RESUMEN
Cell function and activity are regulated through integration of signaling, epigenetic, transcriptional, and metabolic pathways. Here, we introduce INs-seq, an integrated technology for massively parallel recording of single-cell RNA sequencing (scRNA-seq) and intracellular protein activity. We demonstrate the broad utility of INs-seq for discovering new immune subsets by profiling different intracellular signatures of immune signaling, transcription factor combinations, and metabolic activity. Comprehensive mapping of Arginase 1-expressing cells within tumor models, a metabolic immune signature of suppressive activity, discovers novel Arg1+ Trem2+ regulatory myeloid (Mreg) cells and identifies markers, metabolic activity, and pathways associated with these cells. Genetic ablation of Trem2 in mice inhibits accumulation of intra-tumoral Mreg cells, leading to a marked decrease in dysfunctional CD8+ T cells and reduced tumor growth. This study establishes INs-seq as a broadly applicable technology for elucidating integrated transcriptional and intra-cellular maps and identifies the molecular signature of myeloid suppressive cells in tumors.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Neoplasias/patología , ARN Citoplasmático Pequeño/química , Receptores Inmunológicos/metabolismo , Animales , Arginasa/genética , Arginasa/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/metabolismo , ARN Citoplasmático Pequeño/metabolismo , Receptores Inmunológicos/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Microambiente Tumoral , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Lung development and function arises from the interactions between diverse cell types and lineages. Using single-cell RNA sequencing (RNA-seq), we characterize the cellular composition of the lung during development and identify vast dynamics in cell composition and their molecular characteristics. Analyzing 818 ligand-receptor interaction pairs within and between cell lineages, we identify broadly interacting cells, including AT2, innate lymphocytes (ILCs), and basophils. Using interleukin (IL)-33 receptor knockout mice and in vitro experiments, we show that basophils establish a lung-specific function imprinted by IL-33 and granulocyte-macrophage colony-stimulating factor (GM-CSF), characterized by unique signaling of cytokines and growth factors important for stromal, epithelial, and myeloid cell fates. Antibody-depletion strategies, diphtheria toxin-mediated selective depletion of basophils, and co-culture studies show that lung resident basophils are important regulators of alveolar macrophage development and function. Together, our study demonstrates how whole-tissue signaling interaction map on the single-cell level can broaden our understanding of cellular networks in health and disease.
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Basófilos/metabolismo , Comunicación Celular , Impresión Genómica , Macrófagos Alveolares/metabolismo , Transcriptoma , Animales , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interleucina-33/metabolismo , Macrófagos Alveolares/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Análisis de la Célula IndividualRESUMEN
Consecutive exposures to different pathogens are highly prevalent and often alter the host immune response. However, it remains unknown how a secondary bacterial infection affects an ongoing adaptive immune response elicited against primary invading pathogens. We demonstrated that recruitment of Sca-1+ monocytes into lymphoid organs during Salmonella Typhimurium (STm) infection disrupted pre-existing germinal center (GC) reactions. GC responses induced by influenza, plasmodium, or commensals deteriorated following STm infection. GC disruption was independent of the direct bacterial interactions with B cells and instead was induced through recruitment of CCR2-dependent Sca-1+ monocytes into the lymphoid organs. GC collapse was associated with impaired cellular respiration and was dependent on TNFα and IFNγ, the latter of which was essential for Sca-1+ monocyte differentiation. Monocyte recruitment and GC disruption also occurred during LPS-supplemented vaccination and Listeria monocytogenes infection. Thus, systemic activation of the innate immune response upon severe bacterial infection is induced at the expense of antibody-mediated immunity.
Asunto(s)
Infecciones Bacterianas , Listeriosis , Linfocitos B , Centro Germinal , Humanos , MonocitosRESUMEN
In multicellular organisms, dedicated regulatory circuits control cell type diversity and responses. The crosstalk and redundancies within these circuits and substantial cellular heterogeneity pose a major research challenge. Here, we present CRISP-seq, an integrated method for massively parallel single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-pooled screens. We show that profiling the genomic perturbation and transcriptome in the same cell enables us to simultaneously elucidate the function of multiple factors and their interactions. We applied CRISP-seq to probe regulatory circuits of innate immunity. By sampling tens of thousands of perturbed cells in vitro and in mice, we identified interactions and redundancies between developmental and signaling-dependent factors. These include opposing effects of Cebpb and Irf8 in regulating the monocyte/macrophage versus dendritic cell lineages and differential functions for Rela and Stat1/2 in monocyte versus dendritic cell responses to pathogens. This study establishes CRISP-seq as a broadly applicable, comprehensive, and unbiased approach for elucidating mammalian regulatory circuits.
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Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Células Dendríticas/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Monocitos/metabolismoRESUMEN
Innate lymphoid cells (ILCs) are critical modulators of mucosal immunity, inflammation, and tissue homeostasis, but their full spectrum of cellular states and regulatory landscapes remains elusive. Here, we combine genome-wide RNA-seq, ChIP-seq, and ATAC-seq to compare the transcriptional and epigenetic identity of small intestinal ILCs, identifying thousands of distinct gene profiles and regulatory elements. Single-cell RNA-seq and flow and mass cytometry analyses reveal compartmentalization of cytokine expression and metabolic activity within the three classical ILC subtypes and highlight transcriptional states beyond the current canonical classification. In addition, using antibiotic intervention and germ-free mice, we characterize the effect of the microbiome on the ILC regulatory landscape and determine the response of ILCs to microbial colonization at the single-cell level. Together, our work characterizes the spectrum of transcriptional identities of small intestinal ILCs and describes how ILCs differentially integrate signals from the microbial microenvironment to generate phenotypic and functional plasticity.
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Microbioma Gastrointestinal , Inmunidad Innata/genética , Intestinos/inmunología , Intestinos/microbiología , Linfocitos/inmunología , Linfocitos/microbiología , Animales , Secuencia de Bases , Cromatina/metabolismo , Citocinas/inmunología , Epigénesis Genética , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Análisis de la Célula Individual , Transcripción GenéticaRESUMEN
Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems.
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Neoplasias del Tronco Encefálico , Glioma , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/patología , Cromatina/genética , Epigénesis Genética , Glioma/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , MutaciónRESUMEN
The TP53 gene is mutated in approximately 30% of all breast cancer cases. Adipocytes and preadipocytes, which constitute a substantial fraction of the stroma of normal mammary tissue and breast tumors, undergo transcriptional, metabolic, and phenotypic reprogramming during breast cancer development and play an important role in tumor progression. We report here that p53 loss in breast cancer cells facilitates the reprogramming of preadipocytes, inducing them to acquire a unique transcriptional and metabolic program that combines impaired adipocytic differentiation with augmented cytokine expression. This, in turn, promotes the establishment of an inflammatory tumor microenvironment, including increased abundance of Ly6C+ and Ly6G+ myeloid cells and elevated expression of the immune checkpoint ligand PD-L1. We also describe a potential gain-of-function effect of common p53 missense mutations on the inflammatory reprogramming of preadipocytes. Altogether, our study implicates p53 deregulation in breast cancer cells as a driver of tumor-supportive adipose tissue reprogramming, expanding the network of non-cell autonomous mechanisms whereby p53 dysfunction may promote cancer. Further elucidation of the interplay between p53 and adipocytes within the tumor microenvironment may suggest effective therapeutic targets for the treatment of breast cancer patients.
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Neoplasias de la Mama , Proteína p53 Supresora de Tumor , Humanos , Femenino , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias de la Mama/patología , Genes p53 , Tejido Adiposo/metabolismo , Adipocitos/metabolismo , Microambiente Tumoral/genéticaRESUMEN
Bone protrusions provide stable anchoring sites for ligaments and tendons and define the unique morphology of each long bone. Despite their importance, the mechanism by which superstructures are patterned is unknown. Here, we identify components of the genetic program that control the patterning of Sox9+/Scx+ superstructure progenitors in mouse and show that this program includes both global and regional regulatory modules. Using light-sheet fluorescence microscopy combined with genetic lineage labeling, we mapped the broad contribution of the Sox9+/Scx+ progenitors to the formation of bone superstructures. Then, by combining literature-based evidence, comparative transcriptomic analysis and genetic mouse models, we identified Gli3 as a global regulator of superstructure patterning, whereas Pbx1, Pbx2, Hoxa11 and Hoxd11 act as proximal and distal regulators, respectively. Moreover, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work in a coordinated manner. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning, which further supports the notion that long bone development is a modular process.This article has an associated 'The people behind the papers' interview.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Huesos/anatomía & histología , Huesos/embriología , Genes del Desarrollo , Proteínas de Homeodominio/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Desarrollo Óseo/genética , Huesos/metabolismo , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes del Desarrollo/genética , Proteínas de Homeodominio/metabolismo , Ligamentos/anatomía & histología , Ligamentos/embriología , Ligamentos/metabolismo , Masculino , Ratones , Ratones Transgénicos , Especificidad de Órganos/genética , Factor de Transcripción 1 de la Leucemia de Células Pre-B/genética , Factor de Transcripción 1 de la Leucemia de Células Pre-B/metabolismo , Embarazo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Tendones/anatomía & histología , Tendones/embriología , Tendones/metabolismoRESUMEN
Limited outgrowth development of bovine mammary epithelial stem cells transplanted into de-epithelialized mouse fat pads restricts advanced studies on this productive organ's development and renewal. We challenged the mouse-bovine incompatibility by implanting parenchymal adjacent or distant bovine stromal layers (close and far stroma, respectively) into the mouse fat pad to serve as an endogenous niche for transplanted stem cells. The close stroma better supported stem cell take rate and outgrowth development. The diameter of these open duct-like structures represented and occasionally exceeded that of the endogenous ducts and appeared 8.3-fold wider than the capsule-like structures developed in the mouse fat pad after similar cell transplantation. RNA-Seq revealed lower complement activity in this layer, associated with secretion of specific laminins and WNT proteins favoring epithelial outgrowth development. The close stroma appeared genetically more similar to the parenchyma than to the far stroma due to epithelial characteristics, mainly of fibroblasts, including expression of epithelial markers, milk protein genes, and functional mammary claudins. Gene markers and activators of the mesenchymal-to-epithelial transition were highly enriched in the epithelial gene cluster and may contribute to the acquired epithelial properties of this stromal layer.
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Células Epiteliales/metabolismo , Inmunohistoquímica/métodos , Células Madre/metabolismo , Animales , Bovinos , Diferenciación Celular , Ratones , Ratones Endogámicos NODRESUMEN
Recruited blood monocytes contribute to the establishment, perpetuation, and resolution of tissue inflammation. Specifically, in the inflamed intestine, monocyte ablation was shown to ameliorate colitis scores in preclinical animal models. However, the majority of intestinal macrophages that seed the healthy gut are also monocyte derived. Monocyte ablation aimed to curb inflammation would therefore likely interfere with intestinal homeostasis. In this study, we used a TLR2 trans-membrane peptide that blocks TLR2 dimerization that is critical for TLR2/1 and TLR2/6 heterodimer signaling to blunt inflammation in a murine colitis model. We show that although the TLR2 peptide treatment ameliorated colitis, it allowed recruited monocytes to give rise to macrophages that lack the detrimental proinflammatory gene signature and reduced potentially damaging neutrophil infiltrates. Finally, we demonstrate TLR blocking activity of the peptide on in vitro cultured human monocyte-derived macrophages. Collectively, we provide a significantly improved anti-inflammatory TLR2 peptide and critical insights in its mechanism of action toward future potential use in the clinic.
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Colitis/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , Macrófagos/inmunología , Receptor Toll-Like 2/metabolismo , Animales , Células Cultivadas , Dimerización , Modelos Animales de Enfermedad , Homeostasis , Humanos , Inflamación , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
Alzheimer's disease (AD) and dementia in general are age-related diseases with multiple contributing factors, including brain inflammation. Microglia, and specifically those expressing the AD risk gene TREM2, are considered important players in AD, but their exact contribution to pathology remains unclear. In this study, using high-throughput mass cytometry in the 5×FAD mouse model of amyloidosis, we identified senescent microglia that express high levels of TREM2 but also exhibit a distinct signature from TREM2-dependent disease-associated microglia (DAM). This senescent microglial protein signature was found in various mouse models that show cognitive decline, including aging, amyloidosis and tauopathy. TREM2-null mice had fewer microglia with a senescent signature. Treating 5×FAD mice with the senolytic BCL2 family inhibitor ABT-737 reduced senescent microglia, but not the DAM population, and this was accompanied by improved cognition and reduced brain inflammation. Our results suggest a dual and opposite involvement of TREM2 in microglial states, which must be considered when contemplating TREM2 as a therapeutic target in AD.
Asunto(s)
Envejecimiento , Enfermedad de Alzheimer , Encéfalo , Modelos Animales de Enfermedad , Glicoproteínas de Membrana , Microglía , Receptores Inmunológicos , Animales , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Microglía/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/genética , Ratones , Envejecimiento/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Ratones Transgénicos , Senescencia Celular/fisiología , Senescencia Celular/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Calorie restriction enhances stem cell self-renewal in various tissues, including the mammary gland. We hypothesized that similar to their intestinal counterparts, mammary epithelial stem cells are insulated from sensing changes in energy supply, depending instead on niche signaling. The latter was investigated by subjecting cultures of mammary epithelial stem cells for 8 days to in vivo paracrine calorie-restriction signals collected from a 4-day-conditioned medium of individual mammary cell populations. Conditioned medium from calorie-restricted non-epithelial cells induced latent cell propagation and mammosphere formation-established markers of stem cell self-renewal. Combined RNA-Seq, immunohistochemistry and immunofluorescence analyses of the non-epithelial population identified macrophages and secreted CSF1 as the energy sensor and paracrine signal, respectively. Calorie restriction-induced pStat6 expression in macrophages suggested that skewing to the M2 phenotype contributes to the sensing mechanism. Enhancing CSF1 signaling with recombinant protein and interrupting the interaction with its highly expressed receptor in the epithelial stem cells by neutralizing antibodies were both affected stem cell self-renewal. In conclusion, combined in vivo, in vitro and in silico studies identified macrophages and secreted CSF1 as the energy sensor and paracrine transmitter, respectively, of the calorie restriction-induced effect on mammary stem cell self-renewal.
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Restricción Calórica , Células Madre , Anticuerpos Neutralizantes/farmacología , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Macrófagos , Proteínas Recombinantes/farmacologíaRESUMEN
Mammary epithelial stem cells differentiate to create the basal and luminal layers of the gland. Inducing the number of differentiating bovine mammary stem cells may provide compensating populations for the milk-producing cells that die during lactation. Inhibition of mTOR activity by rapamycin signals self-renewal of intestinal stem cells, with similar consequences in the mouse mammary gland and in bovine mammary implants maintained in mice. The implementation of these results in farm animals for better mammary development and production was studied in 3-month-old calves. mTOR activity decreased by ~50% in mammary epithelial cells subjected to 3-week rapamycin administration, with no negative consequences on mammary morphology or ß-casein expression. Subsequently, stem cell self-renewal was induced, reflected by a higher propagation rate of cultures from rapamycin-treated glands compared to respective controls and higher expression of selected markers. Followed by 4-day estrogen and progesterone administration, rapamycin significantly induced proliferation rate. Higher numbers of basal and luminal PCNA+ cells were detected in small ducts near the elongating sites as compared to large ducts, in which only luminal cells were affected. Rapamycin administration resulted in induction of individual milk protein genes' expression, which was negatively correlated to their endogenous levels. The inductive effect of rapamycin on luminal cell number was confirmed in organoid cultures, but milk protein expression decreased, probably due to lack of oscillation in rapamycin levels. In conclusion, intramammary rapamycin administration is an effective methodology to reduce mTOR activity in bovine mammary epithelial cells and consequently, induce stem cell self-renewal. The latent positive effect of rapamycin on epithelial cell proliferation and its potential to improve milk protein expression in calves may have beneficial implications for mature cows.
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Glándulas Mamarias Animales , Proteínas de la Leche , Animales , Bovinos , Proliferación Celular , Autorrenovación de las Células , Células Epiteliales/metabolismo , Femenino , Lactancia , Glándulas Mamarias Animales/metabolismo , Ratones , Proteínas de la Leche/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Breast cancer, the most frequent cancer in women, is generally classified into several distinct histological and molecular subtypes. However, single-cell technologies have revealed remarkable cellular and functional heterogeneity across subtypes and even within individual breast tumors. Much of this heterogeneity is attributable to dynamic alterations in the epigenetic landscape of the cancer cells, which promote phenotypic plasticity. Such plasticity, including transition from luminal to basal-like cell identity, can promote disease aggressiveness. We now report that the tumor suppressor LATS1, whose expression is often downregulated in human breast cancer, helps maintain luminal breast cancer cell identity by reducing the chromatin accessibility of genes that are characteristic of a "basal-like" state, preventing their spurious activation. This is achieved via interaction of LATS1 with the NCOR1 nuclear corepressor and recruitment of HDAC1, driving histone H3K27 deacetylation near NCOR1-repressed "basal-like" genes. Consequently, decreased expression of LATS1 elevates the expression of such genes and facilitates slippage towards a more basal-like phenotypic identity. We propose that by enforcing rigorous silencing of repressed genes, the LATS1-NCOR1 axis maintains luminal cell identity and restricts breast cancer progression.
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Neoplasias de la Mama , Femenino , Humanos , Neoplasias de la Mama/genética , Genes Reguladores , Proteínas Serina-Treonina Quinasas/genética , Mama , Represión Psicológica , Co-Represor 1 de Receptor Nuclear/genéticaRESUMEN
Trametes sp. M23, isolated from biosolids compost was found to decompose humic acids (HA). A low N (LN) medium (C/N, 53) provided suitable conditions for HA degradation, whereas in a high N (HN) medium (C/N, 10), HA was not degraded. In the absence of Mn(2+), HA degradation was similar to that in Mn(2+)-containing medium. In contrast, MnP activity was significantly affected by Mn(2+). Laccase activity exhibited a negative correlation to HA degradation, while LiP activity was not detected. Thus, ligninolytic enzymes activity could provide only a partial explanation for the HA-degradation mechanism. The decolorization of two dyes, Orange II and Brilliant Blue R250, was also determined. Similar to HA degradation, under LN conditions, decolorization occurred independently of the presence of Mn(2+). We investigated the possible involvement of a Fenton-like reaction in HA degradation. The addition of DMSO, an OH-radical scavenger, to LN media resulted in a significant decrease in HA bleaching. The rate of extracellular Fe(3+) reduction was much higher in the LN vs. HN medium. In addition, the rate of reduction was even higher in the presence of HA in the medium. In vitro HA bleaching in non-inoculated media was observed with H(2)O(2) amendment to a final concentration of 200 mM (obtained by 50 mM amendments for 4 days) and Fe(2+) (36 mM). After 4 days of incubation, HA decolorization was similar to the biological treatment. These results support our hypothesis that a Fenton-like reaction is involved in HA degradation by Trametes sp. M23.
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Glucosidasas/metabolismo , Sustancias Húmicas , Trametes/enzimología , Compuestos Azo/metabolismo , Bencenosulfonatos/metabolismo , Coenzimas/metabolismo , Medios de Cultivo/química , Compuestos Ferrosos/metabolismo , Manganeso/metabolismo , Colorantes de Rosanilina/metabolismoRESUMEN
BACKGROUND: For decades, dementia has been characterized by accumulation of waste in the brain and low-grade inflammation. Over the years, emerging studies highlighted the involvement of the immune system in neurodegenerative disease emergence and severity. Numerous studies in animal models of amyloidosis demonstrated the beneficial role of monocyte-derived macrophages in mitigating the disease, though less is known regarding tauopathy. Boosting the immune system in animal models of both amyloidosis and tauopathy, resulted in improved cognitive performance and in a reduction of pathological manifestations. However, a full understanding of the chain of events that is involved, starting from the activation of the immune system, and leading to disease mitigation, remained elusive. Here, we hypothesized that the brain-immune communication pathway that is needed to be activated to combat tauopathy involves monocyte mobilization via the C-C chemokine receptor 2 (CCR2)/CCL2 axis, and additional immune cells, such as CD4+ T cells, including FOXP3+ regulatory CD4+ T cells. METHODS: We used DM-hTAU transgenic mice, a mouse model of tauopathy, and applied an approach that boosts the immune system, via blocking the inhibitory Programmed cell death protein-1 (PD-1)/PD-L1 pathway, a manipulation previously shown to alleviate disease symptoms and pathology. An anti-CCR2 monoclonal antibody (αCCR2), was used to block the CCR2 axis in a protocol that partially eliminates monocytes from the circulation at the time of anti-PD-L1 antibody (αPD-L1) injection, and for the critical period of their recruitment into the brain following treatment. RESULTS: Performance of DM-hTAU mice in short-term and working memory tasks, revealed that the beneficial effect of αPD-L1, assessed 1 month after a single injection, was abrogated following blockade of CCR2. This was accompanied by the loss of the beneficial effect on disease pathology, assessed by measurement of cortical aggregated human tau load using Homogeneous Time Resolved Fluorescence-based immunoassay, and by evaluation of hippocampal neuronal survival. Using both multiparametric flow cytometry, and Cytometry by Time Of Flight, we further demonstrated the accumulation of FOXP3+ regulatory CD4+ T cells in the brain, 12 days following the treatment, which was absent subsequent to CCR2 blockade. In addition, measurement of hippocampal levels of the T-cell chemoattractant, C-X-C motif chemokine ligand 12 (Cxcl12), and of inflammatory cytokines, revealed that αPD-L1 treatment reduced their expression, while blocking CCR2 reversed this effect. CONCLUSIONS: The CCR2/CCL2 axis is required to modify pathology using PD-L1 blockade in a mouse model of tauopathy. This modification involves, in addition to monocytes, the accumulation of FOXP3+ regulatory CD4+ T cells in the brain, and the T-cell chemoattractant, Cxcl12.
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Quimiocina CCL2/metabolismo , Receptores CCR2/metabolismo , Tauopatías/inmunología , Tauopatías/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Quimiocina CCL2/inmunología , Modelos Animales de Enfermedad , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones , Ratones Transgénicos , Monocitos/inmunología , Receptores CCR2/inmunología , Tauopatías/patologíaRESUMEN
Some antibacterial therapies entail sequential treatments with different antibiotics, but whether this approach is optimal for anti-cancer tyrosine kinase inhibitors (TKIs) remains open. EGFR mutations identify lung cancer patients who can derive benefit from TKIs, but most patients develop resistance to the first-, second-, and third-generation drugs. To explore alternatives to such whack-a-mole strategies, we simulated in patient-derived xenograft models the situation of patients receiving first-line TKIs. Monotherapies comprising approved first-line TKIs were compared to combinations with antibodies specific to EGFR and HER2. We observed uniform and strong superiority of all drug combinations over the respective monotherapies. Prolonged treatments, high TKI dose, and specificity were essential for drug-drug cooperation. Blocking pathways essential for mitosis (e.g., FOXM1), along with downregulation of resistance-conferring receptors (e.g., AXL), might underlie drug cooperation. Thus, upfront treatments using combinations of TKIs and antibodies can prevent emergence of resistance and hence might replace the widely applied sequential treatments utilizing next-generation TKIs.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Mutación , Compuestos Orgánicos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
The mechanical challenge of attaching elastic tendons to stiff bones is solved by the formation of a unique transitional tissue. Here, we show that murine tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, under regulation of shared regulatory elements and Krüppel-like factors (KLFs) transcription factors. High-throughput bulk and single-cell RNA sequencing of humeral attachment cells revealed expression of hundreds of chondrogenic and tenogenic genes, which was validated by in situ hybridization and single-molecule ISH. ATAC sequencing showed that attachment cells share accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis revealed enhancer signatures for most of these regions. Transgenic mouse enhancer reporter assays verified the shared activity of some of these enhancers. Finally, integrative chromatin and motif analyses and transcriptomic data implicated KLFs as regulators of attachment cells. Indeed, blocking expression of both Klf2 and Klf4 in developing limb mesenchyme impaired their differentiation.
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Condrocitos/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Tenocitos/metabolismo , Transcriptoma , Animales , Huesos , Femenino , Factor 4 Similar a Kruppel/genética , Factor 4 Similar a Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Secuencias Reguladoras de Ácidos Nucleicos , TendonesRESUMEN
Gaucher disease (GD) is currently the focus of considerable attention due primarily to the association between the gene that causes GD (GBA) and Parkinson's disease. Mouse models exist for the systemic (type 1) and for the acute neuronopathic forms (type 2) of GD. Here we report the generation of a mouse that phenotypically models chronic neuronopathic type 3 GD. Gba-/-;Gbatg mice, which contain a Gba transgene regulated by doxycycline, accumulate moderate levels of the offending substrate in GD, glucosylceramide, and live for up to 10 months, i.e. significantly longer than mice which model type 2 GD. Gba-/-;Gbatg mice display behavioral abnormalities at â¼4 months, which deteriorate with age, along with significant neuropathology including loss of Purkinje neurons. Gene expression is altered in the brain and in isolated microglia, although the changes in gene expression are less extensive than in mice modeling type 2 disease. Finally, bone deformities are consistent with the Gba-/-;Gbatg mice being a genuine type 3 GD model. Together, the Gba-/-;Gbatg mice share pathological pathways with acute neuronopathic GD mice but also display differences that might help understand the distinct disease course and progression of type 2 and 3 patients.
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Enfermedad de Gaucher , Células de Purkinje , Animales , Encéfalo , Modelos Animales de Enfermedad , Enfermedad de Gaucher/genética , Glucosilceramidasa/genética , Humanos , RatonesRESUMEN
Identification of early processes leading to complex tissue pathologies, such as inflammatory bowel diseases, poses a major scientific and clinical challenge that is imperative for improved diagnosis and treatment. Most studies of inflammation onset focus on cellular processes and signaling molecules, while overlooking the environment in which they take place, the continuously remodeled extracellular matrix. In this study, we used colitis models for investigating extracellular-matrix dynamics during disease onset, while treating the matrix as a complete and defined entity. Through the analysis of matrix structure, stiffness and composition, we unexpectedly revealed that even prior to the first clinical symptoms, the colon displays its own unique extracellular-matrix signature and found specific markers of clinical potential, which were also validated in human subjects. We also show that the emergence of this pre-symptomatic matrix is mediated by subclinical infiltration of immune cells bearing remodeling enzymes. Remarkably, whether the inflammation is chronic or acute, its matrix signature converges at pre-symptomatic states. We suggest that the existence of a pre-symptomatic extracellular-matrix is general and relevant to a wide range of diseases.