IFNβ secreted by microglia mediates clearance of myelin debris in CNS autoimmunity
- Magdalena Kocur†1,
- Reiner Schneider†2,
- Ann-Kathrin Pulm†1,
- Jens Bauer1,
- Sonja Kropp1,
- Michael Gliem2,
- Jens Ingwersen2,
- Norbert Goebels2,
- Judith Alferink3, 4,
- Timour Prozorovski2,
- Orhan Aktas†2 and
- Stefanie Scheu†1Email author
© Kocur et al.; licensee BioMed Central. 2015
Received: 30 January 2015
Accepted: 3 February 2015
Published: 3 April 2015
Multiple sclerosis (MS) is a chronic demyelinating disorder of the central nervous system (CNS) leading to progressive neurological disability. Interferon β (IFNβ) represents a standard treatment for relapsing-remitting MS and exogenous administration of IFNβ exhibits protective effects in experimentally induced CNS autoimmunity. Also, genetic deletion of IFNβ in mice leads to an aggravation of disease symptoms in the MS model of experimental autoimmune encephalomyelitis (EAE). However, neither the underlying mechanisms mediating the beneficial effects nor the cellular source of IFNβ have been fully elucidated.
In this report, a subpopulation of activated microglia was identified as the major producers of IFNβ in the CNS at the peak of EAE using an IFNβ-fluorescence reporter mouse model. These IFNβ expressing microglia specifically localized to active CNS lesions and were associated with myelin debris in demyelinated cerebellar organotypic slice cultures (OSCs). In response to IFNβ microglia showed an enhanced capacity to phagocytose myelin in vitro and up-regulated the expression of phagocytosis-associated genes. IFNβ treatment was further sufficient to stimulate association of microglia with myelin debris in OSCs. Moreover, IFNβ-producing microglia mediated an enhanced removal of myelin debris when co-transplanted onto demyelinated OSCs as compared to IFNβ non-producing microglia.
These data identify activated microglia as the major producers of protective IFNβ at the peak of EAE and as orchestrators of IFNβ-induced clearance of myelin debris.
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). More than 20 years ago, interferon β (IFNβ) became the first immunomodulatory substance used in the treatment of MS. IFNβ is currently the most commonly used therapy for relapsing-remitting MS (RRMS), reducing relapses and severity of disease [1,2]. Genetic ablation of IFNβ or its receptor leads to an increased severity of experimental autoimmune encephalomyelitis (EAE) [3,4] a mouse model exhibiting clinical, neuropathological, and immunological disease manifestations of MS . Conversely, induction of endogenous IFNβ by poly (I:C) stimulation suppresses EAE, confirming its protective role in CNS autoimmunity . Today, neither the cellular source of type I IFN in EAE nor its localization relative to responding cells is known due to a lack of sufficiently sensitive in situ tools.
Also, the exact mechanisms underlying the protective effects of IFNβ remain incompletely understood. Various IFNβ-mediated modes of action have been suggested, including (i) downregulation of matrix metalloproteinase 9 thereby reducing tissue damage and inflammation, (ii) prevention of effector cell migration by downregulating the adhesion molecule very late antigen-4 (VLA-4) [7,8], (iii) downregulation of MHC II molecules on antigen presenting cells combined with upregulation of the inhibitory PD-L1 and PD-L2 ligands [9,10], (iv) inhibition of T cell proliferation , (v) the induction of immune cell apoptosis  and (vi) most recently the induction of FoxA1+ T regulatory cells . Removal of myelin debris has been suggested as an essential protective mechanism ameliorating IFNγ-mediated neuroinflammation by downregulating the transcription levels of pro-inflammatory factors like TNF, IL-1β, or iNOS . Reducing inflammation by enhancing phagocytosis efficacy may therefore represent a novel therapeutic approach in the treatment of neuroinflammation as observed in MS. Until now, however, a direct role for IFNβ in phagocytosis of myelin or axonal debris has not been shown and the functional consequences of microglial phagocytosis remain largely unexplored.
Though IFNβ was shown to delay disease progression, adverse side effects such as depression, flu-like symptoms, skin reactions, and bone marrow suppression have limited its use . Also, IFNβ treatment is not always effective, as about 30% to 50% of patients experience breakthrough disease. One reason is production of neutralizing antibodies to IFNβ resulting in reduced or complete loss of therapeutic efficacy . Moreover, there is the risk that a long term systemic “block” of inflammation could affect the elicitation of immune responses required for host defense. Therefore, it is of great interest to identify the cellular source and define the mechanisms associated with IFNβ-mediated protection against neuroinflammation as a first step in the development of cell-specific treatment regimes.
In this study we characterized the cell type responsible for IFNβ production and its impact on microglia effector functions in EAE using a yellow fluorescent protein (YFP) IFNβ reporter mouse model, organotypic slice cultures, and adult microglia cell cultures. We demonstrate that microglia are the primary IFNβ producing cells during the peak phase of EAE. We further show that IFNβ induces localization of microglia in close proximity to myelin debris and subsequently increases microglial phagocytotic activity. These findings and the fact that IFNβ-producing microglia orchestrated the clearance of myelin debris in organotypic cerebellar slice cultures reveal a so far unknown function of IFNβ. Our data further suggest that future IFNβ-based therapies targeting these cells in the CNS can be developed for treatment of demyelinating CNS disorders.
Materials and methods
Mice and EAE induction
Female C57BL/6 N mice were purchased from Charles River. IFNβmob/mob (messenger of IFN beta: IFNβ/YFP reporter mouse) , IFNβ−/−  and IFNAR1−/−  mice were backcrossed for at least 10 generations onto C57BL/6 N background. PLP-EGFP mice were used for indicated organotypic slice culture experiments . All mice were housed under specific pathogen free conditions in the animal research facility of the University of Duesseldorf. Mice at 6–12 weeks of age were used for all experiments. Active EAE was induced by tail-base immunization with 200 μg of MOG35–55 (Biotrend) in complete Freund's adjuvant (CFA), supplemented with Mycobacterium tuberculosis H37RA (10 mg/ml) (Difco Laboratories) and 200 ng pertussis toxin (Sigma) on 0d and 2d. A control group was treated with CFA only and 200 ng pertussis toxin on 0d and 2d. Classification of disease progression: 0 no paralysis; 0.5 partial paralysis of tail; 1.0 paralysis of tail; 1.5 unilateral paralysis of hind legs; 2.0 bilateral paralysis of hind legs; 2.5 bilateral paralysis of hind legs with unilateral weakness of forelegs; 3 tetraparesis (abort criteria); 4 death. All animal experiments were approved by the government of North Rhine-Westphalia (Az.: 84–02.04.2013.A466, Az.: 8.87-50.10.34.08.241).
Organ and CNS mononuclear cell isolation
Mice were anesthetized and thereafter perfused with 50 ml ice cold PBS. For qRT PCR and flow cytometric analysis spleen, spinal cord and brain were removed. For flow cytometric analysis spinal cord and brain were homogenized and digested with collagenase/dispase (Roche) followed by DNase I (Roche) digestion. CNS derived mononuclear cells were isolated from the 30%/70% interface of a Percoll gradient after centrifugation at 800 × g for 25 min at room temperature. For RNA isolation organs were isolated after perfusion with 50 ml PBS and quick-frozen in liquid nitrogen.
We used monoclonal Antibodies against murine CD11b (M1/70), CD86 (GL-1), CD45 (104) from BD Biosciences, CD16/CD32 (2.4G2) from eBioscience for FACS analysis. Antibodies used for the OSC, spinal cord and brain histology: rat-MBP (1:500) from Millipore, rabbit-Iba1 (1:500) from WAKO Chemicals, mouse-Neurofilament (NF-M) (1:1000) from Convance Laboratories Inc., giunea pig-GFAP (1:1000) from SYnaptic Systems, rat-CD68 (1:500) from BioLegend, rat-Mac3 (1:500) from BioLegend, rat-TLR3 (1:500) from BioLegend, rb-pIRF7 (1:400) from Bioss-Antibodies and rabbit-CCR2 (1:500) from Bioss-Antibodies and rtLAMP2 (1:400) von BioLegend. A polyclonal crossreacting anti-GFP antibody was purchased from Abcam. Biotin conjugated donkey-anti-rabbit, as well as normal sera from mouse, rat and donkey were purchased from Jackson Immuno Research. All secondary antibodies conjugated with fluorophores (Cyanine Dye Cy2, Cy3 and Cy5) were purchased from Life Technologies and used in a dilution of 1 to 500.
Flow cytometry and cell sorting
Co-expression of indicated cell surface markers with YFP expression was analyzed on a FACS Canto II (Becton Dickinson). Cells were pregated as DAPI-. Isolated CNS mononuclear cells or primary adult microglia were sorted on a FACS Aria cell sorter (Becton Dickinson) for CD11b and CD45 and YFP reporter allele expression. RNA isolation of ex vivo sorted primary microglia was performed with the mirVana miRNA isolation kit (Ambion Inc). RNA isolation of in vitro sorted primary adult microglia was performed with RNA isolation kit (Fluka).
Mice were anaesthetized with isofluran and placed in a stereotactic frame. The skull was exposed and trepanated for injection of 6 μg poly (I:C) (Amersham) into the lateral ventricle. The bregma coordinates were AP: −0.3 mm, ML: +1.0 mm, and DV −3.0 mm.
For primary adult microglia culture CNS mononuclear cells were isolated from brain and spinal cord of 4–6 week old mice under sterile conditions and cultured in VLE-DMEM (Biochrom) with 10% FCS, 50 μM β-ME and 15% of M-CSF containing supernatant from L929 cells. The protocol was adapted from Ponomarev . Cells were stimulated on d14 with 50 μg/ml poly (I:C), 6 μg/ml CpG2216 (TIB MOLBIOL), 100 ng/ml Lipopolysaccharide (LPS) from Salmonella minnesota R595 (List Biological Laboratories, Inc.), 1 μg/ml Pam3CSK4 (Invivogen) or 100 U/ml mouse recombinant IFNβ (R&D Systems) for 6 h or 24 h as indicated or analysis of phagocytosis capacity was performed with DII-coupled myelin isolated according to Norton and Poduslo .
Mouse BV2 cells [23,24] were maintained on uncoated petri plates in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with Glucose (4.5 g/l), 10% FCS (Invitrogen), 20 mM GlutaMAX (Life Technologies) and penicillin/streptomycin (5 μg/ml) (Life Technologies). Media was changed every 2 days and cells were passaged at a confluence of 80-90% performing trypsinization (Invitrogen).
Immunofluorescence of microglial cells
Primary microglia or BV2 cells were once washed with PBS, fixed with 4% PFA for 15 minutes and again washed 2 times with PBS. Cells were blocked for 1 h with 5% (v/v) horse serum (Sigma Aldrich) in 0.5% (v/v) Triton X-100 in PBS. Primary antibodies were diluted in 2.5% (v/v) horse serum, 0.25% (v/v) Triton X-100 in PBS and incubated overnight at 4°C. After three times washing for 5 minutes with 0.1% (v/v) Triton X-100 in PBS the cells were incubated with fluorescent secondary antibodies, diluted in 2.5% (v/v) horse serum, 0.25% (v/v) Triton X-100 in PBS, for 1 h at RT. Cells were counterstained with Hoechst (Life Technology) and mounted on glass slides with Immuno Mount (DABCOTM).
RNA isolation, cDNA synthesis and qRT-PCR
RNA was isolated with RNA isolation Kit (Fluka or Macherey-Nagel). Purified RNA was digested with DNase I (Roche) to remove trace contaminating genomic DNA. An aliquot corresponding to 0.5 – 3 μg of purified RNA was used for first-strand cDNA synthesis using Superscript III reverse transcriptase and oligo (dT) in a final volume of 20 μl according to the manufacturer’s instruction (Invitrogen Life Technologies). cDNA was used for subsequent PCR. Real-time quantification of genes was performed using a SYBR Green RT-PCR assay (Applied Biosystems, USA). Briefly, each 20 μl SYBR green reaction consisted of 5 μl cDNA, 10 μl SYBR Green PCR-mix (2×), 1 μl forward and reverse primer (5pM) and 4 μl distilled water. PCR was performed with the following cycling conditions: 40 cycles of 10 sec at 95°C, 60 sec at 60°C and a separate dissociation step. Specificity of the PCR product was confirmed by examination of the dissociation reaction plots. A distinct single peak indicated only one DNA sequence was amplified during the RT-PCR. The samples were run in duplicates and the level of expression of each gene was compared with the expression of GAPDH. Amplification, detection of specific gene products and quantitative analysis were performed using an “ABI 7500” sequence detection system (Applied Biosystems, USA).
Organotypic slice cultures
Organotypic slice cultures (OSCs) were generated from 10 days old mice as described before . The cerebellum was cut into 400 μm thick slices using a McIllwain tissue chopper (GaLa Instrumente). OSC were dissociated in ice-cold dissecting medium (Hank’s Balanced Salt Solution (HBSS), Life Technologies) complemented with penicillin/streptomycin (100 U/ml, Life Technologies), 2.5 mg/ml glucose (Sigma Aldrich) and 10 mM kynurenic acid (Sigma Aldrich). OSCs were cultured on Millicell-CM culture plate inserts (Millipore) in culture medium (50% (v/v) MEM, 25% (v/v) HBSS, 25% (v/v) heat-inactivated horse serum, 2 mM glutamine, penicillin/streptomycin (100 U/ml) (all from Life Technologies) and 2.5 mg/ml glucose (Sigma Aldrich) for 3–5 days at 37°C in a humidified atmosphere with 5% CO2, and then demyelinated with lysolecithin (0.5 mg/ml, 16 h). After incubation the lysolecithin containing medium was removed and replaced with fresh medium. At this point OSCs were used for all experiments. In some experiments OSCs were treated with 100 U/ml mouse rIFNβ as indicated. For the usage of PLP-EGFP slices, fluorescent images were taken on indicated time points with an Olympus BX51 microscope at low magnification and in sterile conditions.
Brain and spinal cord were fixed with periodate-lysine-paraformaldehyde (PLP) overnight, incubated in 10% sucrose followed by 20% and 30% sucrose incubation steps. Organs were frozen in TissueTek (Sakura). Endogenous peroxidase activity and biotin were blocked. The staining of YFP was performed with a polyclonal crossreacting anti-GFP antibody overnight with 0.1% Triton at 4°C. Fluorescence was enhanced with TSA Fluorescein (PerkinElmer) according to the manufacturer‘s instructions. Sections were mounted with DAPI containing Vectashield . Imaging was performed on an epifluorescence microscope (Eclipse TE 2000, Nikon) with digital camera (CCD-1300, Vosskuehler) and overlaid using Adobe Photoshop.
For immunocytochemistry cryo sections were stained with luxol fast blue (solvent blue from Sigma Aldrich) for demyelination and nuclear fast red (Sigma Aldrich) for nuclei. Imaging was performed on a fluorescent microscope (Olympus BX51) with a digital camera (Olympus F-View II).
OSCs were washed two times in warm PBS, fixed 40 minutes in 4% paraformaldehyde (PFA) and permeabilized for 1 h with 1% (v/v) Triton X-100 (Sigma Aldrich) in PBS . OSCs were blocked for 2 h with 5% (v/v) horse serum (Sigma Aldrich) in 0.5% (v/v) Triton X-100 in PBS. Primary antibodies were diluted in 2.5% (v/v) horse serum, 0.25% (v/v) Triton X-100 in PBS and incubated for two days at 4°C. After three times washing for 15 minutes with 0.1% (v/v) Triton X-100 in PBS the OSCs were incubated with fluorescent secondary antibodies, diluted in 2.5% (v/v) horse serum, 0.25% (v/v) Triton X-100 in PBS, overnight at 4°C. OSCs were counterstained with Hoechst (Life Technology) and mounted on glass slides with Immuno Mount (DABCO™). Primary and secondary antibodies used are described above. Imaging was performed on a laser scanning confocal microscope (Zeiss Axiovert 200 M/LSM 510) with a digital camera (Zeiss Axiocam) and a fluorescent microscope (Olympus BX51) with a digital camera (Olympus F-View II). All images were overlaid and processed with Adobe Photoshop. For better visualization of transplanted BV-2 cells on OSCs the cells were circled using Adobe Photoshop software. For quantifying the MBP staining intensity images were analyzed by ImageJ software.
All values in the figures are shown as indicated (mean ± SEM or mean ± SD). Statistical significance was assessed using Student’s t test (*p < 0.05; **p < 0.01; ***p < 0.001).
IFNβ is produced primarily by microglia during the effector phase of EAE
IFNβ/YFP producing cells are located within active lesions in the CNS at the peak of disease
Microglia but not astrocytes or neurons are the source of IFNβ production in situ
IFNβ enhances microglia association with myelin debris and their phagocytic activity
Next, we examined whether IFNβ activates the phagocytic machinery in microglia cells. For this, expression of surface receptors and intracellular proteins involved in phagocytosis and phagosome maturation were assessed in WT, IFNβ−/− or IFNAR1−/− primary microglia as well as the microglia derived, immortalized cell line BV2. Multiple phagocytosis associated genes were markedly upregulated in WT microglia in response to IFNβ (Figure 4b, Additional file 5: Figure S5d and S5e), among those, the sensor molecules complement receptor 3 (CR3), signal-regulatory protein alpha (SIRPα) and Fc gamma Receptor 3 (FcγR3), shown to be directly involved in myelin phagocytosis in microglia and macrophages [29,30]. In addition, IFNβ upregulated microglial expression of molecules functionally involved in the general phagocytic machinery like macrophage scavenger receptor 1 (MSR1), CD68 and milk fat globule-EGF factor 8 (Mfg-E8) [31,32]. Furthermore, lysosome-associated membrane protein (LAMP) 1 and LAMP2 expression, both important for phagosome assembly were enhanced in response to IFNβ . MBP positive vesicular structures could be observed within IFNβ-activated, LAMP2+ primary microglia indicating active phagocytosis (Additional file 5: Figure S5f). The fact that most phagocytosis associated genes and Isg56 were also upregulated in IFNβ−/− microglia indicated a limited contribution of endogenously produced IFNβ to this gene activation (Figure 4b, Additional file 5: Figure S5d and S5g). We also verified the induction of Isg56 gene expression in WT and IFNβ−/− microglia (Additional file 5: Figure S5g). None of these genes were found to be upregulated in IFNAR1−/− microglia verifying the specific effect of IFNβ (Figure 4b).
We further studied whether IFNβ affected the capacity of microglia to specifically phagocytose myelin debris. For this microglia cultures from adult WT, IFNβ−/− or IFNAR1−/− mice and BV2 cells were treated with recombinant IFNβ (rIFNβ) or left untreated and incubated with DII-fluorescent labeled myelin debris for measurement of phagocytosis efficiency by flow cytometry. WT and IFNβ−/− microglia as well as BV2 microglia phagocytosed myelin debris more efficiently following treatment with IFNβ (Figure 4c and d, Additional file 5: Figure S5h and S5i). In contrast, IFNAR1−/− microglia did not exhibit increased phagocytotic rates of myelin debris (Figure 4c and d). These data indicate that IFNβ and IFNAR1-mediated signaling is required to stimulate microglial phagocytosis of myelin debris.
To investigate whether IFNβ would also induce phagocytosis in the ex vivo cerebellar slice culture model we used the PLP-EGFP reporter mouse model to monitor the clearance of myelin debris in situ . Thus, we induced demyelination in OSCs from PLP-GFP mice with LPC and treated these cultures with rIFNβ (Figure 4e). By using PLP-EGFP OSCs it was possible to measure the myelin content in the same living slice culture before and after the treatment. The myelin debris content was significantly decreased by rIFNβ as visualized by reduced EGFP-PLP signal in slices after 24 h treatment as compared to the PBS-treated demyelinated control cultures (Figure 4f and g). To verify the IFNβ specific effects in this model we prepared OSCs from WT, IFNβ−/− and IFNAR1−/− mice, induced demyelination with LPC and stimulated them with rIFNβ (Figure 4e). The myelin debris pattern was scored using an earlier established method (Additional file 6: Figure S6) . This revealed that WT and IFNβ−/− slice cultures stimulated by rIFNβ showed significantly less myelin debris than untreated OSCs (Figure 4h and i). IFNAR1−/− OSCs exhibited no increase in the removal of myelin debris in response to rIFNβ, providing additional evidence that IFNβ-dependent mechanisms drive myelin debris removal. In a complementary approach we investigated whether endogenously produced IFNβ also affects removal of myelin debris in the CNS. Here OSCs from WT and IFNβ−/− mice were treated with poly (I:C) and subjected to LPC-induced demyelination. We found a significantly higher intensity of myelin debris in OSCs of IFNβ−/− compared to WT slices which further underscored that IFNβ is functionally involved in the clearance of myelin debris (Additional file 5: Figure S5j and S5k). Myelin debris was detected within the cytoplasm of Iba1+ microglia cells in LPC-treated OSCs by confocal microscopy (Figure 4j). This points to an uptake of myelin debris by microglia induced by IFNβ.
Myelin debris accumulation in the CNS is increased in EAE in the absence of IFNβ or its receptor
IFNβ-producing microglia act as orchestrators of myelin debris removal
The high relevance of IFNβ in the therapy of MS and its pleiotropic protective effects in mice and men are irrevocable. Here we identified the so far ill-defined IFNβ producing cells in CNS autoimmunity as primarily microglia in active lesions within the CNS. We further demonstrate that these microglia orchestrate phagocytosis of myelin debris in a process mediated by and dependent on IFNβ.
Our time course analyses showed that IFNβ production and the expression of the IFN-inducible Isg56 in the CNS starts with the onset of clinical symptoms and increases in parallel with the disease score of MOG-induced EAE. These findings extend previous data showing that IFNβ is produced at the peak of EAE exclusively in the CNS . In peripheral lymphoid organs, however, we found IFNβ upregulated early after MOG-immunization and decreased afterwards. This initial expression of IFNβ is also observed when CFA is administered in the absence of MOG and is therefore the result of the immuno-adjuvant containing heat-inactivated M. tuberculosis that induces a strong innate immune response. Plasmacytoid dendritic cells have been shown to be responsible for this early produced IFNβ  that exacerbates the clinical course of EAE presumably via contributing to the priming of encephalitogenic T cells. Expression of IFNα/β in the effector phase of disease instead was suggested to mediate protective effects by acting directly on myeloid cells .
We identified microglia, but not astrocytes or neurons as the major cellular source of IFNβ in the CNS at peak EAE. The identity of IFNβ producing cells in the course of EAE has been a long-standing topic of debate. In general, different cell types, mainly professional antigen presenting cells, are capable of producing IFNβ in the context of immune activation. Plasmacytoid and classical dendritic cells are capable of IFNβ production after TLR9- and TLR3/MDA5-stimulation, respectively [17,36]. During viral infections in the CNS also neurons have been shown to produce IFNβ . In vitro murine neurons as well as microglia produced IFNβ after poly (I:C) stimulation [38,39]. Other brain resident cells reported to produce predominantly IFNα are astrocytes as shown in Aicardi-Goutières Syndrome, a rare neurodevelopmental disorder . In MS IFNβ production was detected in active lesions in cells defined as macrophages and astrocytes based on their morphology . Also, based on morphological studies it was suggested earlier that IFNβ may be produced by ramified microglia or infiltrating cells in EAE . Our study defines activated microglia as the prominent IFNβ-producing cell type in the CNS as characterized by an intermediate CD45 and high Iba1 expression as well as a hypermorphic-rounded morphology in the IFNβ/YFP fluorescence reporter mouse model [42-44]. It has been suggested that an activated CD45high CD11b+ microglia subset with the capacity to differentiate into macrophages or dendritic-like cells plays an active role in the pathogenesis of EAE [21,45]. However, our data indicate that IFNβ-expressing microglia did not acquire a CD45high CD11b+ phenotype during MOG-EAE excluding that they phenotypically and functionally resemble the earlier described subset. The fact that in our study IFNβ production was identified in microglia not only during EAE but also after intrathecal injection of the molecular pathogen compound poly (I:C) points to a specialized function of these CNS resident phagocytes to produce type I IFNs. Of note, at early timepoints after intrathecal poly (I:C) application Khorooshi et al. describe a quick mobilization of IFNβ producing myeloid cells from the periphery into the CNS (personal communication). The discrepancies between these two studies could be explained by the different modes intrathecal poly (I:C) application was used (intracerebroventricular vs. intracisterna magna), that may cause induction of divergent chemokine patterns driving leukocyte infiltration. However, in this report, also microglia were shown to contribute to IFNβ production. The IFNβ expression capacity by these cells is in line with our findings on IFNβ production by microglia in CNS autoimmunity without prior poly (I:C) stimulation. We could show that IFNβ-producing microglia localized in the proximity of myelin lesions and exhibit a superior capacity to induce phagocytosis of myelin debris in surrounding cells. It is yet unclear which chemotactic factors guide positioning of microglia into areas of demyelination in CNS autoimmunity. It has been suggested that astrocytes direct migration and activation of microglia and macrophages in demyelinating lesions via expression of CCL2 and CXCL10. Correspondingly microglia and immigrating macrophages in MS lesions stained positive for CXCR3 and CCR2 in MS lesions . Recent elegant data defined CCR2 as a selective marker for infiltrating macrophages in the inflamed CNS [47-49]. Our data do not rule out that immigrating myeloid cells contribute to IFNβ production as indicated by (i) detection of IFNβ mRNA in peripheral tissues early after immunization, (ii) CCR2 expression in a subset of IFNβ/YFP+ cells in CNS lesions, and (iii) direct flow cytometric detection of CD45high IFNβ/YFP+ cells.
The most important finding of our study is that strategically positioned IFNβ producing microglia within active CNS lesions exhibit a superior capacity to induce myelin debris removal in surrounding tissue phagocytes. Effective clearance of myelin debris is a critical step in the pathogenesis of MS as well as EAE. While microglia have been attributed important roles in the inflammatory response during infections and CNS autoimmunity  it is still a matter of debate whether microglia represent efficient phagocytes in the CNS . Here we could show that phagocytosis of myelin debris by microglia was dependent on IFNβ and its receptor IFNAR1. These findings are supported by recent studies showing that in vitro microglia deficient in TIR domain containing adapter inducing interferon beta (TRIF)−/− less effectively cleared axonal debris. These microglia further exhibited an increased threshold for activation of interferon-regulated genes, suggesting that IFNβ may upregulate phagocytic activity . In contrast to this, earlier studies suggested that IFNβ suppresses the phagocytosis of myelin debris in vitro . In these studies, phagocytic activity, however, was tested in peritoneal macrophages or CD11b+ cells in the CNS not differentiating between resident microglia and immigrated macrophages from the periphery. The discrepancy to our data can therefore be explained by differences in the phagocytic activity of microglia and macrophages .
The EAE in vivo model exhibits a high variability in disease severity and in the localization of CNS lesions between individual animals. Also the suppressive effect of IFNβ and IFNAR mediated signalling on EAE development might reduce demyelination as well as microglia activation [3,4], and Khorooshi et al., personal communication. To eliminate this variability, the unwanted bias and the influence of peripheral immune responses, we used the model of LPC-induced demyelination on OSCs  allowing a reproducible and controlled evaluation of IFNβ and microglia mediated effects. IFNβ expressing microglia specifically localized to demyelinated regions in OSCs and further showed lower amounts of myelin debris in their direct proximity in comparison to IFNβ non-producers indicative of a more efficient myelin debris removal. This phagocytosis activating effect was confirmed by transfer of IFNβ/YFP producing microglia on demyelinated WT as well as IFNβ−/− OSCs but was not observed on IFNAR1 deficient OSCs. These data point towards a pivotal function of IFNβ producing microglia in the orchestration of phagocytosis of myelin debris by not only neighboring microglia but also immigrating phagocytes in CNS autoimmunity. It remains to be shown, however, whether the effects observed in EAE can be translated into human MS. While myelin phagocytosis has been suggested to contribute to damage processes in MS by the associated oxidative burst, a number of studies have shown beneficial effects for the effective clearing of myelin debris . Phagocytosis of myelin debris by activated microglia was observed in MS lesions  and was essential to promote regeneration . Recently, overexpression of the phagocytosis triggering receptor TREM2 was shown to reduce the severity of clinical symptoms in EAE . Myelin debris was shown to impair remyelination by inhibiting differentiation as well as the recruitment of oligodendrocyte precursor cells after injury . Also, myelin directly inhibited axonal re-growth as it contains several growth inhibitory molecules such as Nogo A . A secondary protective effect of IFNβ-activated myelin phagocytosis might be the induction of a regulatory type of microglia resembling M2 macrophages .
A number of studies indicate that myelin clearance in the CNS after demyelination is protective or ameliorates disease symptoms in EAE and MS. Here, we identify microglia as orchestrators of myelin phagocytosis via production of the protective IFNβ at the peak of CNS autoimmunity. Our findings represent novel insights into the in vivo functions of microglia-derived IFNβ and the feasibility of novel therapeutic approaches for MS specifically targeting CNS microglia.
This work was supported by the Deutsche Forschungsgemeinschaft (SCHE692/3-1, SCHE692/4-1, EXC 1003-CiM) and the Strategic Research Fund of the Heinrich-Heine-University Duesseldorf. For some illustrations the servier medical art database was used.
- Aktas O, Kieseier B, Hartung HP (2010) Neuroprotection, regeneration and immunomodulation: broadening the therapeutic repertoire in multiple sclerosis. Trends Neurosci 33:140–152, doi:10.1016/j.tins.2009.12.002View ArticlePubMedGoogle Scholar
- Paty DW, Li DK (1993) Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. II. MRI analysis results of a multicenter, randomized, double-blind, placebo-controlled trial. UBC MS/MRI Study Group and the IFNB Multiple Sclerosis Study Group. Neurology 43:662–667View ArticlePubMedGoogle Scholar
- Prinz M, Schmidt H, Mildner A, Knobeloch KP, Hanisch UK, Raasch J, Merkler D, Detje C, Gutcher I, Mages J, Lang R, Martin R, Gold R, Becher B, Bruck W, Kalinke U (2008) Distinct and nonredundant in vivo functions of IFNAR on myeloid cells limit autoimmunity in the central nervous system. Immunity 28:675–686, doi:10.1016/j.immuni.2008.03.011View ArticlePubMedGoogle Scholar
- Teige I, Treschow A, Teige A, Mattsson R, Navikas V, Leanderson T, Holmdahl R, Issazadeh-Navikas S (2003) IFN-beta gene deletion leads to augmented and chronic demyelinating experimental autoimmune encephalomyelitis. J Immunol 170:4776–4784View ArticlePubMedGoogle Scholar
- Hohlfeld R, Wekerle H (2001) Immunological update on multiple sclerosis. Curr Opin Neurol 14:299–304View ArticlePubMedGoogle Scholar
- Touil T, Fitzgerald D, Zhang GX, Rostami A, Gran B (2006) Cutting Edge: TLR3 stimulation suppresses experimental autoimmune encephalomyelitis by inducing endogenous IFN-beta. J Immunol 177:7505–7509View ArticlePubMedGoogle Scholar
- Calabresi PA, Pelfrey CM, Tranquill LR, Maloni H, McFarland HF (1997) VLA-4 expression on peripheral blood lymphocytes is downregulated after treatment of multiple sclerosis with interferon beta. Neurology 49:1111–1116View ArticlePubMedGoogle Scholar
- Nelissen I, Ronsse I, Van Damme J, Opdenakker G (2002) Regulation of gelatinase B in human monocytic and endothelial cells by PECAM-1 ligation and its modulation by interferon-beta. J Leukoc Biol 71:89–98PubMedGoogle Scholar
- Wiesemann E, Deb M, Trebst C, Hemmer B, Stangel M, Windhagen A (2008) Effects of interferon-beta on co-signaling molecules: upregulation of CD40, CD86 and PD-L2 on monocytes in relation to clinical response to interferon-beta treatment in patients with multiple sclerosis. Mult Scler 14:166–176, doi:10.1177/1352458507081342View ArticlePubMedGoogle Scholar
- Teige I, Liu Y, Issazadeh-Navikas S (2006) IFN-beta inhibits T cell activation capacity of central nervous system APCs. J Immunol 177:3542–3553View ArticlePubMedGoogle Scholar
- Pette M, Pette DF, Muraro PA, Farnon E, Martin R, McFarland HF (1997) Interferon-beta interferes with the proliferation but not with the cytokine secretion of myelin basic protein-specific, T-helper type 1 lymphocytes. Neurology 49:385–392View ArticlePubMedGoogle Scholar
- Gniadek P, Aktas O, Wandinger KP, Bellmann-Strobl J, Wengert O, Weber A, von Wussow P, Obert HJ, Zipp F (2003) Systemic IFN-beta treatment induces apoptosis of peripheral immune cells in MS patients. J Neuroimmunol 137:187–196View ArticlePubMedGoogle Scholar
- Liu Y, Carlsson R, Comabella M, Wang J, Kosicki M, Carrion B, Hasan M, Wu X, Montalban X, Dziegiel MH, Sellebjerg F, Sorensen PS, Helin K, Issazadeh-Navikas S (2014) FoxA1 directs the lineage and immunosuppressive properties of a novel regulatory T cell population in EAE and MS. Nat Med 20:272–282, doi:10.1038/nm.3485View ArticlePubMedGoogle Scholar
- Liu Y, Hao W, Letiembre M, Walter S, Kulanga M, Neumann H, Fassbender K (2006) Suppression of microglial inflammatory activity by myelin phagocytosis: role of p47-PHOX-mediated generation of reactive oxygen species. J Neurosci 26:12904–12913, doi:10.1523/JNEUROSCI. 2531-06.2006View ArticlePubMedGoogle Scholar
- Munschauer FE 3rd, Kinkel RP (1997) Managing side effects of interferon-beta in patients with relapsing-remitting multiple sclerosis. Clin Ther 19:883–893View ArticlePubMedGoogle Scholar
- Perini P, Calabrese M, Biasi G, Gallo P (2004) The clinical impact of interferon beta antibodies in relapsing-remitting MS. J Neurol 251:305–309, doi:10.1007/s00415-004-0312-8View ArticlePubMedGoogle Scholar
- Scheu S, Dresing P, Locksley RM (2008) Visualization of IFNbeta production by plasmacytoid versus conventional dendritic cells under specific stimulation conditions in vivo. Proc Natl Acad Sci U S A 105:20416–20421, doi:10.1073/pnas.0808537105View ArticlePubMed CentralPubMedGoogle Scholar
- Erlandsson L, Blumenthal R, Eloranta ML, Engel H, Alm G, Weiss S, Leanderson T (1998) Interferon-beta is required for interferon-alpha production in mouse fibroblasts. Curr Biol 8:223–226View ArticlePubMedGoogle Scholar
- Hwang SY, Hertzog PJ, Holland KA, Sumarsono SH, Tymms MJ, Hamilton JA, Whitty G, Bertoncello I, Kola I (1995) A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses. Proc Natl Acad Sci U S A 92:11284–11288View ArticlePubMed CentralPubMedGoogle Scholar
- Sobottka B, Ziegler U, Kaech A, Becher B, Goebels N (2011) CNS live imaging reveals a new mechanism of myelination: the liquid croissant model. Glia 59:1841–1849, doi:10.1002/glia.21228View ArticlePubMedGoogle Scholar
- Ponomarev ED, Shriver LP, Maresz K, Dittel BN (2005) Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J Neurosci Res 81:374–389, doi:10.1002/jnr.20488View ArticlePubMedGoogle Scholar
- Norton WT, Poduslo SE (1973) Myelination in rat brain: method of myelin isolation. J Neurochem 21:749–757View ArticlePubMedGoogle Scholar
- Blasi E, Barluzzi R, Bocchini V, Mazzolla R, Bistoni F (1990) Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. J Neuroimmunol 27:229–237View ArticlePubMedGoogle Scholar
- Bocchini V, Mazzolla R, Barluzzi R, Blasi E, Sick P, Kettenmann H (1992) An immortalized cell line expresses properties of activated microglial cells. J Neurosci Res 31:616–621, doi:10.1002/jnr.490310405View ArticlePubMedGoogle Scholar
- Aktas O, Smorodchenko A, Brocke S, Infante-Duarte C, Schulze Topphoff U, Vogt J, Prozorovski T, Meier S, Osmanova V, Pohl E, Bechmann I, Nitsch R, Zipp F (2005) Neuronal damage in autoimmune neuroinflammation mediated by the death ligand TRAIL. Neuron 46:421–432, doi:10.1016/j.neuron.2005.03.018View ArticlePubMedGoogle Scholar
- Dresing P, Borkens S, Kocur M, Kropp S, Scheu S (2010) A fluorescence reporter model defines "Tip-DCs" as the cellular source of interferon beta in murine listeriosis. PLoS One 5:e15567, doi:10.1371/journal.pone.0015567View ArticlePubMed CentralPubMedGoogle Scholar
- Sierra A, Abiega O, Shahraz A, Neumann H (2013) Janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis. Front Cell Neurosci 7:6, doi:10.3389/fncel.2013.00006View ArticlePubMed CentralPubMedGoogle Scholar
- Birgbauer E, Rao TS, Webb M (2004) Lysolecithin induces demyelination in vitro in a cerebellar slice culture system. J Neurosci Res 78:157–166, doi:10.1002/jnr.20248View ArticlePubMedGoogle Scholar
- Gitik M, Liraz-Zaltsman S, Oldenborg PA, Reichert F, Rotshenker S (2011) Myelin down-regulates myelin phagocytosis by microglia and macrophages through interactions between CD47 on myelin and SIRPalpha (signal regulatory protein-alpha) on phagocytes. J Neuroinflammation 8:24, doi:10.1186/1742-2094-8-24View ArticlePubMed CentralPubMedGoogle Scholar
- Makranz C, Cohen G, Baron A, Levidor L, Kodama T, Reichert F, Rotshenker S (2004) Phosphatidylinositol 3-kinase, phosphoinositide-specific phospholipase-Cgamma and protein kinase-C signal myelin phagocytosis mediated by complement receptor-3 alone and combined with scavenger receptor-AI/II in macrophages. Neurobiol Dis 15:279–286, doi:10.1016/j.nbd.2003.11.007View ArticlePubMedGoogle Scholar
- Liu Y, Yang X, Guo C, Nie P, Liu Y, Ma J (2013) Essential role of MFG-E8 for phagocytic properties of microglial cells. PLoS One 8:e55754, doi:10.1371/journal.pone.0055754View ArticlePubMed CentralPubMedGoogle Scholar
- Thomas CA, Li Y, Kodama T, Suzuki H, Silverstein SC, El Khoury J (2000) Protection from lethal gram-positive infection by macrophage scavenger receptor-dependent phagocytosis. J Exp Med 191:147–156View ArticlePubMed CentralPubMedGoogle Scholar
- Huynh KK, Eskelinen EL, Scott CC, Malevanets A, Saftig P, Grinstein S (2007) LAMP proteins are required for fusion of lysosomes with phagosomes. EMBO J 26:313–324, doi: 10.1038/sj.emboj.7601511View ArticlePubMed CentralPubMedGoogle Scholar
- Skripuletz T, Hackstette D, Bauer K, Gudi V, Pul R, Voss E, Berger K, Kipp M, Baumgartner W, Stangel M (2013) Astrocytes regulate myelin clearance through recruitment of microglia during cuprizone-induced demyelination. Brain 136:147–167, doi:10.1093/brain/aws262View ArticlePubMedGoogle Scholar
- Isaksson M, Ardesjo B, Ronnblom L, Kampe O, Lassmann H, Eloranta ML, Lobell A (2009) Plasmacytoid DC promote priming of autoimmune Th17 cells and EAE. Eur J Immunol 39:2925–2935, doi:10.1002/eji.200839179View ArticlePubMedGoogle Scholar
- Asselin-Paturel C, Boonstra A, Dalod M, Durand I, Yessaad N, Dezutter-Dambuyant C, Vicari A, O'Garra A, Biron C, Briere F, Trinchieri G (2001) Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology. Nat Immunol 2:1144–1150, doi:10.1038/ni736View ArticlePubMedGoogle Scholar
- Delhaye S, Paul S, Blakqori G, Minet M, Weber F, Staeheli P, Michiels T (2006) Neurons produce type I interferon during viral encephalitis. Proc Natl Acad Sci U S A 103:7835–7840, doi:10.1073/pnas.0602460103View ArticlePubMed CentralPubMedGoogle Scholar
- Town T, Jeng D, Alexopoulou L, Tan J, Flavell RA (2006) Microglia recognize double-stranded RNA via TLR3. J Immunol 176:3804–3812View ArticlePubMedGoogle Scholar
- Ward LA, Massa PT (1995) Neuron-specific regulation of major histocompatibility complex class I, interferon-beta, and anti-viral state genes. J Neuroimmunol 58:145–155View ArticlePubMedGoogle Scholar
- van Heteren JT, Rozenberg F, Aronica E, Troost D, Lebon P, Kuijpers TW (2008) Astrocytes produce interferon-alpha and CXCL10, but not IL-6 or CXCL8, in Aicardi-Goutieres syndrome. Glia 56:568–578, doi:10.1002/glia.20639View ArticlePubMedGoogle Scholar
- Traugott U, Lebon P (1988) Multiple sclerosis: involvement of interferons in lesion pathogenesis. Ann Neurol 24:243–251, doi:10.1002/ana.410240211View ArticlePubMedGoogle Scholar
- Imai Y, Ibata I, Ito D, Ohsawa K, Kohsaka S (1996) A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochem Biophys Res Commun 224:855–862, doi:10.1006/bbrc.1996.1112View ArticlePubMedGoogle Scholar
- Sedgwick JD, Schwender S, Imrich H, Dorries R, Butcher GW, ter Meulen V (1991) Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system. Proc Natl Acad Sci U S A 88:7438–7442View ArticlePubMed CentralPubMedGoogle Scholar
- Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308:1314–1318, doi:10.1126/science.1110647View ArticlePubMedGoogle Scholar
- Murphy AC, Lalor SJ, Lynch MA, Mills KH (2010) Infiltration of Th1 and Th17 cells and activation of microglia in the CNS during the course of experimental autoimmune encephalomyelitis. Brain Behav Immun 24:641–651, doi:10.1016/j.bbi.2010.01.014View ArticlePubMedGoogle Scholar
- Tanuma N, Sakuma H, Sasaki A, Matsumoto Y (2006) Chemokine expression by astrocytes plays a role in microglia/macrophage activation and subsequent neurodegeneration in secondary progressive multiple sclerosis. Acta Neuropathol 112:195–204, doi:10.1007/s00401-006-0083-7View ArticlePubMedGoogle Scholar
- Yamasaki R, Lu H, Butovsky O, Ohno N, Rietsch AM, Cialic R, Wu PM, Doykan CE, Lin J, Cotleur AC, Kidd G, Zorlu MM, Sun N, Hu W, Liu L, Lee JC, Taylor SE, Uehlein L, Dixon D, Gu J, Floruta CM, Zhu M, Charo IF, Weiner HL, Ransohoff RM (2014) Differential roles of microglia and monocytes in the inflamed central nervous system. J Exp Med 211:1533–1549, doi:10.1084/jem.20132477View ArticlePubMed CentralPubMedGoogle Scholar
- Mizutani M, Pino PA, Saederup N, Charo IF, Ransohoff RM, Cardona AE (2012) The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood. J Immunol 188:29–36, doi:10.4049/jimmunol.1100421View ArticlePubMed CentralPubMedGoogle Scholar
- Saederup N, Cardona AE, Croft K, Mizutani M, Cotleur AC, Tsou CL, Ransohoff RM, Charo IF (2010) Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice. PLoS One 5:e13693, doi:10.1371/journal.pone.0013693View ArticlePubMed CentralPubMedGoogle Scholar
- Nayak D, Roth TL, McGavern DB (2014) Microglia development and function. Annu Rev Immunol 32:367–402, doi:10.1146/annurev-immunol-032713-120240View ArticlePubMedGoogle Scholar
- Hosmane S, Tegenge MA, Rajbhandari L, Uapinyoying P, Kumar NG, Thakor N, Venkatesan A (2012) Toll/interleukin-1 receptor domain-containing adapter inducing interferon-beta mediates microglial phagocytosis of degenerating axons. J Neurosci 32:7745–7757, doi:10.1523/JNEUROSCI. 0203-12.2012View ArticlePubMed CentralPubMedGoogle Scholar
- Kuhlmann T, Wendling U, Nolte C, Zipp F, Maruschak B, Stadelmann C, Siebert H, Bruck W (2002) Differential regulation of myelin phagocytosis by macrophages/microglia, involvement of target myelin, Fc receptors and activation by intravenous immunoglobulins. J Neurosci Res 67:185–190View ArticlePubMedGoogle Scholar
- Bauer J, Sminia T, Wouterlood FG, Dijkstra CD (1994) Phagocytic activity of macrophages and microglial cells during the course of acute and chronic relapsing experimental autoimmune encephalomyelitis. J Neurosci Res 38:365–375, doi:10.1002/jnr.490380402View ArticlePubMedGoogle Scholar
- Napoli I, Neumann H (2010) Protective effects of microglia in multiple sclerosis. Exp Neurol 225:24–28, doi:10.1016/j.expneurol.2009.04.024View ArticlePubMedGoogle Scholar
- Takahashi K, Prinz M, Stagi M, Chechneva O, Neumann H (2007) TREM2-transduced myeloid precursors mediate nervous tissue debris clearance and facilitate recovery in an animal model of multiple sclerosis. PLoS Med 4:e124, doi:10.1371/journal.pmed.0040124View ArticlePubMed CentralPubMedGoogle Scholar
- Kotter MR, Li WW, Zhao C, Franklin RJ (2006) Myelin impairs CNS remyelination by inhibiting oligodendrocyte precursor cell differentiation. J Neurosci 26:328–332, doi:10.1523/JNEUROSCI. 2615-05.2006View ArticlePubMedGoogle Scholar
- David S, Lacroix S (2003) Molecular approaches to spinal cord repair. Annu Rev Neurosci 26:411–440, doi:10.1146/annurev.neuro.26.043002.094946View ArticlePubMedGoogle Scholar
- Goldmann T, Prinz M (2013) Role of microglia in CNS autoimmunity. Clin Dev Immunol 2013:208093, doi:10.1155/2013/208093View ArticlePubMed CentralPubMedGoogle Scholar
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