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2.
J Crohns Colitis ; 14(6): 778-790, 2020 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31711184

RESUMO

BACKGROUND AND AIMS: Intestinal inflammation in inflammatory bowel diseases [IBD] is thought to be T cell mediated and therefore dependent on the interaction between the T cell receptor [TCR] and human leukocyte antigen [HLA] proteins expressed on antigen presenting cells. The collection of all TCRs in one individual, known as the TCR repertoire, is characterised by enormous diversity and inter-individual variability. It was shown that healthy monozygotic [MZ] twins are more similar in their TCR repertoire than unrelated individuals. Therefore MZ twins, concordant or discordant for IBD, may be useful to identify disease-related and non-genetic factors in the TCR repertoire which could potentially be used as disease biomarkers. METHODS: Employing unique molecular barcoding that can distinguish between polymerase chain reaction [PCR] artefacts and true sequence variation, we performed deep TCRα and TCRß repertoire profiling of the peripheral blood of 28 MZ twin pairs from Denmark and Germany, 24 of whom were discordant and four concordant for IBD. RESULTS: We observed disease- and smoking-associated traits such as sharing, diversity and abundance of specific clonotypes in the TCR repertoire of IBD patients, and particularly in patients with active disease, compared with their healthy twins. CONCLUSIONS: Our findings identified TCR repertoire features specific for smokers and IBD patients, particularly when signs of disease activity were present. These findings are a first step towards the application of TCR repertoire analyses as a valuable tool to characterise inflammatory bowel diseases and to identify potential biomarkers and true disease causes.


Assuntos
Colite Ulcerativa , Doença de Crohn , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Receptores de Antígenos de Linfócitos T alfa-beta/sangue , Adulto , Proteína C-Reativa/análise , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/imunologia , Colite Ulcerativa/fisiopatologia , Doença de Crohn/diagnóstico , Doença de Crohn/imunologia , Doença de Crohn/fisiopatologia , Dinamarca , Fezes , Feminino , Alemanha , Humanos , Complexo Antígeno L1 Leucocitário/análise , Masculino , Gravidade do Paciente , Análise de Sequência de DNA , Fumar/imunologia , Gêmeos Monozigóticos
3.
Nat Immunol ; 20(12): 1644-1655, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31636468

RESUMO

Invariant natural killer T (iNKT) cells recognize activating self and microbial lipids presented by CD1d. CD1d can also bind non-activating lipids, such as sphingomyelin. We hypothesized that these serve as endogenous regulators and investigated humans and mice deficient in acid sphingomyelinase (ASM), an enzyme that degrades sphingomyelin. We show that ASM absence in mice leads to diminished CD1d-restricted antigen presentation and iNKT cell selection in the thymus, resulting in decreased iNKT cell levels and resistance to iNKT cell-mediated inflammatory conditions. Defective antigen presentation and decreased iNKT cells are also observed in ASM-deficient humans with Niemann-Pick disease, and ASM activity in healthy humans correlates with iNKT cell phenotype. Pharmacological ASM administration facilitates antigen presentation and restores the levels of iNKT cells in ASM-deficient mice. Together, these results demonstrate that control of non-agonistic CD1d-associated lipids is critical for iNKT cell development and function in vivo and represents a tight link between cellular sphingolipid metabolism and immunity.


Assuntos
Inflamação/imunologia , Células T Matadoras Naturais/imunologia , Doenças de Niemann-Pick/genética , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/imunologia , Timo/imunologia , Animais , Apresentação do Antígeno , Antígenos CD1d/metabolismo , Diferenciação Celular , Seleção Clonal Mediada por Antígeno , Terapia de Reposição de Enzimas , Humanos , Ativação Linfocitária , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esfingomielina Fosfodiesterase/genética , Esfingomielinas/metabolismo
4.
Front Immunol ; 10: 569, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30972064

RESUMO

The functional plasticity and anti-tumor potential of human γδ T cells have been widely studied. However, the epigenetic regulation of γδ T-cell/tumor cell interactions has been poorly investigated. In the present study, we show that treatment with the histone deacetylase inhibitor Valproic acid (VPA) significantly enhanced the expression and/or release of the NKG2D ligands MICA, MICB and ULBP-2, but not ULBP-1 in the pancreatic carcinoma cell line Panc89 and the prostate carcinoma cell line PC-3. Under in vitro tumor co-culture conditions, the expression of full length and the truncated form of the NKG2D receptor in γδ T cells was significantly downregulated. Furthermore, using a newly established flow cytometry-based method to analyze histone acetylation (H3K9ac) in γδ T cells, we showed constitutive H3K9aclow and inducible H3K9achigh expression in Vδ2 T cells. The detailed analysis of H3K9aclow Vδ2 T cells revealed a significant reversion of TEMRA to TEM phenotype during in vitro co-culture with pancreatic ductal adenocarcinoma cells. Our study uncovers novel mechanisms of how epigenetic modifiers modulate γδ T-cell differentiation during interaction with tumor cells. This information is important when considering combination therapy of VPA with the γδ T-cell-based immunotherapy for the treatment of certain types of cancer.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Linfócitos Intraepiteliais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Ácido Valproico/farmacologia , Acetilação , Linhagem Celular Tumoral , Proteínas Ligadas por GPI/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Histonas/metabolismo , Humanos , Memória Imunológica/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária/imunologia , Masculino , Células PC-3 , Neoplasias Pancreáticas/imunologia , Neoplasias da Próstata/imunologia
5.
Gut ; 68(1): 25-39, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29730603

RESUMO

OBJECTIVE: Vedolizumab, a monoclonal antibody directed against the integrin heterodimer α4ß7, is approved for the treatment of Crohn's disease and ulcerative colitis. The efficacy of vedolizumab has been suggested to result from inhibition of intestinal T cell trafficking although human data to support this conclusion are scarce. We therefore performed a comprehensive analysis of vedolizumab-induced alterations in mucosal and systemic immunity in patients with inflammatory bowel disease (IBD), using anti-inflammatory therapy with the TNFα antibody infliximab as control. DESIGN: Immunophenotyping, immunohistochemistry, T cell receptor profiling and RNA sequencing were performed using blood and colonic biopsies from patients with IBD before and during treatment with vedolizumab (n=18) or, as control, the anti-TNFα antibody infliximab (n=20). Leucocyte trafficking in vivo was assessed using single photon emission computed tomography and endomicroscopy. RESULTS: Vedolizumab was not associated with alterations in the abundance or phenotype of lamina propria T cells and did not affect the mucosal T cell repertoire or leucocyte trafficking in vivo. Surprisingly, however, α4ß7 antibody treatment was associated with substantial effects on innate immunity including changes in macrophage populations and pronounced alterations in the expression of molecules involved in microbial sensing, chemoattraction and regulation of the innate effector response. These effects were specific to vedolizumab, not observed in response to the TNFα antibody infliximab, and associated with inhibition of intestinal inflammation. CONCLUSION: Our findings suggest that modulation of innate immunity contributes to the therapeutic efficacy of vedolizumab in IBD. TRIAL REGISTRATION NUMBER: NCT02694588.


Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Anticorpos Monoclonais Humanizados/uso terapêutico , Fármacos Gastrointestinais/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/imunologia , Adulto , Biópsia , Estudos de Casos e Controles , Feminino , Humanos , Imuno-Histoquímica , Imunofenotipagem , Infliximab/uso terapêutico , Integrinas/antagonistas & inibidores , Masculino , Fenótipo , Estudos Prospectivos , Análise de Sequência de RNA , Linfócitos T/imunologia , Tomografia Computadorizada de Emissão de Fóton Único
6.
BMC Biotechnol ; 17(1): 61, 2017 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-28693542

RESUMO

BACKGROUND: The T-cell receptor (TCR), located on the surface of T cells, is responsible for the recognition of the antigen-major histocompatibility complex, leading to the initiation of an inflammatory response. Analysing the TCR repertoire may help to gain a better understanding of the immune system features and of the aetiology and progression of diseases, in particular those with unknown antigenic triggers. The extreme diversity of the TCR repertoire represents a major analytical challenge; this has led to the development of specialized methods which aim to characterize the TCR repertoire in-depth. Currently, next generation sequencing based technologies are most widely employed for the high-throughput analysis of the immune cell repertoire. RESULTS: Here, we report on the latest methodological advancements in the field by describing and comparing the available tools; from the choice of the starting material and library preparation method, to the sequencing technologies and data analysis. Finally, we provide a practical example and our own experience by reporting some exemplary results from a small internal benchmark study, where current approaches from the literature and the market are employed and compared. CONCLUSIONS: Several valid methods for clonotype identification and TCR repertoire analysis exist, however, a gold standard method for the field has not yet been identified. Depending on the purpose of the scientific study, some approaches may be more suitable than others. Finally, due to possible method specific biases, scientists must be careful when comparing results obtained using different methods.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Linfócitos T/imunologia , Regiões Determinantes de Complementaridade/genética , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Linfócitos T/fisiologia
7.
Front Mol Neurosci ; 10: 118, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28496400

RESUMO

The low-density lipoprotein receptor-related protein 1, LRP1, interacts with APP and affects its processing. This is assumed to be mostly caused by the impact of LRP1 on APP endocytosis. More recently, also an interaction of APP and LRP1 early in the secretory pathway was reported whereat retention of LRP1 in the ER leads to decreased APP cell surface levels and in turn, to reduced Aß secretion. Here, we extended the biochemical and immunocytochemical analyses by showing via live cell imaging analyses in primary neurons that LRP1 and APP are transported only partly in common (one third) but to a higher degree in distinct fast axonal transport vesicles. Interestingly, co-expression of LRP1 and APP caused a change of APP transport velocities, indicating that LRP1 recruits APP to a specific type of fast axonal transport vesicles. In contrast lowered levels of LRP1 facilitated APP transport. We further show that monomeric and dimeric APP exhibit similar transport characteristics and that both are affected by LRP1 in a similar way, by slowing down APP anterograde transport and increasing its endocytosis rate. In line with this, a knockout of LRP1 in CHO cells and in primary neurons caused an increase of monomeric and dimeric APP surface localization and in turn accelerated shedding by meprin ß and ADAM10. Notably, a choroid plexus specific LRP1 knockout caused a much higher secretion of sAPP dimers into the cerebrospinal fluid compared to sAPP monomers. Together, our data show that LRP1 functions as a sorting receptor for APP, regulating its cell surface localization and thereby its processing by ADAM10 and meprin ß, with the latter exhibiting a preference for APP in its dimeric state.

8.
Clin Immunol ; 159(2): 128-33, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25988859

RESUMO

The human host and the intestinal microbiota co-exist in a mutually beneficial relationship, which contributes to host and microbial metabolism as well as maturation of the host's immune system, among many other pathways (Tremaroli and Backhed, 2012; Hooper et al., 2012). At mucosal surfaces, and particularly in the intestine, the commensal microbiota provides 'colonization resistance' to invading pathogens and maintains homeostasis through microbial regulation of mucosal innate and adaptive immunity (Renz et al., 2012). Recent evidence suggests that natural killer T cells (NKT cells), a subgroup of lipid-reactive T cells, play central roles in bidirectional interactions between the host and the commensal microbiota, which govern intestinal homeostasis and prevent inflammation. Here, we provide a brief overview of recently identified pathways of commensal microbial regulation of NKT cells, discuss feedback mechanisms of NKT cell-dependent control of microbial colonization and composition, and highlight the critical role of host-microbial cross-talk for prevention of NKT cell-dependent mucosal inflammation.


Assuntos
Citocinas/imunologia , Microbioma Gastrointestinal/imunologia , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Células T Matadoras Naturais/imunologia , Diferenciação Celular , Homeostase/imunologia , Humanos , Inflamação , Doenças Inflamatórias Intestinais/microbiologia , Mucosa Intestinal/microbiologia , Intestinos/imunologia , Intestinos/microbiologia
9.
Nature ; 509(7501): 497-502, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24717441

RESUMO

The mechanisms by which mucosal homeostasis is maintained are of central importance to inflammatory bowel disease. Critical to these processes is the intestinal epithelial cell (IEC), which regulates immune responses at the interface between the commensal microbiota and the host. CD1d presents self and microbial lipid antigens to natural killer T (NKT) cells, which are involved in the pathogenesis of colitis in animal models and human inflammatory bowel disease. As CD1d crosslinking on model IECs results in the production of the important regulatory cytokine interleukin (IL)-10 (ref. 9), decreased epithelial CD1d expression--as observed in inflammatory bowel disease--may contribute substantially to intestinal inflammation. Here we show in mice that whereas bone-marrow-derived CD1d signals contribute to NKT-cell-mediated intestinal inflammation, engagement of epithelial CD1d elicits protective effects through the activation of STAT3 and STAT3-dependent transcription of IL-10, heat shock protein 110 (HSP110; also known as HSP105), and CD1d itself. All of these epithelial elements are critically involved in controlling CD1d-mediated intestinal inflammation. This is demonstrated by severe NKT-cell-mediated colitis upon IEC-specific deletion of IL-10, CD1d, and its critical regulator microsomal triglyceride transfer protein (MTP), as well as deletion of HSP110 in the radioresistant compartment. Our studies thus uncover a novel pathway of IEC-dependent regulation of mucosal homeostasis and highlight a critical role of IL-10 in the intestinal epithelium, with broad implications for diseases such as inflammatory bowel disease.


Assuntos
Antígenos CD1d/imunologia , Células Epiteliais/imunologia , Imunidade nas Mucosas/imunologia , Interleucina-10/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Animais , Proteínas de Transporte/metabolismo , Colite/imunologia , Colite/patologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Feminino , Proteínas de Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP110/metabolismo , Humanos , Inflamação/imunologia , Inflamação/patologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Interleucina-10/genética , Masculino , Camundongos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Oxazolona , Fator de Transcrição STAT3/metabolismo
10.
Biol Chem ; 395(1): 61-81, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23999493

RESUMO

Lipids are not only a central part of human metabolism but also play diverse and critical roles in the immune system. As such, they can act as ligands of lipid-activated nuclear receptors, control inflammatory signaling through bioactive lipids such as prostaglandins, leukotrienes, lipoxins, resolvins, and protectins, and modulate immunity as intracellular phospholipid- or sphingolipid-derived signaling mediators. In addition, lipids can serve as antigens and regulate immunity through the activation of lipid-reactive T cells, which is the topic of this review. We will provide an overview of the mechanisms of lipid antigen presentation, the biology of lipid-reactive T cells, and their contribution to immunity.


Assuntos
Antígenos/imunologia , Lipídeos/imunologia , Animais , Humanos , Inflamação/imunologia , Transdução de Sinais
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