Edited by: Edgar Meinl, Ludwig Maximilian University of Munich, Germany
Reviewed by: Markus Kipp, Munich Cluster of Systems Neurology, Germany; Tanja Kuhlmann, University Hospital Münster, Germany
This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Immunology
†These authors have contributed equally to this work
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Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS) characterized by the presence of focal demyelinated plaques. Sufficient clearance of myelin and cellular debris is one of the requirements for proper tissue repair and remyelination. The mechanisms underlying the clearance of such debris by phagocytes are not fully understood, but recent findings suggest a prominent role for lipoprotein-lipase (LPL) in this process. Here, we demonstrate that angiopoietin-like 4 (ANGPTL4), a potent inhibitor of LPL, is abundantly expressed in astrocytes in control white matter tissue and its expression is markedly reduced in active MS lesions. We provide evidence that ANGPTL4 inhibits the uptake of myelin-derived lipids by LPL-immunoreactive phagocytes. Taken together, our data suggest that the strong reduction in astrocytic ANGPTL4 expression in active demyelinating MS lesions enables phagocytes to adequately clear myelin debris, setting the stage for remyelination.
MS is an inflammatory demyelinating disease characterized by massive infiltration of monocyte-derived macrophages into the central nervous system (CNS). Infiltrated macrophages, as well as brain-resident activated microglia, produce a variety of cytotoxic factors and cytokines and thereby contribute to CNS damage and associated neurodegeneration. Macrophage depletion in an experimental MS animal model significantly reduces clinical symptoms underscoring the pathogenic role of these cells (
Blocks of formalin-fixed paraffin-embedded post-mortem brains samples were obtained from the VUmc MS Centrum Amsterdam and the Netherlands Brain Bank from 12 MS patients and 3 non-neurological controls. Detailed clinical data are summarized in
Clinical data of MS patients and non-neurological controls.
Ctrl 1 | 87 | NA | F | 07:20 | NA | Cachexia and dehydration |
Ctrl 2 | 83 | NA | M | 05:15 | NA | Unknown |
Ctrl 3 | 51 | NA | F | 05:36 | NA | Unknown |
MS 1 | 61 | RR | F | 10:55 | Unknown | Sepsis |
MS 2 | 39 | RR/SP | F | 08:30 | 8 | Ileus |
MS 3 | 75 | SP | M | 22:20 | 35 | Urosepsis |
MS 4 | 48 | SP | F | 09:20 | 24 | Pneumonia |
MS 5 | 56 | RR | F | 08:55 | 20 | Legal euthanasia |
MS 6 | 61 | RR | F | 10:55 | Unknown | Sepsis |
MS 7 | 41 | PP | M | 07:23 | 14 | Urosepsis and pneumonia |
MS 8 | 49 | RR | M | 08:00 | 25 | Pneumonia |
MS 9 | 51 | SP | M | 11:00 | 18 | Infection |
MS 10 | 44 | SP | M | 10:15 | 21 | Unknown |
MS 11 | 57 | SP | F | 08:40 | 27 | Respiratory insufficiency |
MS 12 | 54 | PP | M | 08:15 | 12 | Legal euthanasia |
Antibody details.
HLA-DR | Mouse | 1:1000 | eBioscience | 14-9956-82 |
PLP | Mouse | 1:3000 | Rio-rad | MCA839G |
ANGPTL4 | Rabbit | 1:300 | Abcam | ab115798 |
GFAP-cy3 | Mouse | 1:300 | Sigma | C9205 |
GFAP | Rabbit | 1:250 | Chemicon | ab1980 |
LPL | Mouse | 1:100 | Abcam | ab21356 |
Iba1 | Goat | 1:100 | Abcam | Ab5076 |
For cellular localization sections were incubated overnight with antibodies applied simultaneously at 4°C. After washing with PBS, secondary antibodies consisting of donkey-anti-mouse Alexa Fluor 488 (1:200, Abcam, ab150105, UK), rabbit-anti-goat Alexa Fluor 647 (1:200, Abcam, ab150143, UK), or goat-anti-rabbit Alexa Fluor 647 (1:200, Abcam, ab150079, UK) were applied for 1 h at room temperature. Fluorescent preparations were embedded and analysis was performed with a Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems, Heidelberg, Germany). GFAP and ANGPTL4 fluorescence intensity were measured by a blinded observer using ImageJ software (freely available from: U.S. National Institutes of Health, Bethesda, MD, USA). Per slide, 3 independent locations were analyzed. Relative ANGPTL4 intensity to GFAP was calculated by dividing ANGPTL4 intensity over GFAP intensity. Next, intensity was normalized to the relative intensity of ANGPTL4 over GFAP in NAWM slides.
Human astrocytoma cells (U373) were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Life Technologies, #11320033, USA) containing 10% fetal bovine serum (FBS; Biowest, S1810-500, USA), and penicillin/streptomycin (50 mg/ml; Life Technologies, #15140122, USA) in 5% CO2 at 37°C.
RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) according to manufacturer's protocol. mRNA concentration and quality (OD 280/260 ratios of 1.8 or higher) was measured using Nanodrop (Nanodrop Technologies, USA). cDNA syntheses was performed using the Reverse Transcription System kit (Applied Biosystems, #4368814, USA) following manufacture's guidelines. Expression was assessed by quantitative RT-PCR using SYBR Green Power mix (Applied Biosystems, #4367659, USA). All primer sequences are listed in
Primer sequences.
P2X7 | Human | GAA CAA TAT CGA CTT CCC CGG | TTA TCG CCT GTT TCT CGG AAG |
CD40 | Human | CAA ATA CTG CGA CCC CAA CCT A | TTT CTG AGG TGC CCT TCT GCT |
CD206 | Human | GTC TTG GGC CAC AGG TGA A | AAG GCG TTT GGA TAG CCA CA |
P2Y12 | Human | ACC AGA GAC TAC AAA ATC ACC C | AGA AAA TCC TCA TCG CCA GG |
LPL | Human | CGA GCG CTC CAT TCA TCT CT | CCA GAT TGT TGC AGC GGT TC |
ANGPTL4 | Human | ATG GCT CAG TGG ACT TCA AC | GCT ATG CAC CTT CTC CAG AC |
18S | Human | TAC CAC ATC CAA GGA AGG CAG CA | TGG AAT TAC CGC GGC TG CTG GCA |
HMBS | Human | CAC GAT CCC GAG ACT CTG CT | TAC TG GCA CAC TGC AGC CTC |
Myelin was isolated and labeled as described previously (
Human blood monocytes were isolated from buffy coats of healthy donors (Sanquin Blood Bank, The Netherlands) using Ficoll (Lymphoprep™, Axis-Shield, #1114544 Norway) and subsequent Percoll density gradient centrifugation. Monocytes were differentiated into macrophages in culture medium (IMDM, Gibco) containing 10% FBS, penicillin (100IU/ml), streptomycin (50 mg/ml) and 50 ng/ml MCSF, for 6 days at 37°C, 5% CO2. The alternatively activated macrophages phenotype was induced by culturing M-CSF differentiated macrophages in the presence of 10 ng/ml human IL-4 (ImmunoTools, #13340043, Germany) for 48 h as described before by (
Human monocytes were cultured on top of a transwell filter (Corning, #3421, USA) with a pore size of 0.3 μm and differentiated into macrophages as described above. Human astrocytoma cells (U373) were plated in a culture plate. After cell adhesion, the transwell filter containing macrophages was transferred to the culture plate containing astrocytes. After 24 h at 37°C in 5% CO2, the transwell filter containing macrophages was discarded and mRNA from astrocytes was isolated as described above.
Alternatively activated macrophages were cultured on glass coverslips and fixed with 4%PFA for 10 min. Oil-Red-O stock solution was prepared by dissolving in 0.25 g Oil-Red-O (Sigma-Aldrich, #O0265, USA) in 50 ml isopropanol. Oil-Red-O working solution was prepared by mixing 3 parts Oil-Red-OO stock solution with 2 parts dH2O, followed by filtration. Coverslips were immersed in Oil-Red-O working solution for 10 min, followed by two rinses with 60% isopropanol and one rinse with dH2O. Nuclear staining was performed using Haematoxylin for 5 min followed by several rinses with dH2O. Quantification of Oil-Red-O staining was performed by extracting Oil-Red-O using 100% isopropanol for 5 min. Absorbance was subsequently measuring at 492 nm. 100% isopropanol was used as background control.
For phagocytosis experiments, alternatively activated macrophages were exposed to 5 μg/ml atto633-labeled myelin for 4 h at 37°C in 5% CO2, with or without addition of 1 μg/ml ANGPTL4 (R&D systems, 4487-AN, USA) or 10 μM Cytochalasin D (Sigma-Aldrich, C8273, USA). Subsequently, cells were thoroughly washed with PBS to remove extracellular myelin. Phagocytosis of fluorescent myelin particles was quantified using intracellular FACS analysis (Calibur flow cytometer, Becton & Dickinson, USA).
Lipoprotein lipase activity on alternatively activated monocyte derived macrophages was measured using a fluorometric assay kit (Abcam, ab204721, UK) according to the manufacturer's instructions.
All data reflect mean ± SEM and all comparisons were statistically tested in GraphPad Prism 5.0. For comparing two experimental groups with normal distribution, unpaired two-tailed Student's
First, we investigated the cellular expression of ANGPTL4 in well-characterized MS lesions. Active white matter lesions were identified by the absence of myelin (proteolipid protein) and the presence of MHC class II+ cells with some MHC class II cells containing myelin proteins. Inactive lesions are characterized with a demyelinated core with little evidence of ongoing inflammation. We observed no clear differences in cellular expression nor intensity of ANGPTL4 immunoreactivity comparing control white matter with normal appearing white matter (NAWM). ANGPTL4 was found to be expressed in normal appearing white matter by cells with an astrocytic morphology. Interestingly we observed a marked decreased expression of ANGPTL4 in active MS lesions compared to NAWM, while in inactive lesions the expression of ANGPTL4 displayed similar immunoreactivity as observed in NAWM (
ANGPTL4 expression is strikingly reduced in active MS lesions.
Since ANGPTL4 is a known inhibitor of lipoprotein-lipase (LPL), we next analyzed the cellular distribution of LPL in MS brain specimens. LPL was weakly expressed in NAWM and abundantly expressed in active lesions, localized to cells with the morphological appearance of macrophages (
LPL is expressed on iba1 positive cells in active MS lesion.
To determine what underlies the observed decrease in astrocytic ANGPTL4 expression, human astrocytes were exposed to myelin for 24 h. Exposure to myelin did not affect ANGPTL4 expression in astrocytes (
Astrocytic ANGPTL4 expression is not influenced by myelin, but is by crosstalk with macrophages.
Recent reports have highlighted the pivotal role of microglial LPL in remyelination (
ANGPTL4 inhibits uptake of myelin derived lipids but not myelin phagocytosis.
We previously demonstrated that angiopoietin-like 4 (ANGPTL4) is expressed by astrocytes in white matter and in gray matter of patients suffering from capillary cerebral amyloid angiopathy (
Phagocytes, i.e., microglia and macrophages, play a critical role in pathogenesis of MS. In MS, phagocytes promote the clearance of cellular debris after myelin damage, which is a prerequisite for remyelination (
We next investigated which factors might be responsible for the decreased expression of ANGPTL4 on astrocytes in active MS lesions. We first cultured astrocytes in the presence of myelin debris, which has been shown to induce a reactive astrocyte profile (
Recent reports have highlighted the pivotal role of microglial LPL in remyelination. LPL hydrolyses triglycerides into glycerol and free fatty acids (FFA). The resulting FFAs can in turn be taken up via scavenger receptors, such as CD36 and CD68 by microglia (
Upon exposure to myelin fragments, the lipid storage in macrophages was dramatically increased as determined by Oil-Red-O staining. Inhibition of LPL in macrophages by addition of recombinant ANGPTL4 or the LPL inhibitor Orlistat significantly reduced the amount of Oil-Red-O positive lipid droplets. Interestingly, we did not observe differences in their capacity to phagocytose myelin. These findings suggest that ANGPTL4 inhibits the breakdown and subsequent uptake of myelin derived lipids by macrophages, but not myelin uptake via phagocytosis. In line with these observations, ANGPTL4 mediated inhibition of LPL has been shown to limit the formation of foam cells in the context of atherosclerosis (
In conclusion, we provide evidence that ANGPTL4 expression is strongly reduced in reactive astrocytes in active MS lesions, which might be a protective mechanism enabling phagocytes to effectively remove myelin debris setting the stage for repair.
VUmc MS Centrum Amsterdam and the Netherlands Brain Bank received permission to perform autopsies for the use of tissue and for access to medical records for research purposes from the Ethical Committee of the VU University Medical Center, Amsterdam, The Netherlands. All patients and controls, or their next of kin, had given informed consent for autopsy and use of brain tissue for research purposes. Buffy coats were obtained from volunteer donors at Sanquin after written informed consent was obtained.
AK with the help of JvH and HdV conceived the study. AK, JvH, MR, and HdV designed the experiments. AK with the help of AC performed most of the experiments. SvdP performed FACS experiments. AK with the help of JvH, MR, and HdV wrote the manuscript, which was reviewed by all authors.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Expert technical support by the AO|2M facility (Advanced Optical Microscopy facility in O|2, VU Medical Center, Amsterdam) was highly appreciated.
The Supplementary Material for this article can be found online at:
Gating strategy. Non-myelin treated monocyte derived macrophages were gated on forward scatter/side scatter (FCS/SSC) dot plot. These events were next visualized using an FSC/myelin-633 dot plot and a gate was placed for atto6330-labeled myelin negative cells. Then atto633-labeled myelin treated monocyte derived macrophages were analyzed to confirm that treated macrophages lay inside the gate.