RESUMO
BACKGROUND & AIMS: The enterohepatic circuit of T cells may be responsible for the development of autoimmune liver disease. We employed transgenic mice to characterize phenotype and migration patterns of CD8 T cells activated in liver and gut. METHODS: We studied the migration of antigen-specific CD8 T cells primed in liver or gut after transfer in wild-type mice or mice that express ovalbumin in liver or gut. We performed transcriptome analysis of these two distinct T cell populations and confirmed our findings by flow cytometry. RESULTS: Specific migration patterns were induced by activation of CD8 T cells in gut or liver. Gut-activated CD8 T cells expressed α4ß7 and CCR9 and migrated to the gut and to the liver. Liver-activated T cells expressed integrins α4, α6, ß1, α4ß7 as well as CD62L, Ly6C, and neuropilin-1 and retained the capability to re-circulate through lymph nodes. Presence of the antigen increased retention of both types of activated T cells in the liver, but migration of liver-activated T cells to the gut was prohibited. CONCLUSIONS: CD8 T cells primed in the liver in vivo are not capable of migrating to the gut, implying that the enterohepatic circuit of CD8 T cells is in fact a one-way road from the gut to the liver. Priming of CD8 T cells in the liver results in a distinct phenotype with attributes of central memory cells and induces a unique homing pattern. Gut-primed T cells preferentially home to the liver, in principle enabling them to induce autoimmune liver disease.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Movimento Celular/imunologia , Colangite Esclerosante/imunologia , Circulação Êntero-Hepática/imunologia , Hepatite Autoimune/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Colangite Esclerosante/genética , Modelos Animais de Doenças , Hepatite Autoimune/genética , Intestinos/imunologia , Fígado/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/genética , Ovalbumina/imunologia , Fenótipo , Transcriptoma/imunologiaRESUMO
BACKGROUND: Toll-like receptors (TLRs) enable innate immune cells to respond to pathogen- and host-derived molecules. The central nervous system (CNS) exhibits most of the TLRs identified with predominant expression in microglia, the major immune cells of the brain. Although individual TLRs have been shown to contribute to CNS disorders, the consequences of multiple activated TLRs on the brain are unclear. We therefore systematically investigated and compared the impact of sole and pairwise TLR activation on CNS inflammation and injury. METHODS: Selected TLRs expressed in microglia and neurons were stimulated with their specific TLR ligands in varying combinations. Cell cultures were then analyzed by immunocytochemistry, FlowCytomix, and ELISA. To determine neuronal injury and neuroinflammation in vivo, C57BL/6J mice were injected intrathecally with TLR agonists. Subsequently, brain sections were analyzed by quantitative real-time PCR and immunohistochemistry. RESULTS: Simultaneous stimulation of TLR4 plus TLR2, TLR4 plus TLR9, and TLR2 plus TLR9 in microglia by their respective specific ligands results in an increased inflammatory response compared to activation of the respective single TLR in vitro. In contrast, additional activation of TLR7 suppresses the inflammatory response mediated by the respective ligands for TLR2, TLR4, or TLR9 up to 24 h, indicating that specific combinations of activated TLRs individually modulate the inflammatory response. Accordingly, the composition of the inflammatory response pattern generated by microglia varies depending on the identity and combination of the activated TLRs engaged. Likewise, neuronal injury occurs in response to activation of only selected TLRs and TLR combinations in vitro. Activation of TLR2, TLR4, TLR7, and TLR9 in the brain by intrathecal injection of the respective TLR ligand into C57BL/6J mice leads to specific expression patterns of distinct TLR mRNAs in the brain and causes influx of leukocytes and inflammatory mediators into the cerebrospinal fluid to a variable extent. Also, the intensity of the inflammatory response and neurodegenerative effects differs according to the respective activated TLR and TLR combinations used in vivo. CONCLUSIONS: Sole and pairwise activation of TLRs modifies the pattern and extent of inflammation and neurodegeneration in the CNS, thereby enabling innate immunity to take account of the CNS diseases' diversity.
Assuntos
Citocinas/metabolismo , Encefalite/metabolismo , Doenças Neurodegenerativas/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Aminoquinolinas/toxicidade , Animais , Células Cultivadas , Chaperonina 60/farmacologia , Encefalite/líquido cefalorraquidiano , Encefalite/induzido quimicamente , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Imiquimode , Lipopeptídeos/toxicidade , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Doenças Neurodegenerativas/líquido cefalorraquidiano , Doenças Neurodegenerativas/induzido quimicamente , Neurônios/efeitos dos fármacos , Óxido Nítrico/metabolismo , Oligodesoxirribonucleotídeos/toxicidade , Fosfopiruvato Hidratase/metabolismo , Receptor 4 Toll-Like/deficiênciaRESUMO
Innate immune receptors represent an evolutionarily ancient system that allows organisms to detect and rapidly respond to pathogen- and host-derived factors. TLRs are predominantly expressed in immune cells and mediate such a response. Although this class of pattern recognition receptors is involved in CNS disorders, the knowledge of ligands leading to activation of TLRs and to subsequent CNS damage is limited. We report in this study that ssRNA causes neurodegeneration and neuroinflammation dependent on TLR7 in the CNS. TLR7 is not only expressed in microglia, the major immune cells of the brain, but also in neurons of the CNS. Extracellularly delivered ssRNA40, an oligoribonucleotide derived from HIV and an established ligand of TLR7, induces neuronal cell death dependent on TLR7 and the central adapter molecule MyD88 in vitro. Activation of caspase-3 is involved in neuronal damage mediated by TLR7. This cell-autonomous neuronal cell death induced by ssRNA40 is amplified in the presence of microglia that mount an inflammatory response to ssRNA40 through TLR7. Intrathecal administration of ssRNA40 causes widespread neurodegeneration in wild-type but not in TLR7(-/-) mice, confirming that neuronal cell death induced by ssRNA40 through TLR7 occurs in vivo. Our results point to a possible mechanism through which extracellularly delivered ssRNA contributes to CNS damage and determine an obligatory role for TLR7 in this pathway.
Assuntos
Líquido Extracelular/imunologia , Líquido Extracelular/virologia , Glicoproteínas de Membrana/fisiologia , Doenças Neurodegenerativas/imunologia , Doenças Neurodegenerativas/virologia , RNA Viral/administração & dosagem , Receptor 7 Toll-Like/fisiologia , Animais , Caspase 3/efeitos adversos , Caspase 3/fisiologia , Morte Celular/genética , Morte Celular/imunologia , Linhagem Celular Tumoral , Células HEK293 , HIV/genética , HIV/imunologia , Humanos , Injeções Espinhais , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/efeitos adversos , Fator 88 de Diferenciação Mieloide/fisiologia , Doenças Neurodegenerativas/patologia , Cultura Primária de Células , RNA Viral/efeitos adversos , RNA Viral/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/deficiência , Receptor 7 Toll-Like/genéticaRESUMO
The chaperone protein Unc-93 homolog B1 (UNC93B1) regulates internalization, trafficking, and stabilization of nucleic acid-sensing Toll-like receptors (TLR) in peripheral immune cells. We sought to determine UNC93B1 expression and its functional relevance in inflammatory and injurious processes in the central nervous system (CNS). We found that UNC93B1 is expressed in various CNS cells including microglia, astrocytes, oligodendrocytes, and neurons, as assessed by PCR, immunocyto-/histochemistry, and flow cytometry. UNC93B1 expression in the murine brain increased during development. Exposure to the microRNA let-7b, a recently discovered endogenous TLR7 activator, but also to TLR3 and TLR4 agonists, led to increased UNC93B1 expression in microglia and neurons. Microglial activation by extracellular let-7b required functional UNC93B1, as assessed by TNF ELISA. Neuronal injury induced by extracellular let-7b was dependent on UNC93B1, as UNC93B1-deficient neurons were unaffected by the microRNA's neurotoxicity in vitro. Intrathecal application of let-7b triggered neurodegeneration in wild-type mice, whereas mice deficient for UNC93B1 were protected against injurious effects on neurons and axons. In summary, our data demonstrate broad UNC93B1 expression in the murine brain and establish this chaperone as a modulator of neuroinflammation and neuronal injury triggered by extracellular microRNA and subsequent induction of TLR signaling.
Assuntos
Sistema Nervoso Central/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , MicroRNAs/genética , Doenças Neuroinflamatórias/etiologia , Doenças Neuroinflamatórias/metabolismo , Neurônios/metabolismo , Animais , Biomarcadores , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Imunofluorescência , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/metabolismo , Degeneração Neural/genética , Degeneração Neural/metabolismo , Doenças Neuroinflamatórias/patologia , Neurônios/efeitos dos fármacos , Organogênese/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
We have previously demonstrated that ROR1 and FMOD (fibromodulin) are two genes upregulated in chronic lymphocytic leukaemia (CLL) cells compared to normal blood B cells. In this study, siRNAs were used to specifically silence ROR1 and FMOD expression in CLL cells, healthy B cells and human fibroblast cell lines. siRNA treatment induced a specific reduction (75-95%) in FMOD and ROR1 mRNA. Western blot analysis with specific antibodies for FMOD and ROR1 demonstrated that the proteins were significantly downregulated 48 h after siRNA treatment. Silencing of FMOD and ROR1 resulted in statistically significant (P ≤ 0·05-0·001) apoptosis of CLL cells but not of B cells from normal donors. Human fibroblast cell lines treated with FMOD and ROR1 siRNA did not undergo apoptosis. This is the first report demonstrating that ROR1 and FMOD may be involved in the survival of CLL cells. ROR1 in particular is further explored as potential target for therapy in CLL.
Assuntos
Apoptose/genética , Proteínas da Matriz Extracelular/genética , Inativação Gênica , Leucemia Linfocítica Crônica de Células B/genética , Proteoglicanas/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Proteínas da Matriz Extracelular/biossíntese , Fibromodulina , Genes Neoplásicos , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteoglicanas/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transfecção , Células Tumorais CultivadasRESUMO
UNLABELLED: Elucidating cellular mechanisms that maintain the intrahepatic immune balance is crucial to our understanding of viral or autoimmune liver diseases and allograft acceptance. Liver sinusoidal endothelial cells (LSECs) play an important role in modifying local immune responses to tolerance in major histocompatibility complex (MHC) I-restricted models, whereas their contribution in the MHCII context is still controversial. In an MHCII chimeric mouse model that excludes MHCII-mediated antigen presentation by professional antigen-presenting cells, we demonstrated that LSECs prime CD4(+) T cells to a CD45RB(low) memory phenotype lacking marker cytokine production for effector cells that was stable in vivo following immunogenic antigen re-encounter. Although these cells, which we term T(LSEC), had the capacity to enter lymph nodes and the liver, they did not function as effector cells either in a delayed-type hypersensitivity reaction or in a hepatitis model. T(LSEC) inhibited the proliferation of naïve CD4(+) T cells in vitro although being CD25(low) and lacking expression of forkhead box protein (FoxP)3. Furthermore, these cells suppressed hepatic inflammation as monitored by alanine aminotransferase levels and cellular infiltrates in a T cell-mediated autoimmune hepatitis model in vivo. CONCLUSION: T(LSEC) first described here might belong to the expanding group of FoxP3(-) regulatory T cells. Our findings strengthen the previously discussed assumption that CD4(+) T cell priming by nonprofessional antigen-presenting cells induces anti-inflammatory rather than proinflammatory phenotypes. Because recruitment of CD4(+) T cells is increased upon hepatic inflammation, T(LSEC) might contribute to shifting antigen-dependent immune responses to tolerance toward exogenous antigens or toward endogenous self-antigens, especially under inflammatory conditions.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Endoteliais/imunologia , Fatores de Transcrição Forkhead/fisiologia , Hepatite Autoimune/prevenção & controle , Fígado/imunologia , Linfócitos T Reguladores/imunologia , Animais , Movimento Celular , Feminino , Interferon gama/biossíntese , Interleucina-10/biossíntese , Camundongos , Camundongos Endogâmicos C57BLRESUMO
This study compares the effect of recombinant Factor VIII Fc fusion protein (rFVIII-Fc) with recombinant FVIII (rFVIII) on monocyte-derived dendritic cells (moDC's). Cells treated with rFVIII-Fc showed morphological changes typical for cell activation, had a significant up-regulation of cell activation markers and produced higher levels of pro-inflammatory cytokines. Even after stimulation with Lipopolysaccharides, the addition of rFVIII-Fc led to increased expression of activation markers, indicating that rFVIII-Fc is capable of amplifying the maturation signal. On the contrary, cultivation of moDC's with rFVIII did not alter cell morphology or increase surface activation marker expression and pro-inflammatory cytokine production. The binding of the Fc domain to the activating Fcγ receptor IIa (FcγRIIa) can cause cell activation. Therefore, the effect of rFVIII-Fc on FcγRIIa was analyzed in detail. Cultivation of moDC's with rFVIII-Fc led to increased phosphorylation of FcγRIIa, which was not detected for rFVIII. Blocking FcγRIIa prior to the cultivation with rFVIII-Fc significantly reduced the activating effect of rFVIII-Fc, indicating that rFVIII-Fc-induced moDC activation was caused by FcγRIIa. Moreover, rFVIII-Fc bound to FCGR2A-transfected human embryonic kidney 293 cells. Taken together, our data present a new mechanism of moDC activation by rFVIII-Fc via FcγRIIa.
RESUMO
Although human endogenous retroviruses (HERVs) represent a substantial proportion of the human genome and some HERVs, such as HERV-K(HML-2), are reported to be involved in neurological disorders, little is known about their biological function. We report that RNA from an HERV-K(HML-2) envelope gene region binds to and activates human Toll-like receptor (TLR) 8, as well as murine Tlr7, expressed in neurons and microglia, thereby causing neurodegeneration. HERV-K(HML-2) RNA introduced into the cerebrospinal fluid (CSF) of either C57BL/6 wild-type mice or APPPS1 mice, a mouse model for Alzheimer's disease (AD), resulted in neurodegeneration and microglia accumulation. Tlr7-deficient mice were protected against neurodegenerative effects but were resensitized toward HERV-K(HML-2) RNA when neurons ectopically expressed murine Tlr7 or human TLR8. Transcriptome data sets of human AD brain samples revealed a distinct correlation of upregulated HERV-K(HML-2) and TLR8 RNA expression. HERV-K(HML-2) RNA was detectable more frequently in CSF from individuals with AD compared with controls. Our data establish HERV-K(HML-2) RNA as an endogenous ligand for species-specific TLRs 7/8 and imply a functional contribution of human endogenous retroviral transcripts to neurodegenerative processes, such as AD.
Assuntos
Doença de Alzheimer , Retrovirus Endógenos , Glicoproteínas de Membrana , RNA Viral , Receptor 7 Toll-Like , Receptor 8 Toll-Like , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Modelos Animais de Doenças , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , RNA Viral/genética , RNA Viral/metabolismo , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/genética , Receptor 8 Toll-Like/metabolismoRESUMO
BACKGROUND & AIMS: V alpha14 invariant natural killer T cells (iNKT) are localized in peripheral tissues such as the liver rather than lymphoid tissues. Therefore, their role in modulating the stimulation of conventional, major histocompatibility complex (MHC)-restricted T-cell responses has remained ambiguous. We here describe a role for V alpha14 iNKT cells in modulating conventional T-cell responses to antigen expressed in liver, using transferrin-mOVA (Tf-mOVA) mice. METHODS: Naïve ovalbumin-specific class I MHC-restricted T cells (OTI) were adoptively transferred into Tf-mOVA mice in the presence or absence of iNKT-cell agonist alpha-galactosylceramide, after which OTI T-cell priming, antigen-specific cytokine production, cytotoxic killing ability, and liver damage were analyzed. RESULTS: Transfer of OTI cells resulted in robust intrahepatic, antigen-specific proliferation of T cells. OTI T cells were activated in liver, and antigen-specific effector function was stimulated by coactivation of Valpha14 iNKT cells using alpha-galactosylceramide. This stimulation was absent in CD1d(-/-)Tf-mOVA mice, which lack V alpha14 iNKT cells, and was prevented when interferon-gamma and tumor necrosis factor-alpha production by V alpha14 iNKT cells was blocked. CONCLUSIONS: CD1d-restricted V alpha14 iNKT cells stimulate intrahepatic CD8 T-cell effector responses to antigen expressed in liver. Our findings elucidate a previously unknown intervention point for targeted immunotherapy to autoimmune and possibly infectious liver diseases.
Assuntos
Antígenos CD1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Hepatite Autoimune/imunologia , Células Matadoras Naturais/imunologia , Fígado/imunologia , Ativação Linfocitária , Serpinas/metabolismo , Transferência Adotiva , Animais , Anticorpos , Antígenos CD1/genética , Antígenos CD1d , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Citotoxicidade Imunológica , Galactosilceramidas/metabolismo , Hepatite Autoimune/genética , Hepatite Autoimune/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Fígado/metabolismo , Fígado/patologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/metabolismo , Serpinas/genética , Transferrina/genética , Transferrina/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
MicroRNAs (miRNAs) are non-coding RNAs originally involved in RNA silencing and post-transcriptional regulation of gene expression. We have shown in previous work that the miRNA let-7b can act as a signalling molecule for Toll-like receptor 7, thereby initiating innate immune pathways and apoptosis in the central nervous system. Here, we investigated whether different members of the miRNA family let-7, abundantly expressed in the brain, are released into the human cerebrospinal fluid (CSF) and whether quantitative differences in let-7 copies exist in neurodegenerative diseases. RNA isolated from CSF of patients with Alzheimer´s disease (AD) and from control patients with frontotemporal lobe dementia (FTLD), major depressive episode (MDE) without clinical or neurobiological signs of AD, and healthy individuals, was reverse transcribed with primers against nine let-7 family members, and miRNAs were quantified and analyzed comparatively by quantitative PCR. let-7 miRNAs were present in CSF from patients with AD, FTLD, MDE, and healthy controls. However, the amount of individual let-7 miRNAs in the CSF varied substantially. CSF from AD patients contained higher amounts of let-7b and let-7e compared to healthy controls, while no differences were observed regarding the other let-7 miRNAs. No increase in let-7b and let-7e was detected in CSF from FTLD patients, while in CSF from MDE patients, let-7b and let-7e copy levels were elevated. In CSF from AD patients, let-7b and let-7e were associated with extracellular vesicles. let-7 family members present in the CSF mediated neurotoxicity in vitro, albeit to a variable extent. Taken together, neurotoxic let-7 miRNAs are differentially and specifically released in AD, but also in MDE patients. Thus, these miRNAs may mirror common neuropathological paths and by this serve to unscramble mechanisms of different neurodegenerative diseases.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Micropartículas Derivadas de Células/metabolismo , Regulação da Expressão Gênica , MicroRNAs/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Transtorno Depressivo Maior/líquido cefalorraquidiano , Feminino , Demência Frontotemporal/líquido cefalorraquidiano , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
A variety of approaches have been used to deliver tumor-associated antigens (TAA) in conjunction with dendritic cells (DC) as cellular adjuvants. DC derived from monocytic precursors have been pulsed with whole tumor antigen using a variety of strategies and have been demonstrated to induce CD4+ and CD8+ antitumor responses. In the present study, monocyte-derived DC have been pulsed with lysate from an allogeneic melanoma cell line, A-375, and used to repeatedly stimulate T cells. The resultant T cells were examined for cytotoxic activity against A-375 targets as well as the HLA A2-positive melanoma cell line DFW. Uptake of FITC-labeled melanoma lysate by DC established that lysate of melanoma cells was efficiently endocytosed. Stimulation with lysate-pulsed DC resulted in strong proliferative responses by T cells, which could be inhibited by antibodies against both MHC class I and class II. T cells stimulated in vitro with lysate-pulsed DC demonstrated potent cytotoxicity against the melanoma targets which were blocked by antibodies against MHC class I. Lysate-pulsed DC also elicited IFN-gamma secretion by T cells as measured in an ELISPOT assay. We have also examined the ability of lysate-pulsed DC to present melanoma-associated antigens to T cells. ELISPOT assays with synthetic peptides of melanoma-associated antigens, such as gp100, mage1, NY-ESO, and MART-1, revealed that lysate-pulsed DC could stimulate T cells in an antigen-specific manner. The results demonstrate that lysate from allogeneic tumor cells may be used as a source of antigens to stimulate tumor-specific T cells in melanoma.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Melanoma/imunologia , Linfócitos T/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/farmacologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Humanos , Isoantígenos/química , Isoantígenos/imunologia , Isoantígenos/farmacologia , Células K562 , Melanoma/química , MonócitosRESUMO
Peripheral macrophages and resident microglia constitute the dominant glioma-infiltrating cells. The tumor induces an immunosuppressive and tumor-supportive phenotype in these glioma-associated microglia/brain macrophages (GAMs). A subpopulation of glioma cells acts as glioma stem cells (GSCs). We explored the interaction between GSCs and GAMs. Using CD133 as a marker of stemness, we enriched for or deprived the mouse glioma cell line GL261 of GSCs by fluorescence-activated cell sorting (FACS). Over the same period of time, 100 CD133(+ )GSCs had the capacity to form a tumor of comparable size to the ones formed by 10,000 CD133(-) GL261 cells. In IL-6(-/-) mice, only tumors formed by CD133(+ )cells were smaller compared with wild type. After stimulation of primary cultured microglia with medium from CD133-enriched GL261 glioma cells, we observed an selective upregulation in microglial IL-6 secretion dependent on Toll-like receptor (TLR) 4. Our results show that GSCs, but not the bulk glioma cells, initiate microglial IL-6 secretion via TLR4 signaling and that IL-6 regulates glioma growth by supporting GSCs. Using human glioma tissue, we could confirm the finding that GAMs are the major source of IL-6 in the tumor context.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptor 4 Toll-Like/biossíntese , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Galinhas , Glioma/patologia , Humanos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/patologia , Células-Tronco Neoplásicas/patologia , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas , Regulação para Cima/fisiologiaRESUMO
Photodynamic therapy (PDT) has emerged as an effective and minimally invasive treatment option for several diseases, including some forms of cancer. However, several drawbacks of the approved photosensitizers (PS), such as insufficient light absorption at therapeutically relevant wavelengths hampered the clinical effectiveness of PDT. Phthalocyanines (Pc) are interesting PS-candidates with a strong light absorption in the favourable red spectral region and a high quantum yield of cancer cell destroying singlet oxygen generation. Here, we evaluated the suitability of tetra-triethyleneoxysulfonyl substituted zinc phthalocyanine (ZnPc) as novel PS for PDT. ZnPc-induced phototoxicity, induction of apoptosis as well as cell cycle arresting effects was studied in the human gastrointestinal cancer cell lines of different origin. Photoactivation of ZnPc-pretreated (1-10 µM) cancer cells was achieved by illumination with a broad band white light source (400-700 nm) at a power density of 10 J/cm(2). Photoactivation of ZnPc-loaded cells revealed strong phototoxic effects, leading to a dose-dependent decrease of cancer cell proliferation of up to almost 100%, the induction of apoptosis and a G1-phase arrest of the cell cycle, which was associated with decrease in cyclin D1 expression. By contrast, ZnPc-treatment without illumination did not induce any cytotoxicity, apoptosis, cell cycle arrest or decreased cell growth. Antiangiogenic effects of ZnPc-PDT were investigated in vivo by performing CAM assays, which revealed a marked degradation of blood vessels and the capillary plexus of the chorioallantoic membrane of fertilized chicken eggs. Based on our data we think that ZnPc may be a promising novel photosensitizer for innovative PDT.
Assuntos
Indóis/administração & dosagem , Indóis/síntese química , Neoplasias Experimentais/tratamento farmacológico , Compostos Organometálicos/administração & dosagem , Compostos Organometálicos/síntese química , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Humanos , Isoindóis , Neoplasias Experimentais/patologia , Fármacos Fotossensibilizantes/síntese química , Resultado do Tratamento , Compostos de ZincoRESUMO
BACKGROUND: Interleukin-17 (IL-17) acts as a key regulator in central nervous system (CNS) inflammation. γδ T cells are an important innate source of IL-17. Both IL-17+ γδ T cells and microglia, the major resident immune cells of the brain, are involved in various CNS disorders such as multiple sclerosis and stroke. Also, activation of Toll-like receptor (TLR) signaling pathways contributes to CNS damage. However, the mechanisms underlying the regulation and interaction of these cellular and molecular components remain unclear. OBJECTIVE: In this study, we investigated the crosstalk between γδ T cells and microglia activated by TLRs in the context of neuronal damage. To this end, co-cultures of IL-17+ γδ T cells, neurons, and microglia were analyzed by immunocytochemistry, flow cytometry, ELISA and multiplex immunoassays. RESULTS: We report here that IL-17+ γδ T cells but not naïve γδ T cells induce a dose- and time-dependent decrease of neuronal viability in vitro. While direct stimulation of γδ T cells with various TLR ligands did not result in up-regulation of CD69, CD25, or in IL-17 secretion, supernatants of microglia stimulated by ligands specific for TLR2, TLR4, TLR7, or TLR9 induced activation of γδ T cells through IL-1ß and IL-23, as indicated by up-regulation of CD69 and CD25 and by secretion of vast amounts of IL-17. This effect was dependent on the TLR adaptor myeloid differentiation primary response gene 88 (MyD88) expressed by both γδ T cells and microglia, but did not require the expression of TLRs by γδ T cells. Similarly to cytokine-primed IL-17+ γδ T cells, IL-17+ γδ T cells induced by supernatants derived from TLR-activated microglia also caused neurotoxicity in vitro. While these neurotoxic effects required stimulation of TLR2, TLR4, or TLR9 in microglia, neuronal injury mediated by bone marrow-derived macrophages did not require TLR signaling. Neurotoxicity mediated by IL-17+ γδ T cells required a direct cell-cell contact between T cells and neurons. CONCLUSION: Taken together, these results point to a crucial role for microglia activated through TLRs in polarization of γδ T cells towards neurotoxic IL-17+ γδ T cells.
Assuntos
Interleucina-17/biossíntese , Microglia/patologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Células Cultivadas , Encefalomielite Autoimune Experimental/imunologia , Ativação Enzimática/imunologia , Interleucina-1beta/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade p19 da Interleucina-23/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Neurônios/imunologia , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/imunologia , Células Th17/imunologia , Receptor 2 Toll-Like/genética , Receptor 7 Toll-Like/genéticaRESUMO
BACKGROUND: The role of regulatory CD4 T cells (Treg) in immune-mediated liver disease is still under debate. It remains disputed whether Treg suppress T cell-mediated hepatitis in vivo and whether hepatic regulatory T cells are functional in patients with autoimmune hepatitis. METHODS: We used TF-OVA mice, which express ovalbumin in hepatocytes, to investigate the impact of Treg in a model of autoimmune hepatitis. Treg isolated from inflamed livers of TF-OVA mice were tested for their functionality in vitro. By employing double transgenic TF-OVAxDEREG (DEpletion of REGulatory T cells) mice we analyzed whether Treg-depletion aggravates autoimmune inflammation in the liver in vivo. RESULTS: CD25+Foxp3+ CD4 T cells accumulated in the liver in the course of CD8 T cell-mediated hepatitis. Treg isolated from inflamed livers were functional to suppress CD8 T-cell proliferation in vitro. Depletion of Treg in TF-OVAxDEREG mice dramatically amplified T cell-mediated hepatitis. Repeated administration of antigen-specific CD8 T cells led to a second wave of inflammation only after depletion of Treg. CONCLUSION: Our data add to the evidence for an important role of Treg in autoimmune hepatitis and show that Treg reduce the severity of T-cell mediated hepatitis in vivo. They constitute a key immune cell population that actively maintains a tolerogenic milieu in the liver and protects the liver against repeated inflammatory challenges.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Hepatite Autoimune/imunologia , Hepatite Autoimune/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Fatores de Transcrição Forkhead/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Linfócitos T Reguladores/imunologiaRESUMO
BACKGROUND: Toll-like receptors (TLR) constitute a highly conserved class of receptors through which the innate immune system responds to both pathogen- and host-derived factors. Although TLRs are involved in a wide range of central nervous system (CNS) disorders including neurodegenerative diseases, the molecular events leading from CNS injury to activation of these innate immune receptors remain elusive. The stress protein heat shock protein 60 (HSP60) released from injured cells is considered an endogenous danger signal of the immune system. In this context, the main objective of the present study was to investigate the impact of extracellular HSP60 on the brain in vivo. RESULTS: We show here that HSP60 injected intrathecally causes neuronal and oligodendrocyte injury in the CNS in vivo through TLR4-dependent signaling. Intrathecal HSP60 results in neuronal cell death, axonal injury, loss of oligodendrocytes, and demyelination in the cerebral cortex of wild-type mice. In contrast both mice lacking TLR4 and the TLR adaptor molecule MyD88 are protected against deleterious effects induced by HSP60. In contrast to the exogenous TLR4 ligand, lipopolysaccharide, intrathecal HSP60 does not induce such a considerable inflammatory response in the brain. In the CNS, endogenous HSP60 is predominantly expressed in neurons and released during brain injury, since the cerebrospinal fluid (CSF) from animals of a mouse stroke model contains elevated levels of this stress protein compared to the CSF of sham-operated mice. CONCLUSIONS: Our data show a direct toxic effect of HSP60 towards neurons and oligodendrocytes in the CNS. The fact that these harmful effects involve TLR4 and MyD88 confirms a molecular pathway mediated by the release of endogenous TLR ligands from injured CNS cells common to many forms of brain diseases that bi-directionally links CNS injury and activation of the innate immune system to neurodegeneration and demyelination in vivo.
Assuntos
Sistema Nervoso Central/metabolismo , Chaperonina 60/metabolismo , Doenças Desmielinizantes/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/metabolismo , Animais , Morte Celular , Células Cultivadas , Chaperonina 60/farmacologia , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Oligodendroglia/metabolismo , Receptores Imunológicos/metabolismoRESUMO
Magnetic activated cell sorting (MACS) is a commonly used cell separation technique. Miltenyi Biotec has developed an efficient MACS protocol for isolation of CD8α(+) T cells from adult mice spleen. However, due to a low percentage of CD8α(+) T cells in spleen of postnatal mice, MACS isolation of CD8α(+) T cells results in a low purity. Our study illustrates how this problem can be solved by performing a CD45R(+) B cell depletion prior to a positive selection of CD8α(+) T cells. This protocol can be used when low frequencies of CD8α(+) T cells are present in a tissue also populated with CD45R(+) B cells, and a high purity is required for downstream applications.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Separação Imunomagnética/métodos , Baço/citologia , Baço/imunologia , Fatores Etários , Animais , Antígenos CD8/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Interferon-ß is an established treatment for patients with multiple sclerosis (MS) but its mechanisms of action are not well understood. Viral infections are a known trigger of MS relapses. Toll-like receptors (TLRs) are key components of the innate immune system, which sense conserved structures of viruses and other pathogens. Effects of interferon-ß on mRNA levels of all known human TLRs (TLR1-10) and the TLR adaptor molecule MyD88 were analyzed in peripheral blood mononuclear cells (PBMCs) of healthy donors by quantitative real-time PCR and by transcriptome analysis in PBMCs of 25 interferon-ß-treated patients with relapsing-remitting MS. Regulation of TLR protein expression by interferon-ß was investigated by flow cytometry of leukocyte subsets of healthy subjects and of untreated, interferon-ß-, or glatiramer acetate-treated patients with MS. Interferon-ß specifically upregulated mRNA expression of TLR3, TLR7, and MyD88 and downregulated TLR9 mRNA in PBMCs of healthy donors as well as in PBMCs of patients with MS. Plasmacytoid dendritic cells (pDCs) were identified as the major cell type responding to interferon-ß with increased expression of TLR7 and MyD88 protein. In line with this, expression of TLR7 protein was increased in pDCs of interferon-ß-treated, but not untreated or glatiramer acetate-treated patients with MS. Interferon-ß-induced upregulation of TLR7 in pDCs is of functional relevance since pre-treatment of PBMCs with interferon-ß resulted in a strongly increased production of interferon-α upon stimulation with the TLR7 agonist loxoribine. Flow cytometry confirmed pDCs as the cellular source of interferon-α production induced by activation of TLR7. Thus, upregulation of TLR7 in pDCs and a consequently increased activation of pDCs by TLR7 ligands represents a novel immunoregulatory mechanism of interferon-ß. We hypothesize that this mechanism could contribute to a reduction of virus-triggered relapses in patients with MS.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon beta/farmacologia , Esclerose Múltipla/genética , Receptor 7 Toll-Like/genética , Adulto , Relação Dose-Resposta a Droga , Feminino , Humanos , Interferon-alfa/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Adulto JovemRESUMO
Toll-like receptors (TLR) are key players of the innate and adaptive immune response in vertebrates. The original protein Toll in Drosophila melanogaster regulates both host defense and morphogenesis during development. Making use of real-time PCR, in situ hybridization, and immunohistochemistry we systematically examined the expression of TLR1-9 and the intracellular adaptor molecules MyD88 and TRIF during development of the mouse brain. Expression of TLR7 and TLR9 in the brain was strongly regulated during different embryonic, postnatal, and adult stages. In contrast, expression of TLR1-6, TLR8, MyD88, and TRIF mRNA displayed no significant changes in the different phases of brain development. Neurons of various brain regions including the neocortex and the hippocampus were identified as the main cell type expressing both TLR7 and TLR9 in the developing brain. Taken together, our data reveal specific expression patterns of distinct TLRs in the developing mouse brain and lay the foundation for further investigation of the pathophysiological significance of these receptors for developmental processes in the central nervous system of vertebrates.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Transcriptoma , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Axônios/metabolismo , Encéfalo/citologia , Células HEK293 , Humanos , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Activation of innate immune receptors by host-derived factors exacerbates CNS damage, but the identity of these factors remains elusive. We uncovered an unconventional role for the microRNA let-7, a highly abundant regulator of gene expression in the CNS, in which extracellular let-7 activates the RNA-sensing Toll-like receptor (TLR) 7 and induces neurodegeneration through neuronal TLR7. Cerebrospinal fluid (CSF) from individuals with Alzheimer's disease contains increased amounts of let-7b, and extracellular introduction of let-7b into the CSF of wild-type mice by intrathecal injection resulted in neurodegeneration. Mice lacking TLR7 were resistant to this neurodegenerative effect, but this susceptibility to let-7 was restored in neurons transfected with TLR7 by intrauterine electroporation of Tlr7(−/−) fetuses. Our results suggest that microRNAs can function as signaling molecules and identify TLR7 as an essential element in a pathway that contributes to the spread of CNS damage.