- Open Access
LRP1 expression in microglia is protective during CNS autoimmunity
© The Author(s). 2016
Received: 28 June 2016
Accepted: 28 June 2016
Published: 11 July 2016
Multiple sclerosis is a devastating neurological disorder characterized by the autoimmune destruction of the central nervous system myelin. While T cells are known orchestrators of the immune response leading to MS pathology, the precise contribution of CNS resident and peripheral infiltrating myeloid cells is less well described. Here, we explore the myeloid cell function of Low-density lipoprotein receptor-related protein-1 (LRP1), a scavenger receptor involved in myelin clearance and the inflammatory response, in the context of Multiple sclerosis. Supporting its central role in Multiple sclerosis pathology, we find that LRP1 expression is increased in Multiple sclerosis lesions in comparison to the surrounding healthy tissue. Using two genetic mouse models, we show that deletion of LRP1 in microglia, but not in peripheral macrophages, negatively impacts the progression of experimental autoimmune encephalomyelitis, an animal model of Multiple sclerosis. We further show that the increased disease severity in experimental autoimmune encephalomyelitis is not due to haplodeficiency of the Cx3cr1 locus. At the cellular level, microglia lacking LRP1 adopt a pro-inflammatory phenotype characterized by amoeboid morphology and increased production of the inflammatory mediator TNF-α. We also show that LRP1 functions as a robust inhibitor of NF-kB activation in myeloid cells via a MyD88 dependent pathway, potentially explaining the increase in disease severity observed in mice lacking LRP1 expression in microglia. Taken together, our data suggest that the function of LRP1 in microglia is to keep these cells in an anti-inflammatory and neuroprotective status during inflammatory insult, including experimental autoimmune encephalomyelitis and potentially in Multiple sclerosis.
Multiple sclerosis (MS) is an inflammatory autoimmune disease characterized by the destruction of myelin in the central nervous system (CNS) and irreversible neurodegeneration . Myelin destruction is initiated by the myelin-reactive T cells, and is further amplified by the inflammatory response of myeloid cells, including the brain resident microglia and the brain infiltrating inflammatory macrophages [2–6].
Although it is clear that microglia and macrophages participate in the MS pathology development, their precise functions remain extremely controversial . On one hand, microglia are proposed to be protective and relatively immune silent in MS, when compared to macrophages , while other studies demonstrate that microglia themselves can contribute to disease initiation. Indeed, mice with a microglia-specific deficiency in the activation of the transcriptional master regulator of inflammation, NF-kB, are protected from EAE .
Low-density lipoprotein receptor-related protein-1 (LRP1), or CD91, is a scavenger receptor involved in the removal of myelin debris, as well as necrotic and apoptotic cells [10–12]. The clearance of myelin debris generated during demyelination episodes is critical for the regenerative capacity of the CNS. Improper myelin debris clearance by microglia has been shown to delay recovery in a mouse model of demyelination . Besides mediating the removal of cellular debris, LRP1 can also directly influence cellular signaling pathways . We have previously shown that LRP1 functions as a broad inhibitor of NF-kB activity and inflammatory mediator production . Because inhibition of NF-kB is known to offer protection during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS [16–20], placement of LRP1 at the crossroads of inflammation and phagocytosis raises the possibility of its involvement as a central regulator of MS pathology.
In this study, we explore the precise contribution of LRP1 during MS, using EAE as an animal model. We show that LRP1 protein expression is increased in the myeloid and astrocytic compartments during active disease in MS, in comparison to the healthy CNS. To elucidate the role of LRP1 in myeloid cells, we induced EAE in mice lacking LRP1 in either the peripheral or resident myeloid cells. To our surprise, deletion of LRP1 in peripheral macrophages had no effect on the disease severity, whereas in mice lacking LRP1 specifically in microglia, disease was intensified, suggesting a protective role for LRP1 in microglia. Further experiments revealed that, at baseline, microglia deficient in LRP1 adopt a pro-inflammatory phenotype and exhibit morphological changes that parallel those observed in microglia under inflammatory conditions. On a mechanistic level, our results suggest that the increased pathology in mice with LRP1 deficient microglia originates from uncontrolled NF-kB activity. Taken together, our results demonstrate that LRP1 has a protective role in microglia and that the alteration of their function can drastically impact EAE outcome.
Materials and methods
The human pathology study was approved by the Institutional Review Board of Mayo Clinic, Rochester, MN. Six clinically and pathologically confirmed multiple sclerosis autopsy cases were used in this study. Formalin-fixed paraffin-embedded 5 μm thick sections were stained for H&E, Luxol fast blue and periodic acid Schiff (LFB/PAS), and Bielschowsky’s silver stain for routine pathology analysis. Immunohistochemistry was performed with the avidin–biotin-complex method according to the manufacturer’s instructions (Vectorlab). The following primary antibodies are used: LRP1 (1:200, Abcam) myelin proteolipid protein (PLP, 1:500, Serotec), myelin-associated glycoprotein (MAG, 1:1000, Abcam), myelin oligodendrocyte glycoprotein (MOG, 1:1000, Abcam), CD68 (1:100, DAKO), and glial fibrillary acidic protein (GFAP, 1:100, DAKO). Steamed antigen retrieval with citrate buffer (pH 6.0) was performed for LRP1, MAG, MOG, CD68, and GFAP. The demyelinating activity in the parenchyma for each block was evaluated in regions derived from the 6 cases, according to the myelin debris in macrophages as previously described .
C57BL/6 mice with loxP sites flanking the LRP1 gene  were crossed with Cx3cr1 cre, Cx3cr1 creER  and LysM cre  mice to generate LRP1 fl/fl Cx3cr1 cre, LRP1 fl/fl Cx3cr1 creER and LRP1 fl/fl LysM cre mice. To induce Cre recombinase activity, 4 to 6-weeks old LRP1 fl/fl Cx3cr1 creER mice were injected twice i.p. with tamoxifen (250 mg/kg in corn oil, Sigma) 1 week apart as described in the literature . For controls, littermates carrying loxP-flanked LRP1 alleles without Cre recombinase expression or age and sex matched mice with Cx3cr1 creER activity without loxP-flanked LRP1 alleles, were used. All animal procedures were approved by the University of Virginia’s Animal Care & Use Committee.
Experimental autoimmune encephalomyelitis
EAE was induced in female mice (8 to 12 weeks) by subcutaneous injection of MOG35-55 peptide (100 μg, CSBio) emulsified in complete Freund’s adjuvant containing Mycobacterium tuberculosis (1 mg/ml, BD). Pertussis toxin (200 ng, List Biologicals) was administered i.p. on the day of and 1d after MOG immunization. For experiments with Cx3cr1 creER mice, EAE was induced 4 weeks after the tamoxifen treatment as previously described . For clinical evaluation, mice were scored daily: 0-no clinical disease, 1-limp tail, 2-hindlimb weakness, 3-hindlimb paralysis, 4-partial front limb paralysis, 5-moribund.
Antigen recall assay
Antigen recall assay was performed as previously described . Briefly, single cell suspensions were prepared from draining inguinal lymph nodes of Cx3cr1creER Lrp1 fl/fl and control mice 7 days post immunization with MOG35-55 peptide. Cells were treated with various concentrations of MOG35-55 peptide as indicated in the manuscript for 24 h. ELISA was used to measure IFN-γ and IL-17A production. To determine proliferation, BrdU (10 μM, BD) was added to the culture media and BrdU incorporation was measured 16 h after treatment by flow cytometry.
Blood brain barrier assay
Six hours prior to the assay, mice were injected i.p. with LPS (6 mg/kg). Heparin (20 U/mouse) was administered i.p. and followed, 30 min later, by 2 % sodium fluorescein (200 μl, NaF). After another 30 min, the mice were perfused, the meninges removed, and the brains weighed and isolated in 50 % trichloroacetic acid overnight at 4 °C. The tissue was finely diced and homogenized. After centrifugation (13,000 x g, 10 min, 4 °C) and neutralization of the acid with NaOH (5 M), 200 μl of the clarified homogenate was plated in 96 well black/clear bottom plates and fluorescence measured. Data is expressed as amount of tracer per gram of tissue.
Cell isolation from the CNS
Brain and/or spinal cords were removed and digested in HBSS containing collagenase IV (2 mg/ml) and DNase (20 U/ml) for 45 min at 37 °C. Cells were isolated using a 30 %/70 % Percoll gradient (GE Healthcare) . For adult primary microglia culture, cells were positively selected using CD11b magnetic microbeads (Miltenyi Biotec) after digestion. For flow cytometry analysis, single cell suspensions were stained with antibody against CD45 (30-F11, eBioscience), CD11b (M1/70, eBioscience) and LRP1 (ab92544, 1:1000, Abcam). Flow cytometry analyses were performed on a 10-color Beckman Coulter Gallios flow cytometer and data were analyzed with FlowJo software (TreeStar).
Anesthetized mice were perfused transcardially with PBS, followed by 4 % paraformaldehyde (PFA) prepared in PBS. Tissues were post-fixed for at least 24 h in 4 % PFA, and transferred to 20 % sucrose solution before sectioning. Cryosections (40 μm) were permeabilized in 0.5 % PBS-Tween for 15 min and washed twice with PBS. After blocking in 5 % serum in PBS for 2 h at room temperature, sections were incubated overnight at 4 °C with antibody against Iba1 (ab5076, 1:400, Abcam), LRP1 (ab92544, 1:250, Abcam) and CD68 (14-0688, 1:50, eBioscience). After washing, sections were incubated with secondary antibodies conjugated to Alexa488 or Alexa647 (Life Technologies). Sections were mounted with Prolong Gold Anti-Fade Reagent containing DAPI (Life Technologies). All sections were imaged with a Leica TCS SP8 confocal microscope and analyzed with ImageJ. Sholl analysis was performed as described .
Slides were deparaffinized using xylenes and an ethanol gradient. Heat induced antigen retrieval was performed for 10 min (Sodium Citrate Buffer, pH 6). Staining for CD68, Iba1, and LRP1 was conducted using ImmPress reagents according to manufacturer’s instructions (Vector lab). Adjacent sections were stained with LFB for demyelinating plaques. Histological analysis was performed by two independent investigators, blinded to the status of LRP1.
Preparation of Bone Marrow Derived Macrophages
Mice were euthanized and dissected to retrieve femurs and tibias. BMDM cells were cultured at 37 °C 5 % CO2 in high glucose DMEM with L-glutamine (GE Life Sciences, SH30022) supplemented with 10 % fetal bovine serum (Atlanta Biologicals, s12450), sodium pyruvate (Life Technologies, 11360-070), 1x Penicillin-Streptomycin (Life Technologies, 151490-122) and 30 % L929-conditioned media. Fresh media was supplemented at day 4, and the cells were replated on day 6. In some experiments, BMDM were treated with LPS (Sigma) and other TLR ligands (InvivoGen) as described in the text.
ELISA analyses for IL-6 and TNF-α were performed as previously described . Antibodies used were: anti-mouse IL-6 (MP5-20 F3, Biolegend) 0.5 μg/mL; biotin anti-mouse IL-6 (MP5-32C11, Biolegend) 1 μg/mL; anti-mouse TNF-α (R&D systems, AF-410-NA) 0.5 μg/mL; biotin anti-mouse TNF-α (R&D systems, BAF410) 0.25 μg/mL.
Proteins were extracted in RIPA buffer supplemented with protease inhibitor cocktail (Roche) and the phosphatase inhibitor sodium orthovanadate (2 mM). After incubation for 15 min on ice, the lysates were centrifuged (13,000 x g, 4 °C) and protein quantified using BCA assay. Protein samples were run on a Protean TGX gel (Bio-Rad) and transferred to a PVDF membrane. After blocking with 5 % milk in TBS-Tween for 1 h at room temperature, membranes were incubated overnight with primary antibody. After washing, membranes were incubated with HRP conjugated secondary antibodies (Pierce) for 1 h at room temperature and developed with Western Lighting Plus ECL (Perkin Elmer).
RNA was extracted using the Isolate II Kit (Bioline) according to manufacturer’s instructions and cDNA prepared with the iScript cDNA synthesis kit (Bio-Rad). qPCR was conducted with TaqMan primers (Applied Biosystems) for LRP1, IL-6, TNF-α, Il-1β, and normalized to GAPDH. Reactions were run on a PikoReal PCR system (Thermo Fisher) with the 2x NO-ROX Sensifast Mix or 2x SYBR Sensifast Mix (Bioline).
LRP1 expression is increased in MS lesions
LRP1 expression in MS: 6 clinical and pathologically confirmed multiple sclerosis autopsy samples were analyzed
Lesion stage from MS autopsy
LRP1 expression in the Lesion
100 % (2/2)
100 % (2/2)
0 % (0/1)
100 % (1/1)
Periplaque White Matter
0 % (0/5)
100 % (5/5)
Non-Affected White Matter
0 % (0/5)
100 % (5/5)
0 % (0/4)
100 % (4/4)
100 % (4/4)
Non-Affected Gray Matter
0 % (0/5)
100 % (5/5)
100 % (5/5)
Microglial LRP1 is protective during experimental autoimmune encephalomyelitis
Next, we generated a new mouse strain to study the role of LRP1 in microglia during EAE. The Lrp1 fl/fl strain  was crossed to the recently developed Cx3cr1 creER strain  to generate Cx3cr1 creER-Lrp1 fl/fl animals. Because CX3CR1 is expressed in microglia and monocytes , tamoxifen treatment initially leads to the deletion of LRP1 in both cell types. However, the pool of circulating monocytes that can give rise to macrophages during inflammation is continuously renewed by progenitors from the bone marrow, while the long-lived brain resident microglia are not replenished and remain LRP1 deleted [2, 9]. Indeed, 1 month after the administration of tamoxifen, we could still detect deletion of LRP1 in microglia, while LRP1 expression in bone marrow derived macrophages was comparable to control cells (Fig. 2d). EAE was induced by immunization with MOG35-55 peptide. LRP1 deletion in microglia results in a significant increase in clinical scores and overall incidence of EAE (Fig. 2e-f). Difference in the clinical score was not due to CX3CR1 haplodeficiency, as induction of EAE in animals that were not treated with tamoxifen showed no significant difference between groups (Additional file 1: Figure S2). Our results suggest that LRP1 expression in microglia has a protective role in EAE, significantly reducing the disease severity.
Demyelination and peripheral immune cell recruitment are elevated in mice lacking LRP1 in microglia
Lymphocyte activation is not affected in the absence of LRP1
Function of the blood brain barrier is intact in mice lacking microglial LRP1
Blood Brain Barrier (BBB) permeability disruption is an essential event in providing access to peripheral lymphocyte infiltration into the CNS during EAE , and microglia are known to modulate the function of the BBB . To investigate if LRP1 deletion in microglia can impact BBB permeability, we induced microgliosis in Cx3cr1 creER-Lrp1 fl/fl and control mice by injecting LPS, as previously described . LPS administration leads to increased extravasation of fluorescein in comparison to the basal level, as expected . However, BBB permeability was not affected by the status of LRP1 expression in microglia (Fig. 4d). These results suggest that LRP1 function in microglia is likely not critical for the maintenance of the BBB function.
Microglial morphology is altered by LRP1 deficiency
Microglia lacking LRP1 have a pro-inflammatory signature
LRP1 blocks NF-kB function
In this study, we explore the function of LRP1 expression in myeloid cells during MS using the mouse model EAE. To begin, we have made the novel observation that LRP1 protein expression is significantly increased in human MS lesions, compared to normal appearing brain tissue. The cellular compartments involved in the increase of LRP1 immunoreactivity included the myeloid cells and astrocytes. This observation is in agreement with our previous work showing that LRP1 expression is upregulated during EAE  and the work of Hendrickx et al., showing that LRP1 transcript is increased at the rim of MS lesions . The LRP1 expression increase by glia is not limited to MS, as previous work has demonstrated a similar pattern during CNS injury and neoplasia . Therefore, although our study centered on LRP1 function in myeloid cells, future studies will be needed to understand the role of LRP1 in astrocytes.
Here, we have explored the contribution of LRP1 expression during EAE in two distinct myeloid cell populations: Peripheral macrophages and microglia. To our surprise, we have discovered that LRP1 deletion in peripheral macrophages had no detectable impact on EAE progression. This result is in stark contrast with the consequence of LRP1 deletion in microglia, which leads to a significant worsening of disease progression. We, and others, have previously demonstrated that macrophages lacking LRP1 expression display a pro-inflammatory phenotype in vitro, characterized by increased production of inflammatory mediators, chemokines and an M1-type macrophage skewing [15, 43, 44], suggesting that the removal of LRP1 in macrophages could impact the progression of EAE in a detrimental manner. Therefore, we were surprised to find that LRP1 deletion in LysM cre-Lrp1 fl/fl mice did not result in increased disease severity in two separate EAE protocols (varying in the amount of mycobacterium used for the preparation of the complete Freund’s adjuvant). One potential explanation for these observations is that the peripheral immune cells are primed for a powerful inflammatory response prior to their entry into the CNS, and the removal of LRP1 does not alter their function in the diseased tissue. Future studies are necessary to distinguish whether LRP1 deletion in peripheral myeloid cells alters their function under homeostatic conditions.
On the contrary, ablation of LRP1 in microglia had a significant impact on EAE disease severity and progression. Microglia are the resident myeloid cells of the CNS and their function in EAE remains actively debated; while some studies suggest that they actively participate in disease initiation and peripheral immune cell recruitment [9, 45], others show that microglia remain relatively inert during EAE . Microglia have also been reported to express higher levels of the LRP1 transcript, compared to the peripheral macrophages, in both EAE and the homeostatic conditions [46, 47]. Therefore, we propose that microglia lacking LRP1 acquire an inflammatory phenotype that leads to disease exacerbation in EAE. In agreement with this hypothesis are the observations that LRP1 deficient microglia appear amoeboid and hypertrophic, even in the absence of ongoing disease. The transition from a ramified morphology to a more compact appearance by microglia has been described by many groups as an hallmark of inflammation [37, 48]. The fact that microglia appear activated in the healthy brain does suggest that they are primed to produce a more robust inflammatory response. Indeed, microglia lacking LRP1 secrete more TNF-α after LPS stimulation in vitro, a key cytokine involved in EAE pathology .
On a mechanistic level, we propose that LRP1 is an inhibitor of NF-kB signaling in myeloid cells, in agreement with our previous studies [15, 44]. Here, for the first time, we show that LRP1 only influences the MyD88 arm of TLR signaling, suggesting that LRP1 only has an effect on the extracellular TLR ligands . Further studies are needed to understand if the role of LRP1 as an NF-kB inhibitor intersects with LRP1 function in the removal of myelin debris. Indeed, the function of LRP1 in the removal of degraded myelin and dying cells could be critical in maintaining CNS homeostasis under normal physiological conditions, as well as in removing the degenerating oligodendrocytes during MS [10, 11]. Proper clearance of the myelin debris is critical for brain function, as the release of degenerated myelin could further propagate inflammation and damage the CNS. Furthermore, myelin-laden macrophages isolated from the brain are known to have decreased inflammatory responses, when compared to macrophages that have not engulfed myelin . The receptors and mechanisms that initiate the anti-inflammatory response to apoptotic cell or myelin engulfment are still poorly understood . Further studies will be needed to understand if LRP1 mediated signaling in microglia can initiate an anti-inflammatory response, thereby exerting protection in EAE and, perhaps, MS.
In conclusion, we demonstrated that LRP1 expression is significantly upregulated by myeloid cells in active MS lesions. To study the role of LRP1 in myeloid cells, we induced EAE in mice lacking LRP1 in microglia or in macrophages and showed that only microglial LRP1 was protective, as animal lacking LRP1 in this compartment experienced a worse clinical outcome. At the mechanistic level, our study demonstrates that LRP1 regulates inflammation by inhibiting NF-kB, a master regulator of inflammation, via a MyD88 dependent pathway.
MS, Multiple sclerosis; CNS, central nervous system; LRP1, Low-density lipoprotein receptor-related protein-1; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; BMDM, bone marrow derived macrophages; BBB, Blood Brain Barrier.
We thank Dr. Sanja Arandjelovic (University of Virginia) for critical reading of the manuscript.
The authors are supported by NIH grants R01 NS083542.
TYC designed, performed and analyzed the research and wrote the paper. YG, SS, AR, DJ, JM and CF performed research. AG designed and analyzed the research and wrote the paper. All authors read and approved the manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
The human pathology study was approved by the Institutional Review Board of Mayo Clinic, Rochester, MN.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Compston A, Coles A. Multiple sclerosis. Lancet. 2008;372(9648):1502–17.View ArticlePubMedGoogle Scholar
- Ajami B et al. Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Nat Neurosci. 2011;14(9):1142–9.View ArticlePubMedGoogle Scholar
- Lucchinetti CF, Bruck W, Lassmann H. Evidence for pathogenic heterogeneity in multiple sclerosis. Ann Neurol. 2004;56(2):308.View ArticlePubMedGoogle Scholar
- Rinner WA, et al. Resident microglia and hematogenous macrophages as phagocytes in adoptively transferred experimental autoimmune encephalomyelitis: an investigation using rat radiation bone marrow chimeras. Glia. 1995;14(4):257–66.View ArticlePubMedGoogle Scholar
- King IL, Dickendesher TL, Segal BM. Circulating Ly-6C+ myeloid precursors migrate to the CNS and play a pathogenic role during autoimmune demyelinating disease. Blood. 2009;113(14):3190–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Nair A, Frederick TJ, Miller SD. Astrocytes in multiple sclerosis: a product of their environment. Cell Mol Life Sci. 2008;65(17):2702–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Bogie JF, Stinissen P, Hendriks JJ. Macrophage subsets and microglia in multiple sclerosis. Acta Neuropathol. 2014;128(2):191–213.View ArticlePubMedGoogle Scholar
- Vainchtein ID, et al. In acute experimental autoimmune encephalomyelitis, infiltrating macrophages are immune activated, whereas microglia remain immune suppressed. Glia. 2014;62(10):1724–35.View ArticlePubMedGoogle Scholar
- Goldmann T, et al. A new type of microglia gene targeting shows TAK1 to be pivotal in CNS autoimmune inflammation. Nat Neurosci. 2013;16(11):1618–26.View ArticlePubMedGoogle Scholar
- Fernandez-Castaneda A, et al. Identification of the low density lipoprotein (LDL) receptor-related protein-1 interactome in central nervous system myelin suggests a role in the clearance of necrotic cell debris. J Biol Chem. 2013;288(7):4538–48.View ArticlePubMedGoogle Scholar
- Gaultier A, et al. Low-density lipoprotein receptor-related protein 1 is an essential receptor for myelin phagocytosis. J Cell Sci. 2009;122(Pt 8):1155–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Gardai SJ, et al. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell. 2005;123(2):321–34.View ArticlePubMedGoogle Scholar
- Lampron A, et al. Inefficient clearance of myelin debris by microglia impairs remyelinating processes. J Exp Med. 2015;212(4):481–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Lillis AP, et al. LDL Receptor-Related Protein 1: Unique Tissue-Specific Functions Revealed by Selective Gene Knockout Studies. Physiol Rev. 2008;88(3):887–918.View ArticlePubMedPubMed CentralGoogle Scholar
- Gaultier A, et al. Regulation of tumor necrosis factor receptor-1 and the IKK-NF-kappaB pathway by LDL receptor-related protein explains the antiinflammatory activity of this receptor. Blood. 2008;111(11):5316–25.View ArticlePubMedPubMed CentralGoogle Scholar
- van Loo G, et al. Inhibition of transcription factor NF-kappaB in the central nervous system ameliorates autoimmune encephalomyelitis in mice. Nat Immunol. 2006;7(9):954–61.View ArticlePubMedGoogle Scholar
- Hilliard B, et al. Experimental autoimmune encephalomyelitis in NF-kappa B-deficient mice:roles of NF-kappa B in the activation and differentiation of autoreactive T cells. J Immunol. 1999;163(5):2937–43.PubMedGoogle Scholar
- Hilliard BA, et al. Critical roles of c-Rel in autoimmune inflammation and helper T cell differentiation. J Clin Invest. 2002;110(6):843–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Pahan K, Schmid M. Activation of nuclear factor-kB in the spinal cord of experimental allergic encephalomyelitis. Neurosci Lett. 2000;287(1):17–20.View ArticlePubMedGoogle Scholar
- Dasgupta S, et al. Antineuroinflammatory effect of NF-kappaB essential modifier-binding domain peptides in the adoptive transfer model of experimental allergic encephalomyelitis. J Immunol. 2004;173(2):1344–54.View ArticlePubMedGoogle Scholar
- Popescu BF, Pirko I, Lucchinetti CF. Pathology of multiple sclerosis: where do we stand? Continuum (Minneap Minn). 2013;19(4 Multiple Sclerosis):901–21.Google Scholar
- Rohlmann A, et al. Sustained somatic gene inactivation by viral transfer of Cre recombinase. Nat Biotechnol. 1996;14(11):1562–5.View ArticlePubMedGoogle Scholar
- Clausen BE, et al. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res. 1999;8(4):265–77.View ArticlePubMedGoogle Scholar
- Cronk JC, et al. Methyl-CpG Binding Protein 2 Regulates Microglia and Macrophage Gene Expression in Response to Inflammatory Stimuli. Immunity. 2015;42(4):679–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Remick DG, et al. Six at six: interleukin-6 measured 6 h after the initiation of sepsis predicts mortality over 3 days. Shock. 2002;17(6):463–7.View ArticlePubMedGoogle Scholar
- Wolf BB, et al. Characterization and immunohistochemical localization of alpha 2-macroglobulin receptor (low-density lipoprotein receptor-related protein) in human brain. Am J Pathol. 1992;141(1):37–42.PubMedPubMed CentralGoogle Scholar
- Ginhoux F, et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010;330(6005):841–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Cho IH, et al. Role of microglial IKKbeta in kainic acid-induced hippocampal neuronal cell death. Brain. 2008;131(Pt 11):3019–33.View ArticlePubMedPubMed CentralGoogle Scholar
- Overton CD, et al. Deletion of macrophage LDL receptor-related protein increases atherogenesis in the mouse. Circ Res. 2007;100(5):670–7.View ArticlePubMedGoogle Scholar
- Stromnes IM, Goverman JM. Active induction of experimental allergic encephalomyelitis. Nat Protoc. 2006;1(4):1810–9.View ArticlePubMedGoogle Scholar
- Yona S, et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity. 2013;38(1):79–91.View ArticlePubMedGoogle Scholar
- Subramanian M, et al. An AXL/LRP-1/RANBP9 complex mediates DC efferocytosis and antigen cross-presentation in vivo. J Clin Invest. 2014;124(3):1296–308.View ArticlePubMedPubMed CentralGoogle Scholar
- Jung S, et al. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol. 2000;20(11):4106–14.View ArticlePubMedPubMed CentralGoogle Scholar
- Bennett J, et al. Blood-brain barrier disruption and enhanced vascular permeability in the multiple sclerosis model EAE. J Neuroimmunol. 2010;229(1-2):180–91.View ArticlePubMedGoogle Scholar
- da Fonseca AC, et al. The impact of microglial activation on blood-brain barrier in brain diseases. Front Cell Neurosci. 2014;8:362.View ArticlePubMedPubMed CentralGoogle Scholar
- Ramirez SH, et al. Activation of cannabinoid receptor 2 attenuates leukocyte-endothelial cell interactions and blood-brain barrier dysfunction under inflammatory conditions. J Neurosci. 2012;32(12):4004–16.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen Z, et al. Lipopolysaccharide-induced microglial activation and neuroprotection against experimental brain injury is independent of hematogenous TLR4. J Neurosci. 2012;32(34):11706–15.View ArticlePubMedPubMed CentralGoogle Scholar
- Selmaj KW, Raine CS. Experimental autoimmune encephalomyelitis: immunotherapy with anti-tumor necrosis factor antibodies and soluble tumor necrosis factor receptors. Neurology. 1995;45(6 Suppl 6):S44–9.View ArticlePubMedGoogle Scholar
- Lu YC, Yeh WC, Ohashi PS. LPS/TLR4 signal transduction pathway. Cytokine. 2008;42(2):145–51.View ArticlePubMedGoogle Scholar
- Yu L, Wang L, Chen S. Endogenous toll-like receptor ligands and their biological significance. J Cell Mol Med. 2010;14(11):2592–603.View ArticlePubMedPubMed CentralGoogle Scholar
- Hendrickx DA, et al. Selective upregulation of scavenger receptors in and around demyelinating areas in multiple sclerosis. J Neuropathol Exp Neurol. 2013;72(2):106–18.View ArticlePubMedGoogle Scholar
- Lopes MB, et al. Expression of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein is increased in reactive and neoplastic glial cells. FEBS Lett. 1994;338(3):301–5.View ArticlePubMedGoogle Scholar
- May P, Bock HH, Nofer JR. Low density receptor-related protein 1 (LRP1) promotes anti-inflammatory phenotype in murine macrophages. Cell Tissue Res. 2013;354(3):887–9.View ArticlePubMedGoogle Scholar
- Staudt ND, et al. Myeloid cell receptor LRP1/CD91 regulates monocyte recruitment and angiogenesis in tumors. Cancer Res. 2013;73(13):3902–12.View ArticlePubMedPubMed CentralGoogle Scholar
- D’Mello C, Le T, Swain MG. Cerebral microglia recruit monocytes into the brain in response to tumor necrosis factoralpha signaling during peripheral organ inflammation. J Neurosci. 2009;29(7):2089–102.View ArticlePubMedGoogle Scholar
- Lewis ND, et al. RNA sequencing of microglia and monocyte-derived macrophages from mice with experimental autoimmune encephalomyelitis illustrates a changing phenotype with disease course. J Neuroimmunol. 2014;277(1-2):26–38.View ArticlePubMedGoogle Scholar
- Butovsky O, et al. Identification of a unique TGF-beta-dependent molecular and functional signature in microglia. Nat Neurosci. 2014;17(1):131–43.View ArticlePubMedGoogle Scholar
- Kozlowski C, Weimer RM. An automated method to quantify microglia morphology and application to monitor activation state longitudinally in vivo. PLoS One. 2012;7(2):e31814.View ArticlePubMedPubMed CentralGoogle Scholar
- Boven LA, et al. Myelin-laden macrophages are anti-inflammatory, consistent with foam cells in multiple sclerosis. Brain. 2006;129(Pt 2):517–26.PubMedGoogle Scholar
- Henson PM, Bratton DL. Antiinflammatory effects of apoptotic cells. J Clin Invest. 2013;123(7):2773–4.View ArticlePubMedPubMed CentralGoogle Scholar