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1.
Nat Immunol ; 2024 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-39438660

RESUMO

Precision medicine in immune-mediated inflammatory diseases (IMIDs) requires a cellular understanding of treatment response. We describe a therapeutic atlas for Crohn's disease (CD) and ulcerative colitis (UC) following adalimumab, an anti-tumour necrosis factor (anti-TNF) treatment. We generated ~1 million single-cell transcriptomes, organised into 109 cell states, from 216 gut biopsies (41 subjects), revealing disease-specific differences. A systems biology-spatial analysis identified granuloma signatures in CD and interferon (IFN)-response signatures localising to T cell aggregates and epithelial damage in CD and UC. Pretreatment differences in epithelial and myeloid compartments were associated with remission outcomes in both diseases. Longitudinal comparisons demonstrated disease progression in nonremission: myeloid and T cell perturbations in CD and increased multi-cellular IFN signalling in UC. IFN signalling was also observed in rheumatoid arthritis (RA) synovium with a lymphoid pathotype. Our therapeutic atlas represents the largest cellular census of perturbation with the most common biologic treatment, anti-TNF, across multiple inflammatory diseases.

2.
PLoS Biol ; 21(2): e3001954, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36745597

RESUMO

Fibroblasts are known for their ability to make and modify the extracellular matrix. However, there is more to them than meets the eye. It is now clear that they help define tissue microenvironments and support immune responses in organs. As technology advances, we have started to uncover the secrets of fibroblasts. In this Essay, we present fibroblasts as not only the builders and renovators of tissue environments but also the rheostat cells for immune circuits. Although they perform location-specific functions, they do not have badges of fixed identity. Instead, they display a spectrum of functional states and can swing between these states depending on the needs of the organ. As fibroblasts participate in a range of activities both in health and disease, finding the key factors that alter their development and functional states will be an important goal to restore homeostasis in maladapted tissues.


Assuntos
Matriz Extracelular , Fibroblastos , Biologia
3.
Nature ; 570(7760): 246-251, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31142839

RESUMO

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune-mediated inflammatory diseases (IMIDs)1,2. However, it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue-driven processes observed in IMIDs, such as inflammation and damage3-5. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of fibroblast activation protein-α (FAPα)+ fibroblasts suppressed both inflammation and bone erosions in mouse models of resolving and persistent arthritis. Single-cell transcriptional analysis identified two distinct fibroblast subsets within the FAPα+ population: FAPα+THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+THY1- destructive fibroblasts restricted to the synovial lining layer. When adoptively transferred into the joint, FAPα+THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation, whereas transfer of FAPα+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell-based therapies aimed at modulating inflammation and tissue damage.


Assuntos
Artrite Reumatoide/patologia , Fibroblastos/patologia , Animais , Osso e Ossos/patologia , Endopeptidases , Feminino , Fibroblastos/classificação , Fibroblastos/metabolismo , Gelatinases/metabolismo , Humanos , Inflamação/patologia , Articulações/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , RNA-Seq , Serina Endopeptidases/metabolismo , Análise de Célula Única , Membrana Sinovial/patologia , Antígenos Thy-1/metabolismo
4.
PLoS Comput Biol ; 18(6): e1010112, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35731827

RESUMO

Cell-cell communication is mediated by many soluble mediators, including over 40 cytokines. Cytokines, e.g. TNF, IL1ß, IL5, IL6, IL12 and IL23, represent important therapeutic targets in immune-mediated inflammatory diseases (IMIDs), such as inflammatory bowel disease (IBD), psoriasis, asthma, rheumatoid and juvenile arthritis. The identification of cytokines that are causative drivers of, and not just associated with, inflammation is fundamental for selecting therapeutic targets that should be studied in clinical trials. As in vitro models of cytokine interactions provide a simplified framework to study complex in vivo interactions, and can easily be perturbed experimentally, they are key for identifying such targets. We present a method to extract a minimal, weighted cytokine interaction network, given in vitro data on the effects of the blockage of single cytokine receptors on the secretion rate of other cytokines. Existing biological network inference methods typically consider the correlation structure of the underlying dataset, but this can make them poorly suited for highly connected, non-linear cytokine interaction data. Our method uses ordinary differential equation systems to represent cytokine interactions, and efficiently computes the configuration with the lowest Akaike information criterion value for all possible network configurations. It enables us to study indirect cytokine interactions and quantify inhibition effects. The extracted network can also be used to predict the combined effects of inhibiting various cytokines simultaneously. The model equations can easily be adjusted to incorporate more complicated dynamics and accommodate temporal data. We validate our method using synthetic datasets and apply our method to an experimental dataset on the regulation of IL23, a cytokine with therapeutic relevance in psoriasis and IBD. We validate several model predictions against experimental data that were not used for model fitting. In summary, we present a novel method specifically designed to efficiently infer cytokine interaction networks from cytokine perturbation data in the context of IMIDs.


Assuntos
Doenças Inflamatórias Intestinais , Psoríase , Citocinas , Humanos , Inflamação , Psoríase/tratamento farmacológico , Receptores de Citocinas
5.
J Pharmacokinet Pharmacodyn ; 49(5): 539-556, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35933452

RESUMO

Physiologically-based pharmacokinetic and cellular kinetic models are used extensively to predict concentration profiles of drugs or adoptively transferred cells in patients and laboratory animals. Models are fit to data by the numerical optimisation of appropriate parameter values. When quantities such as the area under the curve are all that is desired, only a close qualitative fit to data is required. When the biological interpretation of the model that produced the fit is important, an assessment of uncertainties is often also warranted. Often, a goal of fitting PBPK models to data is to estimate parameter values, which can then be used to assess characteristics of the fit system or applied to inform new modelling efforts and extrapolation, to inform a prediction under new conditions. However, the parameters that yield a particular model output may not necessarily be unique, in which case the parameters are said to be unidentifiable. We show that the parameters in three published physiologically-based pharmacokinetic models are practically (deterministically) unidentifiable and that it is challenging to assess the associated parameter uncertainty with simple curve fitting techniques. This result could affect many physiologically-based pharmacokinetic models, and we advocate more widespread use of thorough techniques and analyses to address these issues, such as established Markov Chain Monte Carlo and Bayesian methodologies. Greater handling and reporting of uncertainty and identifiability of fit parameters would directly and positively impact interpretation and translation for physiologically-based model applications, enhancing their capacity to inform new model development efforts and extrapolation in support of future clinical decision-making.


Assuntos
Modelos Biológicos , Animais , Teorema de Bayes , Cadeias de Markov , Método de Monte Carlo , Incerteza
6.
Proc Natl Acad Sci U S A ; 116(27): 13490-13497, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31213547

RESUMO

Resident fibroblasts at sites of infection, chronic inflammation, or cancer undergo phenotypic and functional changes to support leukocyte migration and, in some cases, aggregation into tertiary lymphoid structures (TLS). The molecular programming that shapes these changes and the functional requirements of this population in TLS development are unclear. Here, we demonstrate that external triggers at mucosal sites are able to induce the progressive differentiation of a population of podoplanin (pdpn)-positive stromal cells into a network of immunofibroblasts that are able to support the earliest phases of TLS establishment. This program of events, that precedes lymphocyte infiltration in the tissue, is mediated by paracrine and autocrine signals mainly regulated by IL13. This initial fibroblast network is expanded and stabilized, once lymphocytes are recruited, by the local production of the cytokines IL22 and lymphotoxin. Interfering with this regulated program of events or depleting the immunofibroblasts in vivo results in abrogation of local pathology, demonstrating the functional role of immunofibroblasts in supporting TLS maintenance in the tissue and suggesting novel therapeutic targets in TLS-associated diseases.


Assuntos
Fibroblastos/patologia , Estruturas Linfoides Terciárias/patologia , Animais , Modelos Animais de Doenças , Citometria de Fluxo , Imunofluorescência , Humanos , Interleucina-13/metabolismo , Interleucinas/metabolismo , Linfócitos/patologia , Camundongos , Glândulas Salivares/patologia , Interleucina 22
7.
Gut ; 70(6): 1023-1036, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33037057

RESUMO

OBJECTIVE: Dysregulated immune responses are the cause of IBDs. Studies in mice and humans suggest a central role of interleukin (IL)-23-producing mononuclear phagocytes in disease pathogenesis. Mechanistic insights into the regulation of IL-23 are prerequisite for selective IL-23 targeting therapies as part of personalised medicine. DESIGN: We performed transcriptomic analysis to investigate IL-23 expression in human mononuclear phagocytes and peripheral blood mononuclear cells. We investigated the regulation of IL-23 expression and used single-cell RNA sequencing to derive a transcriptomic signature of hyperinflammatory monocytes. Using gene network correlation analysis, we deconvolved this signature into components associated with homeostasis and inflammation in patient biopsy samples. RESULTS: We characterised monocyte subsets of healthy individuals and patients with IBD that express IL-23. We identified autosensing and paracrine sensing of IL-1α/IL-1ß and IL-10 as key cytokines that control IL-23-producing monocytes. Whereas Mendelian genetic defects in IL-10 receptor signalling induced IL-23 secretion after lipopolysaccharide stimulation, whole bacteria exposure induced IL-23 production in controls via acquired IL-10 signalling resistance. We found a transcriptional signature of IL-23-producing inflammatory monocytes that predicted both disease and resistance to antitumour necrosis factor (TNF) therapy and differentiated that from an IL-23-associated lymphocyte differentiation signature that was present in homeostasis and in disease. CONCLUSION: Our work identifies IL-10 and IL-1 as critical regulators of monocyte IL-23 production. We differentiate homeostatic IL-23 production from hyperinflammation-associated IL-23 production in patients with severe ulcerating active Crohn's disease and anti-TNF treatment non-responsiveness. Altogether, we identify subgroups of patients with IBD that might benefit from IL-23p19 and/or IL-1α/IL-1ß-targeting therapies upstream of IL-23.


Assuntos
Resistência a Medicamentos/genética , Doenças Inflamatórias Intestinais/genética , Interleucina-10/genética , Subunidade p19 da Interleucina-23/biossíntese , Subunidade p19 da Interleucina-23/genética , Monócitos/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Comunicação Autócrina , Células Cultivadas , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Homeostase/genética , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Interleucina-10/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Comunicação Parácrina , Receptores de Interleucina-10/antagonistas & inibidores , Receptores de Interleucina-10/metabolismo , Transdução de Sinais/genética , Transcriptoma , Fator de Necrose Tumoral alfa/efeitos adversos , Adulto Jovem
8.
Immunology ; 163(1): 105-111, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33502012

RESUMO

Whether resident and recruited myeloid cells may impair or aid healing of acute skin wounds remains a debated question. To begin to address this, we examined the importance of CD11c+ myeloid cells in the early activation of skin wound repair. We find that an absence of CD11c+ cells delays wound closure and epidermal proliferation, likely due to defects in the activation of the IL-23-IL-22 axis that is required for wound healing.


Assuntos
Antígenos CD11/deficiência , Células Dendríticas/imunologia , Pele/imunologia , Cicatrização , Ferimentos e Lesões/imunologia , Animais , Antígenos CD11/genética , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Cinética , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pele/metabolismo , Pele/patologia , Ferimentos e Lesões/genética , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologia
9.
Br J Cancer ; 124(10): 1661-1669, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33723392

RESUMO

BACKGROUND: Immune checkpoint blockers (ICBs) activate CD8+ T cells, eliciting both anti-cancer activity and immune-related adverse events (irAEs). The relationship of irAEs with baseline parameters and clinical outcome is unclear. METHODS: Retrospective evaluation of irAEs on survival was performed across primary (N = 144) and secondary (N = 211) independent cohorts of patients with metastatic melanoma receiving single agent (pembrolizumab/nivolumab-sICB) or combination (nivolumab and ipilimumab-cICB) checkpoint blockade. RNA from pre-treatment and post-treatment CD8+ T cells was sequenced and differential gene expression according to irAE development assessed. RESULTS: 58.3% of patients developed early irAEs and this was associated with longer progression-free (PFS) and overall survival (OS) across both cohorts (log-rank test, OS: P < 0.0001). Median survival for patients without irAEs was 16.6 months (95% CI: 10.9-33.4) versus not-reached (P = 2.8 × 10-6). Pre-treatment monocyte and neutrophil counts, but not BMI, were additional predictors of clinical outcome. Differential expression of numerous gene pathway members was observed in CD8+ T cells according to irAE development, and patients not developing irAEs demonstrating upregulated CXCR1 pre- and post-treatment. CONCLUSIONS: Early irAE development post-ICB is associated with favourable survival in MM. Development of irAEs is coupled to expression of numerous gene pathways, suggesting irAE development in-part reflects baseline immune activation.


Assuntos
Doenças Autoimunes/induzido quimicamente , Linfócitos T CD8-Positivos/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/efeitos adversos , Melanoma/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/efeitos adversos , Antineoplásicos Imunológicos/uso terapêutico , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/epidemiologia , Doenças Autoimunes/genética , Autoimunidade/efeitos dos fármacos , Autoimunidade/genética , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/efeitos adversos , Masculino , Melanoma/imunologia , Melanoma/mortalidade , Melanoma/patologia , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Intervalo Livre de Progressão , Estudos Retrospectivos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Análise de Sobrevida , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologia , Resultado do Tratamento , Reino Unido/epidemiologia
10.
Nat Immunol ; 10(10): 1118-24, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19749763

RESUMO

Natural killer (NK) cells are a subset of lymphocytes crucial for innate immunity and modification of adaptive immune responses. In contrast to commitment to the T cell or B cell lineage, little is known about NK cell lineage commitment. Here we show that the basic leucine zipper (bZIP) transcription factor E4BP4 (also called NFIL3) is essential for generation of the NK cell lineage. E4BP4-deficient mice (Nfil3(-/-); called 'E4bp4(-/-)' here) had B cells, T cells and NKT cells but specifically lack NK cells and showed severely impaired NK cell-mediated cytotoxicity. Overexpression of E4bp4 was sufficient to increase NK cell production from hematopoietic progenitor cells. E4BP4 acted in a cell-intrinsic manner 'downstream' of the interleukin 15 receptor (IL-15R) and through the transcription factor Id2. E4bp4(-/-) mice may provide a model for definitive analysis of the contribution of NK cells to immune responses and pathologies.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Diferenciação Celular/imunologia , Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/citologia , Animais , Linfócitos B/imunologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Citotoxicidade Imunológica/genética , Citometria de Fluxo , Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/imunologia , Proteína 2 Inibidora de Diferenciação/metabolismo , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Receptores de Interleucina-15/genética , Receptores de Interleucina-15/imunologia , Receptores de Interleucina-15/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia
11.
Proc Natl Acad Sci U S A ; 115(52): 13204-13209, 2018 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-30530663

RESUMO

Cell communication is primarily regulated by secreted proteins, whose inhomogeneous secretion often indicates physiological disorder. Parallel monitoring of innate protein-secretion kinetics from individual cells is thus crucial to unravel systemic malfunctions. Here, we report a label-free, high-throughput method for parallel, in vitro, and real-time analysis of specific single-cell signaling using hyperspectral photonic crystal resonant technology. Heterogeneity in physiological thrombopoietin expression from individual HepG2 liver cells in response to platelet desialylation was quantified demonstrating how mapping real-time protein secretion can provide a simple, yet powerful approach for studying complex physiological systems regulating protein production at single-cell resolution.


Assuntos
Técnicas Biossensoriais/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Rim/metabolismo , Fótons , Análise de Célula Única/métodos , Trombopoetina/metabolismo , Animais , Técnicas Biossensoriais/métodos , Células Cultivadas , Cricetinae , Células Hep G2 , Humanos , Transdução de Sinais
12.
Nature ; 576(7786): 215-216, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31822827
13.
Blood ; 129(18): 2507-2518, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28202459

RESUMO

Follicular lymphoma (FL) is the most frequent indolent lymphoma and is characterized by the accumulation of germinal center-derived malignant B cells engaged in a bidirectional crosstalk with their supportive microenvironment in invaded lymph nodes (LNs) and bone marrow (BM). T follicular helper (TFH) cells and infiltrating stromal cells have been shown to favor FL B-cell growth, but the mechanisms of their protumoral effect and how the LN/BM microenvironment is converted into a lymphoma-permissive cell niche remain poorly understood. We demonstrated here that FL-infiltrating LN and BM stromal cells overexpressed CXCL12 in situ. Interleukin-4 high (IL-4hi) FL-TFH cells, unlike FL B cells themselves, triggered CXCL12 upregulation in human stromal cell precursors. In agreement, expression of CXCL12 was associated with IL-4 expression and signaling within the FL BM and LN niches. This IL-4/CXCL12 axis was amplified in activated lymphoid stromal cells as shown in our in vitro model of human lymphoid stroma differentiation and in an inducible mouse model of ectopic lymphoid organ formation. Finally, CXCL12 triggered primary FL B-cell activation, migration, and adhesion, a process antagonized by BTK and PI3K inhibitors. These data identified the IL-4/CXCL12 loop as a previously unrecognized pathway involved in lymphoid stroma polarization and as a potential therapeutic target in FL patients.


Assuntos
Medula Óssea/imunologia , Quimiocina CXCL12/imunologia , Interleucina-4/imunologia , Linfonodos/imunologia , Linfoma Folicular/imunologia , Transdução de Sinais/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Medula Óssea/patologia , Movimento Celular/genética , Movimento Celular/imunologia , Quimiocina CXCL12/genética , Feminino , Humanos , Interleucina-4/genética , Linfonodos/patologia , Linfoma Folicular/genética , Linfoma Folicular/patologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Células Estromais/imunologia , Células Estromais/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia
14.
J Biol Chem ; 292(50): 20683-20693, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29066622

RESUMO

Programmed death ligand-1 (PD-L1) is a critical regulator of T cell function contributing to peripheral immune tolerance. Although it has been shown that posttranscriptional regulatory mechanisms control PD-L1 expression in cancer, it remains unknown whether such regulatory loops operate also in non-transformed cells. Here we studied PD-L1 expression in human dermal lymphatic endothelial cells (HDLECs), which play key roles in immunity and cancer. Treatment of HDLECs with the pro-inflammatory cytokines IFN-γ and TNF-α synergistically up-regulated PD-L1 expression. IFN-γ and TNF-α also affected expression of several microRNAs (miRNAs) that have the potential to suppress PD-L1 expression. The most highly up-regulated miRNA following IFN-γ and TNF-α treatment in HDLECs was miR-155, which has a central role in the immune system and cancer. Induction of miR-155 was driven by TNF-α, the effect of which was significantly enhanced by IFN-γ. The PD-L1 3'-UTR contains two functional miR-155-binding sites. Endogenous miR-155 controlled the kinetics and maximal levels of PD-L1 induction upon IFN-γ and TNF-α treatments. We obtained similar findings in dermal fibroblasts, demonstrating that the IFN-γ/TNF-α/miR-155/PD-L1 pathway is not restricted to HDLECs. These results reveal miR-155 as a critical component of an inflammation-induced regulatory loop controlling PD-L1 expression in primary cells.


Assuntos
Antígeno B7-H1/antagonistas & inibidores , Derme/metabolismo , Endotélio Linfático/metabolismo , Regulação da Expressão Gênica , Interferon gama/metabolismo , MicroRNAs/agonistas , Fator de Necrose Tumoral alfa/metabolismo , Regiões 3' não Traduzidas , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Derme/citologia , Derme/imunologia , Endotélio Linfático/citologia , Endotélio Linfático/imunologia , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Interferon gama/genética , Cinética , MicroRNAs/química , MicroRNAs/metabolismo , Microscopia de Fluorescência , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Elementos de Resposta
15.
PLoS Comput Biol ; 13(2): e1005351, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28158307

RESUMO

A calibrated computational model reflects behaviours that are expected or observed in a complex system, providing a baseline upon which sensitivity analysis techniques can be used to analyse pathways that may impact model responses. However, calibration of a model where a behaviour depends on an intervention introduced after a defined time point is difficult, as model responses may be dependent on the conditions at the time the intervention is applied. We present ASPASIA (Automated Simulation Parameter Alteration and SensItivity Analysis), a cross-platform, open-source Java toolkit that addresses a key deficiency in software tools for understanding the impact an intervention has on system behaviour for models specified in Systems Biology Markup Language (SBML). ASPASIA can generate and modify models using SBML solver output as an initial parameter set, allowing interventions to be applied once a steady state has been reached. Additionally, multiple SBML models can be generated where a subset of parameter values are perturbed using local and global sensitivity analysis techniques, revealing the model's sensitivity to the intervention. To illustrate the capabilities of ASPASIA, we demonstrate how this tool has generated novel hypotheses regarding the mechanisms by which Th17-cell plasticity may be controlled in vivo. By using ASPASIA in conjunction with an SBML model of Th17-cell polarisation, we predict that promotion of the Th1-associated transcription factor T-bet, rather than inhibition of the Th17-associated transcription factor RORγt, is sufficient to drive switching of Th17 cells towards an IFN-γ-producing phenotype. Our approach can be applied to all SBML-encoded models to predict the effect that intervention strategies have on system behaviour. ASPASIA, released under the Artistic License (2.0), can be downloaded from http://www.york.ac.uk/ycil/software.


Assuntos
Algoritmos , Modelos Biológicos , Linguagens de Programação , Software , Biologia de Sistemas/métodos , Simulação por Computador
16.
J Immunol ; 195(9): 4257-4263, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26378073

RESUMO

Adaptive immunity critically depends on the functional compartmentalization of secondary lymphoid organs. Mesenchymal stromal cells create and maintain specialized niches that support survival, activation, and expansion of T and B cells, and integrated analysis of lymphocytes and their niche has been instrumental in understanding adaptive immunity. Lymphoid organs are also home to type 3 innate lymphoid cells (ILC3), innate effector cells essential for barrier immunity. However, a specialized stromal niche for ILC3 has not been identified. A novel lineage-tracing approach now identifies a subset of murine fetal lymphoid tissue organizer cells that gives rise exclusively to adult marginal reticular cells. Moreover, both cell types are conserved from mice to humans and colocalize with ILC3 in secondary lymphoid tissues throughout life. In sum, we provide evidence that fetal stromal organizers give rise to adult marginal reticular cells and form a dedicated stromal niche for innate ILC3 in adaptive lymphoid organs.


Assuntos
Imunidade Inata/imunologia , Linfócitos/imunologia , Tecido Linfoide/imunologia , Nicho de Células-Tronco/imunologia , Células Estromais/imunologia , Animais , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Quimiocinas/imunologia , Quimiocinas/metabolismo , Feminino , Feto/citologia , Citometria de Fluxo , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Linfócitos/metabolismo , Tecido Linfoide/citologia , Tecido Linfoide/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Camundongos Transgênicos , Microscopia Confocal , Ligante RANK/genética , Ligante RANK/imunologia , Ligante RANK/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nicho de Células-Tronco/genética , Células Estromais/citologia , Células Estromais/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
17.
Dev Dyn ; 245(1): 34-46, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26447465

RESUMO

BACKGROUND: Lin28 proteins are post-transcriptional regulators of gene expression with multiple roles in development and the regulation of pluripotency in stem cells. Much attention has focussed on Lin28 proteins as negative regulators of let-7 miRNA biogenesis; a function that is conserved in several animal groups and in multiple processes. However, there is increasing evidence that Lin28 proteins have additional roles, distinct from regulation of let-7 abundance. We have previously demonstrated that lin28 proteins have functions associated with the regulation of early cell lineage specification in Xenopus embryos, independent of a lin28/let-7 regulatory axis. However, the nature of lin28 targets in Xenopus development remains obscure. RESULTS: Here, we show that mir-17∼92 and mir-106∼363 cluster miRNAs are down-regulated in response to lin28 knockdown, and RNAs from these clusters are co-expressed with lin28 genes during germ layer specification. Mature miRNAs derived from pre-mir-363 are most sensitive to lin28 inhibition. We demonstrate that lin28a binds to the terminal loop of pre-mir-363 with an affinity similar to that of let-7, and that this high affinity interaction requires to conserved a GGAG motif. CONCLUSIONS: Our data suggest a novel function for amphibian lin28 proteins as positive regulators of mir-17∼92 family miRNAs.


Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Embrião não Mamífero/fisiologia , Camadas Germinativas/metabolismo , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Xenopus , Proteínas de Xenopus/genética
18.
Biochemistry ; 55(36): 5021-7, 2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27559824

RESUMO

Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumor suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity for this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterization of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have engineered a maltose binding protein fusion with Lin28, which binds let-7 miRNA with a Kd of 54.1 ± 4.2 nM, in agreement with previous data on a murine homologue. We show that human Lin28A binds miR-363 with a 1:1 stoichiometry and with a similar, if not higher, affinity (Kd = 16.6 ± 1.9 nM). Further analysis suggests that the interaction of the N-terminal cold shock domain of Lin28A with RNA is salt-dependent, supporting a model in which the cold shock domain allows the protein to sample RNA substrates through transient electrostatic interactions.


Assuntos
MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Polarização de Fluorescência , Humanos , Ligação Proteica
19.
Biomacromolecules ; 17(3): 723-34, 2016 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-26686360

RESUMO

There is a lack of hydrogel materials whose properties can be tuned at the point of use. Biological hydrogels, such as collagen, gelate at physiological temperatures; however, they are not always ideal as scaffolds because of their low mechanical strength. Their mechanics can be improved through cross-linking and chemical modification, but these methods still require further synthesis. We have demonstrated that by combining collagen with a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM), the mechanical properties can be improved while maintaining cytocompatibility. Furthermore, different concentrations of this polymer led to a range of hydrogels with shear moduli ranging from 10(5) Pa down to less than 10(2) Pa, similar to the soft tissues in the body. In addition to variable mechanical properties, the hydrogel blends have a range of micron-scale structures and porosities, which caused adipose-derived stromal cells (ADSCs) to adopt different morphologies when encapsulated within and may therefore be able to direct cell fate.


Assuntos
Resinas Acrílicas/química , Colágeno/química , Hidrogéis/síntese química , Reagentes de Ligações Cruzadas/química , Humanos , Hidrogéis/efeitos adversos , Hidrogéis/química , Fenômenos Mecânicos , Células-Tronco Mesenquimais/efeitos dos fármacos , Resistência à Tração
20.
Semin Immunol ; 24(3): 190-7, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22421575

RESUMO

The development and homeostasis of adaptive and innate lymphocytes is dependent on the stromal cytokine IL-7. The initial priming of immune responses to pathogenic challenges is executed by innate lymphoid cells (ILCs) with programmed capacity to rapidly secrete effector cytokines. How ILCs are controlled by IL-7 in distinct anatomical locale has evolved into a more complex problem as IL-7 receptor is not only expressed on ILCs, but also on surrounding neighbors, including vascular endothelium and mesenchymal cells that compete for limiting IL-7. For the generation of γδ T and B cells IL-7 is required for the production of antigen receptors, and it is likely that IL-7 performs critical function in facilitating ILC effector programming in addition to its regulatory actions on cell survival and proliferation. Most of our current understanding of the highly calibrated regulatory circuits of IL-7 function and IL-7 receptor signaling has derived from studies of adaptive, conventional lymphocytes. Here we highlight recent advances in mapping the gene circuits and cellular interactions that regulate temporospatial activities of IL-7 in diverse macro and micro niches that have direct relevance to deciphering the sphere of impact of IL-7 on ILC differentiation.


Assuntos
Linfócitos B/imunologia , Imunidade Inata , Interleucina-7/imunologia , Linfócitos T/imunologia , Animais , Linfócitos B/citologia , Comunicação Celular , Humanos , Receptores de Interleucina-7/imunologia , Linfócitos T/citologia
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