EnzChek gelatinase assay kit was used to measure gelatinase activity (Molecular Probes, ThermoFisher scientific, Waltham, MA, USA). Briefly, the slides were fixed with 100 % ethanol for 20 minutes. Next, the slides were incubated with the gelatin solution in a final concentration of 20 µg/ml covered with parafilm and incubated in a moister box for 2 hours at 37 °C. To stop the reaction, samples were fixed in 3.7 % formaldehyde for 10 minutes and stained with DAPI for 5 minutes. Fluorescence images were obtained using a Leica DM3000 microscope and quantified with photoshop CS4. Gelatinase activity (green fluorescence) was corrected for total plaque size.

Bone marrow-derived macrophage culture and collagen synthesis by VSMCs

Bone marrow was isolated from femurs and tibias of Kdm6bwt_{and Kdm6b}del_{mice by}

flushing. The cells were cultured in RPMI-1640 with 25mM HEPES and 2mM L-glutamine which was supplemented with 10 % FCS, penicillin (100 U/ml), streptomycin (100 µg/ml) and 15 % L929-conditioned medium as a source of M-CSF for 8 days. On day 8, cells were stimulated with 50 µg/ml oxLDL, 50 µg/ml acLDL (Alfa Aesar, Karlsruhe, Germany) or left unstimulated for 24 hours. Supernatants were collected and used for the collagen production by vascular smooth muscle cells (VSMCs) assays. Primary mouse VSMCs were isolated and cultured in DMEM/F12 with 20 % FCS (Gibco) on 0.1 % gelatin coated plates. 5*104_{VSMCs were plated per well in a gelatin-coated}

24-wells plate. After overnight adherence, cells were starved (0 % FCS) for 48 hours and next incubated with bone marrow-derived macrophage (BMDM) supernatants for 24 hours. Hereafter the supernatant was removed and VSMCs were fixed in 3.7% formaldehyde and stained with 1 % Sirius Red in 0.01 M HCl. Cells were lysed with 0.01 M NaOH and absorption was measured on a plate reader at 544nm. Gelatin was used as a standard for quantification.

Peritoneal macrophages

Four days prior to the sacrifice five mice per group (Kdm6bwt_{or Kdm6b}del_{) were}

injected intraperitoneally with thioglycollate medium (3%, Fisher, Bleiswijk, The Netherlands) as published (19).

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112

Flow cytometry

150 μl of blood was withdrawn from mice via tail vein incision before the start of the diet, five weeks after the diet and right before sacrifice and added to 20 μl of 0,5 M EDTA (Sigma-Aldrich, Gillingham, UK). Blood was withdrawn from mice which were restricted from food for at least 4 hours. The blood was centrifuged (10 min, 4°C, 2000 rpm) to separate the plasma from blood cells and plasma cholesterol and triglyceride levels were enzymatically measured according to the manufacturer's protocol (Roche, Woerden, The Netherlands). Blood was further used for flow cytometry to assess relative leukocyte counts. Red blood cells were lysed by adding 5 ml of erythrocyte lysis buffer (8.4 g of NH4Cl, 0.84 g of NaHCO3 and 0.37 g EDTA) for 15 min at RT. PBS

was added to stop the reaction and cells were spun down. White blood cells were used for flow cytometry. First Fc receptors were blocked with CD16/CD32 blocking antibody (1:100, eBioscience) in FACS buffer (0.5% BSA, 0.01% NaN3 in PBS). Hereafter

cells were incubated with the appropriate antibodies for 30 min at RT (table 1). Cells were washed once and resuspended in FACS buffer. Fluorescence was measured with a BD Canto II and analyzed with FlowJo software. Live cells were gated based on CD45+ expression and the following cell types were distinguished: Monocytes (CD11b+ and Ly6G-), neutrophils (CD11b+ and Ly6G+), B cells (CD19+), and T cells (CD3+).

Antibody Dilution Supplier

CD45 (APC-Cy7) 1:100 Biolegend

CD11b (FITC) 1:100 eBioscience

Ly6G (PE) 1:200 Biolegend

CD19 (PerCp Cy5.5) 1:100 eBioscience

CD3 (FITC) 1:100 eBioscience

Table 1: Antibody specifications

RNA isolation and quantitative PCR analysis

RNA from peritoneal macrophages was isolated with High Pure RNA Isolation kits (Roche, Basel, Switzerland) from 500,000 cells as described and used in our previous study (19). RNA from aortic arches was isolated with the Rneasy mini column kit (Qiagen, Venlo, The Netherlands). 400 ng (PEMs) or 200 ng (aortic arch) of RNA was used for cDNA synthesis with iScript (BioRad, Veenendaal, The Netherlands). qPCR was performed with 4 ng cDNA using Sybr Green Fast on a ViiA7 PCR machine (Applied Biosystems, Bleiswijk, The Netherlands). All genes were normalized to the mean of the two housekeeping genes Ppia and Rplp0. Primer sequences are available on request.

113

RNA sequencing and Bioinformatic analysis

RNA from unstimulated Kdm6bwt_{and Kdm6b}del _{peritoneal foam cells was used for RNA}

sequencing as previously published (19). Bioinformatic analysis was performed as described before (19). Gene expression was called as differential with a multiple testing (Bonferroni) adjusted P < 0.05 and an average RPKM >1 in at least one group.

Statistical analysis

Data represent the mean ± standard error of the mean (SEM). Differences between Kdm6bwt_{and Kdm6b}del _{transplanted mice are analyzed using a unpaired student’s}

t-test or a two-way ANOVA using bonferroni post-hoc t-test analysis for grouped analysis. P-Values <0.05 were considered statistically significant. Data were analyzed using Prism version 5.0 (GraphPad software, La Jolla, California).

Results

Bone marrow of Kdm6bwt_{and Kdm6b}del _{mice was effectively transplanted to Ldlr}

-/-mice

To study the involvement of macrophage Kdm6b in atherosclerosis, bone marrow of Kdm6bwt_{(Cre-) or Kdm6b}del _{(Cre+) mice was transplanted to irradiated Ldlr}-/- _mice.

After reconstitution of the bone marrow, chimeras were fed a high fat diet (HFD) for nine weeks to induce atherosclerosis. The transplanted bone marrow was efficiently reconstituted in both Kdm6bwt_{and Kdm6b}del_{transplanted mice (Figure 1A). Weight}

gain was similar after nine weeks of HFD (Figure 1B). A small but significant decrease in cholesterol and triglyceride levels was observed in Kdm6bdel_{transplanted mice after}

nine weeks, but not after five weeks of diet (Figure 1C-D). This had no effect on the leukocyte composition in the blood as measured by flow cytometry (Figure 1E). Thioglycollate elucidated peritoneal macrophages were isolated and studied ex vivo as previously reported (19). Kdm6b gene expression in peritoneal foam cells was significantly reduced in Kdm6bdel_{transplanted mice, indicating efficient deletion of}

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112

Flow cytometry

150 μl of blood was withdrawn from mice via tail vein incision before the start of the diet, five weeks after the diet and right before sacrifice and added to 20 μl of 0,5 M EDTA (Sigma-Aldrich, Gillingham, UK). Blood was withdrawn from mice which were restricted from food for at least 4 hours. The blood was centrifuged (10 min, 4°C, 2000 rpm) to separate the plasma from blood cells and plasma cholesterol and triglyceride levels were enzymatically measured according to the manufacturer's protocol (Roche, Woerden, The Netherlands). Blood was further used for flow cytometry to assess relative leukocyte counts. Red blood cells were lysed by adding 5 ml of erythrocyte lysis buffer (8.4 g of NH4Cl, 0.84 g of NaHCO3 and 0.37 g EDTA) for 15 min at RT. PBS

was added to stop the reaction and cells were spun down. White blood cells were used for flow cytometry. First Fc receptors were blocked with CD16/CD32 blocking antibody (1:100, eBioscience) in FACS buffer (0.5% BSA, 0.01% NaN3 in PBS). Hereafter

cells were incubated with the appropriate antibodies for 30 min at RT (table 1). Cells were washed once and resuspended in FACS buffer. Fluorescence was measured with a BD Canto II and analyzed with FlowJo software. Live cells were gated based on CD45+ expression and the following cell types were distinguished: Monocytes (CD11b+ and Ly6G-), neutrophils (CD11b+ and Ly6G+), B cells (CD19+), and T cells (CD3+).

Antibody Dilution Supplier

CD45 (APC-Cy7) 1:100 Biolegend

CD11b (FITC) 1:100 eBioscience

Ly6G (PE) 1:200 Biolegend

CD19 (PerCp Cy5.5) 1:100 eBioscience

CD3 (FITC) 1:100 eBioscience

Table 1: Antibody specifications

RNA isolation and quantitative PCR analysis

RNA from peritoneal macrophages was isolated with High Pure RNA Isolation kits (Roche, Basel, Switzerland) from 500,000 cells as described and used in our previous study (19). RNA from aortic arches was isolated with the Rneasy mini column kit (Qiagen, Venlo, The Netherlands). 400 ng (PEMs) or 200 ng (aortic arch) of RNA was used for cDNA synthesis with iScript (BioRad, Veenendaal, The Netherlands). qPCR was performed with 4 ng cDNA using Sybr Green Fast on a ViiA7 PCR machine (Applied Biosystems, Bleiswijk, The Netherlands). All genes were normalized to the mean of the two housekeeping genes Ppia and Rplp0. Primer sequences are available on request.

113

RNA sequencing and Bioinformatic analysis

RNA from unstimulated Kdm6bwt_{and Kdm6b}del _{peritoneal foam cells was used for RNA}

sequencing as previously published (19). Bioinformatic analysis was performed as described before (19). Gene expression was called as differential with a multiple testing (Bonferroni) adjusted P < 0.05 and an average RPKM >1 in at least one group.

Statistical analysis

Data represent the mean ± standard error of the mean (SEM). Differences between Kdm6bwt_{and Kdm6b}del _{transplanted mice are analyzed using a unpaired student’s}

t-test or a two-way ANOVA using bonferroni post-hoc t-test analysis for grouped analysis. P-Values <0.05 were considered statistically significant. Data were analyzed using Prism version 5.0 (GraphPad software, La Jolla, California).

Results

Bone marrow of Kdm6bwt_{and Kdm6b}del _{mice was effectively transplanted to Ldlr}

-/-mice

To study the involvement of macrophage Kdm6b in atherosclerosis, bone marrow of Kdm6bwt_{(Cre-) or Kdm6b}del _{(Cre+) mice was transplanted to irradiated Ldlr}-/- _mice.

After reconstitution of the bone marrow, chimeras were fed a high fat diet (HFD) for nine weeks to induce atherosclerosis. The transplanted bone marrow was efficiently reconstituted in both Kdm6bwt_{and Kdm6b}del_{transplanted mice (Figure 1A). Weight}

gain was similar after nine weeks of HFD (Figure 1B). A small but significant decrease in cholesterol and triglyceride levels was observed in Kdm6bdel_{transplanted mice after}

nine weeks, but not after five weeks of diet (Figure 1C-D). This had no effect on the leukocyte composition in the blood as measured by flow cytometry (Figure 1E). Thioglycollate elucidated peritoneal macrophages were isolated and studied ex vivo as previously reported (19). Kdm6b gene expression in peritoneal foam cells was significantly reduced in Kdm6bdel_{transplanted mice, indicating efficient deletion of}

Kdm6b (Figure 1F).

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114

Figure 1: Bone marrow of Kdm6bwt_{and Kdm6b}del _{mice was effectively transplanted to Ldlr}-/-_{mice. (A)} Chimerism determination by qPCR for the Ldlr-/-_{in the DNA of blood of Kdm6b}wt_{(black) and Kdm6b}del_(white) transplanted mice. (B) Mouse weight in grams at the start of the diet (week 0) and after 9 weeks of high fat diet. (C) Triglyceride and (D) cholesterol levels in the plasma at the start (week 0), middle (week 5), and end of the

diet (week 9). (E) Percentage of blood leukocyte subsets assessed by flow cytometry. (F) Kdm6b mRNA

expression in peritoneal foam cells. n=16-18/group. Data represent mean ± SEM *P<0.05; **P<0.01; ***P<0.001.

Macrophage Kdm6b disruption alters collagen deposition in atherosclerotic lesions of mice

We found that myeloid Kdm6b deficiency does not alter atherosclerotic lesion size after nine weeks of HFD (Figure 2A-B). Plaque phenotype analysis indicated a small but non-significant effect on plaque severity. Kdm6bdel mice had less intimal xanthoma’s and more fibrous cap atheroma’s compared to Kdm6bwt_{mice, indicating that the}

lesions are more advanced in Kdm6bdel_{transplanted mice (Figure 2C). Pathology}

scoring also revealed that collagen content was significantly higher in the Kdm6bdel

transplanted mice compared to wildtype (Figure 2D). Collagen staining by Sirius red on the atherosclerotic lesions confirmed a significant increase in the collagen content of atherosclerotic lesions in Kdm6bdel _{transplanted mice (Figure 2E-F). In addition,}

minimal cap thickness was also increased by approximately 2-fold (Figure 2G). Macrophage and neutrophil content of the plaques was unaltered (Figure 2H-I and data not shown) but necrotic area was enhanced in Kdm6bdel_{transplanted mice}

(Figure 2J-K). These data indicate that atherosclerotic lesions of Kdm6bdel_transplanted

mice are similar in size but have a more advanced phenotype.

115

Figure 2: Myeloid Kdm6b deficiency results in advanced atherosclerotic lesions. (A) Representative toluidine

blue staining of the aortic root of Kdm6bwt_{and Kdm6b}del_{transplanted mice}_{(B) Aortic lesion area presented as} the sum of the three valves per mice (C) Plaque severity scoring by a pathologist (D) Collagen scoring by a

pathologist scoring from few (-) to many (++++). P-value is calculated by a Chi-Square test. (E) Representative

Sirius Red staining to measure collagen. (F) Collagen content as percentage of total lesion area. (G) Minimal cap

thickness in µm measured at the thinnest region of the fibrous cap. (H) Representative MOMA-2 staining for

macrophages. (I) Macrophage area as percentage of total lesion area. (J) Necrotic core area indicated in red on

the toluidine blue staining. (K) Necrotic core are as percentage of total lesion area. Data represent mean ± SEM

*P<0.05; **P<0.01; ***P<0.001.

No clear macrophage phenotype is responsible for the increase in collagen content

Atherosclerotic lesions of Kdm6bdel_{mice have increased collagen content. In contrast,}

pathway analysis previously revealed that pro-fibrotic pathways were downregulated (19). We next assessed the expression of other candidate genes in these foam cells involved in collagen synthesis and breakdown. The expression of transforming growth factor beta (Tgfb1) was slightly enhanced in Kdm6bdel peritoneal foam cells (Figure 3A). Metalloproteinases are responsible for the degradation of collagen and we found

(10)