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1.
Cell ; 168(6): 1086-1100.e10, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28283063

RESUMO

Innate lymphoid cells (ILCs) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCPs). How ILCPs give rise to mature tissue-resident ILCs remains unclear. Here, we identify circulating and tissue ILCPs in humans that fail to express the transcription factors and cytokine outputs of mature ILCs but have these signature loci in an epigenetically poised configuration. Human ILCPs robustly generate all ILC subsets in vitro and in vivo. While human ILCPs express low levels of retinoic acid receptor (RAR)-related orphan receptor C (RORC) transcripts, these cells are found in RORC-deficient patients and retain potential for EOMES+ natural killer (NK) cells, interferon gamma-positive (IFN-γ+) ILC1s, interleukin (IL)-13+ ILC2s, and for IL-22+, but not for IL-17A+ ILC3s. Our results support a model of tissue ILC differentiation ("ILC-poiesis"), whereby diverse ILC subsets are generated in situ from systemically distributed ILCPs in response to local environmental signals.


Assuntos
Linfócitos/citologia , Células-Tronco/citologia , Animais , Antígenos CD34/análise , Diferenciação Celular , Linhagem da Célula , Sangue Fetal/citologia , Feto/citologia , Humanos , Imunidade Inata , Interleucina-17 , Fígado/citologia , Pulmão/citologia , Linfócitos/imunologia , Tecido Linfoide/citologia , Camundongos , Proteínas Proto-Oncogênicas c-kit/análise , Transcrição Gênica
2.
Nat Immunol ; 18(3): 313-320, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28068307

RESUMO

Notch2 and B cell antigen receptor (BCR) signaling determine whether transitional B cells become marginal zone B (MZB) or follicular B (FoB) cells in the spleen, but it is unknown how these pathways are related. We generated Taok3-/- mice, lacking the serine/threonine kinase Taok3, and found cell-intrinsic defects in the development of MZB but not FoB cells. Type 1 transitional (T1) B cells required Taok3 to rapidly respond to ligation by the Notch ligand Delta-like 1. BCR ligation by endogenous or exogenous ligands induced the surface expression of the metalloproteinase ADAM10 on T1 B cells in a Taok3-dependent manner. T1 B cells expressing surface ADAM10 were committed to becoming MZB cells in vivo, whereas T1 B cells lacking expression of ADAM10 were not. Thus, during positive selection in the spleen, BCR signaling causes immature T1 B cells to become receptive to Notch ligands via Taok3-mediated surface expression of ADAM10.


Assuntos
Proteína ADAM10/metabolismo , Imunidade Adaptativa , Secretases da Proteína Precursora do Amiloide/metabolismo , Linfócitos B/fisiologia , Diferenciação Celular , Linhagem da Célula , Centro Germinativo/imunologia , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/genética , Animais , Células Cultivadas , Seleção Clonal Mediada por Antígeno , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Receptor Notch2/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais
3.
Nat Immunol ; 21(7): 724-726, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572239
4.
Eur J Immunol ; 54(6): e2350670, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38593342

RESUMO

Chronic thromboembolic pulmonary hypertension (CTEPH) is a debilitating disease characterized by thrombotic occlusion of pulmonary arteries and vasculopathy, leading to increased pulmonary vascular resistance and progressive right-sided heart failure. Thrombotic lesions in CTEPH contain CD68+ macrophages, and increasing evidence supports their role in disease pathogenesis. Macrophages are classically divided into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages, which are involved in wound healing and tissue repair. Currently, the phenotype of macrophages and their localization within thrombotic lesions of CTEPH are largely unknown. In our study, we subclassified thrombotic lesions of CTEPH patients into developing fresh thrombi (FT) and organized thrombi (OT), based on the degree of fibrosis and remodeling. We used multiplex immunofluorescence histology to identify immune cell infiltrates in thrombotic lesions of CPTEH patients. Utilizing software-assisted cell detection and quantification, increased proportions of macrophages were observed in immune cell infiltrates of OT lesions, compared with FT. Strikingly, the proportions with a CD206+INOS- M2 phenotype were significantly higher in OT than in FT, which mainly contained unpolarized macrophages. Taken together, we observed a shift from unpolarized macrophages in FT toward an expanded population of M2 macrophages in OT, indicating a dynamic role of macrophages during CTEPH pathogenesis.


Assuntos
Hipertensão Pulmonar , Macrófagos , Embolia Pulmonar , Trombose , Humanos , Macrófagos/imunologia , Hipertensão Pulmonar/imunologia , Hipertensão Pulmonar/patologia , Feminino , Masculino , Pessoa de Meia-Idade , Embolia Pulmonar/imunologia , Embolia Pulmonar/patologia , Doença Crônica , Trombose/imunologia , Trombose/patologia , Idoso , Antígenos CD/metabolismo
5.
Immunity ; 45(6): 1285-1298, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-27939673

RESUMO

Allergic disease originates in early life and polymorphisms in interleukin-33 gene (IL33) and IL1RL1, coding for IL-33R and decoy receptor sST2, confer allergy risk. Early life T helper 2 (Th2) cell skewing and allergy susceptibility are often seen as remnants of feto-maternal symbiosis. Here we report that shortly after birth, innate lymphoid type 2 cells (ILC2s), eosinophils, basophils, and mast cells spontaneously accumulated in developing lungs in an IL-33-dependent manner. During the phase of postnatal lung alveolarization, house dust mite exposure further increased IL-33, which boosted cytokine production in ILC2s and activated CD11b+ dendritic cells (DCs). IL-33 suppressed IL-12p35 and induced OX40L in neonatal DCs, thus promoting Th2 cell skewing. Decoy sST2 had a strong preventive effect on asthma in the neonatal period, less so in adulthood. Thus, enhanced neonatal Th2 cell skewing to inhaled allergens results from postnatal hyperactivity of the IL-33 axis during a period of maximal lung remodeling.


Assuntos
Asma/imunologia , Interleucina-33/imunologia , Pulmão/crescimento & desenvolvimento , Pulmão/imunologia , Células Th2/imunologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Hipersensibilidade/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pyroglyphidae/imunologia , Transdução de Sinais/imunologia
6.
Thorax ; 79(9): 811-821, 2024 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-38373824

RESUMO

BACKGROUND: In patients with asthma, respiratory syncytial virus (RSV) infections can cause disease exacerbation by infecting the epithelial layer of the airways, inducing subsequent immune response. The type I interferon antiviral response of epithelial cells upon RSV infection is found to be reduced in asthma in most-but not all-studies. Moreover, the molecular mechanisms causing the differences in the asthmatic bronchial epithelium in response to viral infection are poorly understood. METHODS: Here, we investigated the transcriptional response to RSV infection of primary bronchial epithelial cells (pBECs) from patients with asthma (n=8) and healthy donors (n=8). The pBECs obtained from bronchial brushes were differentiated in air-liquid interface conditions and infected with RSV. After 3 days, cells were processed for single-cell RNA sequencing. RESULTS: A strong antiviral response to RSV was observed for all cell types, for all samples (p<1e-48). Most (1045) differentially regulated genes following RSV infection were found in cells transitioning to secretory cells. Goblet cells from patients with asthma showed lower expression of genes involved in the interferon response (false discovery rate <0.05), including OASL, ICAM1 and TNFAIP3. In multiciliated cells, an impairment of the signalling pathways involved in the response to RSV in asthma was observed. CONCLUSION: Our results highlight that the response to RSV infection of the bronchial epithelium in asthma and healthy airways was largely similar. However, in asthma, the response of goblet and multiciliated cells is impaired, highlighting the need for studying airway epithelial cells at high resolution in the context of asthma exacerbation.


Assuntos
Asma , Células Epiteliais , Células Caliciformes , Infecções por Vírus Respiratório Sincicial , Humanos , Asma/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Células Caliciformes/patologia , Masculino , Feminino , Células Epiteliais/virologia , Células Epiteliais/metabolismo , Mucosa Respiratória/virologia , Mucosa Respiratória/metabolismo , Adulto , Brônquios , Pessoa de Meia-Idade , Células Cultivadas , Vírus Sinciciais Respiratórios , Cílios/patologia , Estudos de Casos e Controles , Molécula 1 de Adesão Intercelular
7.
Eur Respir J ; 64(2)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38901884

RESUMO

BACKGROUND: Individual differences in susceptibility to developing asthma, a heterogeneous chronic inflammatory lung disease, are poorly understood. Whether genetics can predict asthma risk and how genetic variants modulate the complex pathophysiology of asthma are still debated. AIM: To build polygenic risk scores for asthma risk prediction and epigenomically link predictive genetic variants to pathophysiological mechanisms. METHODS: Restricted polygenic risk scores were constructed using single nucleotide variants derived from genome-wide association studies and validated using data generated in the Rotterdam Study, a Dutch prospective cohort of 14 926 individuals. Outcomes used were asthma, childhood-onset asthma, adulthood-onset asthma, eosinophilic asthma and asthma exacerbations. Genome-wide chromatin analysis data from 19 disease-relevant cell types were used for epigenomic polygenic risk score partitioning. RESULTS: The polygenic risk scores obtained predicted asthma and related outcomes, with the strongest associations observed for childhood-onset asthma (2.55 odds ratios per polygenic risk score standard deviation, area under the curve of 0.760). Polygenic risk scores allowed for the classification of individuals into high-risk and low-risk groups. Polygenic risk score partitioning using epigenomic profiles identified five clusters of variants within putative gene regulatory regions linked to specific asthma-relevant cells, genes and biological pathways. CONCLUSIONS: Polygenic risk scores were associated with asthma(-related traits) in a Dutch prospective cohort, with substantially higher predictive power observed for childhood-onset than adult-onset asthma. Importantly, polygenic risk score variants could be epigenomically partitioned into clusters of regulatory variants with different pathophysiological association patterns and effect estimates, which likely represent distinct genetically driven disease pathways. Our findings have potential implications for personalised risk mitigation and treatment strategies.


Assuntos
Asma , Epigenômica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Herança Multifatorial , Polimorfismo de Nucleotídeo Único , Humanos , Asma/genética , Feminino , Masculino , Países Baixos , Pessoa de Meia-Idade , Estudos Prospectivos , Idoso , Adulto , Fatores de Risco , Criança , Idade de Início , Medição de Risco , Estratificação de Risco Genético
8.
J Autoimmun ; 143: 103168, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38350168

RESUMO

OBJECTIVE: Altered B cell receptor (BCR) signaling has been implicated in the pathogenesis of rheumatoid arthritis (RA). Here we aimed to identify signaling aberrations in autoantibody-positive and autoantibody-negative RA patients by performing a comprehensive analysis of the BCR signaling cascade in different B cell subsets. METHODS: We first optimized phosphoflow cytometry for an in-depth analysis of BCR signaling across immunoglobulin isotypes in healthy donors. Subsequently, we compared BCR signaling in circulating B cell subsets from treatment-naïve, newly-diagnosed autoantibody-positive RA and autoantibody-negative RA patients and healthy controls (HCs). RESULTS: We observed subset-specific phosphorylation patterns of the BCR signalosome in circulating B cells from healthy donors. Compared with HCs, autoantibody-positive RA patients displayed enhanced responses to BCR stimulation for multiple signaling proteins, specifically in naïve and IgA+ memory B cells. Whereas in unstimulated healthy donor B cells, the phosphorylation status of individual signaling proteins showed only limited correlation, BCR stimulation enhanced the interconnectivity in phosphorylation within the BCR signalosome. However, this strong interconnectivity within the BCR signalosome in stimulated B cells from HCs was lost in RA, especially in autoantibody-positive RA patients. Finally, we observed strong correlations between SYK and BTK protein expression, and IgA and IgG anti-citrullinated protein antibody concentrations in serum from autoantibody-positive RA patients. CONCLUSION: Collectively, the isotype-specific analysis of multiple key components of the BCR signalosome identified aberrant BCR signaling responses in treatment-naïve autoantibody-positive RA patients, particularly in naïve B cells and IgA+ memory B cells. Our findings support differential involvement of dysregulated BCR signaling in the pathogenesis of autoantibody-positive and autoantibody-negative RA.


Assuntos
Artrite Reumatoide , Autoanticorpos , Humanos , Células B de Memória , Isotipos de Imunoglobulinas , Receptores de Antígenos de Linfócitos B , Imunoglobulina A
9.
Respir Res ; 25(1): 196, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38715030

RESUMO

BACKGROUND: The treatment response to corticosteroids in patients with sarcoidosis is highly variable. CD4+ T cells are central in sarcoid pathogenesis and their phenotype in peripheral blood (PB) associates with disease course. We hypothesized that the phenotype of circulating T cells in patients with sarcoidosis may correlate with the response to prednisone treatment. Therefore, we aimed to correlate frequencies and phenotypes of circulating T cells at baseline with the pulmonary function response at 3 and 12 months during prednisone treatment in patients with pulmonary sarcoidosis. METHODS: We used multi-color flow cytometry to quantify activation marker expression on PB T cell populations in 22 treatment-naïve patients and 21 healthy controls (HCs). Pulmonary function tests at baseline, 3 and 12 months were used to measure treatment effect. RESULTS: Patients with sarcoidosis showed an absolute forced vital capacity (FVC) increase of 14.2% predicted (± 10.6, p < 0.0001) between baseline and 3 months. Good response to prednisone (defined as absolute FVC increase of ≥ 10% predicted) was observed in 12 patients. CD4+ memory T cells and regulatory T cells from patients with sarcoidosis displayed an aberrant phenotype at baseline, compared to HCs. Good responders at 3 months had significantly increased baseline proportions of PD-1+CD4+ memory T cells and PD-1+ regulatory T cells, compared to poor responders and HCs. Moreover, decreased fractions of CD25+ cells and increased fractions of PD-1+ cells within the CD4+ memory T cell population correlated with ≥ 10% FVC increase at 12 months. During treatment, the aberrantly activated phenotype of memory and regulatory T cells reversed. CONCLUSIONS: Increased proportions of circulating PD-1+CD4+ memory T cells and PD-1+ regulatory T cells and decreased proportions of CD25+CD4+ memory T cells associate with good FVC response to prednisone in pulmonary sarcoidosis, representing promising new blood biomarkers for prednisone efficacy. TRIAL REGISTRATION: NL44805.078.13.


Assuntos
Prednisona , Receptor de Morte Celular Programada 1 , Sarcoidose Pulmonar , Linfócitos T Reguladores , Humanos , Masculino , Sarcoidose Pulmonar/tratamento farmacológico , Sarcoidose Pulmonar/sangue , Sarcoidose Pulmonar/imunologia , Sarcoidose Pulmonar/diagnóstico , Feminino , Pessoa de Meia-Idade , Prednisona/uso terapêutico , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Adulto , Resultado do Tratamento , Células T de Memória/efeitos dos fármacos , Células T de Memória/imunologia , Células T de Memória/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Glucocorticoides/uso terapêutico , Capacidade Vital/efeitos dos fármacos , Idoso
10.
BMC Genomics ; 24(1): 722, 2023 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-38030970

RESUMO

Cell type-specific differential gene expression analyses based on single-cell transcriptome datasets are sensitive to the presence of cell-free mRNA in the droplets containing single cells. This so-called ambient RNA contamination may differ between samples obtained from patients and healthy controls. Current ambient RNA correction methods were not developed specifically for single-cell differential gene expression (sc-DGE) analyses and might therefore not sufficiently correct for ambient RNA-derived signals. Here, we show that ambient RNA levels are highly sample-specific. We found that without ambient RNA correction, sc-DGE analyses erroneously identify transcripts originating from ambient RNA as cell type-specific disease-associated genes. We therefore developed a computationally lean and intuitive correction method, Fast Correction for Ambient RNA (FastCAR), optimized for sc-DGE analysis of scRNA-Seq datasets generated by droplet-based methods including the 10XGenomics Chromium platform. FastCAR uses the profile of transcripts observed in libraries that likely represent empty droplets to determine the level of ambient RNA in each individual sample, and then corrects for these ambient RNA gene expression values. FastCAR can be applied as part of the data pre-processing and QC in sc-DGE workflows comparing scRNA-Seq data in a health versus disease experimental design. We compared FastCAR with two methods previously developed to remove ambient RNA, SoupX and CellBender. All three methods identified additional genes in sc-DGE analyses that were not identified in the absence of ambient RNA correction. However, we show that FastCAR performs better at correcting gene expression values attributed to ambient RNA, resulting in a lower frequency of false-positive observations. Moreover, the use of FastCAR in a sc-DGE workflow increases the cell-type specificity of sc-DGE analyses across disease conditions.


Assuntos
Perfilação da Expressão Gênica , RNA , Humanos , RNA/metabolismo , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Transcriptoma , Projetos de Pesquisa , Análise de Célula Única/métodos
11.
J Autoimmun ; : 103120, 2023 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-37863732

RESUMO

RATIONALE: Disease course in sarcoidosis is highly variable. Bronchoalveolar lavage fluid and mediastinal lymph nodes show accumulation of activated T cells with a T-helper (Th)17.1 signature, which correlates with non-resolving sarcoidosis. We hypothesize that the peripheral blood (PB) T cell phenotype may correlate with outcome. OBJECTIVES: To compare frequencies, phenotypes and function of circulating T cell populations in sarcoidosis patients with healthy controls (HCs) and correlate these parameters with outcome. METHODS: We used multi-color flow cytometry to quantify activation marker expression on PB T cell subsets in treatment-naïve patients and HCs. The disease course was determined after 2-year follow-up. Cytokine production was measured after T cell stimulation in vitro. MEASUREMENTS AND MAIN RESULTS: We observed significant differences between patients and HCs in several T cell populations, including CD8+ and CD4+ T cells, Th1/Th17 subsets, CD4+ T memory stem cells, regulatory T cells (Tregs) and γδ T cells. Decreased frequencies of CD4+ T cells and increased frequencies of Tregs and CD8+ γδ T cells correlated with worse outcome. Naïve CD4+ T cells displayed an activated phenotype with increased CD25 expression in patients with active chronic disease at 2-year follow-up. A distinctive Treg phenotype with increased expression of CD25, CTLA4, CD69, PD-1 and CD95 correlated with chronic sarcoidosis. Upon stimulation, both naïve and memory T cells displayed a different cytokine profile in sarcoidosis compared to HCs. CONCLUSIONS: Circulating T cell subpopulations of sarcoidosis patients display phenotypic abnormalities that correlate with disease outcome, supporting a critical role of aberrant T cell activation in sarcoidosis pathogenesis.

12.
Blood ; 137(22): 3079-3092, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33512383

RESUMO

NOTCH1 gain-of-function mutations are recurrent in B-cell chronic lymphocytic leukemia (B-CLL), where they are associated with accelerated disease progression and refractoriness to chemotherapy. The specific role of NOTCH1 in the development and progression of this malignancy is unclear. Here, we assess the impact of loss of Notch signaling and pathway hyperactivation in an in vivo mouse model of CLL (IgH.TEµ) that faithfully replicates many features of the human pathology. Ablation of canonical Notch signaling using conditional gene inactivation of RBP-J in immature hematopoietic or B-cell progenitors delayed CLL induction and reduced incidence of mice developing disease. In contrast, forced expression of a dominant active form of Notch resulted in more animals developing CLL with early disease onset. Comparative analysis of gene expression and epigenetic features of Notch gain-of-function and control CLL cells revealed direct and indirect regulation of cell cycle-associated genes, which led to increased proliferation of Notch gain-of-function CLL cells in vivo. These results demonstrate that Notch signaling facilitates disease initiation and promotes CLL cell proliferation and disease progression.


Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Leucemia Linfocítica Crônica de Células B/genética , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Receptor Notch1/genética
13.
Allergy ; 78(4): 940-956, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36727912

RESUMO

Asthma is a complex and heterogeneous chronic inflammatory disease of the airways. Alongside environmental factors, asthma susceptibility is strongly influenced by genetics. Given its high prevalence and our incomplete understanding of the mechanisms underlying disease susceptibility, asthma is frequently studied in genome-wide association studies (GWAS), which have identified thousands of genetic variants associated with asthma development. Virtually all these genetic variants reside in non-coding genomic regions, which has obscured the functional impact of asthma-associated variants and their translation into disease-relevant mechanisms. Recent advances in genomics technology and epigenetics now offer methods to link genetic variants to gene regulatory elements embedded within non-coding regions, which have started to unravel the molecular mechanisms underlying the complex (epi)genetics of asthma. Here, we provide an integrated overview of (epi)genetic variants associated with asthma, focusing on efforts to link these disease associations to biological insight into asthma pathophysiology using state-of-the-art genomics methodology. Finally, we provide a perspective as to how decoding the genetic and epigenetic basis of asthma has the potential to transform clinical management of asthma and to predict the risk of asthma development.


Assuntos
Asma , Estudo de Associação Genômica Ampla , Humanos , Epigênese Genética , Epigenômica , Asma/genética , Genômica , Predisposição Genética para Doença
14.
Immunity ; 41(2): 191-206, 2014 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-25148023

RESUMO

The zinc-finger transcription factor GATA-3 has received much attention as a master regulator of T helper 2 (Th2) cell differentiation, during which it controls interleukin-4 (IL-4), IL-5, and IL-13 expression. More recently, GATA-3 was shown to contribute to type 2 immunity through regulation of group 2 innate lymphoid cell (ILC2) development and function. Furthermore, during thymopoiesis, GATA-3 represses B cell potential in early T cell precursors, activates TCR signaling in pre-T cells, and promotes the CD4(+) T cell lineage after positive selection. GATA-3 also functions outside the thymus in hematopoietic stem cells, regulatory T cells, CD8(+) T cells, thymic natural killer cells, and ILC precursors. Here we discuss the varied functions of GATA-3 in innate and adaptive immune cells, with emphasis on its activity in T cells and ILCs, and examine the mechanistic basis for the dose-dependent, developmental-stage- and cell-lineage-specific activity of this transcription factor.


Assuntos
Imunidade Adaptativa , Fator de Transcrição GATA3/imunologia , Imunidade Inata , Células Th2/imunologia , Linfócitos B/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Interleucina-13/biossíntese , Interleucina-13/imunologia , Interleucina-4/biossíntese , Interleucina-4/imunologia , Interleucina-5/biossíntese , Interleucina-5/imunologia , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T , Linfócitos T Reguladores/imunologia
15.
J Immunol ; 207(9): 2337-2346, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34561228

RESUMO

TNF is important in immune-mediated inflammatory diseases, including spondyloarthritis (SpA). Transgenic (tg) mice overexpressing transmembrane TNF (tmTNF) develop features resembling human SpA. Furthermore, both tmTNF tg mice and SpA patients develop ectopic lymphoid aggregates, but it is unclear whether these contribute to pathology. Therefore, we characterized the lymphoid aggregates in detail and studied potential alterations in the B and T cell lineage in tmTNF tg mice. Lymphoid aggregates developed in bone marrow (BM) of vertebrae and near the ankle joints prior to the first SpA features and displayed characteristics of ectopic lymphoid structures (ELS) including presence of B cells, T cells, germinal centers, and high endothelial venules. Detailed flow cytometric analyses demonstrated more germinal center B cells with increased CD80 and CD86 expression, along with significantly more T follicular helper, T follicular regulatory, and T regulatory cells in tmTNF tg BM compared with non-tg controls. Furthermore, tmTNF tg mice exhibited increased IgA serum levels and significantly more IgA+ plasma cells in the BM, whereas IgA+ plasma cells in the gut were not significantly increased. In tmTNF tg × TNF-RI-/- mice, ELS were absent, consistent with reduced disease symptoms, whereas in tmTNF tg × TNF-RII-/- mice, ELS and clinical symptoms were still present. Collectively, these data show that tmTNF overexpression in mice results in osteitis and ELS formation in BM, which may account for the increased serum IgA levels that are also observed in human SpA. These effects are mainly dependent on TNF-RI signaling and may underlie important aspects of SpA pathology.


Assuntos
Linfócitos B/imunologia , Medula Óssea/metabolismo , Centro Germinativo/imunologia , Proteínas de Membrana/metabolismo , Osteíte/imunologia , Espondilite Anquilosante/imunologia , Linfócitos T/imunologia , Estruturas Linfoides Terciárias/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Medula Óssea/patologia , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imunoglobulina A/metabolismo , Proteínas de Membrana/genética , Camundongos , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética
16.
Infect Immun ; 90(6): e0067421, 2022 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-35587199

RESUMO

Peritonitis and abdominal sepsis remain major health problems and challenge for clinicians. Bruton's tyrosine kinase (Btk) is a versatile signaling protein involved in the regulation of B cell development and function, as well as innate host defense. In the current study, we aimed to explore the role of Btk in the host response during peritonitis and sepsis in mice induced by a gradually growing pathogenic strain of Escherichia coli bacteria. We found that Btk deficiency ameliorated antibacterial host defense during the late stage of E. coli-induced peritonitis. Btk was not required for cytokine and chemokine release in response to either E. coli or lipopolysaccharide and did not impact organ damage evoked by E. coli. Btk deficiency also did not alter neutrophil influx to the primary site of infection. However, the absence of Btk modestly enhanced phagocytosis of E. coli by neutrophils. These results indicate that Btk-mediated signaling is superfluous for inflammatory responses and remarkably detrimental for antibacterial defense during E. coli-induced peritonitis.


Assuntos
Anti-Infecciosos , Infecções por Escherichia coli , Peritonite , Sepse , Tirosina Quinase da Agamaglobulinemia/metabolismo , Animais , Antibacterianos , Escherichia coli/metabolismo , Camundongos
17.
Eur J Immunol ; 51(9): 2251-2265, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34323286

RESUMO

Bruton's tyrosine kinase (Btk) is a crucial signaling molecule in BCR signaling and a key regulator of B- cell differentiation and function. Btk inhibition has shown impressive clinical efficacy in various B-cell malignancies. However, it remains unknown whether inhibition additionally induces changes in BCR signaling due to feedback mechanisms, a phenomenon referred to as BCR rewiring. In this report, we studied the impact of Btk activity on major components of the BCR signaling pathway in mice. As expected, NF-κB and Akt/S6 signaling was decreased in Btk-deficient B cells. Unexpectedly, phosphorylation of several proximal signaling molecules, including CD79a, Syk, and PI3K, as well as the key Btk-effector PLCγ2 and the more downstream kinase Erk, were significantly increased. This pattern of BCR rewiring was essentially opposite in B cells from transgenic mice overexpressing Btk. Importantly, prolonged Btk inhibitor treatment of WT mice or mice engrafted with leukemic B cells also resulted in increased phosho-CD79a and phospho-PLCγ2 in B cells. Our findings show that Btk enzymatic function determines phosphorylation of proximal and distal BCR signaling molecules in B cells. We conclude that Btk inhibitor treatment results in rewiring of BCR signaling, which may affect both malignant and healthy B cells.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Linfócitos B/imunologia , Antígenos CD79/metabolismo , Fosfolipase C gama/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Tirosina Quinase da Agamaglobulinemia/genética , Animais , Antineoplásicos/farmacologia , Linfócitos B/citologia , Benzamidas/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imunoglobulina M/imunologia , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazinas/farmacologia , Transdução de Sinais/imunologia , Quinase Syk/metabolismo
18.
Respir Res ; 23(1): 336, 2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36494786

RESUMO

BACKGROUND: The global initiative for chronic obstructive lung disease (GOLD) 2020 emphasizes that there is only a weak correlation between FEV1, symptoms and impairment of the health status of patients with chronic obstructive pulmonary disease (COPD). Various studies aimed to identify COPD phenotypes by cluster analyses, but behavioral aspects besides smoking were rarely included. METHODS: The aims of the study were to investigate whether (i) clustering analyses are in line with the classification into GOLD ABCD groups; (ii) clustering according to Burgel et al. (Eur Respir J. 36(3):531-9, 2010) can be reproduced in a real-world COPD cohort; and (iii) addition of new behavioral variables alters the clustering outcome. Principal component and hierarchical cluster analyses were applied to real-world clinical data of COPD patients newly referred to secondary care (n = 155). We investigated if the obtained clusters paralleled GOLD ABCD subgroups and determined the impact of adding several variables, including quality of life (QOL), fatigue, satisfaction relationship, air trapping, steps per day and activities of daily living, on clustering. RESULTS: Using the appropriate corresponding variables, we identified clusters that largely reflected the GOLD ABCD groups, but we could not reproduce Burgel's clinical phenotypes. Adding six new variables resulted in the formation of four new clusters that mainly differed from each other in the following parameters: number of steps per day, activities of daily living and QOL. CONCLUSIONS: We could not reproduce previously identified clinical COPD phenotypes in an independent population of COPD patients. Our findings therefore indicate that COPD phenotypes based on cluster analysis may not be a suitable basis for treatment strategies for individual patients.


Assuntos
Doença Pulmonar Obstrutiva Crônica , Qualidade de Vida , Humanos , Medicina de Precisão , Atividades Cotidianas , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Doença Pulmonar Obstrutiva Crônica/terapia , Testes de Função Respiratória
19.
J Immunol ; 204(10): 2852-2863, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32253241

RESUMO

BCR signaling, involving phosphorylation of various downstream molecules, including kinases, lipases, and linkers, is crucial for B cell selection, survival, proliferation, and differentiation. Phosphoflow cytometry (phosphoflow) is a single-cell-based technique to measure phosphorylated intracellular proteins, providing a more quantitative read-out than Western blotting. Recent advances in phosphoflow basically allow simultaneous analysis of protein phosphorylation in B cell (sub)populations, without prior cell sorting. However, fixation and permeabilization procedures required for phosphoflow often affect cell surface epitopes or mAb conjugates, precluding the evaluation of the phosphorylation status of signaling proteins across different B cell subpopulations present in a single sample. In this study, we report a versatile phosphoflow protocol allowing extensive staining of B cell subpopulations in human peripheral blood or various anatomical compartments in the mouse, starting from freshly isolated or frozen cell suspensions. Both human and mouse B cell subpopulations showed different basal and BCR stimulation-induced phosphorylation levels of downstream signaling proteins. For example, peritoneal B-1 cells and splenic marginal zone B cells exhibited significantly increased basal (ex vivo) signaling and increased responsiveness to in vitro BCR stimulation compared with peritoneal B-2 cells and splenic follicular B cells, respectively. In addition, whereas stimulation with anti-IgM or anti-Igκ L chain Abs resulted in strong pCD79a and pPLCγ2 signals, IgD stimulation only induced CD79a but not pPLCγ2 phosphorylation. In summary, the protocol is user friendly and quantifies BCR-mediated phosphorylation with high sensitivity at the single-cell level, in combination with extensive staining to identify individual B cell development and differentiation stages.


Assuntos
Subpopulações de Linfócitos B/fisiologia , Linfócitos B/fisiologia , Citometria de Fluxo/métodos , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Antígenos CD79/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Imunoglobulina D/metabolismo , Imunoglobulina M/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipase C gama/metabolismo , Fosforilação , Transdução de Sinais , Análise de Célula Única
20.
J Immunol ; 204(9): 2600-2611, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32213568

RESUMO

Accurately tuned expression levels of the transcription factor GATA-3 are crucial at several stages of T cell and innate lymphoid cell development and differentiation. Moreover, several lines of evidence suggest that Gata3 expression might provide a reliable molecular marker for the identification of elusive progenitor cell subsets at the earliest stages of T lineage commitment. To be able to faithfully monitor Gata3 expression noninvasively at the single-cell level, we have generated a novel strain of knock-in reporter mice, termed GATIR, by inserting an expression cassette encoding a bright fluorescent marker into the 3'-untranslated region of the endogenous Gata3 locus. Importantly, in contrast to three previously published strains of Gata3 reporter mice, GATIR mice preserve physiological Gata3 expression on the targeted allele. In this study, we show that GATIR mice faithfully reflect endogenous Gata3 expression without disturbing the development of GATA-3-dependent lymphoid cell populations. We further show that GATIR mice provide an ideal tool for noninvasive monitoring of Th2 polarization and straightforward identification of innate lymphoid cell 2 progenitor populations. Finally, as our reporter is non-gene-destructive, GATIR mice can be bred to homozygosity, not feasible with previously published strains of Gata3 reporter mice harboring disrupted alleles. The availability of hetero- and homozygous Gata3 reporter mice with an exceptionally bright fluorescent marker, allowed us to visualize allelic Gata3 expression in individual cells simply by flow cytometry. The unambiguous results obtained provide compelling evidence against previously postulated monoallelic Gata3 expression in early T lineage and hematopoietic stem cell subsets.


Assuntos
Fator de Transcrição GATA3/genética , Genes Reporter/genética , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/imunologia , Alelos , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Feminino , Citometria de Fluxo/métodos , Corantes Fluorescentes/metabolismo , Fator de Transcrição GATA3/imunologia , Técnicas de Introdução de Genes/métodos , Genes Reporter/imunologia , Células-Tronco Hematopoéticas/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Linfócitos/imunologia , Células Progenitoras Linfoides/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia
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