Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.515
Filtrar
1.
J Vis Exp ; (167)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33491676

RESUMO

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0. Here, we describe a flow cytometric method for capturing a "snapshot" of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1. Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Ciclo Celular , DNA/metabolismo , Epitopos/imunologia , Citometria de Fluxo/métodos , Antígeno Ki-67/metabolismo , Vacinação , Animais , Células da Medula Óssea/citologia , Análise de Dados , Feminino , Humanos , Linfonodos/citologia , Camundongos Endogâmicos BALB C , Baço/citologia , Coloração e Rotulagem
3.
PLoS One ; 15(10): e0239517, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33021999

RESUMO

Vascularized lymph node (VLN) transfer is an emerging strategy to re-establish lymphatic drainage in chronic lymphedema. However, the biological processes underlying lymph node integration remain elusive. This study introduces an experimental approach facilitating the analysis of short-term molecular and cellular effects of ischemia/reperfusion on VLN flaps. Lymph node flaps were dissected pedicled on the lateral thoracic vessels in 44 Lewis rats. VLN flaps were exposed to 45 or 120 minutes ischemia by in situ clamping of the vascular pedicle with subsequent reperfusion for 24 hours. Flaps not exposed to ischemia/reperfusion served as controls. Lymph nodes and the perinodal adipose tissue were separately analyzed by Western blot for the expression of lymphangiogenic and angiogenic growth factors. Moreover, morphology, microvessel density, proliferation, apoptosis and immune cell infiltration of VLN flaps were further assessed by histology and immunohistochemistry. Ischemia for 120 minutes was associated with a markedly reduced cellularity of lymph nodes but not of the perinodal adipose tissue. In line with this, ischemic lymph nodes exhibited a significantly lower microvessel density and an increased expression of VEGF-D and VEGF-A. However, VEGF-C expression was not upregulated. In contrast, analyses of the perinodal adipose tissue revealed a more subtle decrease of microvessel density, while only the expression of VEGF-D was increased. Moreover, after 120 minutes ischemia, lymph nodes but not the perinodal adipose tissue exhibited significantly higher numbers of proliferating and apoptotic cells as well as infiltrated macrophages and neutrophilic granulocytes compared with non-ischemic flaps. Taken together, lymph nodes of VLN flaps are highly susceptible to ischemia/reperfusion injury. In contrast, the perinodal adipose tissue is less prone to ischemia/reperfusion injury.


Assuntos
Linfonodos/irrigação sanguínea , Linfonodos/cirurgia , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Retalhos Cirúrgicos , Animais , Apoptose , Linfonodos/citologia , Microvasos/fisiopatologia , Estresse Oxidativo , Ratos , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/cirurgia
4.
Proc Natl Acad Sci U S A ; 117(38): 23674-23683, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32907933

RESUMO

The gut microbiome has garnered attention as an effective target to boost immunity and improve cancer immunotherapy. We found that B cell-defective (BCD) mice, such as µ-membrane targeted deletion (µMT) and activation-induced cytidine deaminase (AID) knockouts (KOs), have elevated antitumor immunity under specific pathogen-free but not germ-free conditions. Microbial dysbiosis in these BCD mice enriched the type I IFN (IFN) signature in mucosal CD8+ T cells, resulting in up-regulation of the type I IFN-inducible protein stem cell antigen-1 (Sca-1). Among CD8+ T cells, naïve cells predominantly circulate from the gut to the periphery, and those that had migrated from the mesenteric lymph nodes (mLNs) to the periphery had significantly higher expression of Sca-1. The gut-educated Sca-1+ naïve subset is endowed with enhanced mitochondrial activity and antitumor effector potential. The heterogeneity and functional versatility of the systemic naïve CD8+ T cell compartment was revealed by single-cell analysis and functional assays of CD8+ T cell subpopulations. These results indicate one of the potential mechanisms through which microbial dysbiosis regulates antitumor immunity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Microbioma Gastrointestinal/imunologia , Interferon Tipo I/imunologia , Neoplasias Experimentais/imunologia , Animais , Antígenos Ly/imunologia , Antígenos Ly/metabolismo , Linfócitos B , Linhagem Celular Tumoral , Células Cultivadas , Disbiose/imunologia , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Interferon Tipo I/metabolismo , Linfonodos/citologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/imunologia
5.
Life Sci ; 257: 118117, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32693243

RESUMO

AIMS: B cells can promote or inhibit immune responses against breast cancer. We investigated changes in the frequency of B cells with stimulatory or regulatory capacity in breast tumor draining lymph nodes during cancer progression. MAIN METHODS: We isolated mononuclear cells from fresh axillary lymph nodes (LNs) of 44 patients with breast cancer and stained lymphocytes with antibodies against CD19, CD80, CD86, CD39 and CD73. To assess programmed death-1 (PD-1) and PD-ligand 1 (PD-L1) expression, lymphocytes were briefly stimulated, stained for CD19, PD-1 and PD-L1, and examined with flow cytometry. KEY FINDINGS: The frequency of CD80+ B cells was higher in nonmetastatic lymph nodes, while the percentage of CD86+ B cells showed a positive relationship with higher tumor grade and higher numbers of involved LNs. A small proportion of unstimulated B cells expressed PD-1 or PD-L1 but these molecules were rapidly upregulated on B cells following activation. The frequency of stimulated PD-L1+ B cells showed an inverse association with estrogen and progesterone receptor expression and a nonsignificant positive association with tumor grade. In addition, the percentage of unstimulated PD-1+ B cells was higher in patients with higher-grade tumors. CD73 expression on B cells was associated with lower numbers of involved LNs, and the frequency of CD39+ B cells was higher in patients with larger tumors. SIGNIFICANCE: CD86+, CD39+, PD-1+ and PD-L1+ B cells showed associations with poor prognostic factors, therefore their potential role in the suppression of the immune responses against breast cancer should be evaluated in greater detail.


Assuntos
Subpopulações de Linfócitos B/patologia , Linfócitos B Reguladores/patologia , Neoplasias da Mama/imunologia , Linfonodos/patologia , Adulto , Idoso , Apirase/imunologia , Axila , Subpopulações de Linfócitos B/imunologia , Linfócitos B Reguladores/imunologia , Antígeno B7-2/imunologia , Antígeno B7-H1/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Feminino , Citometria de Fluxo , Humanos , Linfonodos/citologia , Linfonodos/imunologia , Pessoa de Meia-Idade , Prognóstico , Receptor de Morte Celular Programada 1/metabolismo
6.
Nat Commun ; 11(1): 3272, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601304

RESUMO

Tumor-draining lymph node (TDLN) invasion by metastatic cells in breast cancer correlates with poor prognosis and is associated with local immunosuppression, which can be partly mediated by regulatory T cells (Tregs). Here, we study Tregs from matched tumor-invaded and non-invaded TDLNs, and breast tumors. We observe that Treg frequencies increase with nodal invasion, and that Tregs express higher levels of co-inhibitory/stimulatory receptors than effector cells. Also, while Tregs show conserved suppressive function in TDLN and tumor, conventional T cells (Tconvs) in TDLNs proliferate and produce Th1-inflammatory cytokines, but are dysfunctional in the tumor. We describe a common transcriptomic signature shared by Tregs from tumors and nodes, including CD80, which is significantly associated with poor patient survival. TCR RNA-sequencing analysis indicates trafficking between TDLNs and tumors and ongoing Tconv/Treg conversion. Overall, TDLN Tregs are functional and express a distinct pattern of druggable co-receptors, highlighting their potential as targets for cancer immunotherapy.


Assuntos
Linfonodos/patologia , Metástase Linfática/imunologia , Linfócitos T Reguladores/imunologia , Antígeno B7-1/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Imunossupressão , Linfonodos/citologia , Linfonodos/imunologia , Metástase Linfática/patologia , Linfócitos T Reguladores/metabolismo
7.
J Leukoc Biol ; 107(6): 1107-1113, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32293062

RESUMO

The ability of a cell to migrate, adhere, and change its morphology is determinant in developing its functions; these capacities reach their maximum relevance in immune cells. For an efficient immune response, immune cells must localize in the right place at the right time; that implies crossing tissue barriers and migrating in the interstitial space of the tissues at high velocities. The dependency on trafficking abilities is even higher for B cells, one of the arms of the adaptive immune system, considering that they must encounter specific antigens for their clonal receptor in the enormous tissue volume of the secondary lymphoid organs (spleen, lymph nodes, Peyer patches). The regulated interplay between cell motility and cell adhesion allows B cells to reach distinct lymphoid tissues and, within them, to explore the stromal cell networks where antigen might be exposed. In this meeting-invited review, I summarize the current knowledge on the molecular cues and mechanisms that shapes B cell dynamics at the initial phase of the humoral immune response, including homeostatic chemoattractants and innate/inflammatory stimuli. I also revised the B cell behavior alterations caused by BCR recognition of antigen and the molecular mechanisms involved.


Assuntos
Linfócitos B/imunologia , Homeostase/imunologia , Imunidade Humoral , Linfonodos/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Linfócitos B/citologia , Adesão Celular , Movimento Celular , Expressão Gênica , Humanos , Imunidade Inata , Sinapses Imunológicas , Linfonodos/citologia , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Receptores de Antígenos de Linfócitos B/genética , Baço/citologia , Baço/imunologia , Células Estromais/citologia , Células Estromais/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
8.
PLoS Biol ; 18(4): e3000704, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251437

RESUMO

Lymph nodes (LNs) are highly organized secondary lymphoid organs that mediate adaptive immune responses to antigens delivered via afferent lymphatic vessels. Lymphatic endothelial cells (LECs) line intranodal lymphatic sinuses and organize lymph and antigen distribution. LECs also directly regulate T cells, mediating peripheral tolerance to self-antigens, and play a major role in many diseases, including cancer metastasis. However, little is known about the phenotypic and functional heterogeneity of LN LECs. Using single-cell RNA sequencing, we comprehensively defined the transcriptome of LECs in murine skin-draining LNs and identified new markers and functions of distinct LEC subpopulations. We found that LECs residing in the subcapsular sinus (SCS) have an unanticipated function in scavenging of modified low-density lipoprotein (LDL) and also identified a specific cortical LEC subtype implicated in rapid lymphocyte egress from LNs. Our data provide new, to our knowledge, insights into the diversity of LECs in murine LNs and a rich resource for future studies into the regulation of immune responses by LN LECs.


Assuntos
Linfonodos/citologia , Análise de Célula Única/métodos , Animais , Biomarcadores/metabolismo , Células Endoteliais/citologia , Endotélio Linfático/citologia , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Integrina alfa2/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Receptores CCR/genética , Receptores CCR/metabolismo , Análise de Sequência de RNA , Proteínas de Transporte Vesicular/genética
9.
Nat Commun ; 11(1): 1114, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111837

RESUMO

Little is known regarding lymph node (LN)-homing of immune cells via afferent lymphatics. Here, we show, using a photo-convertible Dendra-2 reporter, that recently activated CD4 T cells enter downstream LNs via afferent lymphatics at high frequencies. Intra-lymphatic immune cell transfer and live imaging data further show that activated T cells come to an instantaneous arrest mediated passively by the mechanical 3D-sieve barrier of the LN subcapsular sinus (SCS). Arrested T cells subsequently migrate randomly on the sinus floor independent of both chemokines and integrins. However, chemokine receptors are imperative for guiding cells out of the SCS, and for their subsequent directional translocation towards the T cell zone. By contrast, integrins are dispensable for LN homing, yet still contribute by increasing the dwell time within the SCS and by potentially enhancing T cell sensing of chemokine gradients. Together, these findings provide fundamental insights into mechanisms that control homing of lymph-derived immune cells.


Assuntos
Linfócitos T CD4-Positivos/fisiologia , Movimento Celular/imunologia , Quimiocinas/metabolismo , Integrinas/metabolismo , Linfonodos/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Endotélio Linfático/fisiologia , Integrinas/genética , Linfa/citologia , Linfonodos/citologia , Ativação Linfocitária , Camundongos , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores de Retorno de Linfócitos/metabolismo
10.
J Leukoc Biol ; 108(1): 83-91, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32170880

RESUMO

Eosinophils are traditionally associated with allergic and parasitic inflammation. More recently, eosinophils have also been shown to have roles in diverse processes including development, intestinal health, thymic selection, and B-cell survival with the majority of these insights being derived from murine models and in vitro assays. Despite this, tools to measure the dynamic activity of eosinophils in situ have been lacking. Intravital microscopy is a powerful tool that enables direct visualization of leukocytes and their dynamic behavior in real-time in a wide range of processes in both health and disease. Until recently eosinophil researchers have not been able to take full advantage of this technology due to a lack of tools such as genetically encoded reporter mice. This mini-review examines the history of intravital microscopy with a focus on eosinophils. The development and use of eosinophil-specific Cre (EoCre) mice to create GFP and tdTomato fluorescent reporter animals is also described. Genetically encoded eosinophil reporter mice combined with intravital microscopy provide a powerful tool to add to the toolbox of technologies that will help us unravel the mysteries still surrounding this cell.


Assuntos
Eosinófilos/citologia , Microscopia Intravital , Animais , Ceco/citologia , Corantes Fluorescentes/metabolismo , Genes Reporter , Intestino Delgado/citologia , Pulmão/citologia , Linfonodos/citologia , Camundongos Endogâmicos C57BL , Músculos/citologia
11.
Cytotherapy ; 22(1): 6-15, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32005355

RESUMO

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths with high recurrence after surgery due to a paucity of effective post-surgical adjuvant treatments. DC vaccines can activate multiple anti-tumor immune responses but have not been explored for post-surgery PDAC recurrence. Intraperitoneal (IP) delivery may allow increased DC vaccine dosage and migration to lymph nodes. Here, we investigated the role of prophylactic DC vaccination controlling PDAC tumor growth with IP delivery as an administration route for DC vaccination. METHODS: DC vaccines were generated using ex vivo differentiation and maturation of bone marrow-derived precursors. Twenty mice were divided into four groups (n = 5) and treated with DC vaccines, unpulsed mature DCs, Panc02 lysates or no treatment. After tumor induction, mice underwent three magnetic resonance imaging scans to track tumor growth. Apparent diffusion coefficient (ADC), a quantitative magnetic resonance imaging measurement of tumor microstructure, was calculated. Survival was tracked. Tumor tissue was collected after death and stained with hematoxylin and eosin, Masson's trichrome, terminal deoxynucleotidyl transferase dUTP nick end labeling and anti-CD8 stains for histology. RESULTS: DC-vaccinated mice demonstrated stronger anti-tumor cytotoxicity compared with control groups on lactate dehydrogenase assay. DC vaccine mice also demonstrated decreased tumor volume, prolonged survival and increased ΔADC compared with control groups. On histology, the DC vaccine group had increased apoptosis, increased CD8+ T cells and decreased collagen. ΔADC negatively correlated with % collagen in tumor tissues. DISCUSSION: Prophylactic DC vaccination may inhibit PDAC tumor growth during recurrence and prolong survival. ΔADC may be a potential imaging biomarker that correlates with tumor histological features.


Assuntos
Vacinas Anticâncer/imunologia , Carcinoma Ductal Pancreático/terapia , Células Dendríticas/imunologia , Células Dendríticas/transplante , Neoplasias Pancreáticas/terapia , Adenocarcinoma/terapia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Recidiva Local de Neoplasia/prevenção & controle , Vacinação
12.
Nat Rev Immunol ; 20(9): 566-578, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32094869

RESUMO

The influx and efflux of cells and antigens to and from the draining lymph nodes largely take place through the subcapsular, cortical and medullary sinus systems. Recent analyses in mice and humans have revealed unexpected diversity in the lymphatic endothelial cells, which form the distinct regions of the sinuses. As a semipermeable barrier, the lymphatic endothelial cells regulate the sorting of lymph-borne antigens to the lymph node parenchyma and can themselves serve as antigen-presenting cells. The leukocytes entering the lymph node via the sinus system and the lymphocytes egressing from the parenchyma migrate through the lymphatic endothelial cell layer. The sinus lymphatic endothelial cells also orchestrate the organogenesis of lymph nodes, and they undergo bidirectional signalling with other sinus-resident cells, such as subcapsular sinus macrophages, to generate a unique lymphatic niche. In this Review, we consider the structural and functional basis of how the lymph node sinus system coordinates immune responses under physiological conditions, and in inflammation and cancer.


Assuntos
Células Endoteliais/imunologia , Endotélio Linfático/citologia , Linfonodos/citologia , Animais , Endotélio Linfático/imunologia , Humanos , Imunidade Celular , Inflamação/imunologia , Linfonodos/imunologia , Neoplasias/imunologia
13.
Cell ; 180(4): 749-763.e13, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32059780

RESUMO

Immune responses in diverse tissue sites are critical for protective immunity and homeostasis. Here, we investigate how tissue localization regulates the development and function of human natural killer (NK) cells, innate lymphocytes important for anti-viral and tumor immunity. Integrating high-dimensional analysis of NK cells from blood, lymphoid organs, and mucosal tissue sites from 60 individuals, we identify tissue-specific patterns of NK cell subset distribution, maturation, and function maintained across age and between individuals. Mature and terminally differentiated NK cells with enhanced effector function predominate in blood, bone marrow, spleen, and lungs and exhibit shared transcriptional programs across sites. By contrast, precursor and immature NK cells with reduced effector capacity populate lymph nodes and intestines and exhibit tissue-resident signatures and site-specific adaptations. Together, our results reveal anatomic control of NK cell development and maintenance as tissue-resident populations, whereas mature, terminally differentiated subsets mediate immunosurveillance through diverse peripheral sites. VIDEO ABSTRACT.


Assuntos
Envelhecimento/imunologia , Células Matadoras Naturais/citologia , Linfopoese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/genética , Antígenos CD/metabolismo , Células Cultivadas , Criança , Feminino , Humanos , Imunidade Inata , Mucosa Intestinal/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/fisiologia , Pulmão/citologia , Linfonodos/citologia , Masculino , Pessoa de Meia-Idade , Baço/citologia
14.
Nat Immunol ; 21(3): 343-353, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066951

RESUMO

Gastrointestinal microbiota and immune cells interact closely and display regional specificity; however, little is known about how these communities differ with location. Here, we simultaneously assess microbiota and single immune cells across the healthy, adult human colon, with paired characterization of immune cells in the mesenteric lymph nodes, to delineate colonic immune niches at steady state. We describe distinct helper T cell activation and migration profiles along the colon and characterize the transcriptional adaptation trajectory of regulatory T cells between lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal expansion and mutational frequency from the cecum to the sigmoid colon and link this to the increasing number of reactive bacterial species.


Assuntos
Colo/imunologia , Colo/microbiologia , Microbioma Gastrointestinal/imunologia , Adulto , Linfócitos B/imunologia , Colo/citologia , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Linfonodos/citologia , Linfonodos/imunologia , Ativação Linfocitária , Especificidade de Órgãos , RNA-Seq , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Transcriptoma
15.
J Immunol ; 204(5): 1395-1407, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31953352

RESUMO

Activation of adaptive immunity is a complex process coordinated at multiple levels in both time and the three-dimensional context of reactive lymph nodes (LNs). Although microscopy-based visualization of its spatiotemporal dynamics unravels complexities of developing immune response, such approach is highly limited by light-obstructing nature of tissue components. Recently, tissue optical clearing (TOC) techniques were established to bypass this obstacle and now allow to image and quantify the entire murine organs with cellular resolution. However, the spectrum of TOC is represented by wide variety of chemically distinct methods, each having certain advantages and disadvantages that were unsatisfactorily compared for suitability to LNs clearing. In this study, we have systematically tested 13 typical TOC techniques and assessed their impact on a number of critical factors such as LN transparency, imaging depth, change in size, compatibility with proteinaceous fluorophores, immunostaining, H&E staining, and light-sheet fluorescence microscopy. Based on the detailed data specific to TOC process of murine LNs, we provide a reliable reference for most suitable methods in an application-dependent manner.


Assuntos
Imageamento Tridimensional , Linfonodos/citologia , Animais , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Linfonodos/imunologia , Camundongos , Microscopia de Fluorescência
16.
PLoS Negl Trop Dis ; 14(1): e0006596, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31923234

RESUMO

An association between increased susceptibility to infectious diseases and obesity has been described as a result of impaired immunity in obese individuals. It is not clear whether a similar linkage can be drawn between obesity and parasitic diseases. To evaluate the effect of obesity in the immune response to cutaneous Leishmania major infection, we studied the ability of C57BL/6 mice fed a hypercaloric diet (HSB) to control leishmaniasis. Mice with diet-induced obesity presented thicker lesions with higher parasite burden and a more intense inflammatory infiltrate in the infected ear after infection with L. major. There was no difference between control and obese mice in IFN-gamma or IL-4 production by auricular draining lymph node cells, but obese mice produced higher levels of IgG1 and IL-17. Peritoneal macrophages from obese mice were less efficient to kill L. major when infected in vitro than macrophages from control mice. In vitro stimulation of macrophages with IL-17 decreased their capacity to kill the parasite. Moreover, macrophages from obese mice presented higher arginase activity. To confirm the role of IL-17 in the context of obesity and infection, we studied lesion development in obese IL-17R-/- mice infected with L. major and found no difference in skin lesions and the leukocyte accumulation in the draining lymph node is redcuced in knockout mice compared between obese and lean animals. Our results indicate that diet-induced obesity impairs resistance to L. major in C57BL/6 mice and that IL-17 is involved in lesion development.


Assuntos
Leishmania major/patogenicidade , Leishmaniose Cutânea/imunologia , Obesidade , Animais , Dieta/efeitos adversos , Orelha/parasitologia , Feminino , Interferon gama , Interleucina-17 , Leishmaniose Cutânea/parasitologia , Linfonodos/citologia , Macrófagos Peritoneais/parasitologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Risco
17.
Nat Commun ; 11(1): 519, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980640

RESUMO

Fibroblastic reticular cells (FRCs) are immunologically specialized myofibroblasts of lymphoid organ, and FRC maturation is essential for structural and functional properties of lymph nodes (LNs). Here we show that YAP and TAZ (YAP/TAZ), the final effectors of Hippo signaling, regulate FRC commitment and maturation. Selective depletion of YAP/TAZ in FRCs impairs FRC growth and differentiation and compromises the structural organization of LNs, whereas hyperactivation of YAP/TAZ enhances myofibroblastic characteristics of FRCs and aggravates LN fibrosis. Mechanistically, the interaction between YAP/TAZ and p52 promotes chemokine expression that is required for commitment of FRC lineage prior to lymphotoxin-ß receptor (LTßR) engagement, whereas LTßR activation suppresses YAP/TAZ activity for FRC maturation. Our findings thus present YAP/TAZ as critical regulators of commitment and maturation of FRCs, and hold promise for better understanding of FRC-mediated pathophysiologic processes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Fibroblastos/metabolismo , Linfonodos/citologia , Transativadores/metabolismo , Adipócitos/metabolismo , Animais , Quimiocinas/metabolismo , Fibroblastos/ultraestrutura , Linfonodos/ultraestrutura , Receptor beta de Linfotoxina/metabolismo , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo
18.
Nat Commun ; 11(1): 404, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964880

RESUMO

Although fibroblast heterogeneity is recognized in primary tumors, both its characterization in and its impact on metastases remain unknown. Here, combining flow cytometry, immunohistochemistry and RNA-sequencing on breast cancer samples, we identify four Cancer-Associated Fibroblast (CAF) subpopulations in metastatic lymph nodes (LN). Two myofibroblastic subsets, CAF-S1 and CAF-S4, accumulate in LN and correlate with cancer cell invasion. By developing functional assays on primary cultures, we demonstrate that these subsets promote metastasis through distinct functions. While CAF-S1 stimulate cancer cell migration and initiate an epithelial-to-mesenchymal transition through CXCL12 and TGFß pathways, highly contractile CAF-S4 induce cancer cell invasion in 3-dimensions via NOTCH signaling. Patients with high levels of CAFs, particularly CAF-S4, in LN at diagnosis are prone to develop late distant metastases. Our findings suggest that CAF subset accumulation in LN is a prognostic marker, suggesting that CAF subsets could be examined in axillary LN at diagnosis.


Assuntos
Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/metabolismo , Metástase Linfática/patologia , Miofibroblastos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Axila , Neoplasias da Mama/mortalidade , Neoplasias da Mama/terapia , Fibroblastos Associados a Câncer/patologia , Proliferação de Células , Separação Celular , Quimiocina CXCL12/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Linfonodos/citologia , Linfonodos/patologia , Pessoa de Meia-Idade , Miofibroblastos/patologia , Invasividade Neoplásica/patologia , Cultura Primária de Células , Prognóstico , Intervalo Livre de Progressão , Receptores Notch/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas , Microambiente Tumoral
19.
Am J Physiol Endocrinol Metab ; 318(4): E480-E491, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961709

RESUMO

Probiotic bacteria can protect from ovariectomy (ovx)-induced bone loss in mice. Akkermansia muciniphila is considered to have probiotic potential due to its beneficial effect on obesity and insulin resistance. The purpose of the present study was to determine if treatment with pasteurized Akkermansia muciniphila (pAkk) could prevent ovx-induced bone loss. Mice were treated with vehicle or pAkk for 4 wk, starting 3 days before ovx or sham surgery. Treatment with pAkk reduced fat mass accumulation confirming earlier findings. However, treatment with pAkk decreased trabecular and cortical bone mass in femur and vertebra of gonadal intact mice and did not protect from ovx-induced bone loss. Treatment with pAkk increased serum parathyroid hormone (PTH) levels and increased expression of the calcium transporter Trpv5 in kidney suggesting increased reabsorption of calcium in the kidneys. Serum amyloid A 3 (SAA3) can suppress bone formation and mediate the effects of PTH on bone resorption and bone loss in mice and treatment with pAkk increased serum levels of SAA3 and gene expression of Saa3 in colon. Moreover, regulatory T cells can be protective of bone and pAkk-treated mice had decreased number of regulatory T cells in mesenteric lymph nodes and bone marrow. In conclusion, treatment with pAkk protected from ovx-induced fat mass gain but not from bone loss and reduced bone mass in gonadal intact mice. Our findings with pAkk differ from some probiotics that have been shown to protect bone mass, demonstrating that not all prebiotic and probiotic factors have the same effect on bone.


Assuntos
Tecido Adiposo/crescimento & desenvolvimento , Microbioma Gastrointestinal/fisiologia , Osteoporose/metabolismo , Probióticos/farmacologia , Verrucomicrobia/metabolismo , Tecido Adiposo/metabolismo , Animais , Canais de Cálcio/metabolismo , Colo/efeitos dos fármacos , Colo/microbiologia , Feminino , Fêmur/efeitos dos fármacos , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Hormônio Paratireóideo/metabolismo , Pasteurização , Proteína Amiloide A Sérica/metabolismo , Coluna Vertebral/efeitos dos fármacos , Linfócitos T Reguladores , Canais de Cátion TRPV/metabolismo
20.
Clin Exp Immunol ; 199(2): 119-130, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31626717

RESUMO

Generation of antigen-specific humoral responses following vaccination or infection requires the maturation and function of highly specialized immune cells in secondary lymphoid organs (SLO), such as lymph nodes or tonsils. Factors that orchestrate the dynamics of these cells are still poorly understood. Currently, experimental approaches that enable a detailed description of the function of the immune system in SLO have been mainly developed and optimized in animal models. Conversely, methodological approaches in humans are mainly based on the use of blood-associated material because of the challenging access to tissues. Indeed, only few studies in humans were able to provide a discrete description of the complex network of cytokines, chemokines and lymphocytes acting in tissues after antigenic challenge. Furthermore, even fewer data are currently available on the interaction occurring within the complex micro-architecture of the SLO. This information is crucial in order to design particular vaccination strategies, especially for patients affected by chronic and immune compromising medical conditions who are under-vaccinated or who respond poorly to immunizations. Analysis of immune cells in different human tissues by high-throughput technologies, able to obtain data ranging from gene signature to protein expression and cell phenotypes, is needed to dissect the peculiarity of each immune cell in a definite human tissue. The main aim of this review is to provide an in-depth description of the current available methodologies, proven evidence and future perspectives in the analysis of immune mechanisms following immunization or infections in SLO.


Assuntos
Citocinas/imunologia , Imunoterapia Adotiva , Linfonodos/imunologia , Linfócitos/imunologia , Vacinação , Animais , Humanos , Linfonodos/citologia , Linfócitos/citologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA