RESUMO
Extracellular vesicles (EVs) are lipid-bound particles produced by a wide variety of cells from different biological species. EVs can carry molecules, such as nucleic acids and metabolites, and are involved in cell functioning, communication, and signaling. Recent literature reported that pathogenic or commensal yeast strains can produce EVs targeting the host's immune system and exerting immunomodulatory actions. In humans, yeast EVs can be endocytosed by dendritic cells (DCs), characterized by phagocyting and migrating capabilities with the role of capturing antigens to present to T lymphocytes, triggering the immune response. Physiological or disease-associated immunosenescence impairs both DC functionality and gut microbiota; thus investigating the interaction between commensal microorganisms and the host's immune system would help elucidate the impact of aging on the immune system-microbiota interplay. We hereby present a protocol for the incubation of in vitro-generated human monocyte-derived DCs with EVs purified from different yeast strains isolated from fermented milk. The protocol includes flow cytometry analysis on DC activation markers and endocytosis assay.
Assuntos
Células Dendríticas , Vesículas Extracelulares , Monócitos , Humanos , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Monócitos/metabolismo , Monócitos/imunologia , Monócitos/microbiologia , Citometria de Fluxo/métodos , Endocitose , Leveduras/metabolismo , Saccharomyces cerevisiae/metabolismo , Células CultivadasRESUMO
Introduction: Cytokine release syndrome (CRS) is one of the leading causes of mortality in patients with COVID-19 caused by the SARS-CoV-2 coronavirus. However, the mechanism of CRS induced by SARS-CoV-2 is vague. Methods: Using spike protein combined with IL-2, IFN-γ, and TNF-α to stimulate human peripheral blood mononuclear cells (PBMCs) to secrete CRS-related cytokines, the content of cytokines in the supernatant was detected, and the effects of NK, T, and monocytes were analyzed. Results: This study shows that dendritic cells loaded with spike protein of SARS-CoV-2 stimulate T cells to release much more interleukin-2 (IL-2,) which subsequently cooperates with spike protein to facilitate PBMCs to release IL-1ß, IL-6, and IL-8. These effects are achieved via IL-2 stimulation of NK cells to release tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), as well as T cells to release IFN-γ Mechanistically, IFN-γ and TNF-α enhance the transcription of CD40, and the interaction of CD40 and its ligand stabilizes the membrane expression of toll-like receptor 4 (TLR4) that serves as a receptor of spike protein on the surface of monocytes. As a result, there is a constant interaction between spike protein and TLR4, leading to continuous activation of nuclear factor-κ-gene binding (NF-κB). Furthermore, TNF-α also activates NF-κB signaling in monocytes, which further cooperates with IFN-γ and spike protein to modulate NF-κB-dependent transcription of CRS-related inflammatory cytokines. Discussion: Targeting TNF-α/IFN-γ in combination with TLR4 may represent a promising therapeutic approach for alleviating CRS in individuals with COVID-19.
Assuntos
COVID-19 , Síndrome da Liberação de Citocina , Interleucina-2 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Linfócitos T , Humanos , Glicoproteína da Espícula de Coronavírus/imunologia , Interleucina-2/metabolismo , Interleucina-2/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Síndrome da Liberação de Citocina/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interferon gama/metabolismo , Interferon gama/imunologia , Receptor 4 Toll-Like/metabolismo , NF-kappa B/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Citocinas/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Recent advances in cancer research have highlighted the pivotal role of tertiary lymphoid structures (TLSs) in modulating immune responses, particularly in breast cancer (BRCA). Here, we performed an integrated analysis of bulk transcriptome data from over 6000 BRCA samples using biological network-based computational strategies and machine learning (ML) methods, and identified LGALS2 as a key marker within TLSs. Single-cell sequencing and spatial transcriptomics uncover the role of LGALS2 in TLS-associated dendritic cells (DCs) stimulation and reveal the complexity of the tumor microenvironment (TME) at both the macro and micro levels. Elevated LGALS2 expression correlates with prolonged survival, which is associated with a robust immune response marked by diverse immune cell infiltration and active anti-tumor pathways leading to a 'hot' tumor microenvironment. The colocalization of LGALS2 with TLS-associated DCs and its role in immune activation in BRCA were confirmed by hematoxylin-eosin (HE), immunohistochemistry (IHC), and in vivo validation analyses. The identification of LGALS2 as a key factor in BRCA not only highlights its therapeutic potential in novel TLS-directed immunotherapy but also opens new avenues in patient stratification and treatment selection, ultimately improving clinical management.
Assuntos
Neoplasias da Mama , Células Dendríticas , Galectina 2 , Imunoterapia , Estruturas Linfoides Terciárias , Microambiente Tumoral , Humanos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Feminino , Microambiente Tumoral/imunologia , Imunoterapia/métodos , Estruturas Linfoides Terciárias/imunologia , Estruturas Linfoides Terciárias/patologia , Galectina 2/genética , Galectina 2/metabolismo , Animais , Biomarcadores Tumorais , Regulação Neoplásica da Expressão Gênica , Camundongos , Análise de Célula Única , PrognósticoRESUMO
Interferon regulatory factor-8 (IRF8) is the lineage determining transcription factor for the type one classical dendritic cell (cDC1) subset, a terminal selector for plasmacytoid dendritic cells and important for the function of monocytes. Studies of Irf8 gene regulation have identified several enhancers controlling its activity during development of progenitors in the bone marrow that precisely regulate expression at distinct developmental stages. Each enhancer responds to distinct transcription factors that are expressed at each stage. IRF8 is first expressed in early progenitors that form the monocyte dendritic cell progenitor (MDP) in response to induction of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) acting at the Irf8 +56 kb enhancer. IRF8 levels increase further as the MDP transits into the common dendritic cell progenitor (CDP) in response to E protein activity at the Irf8 +41 kb enhancer. Upon Nfil3-induction in CDPs leading to specification of the cDC1 progenitor, abrupt induction of BATF3 forms the JUN/BATF3/IRF8 heterotrimer that activates the Irf8 +32 kb enhancer that sustains Irf8 autoactivation throughout the cDC1 lifetime. Deletions of each of these enhancers has revealed their stage dependent activation. Surprisingly, studies of compound heterozygotes for each combination of enhancer deletions revealed that activation of each subsequent enhancer requires the successful activation of the previous enhancer in strictly cis-dependent mechanism. Successful progression of enhancer activation is finely tuned to alter the functional accessibility of subsequent enhancers to factors active in the next stage of development. The molecular basis for these phenomenon is still obscure but could have implications for genomic regulation in a broader developmental context.
Assuntos
Células Dendríticas , Elementos Facilitadores Genéticos , Fatores Reguladores de Interferon , Humanos , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Elementos Facilitadores Genéticos/genética , Diferenciação Celular , Regulação da Expressão Gênica , Super IntensificadoresRESUMO
Introduction: Immunogenicity, the unwanted immune response triggered by therapeutic antibodies, poses significant challenges in biotherapeutic development. This response can lead to the production of anti-drug antibodies, potentially compromising the efficacy and safety of treatments. The internalization of therapeutic antibodies into dendritic cells (DCs) is a critical factor influencing immunogenicity. Using monoclonal antibodies, with differences in non-specific cellular uptake, as tools to explore the impact on the overall risk of immunogenicity, this study explores how internalization influences peptide presentation and subsequently T cell activation. Materials and methods: To investigate the impact of antibody internalization on immunogenicity, untargeted toolantibodies with engineered positive or negative charge patches were utilized. Immature monocyte-derived DCs (moDCs), known for their physiologically relevant high endocytic activity, were employed for internalization assays, while mature moDCs were used for MHC-II associated peptide proteomics (MAPPs) assays. In addition to the lysosomal accumulation and peptide presentation, subsequent CD4+ T cell activation has been assessed. Consequently, a known CD4+ T cell epitope from ovalbumin was inserted into the tool antibodies to evaluate T cell activation on a single, shared epitope. Results: Antibodies with positive charge patches exhibited higher rates of lysosomal accumulation and epitope presentation compared to those with negative charge patches or neutral surface charge. Furthermore, a direct correlation between internalization rate and presentation on MHC-II molecules could be established. To explore the link between internalization, peptide presentation and CD4+ T cell activation, tool antibodies containing the same OVA epitope were used. Previous observations were not altered by the insertion of the OVA epitope and ultimately, an enhanced CD4+ T cell response correlated with increased internalization in DCs and peptide presentation. Discussion: These findings demonstrate that the biophysical properties of therapeutic antibodies, particularly surface charge, play a crucial role in their internalization into DCs. Antibodies internalized faster and processed by DCs, are also more prone to be presented on their surface leading to a higher risk of triggering an immune response. These insights underscore the importance of considering antibody surface charge and other properties that enhance cellular accumulation during the preclinical development of biotherapeutics to mitigate immunogenicity risks.
Assuntos
Apresentação de Antígeno , Células Dendríticas , Ativação Linfocitária , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Apresentação de Antígeno/imunologia , Ativação Linfocitária/imunologia , Anticorpos Monoclonais/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Fatores de Risco , Endocitose/imunologia , Ovalbumina/imunologiaRESUMO
Dendritic cells (DCs) play a crucial role in orchestrating immune responses, particularly in promoting IFNγ-producing-CD8 cytotoxic T lymphocytes (CTLs) and IFNγ-producing-CD4 T helper 1 (Th1) cells, which are essential for defending against viral infections. Additionally, the nuclear envelope protein lamin A/C has been implicated in T cell immunity. Nevertheless, the intricate interplay between innate and adaptive immunity in response to viral infections, particularly the role of lamin A/C in DC functions within this context, remains poorly understood. In this study, we demonstrate that mice lacking lamin A/C in myeloid LysM promoter-expressing cells exhibit a reduced capacity to induce Th1 and CD8 CTL responses, leading to impaired clearance of acute primary Vaccinia virus (VACV) infection. Remarkably, in vitro-generated granulocyte macrophage colony-stimulating factor bone marrow-derived DCs (GM-CSF BMDCs) show high levels of lamin A/C. Lamin A/C absence on GM-CSF BMDCs does not affect the expression of costimulatory molecules on the cell membrane but it reduces the cellular ability to form immunological synapses with naïve CD4 T cells. Lamin A/C deletion induces alterations in NFκB nuclear localization, thereby influencing NF-κB-dependent transcription. Furthermore, lamin A/C ablation modifies the gene accessibility of BMDCs, predisposing these cells to mount a less effective antiviral response upon TLR stimulation. This study highlights the critical role of DCs in interacting with CD4 T cells during antiviral responses and proposes some mechanisms through which lamin A/C may modulate DC function via gene accessibility and transcriptional regulation.
Assuntos
Células Dendríticas , Lamina Tipo A , Camundongos Endogâmicos C57BL , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Animais , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Camundongos , NF-kappa B/metabolismo , Vaccinia virus/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Camundongos Knockout , Vacínia/imunologia , Células Th1/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Sinapses Imunológicas/metabolismo , Sinapses Imunológicas/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
We identified a novel mouse plasmacytoid dendritic cell (pDC) lineage derived from the common lymphoid progenitors (CLPs) that is dependent on expression of Bcl11a. These CLP-derived pDCs, which we refer to as 'B-pDCs', have a unique gene expression profile that includes hallmark B cell genes, normally not expressed in conventional pDCs. Despite expressing most classical pDC markers such as SIGLEC-H and PDCA1, B-pDCs lack IFN-α secretion, exhibiting a distinct inflammatory profile. Functionally, B-pDCs induce T cell proliferation more robustly than canonical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs, along with another homogeneous subpopulation of myeloid-derived pDCs, display elevated levels of the cell surface receptor tyrosine kinase AXL, mirroring human AXL+ transitional DCs in function and transcriptional profile. Murine B-pDCs therefore represent a phenotypically and functionally distinct CLP-derived DC lineage specialized in T cell activation and previously not described in mice.
Assuntos
Células Dendríticas , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Camundongos , Ativação Linfocitária , Linfócitos T/imunologia , Linfócitos T/metabolismo , Camundongos Endogâmicos C57BL , Perfilação da Expressão Gênica , Células Progenitoras Linfoides/metabolismo , Células Progenitoras Linfoides/citologia , Linhagem da CélulaRESUMO
Homologous animal cell product was obtained in protocol developed for female BALB/c mice. Dendritic cell (DC) migration from the injection site into the draining lymph nodes was evaluated. The number of DC labeled with carboxyfluorescein succinimidyl ester (CFSE) in draining lymph nodes increased from 5.3% (16 h) to 13.3% (48 h) (p=0.028) with a maximum at 72 h (15.4%, p=0.003). The immunophenotype of CFSE-DC detected in murine lymph nodes corresponded to the immunophenotype of mature vaccine DCs: they expressed differentiation markers CD11c, CD80, CD83, and CD86 (p>0.05 vs initial DC).
Assuntos
Vacinas Anticâncer , Células Dendríticas , Linfonodos , Camundongos Endogâmicos BALB C , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Vacinas Anticâncer/imunologia , Camundongos , Linfonodos/imunologia , Linfonodos/metabolismo , Succinimidas , Antígenos CD/imunologia , Antígenos CD/metabolismo , Fluoresceínas , Antígeno CD11c/metabolismo , Antígeno CD11c/imunologia , Antígeno B7-2/metabolismo , Antígeno B7-2/imunologia , Antígeno B7-1/metabolismo , Antígeno B7-1/imunologia , Antígeno CD83 , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Diferenciação Celular , Distribuição Tecidual , Imunofenotipagem , Movimento CelularRESUMO
Skin psoriasis is defined as receiving external stimulation to activate skin dendritic cells (DCs) which can release interleukin 23 (IL-23) to interlink the innate and adaptive immunity as well as induce T helper 17 (Th17) cell differentiation leading to elevated production of interleukin 17 (IL-17) for keratinocytes over production. This autoimmune loop in psoriasis pathogenesis is influenced by G protein-coupled receptor (GPCR) signalling transduction, and in particular, function of adhesion molecule GPR97 in psoriasis endures to be utterly addressed. In this research, our team allocated GPR97 depletion (GPR97-/-), GPR97 conditional depletion on dendritic cell (DC-cKO), and keratin 14-conditional knockout (K14-cKO) mice models to explore the function of GPR97 which influences keratinocytes and skin immunity. It was found that significantly aggravated psoriasis-like lesion in GPR97-/- mice. In addition, hyperproliferative keratinocytes as well as accumulation of DCs and Th17 cells were detected in imiquimod (IMQ)-induced GPR97-/- mice, which was consistent with the results in DC-cKO and K14-cKO psoriasis model. Additional investigations indicated that beclomethasone dipropionate (BDP), an agonist of GPR97, attenuated the psoriasis-like skin disease and restricted HaCaT cells abnormal proliferation as well as Th17 cells differentiation. Particularly, we found that level of NF-κB p65 was increased in GPR97-/- DCs and BDP could inhibit p65 activation in DCs. Role of GPR97 is indispensable and this adhesion receptor may affect immune cell enrichment and function in skin and alter keratinocytes proliferation as well as differentiation in psoriasis.
Assuntos
Imiquimode , Interleucina-17 , Interleucina-23 , Queratinócitos , Camundongos Knockout , Psoríase , Receptores Acoplados a Proteínas G , Transdução de Sinais , Células Th17 , Animais , Psoríase/induzido quimicamente , Psoríase/patologia , Psoríase/genética , Psoríase/metabolismo , Interleucina-17/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Camundongos , Queratinócitos/metabolismo , Queratinócitos/patologia , Células Th17/imunologia , Células Th17/metabolismo , Interleucina-23/metabolismo , Células Dendríticas/metabolismo , Camundongos Endogâmicos C57BL , Humanos , Modelos Animais de Doenças , Pele/patologia , Pele/metabolismoRESUMO
In allergen-specific immunotherapy, adjuvants are explored for modulating allergen-specific Th2 immune responses to re-establish clinical tolerance. One promising class of adjuvants are ß-glucans, which are naturally derived sugar structures and components of dietary fibers that activate C-type lectin (CLR)-, "Toll"-like receptors (TLRs), and complement receptors (CRs). We characterized the immune-modulating properties of six commercially available ß-glucans, using immunological (receptor activation, cytokine secretion, and T cell modulating potential) as well as metabolic parameters (metabolic state) in mouse bone marrow-derived myeloid dendritic cells (mDCs). All tested ß-glucans activated the CLR Dectin-1a, whereas TLR2 was predominantly activated by Zymosan. Further, the tested ß-glucans differentially induced mDC-derived cytokine secretion and activation of mDC metabolism. Subsequent analyses focusing on Zymosan, Zymosan depleted, ß-1,3 glucan, and ß-1,3 1,6 glucan revealed robust mDC activation with the upregulation of the cluster of differentiation 40 (CD40), CD80, CD86, and MHCII to different extents. ß-glucan-induced cytokine secretion was shown to be, in part, dependent on the activation of the intracellular Dectin-1 adapter molecule Syk. In co-cultures of mDCs with Th2-biased CD4+ T cells isolated from birch allergen Bet v 1 plus aluminum hydroxide (Alum)-sensitized mice, these four ß-glucans suppressed allergen-induced IL-5 secretion, while only Zymosan and ß-1,3 glucan significantly suppressed allergen-induced interferon gamma (IFNγ) secretion, suggesting the tested ß-glucans to have distinct effects on mDC T cell priming capacity. Our experiments indicate that ß-glucans have distinct immune-modulating properties, making them interesting adjuvants for future allergy treatment.
Assuntos
Citocinas , Células Dendríticas , Lectinas Tipo C , beta-Glucanas , Animais , Células Dendríticas/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , beta-Glucanas/farmacologia , beta-Glucanas/química , Camundongos , Lectinas Tipo C/metabolismo , Citocinas/metabolismo , Adjuvantes Imunológicos/farmacologia , Zimosan/farmacologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Receptor 2 Toll-Like/metabolismo , Camundongos Endogâmicos C57BL , Quinase Syk/metabolismoRESUMO
The development, differentiation, and function of immune cells are precisely regulated by transcription factors. The E26 transformation-specific (ETS) transcription factor family is involved in various physiological and pathological processes by regulating cell proliferation, differentiation, and apoptosis. Emerging evidence has suggested that ETS family proteins are intimately involved in the development and function of immune cells. This review summarizes the role of the ETS family in immune cells and immune-related disorders. Seven transcription factors within the ETS family, including PU.1, ETV5, ETV6, ETS1/2, ELK3, and ELF1, play essential roles in the development and function of T cells, B cells, macrophages, neutrophils, and dendritic cells. Furthermore, they are involved in the occurrence and development of immune-related diseases, including tumors, allergies, autoimmune diseases, and arteriosclerosis. This review is conducive to a comprehensive overview of the role of the ETS family in immune cells, and thus is informative for the development of novel therapeutic strategies targeting the ETS family for immune-related diseases.
Assuntos
Proteínas Proto-Oncogênicas c-ets , Humanos , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Animais , Doenças do Sistema Imunitário/imunologia , Doenças do Sistema Imunitário/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismoRESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma tumors exhibit resistance to chemotherapy, targeted therapies, and even immunotherapy. Dendritic cells use glucose to support their effector functions and play a key role in anti-tumor immunity by promoting cytotoxic CD8+ T cell activity. However, the effects of glucose and lactate levels on dendritic cells in pancreatic ductal adenocarcinoma are unclear. In this study, we aimed to clarify how glucose and lactate can impact the dendritic cell antigen-presenting function and elucidate the relevant mechanisms. METHODS: Glycolytic activity and immune cell infiltration in pancreatic ductal adenocarcinoma were evaluated using patient-derived organoids and resected specimens. Cell lines with increased or decreased glycolysis were established from KPC mice. Flow cytometry and single-cell RNA sequencing were used to evaluate the impacts on the tumor microenvironment. The effects of glucose and lactate on the bone marrow-derived dendritic cell antigen-presenting function were detected by flow cytometry. RESULTS: The pancreatic ductal adenocarcinoma tumor microenvironment exhibited low glucose and high lactate concentrations from varying levels of glycolytic activity in cancer cells. In mouse transplantation models, tumors with increased glycolysis showed enhanced myeloid-derived suppressor cell infiltration and reduced dendritic cell and CD8+ T cell infiltration, whereas tumors with decreased glycolysis displayed the opposite trends. In three-dimensional co-culture, increased glycolysis in cancer cells suppressed the antigen-presenting function of bone marrow-derived dendritic cells. In addition, low-glucose and high-lactate media inhibited the antigen-presenting and mitochondrial functions of bone marrow-derived dendritic cells. CONCLUSIONS: Our study demonstrates the impact of dynamic glycolytic reprogramming on the composition of immune cells in the tumor microenvironment of pancreatic ductal adenocarcinoma, especially on the antigen-presenting function of dendritic cells.
Assuntos
Carcinoma Ductal Pancreático , Células Dendríticas , Glicólise , Neoplasias Pancreáticas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/metabolismo , Animais , Camundongos , Humanos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral , Reprogramação Celular , Linhagem Celular TumoralRESUMO
Tumor-associated neutrophil (TAN) effects on glioblastoma (GBM) biology remain under-characterized. We show here that neutrophils with dendritic features-including morphological complexity, expression of antigen presentation genes, and the ability to process exogenous peptide and stimulate major histocompatibility complex (MHC)II-dependent T cell activation-accumulate intratumorally and suppress tumor growth in vivo. Trajectory analysis of patient TAN scRNA-seq identifies this "hybrid" dendritic-neutrophil phenotype as a polarization state that is distinct from canonical cytotoxic TANs, and which differentiates from local precursors. These hybrid-inducible immature neutrophils-which we identified in patient and murine glioblastomas-arise not from circulation, but from local skull marrow. Through labeled skull flap transplantation and targeted ablation, we characterize calvarial marrow as a contributor of antitumoral myeloid antigen-presenting cells (APCs), including TANs, which elicit T cell cytotoxicity and memory. As such, agents augmenting neutrophil egress from skull marrow-such as intracalvarial AMD3100, whose survival-prolonging effect in GBM we report-present therapeutic potential.
Assuntos
Neoplasias Encefálicas , Diferenciação Celular , Células Dendríticas , Glioblastoma , Neutrófilos , Humanos , Animais , Camundongos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Glioblastoma/patologia , Glioblastoma/imunologia , Glioblastoma/genética , Glioblastoma/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Crânio/patologia , Crânio/imunologia , Medula Óssea/patologia , Medula Óssea/imunologia , Camundongos Endogâmicos C57BL , Linhagem Celular TumoralRESUMO
Clostridioides difficile is a spore-forming pathogen and the most common cause of healthcare-associated diarrhea and colitis in the United States. Besides producing the main virulence factors, toxin A (TcdA) and toxin B (TcdB), many of the common clinical strains encode the C. difficile transferase (CDT) binary toxin. The role of CDT in the context of C. difficile infection (CDI) is poorly understood. Inflammation is a hallmark of CDI and multiple mechanisms of inflammasome activation have been reported for TcdA, TcdB, and the organism. Some studies have suggested that CDT contributes to this inflammation through a TLR2-dependent priming mechanism that leads to the suppression of protective eosinophils. Here, we show that CDT does not prime but instead activates the inflammasome in bone marrow-derived dendritic cells (BMDCs). In bone marrow-derived macrophages (BMDMs), the cell binding and pore-forming component of the toxin, CDTb, alone activates the inflammasome and is dependent on K+ efflux. The activation is not observed in the presence of CDTa and is not observed in BMDMs derived from Nlrp3-/- mice suggesting the involvement of the NLRP3 inflammasome. However, we did not observe evidence of CDT-dependent inflammasome priming or activation in vivo. Mice were infected with R20291 and an isogenic CRISPR/Cas9-generated R20291 ΔcdtB strain of C. difficile. While CDT contributes to increased weight loss and cecal edema at 2 days post infection, the relative levels of inflammasome-associated cytokines, IL-1ß and IL-18, in the cecum and distal colon are unchanged. We also saw CDT-dependent weightloss in Nlrp3-/- mice, suggesting that the increased weightloss associated with the presence of CDT is not a result of NLRP3-dependent inflammasome activation. This study highlights the importance of studying gene deletions in the context of otherwise fully isogenic strains and the challenge of translating toxin-specific cellular responses into a physiological context, especially when multiple toxins are acting at the same time.
Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Clostridioides difficile , Infecções por Clostridium , Inflamassomos , Inflamação , Camundongos Endogâmicos C57BL , Animais , Camundongos , Clostridioides difficile/patogenicidade , Toxinas Bacterianas/metabolismo , Inflamação/metabolismo , Inflamassomos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Infecções por Clostridium/imunologia , Infecções por Clostridium/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , ADP Ribose Transferases/metabolismo , ADP Ribose Transferases/genética , Macrófagos/metabolismo , Macrófagos/imunologia , Camundongos Knockout , EnterotoxinasRESUMO
In this study, a new secobutanolide, named secosubamolide B (3), along with three previously known secobutanolides (1, 2, and 4), were successfully isolated from a methanol extract of the stem of Lindera obtusiloba. The chemical structures of these compounds were elucidated through the analysis of spectroscopic data, and then compared with the existing literature to confirm their identities. Furthermore, the anti-inflammatory effect of these isolated compounds on bone marrow-derived dendritic cells stimulated by lipopolysaccharide (LPS) was evaluated. Compounds 1-3 showed the significant suppression of LPS-triggered IL-6 and IL-12 p40 production, with IC50 values between 1.8 and 24.1 µM. These findings may provide a scientific foundation for developing anti-inflammatory agents from L. obtusiloba.
Assuntos
Anti-Inflamatórios , Lindera , Lipopolissacarídeos , Caules de Planta , Lindera/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Animais , Camundongos , Lipopolissacarídeos/farmacologia , Caules de Planta/química , Interleucina-6/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Estrutura Molecular , Subunidade p40 da Interleucina-12/metabolismoRESUMO
OBJECTIVE: To assess the impact of the IFIT3/TBK1 signalling pathway in activating plasmacytoid dendritic cells (pDCs) and its role in the development of SSc. METHODS: Utilized single-cell RNA sequencing (scRNA-seq) and high-throughput transcriptome RNA sequencing to reveal the differential abundance of pDCs and the role of the key gene IFIT3 in SSc. Conducted in vitro cell experiments to evaluate the effect of IFIT3/TBK1 signalling pathway intervention on pDC activation cytokine release and fibroblast function. Constructed an IFIT3-/- mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing to assess the potential benefits of intervening in the IFIT3/TBK1 signalling pathway on skin and lung fibrosis in the SSc mouse model. RESULTS: The IFIT3/TBK1 signalling pathway plays a crucial role in activating pDCs, with IFIT3 acting as an upstream regulator of TBK1. Intervention in the IFIT3/TBK1 signalling pathway can inhibit pDC activation cytokine release and impact fibroblast function. The IFIT3-/- mouse model shows potential benefits of targeting the IFIT3/TBK1 signalling pathway in reducing skin and lung fibrosis in the SSc mouse model. CONCLUSION: This study provides new insights into potential therapeutic targets for SSc, highlighting the critical role of the IFIT3/TBK1 signalling pathway in SSc development. HIGHLIGHTS: This study elucidates the pivotal role of plasmacytoid dendritic cells (pDCs) in systemic sclerosis (SSc). This study identified the key regulatory gene involved in systemic sclerosis (SSc) as IFIT3. This study has found that IFIT3 functions as an upstream regulatory factor, activating TBK1. This study provides Evidence of the regulatory effects of the IFIT3/TBK1 pathway on plasmacytoid dendritic cells (pDCs). This study validated the therapeutic potential using the IFIT3-/- mouse model.
Assuntos
Células Dendríticas , Modelos Animais de Doenças , Proteínas Serina-Treonina Quinases , Escleroderma Sistêmico , Animais , Feminino , Masculino , Camundongos , Células Dendríticas/metabolismo , Camundongos Knockout , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/genética , Transdução de SinaisRESUMO
Manipulation of immune cell functions, independently of direct infection of these cells, emerges as a key process in viral pathophysiology. Chronic infection by Human T-cell Leukemia Virus type 1 (HTLV-1) is associated with immune dysfunctions, including misdirected responses of dendritic cells (DCs). Here, we interrogate the ability of transformed HTLV-1-infected T cells to manipulate human DC functions. We show that exposure to transformed HTLV-1-infected T cells induces a biased and peculiar transcriptional signature in monocyte-derived DCs, associated with an inefficient maturation and a poor responsiveness to subsequent stimulation by a TLR4 agonist. This poor responsiveness is also associated with a unique transcriptional landscape characterized by a set of genes whose expression is either conferred, impaired or abolished by HTLV-1 pre-exposure. Induction of this functional impairment requires several hours of coculture with transformed HTLV-1-infected cells, and associated mechanisms driven by viral capture, cell-cell contacts, and soluble mediators. Altogether, this cross-talk between infected T cells and DCs illustrate how HTLV-1 might co-opt communications between cells to induce a unique local tolerogenic immune microenvironment suitable for its own persistence.
Assuntos
Células Dendríticas , Infecções por HTLV-I , Vírus Linfotrópico T Tipo 1 Humano , Humanos , Células Dendríticas/imunologia , Células Dendríticas/virologia , Células Dendríticas/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Infecções por HTLV-I/genética , Linfócitos T/imunologia , Linfócitos T/virologia , Reprogramação CelularRESUMO
Cancer vaccine development is inhibited by a lack of strategies for directing dendritic cell (DC) induction of effective tumor-specific cellular immunity. Pathogen engagement of DC lectins and toll-like receptors (TLRs) is thought to shape immunity by directing T cell function. Controlling downstream responses, however, remains a major challenge. A critical goal in advancing vaccine development involves the identification of receptors that drive type 1 cellular immunity. The immune system monitors cells for aberrant glycosylation (a sign of a foreign entity), but potent activation occurs when a second signal, such as single-stranded RNA or lipopolysaccharide, is present to activate TLR signaling. To exploit dual signaling, we engineered a glycan-costumed virus-like particle (VLP) vaccine that displays a DC-SIGN-selective aryl mannose ligand and encapsulates TLR7 agonists. These VLPs deliver programmable peptide antigens to induce robust DC activation and type 1 cellular immunity. In contrast, VLPs lacking this critical DC-SIGN ligand promoted DC-mediated humoral immunity, offering limited tumor control. Vaccination with glycan-costumed VLPs generated tumor antigen-specific Th1 CD4+ and CD8+ T cells that infiltrated solid tumors, significantly inhibiting tumor growth in a murine melanoma model. The tailored VLPs also afforded protection against the reintroduction of tumor cells. Thus, DC lectin-driven immune reprogramming, combined with the modular programmability of VLP platforms, provides a promising framework for directing cellular immunity to advance cancer immunotherapies and vaccines.
Assuntos
Vacinas Anticâncer , Células Dendríticas , Lectinas Tipo C , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Animais , Camundongos , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/química , Lectinas Tipo C/metabolismo , Lectinas Tipo C/imunologia , Camundongos Endogâmicos C57BL , Humanos , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas de Partículas Semelhantes a Vírus/imunologia , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Carboidratos/química , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/imunologia , Polissacarídeos/química , Imunidade CelularRESUMO
Dendritic cells (DCs) serve as key regulators in tumor immunity, with activated DCs potentiating antitumor responses through the secretion of pro-inflammatory cytokines and the expression of co-stimulatory molecules. Most current studies focus on the relationship between DC subgroups and clear-cell renal-cell carcinoma (ccRCC), but there is limited research on the connection between DCs and ccRCC from the perspective of immune activation. In this study, activated DC genes were identified in both bulk and single-cell RNA-seq data. A prognostic model related to activated DCs was constructed using univariate, multivariate Cox regression and LASSO regression. The prognostic model was validated in three external validation sets: GSE167573, ICGC, and E-MTAB-1980. The prognostic model consists of five genes, PLCB2, XCR1, IFNG, HLA-DQB2, and SMIM24. The expression of these genes was validated in tissue samples using qRT-PCR. Stratified analysis revealed that the prognostic model was able to better predict outcomes in advanced ccRCC patients. The risk scores were associated with tumor progression, tumor mutation burden, immune cell infiltration, and adverse outcomes of immunotherapy. Notably, there was a strong correlation between the expression of the five genes and the sensitivity to JQ1, a BET inhibitor. Molecular docking indicated high-affinity binding of the proteins encoded by these genes with JQ1. In conclusion, our study reveals the crucial role of activated DCs in ccRCC, offering new insights into predicting immune response, targeted therapy effectiveness, and prognosis for ccRCC patients.
Assuntos
Carcinoma de Células Renais , Células Dendríticas , Neoplasias Renais , RNA-Seq , Análise de Célula Única , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/imunologia , Humanos , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Neoplasias Renais/imunologia , Neoplasias Renais/metabolismo , Prognóstico , Análise de Célula Única/métodos , Regulação Neoplásica da Expressão Gênica , Biomarcadores Tumorais/genética , Masculino , Feminino , Análise da Expressão Gênica de Célula ÚnicaRESUMO
Inflammatory Bowel Diseases (IBD), which encompass ulcerative colitis (UC) and Crohn's disease (CD), are characterized by chronic inflammation and tissue damage of the gastrointestinal tract. This study aimed to uncover novel disease-gene signatures, dysregulated pathways, and the immune cell infiltration landscape of inflamed tissues. Eight publicly available transcriptomic datasets, including inflamed and non-inflamed tissues from CD and UC patients were analyzed. Common differentially expressed genes (DEGs) were identified through meta-analysis, revealing 180 DEGs. DEGs were implicated in leukocyte transendothelial migration, PI3K-Akt, chemokine, NOD-like receptors, TNF signaling pathways, and pathways in cancer. Protein-protein interaction network and cluster analysis identified 14 central IBD players, which were validated using eight external datasets. Disease module construction using the NeDRex platform identified nine out of 14 disease-associated genes (CYBB, RAC2, GNAI2, ITGA4, CYBA, NCF4, CPT1A, NCF2, and PCK1). Immune infiltration profile assessment revealed a significantly higher degree of infiltration of neutrophils, activated dendritic cells, plasma cells, mast cells (resting/activated), B cells (memory/naïve), regulatory T cells, and M0 and M1 macrophages in inflamed IBD tissue. Collectively, this study identified the immune infiltration profile and nine disease-associated genes as potential modulators of IBD pathogenesis, offering insights into disease molecular mechanisms, and highlighting potential disease modulators and immune cell dynamics.