RESUMO
Intracellular surveillance for systemic microbial components during homeostasis and infections governs host physiology and immunity. However, a long-standing question is how circulating microbial ligands become accessible to intracellular receptors. Here we show a role for host-derived extracellular vesicles (EVs) in this process; human and murine plasma-derived and cell culture-derived EVs have an intrinsic capacity to bind bacterial lipopolysaccharide (LPS). Remarkably, circulating host EVs capture blood-borne LPS in vivo, and the LPS-laden EVs confer cytosolic access for LPS, triggering non-canonical inflammasome activation of gasdermin D and pyroptosis. Mechanistically, the interaction between the lipid bilayer of EVs and the lipid A of LPS underlies EV capture of LPS, and the intracellular transfer of LPS by EVs is mediated by CD14. Overall, this study demonstrates that EVs capture and escort systemic LPS to the cytosol licensing inflammasome responses, uncovering EVs as a previously unrecognized link between systemic microbial ligands and intracellular surveillance.
Assuntos
Vesículas Extracelulares , Inflamassomos , Humanos , Animais , Camundongos , Inflamassomos/metabolismo , Lipopolissacarídeos , Caspases/metabolismo , Piroptose , Citosol , Vesículas Extracelulares/metabolismoRESUMO
The meninges surround the brain and spinal cord, affording physical protection while also serving as a niche of neuroimmune activity. Though possessing stromal qualities, its complex cellular and extracellular makeup has yet to be elaborated, and it remains unclear whether the meninges vary along the neuroaxis. Hence, studies were carried-out to elucidate the protein composition and structural organization of brain and spinal cord meninges in normal, adult Biozzi ABH mice. First, shotgun, bottom-up proteomics was carried-out. Prominent proteins at both brain and spinal levels included Type II collagen and Type II keratins, representing extracellular matrix (ECM) and cytoskeletal categories, respectively. While the vast majority of total proteins detected was shared between both meningeal locales, more were uniquely detected in brain than in spine. This pattern was also seen when total proteins were subdivided by cellular compartment, except in the case of the ECM category where brain and spinal meninges each had near equal number of unique proteins, and Type V and type III collagen registered exclusively in the spine. Quantitative analysis revealed differential expression of several collagens and cytoskeletal proteins between brain and spinal meninges. High-resolution immunofluorescence and immunogold-scanning electronmicroscopy on sections from whole brain and spinal cord - still encased within bone -identified major proteins detected by proteomics, and highlighted their association with cellular and extracellular elements of variously shaped arachnoid trabeculae. Western blotting aligned with the proteomic and immunohistological analyses, reinforcing differential appearance of proteins in brain vs spinal meninges. Results could reflect regional distinctions in meninges that govern protective and/or neuroimmune functions.
Assuntos
Meninges , Proteômica , Camundongos , Animais , Camundongos Biozzi , Meninges/metabolismo , Medula Espinal/metabolismo , EncéfaloRESUMO
BACKGROUND: Peripheral nerve injuries stimulate the regenerative capacity of injured neurons through a neuroimmune phenomenon termed the conditioning lesion (CL) response. This response depends on macrophage accumulation in affected dorsal root ganglia (DRGs) and peripheral nerves. The macrophage chemokine CCL2 is upregulated after injury and is allegedly required for stimulating macrophage recruitment and pro-regenerative signaling through its receptor, CCR2. In these tissues, CCL2 is putatively produced by neurons in the DRG and Schwann cells in the distal nerve. METHODS: Ccl2fl/fl mice were crossed with Advillin-Cre, P0-Cre, or both to create conditional Ccl2 knockouts (CKOs) in sensory neurons, Schwann cells, or both to hypothetically remove CCL2 and macrophages from DRGs, nerves or both. CCL2 was localized using Ccl2-RFPfl/fl mice. CCL2-CCR2 signaling was further examined using global Ccl2 KOs and Ccr2gfp knock-in/knock-outs. Unilateral sciatic nerve transection was used as the injury model, and at various timepoints, chemokine expression, macrophage accumulation and function, and in vivo regeneration were examined using qPCR, immunohistochemistry, and luxol fast blue staining. RESULTS: Surprisingly, in all CKOs, DRG Ccl2 gene expression was decreased, while nerve Ccl2 was not. CCL2-RFP reporter mice revealed CCL2 expression in several cell types beyond the expected neurons and Schwann cells. Furthermore, macrophage accumulation, myelin clearance, and in vivo regeneration were unaffected in all CKOs, suggesting CCL2 may not be necessary for the CL response. Indeed, Ccl2 global knockout mice showed normal macrophage accumulation, myelin clearance, and in vivo regeneration, indicating these responses do not require CCL2. CCR2 ligands, Ccl7 and Ccl12, were upregulated after nerve injury and perhaps could compensate for the absence of Ccl2. Finally, Ccr2gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. Ccr2gfp/gfp KOs showed a 50% decrease in macrophages in the distal nerve compared to controls with a relative increase in resident macrophages. In the DRG there was a small but insignificant decrease in macrophages. CONCLUSIONS: CCL2 is not necessary for macrophage accumulation, myelin clearance, and axon regeneration in the peripheral nervous system. Without CCL2, other CCR2 chemokines, resident macrophage proliferation, and CCR2-independent monocyte recruitment can compensate and allow for normal macrophage accumulation.
Assuntos
Quimiocina CCL2 , Macrófagos , Traumatismos dos Nervos Periféricos , Animais , Axônios/imunologia , Axônios/patologia , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Quimiocinas/imunologia , Quimiocinas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/imunologia , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/patologiaRESUMO
Neutrophils are critical for inflammation and innate immunity, and their adhesion to vascular endothelium is a crucial step in neutrophil recruitment. Mitofusin-2 (MFN2) is required for neutrophil adhesion, but molecular details are unclear. Here, we demonstrated that ß2 -integrin-mediated slow-rolling and arrest, but not PSGL-1-mediated cell rolling, are defective in MFN2-deficient neutrophil-like HL60 cells. This adhesion defect is associated with reduced expression of fMLP (N-formylmethionyl-leucyl-phenylalanine) receptor FPR1 as well as the inhibited ß2 integrin activation, as assessed by conformation-specific monoclonal antibodies. MFN2 deficiency also leads to decreased actin polymerization, which is important for ß2 integrin activation. Mn2+ -induced cell spreading is also inhibited after MFN2 knockdown. MFN2 deficiency limited the maturation of ß2 integrin activation during the neutrophil-directed differentiation of HL60 cells, which is indicated by CD35 and CD87 markers. MFN2 knockdown in ß2-integrin activation-matured cells (CD87high population) also inhibits integrin activation, indicating that MFN2 directly affects ß2 integrin activation. Our study illustrates the function of MFN2 in leukocyte adhesion and may provide new insights into the development and treatment of MFN2 deficiency-related diseases.
Assuntos
Antígenos CD18 , Neutrófilos , Antígenos CD18/metabolismo , Adesão Celular , N-Formilmetionina Leucil-Fenilalanina , Infiltração de NeutrófilosRESUMO
BACKGROUND: Tight junctions (TJs) are membrane specializations characteristic of barrier-forming membranes, which function to seal the aqueous pathway between endothelial cells or epithelial cells and, thereby, obstruct intercellular solute and cellular movement. However, previous work from our laboratory found that claudin-5 (CLN-5), a TJ protein prominent at the blood-brain barrier (BBB), was also detected, ectopically, on leukocytes (CLN-5+) in the blood and central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a neuroinflammatory, demyelinating disease that is a model for multiple sclerosis. CLN-5 was further shown to be transferred from endothelial cells to circulating leukocytes during disease, prompting consideration this action is coupled to leukocyte transendothelial migration (TEM) into the CNS by fostering transient interactions between corresponding leukocyte and endothelial junctional proteins at the BBB. METHODS: To begin clarifying the significance of CLN-5+ leukocytes, flow cytometry was used to determine their appearance in the blood and CNS during EAE. RESULTS: Flow cytometric analysis revealed CLN-5+ populations among CD4 and CD8 T cells, B cells, monocytes and neutrophils, and these appeared with varying kinetics and to different extents in both blood and CNS. CLN-5 levels on circulating T cells further correlated highly with activation state. And, the percentage of CLN-5+ cells among each of the subtypes analyzed was considerably higher in CNS tissue than in blood, consistent with the interpretation that CLN-5+ leukocytes gain preferred access to the CNS. CONCLUSION: Several leukocyte subtypes variably acquire CLN-5 in blood before they enter the CNS, an event that may represent a novel mechanism to guide leukocytes to sites for paracellular diapedesis across the BBB.
Assuntos
Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Claudina-5/genética , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Leucócitos/patologia , Animais , Barreira Hematoencefálica/metabolismo , Claudina-5/sangue , Claudina-5/metabolismo , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neuroinflamatórias/genética , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Proteínas de Junções Íntimas/metabolismoRESUMO
BACKGROUND: Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by both inflammatory demyelination and impaired remyelination. Studies indicate that Toll-like receptor 2 (TLR2) signaling contributes to both the inflammatory component and the defective remyelination in MS. While most MS therapeutics target adaptive immunity, we recently reported that reducing TLR2 signaling in innate immune cells by inducing TLR2 tolerance attenuates adoptively transferred experimental autoimmune encephalomyelitis. Given that previous reports suggest TLR2 signaling also inhibits myelin repair, the objective of this study was to assess how reducing TLR2 signaling through TLR2 tolerance induction affects CNS myelin repair. METHODS: Chow containing 0.2% cuprizone was fed to male and female wild-type (WT) C57BL/6 mice or TLR2-deficient (TLR2-/-) mice for 5 weeks to induce demyelination. During a 2-week remyelination period following discontinuation of cuprizone, WT mice received either low dose TLR2 ligands to induce systemic TLR2 tolerance or vehicle control (VC). Remyelination was evaluated via electron microscopy and immunohistochemical analysis of microglia and oligodendrocytes in the corpus callosum. Statistical tests included 2-way ANOVA and Mann-Whitney U analyses. RESULTS: Inducing TLR2 tolerance in WT mice during remyelination significantly enhanced myelin recovery, restoring unmyelinated axon frequency and myelin thickness to baseline levels compared to VC-treated mice. Mechanistically, enhanced remyelination in TLR2 tolerized mice was associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. This result was confirmed in vitro by inducing TLR2 tolerance in WT microglia cultures. TLR2-/- mice, without TLR2 tolerance induction, also significantly enhanced myelin recovery compared to WT mice, adding confirmation that reduced TLR2 signaling is associated with enhanced remyelination. DISCUSSION: Our results suggest that reducing TLR2 signaling in vivo by inducing TLR2 tolerance significantly enhances myelin repair. Furthermore, the enhanced remyelination resulting from TLR2 tolerance induction is associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. While deletion of TLR2 would be an impractical approach in vivo, reducing innate immune signaling through TLR2 tolerance induction may represent a novel, two-pronged approach for treating both inflammatory and myelin repair components of MS.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Lipopeptídeos/uso terapêutico , Microglia/metabolismo , Oligodendroglia/metabolismo , Remielinização/fisiologia , Receptor 2 Toll-Like/metabolismo , Animais , Encefalomielite Autoimune Experimental/tratamento farmacológico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Resultado do TratamentoRESUMO
BACKGROUND: Immune cell trafficking into the CNS is considered to contribute to pathogenesis in MS and its animal model, EAE. Disruption of the blood-brain barrier (BBB) is a hallmark of these pathologies and a potential target of therapeutics. Human embryonic stem cell-derived mesenchymal stem/stromal cells (hES-MSCs) have shown superior therapeutic efficacy, compared to bone marrow-derived MSCs, in reducing clinical symptoms and neuropathology of EAE. However, it has not yet been reported whether hES-MSCs inhibit and/or repair the BBB damage associated with neuroinflammation that accompanies EAE. METHODS: BMECs were cultured on Transwell inserts as a BBB model for all the experiments. Disruption of BBB models was induced by TNF-α, a pro-inflammatory cytokine that is a hallmark of acute and chronic neuroinflammation. RESULTS: Results indicated that hES-MSCs reversed the TNF-α-induced changes in tight junction proteins, permeability, transendothelial electrical resistance, and expression of adhesion molecules, especially when these cells were placed in direct contact with BMEC. CONCLUSIONS: hES-MSCs and/or products derived from them could potentially serve as novel therapeutics to repair BBB disturbances in MS.
Assuntos
Barreira Hematoencefálica/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/efeitos dos fármacos , Linhagem Celular Transformada , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Modelos BiológicosRESUMO
Extracellular vesicles (EVs) are heterogeneous, nano-sized vesicles that are shed into the blood and other body fluids, which disperse a variety of bioactive molecules (e.g., protein, mRNA, miRNA, DNA and lipids) to cellular targets over long and short distances. EVs are thought to be produced by nearly every cell type, however this review will focus specifically on EVs that originate from cells at the interface of CNS barriers. Highlighted topics include, EV biogenesis, the production of EVs in response to neuroinflammation, role in intercellular communication and their utility as a therapeutic platform. In this review, novel concepts regarding the use of EVs as biomarkers for BBB status and as facilitators for immune neuroinvasion are also discussed. Future directions and prospective are covered along with important unanswered questions in the field of CNS endothelial EV biology.
Assuntos
Sistema Nervoso Central/irrigação sanguínea , Sistema Nervoso Central/metabolismo , Vesículas Extracelulares/metabolismo , Inflamação/metabolismo , Exossomos/metabolismo , HumanosRESUMO
Laser-capture microdissection (LCM) coupled to downstream RNA analysis poses unique difficulties for the evaluation of mineralized tissues. A rapid protocol was thus developed to enable sufficient integrity of bone and cartilage tissue for reliable sectioning, while minimizing RNA loss associated with prolonged decalcification and purification steps. Specifically, the protocol involves pump-assisted, cardiac perfusion-fixation with paraformaldehyde, and moderate digestion of LCM-acquired tissue with proteinase K followed by DNase treatment and separation of RNA using magnetic beads. Reverse transcription and cDNA synthesis are performed immediately after RNA purification, without need for further protein removal.
Assuntos
Osso e Ossos/metabolismo , Cartilagem/metabolismo , Modelos Animais de Doenças , Células-Tronco Pluripotentes Induzidas/metabolismo , Microdissecção e Captura a Laser/métodos , Células-Tronco Mesenquimais/metabolismo , RNA/análise , Crânio/metabolismo , Animais , Osso e Ossos/patologia , Cartilagem/patologia , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Mesenquimais/patologia , Camundongos , Perfusão , RNA/genética , RNA/isolamento & purificação , Crânio/patologiaRESUMO
Experimental autoimmune encephalomyelitis (EAE) induced by active immunization of C57BL/6 mice with peptide from myelin oligodendrocyte protein (MOG35-55), is a neuroinflammatory, demyelinating disease widely recognized as an animal model of multiple sclerosis (MS). Typically, EAE presents with an ascending course of paralysis, and inflammation that is predominantly localized to the spinal cord. Recent studies have further indicated that inflammation - in both MS and EAE - might initiate within the meninges and propagate from there to the underlying parenchyma. However, the patterns of inflammation within the respective meningeal and parenchymal compartments along the length of the spinal cord, and the progression with which these patterns develop during EAE, have yet to be detailed. Such analysis could hold key to identifying factors critical for spreading, as well as constraining, inflammation along the neuraxis. To address this issue, high-resolution 3-dimensional (3D) confocal microscopy was performed to visualize, in detail, the sequence of leukocyte infiltration at distinct regions of the spinal cord. High quality virtual slide scanning for imaging the entire spinal cord using epifluorescence was further conducted to highlight the directionality and relative degree of inflammation. Meningeal inflammation was found to precede parenchymal inflammation at all levels of the spinal cord, but did not develop equally or simultaneously throughout the subarachnoid space (SAS) of the meninges. Instead, meningeal inflammation was initially most obvious in the caudal SAS, from which it progressed to the immediate underlying parenchyma, paralleling the first signs of clinical disease in the tail and hind limbs. Meningeal inflammation could then be seen to extend in the caudal-to-rostral direction, followed by a similar, but delayed, trajectory of parenchymal inflammation. To additionally determine whether the course of ascending paralysis and leukocyte infiltration during EAE is reflected in differences in inflammatory gene expression by meningeal and parenchymal microvessels along the spinal cord, laser capture microdissection (LCM) coupled with gene expression profiling was performed. Expression profiles varied between these respective vessel populations at both the cervical and caudal levels of the spinal cord during disease progression, and within each vessel population at different levels of the cord at a given time during disease. These results reinforce a significant role for the meninges in the development and propagation of central nervous system inflammation associated with MS and EAE.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Meninges/imunologia , Tecido Parenquimatoso/imunologia , Animais , Vértebras Cervicais , Progressão da Doença , Encefalomielite Autoimune Experimental/patologia , Feminino , Expressão Gênica , Inflamação/patologia , Inflamação/fisiopatologia , Leucócitos/imunologia , Leucócitos/patologia , Vértebras Lombares , Meninges/patologia , Camundongos Endogâmicos C57BL , Microvasos/imunologia , Microvasos/patologia , Glicoproteína Mielina-Oligodendrócito , Tecido Parenquimatoso/patologia , Fragmentos de Peptídeos , Medula Espinal/imunologia , Medula Espinal/patologiaRESUMO
BACKGROUND: The mechanism of leukocyte transendothelial migration (TEM) across the highly restrictive blood-brain barrier (BBB) remains enigmatic, with paracellular TEM thought to require leukocytes to somehow navigate the obstructive endothelial tight junctions (TJs). Transient interactions between TJ proteins on the respective leukocyte and endothelial surfaces have been proposed as one mechanism for TEM. Given the expanding role of extracellular vesicles (EVs) in intercellular communication, we investigated whether EVs derived from brain microvascular endothelial cells (BMEC) of the BBB may play a role in transferring a major TJ protein, claudin-5 (CLN-5), to leukocytes as a possible basis for such a mechanism during neuroinflammation. METHODS: High-resolution 3D confocal imaging was used to highlight CLN-5 immunoreactivity in the central nervous system (CNS) and on leukocytes of mice with the neuroinflammatory condition experimental autoimmune encephalomyelitis (EAE). Both Western blotting of circulating leukocytes from wild-type mice and fluorescence imaging of leukocyte-associated eGFP-CLN-5 in the blood and CNS of endothelial-targeted, Tie-2-eGFP-CLN-5 transgenic mice were used to confirm the presence of CLN-5 protein on these cells. EVs were isolated from TNF-α-stimulated BMEC cultures and blood plasma of Tie-2-eGFP-CLN-5 mice with EAE and evaluated for CLN-5 protein by Western blotting and fluorescence-activated cell sorting (FACS), respectively. Confocal imaging and FACS were used to detect binding of endothelial-derived EVs from these two sources to leukocytes in vitro. Serial electron microscopy (serial EM) and 3D contour-based surface reconstruction were employed to view EV-like structures at the leukocyte:BBB interface in situ in inflamed CNS microvessels. RESULTS: A subpopulation of leukocytes immunoreactive for CLN-5 on their surface was seen to infiltrate the CNS of mice with EAE and reside in close apposition to inflamed vessels. Confocal imaging of immunostained samples and Western blotting established the presence of CLN-5+ leukocytes in blood as well, implying these cells are present prior to TEM. Moreover, imaging of inflamed CNS vessels and the associated perivascular cell infiltrates from Tie-2-eGFP-CLN-5 mice with EAE revealed leukocytes bearing the eGFP label, further supporting the hypothesis CLN-5 is transferred from endothelial cells to circulating leukocytes in vivo. Western blotting of BMEC-derived EVs, corresponding in size to both exosomes and microvesicles, and FACS analysis of plasma-derived EVs from Tie-2-eGFP-CLN-5 mice with EAE validated expression of CLN-5 by EVs of endothelial origin. Confocal imaging and FACS further revealed both PKH-67-labeled EVs from cultured BMECs and eGFP-CLN-5+ EVs from plasma of Tie-2-eGFP-CLN-5 mice with EAE can bind to leukocytes. Lastly, serial EM and 3D contour-based surface reconstruction revealed a close association of EV-like structures between the marginating leukocytes and BMECs in situ during EAE. CONCLUSIONS: During neuroinflammation, CLN-5+ leukocytes appear in the CNS, and both CLN-5+ leukocytes and CLN-5+ EVs are detected in the blood. As endothelial cells transfer CLN-5+ to leukocytes in vivo, and EVs released from BMEC bind to leukocytes in vitro, EVs may serve as the vehicles to transfer CLN-5 protein at sites of leukocyte:endothelial contact along the BBB. This action may be a prelude to facilitate TEM through the formation of temporary TJ protein bridges between these two cell types.
Assuntos
Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/patologia , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Vesículas Extracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Células Cultivadas , Sistema Nervoso Central/diagnóstico por imagem , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/imunologia , Células Endoteliais/ultraestrutura , Endotélio Vascular/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Feminino , Leucócitos/metabolismo , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidadeRESUMO
The choroid plexus (CP) is considered to be a point of leukocyte entry into the CNS during normal immune surveillance and in neuroinflammatory diseases. The structural and functional alterations within the CP that support this migration are not understood. We used quantitative, high-resolution, 3-dimensional (3-D) fluorescence imaging to analyze CP alterations associated with inflammatory responses in C57/Bl6 mice after the induction of experimental autoimmune encephalomyelitis by immunization with myelin oligodendrocyte glycoprotein (MOG) and complete Freund adjuvant/pertussis toxin (MOG-CFA/PTX) or adjuvants alone (CFA-PTX). The MOG-CFA/PTX and CFA/PTX produced similar effects, although those caused by the former were consistently more marked. Both treatments resulted in the accumulation of serum immunoglobulin G and leukocytes in the CP stroma, consistent with elevated stromal capillary permeability. They also provoked distortions and diminished immunostaining patterns of the tight junction adaptor protein ZO-1 in the choroidal epithelium but no obvious change in the patterns of the tight junction associated protein claudin-2. Only MOG-CFA/PTX triggered visible extravasation of immunoglobulin G and leukocytes across the choroidal epithelium. Our results suggest that CFA/PTX primes the CP for neuroinflammation by inducing structural changes that are exacerbated when there is an immune response to MOG and reinforce the CP as a gateway for leukocytes to enter the CNS by accessing the CSF and leptomeninges.
Assuntos
Permeabilidade Capilar/fisiologia , Plexo Corióideo/metabolismo , Imunoglobulina G/metabolismo , Leucócitos/metabolismo , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo , Sequência de Aminoácidos , Animais , Plexo Corióideo/imunologia , Feminino , Imunoglobulina G/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Leucócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas de Junções Íntimas/imunologia , Junções Íntimas/imunologiaRESUMO
Current therapies for multiple sclerosis (MS) are largely palliative, not curative. Mesenchymal stem cells (MSCs) harbor regenerative and immunosuppressive functions, indicating a potential therapy for MS, yet the variability and low potency of MSCs from adult sources hinder their therapeutic potential. MSCs derived from human embryonic stem cells (hES-MSCs) may be better suited for clinical treatment of MS because of their unlimited and stable supply. Here, we show that hES-MSCs significantly reduce clinical symptoms and prevent neuronal demyelination in a mouse experimental autoimmune encephalitis (EAE) model of MS, and that the EAE disease-modifying effect of hES-MSCs is significantly greater than that of human bone-marrow-derived MSCs (BM-MSCs). Our evidence also suggests that increased IL-6 expression by BM-MSCs contributes to the reduced anti-EAE therapeutic activity of these cells. A distinct ability to extravasate and migrate into inflamed CNS tissues may also be associated with the robust therapeutic effects of hES-MSCs on EAE.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Embrionárias/citologia , Encefalomielite Autoimune Experimental/terapia , Células-Tronco Mesenquimais/citologia , Esclerose Múltipla/patologia , Esclerose Múltipla/terapia , Animais , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Humanos , Transplante de Células-Tronco Mesenquimais , CamundongosRESUMO
BACKGROUND: The chemokine CCL2 is a critical mediator of neuroinflammation in diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). CCL2 drives mononuclear cell infiltration into the central nervous system (CNS), alters expression and distribution of microvascular endothelial tight junction proteins, and disrupts the blood-brain and blood-spinal cord barriers. Immunohistochemistry has consistently revealed astrocytes to be a source of this chemokine during neuroinflammation, while providing less uniform evidence that CNS endothelial cells may also express CCL2. Moreover, the relative contributions of these cell types to the CNS pool of CCL2 during MS/EAE are unclear and the aim of this study was to investigate this further. METHODS: CCL2 gene expression was determined by qRT-PCR in different populations of CNS cells at different times following EAE induced by immunization with MOG35-55 peptide and adjuvants, or after injection with adjuvants alone. CNS cells types were isolated by two different protocols: bulk isolation to yield crude microvascular and parenchymal fractions (containing astrocytes, other glia, and neurons), or laser capture microdissection (LCM) to acquire more precisely microvascular endothelial cells, astrocytes or other parenchymal cells. RESULTS: Both CNS microvessel and parenchymal populations prepared by crude bulk isolation showed up-regulation of CCL2 mRNA following MOG immunization or injection of adjuvants alone. More exact dissection by LCM revealed microvascular endothelial cells and astrocytes to be the specific sources of CCL2 gene induction following MOG immunization, while only astrocytes showed elevated CCL2 mRNA in response to just adjuvants. Astrocytes displayed the greatest degree of stimulation of CCL2 gene expression following EAE induction. CONCLUSIONS: High-precision LCM affirmed both microvascular endothelial cells and astrocytes as the major CNS sources of CCL2 gene expression during EAE. Given the high accessibility of the CNS microvascular endothelium, endothelial-derived CCL2 could prove a viable target for therapeutic intervention in neuroinflammatory disease.
RESUMO
The trafficking of cytotoxic CD8(+) T lymphocytes across the lining of the cerebral vasculature is key to the onset of the chronic neuro-inflammatory disorder multiple sclerosis. However, the mechanisms controlling their final transmigration across the brain endothelium remain unknown. Here, we describe that CD8(+) T lymphocyte trafficking into the brain is dependent on the activity of the brain endothelial adenosine triphosphate-binding cassette transporter P-glycoprotein. Silencing P-glycoprotein activity selectively reduced the trafficking of CD8(+) T cells across the brain endothelium in vitro as well as in vivo. In response to formation of the T cell-endothelial synapse, P-glycoprotein was found to regulate secretion of endothelial (C-C motif) ligand 2 (CCL2), a chemokine that mediates CD8(+) T cell migration in vitro. Notably, CCL2 levels were significantly enhanced in microvessels isolated from human multiple sclerosis lesions in comparison with non-neurological controls. Endothelial cell-specific elimination of CCL2 in mice subjected to experimental autoimmune encephalomyelitis also significantly diminished the accumulation of CD8(+) T cells compared to wild-type animals. Collectively, these results highlight a novel (patho)physiological role for P-glycoprotein in CD8(+) T cell trafficking into the central nervous system during neuro-inflammation and illustrate CCL2 secretion as a potential link in this mechanism.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Encéfalo/imunologia , Linfócitos T CD8-Positivos/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Esclerose Múltipla/imunologia , Migração Transendotelial e Transepitelial/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Barreira Hematoencefálica/fisiologia , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Linfócitos T CD4-Positivos/fisiologia , Linhagem Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/patologia , Microvasos/fisiopatologia , Esclerose Múltipla/patologia , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
BACKGROUND: Expression of chemokine CCL2 in the normal central nervous system (CNS) is nearly undetectable, but is significantly upregulated and drives neuroinflammation during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis which is considered a contributing factor in the human disease. As astrocytes and brain microvascular endothelial cells (BMEC) forming the blood-brain barrier (BBB) are sources of CCL2 in EAE and other neuroinflammatory conditions, it is unclear if one or both CCL2 pools are critical to disease and by what mechanism(s). METHODS: Mice with selective CCL2 gene knockout (KO) in astrocytes (Astro KO) or endothelial cells (Endo KO) were used to evaluate the respective contributions of these sources to neuroinflammation, i.e., clinical disease progression, BBB damage, and parenchymal leukocyte invasion in a myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE model. High-resolution 3-dimensional (3D) immunofluorescence confocal microscopy and colloidal gold immuno-electron microscopy were employed to confirm sites of CCL2 expression, and 3D immunofluorescence confocal microscopy utilized to assess inflammatory responses along the CNS microvasculature. RESULTS: Cell-selective loss of CCL2 immunoreactivity was demonstrated in the respective KO mice. Compared to wild-type (WT) mice, Astro KO mice showed reduced EAE severity but similar onset, while Endo KO mice displayed near normal severity but significantly delayed onset. Neither of the KO mice showed deficits in T cell proliferation, or IL-17 and IFN-γ production, following MOG35-55 exposure in vitro, or altered MOG-major histocompatibility complex class II tetramer binding. 3D confocal imaging further revealed distinct actions of the two CCL2 pools in the CNS. Astro KOs lacked the CNS leukocyte penetration and disrupted immunostaining of CLN-5 at the BBB seen during early EAE in WT mice, while Endo KOs uniquely displayed leukocytes stalled in the microvascular lumen. CONCLUSIONS: These results point to astrocyte and endothelial pools of CCL2 each regulating different stages of neuroinflammation in EAE, and carry implications for drug delivery in neuroinflammatory disease.
Assuntos
Astrócitos/patologia , Quimiocina CCL2/metabolismo , Encefalomielite Autoimune Experimental/patologia , Endotélio/patologia , Imageamento Tridimensional , Microscopia Confocal , Animais , Sistema Nervoso Central/patologia , Quimiocina CCL2/deficiência , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Camundongos , Camundongos Knockout , Microvasos/patologia , Glicoproteína Mielina-Oligodendrócito , Fragmentos de PeptídeosRESUMO
Tight junctions (TJs) feature critically in maintaining the integrity of the blood-brain barrier (BBB), and undergo significant disruption during neuroinflammatory diseases. Accordingly, the expression and distribution of CLN-5, a prominent TJ protein in central nervous system (CNS) microvessels and BBB determinant, has been shown to parallel physiological and pathophysiological changes in microvascular function. However, efforts to quantify CLN-5 within the CNS microvasculature in situ, by using conventional two-dimensional immunohistochemical analysis of thin sections, are encumbered by the tortuosity of capillaries and distorted diameters of inflamed venules. Herein, we describe a novel contour-based 3D image visualization and quantification method, employing high-resolution confocal z-stacks from thick immunofluorescently-stained thoraco-lumbar spinal cord cryosections, to analyze CLN-5 along the junctional regions of different-sized CNS microvascular segments. Analysis was performed on spinal cords of both healthy mice, and mice experiencing experimental autoimmune encephalomyelitis (EAE), an animal model of the neuroinflammatory disease multiple sclerosis. Results indicated that, under normal conditions, the density of CLN-5 staining (CLN-5 intensity/ endothelial surface area) was greatest in the capillaries and smaller venules, and least in the larger venules. This heterogeneity in junctional CLN-5 staining was exacerbated during EAE, as spinal venules revealed a significant loss of junctional CLN-5 staining that was associated with focal leukocyte extravasation, while adjacent capillaries exhibited neither CLN-5 loss nor infiltrating leukocytes. However, despite only venules displaying these behaviors, both capillaries and venules evidenced leakage of IgG during disease, further underscoring the heterogeneity of the inflammatory response in CNS microvessels. This method should be readily adaptable to analyzing other junctional proteins of the CNS and peripheral microvasculature, and serve to highlight their role(s) in health and disease.
Assuntos
Barreira Hematoencefálica , Claudina-5/análise , Encefalomielite Autoimune Experimental/patologia , Endotélio Vascular/química , Imageamento Tridimensional/métodos , Microscopia Confocal/métodos , Microvasos/química , Medula Espinal/irrigação sanguínea , Junções Íntimas/química , Animais , Capilares/química , Capilares/ultraestrutura , Permeabilidade Capilar , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/imunologia , Endotélio Vascular/ultraestrutura , Feminino , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Esclerose Múltipla , Junções Íntimas/ultraestrutura , Vênulas/química , Vênulas/ultraestruturaRESUMO
BACKGROUND: There is increasing awareness that, aside from producing cerebrospinal fluid, the choroid plexus (CP) might be a key regulator of immune activity in the central nervous system (CNS) during neuroinflammation. Specifically, the CP has recently been posited to control entry of sentinel T cells into the uninflamed CNS during the early stages of neuroinflammatory diseases, like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). As the CP is compartmentalized into a stromal core containing fenestrated capillaries devoid of typical blood-brain barrier properties, surrounded by a tight junction-expressing choroidal epithelium, each of these compartments might mount unique responses that instigate the neuroinflammatory process. METHODS: To discern responses of the respective CP stromal capillary and choroidal epithelial tissues during evolving neuroinflammation, we investigated morphology and in situ expression of 93 immune-related genes during early stages of EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Specifically, 3-D immunofluorescent imaging was employed to gauge morphological changes, and laser capture microdissection was coupled to an Immune Panel TaqMan Low Density Array to detail alterations in gene expression patterns at these separate CP sites on days 9 and 15 post-immunization (p.i.). To resolve CP effects due to autoimmunity against MOG peptide, from those due to complete Freund's adjuvant (CFA) and pertussis toxin (PTX) included in the immunization, analysis was performed on MOG-CFA/PTX-treated, CFA/PTX-treated, and naïve cohorts. RESULTS: The CP became swollen and displayed significant molecular changes in response to MOG-CFA/PTX immunization. Both stromal capillary and choroidal epithelial tissues mounted vigorous, yet different, changes in expression of numerous genes over the time course analyzed - including those encoding adhesion molecules, cytokines, chemokines, statins, interleukins, T cell activation markers, costimulatory molecules, cyclooxygenase, pro-inflammatory transcription factors and pro-apoptotic markers. Moreover, CFA/PTX-treatment, alone, resulted in extensive, though less robust, alterations in both CP compartments. CONCLUSIONS: MOG-CFA/PTX immunization significantly affects CP morphology and stimulates distinct expression patterns of immune-related genes in CP stromal capillary and epithelial tissues during evolving EAE. CFA/PTX treatment, alone, causes widespread gene alterations that could prime the CP to unlock the CNS to T cell infiltration during neuroinflammatory disease.
RESUMO
BACKGROUND: Production of the chemokine CCL2 by cells of the neurovascular unit (NVU) drives critical aspects of neuroinflammation. Suppression of CCL2 therefore holds promise in treating neuroinflammatory disease. Accordingly, we sought to determine if the compound bindarit, which inhibits CCL2 synthesis, could repress the three NVU sources of CCL2 most commonly reported in neuroinflammation--astrocytes, microglia and brain microvascular endothelial cells (BMEC)--as well as modify the clinical course of neuroinflammatory disease. METHODS: The effect of bindarit on CCL2 expression by cultured murine astrocytes, microglia and BMEC was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Bindarit action on mouse brain and spinal cord in vivo was similarly investigated by qRT-PCR following LPS injection in mice. And to further gauge the potential remedial effects of bindarit on neuroinflammatory disease, its impact on the clinical course of experimental autoimmune encephalomyelitis (EAE) in mice was also explored. RESULTS: Bindarit repressed CCL2 expression by all three cultured cells, and antagonized upregulated expression of CCL2 in both brain and spinal cord in vivo following LPS administration. Bindarit also significantly modified the course and severity of clinical EAE, diminished the incidence and onset of disease, and evidenced signs of disease reversal. CONCLUSION: Bindarit was effective in suppressing CCL2 expression by cultured NVU cells as well as brain and spinal cord tissue in vivo. It further modulated the course of clinical EAE in both preventative and therapeutic ways. Collectively, these results suggest that bindarit might prove an effective treatment for neuroinflammatory disease.