ABSTRACT
Astrocytes, the most numerous cells of the central nervous system, exert critical functions for brain homeostasis. To this purpose, astrocytes generate a highly interconnected intercellular network allowing rapid exchange of ions and metabolites through gap junctions, adjoined channels composed of hexamers of connexin (Cx) proteins, mainly Cx43. Functional alterations of Cxs and gap junctions have been observed in several neuroinflammatory/neurodegenerative diseases. In the rare leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), astrocytes show defective control of ion/fluid exchanges causing brain edema, fluid cysts, and astrocyte/myelin vacuolation. MLC is caused by mutations in MLC1, an astrocyte-specific protein of elusive function, and in GlialCAM, a MLC1 chaperon. Both proteins are highly expressed at perivascular astrocyte end-feet and astrocyte-astrocyte contacts where they interact with zonula occludens-1 (ZO-1) and Cx43 junctional proteins. To investigate the possible role of Cx43 in MLC pathogenesis, we studied Cx43 properties in astrocytoma cells overexpressing wild type (WT) MLC1 or MLC1 carrying pathological mutations. Using biochemical and electrophysiological techniques, we found that WT, but not mutated, MLC1 expression favors intercellular communication by inhibiting extracellular-signal-regulated kinase 1/2 (ERK1/2)-mediated Cx43 phosphorylation and increasing Cx43 gap-junction stability. These data indicate MLC1 regulation of Cx43 in astrocytes and Cx43 involvement in MLC pathogenesis, suggesting potential target pathways for therapeutic interventions.
Subject(s)
Astrocytes/metabolism , Cell Communication , Connexin 43/metabolism , Cysts/metabolism , Cysts/pathology , Gap Junctions/metabolism , Hereditary Central Nervous System Demyelinating Diseases/metabolism , Hereditary Central Nervous System Demyelinating Diseases/pathology , Membrane Proteins/metabolism , Cell Line, Tumor , Cytosol/metabolism , Humans , MAP Kinase Signaling System , Membrane Proteins/genetics , Models, Biological , Mutation/genetics , Phosphorylation , Protein Stability , Protein TransportABSTRACT
PURPOSE: Human papillomaviruses (HPVs) are the causative agents of cervical cancer and are also associated with other types of cancers. HPVs can modulate microRNAs (miRNAs) expressed by infected cells. The production of extracellular vesicles is deregulated in cancer, and their cargo delivered to the microenvironment can promote tumorigenesis. The involvement of HPV oncoproteins on miRNA expression in cells and exosomes was analyzed in keratinocytes transduced with E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 (K16 and K38). METHODS: MiRNAs were investigated through the TaqMan Array Human MicroRNA Cards, followed by real-time RT-PCR assay for specific miRNAs. Selected miRNA targets were analyzed by Western blot and correlated to the HPV oncoproteins by specifically silencing E6 and E7 expression. Exosomes, isolated from K16 and K38 supernatants by differential centrifugations, were quantified through the vesicle-associated acetylcholinesterase activity. RESULTS: MiRNAs deregulated in K16 and K38 cells were identified. HPV-16 and/or HPV-38 E6 and E7 single proteins can modify the expression of selected miRNAs involved in the tumorigenesis, in particular miR-18a, -19a, -34a and -590-5p. The analysis of the content of exosomes isolated from HPV-positive cells revealed the presence of E6 and E7 mRNAs and few miRNAs. MiR-222, a key miRNA deregulated in many cancers, was identified in exosomes from K16 cells. CONCLUSIONS: HPV E6 and/or E7 oncoprotein expression can induce the deregulation of some miRNAs. Through the production and function of exosomes, HPV oncogenes as well as HPV-deregulated miRNAs can potentiate the virus oncogenic effects in the tumor cell microenvironment.
Subject(s)
Carcinogenesis , MicroRNAs/genetics , Neoplasms/genetics , Oncogene Proteins, Viral/metabolism , Humans , Neoplasms/virologyABSTRACT
We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.
Subject(s)
Drug Carriers/administration & dosage , Exosomes/metabolism , Papillomavirus E7 Proteins/immunology , Papillomavirus Vaccines/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , Mice, Inbred C57BL , Papillomavirus E7 Proteins/administration & dosage , Papillomavirus Vaccines/administration & dosage , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
UNLABELLED: Resting CD4+ T lymphocytes resist human immunodeficiency virus (HIV) infection. Here, we provide evidence that exosomes from HIV-1-infected cells render resting human primary CD4+ T lymphocytes permissive to HIV-1 replication. These results were obtained with transwell cocultures of HIV-1-infected cells with quiescent CD4+ T lymphocytes in the presence of inhibitors of exosome release and were confirmed using exosomes purified from supernatants of HIV-1-infected primary CD4+ T lymphocytes. We found that the expression of HIV-1 Nef in exosome-producing cells is both necessary and sufficient for cell activation as well as HIV-1 replication in target CD4+ T lymphocytes. We also identified a Nef domain important for the effects we observed, i.e., the 62EEEE65 acidic cluster domain. In addition, we observed that ADAM17, i.e., a disintegrin and metalloprotease converting pro-tumor necrosis factor alpha (TNF-α) in its mature form, associates with exosomes from HIV-1-infected cells, and plays a key role in the HIV-1 replication in quiescent CD4+ T lymphocytes. Treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in resting CD4+ T lymphocytes. TNF-α is the downstream effector of ADAM17 since the treatment of resting lymphocytes with anti-TNF-α antibodies blocked the HIV-1 replication. The data presented here are consistent with a model where Nef induces intercellular communication through exosomes to activate bystander quiescent CD4+ T lymphocytes, thus stimulating viral spread. IMPORTANCE: Overall, our findings support the idea that HIV evolved to usurp the exosome-based intercellular communication network to favor its spread in infected hosts.
Subject(s)
ADAM Proteins/genetics , CD4-Positive T-Lymphocytes/virology , Exosomes/immunology , HIV-1/genetics , nef Gene Products, Human Immunodeficiency Virus/genetics , ADAM Proteins/antagonists & inhibitors , ADAM Proteins/immunology , ADAM17 Protein , Antibodies/pharmacology , CD4-Positive T-Lymphocytes/immunology , Cell Communication , Cells, Cultured , Diffusion Chambers, Culture , Enzyme Inhibitors/pharmacology , Exosomes/chemistry , Gene Expression Regulation , HEK293 Cells , HIV-1/immunology , Humans , Lymphocyte Activation , Protein Structure, Tertiary , Signal Transduction , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Virus Replication , nef Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
BACKGROUND: A relevant burden of defective HIV-1 genomes populates PBMCs from HIV-1 infected patients, especially during HAART treatment. These viral genomes, although unable to codify for infectious viral particles, can express viral proteins which may affect functions of host cells as well as bystander ones. Cells expressing defective HIV-1 have a lifespan longer than that of cells producing infectious particles. Hence, their interaction with other cell types, including resting lymphocytes, is expected to occur frequently in tissues where HIV actively replicates. We investigated the effects of the expression of a prototype of functionally defective HIV-1 on bystander, unstimulated CD4+ T lymphocytes. RESULTS: We observed that unstimulated human primary CD4+ T lymphocytes were activated and became permissive for HIV-1 replication when co-cultivated with cells expressing a functionally defective HIV-1 (F12/Hut-78 cells). This effect depended on the presence in F12/Hut-78 supernatants of nanovesicles we identified as exosomes. By inspecting the underlying mechanism, we found that ADAM17, i.e., a disintegrin and metalloprotease converting pro-TNF-α in its mature form, associated with exosomes from F12/Hut-78 cells, and played a key role in the HIV-1 replication in unstimulated CD4+ T lymphocytes. In fact, the treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in unstimulated CD4+ T lymphocytes. TNF-α appeared to be the downstream effector of ADAM17 since the treatment of unstimulated lymphocytes with antibodies against TNF-α or its receptors blocked the HIV-1 replication. Finally, we found that the expression of NefF12 in exosome-producing cells was sufficient to induce the susceptibility to HIV-1 infection in unstimulated CD4+ T lymphocytes. CONCLUSIONS: Exosomes from cells expressing a functionally defective mutant can induce cell activation and HIV-1 susceptibility in unstimulated CD4+ T lymphocytes. This evidence highlights the relevance for AIDS pathogenesis of the expression of viral products from defective HIV-1 genomes.
Subject(s)
CD4-Positive T-Lymphocytes/virology , Exosomes , HIV-1/physiology , Lymphocyte Activation , Virus Replication , ADAM Proteins/physiology , ADAM17 Protein , CD4-Positive T-Lymphocytes/immunology , Cells, Cultured , Humans , Interleukin-2/biosynthesis , Receptors, Tumor Necrosis Factor, Type I/antagonists & inhibitors , Receptors, Tumor Necrosis Factor, Type II/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
HIV budding requires the interaction with cell factors involved in the biogenesis of exosomes. This implies the possibility that viral products undergo exosome incorporation. While this has been already described for both Gag and Nef HIV-1 proteins, no conclusive results on HIV genome have been produced so far. Here, we report that unspliced, but not single or double spliced, HIV-1 RNA species are incorporated in exosomes. Deletion mutant analysis indicated that the presence of a stretch of sequences within the 5' end of the Gag p17 open reading frame is sufficient for HIV-1 RNA exosome incorporation. These sequences were found associating with exosomes also out of the HIV-1 context, thus indicating that the diversion towards the vesicular compartment can occur without need of additional HIV-1 sequences. Finally, the incorporation of genomic HIV-1 RNA in exosomes significantly increased when producer cells express HIV-1 defective for viral genome packaging. Manipulating infected cells to favour the selective incorporation in exosomes of genomic HIV-1 RNA might have therapeutic implications.
Subject(s)
Exosomes/metabolism , HIV-1/physiology , RNA, Viral/metabolism , Virus Assembly , Virus Release , Biological Transport , Cell Line , DNA Mutational Analysis , Humans , Sequence DeletionABSTRACT
BACKGROUND: Possible interactions between nervous and immune systems in neuro-psychiatric disorders remain elusive. Levels of brain dopamine transporter (DAT) have been implicated in several impulse-control disorders, like attention deficit / hyperactivity disorder (ADHD) and obsessive-compulsive disorder (OCD). Here, we assessed the interplay between DAT auto-immunity and behavioural / neurochemical phenotype. METHODS: Male CD-1 mice were immunized with DAT peptide fragments (DAT-i), or vehicle alone (VEH), to generate elevated circulating levels of DAT auto-antibodies (aAbs). Using an operant delay-of-reward task (20 min daily sessions; timeout 25 sec), mice had a choice between either an immediate small amount of food (SS), or a larger amount of food after a delay (LL), which increased progressively across sessions (from 0 to 150 sec). RESULTS: DAT-i mice exhibited spontaneous hyperactivity (2 h-longer wake-up peak; a wake-up attempt during rest). Two sub-populations differing in behavioural flexibility were identified in the VEH control group: they showed either a clear-cut decision to select LL or clear-cut shifting towards SS, as expected. Compared to VEH controls, choice-behaviour profile of DAT-i mice was markedly disturbed, together with long-lasting alterations of the striatal monoamines. Enhanced levels of DA metabolite HVA in DAT-i mice came along with slower acquisition of basal preferences and with impaired shifting; elevation also in DOPAC levels was associated with incapacity to change a rigid selection strategy. This scarce flexibility of performance is indicative of a poor adaptation to task contingencies. CONCLUSIONS: Hyperactivity and reduced cognitive flexibility are patterns of behaviour consistent with enduring functional impairment of striatal regions. It is yet unclear how anti-DAT antibodies could enter or otherwise affect these brain areas, and which alterations in DAT activity exactly occurred after immunization. Present neuro-behavioural alterations, coming along with an experimentally-induced rise of circulating DAT-directed aAbs, open the issue of a potential role for auto-immunity in vulnerability to impulse-control disorders.
Subject(s)
Behavior, Animal/physiology , Cognition/physiology , Corpus Striatum/physiopathology , Dopamine Plasma Membrane Transport Proteins/metabolism , Hyperkinesis/physiopathology , Peptide Fragments/pharmacology , Animals , Cognition/drug effects , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Exploratory Behavior/physiology , Hyperkinesis/metabolism , Immunization , Male , Mice , Peptide Fragments/metabolism , RewardABSTRACT
Multiple sclerosis (MS) is a demyelinating disease that causes major neurological disability in young adults. A definitive diagnosis at the time of the first episode is still lacking, but since early treatment leads to better prognosis, the search for early biomarkers is needed. Here we characterized the transcriptome of the choroid plexus (CP), which is part of the blood-brain barriers (BBBs) and the major site of cerebrospinal fluid production, in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. In addition, cerebrospinal fluid samples from two cohorts of patients with MS and with optic neuritis (ON) were analyzed to confirm the clinical relevance of the findings. Genes encoding for adhesion molecules, chemokines and cytokines displayed the most altered expression, supporting the role of CP as a site of immune-brain interaction in MS. The gene encoding for lipocalin 2 was the most up-regulated; notably, the cerebrospinal fluid lipocalin 2 levels coincided with the active phases of the disease. Immunostaining revealed that neutrophils infiltrating the CP were the source of the increased lipocalin 2 expression in this structure. However, within the brain, lipocalin 2 was also detected in astrocytes, particularly in regions typically affected in patients with MS. The increase of lipocalin 2 in the cerebrospinal fluid and in astrocytes was reverted by natalizumab treatment. Most importantly, the results obtained in the murine model were translatable into humans since patients from two different cohorts presented increased cerebrospinal fluid lipocalin 2 levels. The findings support lipocalin 2 as a valuable molecule for the diagnostic/monitoring panel of MS.
ABSTRACT
Fine regulation of the innate immune response following brain injury or infection is important to avoid excessive activation of microglia and its detrimental consequences on neural cell viability and function. To get insights on the molecular networks regulating microglia activation, we analyzed expression, regulation and functional relevance of tumor necrosis factor receptors (TNFR) 2 in cultured mouse microglia. We found that microglia upregulate TNFR2 mRNA and protein and shed large amounts of soluble TNFR2, but not TNFR1, in response to pro-inflammatory stimuli and through activation of TNFR2 itself. By microarray analysis, we demonstrate that TNFR2 stimulation in microglia regulates expression of genes involved in immune processes, including molecules with anti-inflammatory and neuroprotective function like granulocyte colony-stimulating factor, adrenomedullin and IL-10. In addition, we identify IFN-γ as a regulator of the balance between pro- and anti-inflammatory/neuroprotective factors induced by TNFR2 stimulation. These data indicate that, through TNFR2, microglia may contribute to the counter-regulatory response activated in neuropathological conditions.
Subject(s)
Inflammation/immunology , Microglia/metabolism , Receptors, Tumor Necrosis Factor, Type II/metabolism , Signal Transduction/physiology , Animals , Cells, Cultured , Gene Expression Regulation , Granulocyte Colony-Stimulating Factor/metabolism , Interferon-gamma/immunology , Interleukin-10/metabolism , Mice , Microarray Analysis , Microglia/cytology , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Receptors, Tumor Necrosis Factor, Type II/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
A cardinal feature of multiple sclerosis (MS) is the persistent intrathecal synthesis of antibodies. Our previous finding that a large fraction of B cells infiltrating the MS brain are infected with Epstein-Barr virus (EBV) raises the possibility that this virus, because of its ability to establish a latent infection in B cells and interfere with their differentiation, contributes to B-cell dysregulation in MS. The aim of this study was to gain further insight into EBV latency programs and their relationship to B-cell activation in the MS brain. Immunohistochemical analysis of postmortem MS brain samples harboring large EBV deposits revealed that most B cells in white matter lesions, meninges, and ectopic B-cell follicles are CD27+ antigen-experienced cells and coexpress latent membrane protein 1 and latent membrane protein 2A, 2 EBV-encoded proteins that provide survival and maturation signals to B cells. By combining laser-capture microdissection with preamplification reverse transcription-polymerase chain reaction techniques, EBV latency transcripts (latent membrane protein 2A, EBV nuclear antigen 1) were detected in all MS brain samples analyzed. We also found that B cell-activating factor of the tumor necrosis factor family is expressed in EBV-infected B cells in acute MS lesions and ectopic B-cell follicles. These findings support a role for EBV infection in B-cell activation in the MS brain and suggest that B cell-activating factor of the tumor necrosis factor family produced by EBV-infected B cells may contribute to this process resulting in viral persistence and, possibly, disruption of B-cell tolerance.
Subject(s)
B-Cell Activating Factor/metabolism , B-Lymphocytes/metabolism , Brain/cytology , Herpesvirus 4, Human/pathogenicity , Multiple Sclerosis/pathology , Multiple Sclerosis/virology , Antigens, CD/metabolism , Cell Line, Transformed , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/pathology , Epstein-Barr Virus Nuclear Antigens/metabolism , Humans , Microdissection/methods , Microscopy, Confocal , Multiple Sclerosis/complications , Postmortem Changes , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolismABSTRACT
Brief maternal separations of neonatal animals can exert long-lasting effects on the reactivity of the neuroendocrine system. The aim of the present study was to investigate whether manipulations of the mother-infant interaction could affect susceptibility to immune-mediated diseases, such experimental autoimmune encephalomyelitis (EAE), and whether this effect would be mediated by changes in leptin which has been shown to regulate disease susceptibility and severity at adulthood. Given the different gender susceptibility to EAE previously described, we tested also whether early experiences could differentially affect the two genders. To this purpose, female and male C56BL/6 mice were subjected to handling (15 min daily) postnatally, from day 2 until day 14. All subjects were weaned at 21 days. At 7 weeks of age mice were immunized with MOG(35-55) to actively induce EAE. We thus determined the effect of neonatal handling on plasma concentrations of testosterone in male mice and leptin in both genders at different times post EAE induction. Our results show that early experiences influence susceptibility to EAE in a gender-specific manner, early manipulations resulting in an enhancement of sex-related differences in susceptibility. These effects were associated with changes in the testosterone profile of male subjects. Changes in leptin levels during the preclinical stage of EAE may predict a more severe disease course.
Subject(s)
Disease Susceptibility , Encephalomyelitis, Autoimmune, Experimental/etiology , Handling, Psychological , Animals , Female , Leptin/blood , Leptin/physiology , Male , Mice , Mice, Inbred C57BL , Sex Characteristics , Testosterone/bloodABSTRACT
We evaluated oligoclonal IgG band (OCB) patterns obtained by analyzing paired cerebrospinal fluid (CSF) and serum samples of 77 patients with acute demyelinating encephalomyelitis (ADEM) and 411 patients with multiple sclerosis (MS). OCBs were searched with isoelectric focusing and capillary immunoblotting. CSF-restricted OCBs were found in 89% of MS patients and 10% of ADEM patients (p<0.0001). Identical serum and CSF OCBs ('mirror pattern'), or no OCBs, were detected in 10% of MS patients and 84% of ADEM patients (p<0.0001). OCBs were also analyzed in 27 mice with proteolipid protein-induced experimental autoimmune encephalomyelitis (EAE). In this animal model, the 'mirror pattern' was the most frequently detected pattern (74%), with the immunizing antigen being the main OCB target. These results indicate that CSF analysis can help differentiate between MS and ADEM and that, similarly to EAE, the 'mirror pattern' observed in ADEM accounts for a predominantly systemic immune activation.
Subject(s)
Encephalomyelitis, Acute Disseminated/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Multiple Sclerosis/immunology , Oligoclonal Bands/cerebrospinal fluid , Animals , Chi-Square Distribution , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Female , Follow-Up Studies , Humans , Male , Mice , Middle Aged , Myelin Proteolipid ProteinABSTRACT
Lymphoid chemokines play an essential role in the establishment and maintenance of lymphoid tissue microarchitecture and have been implicated in the formation of tertiary (or ectopic) lymphoid tissue in chronic inflammatory conditions. Here, we review recent advances in lymphoid chemokine research in central nervous system inflammation, focusing on multiple sclerosis and the animal model experimental autoimmune encephalomyelitis. We also highlight how the study of lymphoid chemokines, particularly CXCL13, has led to the identification of intrameningeal B-cell follicles in the multiple sclerosis brain paving the way to the discovery that these abnormal structures are highly enriched in Epstein-Barr virus-infected B cells and plasma cells.
Subject(s)
Central Nervous System/immunology , Chemokines/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Lymphocytes/immunology , Animals , Chemokine CXCL13/metabolism , Disease Models, Animal , HumansABSTRACT
We have recently shown that de novo formation of lymphoid structures resembling B-cell follicles occurs in the inflamed central nervous system (CNS) meninges in a subset of patients with secondary progressive multiple sclerosis and in SJL mice with relapsing-remitting experimental autoimmune encephalomyelitis (EAE). Because lymphotoxin (LT) alpha(1)beta(2) is essential for lymphoid tissue organization, we used real-time PCR to examine LTbeta and LTbeta receptor (LTbetaR) gene expression in the CNS of SJL mice immunized with PLP 139-151 peptide. Moreover, we used the decoy receptor LTbetaR-immunoglobulin fusion protein to block the interaction of lymphotoxin (LT) alpha(1)beta(2) with the LTbeta receptor (LTbetaR) in mice with established EAE and evaluate the effect of systemic and local treatments with the fusion protein on disease progression, CNS lymphocytic infiltration and formation of meningeal B-cell follicles. The present findings indicate that both LTbeta and LTbetaR are upregulated at EAE onset and during subsequent relapses and that systemic and local blockade of the LT pathway with LTbetaR-Ig results in protracted and transient inhibition of EAE clinical signs, respectively. LTbetaR-Ig treatment also reduces T- and B-cell infiltration and prevents the induction of the chemokines CXCL10 and CXCL13 and the formation of organized ectopic follicles in the EAE-affected CNS. Targeting of molecules involved in lymphoid organogenesis could represent a valid strategy to inhibit CNS inflammation and formation of ectopic follicles, which may play a role in maintaining an abnormal, intrathecal humoral immune response in CNS autoimmune disease.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/prevention & control , Immunoglobulin G/administration & dosage , Inflammation/immunology , Lymphotoxin beta Receptor/metabolism , Meninges/immunology , Recombinant Fusion Proteins/administration & dosage , Animals , B-Lymphocytes/immunology , Central Nervous System/immunology , Central Nervous System/metabolism , Central Nervous System/pathology , Chemokine CXCL10 , Chemokine CXCL13 , Chemokines, CXC/biosynthesis , Chemokines, CXC/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Gene Expression , Gene Expression Profiling , Humans , Immunoglobulin G/immunology , Immunohistochemistry , Inflammation/pathology , Injections, Intraventricular , Lymphotoxin beta Receptor/genetics , Lymphotoxin-beta/antagonists & inhibitors , Lymphotoxin-beta/genetics , Mice , Microscopy, Confocal , Myelin Proteolipid Protein/immunology , Peptide Fragments/immunology , RNA, Messenger/analysis , Recombinant Fusion Proteins/immunology , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/immunologyABSTRACT
In the present study, we use modified CDR3 beta-chain spectratyping (immunoscope) to dissect the effect of Mycobacterium tuberculosis (MT)-derived proteins on individual PLP139-151-specific cells in the SJL mouse strain. In this model, the immunoscope technique allows the characterization of a public TCR that involves rearrangement of Vbeta10 and Jbeta1.1 and a semi-private TCR characterized by rearrangement of Vbeta4 and Jbeta1.6. Both rearrangements are specific for PLP139-151 and sequences of the CDR3 region of the two beta-chains show a conserved motif for the public rearrangement and related but more variable sequences for the semi-private rearrangement. MT-derived proteins promote increase of IFN-gamma-secreting cells. However, we observe that the presence and amount of MT used during immunization have no effect on the frequency of usage, polarization and in vivo expansion of cells carrying the studied rearrangements. Rather, the strong Th1-promoting effect of adjuvant is possibly due to recruitment toward Th1 of a wider spectrum of TCR repertoires. Therefore, instead of having a comprehensive effect on the entire repertoire, MT modulates the immune response by affecting a subset of antigen-specific T cells whose polarization can be adapted to the environment. This step establishes the final balance between Th1 and Th2 and may be essential for the enhancement or protection of disease.
Subject(s)
Adjuvants, Immunologic/chemistry , Autoantigens/immunology , Central Nervous System/immunology , Mycobacterium tuberculosis/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Animals , Bacterial Proteins/immunology , CD4-Positive T-Lymphocytes/metabolism , Central Nervous System/metabolism , Female , Gene Rearrangement, T-Lymphocyte , Immunization , Interferon-gamma/metabolism , Mice , Mice, Inbred Strains , Myelin Proteolipid Protein/immunology , Peptide Fragments/immunology , Th1 Cells/metabolism , Th2 Cells/metabolismABSTRACT
Given the abnormalities in B-cell activity occurring in the central nervous system (CNS) of patients with multiple sclerosis (MS), we have explored the possibility that CNS inflammation induced in mouse models of experimental autoimmune encephalomyelitis (EAE) triggers expression of molecules that control the development and functional organization of lymphoid follicles, the sites where B-cell responses are initiated. By reverse transcription-polymerase chain reaction (RT-PCR), we find that gene expression of CXCL13, a chemokine involved in B-cell recruitment into lymphoid follicles, and BAFF, a key regulator of B-cell survival, is markedly and persistently upregulated in the CNS of mice with relapsing-remitting and chronic-relapsing EAE. Using immunohistochemical techniques, we also show the presence of lymphoid follicle-like structures containing B cells and a reticulum of CXCL13+ and FDC-M1+ follicular dendritic cells within the meninges of several mice undergoing progressive relapsing EAE. These observations indicate that, under chronic inflammatory conditions, the less immunoprivileged meningeal compartment is the site where ectopic lymphoid follicles preferentially develop and where pathogenic B-cell responses could be sustained in autoimmune disorders of the CNS.
Subject(s)
Central Nervous System/metabolism , Chemokines, CXC/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Lymphocytes/metabolism , Membrane Proteins/metabolism , Meninges/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , B-Cell Activating Factor , CD4 Antigens/metabolism , Chemokine CXCL13 , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/etiology , Female , Glycoproteins/toxicity , Immunization/methods , Immunohistochemistry , Ki-67 Antigen/metabolism , Leukocyte Common Antigens/metabolism , Membrane Proteins/genetics , Meninges/drug effects , Mice , Mice, Inbred Strains , Myelin Proteolipid Protein/toxicity , Myelin-Oligodendrocyte Glycoprotein , Peptide Fragments/toxicity , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction/methods , Species Specificity , Spinal Cord/metabolism , Time Factors , Tumor Necrosis Factor-alpha/geneticsABSTRACT
The simultaneous presence of dendritic, T- and B-cells in the central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, suggests that interactions among these cell types might be instrumental in the local induction and maintenance of autoimmune reactions. In this study, we explored the possibility that such aberrant leukocyte recruitment in the CNS could be sustained by "lymphoid" chemokines which orchestrate dendritic cell and lymphocyte homing to lymphoid organs. Transcripts for CCL19 and CCL21 and their common receptor CCR7 were induced in the CNS of mice undergoing relapsing-remitting and chronic-relapsing EAE. While CCL21 immunoreactivity was confined to the endothelium of some inflamed blood vessels, CCL19 was expressed by many infiltrating leukocytes and some astrocytes and microglia in the CNS parenchyma. CCR7+ cells accumulated in inflammatory lesions during EAE progression, when abundant infiltration of the CNS by mature dendritic cells, B-cells and cells expressing naive T-cell markers also occurred. These findings suggest that CCL19 and CCL21 produced in the EAE-affected CNS may be critical for the homing of antigen presenting cells and lymphocytes, resulting in continuous local antigenic stimulation and maintenance of chronic neuroinflammation.
Subject(s)
Chemokines, CC/biosynthesis , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Animals , Astrocytes/immunology , B-Lymphocytes/immunology , Brain/immunology , Brain/pathology , Chemokine CCL19 , Chemokine CCL21 , Dendritic Cells/immunology , Female , Immunohistochemistry , Microglia/immunology , RNA, Messenger/analysis , Receptors, CCR7 , Receptors, Chemokine/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Spinal Cord/immunology , Spinal Cord/pathology , T-Lymphocytes/immunology , Time FactorsABSTRACT
Macrophage inflammatory protein-3alpha/CCL20 is a recently identified chemokine that binds to CCR6 and acts as a chemoattractant for memory/differentiated T-cells, B-cells, and immature dendritic cells. We have previously reported that CCL20 and CCR6 mRNAs are expressed in the CNS of SJL mice with experimental autoimmune encephalomyelitis (EAE) and that CCL20 is produced by CNS-infiltrating leukocytes at disease onset and, additionally, by intraparenchymal astrocyte-like cells during disease relapses. In this study, we provide further immunohistochemical evidence that astrocytes represent the main CNS source of CCL20 during EAE. Moreover, we show that the proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha, but not interferon-gamma, induce expression of CCL20 mRNA and secretion of CCL20 protein in cultures of mouse brain-derived astrocytes. We also show that supernatants from cytokine-activated astrocytes stimulate the migration of polarized T helper cells and that this effect is partially inhibited by anti-CCL20 antibody. These findings suggest that, through secretion of CCL20, astrocytes could play an important role in orchestrating the recruitment of specific leukocyte subsets to the inflamed CNS and in regulating CNS-targeted immune responses.
Subject(s)
Astrocytes/immunology , Chemokines, CC/genetics , Chemokines, CC/metabolism , Encephalomyelitis, Autoimmune, Experimental/immunology , Macrophage Inflammatory Proteins/genetics , Macrophage Inflammatory Proteins/metabolism , Receptors, Chemokine , Animals , Astrocytes/cytology , Astrocytes/metabolism , Cell Movement/immunology , Cells, Cultured , Chemokine CCL20 , Female , Gene Expression/immunology , Immunohistochemistry , In Vitro Techniques , Mice , Mice, Inbred Strains , RNA, Messenger/analysis , Receptors, CCR6 , Th1 Cells/cytology , Th1 Cells/immunology , Th2 Cells/cytology , Th2 Cells/immunologyABSTRACT
Macrophage-derived chemokine (MDC/CCL22) and its receptor CCR4 have been implicated in chronic inflammatory processes and in the homing of monocytes, Th2 cells and regulatory T-cell subsets. Here, we demonstrate that MDC and CCR4 mRNAs are expressed in the central nervous system (CNS) of mice developing relapsing-remitting and chronic-relapsing forms of experimental autoimmune encephalomyelitis (EAE). By immunohistochemistry, we show that MDC is produced by CNS-infiltrating leukocytes and intraparenchymal microglia, whereas CCR4 is expressed on some invading leukocytes. Upon in vitro activation, mouse microglia express MDC transcripts and secrete bioactive MDC that induces chemotaxis of Th2, but not Th1 cells. We suggest that MDC produced by microglia could regulate Th1-mediated CNS inflammation by facilitating the homing of Th2 and, possibly, regulatory T cells into the lesion site.