Interferon regulatory factor 8/interferon consensus sequence binding protein is a critical transcription factor for the physiological phenotype of microglia
- Makoto Horiuchi†1, 2,
- Kouji Wakayama†3,
- Aki Itoh1, 2,
- Kumi Kawai4,
- David Pleasure1, 2,
- Keiko Ozato5 and
- Takayuki Itoh1, 2Email author
© Horiuchi et al.; licensee BioMed Central Ltd. 2012
Received: 16 May 2012
Accepted: 13 August 2012
Published: 28 September 2012
Recent fate-mapping studies establish that microglia, the resident mononuclear phagocytes of the CNS, are distinct in origin from the bone marrow-derived myeloid lineage. Interferon regulatory factor 8 (IRF8, also known as interferon consensus sequence binding protein) plays essential roles in development and function of the bone marrow-derived myeloid lineage. However, little is known about its roles in microglia.
The CNS tissues of IRF8-deficient mice were immunohistochemically analyzed. Pure microglia isolated from wild-type and IRF8-deficient mice were studied in vitro by proliferation, immunocytochemical and phagocytosis assays. Microglial response in vivo was compared between wild-type and IRF8-deficient mice in the cuprizon-induced demyelination model.
Our analysis of IRF8-deficient mice revealed that, in contrast to compromised development of IRF8-deficient bone marrow myeloid lineage cells, development and colonization of microglia are not obviously affected by loss of IRF8. However, IRF8-deficient microglia demonstrate several defective phenotypes. In vivo, IRF8-deficient microglia have fewer elaborated processes with reduced expression of IBA1/AIF1 compared with wild-type microglia, suggesting a defective phenotype. IRF8-deficient microglia are significantly less proliferative in mixed glial cultures than wild-type microglia. Unlike IRF8-deficient bone marrow myeloid progenitors, exogenous macrophage colony stimulating factor (colony stimulating factor 1) (M-CSF (CSF1)) restores their proliferation in mixed glial cultures. In addition, IRF8-deficient microglia exhibit an exaggerated growth response to exogenous granulocyte-macrophage colony stimulating factor (colony stimulating factor 2) (GM-CSF (CSF2)) in the presence of other glial cells. IRF8-deficient microglia also demonstrate altered cytokine expressions in response to interferon-gamma and lipopolysaccharide in vitro. Moreover, the maximum phagocytic capacity of IRF8-deficient microglia is reduced, although their engulfment of zymosan particles is not overtly impaired. Defective scavenging activity of IRF8-deficient microglia was further confirmed in vivo in the cuprizone-induced demyelination model in mice.
This study is the first to demonstrate the essential contribution of IRF8-mediated transcription to a broad range of microglial phenotype. Microglia are distinct from the bone marrow myeloid lineage with respect to their dependence on IRF8-mediated transcription.
KeywordsMicroglia Interferon regulatory factor Phagocytosis Cytokine Cuprizone-induced demyelination
Microglia, the resident mononuclear phagocyte (MP) system of the CNS, play principal roles in the innate immune response to tissue damage and invasion of foreign organisms, and also in initiation and modulation of the adaptive immune response [1–3]. Although microglia share many common features of MPs with bone marrow (BM)-derived monocyte lineage cells and tissue macrophages of monocyte origin, recent studies have demonstrated that the contribution of BM-derived myeloid cells to microglial homeostasis in the steady state is a far less frequent event than was previously believed [4, 5]. Only under some abnormal conditions such as tissue alteration following irradiation to the CNS, do a subset of circulating monocytes (Ly-6C/Gr-1hi CCR2+) become engrafted as BM-derived microglia (BMDM) through the CNS vasculature . Moreover, in vivo fate mapping studies have established that microglia are an ontogenically distinct population in the MP system. Microglial precursors arise from primitive extra-embryonic hematopoiesis in the yolk sac prior to the onset of blood circulation. These primitive MPs migrate into the CNS through blood vessels independently of the second wave of hematopoiesis within the embryo proper, which leads to the adult (definitive) hematopoiesis in the BM . Once having colonized the CNS, embryonic microglia are highly proliferative and strongly dependent on colony stimulating factor 1 receptor (CSF1R)-mediated signaling for development during embryogenesis. In mice lacking CSF1R, the number of microglia is greatly reduced [6, 7], whereas circulating monocytes are present, indicating that, in contrast to microglia, the BM-derived monocytes are less dependent on CSF1R-mediated signaling for their development . These accumulating pieces of evidence further support the notion that microglia are maintained by self-renewal as a distinct MP population without continuous replenishment by systemic BM-derived precursors or myeloid cells, although it remains to be clarified to what extent microglia share common molecular mechanisms with BM-derived MPs in their development, maintenance, and functioning.
The molecular basis of myeloid development in the BM hematopoiesis has been intensively studied. Among the molecules known to be involved in myeloid development to date, interferon regulatory factor-8 (IRF8, also known as interferon consensus sequence binding protein (ICSBP)), a member of the interferon regulatory factor family, acts as an essential transcription factor for differentiation and maturation of BM-derived MPs in the myeloid lineage as well as for B cell development from hematopoietic stem cells [8–10]. IRF8 limits the size of the bipotential granulocyte-macrophage progenitor pool, and directs these progenitor cells to differentiate into the monocyte lineage by stimulating expression of genes critical for MP differentiation and by repressing a series of genes required for granulocytic differentiation . IRF8-deficient BM-derived MPs are not generated efficiently in IRF8-deficient mice, and are also defective in production of cytokines and reactive oxygen species [12–14]. IRF8 is constitutively expressed in microglia as well . Since microglia arise from mesodermal myeloid progenitors in the primitive hematopoiesis, which is distinct from the development of BM-derived MPs, it is essential to determine how IRF8 regulates development of microglia to understand the molecular basis of distinct development of microglia and BM-derived MPs. Moreover, given the essential roles for IRF8 in the functional phenotypes of BM-derived MPs, constitutive expression of IRF8 could also be critical for microglial functions. Indeed, a recent study has demonstrated an essential role for IRF8 in activation of microglia in the spinal cord following peripheral nerve injury .
In this study, we performed a comprehensive study of the microglia of constitutional IRF8-null mice, and further demonstrate essential roles for IRF8 in physiological phenotype and function of microglia.
Materials and methods
The mouse strain with targeted null mutation in the Irf8 gene in this study was reported previously . The strain was backcrossed onto the C57BL/6J strain for more than 5 generations. Animals were housed in standard laboratory cages with unrestricted access to food and water, and maintained under 12 h light/dark cycles. All procedures using the animals were approved by the Institutional Animal Care and Use Committee of the University of California, Davis.
Reagents and chemicals
All reagents and culture media used in this study were purchased from SIGMA (St. Louis, MO, USA) and Invitrogen (Carlsbad, CA, USA), respectively, except for the following products. Mouse anti-β-actin antibody was from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-IBA1/AIF1 antibody was from Wako Chemicals USA (Richmond, VA, USA). Rabbit anti-IRF8 antibody was reported previously .
Mixed glial culture
Whole brains from 0 to 2-day-old mice were dissected and diced into 2-mm cubes. After cleaning off meninges, and vessels including choroidal plexus, the brain chunks were digested by 20 units/ml papain in Earle’s buffered salt solution supplemented with 1 mM L-cystaine, 0.5 mM EDTA, 0.36% (w/v) D-glucose, and 250 units/ml DNase at 33°C for 90 min. Papain was inactivated by 1.5 mg/ml ovomucoid in PBS containing 1.5 mg/ml bovine serum albumin (BSA) and 250 units/ml DNase. The softened chunks were gently triturated by passing through a 1-ml serological pipette several times. The resulting suspension was left for 1 minute to allow undissociated tissues to settle down on the bottom, and then the supernatant containing dissociated cells was collected to another tube. After repeating this trituration step three times, the obtained cell suspension was spun at 220 × g for 15 minutes. Cells were resuspended in the MG medium; 5% (v/v) heat-inactivated fetal bovine serum, 1× N2 NeuroPlex supplement (Gemini Bio-Products, West Sacramento, CA), 100 units/ml penicillin, and 100 μg/ml streptomycin in high glucose Dulbecco’s modified Eagle’s medium (DMEM), and plated on a poly-D-lysine-coated T75 tissue culture flask. In some experiments, 20 ng/ml of either macrophage colony stimulating factor (M-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF) was added to the culture medium at 5 days in vitro, by which time most cultures had reached confluency. Cultures were fed with fresh medium every other day.
Magnetic cell sorting (MACS) to isolate CD11b+mouse microglia
Mixed glial cells in a T75 flask were maintained in the presence of 20 ng/ml M-CSF from day 5 to day 9 in vitro. Then cells were washed with 5 ml Ca++- and Mg++-free Hanks’ balanced salt solution (HBSS-) and then 1 ml trypsin-EDTA (0.05% (v/v) and 0.53 mM, respectively, in HBSS-) was added. After a 3 minute-trypsinization at 37°C, 0.5 ml of trypsin inhibitor-BSA-DNase (0.25 mg/ml, 1.44 mg/ml and 60 units/ml, respectively, in DMEM.) was added, and detached cells were collected into a 50-ml tube containing 3.5 ml MG medium in addition to the medium saved from the cultures and from the HBSS- that had been used for wash. Cells were collected by centrifugation at 520 × g for 5 minutes, resuspended into a mixture of 40 μl CD11b microbeads (catalog number: 130-093-634, Miltenyi Biotech, Auburn, CA, USA) and 160 μl ice-cold MACS buffer (0.5% BSA, 2 mM EDTA in PBS, pH 7.2), and incubated at 4°C for 15 minutes. After a wash with 20 ml ice-cold MACS buffer, cells were resuspended into 500 μl ice-cold MACS buffer, and then applied to a MACS separation column LS (Miltenyi Biotech) in a magnetic field. The column was washed with 3 ml ice-cold MACS buffer three times and removed from the magnetic field. Magnetic labeled cells were eluted with 5 ml MACS buffer. After centrifugation at 520 × g for 5 minutes, cells were plated on to poly-D-lysine-coated culture ware at 1.25 × 103 cells/cm2 in MG medium.
Purified microglia were incubated with rat anti-CD11b antibody (1:50; AdB Serotec, Raleigh, NC, USA) for 30 minutes. After washing with PBS three times, cells were incubated with Alexa Fluor™ 488-conjugated goat anti-rat IgG for 30 minutes. Cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, and then permeabilized with ice-cold 100% methanol for 20 minutes. Nuclei were counterstained with 4,6-diamidio-2-phenylindole (0.5 μg/ml, DAPI).
Protein lysates were prepared in the lysis buffer as described previously . Twenty μg of protein from each sample were size-fractioned by SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane (Schleicher > Schnell, Keene, NH, USA) and probed with primary antibodies for IRF8 (1:5000) and AIF1/IBA1 (1:5000) for 1 h. Full range recombinant Rainbow Molecular Weight Markers (Amersham Biosciences, Piscataway, NJ, USA) were used as a reference for molecular sizes. Immunoreactive signals were detected by enhanced chemiluminescence according to the manufacture’s protocol (Amersham Biosciences). Equal protein loading was confirmed by subsequent probing with the mouse monoclonal antibody against β-actin (1:1000) in each experiment.
List of the fluorochrome-labeled monoclonal antibodies used for flow cytometry
Rat IgG2b, κ
Rat IgG2b, κ
Rat IgG2a, κ
Ar Ham IgG1, λ2
Rat IgG2a, κ
EdU (5-ethynyl-2′-deoxyuridine)-incorporation assay
Mixed glial cultures were incubated with 10 μM EdU for 6 h. Then, trypsinized cells were collected and stained with FITC-conjugated rat anti-CD11b antibody (1:100; BD Bioscience) in PBS containing 1% (w/v) BSA and Fc-blocker (1:40) for 30 minutes at room temperature. After washing with PBS containing 1% (w/v) BSA three times, cells were fixed with 4% (w/v) paraformaldehyde and permeabilized with saponin-based permeabilizing buffer. EdU was detected using a pacific blue-conjugated azide (Invitrogen) according to the manufacturer’s instruction. Samples were acquired by Cyan-ADP flow cytometry, and the number of EdU-positive cells in either CD11b-positive or negative cell population was separately counted by gating with Summit software.
For purified microglial cultures, EdU (10 μM as a final concentration) was added to pure microglia plated on poly-D-lysine-coated 12 mm-diameter round cover slips at 5×104 cells/cover slip at 18 h after addition of MG alone, or MG medium supplemented with 20 ng/ml M-CSF or GM-CSF. After incubation for 6 h, cells were fixed and EdU incorporated into the nucleus was detected with azide conjugated with Alexa Fluor™ 594 according to the manufacturer’s instruction (Click-iT™ EdU cell proliferation assay, Invitrogen). All nuclei were counted with the aid of DAPI.
Phagocytosis assay by flow cytometry
Microglia plated on a 24-well plate were incubated with Alexa Fluor™ 488-conjugated zymosan A particles (Invitrogen) at 2×106 particles/ml in the MG medium. After wash with 0.5 ml HBSS- twice, cells were detached by trypsinization and collected into a 15-ml tube. After centrifugation at 520 × g for 5 minutes, cells were resuspended into 0.5 ml 0.1% (w/v) BSA in PBS, and then analyzed by CyAn-ADP flow cytometer. Mean fluorescent intensity (MFI) was calculated with Summit software.
Real-time PCR (qPCR) analyses were performed by MX3005P (Stratagene, La Jolla, CA, USA) using TaqMan™ Assay-on-Demand™ assay kits (assay nos.: Mm01250092_g1, Mm01270606_m1, Mm00479862_g1, Mm00439546_s1 and Mm00434174_m1 for detection of IRF8, PU.1/SFPI1, AIF1/IBA1, IFNB1, and IL12B cDNA, respectively). For standardization, β-actin cDNA levels were also quantified with the kit (Mm00607939_s1), and the absolute cDNA amounts were expressed as ratios to β-actin cDNA. We analyzed each gene using total RNA samples from at least triplicated independent experiments.
Mice were perfusion-fixed with 4% (w/v) paraformaldehyde in PBS, and tissues were processed as reported previously . Tissue sections (6 μm-thick) were prepared by a cryostat (CM1950, Leica Microsystems, Watzlar, Germany); rinsed in PBS; incubated for 30 minutes at room temperature in a blocking solution containing 0.4% (w/w) Triton X-100, 10% (v/v) donkey serum, 15 mM HEPES, 0.02% (w/v) sodium azide in 1x minimum essential medium; and incubated with primary antibodies in the blocking solution overnight at 4°C. The following primary antibodies were used: rat monoclonal anti-CD11b (M1/184.108.40.206, 1:50; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), rabbit polyclonal anti-PU.1/SFPI1 (#2266, 1:100; Cell Signaling, Danvers, MA), rabbit polyclonal anti-IBA1/AIF1 (No. 019-19741, 1:1000; Wako Chemicals USA, Richmond, VA, USA), rat monoclonal anti-F4/80 antigen (BM8, 1:100; eBioscience, San Diego, CA, USA) and rabbit polyclonal anti-CD68 (No. 250594, 1:200; AB Biotec, San Diego, CA, USA). Then, the sections were washed with PBS, incubated for 1 h in appropriate rhodamine, or Dylight 488-conjugated secondary antibodies (1:500, Jackson Immunoresearch, West Grove, PA, USA) at room temperature, and mounted with ProLong Gold antifade reagent containing DAPI (Invitrogen).
Cuprizone-induced demyelination in mice
Demyelination was induced by feeding wild-type (Irf8+/+) and IRF8-deficient (Irf8-/-) male mice at 8 to 10 weeks of age a diet of pellet feed (Harlan Laboratories, Madison, MI, USA) containing 0.25% (w/w) cuprizone (Sigma) up to 6 weeks. Animals were sacrificed at the indicated time points for histological analyses.
Myelin staining and Oil Red O staining
After fixation with paraformaldehyde as described above, brains were embedded in paraffin. Five μm-thick coronal brain sections between bregma -1.2mm and -2.2mm were stained with Luxol fast blue (LFB) (Sigma) for myelin together with periodic acid Schiff (PAS) for microglia and demyelinated axons. For Oil Red O staining, mice were perfusion fixed by 10% formalin. Brains were removed and postfixed, and then 6 μm-thick cryosections were prepared. NovaUltra™ Oil Red O Stain Kit (IHCWORLD, Woodstock, MD, USA) was used to stain undigested myelin lipids according to the manufacture’s instruction.
Unless otherwise noted, data are presented as mean ± SD, and statistical significance was determined by two-tail ANOVA followed by Student-Newman-Keuls post hoc test. Results were considered significant when P < 0.05.
IRF8-deficient microglia populated the CNS normally, but demonstrated less complex morphology with less IBA1 expression than wild-type microglia
IRF8-deficient microglia did not proliferate well in mixed glial cultures, but demonstrated a hyperproliferative response to GM-CSF
IRF8-deficient microglia demonstrated a reduced maximum phagocytic capacity in vitro
Like their myeloid cousins, microglia are characterized by their motility and phagocytic activity. Motility of Irf8+/+ and Irf8-/- microglia was analyzed in vitro by the same time-lapse video imaging as used in our previous study . In vitro motility analysis failed to reveal significant differences between the two genotypes (data not shown).
IRF8 was necessary to maintain the basal transcriptional level of AIF1/IBA1 in microglia, but not for its induction by interferon-γ
Altered cytokine induction in IRF8-deficient microglia in response to LPS and IFNγ
IRF8-deficient microglia demonstrated a delayed response to tissue damage and a defect in clearing damaged tissues in the cuprizone demyelination model
The first finding in this study is that, even in the absence of IRF8, microglia can develop and colonize the CNS. This contrasts clearly to inefficient development of BM-derived MPs and accumulation of myeloid progenitor cells in Irf8-/- mice , and indicates that IRF8-mediated transcriptional regulation is not necessary for development of microglia in the primitive hematopoiesis and for their migration into the embryonic CNS. On the other hand, loss of PU.1/SFPI1 blocks MP development as early as the primitive hematopoiesis in the yolk sac, resulting in lack of tissue residential MPs including microglia, liver Kupffer cells, and lung alveolar macrophages . IRF8 is known to associate with PU.1/SFPI1 to activate transcription of some genes by interacting with the composite Ets/IRF-cis elements in the promoter region [32, 33]. However, our result indicates that PU.1/SFPI1 does not require IRF8 to exert its critical roles in development of microglia. After migration into the CNS, a large proportion of the adult microglial cell population arises during the first two postnatal weeks by an intense in situ proliferation in proportion to the growth of the CNS tissues . Based on the observations on the mutant mice lacking CSF1R [7, 35] or one of its ligands, M-CSF [36, 37], this perinatal expansion of the microglial population size is largely dependent on mitotic signals mediated by the M-CSF receptor CSF1R/CD115. We can conclude from our in vivo and in vitro experiments that, even in the absence of IRF8, microglia can respond to M-CSF and proliferate.
However, colonized IRF8-deficent microglia are not phenotypically and functionally equivalent to wild-type microglia. An obvious difference is that the processes of Irf8-/- microglia in the steady state are less extended into the tissues compared to those of Irf8+/+ microglia. Recent two-photon in vivo or ex vivo imaging has revealed that, although ramified microglia in the steady state have been referred as ‘resting’ microglia, their processes are highly dynamic to probe changes actively in their microenvironment  and to monitor neuronal synaptic activities . If microglia detect tissue damage, their processes can quickly converge to form a barrier to contain a damaged area from the surrounding healthy tissue [40, 41]. Our observation suggests that Irf8-/- microglia may be less capable of sensing tissue damage due to their less extended processes. IRF8-deficent microglia will provide a unique model for further understanding of process dynamics and sensing capability of microglia.
Reduced expression of AIF1/IBA1 is also a prominent feature of Irf8-/- microglia . As far as we could examine, however, AIF1/IBA1 is the only microglial marker affected by loss of IRF8, indicating again that a major part of microglial development occurs normally in the absence of IRF8. Our analysis of AIF1/IBA1 mRNA and protein levels demonstrated that IRF8 is required to maintain the constitutive expression of AIF1/IBA1 in microglia, whereas AIF1/IBA1 is still inducible in Irf8-/- microglia by IFNγ. AIF1/IBA1, a 17-kDa protein bearing two EF-hand Ca2+ binding motifs, is known to contribute to the plasma membrane and cytoskeleton dynamics tightly linked to motility and phagocytosis of microglia . It is thereby reasonable to speculate that impaired process formation in Irf8-/- microglia might be a consequence of reduced AIF1/IBA1 expression. Despite the report that AIF1/IBA1 is involved in the formation of phagocytic cups in a microglial cell line , however, engulfment of zymosan particles occurred in Irf8-/- microglia in the same time frame as that in Irf8+/+ microglia, indicating that the molecular mechanisms underlying a series of microglial responses from chemotaxis to phagocytosis  are not impaired by the reduction of AIF1/IBA1 protein. A hypothetical explanation for this discrepancy would be that the trace amount of AIF1/IBA1 protein in Irf8-/- microglia might be sufficient for the phagocytic responses. Alternatively, AIF1L/IBA2, a homolog of AIF1/IBA1, might compensate the reduced AIF1/IBA1 . Basal expression of AIF1/IBA1 mRNA in Irf8-/- microglia, though it is five-fold reduced, is likely to be attributable to PU.1/SFPI1 that was expressed at the levels similar to those in Irf8+/+ microglia, because PU.1/SFPI1 has been shown to bind to the promoter region of the aif1/iba1 gene and act as a transcriptional activator . Further studies will be necessary to determine whether IRF8 enhances expression of AIF1/IBA1 directly or indirectly in cooperation with PU.1/SFPI1 in microglia.
In contrast to normal zymosan engulfment, the maximum phagocytic capacity of Irf8-/- microglia is reduced as determined by the amount of zymosan particles engulfed in each microglia. After engulfment, phagosomes containing internalized materials such as bacteria and apoptotic cell corpse undergo a maturation process along with sequential changes in integral membrane proteins and progressive acidification, and eventually fuse with lysosome structures to form phagolysosomes, leading to degradation of the contents [46, 47]. IRF8-mediated transcriptional regulation might be involved in this highly organized maturation process of phagosomes, or cell structural changes associated with the phagocytic activities. Defective scavenging activity of Irf8-/- microglia was also observed in our in vivo experiment using the cuprizone-induced demyelination model. More myelin debris rich in lipids remained in the corpus callosum of Irf8-/- mice. However, this in vivo result may be attributable not only to reduced phagocytic capacity of Irf8-/- microglia, but also to delayed accumulation of Irf8-/- microglia in the demyelinating lesions.
After development of the CNS, microglia continue to cycle slowly , and are capable of accelerated proliferation particularly in response to tissue damages [30, 49, 50]. Although the molecular basis of this proliferative response associated with microglial activation might differ depending on the pathological conditions, CSF1R/CD115-mediated signaling is principally involved in microglial proliferation in some types of CNS lesions such as facial motoneuron death after axotomy [51, 52]. Microglia also proliferate in vitro in response to various cytokines including M-CSF, GM-CSF, interleukins-3, -4, and -5 [53–57]. An interesting finding in our in vitro experiments was that Irf8-/- microglia demonstrated significantly reduced proliferation in mixed glial cultures, whereas exogenous M-CSF restored proliferation of Irf8-/- microglia at a rate comparable to that of Irf8+/+ microglia. Exogenous M-CSF also enhanced proliferation of purified CD11b+Irf8-/- microglia, but to a lesser extent compared with purified CD11b+Irf8+/+ microglia, suggesting that IRF8-dependent transcription in microglia is required for their normal proliferative response to M-CSF at a physiological concentration. Since a previous in vitro study using M-CSF-deficient mice indicated that M-CSF derived from astroglia principally contributes to microglial proliferation in mixed glial cultures , however, IRF8-dependent mechanisms in astroglia might be involved in secretion of colony stimulating factors including M-CSF from astroglia. In addition, the reduced proliferative response of Irf8-/- microglia in the mixed glial cultures could at least partly account for delayed accumulation of Irf8-/- microglia in the cuprizone-induced demyelinated lesions.
GM-CSF is known to be a potent mitogen for wild-type microglia in vitro as well [53, 56, 59]. In mixed glial cultures, Irf8-/- microglia demonstrated a hyperproliferative response to GM-CSF compared with Irf8+/+ microglia, which is quite likely to be identical to that observed with Irf8-/- BM-derived myeloid progenitor cells . However, our results indicated that this hyperproliferative response of Irf8-/- microglia requires the presence of other glial cells, because exogenous GM-CSF failed to enhance proliferation of Irf8-/- microglia after isolation from other glial cells. A straightforward explanation for these findings is that GM-CSF acts indirectly on microglia through GM-CSF-mediated production of other mitogenic factor(s) from astroglia, and that IRF8 is involved in the proliferative response of microglia to these factors. Since some prior studies have pointed out that, in the absence of astroglia, GM-CSF failed to induce proliferation of microglia particularly from adult animals [60, 61], it is also conceivable that GM-CSF-induced proliferation of microglia is dependent on their maturational and activation stages. Further studies will determine whether microglia share the same IRF8-dependent intracellular signaling pathways leading to the mitotic response to GM-CSF as BM-derived myeloid progenitor cells [62, 63]. GM-CSF also induces a phenotypic skew of microglia towards DC-like cells, but not towards granulocytes . We observed that this phenotypic skew was more apparent in Irf8-/- microglia than in Irf8+/+ microglia. Given that IRF8 positively regulates development of diverse BM-derived DC subsets in a subset-selective manner , roles for IRF8 in microglial differentiation towards a DC phenotype in the presence of GM-CSF might be distinct from those in BM-derived common DC progenitors.
We also confirmed that Irf8-/- microglia have the same abnormality in the transcriptional induction of IFNB1 and IL12B as that reported in Irf8-/- BM-derived myeloid lineage cells. Our quantitative and kinetic analysis of IFNB1 mRNA clearly demonstrated that IRF8 positively regulates LPS-mediated (Toll-like receptor 4-mediated) acute induction of IFNB1 mRNA, whereas it suppresses IFNγ-mediated delayed transcriptional induction in microglia. Recently, Li et al. reported that, using human monocytes, IRF8 positively regulates rapid and robust induction of IFNβ in cooperation with IRF3 and PU.1, and that this mode of induction is characteristic to monocytes . Our result is in good agreement with their findings, indicating that microglia utilize the same transcriptional machinery in the IFNB1 promoter as BM-derived monocytes. IRF8 is also an essential transcription factor for induction of IL12 p40 subunit in BM-derived MPs [14, 26]. Although our result confirmed the same positive regulatory role for IRF8 in microglia, we also noticed that LPS-mediated IL12B mRNA induction still occurred in the absence of IRF8 at ten-fold lower levels, suggesting the presence of a compensatory mechanism in microglia.
Our study provides further evidence for differences between microglia and BM-derived MPs with respect to the dependence of their development and physiological phenotypes on IRF8-mediated transcriptional regulation. Detailed gene expression analysis of Irf8+/+ and Irf8-/- microglia, and Irf8+/+ and Irf8-/- BM-derived MPs will be necessary to clarify the downstream events leading to the difference in gene expression profiles between microglia and BM-derived MPs [65, 66].
In conclusion, our comprehensive analyses of IRF8-deficient microglia demonstrate that IRF8 is a critical transcription factor for a broad range of microglial functions, some of which are unique in microglia. In line with our findings, recent studies have revealed direct association of IRF8 with human disease conditions [67, 68]. This study warrants future studies to enhance understanding of critical contributions of IRF8-mediated transcriptional regulation to physiological and pathological roles of microglia.
Bovine serum albumin
Colony stimulating factor 1 receptor
Dulbecco’s modified Eagle’s medium
Granulocyte colony stimulating factor
Granulocyte-macrophage colony stimulating factor
Hanks’ balanced salt solution
interferon consensus sequence binding protein
Interferon regulatory factor
Luxol fast blue
Magnetic cell sorting
Macrophage colony stimulating factor
Mean fluorescent intensity
Periodic acid Schiff
This work was supported by Research Grant of Shriners Hospitals for Children 85400 (to TI) and by US National Institute of Health grant NS025044 (to DP and TI). The authors thank Maksim Gusev for his technical assistance.
- Kettenmann H, Hanisch UK, Noda M, Verkhratsky A: Physiology of microglia. Physiol Rev. 2011, 91: 461-553. 10.1152/physrev.00011.2010.View ArticlePubMedGoogle Scholar
- Prinz M, Mildner A: Microglia in the CNS: immigrants from another world. Glia. 2011, 59: 177-187. 10.1002/glia.21104.View ArticlePubMedGoogle Scholar
- Ransohoff RM, Cardona AE: The myeloid cells of the central nervous system parenchyma. Nature. 2010, 468: 253-262. 10.1038/nature09615.View ArticlePubMedGoogle Scholar
- Ajami B, Bennett JL, Krieger C, Tetzlaff W, Rossi FM: Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat Neurosci. 2007, 10: 1538-1543. 10.1038/nn2014.View ArticlePubMedGoogle Scholar
- Mildner A, Schmidt H, Nitsche M, Merkler D, Hanisch UK, Mack M, Heikenwalder M, Brück W, Priller J, Prinz M: Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nat Neurosci. 2007, 10: 1544-1553. 10.1038/nn2015.View ArticlePubMedGoogle Scholar
- Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF, Conway SJ, Ng LG, Stanley ER, Samokhvalov IM, Merad M: Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010, 330: 841-845. 10.1126/science.1194637.PubMed CentralView ArticlePubMedGoogle Scholar
- Erblich B, Zhu L, Etgen AM, Dobrenis K, Pollard JW: Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits. PLoS One. 2011, 6: e26317-10.1371/journal.pone.0026317.PubMed CentralView ArticlePubMedGoogle Scholar
- Ozato K, Tailor P, Kubota T: The interferon regulatory factor family in host defense: mechanism of action. J Biol Chem. 2007, 282: 20065-20069. 10.1074/jbc.R700003200.View ArticlePubMedGoogle Scholar
- Tamura T, Yanai H, Savitsky D, Taniguchi T: The IRF family transcription factors in immunity and oncogenesis. Annu Rev Immunol. 2008, 26: 535-584. 10.1146/annurev.immunol.26.021607.090400.View ArticlePubMedGoogle Scholar
- Wang H, Morse HC: IRF8 regulates myeloid and B lymphoid lineage diversification. Immunol Res. 2009, 43: 109-117. 10.1007/s12026-008-8055-8.PubMed CentralView ArticlePubMedGoogle Scholar
- Tamura T, Ozato K: ICSBP/IRF-8: its regulatory roles in the development of myeloid cells. J Interferon Cytokine Res. 2002, 22: 145-152. 10.1089/107999002753452755.View ArticlePubMedGoogle Scholar
- Contursi C, Wang IM, Gabriele L, Gadina M, O’Shea J, Morse HC, Ozato K: IFN consensus sequence binding protein potentiates STAT1-dependent activation of IFNγ-responsive promoters in macrophages. Proc Natl Acad Sci USA. 2000, 97: 91-96. 10.1073/pnas.97.1.91.PubMed CentralView ArticlePubMedGoogle Scholar
- Fehr T, Schoedon G, Odermatt B, Holtschke T, Schneemann M, Bachmann MF, Mak TW, Horak I, Zinkernagel RM: Crucial role of interferon consensus sequence binding protein, but neither of interferon regulatory factor 1 nor of nitric oxide synthesis for protection against murine listeriosis. J Exp Med. 1997, 185: 921-931. 10.1084/jem.185.5.921.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang IM, Contursi C, Masumi A, Ma X, Trinchieri G, Ozato K: An IFN-γ-inducible transcription factor, IFN consensus sequence binding protein (ICSBP), stimulates IL-12 p40 expression in macrophages. J Immunol. 2000, 165: 271-279.View ArticlePubMedGoogle Scholar
- Ellis SL, Gysbers V, Manders PM, Li W, Hofer MJ, Müller M, Campbell IL: The cell-specific induction of CXC chemokine ligand 9 mediated by IFN-γ in microglia of the central nervous system is determined by the myeloid transcription factor PU.1. J Immunol. 2010, 185: 1864-1877. 10.4049/jimmunol.1000900.PubMed CentralView ArticlePubMedGoogle Scholar
- Masuda T, Tsuda M, Yoshinaga R, Tozaki-Saitoh H, Ozato K, Tamura T, Inoue K: IRF8 is a critical transcription factor for transforming microglia into a reactive phenotype. Cell Rep. 2012, 1: 334-340. 10.1016/j.celrep.2012.02.014.PubMed CentralView ArticlePubMedGoogle Scholar
- Holtschke T, Löhler J, Kanno Y, Fehr T, Giese N, Rosenbauer F, Lou J, Knobeloch KP, Gabriele L, Waring JF, Bachmann MF, Zinkernagel RM, Morse HC, Ozato K, Horak I: Immunodeficiency and chronic myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP gene. Cell. 1996, 87: 307-317. 10.1016/S0092-8674(00)81348-3.View ArticlePubMedGoogle Scholar
- Nelson N, Kanno Y, Hong C, Contursi C, Fujita T, Fowlkes BJ, O’Connell E, Hu-Li J, Paul WE, Jankovic D, Sher AF, Coligan JE, Thornton A, Appella E, Yang Y, Ozato K: Expression of IFN regulatory factor family proteins in lymphocytes. Induction of Stat-1 and IFN consensus sequence binding protein expression by T cell activation. J Immunol. 1996, 156: 3711-3720.PubMedGoogle Scholar
- Horiuchi M, Itoh A, Pleasure D, Itoh T: MEK-ERK signaling is involved in interferon-γ-induced death of oligodendroglial progenitor cells. J Biol Chem. 2006, 281: 20095-20106. 10.1074/jbc.M603179200.View ArticlePubMedGoogle Scholar
- Kawai K, Itoh T, Itoh A, Horiuchi M, Wakayama K, Bannerman P, Garbern JY, Pleasure D, Lindsten T: Maintenance of the relative proportion of oligodendrocytes to axons even in the absence of BAX and BAK. Eur J Neurosci. 2009, 30: 2030-2041. 10.1111/j.1460-9568.2009.06988.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Suzumura A, Mezitis SG, Gonatas NK, Silberberg DH: MHC antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of Ia antigen expression by gamma-interferon. J Neuroimmunol. 1987, 15: 263-278. 10.1016/0165-5728(87)90121-4.View ArticlePubMedGoogle Scholar
- Scheller M, Foerster J, Heyworth CM, Waring JF, Löhler J, Gilmore GL, Shadduck RK, Dexter TM, Horak I: Altered development and cytokine responses of myeloid progenitors in the absence of transcription factor, interferon consensus sequence binding protein. Blood. 1999, 94: 3764-3771.PubMedGoogle Scholar
- Horiuchi M, Lindsten T, Pleasure D, Itoh T: Differing in vivo survival dependency of mouse and rat NG2+ oligodendroglial progenitor cells. J Neurosci Res. 2010, 88: 957-970.PubMed CentralPubMedGoogle Scholar
- Panchuk-Voloshina N, Haugland RP, Bishop-Stewart J, Bhalgat MK, Millard PJ, Mao F, Leung WY, Haugland RP: Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem. 1999, 47: 1179-1188. 10.1177/002215549904700910.View ArticlePubMedGoogle Scholar
- Nelson N, Marks MS, Driggers PH, Ozato K: Interferon consensus sequence-binding protein, a member of the interferon regulatory factor family, suppresses interferon-induced gene transcription. Mol Cell Biol. 1993, 13: 588-599.PubMed CentralView ArticlePubMedGoogle Scholar
- Scharton-Kersten T, Contursi C, Masumi A, Sher A, Ozato K: Interferon consensus sequence binding protein-deficient mice display impaired resistance to intracellular infection due to a primary defect in interleukin 12 p40 induction. J Exp Med. 1997, 186: 1523-1534. 10.1084/jem.186.9.1523.PubMed CentralView ArticlePubMedGoogle Scholar
- Li P, Wong JJ, Sum C, Sin WX, Ng KQ, Koh MB, Chin KC: IRF8 and IRF3 cooperatively regulate rapid interferon-β induction in human blood monocytes. Blood. 2011, 117: 2847-2854. 10.1182/blood-2010-07-294272.View ArticlePubMedGoogle Scholar
- Kondo A, Nakano T, Suzuki K: Blood-brain barrier permeability to horseradish peroxidase in twitcher and cuprizone-intoxicated mice. Brain Res. 1987, 425: 186-190. 10.1016/0006-8993(87)90499-9.View ArticlePubMedGoogle Scholar
- McMahon EJ, Suzuki K, Matsushima GK: Peripheral macrophage recruitment in cuprizone-induced CNS demyelination despite an intact blood-brain barrier. J Neuroimmunol. 2002, 130: 32-45. 10.1016/S0165-5728(02)00205-9.View ArticlePubMedGoogle Scholar
- Remington LT, Babcock AA, Zehntner SP, Owens T: Microglial recruitment, activation, and proliferation in response to primary demyelination. Am J Pathol. 2007, 170: 1713-1724. 10.2353/ajpath.2007.060783.PubMed CentralView ArticlePubMedGoogle Scholar
- McKercher SR, Torbett BE, Anderson KL, Henkel GW, Vestal DJ, Baribault H, Klemsz M, Feeney AJ, Wu GE, Paige CJ, Maki RA: Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 1996, 15: 5647-5658.PubMed CentralPubMedGoogle Scholar
- Kanno Y, Levi BZ, Tamura T, Ozato K: Immune cell-specific amplification of interferon signaling by the IRF-4/8-PU.1 complex. J Interferon Cytokine Res. 2005, 25: 770-779. 10.1089/jir.2005.25.770.View ArticlePubMedGoogle Scholar
- Huang W, Horvath E, Eklund EA: PU.1, interferon regulatory factor (IRF) 2, and the interferon consensus sequence-binding protein (ICSBP/IRF8) cooperate to activate NF1 transcription in differentiating myeloid cells. J Biol Chem. 2007, 282: 6629-6643.View ArticlePubMedGoogle Scholar
- Dalmau I, Vela JM, González B, Finsen B, Castellano B: Dynamics of microglia in the developing rat brain. J Comp Neurol. 2003, 458: 144-157. 10.1002/cne.10572.View ArticlePubMedGoogle Scholar
- Dai XM, Ryan GR, Hapel AJ, Dominguez MG, Russell RG, Kapp S, Sylvestre V, Stanley ER: Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects. Blood. 2002, 99: 111-120. 10.1182/blood.V99.1.111.View ArticlePubMedGoogle Scholar
- Cohen PE, Zhu L, Nishimura K, Pollard JW: Colony-stimulating factor 1 regulation of neuroendocrine pathways that control gonadal function in mice. Endocrinology. 2002, 143: 1413-1422. 10.1210/en.143.4.1413.View ArticlePubMedGoogle Scholar
- Wegiel J, Wisniewski HM, Dziewiatkowski J, Tarnawski M, Kozielski R, Trenkner E, Wiktor-Jedrzejczak W: Reduced number and altered morphology of microglial cells in colony stimulating factor-1-deficient osteopetrotic op/op mice. Brain Res. 1998, 804: 135-139. 10.1016/S0006-8993(98)00618-0.View ArticlePubMedGoogle Scholar
- Nimmerjahn A, Kirchhoff F, Helmchen F: Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science. 2005, 308: 1314-1318. 10.1126/science.1110647.View ArticlePubMedGoogle Scholar
- Wake H, Moorhouse AJ, Jinno S, Kohsaka S, Nabekura J: Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. J Neurosci. 2009, 29: 3974-3980. 10.1523/JNEUROSCI.4363-08.2009.View ArticlePubMedGoogle Scholar
- Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, Dustin ML, Gan WB: ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci. 2005, 8: 752-758. 10.1038/nn1472.View ArticlePubMedGoogle Scholar
- Hines DJ, Hines RM, Mulligan SJ, Macvicar BA: Microglia processes block the spread of damage in the brain and require functional chloride channels. Glia. 2009, 57: 1610-1618. 10.1002/glia.20874.View ArticlePubMedGoogle Scholar
- Ohsawa K, Imai Y, Kanazawa H, Sasaki Y, Kohsaka S: Involvement of Iba1 in membrane ruffling and phagocytosis of macrophages/microglia. J Cell Sci. 2000, 113: 3073-3084.PubMedGoogle Scholar
- Heinrich V, Lee CY: Blurred line between chemotactic chase and phagocytic consumption: an immunophysical single-cell perspective. J Cell Sci. 2011, 124: 3041-3051. 10.1242/jcs.086413.PubMed CentralView ArticlePubMedGoogle Scholar
- Schulze JO, Quedenau C, Roske Y, Adam T, Schüler H, Behlke J, Turnbull AP, Sievert V, Scheich C, Mueller U, Heinemann U, Büssow K: Structural and functional characterization of human Iba proteins. FEBS J. 2008, 275: 4627-4640. 10.1111/j.1742-4658.2008.06605.x.View ArticlePubMedGoogle Scholar
- Sibinga NE, Feinberg MW, Yang H, Werner F, Jain MK: Macrophage-restricted and interferon γ-inducible expression of the allograft inflammatory factor-1 gene requires Pu.1. J Biol Chem. 2002, 277: 16202-16210. 10.1074/jbc.M200935200.View ArticlePubMedGoogle Scholar
- Fullard JF, Kale A, Baker NE: Clearance of apoptotic corpses. Apoptosis. 2009, 14: 1029-1037. 10.1007/s10495-009-0335-9.View ArticlePubMedGoogle Scholar
- Kinchen JM, Ravichandran KS: Phagosome maturation: going through the acid test. Nat Rev Mol Cell Biol. 2008, 9: 781-795. 10.1038/nrm2515.PubMed CentralView ArticlePubMedGoogle Scholar
- Lawson LJ, Perry VH, Gordon S: Turnover of resident microglia in the normal adult mouse brain. Neuroscience. 1992, 48: 405-415. 10.1016/0306-4522(92)90500-2.View ArticlePubMedGoogle Scholar
- Dissing-Olesen L, Ladeby R, Nielsen HH, Toft-Hansen H, Dalmau I, Finsen B: Axonal lesion-induced microglial proliferation and microglial cluster formation in the mouse. Neuroscience. 2007, 149: 112-122. 10.1016/j.neuroscience.2007.06.037.View ArticlePubMedGoogle Scholar
- Graeber MB, López-Redondo F, Ikoma E, Ishikawa M, Imai Y, Nakajima K, Kreutzberg GW, Kohsaka S: The microglia/macrophage response in the neonatal rat facial nucleus following axotomy. Brain Res. 1998, 813: 241-253. 10.1016/S0006-8993(98)00859-2.View ArticlePubMedGoogle Scholar
- Raivich G, Moreno-Flores MT, Möller JC, Kreutzberg GW: Inhibition of posttraumatic microglial proliferation in a genetic model of macrophage colony-stimulating factor deficiency in the mouse. Eur J Neurosci. 1994, 6: 1615-1618. 10.1111/j.1460-9568.1994.tb00552.x.View ArticlePubMedGoogle Scholar
- Yamamoto S, Nakajima K, Kohsaka S: Macrophage-colony stimulating factor as an inducer of microglial proliferation in axotomized rat facial nucleus. J Neurochem. 2010, 115: 1057-1067. 10.1111/j.1471-4159.2010.06996.x.View ArticlePubMedGoogle Scholar
- Giulian D, Ingleman JE: Colony-stimulating factors as promoters of ameboid microglia. J Neurosci. 1988, 8: 4707-4717.PubMedGoogle Scholar
- Ganter S, Northoff H, Männel D, Gebicke-Härter PJ: Growth control of cultured microglia. J Neurosci Res. 1992, 33: 218-230. 10.1002/jnr.490330205.View ArticlePubMedGoogle Scholar
- Ringheim GE: Mitogenic effects of interleukin-5 on microglia. Neurosci Lett. 1995, 201: 131-134. 10.1016/0304-3940(95)12153-6.View ArticlePubMedGoogle Scholar
- Suzumura A, Sawada M, Yamamoto H, Marunouchi T: Effects of colony stimulating factors on isolated microglia in vitro. J Neuroimmunol. 1990, 30: 111-120. 10.1016/0165-5728(90)90094-4.View ArticlePubMedGoogle Scholar
- Suzumura A, Sawada M, Itoh Y, Marunouchi T: Interleukin-4 induces proliferation and activation of microglia but suppresses their induction of class II major histocompatibility complex antigen expression. J Neuroimmunol. 1994, 53: 209-218. 10.1016/0165-5728(94)90031-0.View ArticlePubMedGoogle Scholar
- Blevins G, Fedoroff S: Microglia in colony-stimulating factor 1-deficient op/op mice. J Neurosci Res. 1995, 40: 535-544. 10.1002/jnr.490400412.View ArticlePubMedGoogle Scholar
- Lee SC, Liu W, Brosnan CF, Dickson DW: GM-CSF promotes proliferation of human fetal and adult microglia in primary cultures. Glia. 1994, 12: 309-318. 10.1002/glia.440120407.View ArticlePubMedGoogle Scholar
- Santambrogio L, Belyanskaya SL, Fischer FR, Cipriani B, Brosnan CF, Ricciardi-Castagnoli P, Stern LJ, Strominger JL, Riese R: Developmental plasticity of CNS microglia. Proc Natl Acad Sci USA. 2001, 98: 6295-6300. 10.1073/pnas.111152498.PubMed CentralView ArticlePubMedGoogle Scholar
- Ponomarev ED, Novikova M, Maresz K, Shriver LP, Dittel BN: Development of a culture system that supports adult microglial cell proliferation and maintenance in the resting state. J Immunol Methods. 2005, 300: 32-46. 10.1016/j.jim.2005.02.011.View ArticlePubMedGoogle Scholar
- Tamura T, Kong HJ, Tunyaplin C, Tsujimura H, Calame K, Ozato K: ICSBP/IRF-8 inhibits mitogenic activity of p210 Bcr/Abl in differentiating myeloid progenitor cells. Blood. 2003, 102: 4547-4554. 10.1182/blood-2003-01-0291.View ArticlePubMedGoogle Scholar
- Zhu C, Saberwal G, Lu Y, Platanias LC, Eklund EA: The interferon consensus sequence-binding protein activates transcription of the gene encoding neurofibromin 1. J Biol Chem. 2004, 279: 50874-50885. 10.1074/jbc.M405736200.View ArticlePubMedGoogle Scholar
- Gabriele L, Ozato K: The role of the interferon regulatory factor (IRF) family in dendritic cell development and function. Cytokine Growth Factor Rev. 2007, 18: 503-510. 10.1016/j.cytogfr.2007.06.008.View ArticlePubMedGoogle Scholar
- Donnou S, Fisson S, Mahe D, Montoni A, Couez D: Identification of new CNS-resident macrophage subpopulation molecular markers for the discrimination with murine systemic macrophages. J Neuroimmunol. 2005, 169: 39-49. 10.1016/j.jneuroim.2005.07.016.View ArticlePubMedGoogle Scholar
- Schmid CD, Melchior B, Masek K, Puntambekar SS, Danielson PE, Lo DD, Sutcliffe JG, Carson MJ: Differential gene expression in LPS/IFNγ activated microglia and macrophages: in vitro versus in vivo. J Neurochem. 2009, 1: 117-125.View ArticleGoogle Scholar
- De Jager PL, Jia X, Wang J, de Bakker PI, Ottoboni L, Aggarwal NT, Piccio L, Raychaudhuri S, Tran D, Aubin C, Briskin R, Romano S, International MS Genetics C, Baranzini SE, McCauley JL, Pericak-Vance MA, Haines JL, Gibson RA, Naeglin Y, Uitdehaag B, Matthews PM, Kappos L, Polman C, McArdle WL, Strachan DP, Evans D, Cross AH, Daly MJ, Compston A, Sawcer SJ, Weiner HL, Hauser SL, Hafler DA, Oksenberg JR: Meta-analysis of genome scans and replication identify CD6, IRF8 and TNFRSF1A as new multiple sclerosis susceptibility loci. Nat Genet. 2009, 41: 776-782. 10.1038/ng.401.PubMed CentralView ArticlePubMedGoogle Scholar
- Hambleton S, Salem S, Bustamante J, et al: IRF8 mutations and human dendritic-cell immunodeficiency. N Engl J Med. 2011, 365: 127-138. 10.1056/NEJMoa1100066.PubMed CentralView ArticlePubMedGoogle Scholar
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