RESUMO
Macrophages sense pathogens and orchestrate specific immune responses. Stimulus specificity is thought to be achieved through combinatorial and dynamical coding by signaling pathways. While NFκB dynamics are known to encode stimulus information, dynamical coding in other signaling pathways and their combinatorial coordination remain unclear. Here, we established live-cell microscopy to investigate how NFκB and p38 dynamics interface in stimulated macrophages. Information theory and machine learning revealed that p38 dynamics distinguish cytokine TNF from pathogen-associated molecular patterns and high doses from low, but contributed little to information-rich NFκB dynamics when both pathways are considered. This suggests that immune response genes benefit from decoding immune signaling dynamics or combinatorics, but not both. We found that the heterogeneity of the two pathways is surprisingly uncorrelated. Mathematical modeling revealed potential sources of uncorrelated heterogeneity in the branched pathway network topology and predicted it to drive gene expression variability. Indeed, genes dependent on both p38 and NFκB showed high scRNAseq variability and bimodality. These results identify combinatorial signaling as a mechanism to restrict NFκB-AND-p38-responsive inflammatory cytokine expression to few cells.
Assuntos
Inflamação , Macrófagos , NF-kappa B , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Macrófagos/metabolismo , Macrófagos/imunologia , Inflamação/metabolismo , Inflamação/genética , Inflamação/imunologia , Animais , Camundongos , Citocinas/metabolismo , Aprendizado de Máquina , Regulação da Expressão Gênica , Humanos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Trans-differentiation from an adenocarcinoma to a small cell neuroendocrine state is associated with therapy resistance in multiple cancer types. To gain insight into the underlying molecular events of the trans-differentiation, we perform a multi-omics time course analysis of a pan-small cell neuroendocrine cancer model (termed PARCB), a forward genetic transformation using human prostate basal cells and identify a shared developmental, arc-like, and entropy-high trajectory among all transformation model replicates. Further mapping with single cell resolution reveals two distinct lineages defined by mutually exclusive expression of ASCL1 or ASCL2. Temporal regulation by groups of transcription factors across developmental stages reveals that cellular reprogramming precedes the induction of neuronal programs. TFAP4 and ASCL1/2 feedback are identified as potential regulators of ASCL1 and ASCL2 expression. Our study provides temporal transcriptional patterns and uncovers pan-tissue parallels between prostate and lung cancers, as well as connections to normal neuroendocrine cell states.
Assuntos
Carcinoma de Células Pequenas , Neoplasias Pulmonares , Neoplasias da Próstata , Carcinoma de Pequenas Células do Pulmão , Masculino , Humanos , Neoplasias Pulmonares/genética , Carcinoma de Células Pequenas/genética , Fatores de Transcrição/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transdiferenciação Celular/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Carcinoma de Pequenas Células do Pulmão/genéticaRESUMO
Immune sentinel macrophages initiate responses to pathogens via hundreds of immune response genes. Each immune threat demands a tailored response, suggesting that the capacity for stimulus-specific gene expression is a key functional hallmark of healthy macrophages. To quantify this property, termed "stimulus-response specificity" (SRS), we developed a single-cell experimental workflow and analytical approaches based on information theory and machine learning. We found that the response specificity of macrophages is driven by combinations of specific immune genes that show low cell-to-cell heterogeneity and are targets of separate signaling pathways. The "response specificity profile," a systematic comparison of multiple stimulus-response distributions, was distinctly altered by polarizing cytokines, and it enabled an assessment of the functional state of macrophages. Indeed, the response specificity profile of peritoneal macrophages from old and obese mice showed characteristic differences, suggesting that SRS may be a basis for measuring the functional state of innate immune cells. A record of this paper's transparent peer review process is included in the supplemental information.
Assuntos
Citocinas , Macrófagos , Animais , Camundongos , Citocinas/metabolismo , Transdução de SinaisRESUMO
The genomic positions of nucleosomes are a defining feature of the cell's epigenomic state, but signal-dependent transcription factors (SDTFs), upon activation, bind to specific genomic locations and modify nucleosome positioning. Here we leverage SDTFs as perturbation probes to learn about nucleosome dynamics in living cells. We develop Markov models of nucleosome dynamics and fit them to time course sequencing data of DNA accessibility. We find that (1) the dynamics of DNA unwrapping are significantly slower in cells than reported from cell-free experiments, (2) only models with cooperativity in wrapping and unwrapping fit the available data, (3) SDTF activity produces the highest eviction probability when its binding site is adjacent to but not on the nucleosome dyad, and (4) oscillatory SDTF activity results in high location variability. Our work uncovers the regulatory rules governing SDTF-induced nucleosome dynamics in live cells, which can predict chromatin accessibility alterations during inflammation at single-nucleosome resolution.
Assuntos
Epigenoma , Nucleossomos , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Macrophages are first responders for the immune system. In this role, they have both effector functions for neutralizing pathogens and sentinel functions for alerting other immune cells of diverse pathologic threats, thereby initiating and coordinating a multipronged immune response. Macrophages are distributed throughout the body-they circulate in the blood, line the mucosal membranes, reside within organs, and survey the connective tissue. Several reviews have summarized their diverse roles in different physiological scenarios and in the initiation or amplification of different pathologies. In this review, we propose that both the effector and the sentinel functions of healthy macrophages rely on three hallmark properties: response specificity, context dependence, and stimulus memory. When these hallmark properties are diminished, the macrophage's biological functions are impaired, which in turn results in increased risk for immune dysregulation, manifested by immune deficiency or autoimmunity. We review the evidence and the molecular mechanisms supporting these functional hallmarks.
Assuntos
Imunidade Inata , Macrófagos , Animais , HumanosRESUMO
Immune cells infiltrate the peripheral nervous system (PNS) after injury and with autoimmunity, but their net effect is divergent. After injury, immune cells are reparative, while in inflammatory neuropathies (e.g., Guillain Barré Syndrome and chronic inflammatory demyelinating polyneuropathy), immune cells are proinflammatory and promote autoimmune demyelination. An understanding of immune cell phenotypes that distinguish these conditions may, therefore, reveal new therapeutic targets for switching immune cells from an inflammatory role to a reparative state. In an autoimmune regulator (Aire)-deficient mouse model of inflammatory neuropathy, we used single-cell RNA sequencing of sciatic nerves to discover a transcriptionally heterogeneous cellular landscape, including multiple myeloid, innate lymphoid, and lymphoid cell types. Analysis of cell-cell ligand-receptor interactions uncovered a macrophage-mediated tumor necrosis factor-α (TNF-α) signaling axis that is induced by interferon-γ and required for initiation of autoimmune demyelination. Developmental trajectory visualization suggested that TNF-α signaling is associated with metabolic reprogramming of macrophages and polarization of macrophages from a reparative state in injury to a pathogenic, inflammatory state in autoimmunity. Autocrine TNF-α signaling induced macrophage expression of multiple genes (Clec4e, Marcksl1, Cxcl1, and Cxcl10) important in immune cell activation and recruitment. Genetic and antibody-based blockade of TNF-α/TNF-α signaling ameliorated clinical neuropathy, peripheral nerve infiltration, and demyelination, which provides preclinical evidence that the TNF-α axis may be effectively targeted to resolve inflammatory neuropathies.
Assuntos
Doenças Neuroinflamatórias/etiologia , Doenças Neuroinflamatórias/metabolismo , Doenças do Sistema Nervoso Periférico/etiologia , Doenças do Sistema Nervoso Periférico/metabolismo , Poliendocrinopatias Autoimunes/complicações , Fator de Necrose Tumoral alfa/metabolismo , Transferência Adotiva , Animais , Anticorpos Monoclonais/farmacologia , Comunicação Autócrina , Biomarcadores , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/patologia , Comunicação Parácrina , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/patologia , Poliendocrinopatias Autoimunes/genética , Receptores do Fator de Necrose Tumoral/deficiência , Nervo Isquiático/imunologia , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Immune sentinel cells initiate immune responses to pathogens and tissue injury and are capable of producing highly stimulus-specific responses. Insight into the mechanisms underlying such specificity has come from the identification of regulatory factors and biochemical pathways, as well as the definition of signaling circuits that enable combinatorial and temporal coding of information. Here, we review the multi-layered molecular mechanisms that underlie stimulus-specific gene expression in macrophages. We categorize components of inflammatory and anti-pathogenic signaling pathways into five layers of regulatory control and discuss unifying mechanisms determining signaling characteristics at each layer. In this context, we review mechanisms that enable combinatorial and temporal encoding of information, identify recurring regulatory motifs and principles, and present strategies for integrating experimental and computational approaches toward the understanding of signaling specificity in innate immunity.
Assuntos
Imunidade Inata/imunologia , Macrófagos/imunologia , Animais , HumanosRESUMO
The epigenome of macrophages can be reprogrammed by extracellular cues, but the extent to which different stimuli achieve this is unclear. Nuclear factor κB (NF-κB) is a transcription factor that is activated by all pathogen-associated stimuli and can reprogram the epigenome by activating latent enhancers. However, we show that NF-κB does so only in response to a subset of stimuli. This stimulus specificity depends on the temporal dynamics of NF-κB activity, in particular whether it is oscillatory or non-oscillatory. Non-oscillatory NF-κB opens chromatin by sustained disruption of nucleosomal histone-DNA interactions, enabling activation of latent enhancers that modulate expression of immune response genes. Thus, temporal dynamics can determine a transcription factor's capacity to reprogram the epigenome in a stimulus-specific manner.
Assuntos
Epigenoma , Macrófagos/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Histonas/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos/imunologia , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , Nucleossomos/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Macrophages initiate inflammatory responses via the transcription factor NFκB. The temporal pattern of NFκB activity determines which genes are expressed and thus, the type of response that ensues. Here, we examined how information about the stimulus is encoded in the dynamics of NFκB activity. We generated an mVenus-RelA reporter mouse line to enable high-throughput live-cell analysis of primary macrophages responding to host- and pathogen-derived stimuli. An information-theoretic workflow identified six dynamical features-termed signaling codons-that convey stimulus information to the nucleus. In particular, oscillatory trajectories were a hallmark of responses to cytokine but not pathogen-derived stimuli. Single-cell imaging and RNA sequencing of macrophages from a mouse model of Sjögren's syndrome revealed inappropriate responses to stimuli, suggestive of confusion of two NFκB signaling codons. Thus, the dynamics of NFκB signaling classify immune threats through six signaling codons, and signal confusion based on defective codon deployment may underlie the etiology of some inflammatory diseases.
Assuntos
Códon/genética , Macrófagos/fisiologia , NF-kappa B/genética , Transdução de Sinais/genética , Animais , Células Cultivadas , Citocinas/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Inflamação/genética , Camundongos , Camundongos Endogâmicos C57BL , Síndrome de Sjogren/genética , Fator de Transcrição RelA/genéticaRESUMO
Melanoma dedifferentiation has been reported to be a state of cellular resistance to targeted therapies and immunotherapies as cancer cells revert to a more primitive cellular phenotype. Here, we show that, counterintuitively, the biopsies of patient tumors that responded to anti-programmed cell death 1 (anti-PD-1) therapy had decreased expression of melanocytic markers and increased neural crest markers, suggesting treatment-induced dedifferentiation. When modeling the effects in vitro, we documented that melanoma cell lines that were originally differentiated underwent a process of neural crest dedifferentiation when continuously exposed to IFN-γ, through global chromatin landscape changes that led to enrichment in specific hyperaccessible chromatin regions. The IFN-γ-induced dedifferentiation signature corresponded with improved outcomes in patients with melanoma, challenging the notion that neural crest dedifferentiation is entirely an adverse phenotype.
Assuntos
Biomarcadores Tumorais , Desdiferenciação Celular/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Interferon gama/metabolismo , Melanoma , Proteínas de Neoplasias , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismoRESUMO
Pathogen-derived lipopolysaccharide (LPS) and cytokine tumor necrosis factor (TNF) activate NFκB with distinct duration dynamics, but how immune response genes decode NFκB duration to produce stimulus-specific expression remains unclear. Here, detailed transcriptomic profiling of combinatorial and temporal control mutants identified 81 genes that depend on stimulus-specific NFκB duration for their stimulus-specificity. Combining quantitative experimentation with mathematical modeling, we found that for some genes a long mRNA half-life allowed effective decoding, but for many genes this was insufficient to account for the data; instead, we found that chromatin mechanisms, such as a slow transition rate between inactive and RelA-bound enhancer states, could also decode NFκB dynamics. Chromatin-mediated decoding is favored by genes acting as immune effectors (e.g., tissue remodelers and T cell recruiters) rather than immune regulators (e.g., signaling proteins and monocyte recruiters). Overall, our results delineate two gene regulatory strategies that decode stimulus-specific NFκB dynamics and determine distinct biological functions.
Assuntos
Regulação da Expressão Gênica/genética , Expressão Gênica/genética , Lipopolissacarídeos/metabolismo , NF-kappa B/metabolismo , HumanosRESUMO
Small-cell neuroendocrine cancers (SCNCs) are an aggressive cancer subtype. Transdifferentiation toward an SCN phenotype has been reported as a resistance route in response to targeted therapies. Here, we identified a convergence to an SCN state that is widespread across epithelial cancers and is associated with poor prognosis. More broadly, non-SCN metastases have higher expression of SCN-associated transcription factors than non-SCN primary tumors. Drug sensitivity and gene dependency screens demonstrate that these convergent SCNCs have shared vulnerabilities. These common vulnerabilities are found across unannotated SCN-like epithelial cases, small-round-blue cell tumors, and unexpectedly in hematological malignancies. The SCN convergent phenotype and common sensitivity profiles with hematological cancers can guide treatment options beyond tissue-specific targeted therapies.
Assuntos
Carcinoma de Células Pequenas/diagnóstico , Carcinoma de Células Pequenas/etiologia , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/etiologia , Tumores Neuroendócrinos/diagnóstico , Tumores Neuroendócrinos/etiologia , Fenótipo , Carcinoma de Células Pequenas/tratamento farmacológico , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Suscetibilidade a Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Mutação , Tumores Neuroendócrinos/tratamento farmacológico , TranscriptomaRESUMO
The use of potent therapies inhibiting critical oncogenic pathways active in epithelial cancers has led to multiple resistance mechanisms, including the development of highly aggressive, small cell neuroendocrine carcinoma (SCNC). SCNC patients have a dismal prognosis due in part to a limited understanding of the molecular mechanisms driving this malignancy and the lack of effective treatments. Here, we demonstrate that a common set of defined oncogenic drivers reproducibly reprograms normal human prostate and lung epithelial cells to small cell prostate cancer (SCPC) and small cell lung cancer (SCLC), respectively. We identify shared active transcription factor binding regions in the reprogrammed prostate and lung SCNCs by integrative analyses of epigenetic and transcriptional landscapes. These results suggest that neuroendocrine cancers arising from distinct epithelial tissues may share common vulnerabilities that could be exploited for the development of drugs targeting SCNCs.
Assuntos
Carcinogênese/genética , Carcinoma Neuroendócrino/patologia , Reprogramação Celular/genética , Neoplasias Pulmonares/patologia , Pulmão/patologia , Próstata/patologia , Neoplasias da Próstata/patologia , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma Neuroendócrino/genética , Linhagem Celular Tumoral , Linhagem da Célula , Técnicas de Reprogramação Celular , Sistemas de Liberação de Medicamentos , Células Epiteliais/patologia , Epitélio/patologia , Humanos , Masculino , Neoplasias da Próstata/genética , Proteína do Retinoblastoma/genética , Carcinoma de Pequenas Células do Pulmão/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Cancer progression to an aggressive phenotype often co-opts aspects of stem cell biology. Here, we developed gene signatures for normal human stem cell populations to understand the relationship between epithelial cancers and stem cell transcriptional programs. Using a pan-cancer approach, we reveal that aggressive epithelial cancers are enriched for a transcriptional signature shared by epithelial adult stem cells. The adult stem cell signature selected for epithelial cancers with worse overall survival and alterations of oncogenic drivers. Lethal small cell neuroendocrine lung, prostate, and bladder cancers transcriptionally converged onto the adult stem cell signature and not other stem cell signatures tested. We found that DNA methyltransferase expression correlated with adult stem cell signature status and was enriched in small cell neuroendocrine cancers. DNA methylation analysis uncovered a shared epigenomic profile between small cell neuroendocrine cancers. These pan-cancer findings establish a molecular link between human adult stem cells and aggressive epithelial cancers.
Assuntos
Células-Tronco Adultas/metabolismo , Células Epiteliais/metabolismo , Neoplasias Pulmonares/genética , Neoplasias da Próstata/genética , Transcriptoma , Neoplasias da Bexiga Urinária/genética , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Neoplasias da Próstata/metabolismo , Neoplasias da Bexiga Urinária/metabolismoRESUMO
Malignant transformation can result in melanoma cells that resemble different stages of their embryonic development. Our gene expression analysis of human melanoma cell lines and patient tumors revealed that melanoma follows a two-dimensional differentiation trajectory that can be subclassified into four progressive subtypes. This differentiation model is associated with subtype-specific sensitivity to iron-dependent oxidative stress and cell death known as ferroptosis. Receptor tyrosine kinase-mediated resistance to mitogen-activated protein kinase targeted therapies and activation of the inflammatory signaling associated with immune therapy involves transitions along this differentiation trajectory, which lead to increased sensitivity to ferroptosis. Therefore, ferroptosis-inducing drugs present an orthogonal therapeutic approach to target the differentiation plasticity of melanoma cells to increase the efficacy of targeted and immune therapies.