RESUMO
Antibody-based blocking of vascular endothelial growth factor (VEGF) reduces choroidal neovascularization (CNV) and retinal edema, rescuing vision in patients with neovascular age-related macular degeneration (nAMD). However, poor response and resistance to anti-VEGF treatment occurs. We report that targeting the Notch ligand Jagged1 by a monoclonal antibody reduces neovascular lesion size, number of activated phagocytes and inflammatory markers and vascular leakage in an experimental CNV mouse model. Additionally, we demonstrate that Jagged1 is expressed in mouse and human eyes, and that Jagged1 expression is independent of VEGF signaling in human endothelial cells. When anti-Jagged1 was combined with anti-VEGF in mice, the decrease in lesion size exceeded that of either antibody alone. The therapeutic effect was solely dependent on blocking, as engineering antibodies to abolish effector functions did not impair the therapeutic effect. Targeting of Jagged1 alone or in combination with anti-VEGF may thus be an attractive strategy to attenuate CNV-bearing diseases.
Assuntos
Neovascularização de Coroide , Fator A de Crescimento do Endotélio Vascular , Humanos , Camundongos , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais/metabolismo , Neovascularização de Coroide/patologia , Anticorpos Bloqueadores/uso terapêutico , Transdução de Sinais/fisiologia , Modelos Animais de Doenças , Inibidores da Angiogênese/uso terapêuticoRESUMO
BACKGROUND: The pathophysiology in atopic dermatitis (AD) is not fully understood, but immune dysfunction, skin barrier defects, and alterations of the skin microbiota are thought to play important roles. AD skin is frequently colonized with Staphylococcus aureus (S. aureus) and microbial diversity on lesional skin (LS) is reduced compared to on healthy skin. Treatment with narrow-band ultraviolet B (nb-UVB) leads to clinical improvement of the eczema and reduced abundance of S. aureus. However, in-depth knowledge of the temporal dynamics of the skin microbiota in AD in response to nb-UVB treatment is lacking and could provide important clues to decipher whether the microbial changes are primary drivers of the disease, or secondary to the inflammatory process. OBJECTIVES: To map the temporal shifts in the microbiota of the skin, nose, and throat in adult AD patients after nb-UVB treatment. METHODS: Skin swabs were taken from lesional AD skin (n = 16) before and after 3 treatments of nb-UVB, and after 6-8 weeks of full-body treatment. We also obtained samples from non-lesional skin (NLS) and from the nose and throat. All samples were characterized by 16S rRNA gene sequencing. RESULTS: We observed shifts towards higher diversity in the microbiota of lesional AD skin after 6-8 weeks of treatment, while the microbiota of NLS and of the nose/throat remained unchanged. After only 3 treatments with nb-UVB, there were no significant changes in the microbiota. CONCLUSION: Nb-UVB induces changes in the skin microbiota towards higher diversity, but the microbiota of the nose and throat are not altered.
Assuntos
Dermatite Atópica/microbiologia , Dermatite Atópica/radioterapia , Microbiota/efeitos da radiação , Pele/microbiologia , Terapia Ultravioleta , Adulto , Idoso , Biodiversidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nariz/microbiologia , Faringe/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/efeitos da radiação , Resultado do Tratamento , Adulto JovemRESUMO
Sturge-Weber syndrome (SWS) is a neurocutaneous disorder characterized by vascular malformations affecting skin, eyes and leptomeninges of the brain, which can lead to glaucoma, seizures and intellectual disability. The discovery of a disease-causing somatic missense mutation in the GNAQ gene, encoding an alpha chain of heterotrimeric G-proteins, has initiated efforts to understand how G-proteins contribute to SWS pathogenesis. The mutation is predominantly detected in endothelial cells and is currently believed to affect downstream MAPK signalling. In this study of six Norwegian patients with classical SWS, we aimed to identify somatic mutations through deep sequencing of DNA from skin biopsies. Surprisingly, one patient was negative for the GNAQ mutation, but instead harbored a somatic mutation in GNB2 (NM_005273.3:c.232A>G, p.Lys78Glu), which encodes a beta chain of the same G-protein complex. The positions of the mutant amino acids in the G-protein are essential for complex reassembly. Therefore, failure of reassembly and continuous signalling is a likely consequence of both mutations. Ectopic expression of mutant proteins in endothelial cells revealed that expression of either mutant reduced cellular proliferation, yet regulated MAPK signalling differently, suggesting that dysregulated MAPK signalling cannot fully explain the SWS phenotype. Instead, both mutants reduced synthesis of Yes-associated protein (YAP), a transcriptional co-activator of the Hippo signalling pathway, suggesting a key role for this pathway in the vascular pathogenesis of SWS. The discovery of the GNB2 mutation sheds novel light on the pathogenesis of SWS and suggests that future research on targets of treatment should be directed towards the YAP, rather than the MAPK, signalling pathway.
Assuntos
Proteínas de Ligação ao GTP/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação , Síndrome de Sturge-Weber/diagnóstico , Síndrome de Sturge-Weber/genética , Adolescente , Adulto , Criança , Análise Mutacional de DNA , Proteínas de Ligação ao GTP/química , Frequência do Gene , Estudos de Associação Genética/métodos , Humanos , Pessoa de Meia-Idade , Modelos Moleculares , Nortriptilina , Fenótipo , Conformação Proteica , Subunidades Proteicas/genética , Relação Estrutura-Atividade , Sequenciamento do Exoma , Adulto JovemRESUMO
The Notch signaling pathway regulates developmental cell-fate decisions and has recently also been linked to inflammatory diseases. Although therapies targeting Notch signaling in inflammation in theory are attractive, their design and implementation have proven difficult, at least partly due to the broad involvement of Notch signaling in regenerative and homeostatic processes. In this review, we summarize the supporting role of Notch signaling in various inflammation-driven diseases, and highlight efforts to intervene with this pathway by targeting Notch ligands and/or receptors with distinct therapeutic strategies, including antibody designs. We discuss this in light of lessons learned from Notch targeting in cancer treatment. Finally, we elaborate on the impact of individual Notch members in inflammation, which may lay the foundation for development of therapeutic strategies in chronic inflammatory diseases.
Assuntos
Anti-Inflamatórios/uso terapêutico , Anticorpos/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Autoimunidade/efeitos dos fármacos , Inflamação/tratamento farmacológico , Receptores Notch/antagonistas & inibidores , Anti-Inflamatórios/efeitos adversos , Anticorpos/efeitos adversos , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doença Crônica , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Terapia de Alvo Molecular , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
Interleukin (IL)-33 is a cytokine that appears to mediate fibrosis by signaling via its receptor ST2 (IL-33R/IL1RL1). It is also, however, a protein that after synthesis is sorted to the cell nucleus, where it appears to affect chromatin folding. Here we describe a novel role for nuclear IL-33 in regulating the fibroblast phenotype in murine kidney fibrosis driven by unilateral ureteral obstruction. Transcriptional profiling of IL-33-deficient kidneys 24 h after ligation revealed enhanced expression of fibrogenic genes and enrichment of gene sets involved in extracellular matrix formation and remodeling. These changes relied on intracellular effects of IL-33, because they were not reproduced by treatment with a neutralizing antibody to IL-33 that prevents IL-33R/ST2L receptor signaling nor were they observed in IL-33R/ST2-deficient kidneys. To further explore the intracellular function of IL-33, we established transcription profiles of human fibroblasts, observing that knockdown of IL-33 skewed the transcription profile from an inflammatory towards a myofibroblast phenotype, reflected in higher levels of COL3A1, COL5A1 and transgelin protein, as well as lower expression levels of IL6, CXCL8, CLL7 and CCL8. In conclusion, our findings suggest that nuclear IL-33 in fibroblasts dampens the initial profibrotic response until persistent stimuli, as enforced by UUO, can override this protective mechanism.
Assuntos
Núcleo Celular/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Interleucina-33/metabolismo , Animais , Núcleo Celular/genética , Quimiocinas/genética , Quimiocinas/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Matriz Extracelular/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Rim/citologia , Rim/metabolismo , Camundongos Endogâmicos C57BL , FenótipoRESUMO
Phototherapy with narrow-band Ultraviolet B (nb-UVB) is a major therapeutic option in atopic dermatitis (AD), yet knowledge of the early molecular responses to this treatment is lacking. The objective of this study was to map the early transcriptional changes in AD skin in response to nb-UVB treatment. Adult patients (n = 16) with AD were included in the study and scored with validated scoring tools. AD skin was irradiated with local nb-UVB on day 0, 2 and 4. Skin biopsies were taken before and after treatment (day 0 and 7) and analysed for genome-wide modulation of transcription. When examining the early response after three local UVB treatments, gene expression analysis revealed 77 significantly modulated transcripts (30 down- and 47 upregulated). Among them were transcripts related to the inflammatory response, melanin synthesis, keratinization and epidermal structure. Interestingly, the pro-inflammatory cytokine IL-36γ was reduced after treatment, while the anti-inflammatory cytokine IL-37 increased after treatment with nb-UVB. There was also a modulation of several other mediators involved in inflammation, among them defensins and S100 proteins. This is the first study of early transcriptomic changes in AD skin in response to nb-UVB. We reveal robust modulation of a small group of inflammatory and anti-inflammatory targets, including the IL-1 family members IL36γ and IL-37, which is evident before any detectable changes in skin morphology or immune cell infiltrates. These findings provide important clues to the molecular mechanisms behind the treatment response and shed light on new potential treatment targets.
Assuntos
Dermatite Atópica/genética , Dermatite Atópica/radioterapia , Interleucina-1/genética , Transcrição Gênica/efeitos da radiação , Terapia Ultravioleta , Adulto , Idoso , Defensinas/genética , Dermatite Atópica/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas S100/genética , Fatores de Tempo , Raios Ultravioleta , Adulto JovemRESUMO
Splenic red pulp macrophages (RPMs) contribute to erythrocyte homeostasis and are required for iron recycling. Heme induces the expression of SPIC transcription factor in monocyte-derived macrophages and promotes their differentiation into RPM precursors, pre-RPMs. However, the requirements for differentiation into mature RPMs remain unknown. Here, we have demonstrated that interleukin (IL)-33 associated with erythrocytes and co-cooperated with heme to promote the generation of mature RPMs through activation of the MyD88 adaptor protein and ERK1/2 kinases downstream of the IL-33 receptor, IL1RL1. IL-33- and IL1RL1-deficient mice showed defective iron recycling and increased splenic iron deposition. Gene expression and chromatin accessibility studies revealed a role for GATA transcription factors downstream of IL-33 signaling during the development of pre-RPMs that retained full potential to differentiate into RPMs. Thus, IL-33 instructs the development of RPMs as a response to physiological erythrocyte damage with important implications to iron recycling and iron homeostasis.
Assuntos
Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Interleucina-33/imunologia , Ferro/metabolismo , Macrófagos/imunologia , Transdução de Sinais/imunologia , Baço/metabolismo , Animais , Eritrócitos/imunologia , Eritrócitos/metabolismo , Heme/imunologia , Heme/metabolismo , Homeostase/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/genética , Interleucina-33/metabolismo , Macrófagos/metabolismo , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Baço/citologiaRESUMO
Inhibition of the key glycolytic activator 6-phosphofructokinase 2/fructose-2,6-bisphosphatase-3 (PFKFB3) by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) strongly attenuates pathological angiogenesis in cancer and inflammation. In addition to modulating endothelial proliferation and migration, 3PO also dampens proinflammatory activation of endothelial cells and experimental inflammation in vivo, suggesting a potential for 3PO in the treatment of chronic inflammation. The aim of our study was to explore if the anti-inflammatory action of 3PO in human endothelial cells was mediated by inhibition of PFKFB3 and glycolysis and assess if other means of PFKFB3 inhibition reduced inflammatory activation in a similar manner. We found that 3PO caused a rapid and transient reduction in IL-1ß- and TNF-induced phosphorylation of both IKKα/ß and JNK, thus inhibiting signaling through the NFκB and the stress-activated kinase pathways. However, in contrast to 3PO-treatment, neither shRNA-mediated silencing of PFKFB3 nor treatment with the alternative PFKFB3 inhibitor 7,8-dihydroxy-3-(4-hydroxy-phenyl)-chromen-4-one (YN1) prevented cytokine-induced NFκB signaling and upregulation of the adhesion molecules VCAM-1 and E-selectin, implying off target effects of 3PO. Collectively, our results suggest that the anti-inflammatory action of 3PO in human endothelial cells is not limited to inhibition of PFKFB3 and cellular glycolysis.
Assuntos
Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosfofrutoquinase-2/metabolismo , Piridinas/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: Endothelial upregulation of adhesion molecules serves to recruit leukocytes to inflammatory sites and appears to be promoted by NOTCH1; however, current models based on interactions between active NOTCH1 and NF-κB components cannot explain the transcriptional selectivity exerted by NOTCH1 in this context. APPROACH AND RESULTS: Observing that Cre/Lox-induced conditional mutations of endothelial Notch modulated inflammation in murine contact hypersensitivity, we found that IL (interleukin)-1ß stimulation induced rapid recruitment of RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A) to genomic sites occupied by NOTCH1-RBPJ (recombination signal-binding protein for immunoglobulin kappa J region) and that NOTCH1 knockdown reduced histone H3K27 acetylation at a subset of NF-κB-directed inflammatory enhancers. CONCLUSIONS: Our findings reveal that NOTCH1 signaling supports the expression of a subset of inflammatory genes at the enhancer level and demonstrate how key signaling pathways converge on chromatin to coordinate the transition to an infla mmatory endothelial phenotype.
Assuntos
Células Endoteliais/efeitos dos fármacos , Histonas/metabolismo , Inflamação/prevenção & controle , Interleucina-1beta/farmacologia , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/metabolismo , Acetilação , Animais , Apendicite/metabolismo , Apendicite/patologia , Células Cultivadas , Dermatite de Contato/genética , Dermatite de Contato/metabolismo , Dermatite de Contato/patologia , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Receptor Notch1/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
IL-33, required for viral clearance by cytotoxic T cells, is generally expressed in vascular endothelial cells in healthy human tissues. We discovered that endothelial IL-33 expression was stimulated as a response to adenoviral transduction. This response was dependent on MRE11, a sensor of DNA damage that can also be activated by adenoviral DNA, and on IRF1, a transcriptional regulator of cellular responses to viral invasion and DNA damage. Accordingly, we observed that endothelial cells responded to adenoviral DNA by phosphorylation of ATM and CHK2 and that depletion or inhibition of MRE11, but not depletion of ATM, abrogated IL-33 stimulation. In conclusion, we show that adenoviral transduction stimulates IL-33 expression in endothelial cells in a manner that is dependent on the DNA-binding protein MRE11 and the antiviral factor IRF1 but not on downstream DNA damage response signaling.
Assuntos
Infecções por Adenoviridae/imunologia , Dano ao DNA/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Interleucina-33/imunologia , Adenoviridae , Infecções por Adenoviridae/metabolismo , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Immunoblotting , Fator Regulador 1 de Interferon/imunologia , Fator Regulador 1 de Interferon/metabolismo , Interleucina-33/biossíntese , Proteína Homóloga a MRE11 , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
Abnormal tumor vessels promote metastasis and impair chemotherapy. Hence, tumor vessel normalization (TVN) is emerging as an anti-cancer treatment. Here, we show that tumor endothelial cells (ECs) have a hyper-glycolytic metabolism, shunting intermediates to nucleotide synthesis. EC haplo-deficiency or blockade of the glycolytic activator PFKFB3 did not affect tumor growth, but reduced cancer cell invasion, intravasation, and metastasis by normalizing tumor vessels, which improved vessel maturation and perfusion. Mechanistically, PFKFB3 inhibition tightened the vascular barrier by reducing VE-cadherin endocytosis in ECs, and rendering pericytes more quiescent and adhesive (via upregulation of N-cadherin) through glycolysis reduction; it also lowered the expression of cancer cell adhesion molecules in ECs by decreasing NF-κB signaling. PFKFB3-blockade treatment also improved chemotherapy of primary and metastatic tumors.
Assuntos
Cisplatino/administração & dosagem , Células Epiteliais/metabolismo , Neoplasias/metabolismo , Fosfofrutoquinase-2/antagonistas & inibidores , Tamoxifeno/administração & dosagem , Animais , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cisplatino/farmacologia , Sinergismo Farmacológico , Tratamento Farmacológico , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Tamoxifeno/farmacologiaRESUMO
Interleukin 33 (IL-33) is a cytokine preferentially elevated in acute ulcerative colitis (UC), inferring a role in its pathogenesis. The role of IL-33 in intestinal inflammation is incompletely understood, with both pro-inflammatory and regulatory properties described. There are also conflicting reports on cellular sources and subcellular location of IL-33 in the colonic mucosa, justifying a closer look at IL-33 expression in well-defined clinical stages of UC. A total of 50 study participants (29 UC patients and 21 healthy controls) were included from a prospective cohort of inflammatory bowel disease patients treated to disease remission with infliximab, a tumour necrosis factor alpha (TNF) inhibitor. To our knowledge this is the first study examining mucosal IL-33 expression before and after anti-TNF therapy. In colonic mucosal biopsies we found a 3-fold increase in IL-33 gene expression comparing acute UC to healthy controls (p < 0.01). A significant reduction of IL33 between acute UC and disease remission was observed when TNF normalised in the mucosa (p = 0.02). Immunostaining revealed IL-33 in the nuclei of epithelial cells of scattered colonic crypts in acute disease, while at disease remission, IL-33 was undetectable, a novel finding suggesting that enterocyte-derived IL-33 is induced and maintained by inflammatory mediators.
Assuntos
Colite Ulcerativa/genética , Expressão Gênica , Interleucina-33/genética , Mucosa Intestinal/metabolismo , Adulto , Idoso , Biomarcadores , Estudos de Casos e Controles , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Feminino , Humanos , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Interleucina-33/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Adoptive cell therapy (ACT) trials to date have focused on transfer of autologous tumor-specific cytotoxic CD8+ T cells; however, the potential of CD4+ T helper (Th) cells for ACT is gaining interest. While encouraging results have been reported with IFNγ-producing Th1 cells, tumor-specific Th2 cells have been largely neglected for ACT due to their reported tumor-promoting properties. In this study, we tested the efficacy of idiotype-specific Th2 cells for the treatment of mice with MHC class II-negative myeloma. Th2 ACT efficiently eradicated subcutaneous myeloma in an antigen-specific fashion. Transferred Th2 cells persisted in vivo and conferred long-lasting immunity. Cancer eradication mediated by tumor-specific Th2 cells did not require B cells, natural killer T cells, CD8+ T cells, or IFNγ. Th2 ACT was also curative against B-cell lymphoma. Upon transfer, Th2 cells induced a type II inflammation at the tumor site with massive infiltration of M2-type macrophages producing arginase. In vivo blockade of arginase strongly inhibited Th2 ACT, consistent with a key role of arginase and M2 macrophages in myeloma elimination by Th2 cells. These results illustrate that cancer eradication may be achieved by induction of a tumor-specific Th2 inflammatory immune response at the tumor site. Thus, ACT with tumor-specific Th2 cells may represent a highly efficient immunotherapy protocol against cancer. Cancer Res; 76(23); 6864-76. ©2016 AACR.
Assuntos
Imunidade Celular/imunologia , Imunoterapia Adotiva/métodos , Inflamação/imunologia , Células Th2/imunologia , Transferência Adotiva , Animais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Camundongos TransgênicosRESUMO
UNLABELLED: Cholangiocytes express antigen-presenting molecules, but it has been unclear whether they can present antigens. Natural killer T (NKT) cells respond to lipid antigens presented by the major histocompatibility complex class I-like molecule CD1d and are abundant in the liver. We investigated whether cholangiocytes express CD1d and present lipid antigens to NKT cells and how CD1d expression varies in healthy and diseased bile ducts. Murine and human cholangiocyte cell lines as well as human primary cholangiocytes expressed CD1d as determined by flow cytometry and western blotting. Murine cholangiocyte cell lines were able to present both exogenous and endogenous lipid antigens to invariant and noninvariant NKT cell hybridomas and primary NKT cells in a CD1d-dependent manner. A human cholangiocyte cell line, cholangiocarcinoma cell lines, and human primary cholangiocytes also presented exogenous CD1d-restricted antigens to invariant NKT cell clones. CD1d expression was down-regulated in the biliary epithelium of patients with late primary sclerosing cholangitis, primary biliary cirrhosis, and alcoholic cirrhosis compared to healthy controls. CONCLUSIONS: Cholangiocytes express CD1d and present antigens to NKT cells and CD1d expression is down-regulated in diseased biliary epithelium, findings which show that the biliary epithelium can activate an important lymphocyte subset of the liver. This is a potentially important immune pathway in the biliary system, which may be capable of regulating inflammation in the context of biliary disease.
Assuntos
Ductos Biliares/imunologia , Ativação Linfocitária , Células T Matadoras Naturais/imunologia , Animais , Antígenos CD1d/fisiologia , Células Cultivadas , Células Epiteliais/imunologia , Epitélio/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
IL-33 is a novel IL-1 family member with a putative role in inflammatory skin disorders and a complex biology. Therefore, recent conflicting data regarding its function in experimental models justify a close assessment of its tissue expression and regulation. Indeed, we report here that there are strong species differences in the expression and regulation of epidermal IL-33. In murine epidermis, IL-33 behaved similar to an alarmin, being constitutively expressed in keratinocyte nuclei and rapidly lost during acute inflammation. By contrast, human and porcine IL-33 were weakly expressed or absent in keratinocytes of noninflamed skin but induced during acute inflammation. To this end, we observed that expression of IL-33 in human keratinocytes but not murine keratinocytes was strongly induced by IFN-γ, and this upregulation completely depended on the presence of EGFR ligands. Accordingly, IFN-γ increased the expression of IL-33 in the basal layers of the epidermis in human ex vivo skin cultures only, despite good evidence of IFN-γ activity in cultures from both species. Together these findings demonstrate that a full understanding of IL-33 function in clinical settings must take species-specific differences into account.
Assuntos
Dermatite/genética , Epiderme/imunologia , Regulação da Expressão Gênica , Inflamação/genética , Interleucinas/genética , Adulto , Animais , Biópsia por Agulha , Western Blotting , Dermatite/fisiopatologia , Modelos Animais de Doenças , Epiderme/metabolismo , Epiderme/patologia , Feminino , Homeostase/genética , Homeostase/fisiologia , Humanos , Imuno-Histoquímica , Inflamação/fisiopatologia , Interleucina-33 , Queratinócitos/imunologia , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estudos de Amostragem , Especificidade da Espécie , Sus scrofa , Suínos , Técnicas de Cultura de TecidosRESUMO
Vascular endothelial cells present luminal chemokines that arrest rolling leukocytes by activating integrins. It appears that several chemokines must form higher-order oligomers to elicit proper in vivo effects, as mutants restricted to forming dimers have lost the ability to recruit leukocytes to sites of inflammation. Here, we show for the first time that the chemokine RANTES/CCL5 binds to the surface of human endothelial cells in a regular filamentous pattern. Furthermore, the filaments bound to the surface in a heparan sulfate-dependent manner. By electron microscopy we observed labeling for RANTES on membrane projections as well as on the remaining plasma membrane. Mutant constructs of RANTES restricted either in binding to heparin, or in forming dimers or tetramers, appeared either in a granular, non-filamentous pattern or were not detectable on the cell surface. The RANTES filaments were also present after exposure to flow, suggesting that they can be present in vivo. Taken together with the lacking in vivo or in vitro effects of RANTES mutants, we suggest that the filamentous structures of RANTES may be of physiological importance in leukocyte recruitment.
Assuntos
Biopolímeros/metabolismo , Quimiocina CCL5/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Heparitina Sulfato/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Interferon gama/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Human mesenchymal stromal cells (hMSCs) show great potential for clinical and experimental use due to their capacity to self-renew and differentiate into multiple mesenchymal lineages. However, disadvantages of primary cultures of hMSCs are the limited in vitro lifespan, and the variable properties of cells from different donors and over time in culture. In this article, we describe the generation of a telomerase-immortalized nontumorigenic human bone marrow-derived stromal mesenchymal cell line, and its detailed characterization after long-term culturing (up to 155 population doublings). The resulting cell line, iMSC#3, maintained a fibroblast-like phenotype comparable to early passages of primary hMSCs, and showed no major differences from hMSCs regarding surface marker expression. Furthermore, iMSC#3 had a normal karyotype, and high-resolution array comparative genomic hybridization confirmed normal copy numbers. The gene expression profiles of immortalized and primary hMSCs were also similar, whereas the corresponding DNA methylation profiles were more diverse. The cells also had proliferation characteristics comparable to primary hMSCs and maintained the capacity to differentiate into osteoblasts and adipocytes. A detailed characterization of the mRNA and microRNA transcriptomes during adipocyte differentiation also showed that the iMSC#3 recapitulates this process at the molecular level. In summary, the immortalized mesenchymal cells represent a valuable model system that can be used for studies of candidate genes and their role in differentiation or oncogenic transformation, and basic studies of mesenchymal biology.
Assuntos
Adipócitos/citologia , Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células-Tronco Mesenquimais/citologia , Linhagem Celular , Transformação Celular Neoplásica/metabolismo , Hibridização Genômica Comparativa , HumanosRESUMO
Tumor-specific Th1 cells can activate tumor-infiltrating macrophages that eliminate MHC class II negative (MHC II(NEG)) tumor cells. Activated M1-like macrophages lack antigen (Ag) receptors, and are presumably unable to discriminate and thus kill both Ag-positive (Ag(POS)) and Ag-negative (Ag(NEG)) tumor cells (bystander killing). The lack of specificity of macrophage-mediated cytotoxicity might be of clinical importance as it could provide a means of avoiding tumor escape. Here, we have tested this idea using mixed populations of Ag(POS) and Ag(NEG) tumor cells in a TCR-transgenic model in which CD4(+) T cells recognize a secreted tumor-specific antigen. Surprisingly, while Ag(POS) tumor cells were recognized and rejected, Ag(NEG) cells grew unimpeded and formed tumors. We further demonstrated that macrophage-mediated cytotoxicity was spatially restricted to areas dominated by Ag(POS) tumor cells, sparing Ag(NEG) tumor cells in the vicinity. As a consequence, macrophage tumoricidal activity did not confer bystander killing in vivo. The present results offer novel insight into the mechanisms of indirect Th1-mediated elimination of MHC II(NEG) tumor cells.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Imunidade Celular/fisiologia , Macrófagos/imunologia , Neoplasias Experimentais/imunologia , Células Th1/imunologia , Animais , Antígenos de Histocompatibilidade Classe II/genética , Camundongos , Camundongos SCID , Camundongos Transgênicos , Neoplasias Experimentais/genéticaRESUMO
OBJECTIVE: Interleukin (IL)-33 is a nuclear protein that is released from stressed or damaged cells to act as an alarmin. We investigated the effects of IL-33 on endothelial cells, using the prototype IL-1 family member, IL-1ß, as a reference. METHODS AND RESULTS: Human umbilical vein endothelial cells were stimulated with IL-33 or IL-1ß, showing highly similar phosphorylation of signaling molecules, induction of adhesion molecules, and transcription profiles. However, intradermally injected IL-33 elicited significantly less proinflammatory endothelial activation when compared with IL-1ß and led us to observe that quiescent endothelial cells (ppRb(low)p27(high)) were strikingly resistant to IL-33. Accordingly, the IL-33 receptor was preferentially expressed in nonquiescent cells of low-density cultures, corresponding to selective induction of adhesion molecules and chemokines. Multiparameter phosphoflow cytometry confirmed that signaling driven by IL-33 was stronger in nonquiescent cells. Manipulation of nuclear IL-33 expression by siRNA or adenoviral transduction revealed no functional link between nuclear, endogenous IL-33, and exogenous IL-33 responsiveness. CONCLUSIONS: In contrast to other inflammatory cytokines, IL-33 selectively targets nonquiescent endothelial cells. By this novel concept, quiescent cells may remain nonresponsive to a proinflammatory stimulus that concomitantly triggers a powerful response in cells that have been released from contact inhibition.
Assuntos
Proliferação de Células , Dermatite/imunologia , Células Endoteliais/imunologia , Mediadores da Inflamação/metabolismo , Interleucinas/metabolismo , Pele/irrigação sanguínea , Adenoviridae/genética , Animais , Biópsia , Células Cultivadas , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Dermatite/patologia , Selectina E/metabolismo , Células Endoteliais/patologia , Feminino , Citometria de Fluxo , Vetores Genéticos , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Interleucina-1beta/metabolismo , Interleucina-33 , Interleucinas/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neovascularização Fisiológica , Fosforilação , Interferência de RNA , Receptores de Interleucina/metabolismo , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Transcrição Gênica , Transdução Genética , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The molecular mechanisms that drive expression of the alarmin interleukin-33 (IL-33) in endothelial cells are unknown. Because nuclear IL-33 is a marker of endothelial cell quiescence (corroborated in this study by coexpression of cyclin-dependent kinase inhibitor p27(Kip1)), we hypothesized that Notch signaling might be involved in regulating IL-33 expression. Activation of Notch1 by immobilized Notch ligands was sufficient to induce nuclear IL-33 expression in cultured endothelial cells. Conversely, IL-33 expression was inhibited by the γ-secretase inhibitor DAPT or by inhibiting the function of Dll4, Jagged1, Notch1, or the canonical Notch transcription factor RBP-Jκ. Insensitivity to cycloheximide indicated that IL-33 was a direct target of Notch signaling, well in line with the identification of several conserved RBP-Jκ binding sites in the IL33 gene. The in vivo expression of Dll4 but not of Jagged1 was well correlated with expression of IL-33 in quiescent vessels, and subcutaneous injection of DAPT in healthy skin reduced IL-33 expression, indicating that Notch signaling was involved. On the other hand, loss of IL-33 during angiogenesis occurred despite sustained Dll4 and Notch1 expression, suggesting that other signals may override the IL-33-driving signal in this context. Taken together, our data demonstrate that endothelial nuclear IL-33 is induced by Notch and that Dll4 may be the dominant ligand responsible for this signaling in vivo.