RESUMEN
Maintenance of tissue homeostasis is dependent on the communication between stem cells and supporting cells in the same niche. Regulatory T cells (Treg cells) are emerging as a critical component of the stem-cell niche for supporting their differentiation. How Treg cells sense dynamic signals in this microenvironment and communicate with stem cells is mostly unknown. In the present study, by using hair follicles (HFs) to study Treg cell-stem cell crosstalk, we show an unrecognized function of the steroid hormone glucocorticoid in instructing skin-resident Treg cells to facilitate HF stem-cell (HFSC) activation and HF regeneration. Ablation of the glucocorticoid receptor (GR) in Treg cells blocks hair regeneration without affecting immune homeostasis. Mechanistically, GR and Foxp3 cooperate in Treg cells to induce transforming growth factor ß3 (TGF-ß3), which activates Smad2/3 in HFSCs and facilitates HFSC proliferation. The present study identifies crosstalk between Treg cells and HFSCs mediated by the GR-TGF-ß3 axis, highlighting a possible means of manipulating Treg cells to support tissue regeneration.
Asunto(s)
Glucocorticoides , Folículo Piloso , Glucocorticoides/metabolismo , Cabello/metabolismo , Folículo Piloso/metabolismo , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta3/metabolismoRESUMEN
Regulatory T (Treg) cells play a pivotal role in suppressing auto-reactive T cells and maintaining immune homeostasis. Treg cell development and function are dependent on the transcription factor Foxp3. Here, we performed a genome-wide CRISPR loss-of-function screen to identify Foxp3 regulators in mouse primary Treg cells. Foxp3 regulators were enriched in genes encoding subunits of the SWI/SNF nucleosome-remodeling and SAGA chromatin-modifying complexes. Among the three SWI/SNF-related complexes, the Brd9-containing non-canonical (nc) BAF complex promoted Foxp3 expression, whereas the PBAF complex was repressive. Chemical-induced degradation of Brd9 led to reduced Foxp3 expression and reduced Treg cell function in vitro. Brd9 ablation compromised Treg cell function in inflammatory disease and tumor immunity in vivo. Furthermore, Brd9 promoted Foxp3 binding and expression of a subset of Foxp3 target genes. Our findings provide an unbiased analysis of the genetic networks regulating Foxp3 and reveal ncBAF as a target for therapeutic manipulation of Treg cell function.
Asunto(s)
Sistemas CRISPR-Cas/genética , Factores de Transcripción Forkhead/metabolismo , Neoplasias/inmunología , Linfocitos T Reguladores/metabolismo , Factores de Transcripción/metabolismo , Animales , Autoinmunidad/inmunología , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleosomas/inmunología , ARN Guía de Kinetoplastida/genética , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Factores de Transcripción/genéticaRESUMEN
The homeostasis of multicellular organisms requires terminally differentiated cells to preserve their lineage specificity. However, it is unclear whether mechanisms exist to actively protect cell identity in response to environmental cues that confer functional plasticity. Regulatory T (Treg) cells, specified by the transcription factor Foxp3, are indispensable for immune system homeostasis. Here, we report that conserved noncoding sequence 2 (CNS2), a CpG-rich Foxp3 intronic cis-element specifically demethylated in mature Tregs, helps maintain immune homeostasis and limit autoimmune disease development by protecting Treg identity in response to signals that shape mature Treg functions and drive their initial differentiation. In activated Tregs, CNS2 helps protect Foxp3 expression from destabilizing cytokine conditions by sensing TCR/NFAT activation, which facilitates the interaction between CNS2 and Foxp3 promoter. Thus, epigenetically marked cis-elements can protect cell identity by sensing key environmental cues central to both cell identity formation and functional plasticity without interfering with initial cell differentiation.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Linfocitos T Reguladores/citología , Animales , Secuencia de Bases , Diferenciación Celular , Secuencia Conservada , Islas de CpG , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Expresión Génica , Trastornos Linfoproliferativos/inmunología , Trastornos Linfoproliferativos/metabolismo , Ratones , Datos de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos , Linfocitos T Reguladores/metabolismoRESUMEN
Dermal fibroblasts (dFBs) resist infection by locally differentiating into adipocytes and producing cathelicidin antimicrobial peptide in response to Staphylococcus aureus (S. aureus). Here, we show that neonatal skin was enriched with adipogenic dFBs and immature dermal fat that highly expressed cathelicidin. The pool of adipogenic and antimicrobial dFBs declined after birth, leading to an age-dependent loss of dermal fat and a decrease in adipogenesis and cathelidicin production in response to infection. Transforming growth factor beta (TGF-ß), which acted on uncommitted embryonic and adult dFBs and inhibited their adipogenic and antimicrobial function, was identified as a key upstream regulator of this process. Furthermore, inhibition of the TGF-ß receptor restored the adipogenic and antimicrobial function of dFBs in culture and increased resistance of adult mice to S. aureus infection. These results provide insight into changes that occur in the skin innate immune system between the perinatal and adult periods of life.
Asunto(s)
Envejecimiento/inmunología , Fibroblastos/fisiología , Piel/metabolismo , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/fisiología , Grasa Subcutánea/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adipocitos/metabolismo , Adipogénesis , Animales , Antiinfecciosos/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Células Cultivadas , Embrión de Mamíferos , Humanos , Inmunidad Innata , Ratones , CatelicidinasRESUMEN
Decades of work have elucidated cytokine signalling and transcriptional pathways that control T cell differentiation and have led the way to targeted biologic therapies that are effective in a range of autoimmune, allergic and inflammatory diseases. Recent evidence indicates that obesity and metabolic disease can also influence the immune system1-7, although the mechanisms and effects on immunotherapy outcomes remain largely unknown. Here, using two models of atopic dermatitis, we show that lean and obese mice mount markedly different immune responses. Obesity converted the classical type 2 T helper (TH2)-predominant disease associated with atopic dermatitis to a more severe disease with prominent TH17 inflammation. We also observed divergent responses to biologic therapies targeting TH2 cytokines, which robustly protected lean mice but exacerbated disease in obese mice. Single-cell RNA sequencing coupled with genome-wide binding analyses revealed decreased activity of nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) in TH2 cells from obese mice relative to lean mice. Conditional ablation of PPARγ in T cells revealed that PPARγ is required to focus the in vivo TH response towards a TH2-predominant state and prevent aberrant non-TH2 inflammation. Treatment of obese mice with a small-molecule PPARγ agonist limited development of TH17 pathology and unlocked therapeutic responsiveness to targeted anti-TH2 biologic therapies. These studies reveal the effects of obesity on immunological disease and suggest a precision medicine approach to target the immune dysregulation caused by obesity.
Asunto(s)
Dermatitis Atópica , PPAR gamma , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inflamación/metabolismo , Ratones , Obesidad/metabolismo , PPAR gamma/agonistas , PPAR gamma/metabolismo , Medicina de Precisión , Análisis de Secuencia de ARN , Células Th2/metabolismoRESUMEN
We conducted this study aimed to evaluate the analgesic effect of dexmedetomidine in thoracoscopic surgery on postoperative wound pain, and to provide a reference for clinical use of the drug. We searched PubMed, Embase, Cochrane Library, Web of Science, Wanfang, Chinese Biomedical Literature Database and China National Knowledge Infrastructure databases, and supplemented with manual searching. We searched from database inception to October 2023, to collect the randomised controlled trials (RCTs) on dexmedetomidine application in thoracoscopic surgery. Two researchers screened all the literature according to the inclusion and exclusion criteria and the literature included in the study was evaluated for quality, extracted information and required data. Stata 17.0 software was employed for data analysis and the outcomes were 2 6, 12, 24 and 48 h postoperative wound visual analog scores (VAS). Twenty-four RCTs totalling 2246 patients undergoing thoracoscopic surgery were finally included. The analysis revealed dexmedetomidine applied to thoracoscopic surgery significantly reduced the postoperative wound VAS scores at 2 h (SMD: -0.96, 95% CI: -1.57 to -0.36, p = 0.002), 6 h (SMD: -0.98, 95% CI: -1.27 to -0.69, p < 0.001), 12 h (SMD: -1.19, 95% CI: -1.44 to -0.94, p < 0.001), 24 h (SMD: -0.91, 95% CI: -1.16 to -0.66, p < 0.001) and 48 h (SMD: -0.75, 95% CI: -1.02 to -0.48, p < 0.001). Our results suggest dexmedetomidine applied to thoracoscopic surgery can significantly reduce postoperative wound pain, which is worthy of clinical application.
Asunto(s)
Analgésicos no Narcóticos , Dexmedetomidina , Dolor Postoperatorio , Toracoscopía , Dexmedetomidina/uso terapéutico , Humanos , Dolor Postoperatorio/tratamiento farmacológico , Analgésicos no Narcóticos/uso terapéutico , Toracoscopía/métodos , Masculino , Adulto , Femenino , Persona de Mediana Edad , Ensayos Clínicos Controlados Aleatorios como Asunto , Anciano , Herida Quirúrgica/tratamiento farmacológico , Dimensión del DolorRESUMEN
T helper 17 (Th17) cells produce interleukin-17 (IL-17) cytokines and drive inflammatory responses in autoimmune diseases such as multiple sclerosis. The differentiation of Th17 cells is dependent on the retinoic acid receptor-related orphan nuclear receptor RORγt. Here, we identify REV-ERBα (encoded by Nr1d1), a member of the nuclear hormone receptor family, as a transcriptional repressor that antagonizes RORγt function in Th17 cells. REV-ERBα binds to ROR response elements (RORE) in Th17 cells and inhibits the expression of RORγt-dependent genes including Il17a and Il17f Furthermore, elevated REV-ERBα expression or treatment with a synthetic REV-ERB agonist significantly delays the onset and impedes the progression of experimental autoimmune encephalomyelitis (EAE). These results suggest that modulating REV-ERBα activity may be used to manipulate Th17 cells in autoimmune diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple/inmunología , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Células Th17/inmunología , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Sitios Genéticos , Células HEK293 , Humanos , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-17/metabolismo , Ratones , Ratones Transgénicos , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/genética , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/agonistas , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Pirrolidinas/farmacología , Pirrolidinas/uso terapéutico , RNA-Seq , Elementos de Respuesta/genética , Células Th17/metabolismo , Tiofenos/farmacología , Tiofenos/uso terapéuticoRESUMEN
Because the deletion of self-reactive T cells is incomplete, thymic development of natural Foxp3+CD4+ regulatory T cells (Treg cells) is required for preventing autoimmunity. However, the function of T cell antigen receptor (TCR) specificity in thymic Treg cell development remains controversial. To address this issue, we generated a transgenic line expressing a naturally occurring Treg cell-derived TCR. Unexpectedly, we found that efficient thymic Treg cell development occurred only when the antigen-specific Treg cell precursors were present at low clonal frequency (o1%) in a normal thymus. Using retroviral vectors and bone marrow chimeras, we observed similar activity with two other Treg cell-derived TCRs. Our data demonstrate that thymic Treg cell development is a 'TCR-instructive' process involving a niche that can be saturable at much lower clonal frequencies than is the niche for positive selection.
Asunto(s)
Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Timo/inmunología , Animales , Células de la Médula Ósea/inmunología , Diferenciación Celular , Quimera/inmunología , Factores de Transcripción Forkhead/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Age-associated insulin resistance (IR) and obesity-associated IR are two physiologically distinct forms of adult-onset diabetes. While macrophage-driven inflammation is a core driver of obesity-associated IR, the underlying mechanisms of the obesity-independent yet highly prevalent age-associated IR are largely unexplored. Here we show, using comparative adipo-immune profiling in mice, that fat-resident regulatory T cells, termed fTreg cells, accumulate in adipose tissue as a function of age, but not obesity. Supporting the existence of two distinct mechanisms underlying IR, mice deficient in fTreg cells are protected against age-associated IR, yet remain susceptible to obesity-associated IR and metabolic disease. By contrast, selective depletion of fTreg cells via anti-ST2 antibody treatment increases adipose tissue insulin sensitivity. These findings establish that distinct immune cell populations within adipose tissue underlie ageing- and obesity-associated IR, and implicate fTreg cells as adipo-immune drivers and potential therapeutic targets in the treatment of age-associated IR.
Asunto(s)
Tejido Adiposo/citología , Tejido Adiposo/inmunología , Envejecimiento/inmunología , Resistencia a la Insulina/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Animales , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Macrófagos/inmunología , Masculino , Síndrome Metabólico/inmunología , Síndrome Metabólico/metabolismo , Ratones , Obesidad/metabolismoRESUMEN
Effector CD4+ T cell subsets, whose differentiation is facilitated by distinct cytokine cues, amplify the corresponding type of inflammatory response. Regulatory T (Treg) cells integrate environmental cues to suppress particular types of inflammation. In this regard, STAT3, a transcription factor essential for T helper 17 (Th17) cell differentiation, is necessary for Treg cell-mediated control of Th17 cell responses. Here, we showed that anti-inflammatory interleukin-10 (IL-10), and not proinflammatory IL-6 and IL-23 cytokine signaling, endowed Treg cells with the ability to suppress pathogenic Th17 cell responses. Ablation of the IL-10 receptor in Treg cells resulted in selective dysregulation of Th17 cell responses and colitis similar to that observed in mice harboring STAT3-deficient Treg cells. Thus, Treg cells limit Th17 cell inflammation by serving as principal amplifiers of negative regulatory circuits operating in immune effector cells.
Asunto(s)
Colitis/inmunología , Interleucina-10/inmunología , Transducción de Señal , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Animales , Linaje de la Célula , Colitis/patología , Interleucina-10/metabolismo , Ratones , Ratones Noqueados , Fosforilación , Receptores de Interleucina-10/deficiencia , Receptores de Interleucina-10/inmunología , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT3/metabolismo , Linfocitos T Reguladores/citologíaRESUMEN
We report here that mouse macrophages undergo receptor-interacting kinase-3 (RIP3)-dependent but TNF-α-independent necrosis when Toll-like receptors (TLR) 3 and 4 are activated by poly(I:C) and LPS, respectively. An adaptor protein, Toll/IL-1 receptor domain-containing adapter inducing IFN-ß (TRIF/TICAM-1), which is dispensable for TNF-α-induced necrosis, forms a complex with RIP3 upon TLR3/TLR4 activation and is essential for TLR3/TLR4-induced necrosis. Mice without RIP3 or functional TRIF did not show macrophage loss and elevation of inflammatory cytokines when they were exposed to LPS. Necrosis in mouse macrophages induced by either TNFR or TLR3/TLR4 is executed by reactive oxygen species. Taken together, these data indicate that there are multiple upstream necrosis-initiating signaling pathways converging on the RIP3 during an innate immune response to viral and bacterial infections in mammals.
Asunto(s)
Macrófagos/inmunología , Macrófagos/patología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal/inmunología , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Citocinas/biosíntesis , Activación Enzimática/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Modelos Inmunológicos , FN-kappa B/metabolismo , Necrosis , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Especies Reactivas de Oxígeno/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Transducción de Señal/efectos de los fármacosRESUMEN
Using copper-ionophores to translocate extracellular copper into mitochondria is a clinically validated anticancer strategy that has been identified as a new type of regulated cell death termed "cuproptosis." This study reports a mitochondria-targeting Cu(I) complex, Cu(I)Br(PPh3)3 (CBP), consisting of a cuprous ion coordinated by three triphenylphosphine moieties and a Br atom. CBP exhibited antitumor and antimetastatic efficacy in vitro and in vivo by specifically targeting mitochondria instigating mitochondrial dysfunction. The cytotoxicity of CBP could only be reversed by a copper chelator rather than inhibitors of the known cell death, indicating copper-dependent cytotoxicity. Furthermore, CBP induced the oligomerization of lipoylated proteins and the loss of Fe-S cluster proteins, consistent with characteristic features of cuproptosis. Additionally, CBP induced remarkable intracellular generation of reactive oxygen species (ROS) through a Fenton-like reaction, indicating a complex antitumor mechanism. This is a proof-of-concept study exploiting the antitumor activity and mechanism of the Cu(I)-based mitochondria-targeting therapy.
Asunto(s)
Antineoplásicos , Complejos de Coordinación , Cobre , Mitocondrias , Especies Reactivas de Oxígeno , Cobre/química , Cobre/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Animales , Especies Reactivas de Oxígeno/metabolismo , Complejos de Coordinación/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/síntesis química , Ratones , Línea Celular TumoralRESUMEN
Toll-like receptor 3 (TLR3) and cytosolic RIG-I-like helicases (RIG-I and MDA5) sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN) through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-ß (TRIF), and mitochondrial antiviral signaling protein (MAVS), respectively. Previously, we demonstrated that hepatitis A virus (HAV), a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro), that is derived by auto-processing of the P3 (3ABCD) segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C)-stimulated dimerization of IFN regulatory factor 3 (IRF-3), IRF-3 translocation to the nucleus, and IFN-ß promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro) protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro) and downstream 3D(pol) sequence, but not 3D(pol) polymerase activity. Cleavage occurs at two non-canonical 3C(pro) recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol) sequence modulates the substrate specificity of the upstream 3C(pro) protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro). HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Cisteína Endopeptidasas/metabolismo , Virus de la Hepatitis A/enzimología , ARN Viral/genética , Receptor Toll-Like 3/metabolismo , Proteínas Virales/metabolismo , Proteasas Virales 3C , Proteínas Adaptadoras del Transporte Vesicular/genética , Línea Celular , Cisteína Endopeptidasas/genética , Virus de la Hepatitis A/aislamiento & purificación , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , Luciferasas/metabolismo , Plásmidos/genética , Transducción de Señal , Especificidad por Sustrato , Receptor Toll-Like 3/genética , Transfección/métodos , Proteínas Virales/genéticaRESUMEN
Ferroptosis is an oxidative damage-related, iron-dependent regulated cell death with intracellular lipid peroxide accumulation, which is associated with many physiological and pathological processes. It exhibits unique features that are morphologically, biochemically, and immunologically distinct from other regulated cell death forms. Ferroptosis is regulated by iron metabolism, lipid metabolism, anti-oxidant defense systems, as well as various signal pathways. Hypoxia, which is found in a group of physiological and pathological conditions, can affect multiple cellular functions by activation of the hypoxia-inducible factor (HIF) signaling and other mechanisms. Emerging evidence demonstrated that hypoxia regulates ferroptosis in certain cell types and conditions. In this review, we summarize the basic mechanisms and regulations of ferroptosis and hypoxia, as well as the regulation of ferroptosis by hypoxia in physiological and pathological conditions, which may contribute to the numerous diseases therapies.
Asunto(s)
Ferroptosis , Muerte Celular Regulada , Humanos , Transducción de Señal , Hipoxia , HierroRESUMEN
Chromatin conformation reorganization is emerging as an important layer of regulation for gene expression and lineage specification. Yet, how lineage-specific transcription factors contribute to the establishment of cell type-specific 3D chromatin architecture in the immune cells remains unclear, especially for the late stages of T cell subset differentiation and maturation. Regulatory T cells (Treg) are mainly generated in the thymus as a subpopulation of T cells specializing in suppressing excessive immune responses. Here, by comprehensively mapping 3D chromatin organization during Treg cell differentiation, we show that Treg-specific chromatin structures were progressively established during its lineage specification, and highly associated with Treg signature gene expression. Additionally, the binding sites of Foxp3, a Treg lineage specifying transcription factor, were highly enriched at Treg-specific chromatin loop anchors. Further comparison of the chromatin interactions between wide-type Tregs versus Treg cells from Foxp3 knock-in/knockout or newly-generated Foxp3 domain-swap mutant mouse revealed that Foxp3 was essential for the establishment of Treg-specific 3D chromatin architecture, although it was not dependent on the formation of the Foxp3 domain-swapped dimer. These results highlighted an underappreciated role of Foxp3 in modulating Treg-specific 3D chromatin structure formation.
RESUMEN
Chromatin conformation reorganization is emerging as an important layer of regulation for gene expression and lineage specification. Yet, how lineage-specific transcription factors contribute to the establishment of cell type-specific 3D chromatin architecture in the immune cells remains unclear, especially for the late stages of T cell subset differentiation and maturation. Regulatory T cells (Treg) are mainly generated in the thymus as a subpopulation of T cells specializing in suppressing excessive immune responses. Here, by comprehensively mapping 3D chromatin organization during Treg cell differentiation, we show that Treg-specific chromatin structures were progressively established during its lineage specification, and highly associated with Treg signature gene expression. Additionally, the binding sites of Foxp3, a Treg lineage specifying transcription factor, were highly enriched at Treg-specific chromatin loop anchors. Further comparison of the chromatin interactions between wide-type Tregs versus Treg cells from Foxp3 knock-in/knockout or newly-generated Foxp3 domain-swap mutant mouse revealed that Foxp3 was essential for the establishment of Treg-specific 3D chromatin architecture, although it was not dependent on the formation of the Foxp3 domain-swapped dimer. These results highlighted an underappreciated role of Foxp3 in modulating Treg-specific 3D chromatin structure formation.
Asunto(s)
Cromatina , Linfocitos T Reguladores , Ratones , Animales , Cromatina/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Diferenciación Celular/genéticaRESUMEN
Signaling through the second messengers calcium and diacylglycerol (DAG) is a critical element in many biological systems. Integration of calcium and DAG signals has been suggested to occur primarily through protein kinase C family members, which bind both calcium and DAG. However, an alternative pathway may involve members of the CalDAG-GEF/RasGRP protein family, which have structural features (calcium-binding EF hands and DAG-binding C1 domains) that suggest they can function in calcium and DAG signal integration. To gain insight into the signaling systems that may be regulated by CalDAG-GEF/RasGRP family members, we have focused on CalDAG-GEFI, which is expressed preferentially in the brain and blood. Through genetic ablation in the mouse, we have found that CalDAG-GEFI is crucial for signal integration in platelets. Mouse platelets that lack CalDAG-GEFI are severely compromised in integrin-dependent aggregation as a consequence of their inability to signal through CalDAG-GEFI to its target, the small GTPase Rap1. These results suggest that analogous signaling defects are likely to occur in the central nervous system when CalDAG-GEFI is absent or compromised in function.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Agregación Plaquetaria/fisiología , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/fisiología , Trombosis/fisiopatología , Animales , Recuento de Células Sanguíneas , Calcio/metabolismo , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Diglicéridos/metabolismo , Citometría de Flujo , Genotipo , Factores de Intercambio de Guanina Nucleótido/fisiología , Inmunohistoquímica , Ratones , Ratones Noqueados , Trombosis/metabolismo , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications.
Asunto(s)
Expresión Génica , Marcación de Gen , Transgenes , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/patología , Colesterol/sangre , Doxiciclina/administración & dosificación , Doxiciclina/metabolismo , Células Madre Embrionarias/fisiología , Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Elementos Aisladores , Ratones , Ratones Transgénicos , Mutagénesis Insercional , Retroviridae/genética , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
BACKGROUND & AIMS: Although hepatitis C virus (HCV) is a common cause of cirrhosis and liver cancer, efforts to understand the pathogenesis of HCV infection have been limited by the low abundance of viral proteins expressed within the liver, which hinders the detection of infected cells in situ. This study evaluated the ability of advanced optical imaging techniques to determine the extent and distribution of HCV-infected cells within the liver. METHODS: We combined 2-photon microscopy with virus-specific, fluorescent, semiconductor quantum dot probes to determine the proportion of hepatocytes that were infected with virus in frozen sections of liver tissue obtained from patients with chronic HCV infection. RESULTS: Viral core and nonstructural protein 3 antigens were detected readily in liver tissues from patients with chronic infection without confounding tissue autofluorescence. Specificity was confirmed by blocking with specific antibodies and by tissue colocalization of distinct viral antigens. Between 7% and 20% of hepatocytes were infected in patients with plasma viral RNA loads of 10(5) IU/mL or greater. Infected cells were in clusters, which suggested spread of the virus from cell to cell. Double-stranded RNA, a product of viral replication, was abundant within cells at the center of such clusters, but often scarce in cells at the periphery, consistent with more recent infection of cells at the periphery. CONCLUSIONS: Two-photon microscopy provides unprecedented sensitivity for the detection of HCV proteins and double-stranded RNA. Studies using this technology indicate that HCV infection is a dynamic process that involves a limited number of hepatocytes. HCV spread between cells is likely to be constrained by host responses.
Asunto(s)
Hepacivirus/química , Antígenos de la Hepatitis C/análisis , Hepatitis C/diagnóstico , Hígado/virología , Microscopía de Fluorescencia por Excitación Multifotónica , Puntos Cuánticos , Proteínas del Núcleo Viral/análisis , Proteínas no Estructurales Virales/análisis , Biopsia , Femenino , Hepacivirus/enzimología , Hepacivirus/genética , Hepacivirus/inmunología , Hepatitis C/patología , Humanos , Hígado/patología , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , ARN Bicatenario/análisis , ARN Viral/sangre , Sensibilidad y Especificidad , Carga ViralRESUMEN
Toll-like receptor-3 (TLR3) senses double-stranded RNA, initiating signaling that activates NF-kappaB and interferon regulatory factor 3 (IRF-3), thereby inducing the synthesis of proinflammatory cytokines, type I interferons, and numerous interferon-stimulated genes (ISGs). This pathway has not been extensively investigated in human hepatocytes, and its role in sensing and protecting against hepatitis virus infections is uncertain. We show here that primary human hepatocytes express TLR3 and robustly upregulate ISGs upon poly(I.C) stimulation. We also show that TLR3 senses hepatitis C virus (HCV) infection when expressed in permissive hepatoma cells, acting independently of retinoic acid-inducible gene I and inducing IRF-3 activation and the synthesis of ISGs that restrict virus replication. In turn, HCV infection reduces the abundance of TRIF, an essential TLR3 adaptor, and impairs poly(I.C)-induced signaling. The induction and disruption of TLR3 signaling by HCV may be important factors in determining the outcome of infection and the ability of HCV to establish persistent infections.