RESUMO
In vertebrates, olfactory receptors localize on multiple cilia elaborated on dendritic knobs of olfactory sensory neurons (OSNs). Although olfactory cilia dysfunction can cause anosmia, how their differentiation is programmed at the transcriptional level has remained largely unexplored. We discovered in zebrafish and mice that Foxj1, a forkhead domain-containing transcription factor traditionally linked with motile cilia biogenesis, is expressed in OSNs and required for olfactory epithelium (OE) formation. In keeping with the immotile nature of olfactory cilia, we observed that ciliary motility genes are repressed in zebrafish, mouse, and human OSNs. Strikingly, we also found that besides ciliogenesis, Foxj1 controls the differentiation of the OSNs themselves by regulating their cell type-specific gene expression, such as that of olfactory marker protein (omp) involved in odor-evoked signal transduction. In line with this, response to bile acids, odors detected by OMP-positive OSNs, was significantly diminished in foxj1 mutant zebrafish. Taken together, our findings establish how the canonical Foxj1-mediated motile ciliogenic transcriptional program has been repurposed for the biogenesis of immotile olfactory cilia, as well as for the development of the OSNs.
Assuntos
Neurônios Receptores Olfatórios , Peixe-Zebra , Animais , Humanos , Camundongos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Cílios/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Mucosa OlfatóriaRESUMO
Most motile cilia have a stereotyped structure of nine microtubule outer doublets and a single central pair of microtubules. The central pair of microtubules are surrounded by a set of proteins, termed the central pair apparatus. A specific kinesin, Klp1 projects from the central pair and contributes to ciliary motility in Chlamydomonas. The vertebrate ortholog, Kif9, is required for beating in mouse sperm flagella, but the mechanism of Kif9/Klp1 function remains poorly defined. Here, using Xenopus epidermal multiciliated cells, we show that Kif9 is necessary for ciliary motility and the proper distal localization of not only central pair proteins, but also radial spokes and dynein arms. In addition, single-molecule assays in vitro reveal that Xenopus Kif9 is a long-range processive motor, although it does not mediate long-range movement in ciliary axonemes in vivo. Together, our data suggest that Kif9 is integral for ciliary beating and is necessary for proper axonemal distal end integrity.
Assuntos
Axonema , Cílios , Cinesinas , Animais , Axonema/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , XenopusRESUMO
Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5 Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder.
Assuntos
Variação Genética/genética , Precursores de Proteínas/genética , Proteína C Associada a Surfactante Pulmonar/genética , Proteínas Associadas a Surfactantes Pulmonares/genética , Proteínas rab5 de Ligação ao GTP/genética , Células Epiteliais Alveolares/metabolismo , Animais , Caenorhabditis elegans/genética , Humanos , Pulmão/metabolismo , Doenças Pulmonares Intersticiais/genética , Surfactantes Pulmonares/metabolismoRESUMO
BACKGROUND: Latent TGFß binding protein-2 (LTBP2) is a fibrillin 1 binding component of the microfibril. LTBP2 is the only LTBP protein that does not bind any isoforms of TGFß, although it may interfere with the function of other LTBPs or interact with other signaling pathways. RESULTS: Here, we investigate mice lacking Ltbp2 (Ltbp2-/- ) and identify multiple phenotypes that impact bodyweight and fat mass, and affect bone and skin development. The alterations in skin and bone development are particularly noteworthy since the strength of these tissues is differentially affected by loss of Ltbp2. Interestingly, some tissues that express high levels of Ltbp2, such as the aorta and lung, do not have a developmental or homeostatic phenotype. CONCLUSIONS: Analysis of these mice show that LTBP2 has complex effects on development through direct effects on the extracellular matrix (ECM) or on signaling pathways that are known to regulate the ECM.
Assuntos
Proteínas de Transporte , Matriz Extracelular , Animais , Camundongos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Matriz Extracelular/metabolismo , Fenótipo , Fator de Crescimento Transformador beta/metabolismo , Isoformas de Proteínas/metabolismo , Ligação ProteicaRESUMO
Nasal nitric oxide (nNO) is low in most patients with primary ciliary dyskinesia (PCD). Decreased ciliary motion could lead to antigen stasis, increasing oxidant production and NO oxidation in the airways. This could both decrease gas phase NO and increase nitrosative stress. We studied primary airway epithelial cells from healthy controls (HCs) and patients with PCD with several different genotypes. We measured antigen clearance in fenestrated membranes exposed apically to the fluorescently labeled antigen Dermatophagoides pteronyssinus (Derp1-f). We immunoblotted for 3-nitrotyrosine (3-NT) and for oxidative response enzymes. We measured headspace NO above primary airway cells without and with a PCD-causing genotype. We measured nNO and exhaled breath condensate (EBC) H2O2 in vivo. Apical Derp1-f was cleared from HC better than from PCD cells. DUOX1 expression was lower in HC than in PCD cells at baseline and after 24-h Derp1-f exposure. HC cells had less 3-NT and NO3- than PCD cells. However, NO consumption by HC cells was less than that by PCD cells; NO loss was prevented by superoxide dismutase (SOD) and by apocynin. nNO was higher in HCs than in patients with PCD. EBC H2O2 was lower in HC than in patients with PCD. The PCD airway epithelium does not optimally clear antigens and is subject to oxidative and nitrosative stress. Oxidation associated with antigen stasis could represent a therapeutic target in PCD, one with convenient monitoring biomarkers.NEW & NOTEWORTHY The PCD airway epithelium does not optimally clear antigens, and antigen exposure can lead to NO oxidation and nitrosative stress. Oxidation caused by antigen stasis could represent a therapeutic target in PCD, and there are convenient monitoring biomarkers.
Assuntos
Transtornos da Motilidade Ciliar , Síndrome de Kartagener , Humanos , Peróxido de Hidrogênio , Estresse Nitrosativo , Testes Respiratórios , Óxido Nítrico/metabolismo , Biomarcadores/metabolismo , Síndrome de Kartagener/metabolismoRESUMO
Common respiratory diseases continue to represent a major public health problem, and much of the morbidity and mortality is due to airway inflammation and mucus production. Previous studies indicated a role for mitogen-activated protein kinase 14 (MAPK14) in this type of disease, but clinical trials are unsuccessful to date. Our previous work identified a related but distinct kinase known as MAPK13 that is activated in respiratory airway diseases and is required for mucus production in human cell-culture models. Support for MAPK13 function in these models came from effectiveness of MAPK13 versus MAPK14 gene-knockdown and from first-generation MAPK13-14 inhibitors. However, these first-generation inhibitors were incompletely optimized for blocking activity and were untested in vivo. Here we report the next generation and selection of a potent MAPK13-14 inhibitor (designated NuP-3) that more effectively downregulates type-2 cytokine-stimulated mucus production in air-liquid interface and organoid cultures of human airway epithelial cells. We also show that NuP-3 treatment prevents respiratory airway inflammation and mucus production in new minipig models of airway disease triggered by type-2 cytokine challenge or respiratory viral infection. The results thereby provide the next advance in developing a small-molecule kinase inhibitor to address key features of respiratory disease.NEW & NOTEWORTHY This study describes the discovery of a potent mitogen-activated protein kinase 13-14 (MAPK13-14) inhibitor and its effectiveness in models of respiratory airway disease. The findings thereby provide a scheme for pathogenesis and therapy of lung diseases [e.g., asthma, chronic obstructive pulmonary disease (COPD), Covid-19, postviral, and allergic respiratory disease] and related conditions that implicate MAPK13-14 function. The findings also refine a hypothesis for epithelial and immune cell functions in respiratory disease that features MAPK13 as a possible component of this disease process.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno , Doença Pulmonar Obstrutiva Crônica , Animais , Humanos , Suínos , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Porco Miniatura/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Muco/metabolismo , Citocinas/metabolismo , Proteína Quinase 13 Ativada por Mitógeno/metabolismoRESUMO
Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive inflammatory lung disease without effective molecular markers of disease activity or treatment responses. Monocyte and interstitial macrophages that express the C-C motif CCR2 (chemokine receptor 2) are active in IPF and central to fibrosis.Objectives: To phenotype patients with IPF for potential targeted therapy, we developed 64Cu-DOTA-ECL1i, a radiotracer to noninvasively track CCR2+ monocytes and macrophages using positron emission tomography (PET).Methods: CCR2+ cells were investigated in mice with bleomycin- or radiation-induced fibrosis and in human subjects with IPF. The CCR2+ cell populations were localized relative to fibrotic regions in lung tissue and characterized using immunolocalization, single-cell mass cytometry, and Ccr2 RNA in situ hybridization and then correlated with parallel quantitation of lung uptake by 64Cu-DOTA-ECL1i PET.Measurements and Main Results: Mouse models established that increased 64Cu-DOTA-ECL1i PET uptake in the lung correlates with CCR2+ cell infiltration associated with fibrosis (n = 72). As therapeutic models, the inhibition of fibrosis by IL-1ß blockade (n = 19) or antifibrotic pirfenidone (n = 18) reduced CCR2+ macrophage accumulation and uptake of the radiotracer in mouse lungs. In lung tissues from patients with IPF, CCR2+ cells concentrated in perifibrotic regions and correlated with radiotracer localization (n = 21). Human imaging revealed little lung uptake in healthy volunteers (n = 7), whereas subjects with IPF (n = 4) exhibited intensive signals in fibrotic zones.Conclusions: These findings support a role for imaging CCR2+ cells within the fibrogenic niche in IPF to provide a molecular target for personalized therapy and monitoring.Clinical trial registered with www.clinicaltrials.gov (NCT03492762).
Assuntos
Biomarcadores/química , Fibrose Pulmonar Idiopática/fisiopatologia , Pulmão/diagnóstico por imagem , Pulmão/fisiopatologia , Macrófagos/fisiologia , Monócitos/fisiologia , Receptores CCR2/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Imagem Molecular , Tomografia por Emissão de PósitronsRESUMO
RATIONALE: Paradigm shifting studies have revealed that the heart contains functionally diverse populations of macrophages derived from distinct embryonic and adult hematopoietic progenitors. Under steady-state conditions, the heart is largely populated by CCR2- (C-C chemokine receptor type 2) macrophages of embryonic descent. After tissue injury, a dramatic shift in macrophage composition occurs whereby CCR2+ monocytes are recruited to the heart and differentiate into inflammatory CCR2+ macrophages that contribute to heart failure progression. Currently, there are no techniques to noninvasively detect CCR2+ monocyte recruitment into the heart and thus identify patients who may be candidates for immunomodulatory therapy. OBJECTIVE: To develop a noninvasive molecular imaging strategy with high sensitivity and specificity to visualize inflammatory monocyte and macrophage accumulation in the heart. METHODS AND RESULTS: We synthesized and tested the performance of a positron emission tomography radiotracer (68Ga-DOTA [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-ECL1i [extracellular loop 1 inverso]) that allosterically binds to CCR2. In naive mice, the radiotracer was quickly cleared from the blood and displayed minimal retention in major organs. In contrast, biodistribution and positron emission tomography demonstrated strong myocardial tracer uptake in 2 models of cardiac injury (diphtheria toxin induced cardiomyocyte ablation and reperfused myocardial infarction). 68Ga-DOTA-ECL1i signal localized to sites of tissue injury and was independent of blood pool activity as assessed by quantitative positron emission tomography and ex vivo autoradiography. 68Ga-DOTA-ECL1i uptake was associated with CCR2+ monocyte and CCR2+ macrophage infiltration into the heart and was abrogated in CCR2-/- mice, demonstrating target specificity. Autoradiography demonstrated that 68Ga-DOTA-ECL1i specifically binds human heart failure specimens and with signal intensity associated with CCR2+ macrophage abundance. CONCLUSIONS: These findings demonstrate the sensitivity and specificity of 68Ga-DOTA-ECL1i in the mouse heart and highlight the translational potential of this agent to noninvasively visualize CCR2+ monocyte recruitment and inflammatory macrophage accumulation in patients.
Assuntos
Coração/diagnóstico por imagem , Macrófagos/fisiologia , Monócitos/fisiologia , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/patologia , Animais , Movimento Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Imagem Molecular , Tomografia por Emissão de Pósitrons , Receptores CCR2/análiseRESUMO
Motile cilia are characterized by dynein motor units, which preassemble in the cytoplasm before trafficking into the cilia. Proteins required for dynein preassembly were discovered by finding human mutations that result in absent ciliary motors, but little is known about their expression, function, or interactions. By monitoring ciliogenesis in primary airway epithelial cells and MCIDAS-regulated induced pluripotent stem cells, we uncovered two phases of expression of preassembly proteins. An early phase, composed of HEATR2, SPAG1, and DNAAF2, preceded other preassembly proteins and was independent of MCIDAS regulation. The early preassembly proteins colocalized within perinuclear foci that also contained dynein arm proteins. These proteins also interacted based on immunoprecipitation and Förster resonance energy transfer (FRET) studies. FRET analysis of HEAT domain deletions and human mutations showed that HEATR2 interacted with itself and SPAG1 at multiple HEAT domains, while DNAAF2 interacted with SPAG1. Human mutations in HEATR2 did not affect this interaction, but triggered the formation of p62/Sequestosome-1-positive aggregates containing the early preassembly proteins, suggesting that degradation of an early preassembly complex is responsible for disease and pointing to key regions required for HEATR2 scaffold stability. We speculate that HEATR2 is an early scaffold for the initiation of dynein complex assembly in motile cilia.
Assuntos
Antígenos de Superfície/metabolismo , Cílios/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas/metabolismo , Mucosa Respiratória/fisiologia , Animais , Antígenos de Superfície/genética , Dineínas do Axonema , Células Cultivadas , Proteínas de Ligação ao GTP/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Mutação , Fenótipo , Proteínas/genética , Mucosa Respiratória/citologiaRESUMO
Single-cell RNA sequencing (scRNASeq) has advanced our understanding of lung biology, but its utility is limited by the need for fresh samples, loss of cell types by death or inadequate dissociation, and transcriptional stress responses induced during tissue digestion. Single-nucleus RNA sequencing (snRNASeq) has addressed these deficiencies in other tissues, but no protocol exists for lung tissue. We present a snRNASeq protocol and compare its results with those of scRNASeq. Two nuclear suspensions were prepared in lysis buffer on ice while one cell suspension was generated using enzymatic and mechanical dissociation. Cells and nuclei were processed using the 10× Genomics platform, and sequencing data were analyzed by Seurat. A total of 16,110 single-nucleus and 11,934 single-cell transcriptomes were generated. Gene detection rates were equivalent in snRNASeq and scRNASeq (â¼1,700 genes and 3,000 unique molecular identifiers per cell) when mapping intronic and exonic reads. In the combined data, 89% of epithelial cells were identified by snRNASeq versus 22.2% of immune cells. snRNASeq transcriptomes are enriched for transcription factors and signaling proteins, with reduction in mitochondrial and stress-response genes. Both techniques improved mesenchymal cell detection over previous studies. Homeostatic signaling relationships among alveolar cell types were defined by receptor-ligand mapping using snRNASeq data, revealing interplay among epithelial, mesenchymal, and capillary endothelial cells. snRNASeq can be applied to archival murine lung samples, improves dissociation bias, eliminates artifactual gene expression, and provides similar gene detection compared with scRNASeq.
Assuntos
Transtornos Dissociativos/genética , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Análise de Sequência de RNA , Animais , Núcleo Celular/metabolismo , Transtornos Dissociativos/metabolismo , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Pulmão/metabolismo , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA/métodosRESUMO
The complement system provides host defense against pathogens and environmental stress. C3, the central component of complement, is present in the blood and increases in BAL fluid after injury. We recently discovered that C3 is taken up by certain cell types and cleaved intracellularly to C3a and C3b. C3a is required for CD4+ T-cell survival. These observations made us question whether complement operates at environmental interfaces, particularly in the respiratory tract. We found that airway epithelial cells (AECs, represented by both primary human tracheobronchial cells and BEAS-2B [cell line]) cultured in C3-free media were unique from other cell types in that they contained large intracellular stores of de novo synthesized C3. A fraction of this protein reduced ("storage form") but the remainder did not, consistent with it being pro-C3 ("precursor form"). These two forms of intracellular C3 were absent in CRISPR knockout-induced C3-deficient AECs and decreased with the use of C3 siRNA, indicating endogenous generation. Proinflammatory cytokine exposure increased both stored and secreted forms of C3. Furthermore, AECs took up C3 from exogenous sources, which mitigated stress-associated cell death (e.g., from oxidative stress or starvation). C3 stores were notably increased within AECs in lung tissues from individuals with different end-stage lung diseases. Thus, at-risk cells furnish C3 through biosynthesis and/or uptake to increase locally available C3 during inflammation, while intracellularly, these stores protect against certain inducers of cell death. These results establish the relevance of intracellular C3 to airway epithelial biology and suggest novel pathways for complement-mediated host protection in the airway.
Assuntos
Brônquios/citologia , Complemento C3/metabolismo , Células Epiteliais/fisiologia , Morte Celular , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Complemento C3/genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Caliciformes/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Pneumopatias/metabolismo , Pneumopatias/patologia , Estresse FisiológicoRESUMO
Epithelial cells that line lung airways produce and secrete proteins with important roles in barrier function and host defense. Secretion of airway goblet cells is controlled by autophagy proteins during inflammatory conditions, resulting in accumulation of mucin proteins. We hypothesized that autophagy proteins would also be important in the function of club cells, dominant secretory airway epithelial cells that are dysregulated in chronic lung disease. We found that in the absence of an inflammatory stimulus, mice with club cells deficient for the autophagy protein Atg5 had a markedly diminished expression of secreted host defense proteins secretoglobulin family 1A, member 1 (Scgb1a1) and surfactant proteins A1 and D (Sftpa1 and Sftpd), as well as abnormal club cell morphology. Adult mice with targeted loss of Atg5 also showed diminished levels of host defense proteins in regenerating cells following ablation with naphthalene. A mouse strain with global deficiency of Atg16-like 1 (Atg16l1), an Atg5 binding partner, had a similar loss of host defense proteins and abnormal club cell morphology. Cigarette smoke exposure reduced levels of Scgb1a1 in wild-type mice as expected. Smoke exposure was not required to trigger club cell abnormalities in mice bearing the human ATG16 variant Atg16l1T300A/T300A, which had low Scgb1a1 levels independent of this environmental stress. Evaluation of lung tissues from former smokers with severe chronic obstructive pulmonary disease showed evidence of reduced autophagy and SCGB1A1 expression in club cells. Thus, autophagy proteins are required for the function of club cells, independent of the cellular stress of cigarette smoke, with roles that appear to be distinct from those of other secretory cell types.
Assuntos
Autofagia/fisiologia , Células Epiteliais/metabolismo , Células Caliciformes/metabolismo , Pulmão/metabolismo , Animais , Bronquíolos/metabolismo , Feminino , Humanos , Masculino , Camundongos Transgênicos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Mucosa Respiratória/metabolismoRESUMO
OBJECTIVE: Aortic arch transplants have advanced our understanding of processes that contribute to progression and regression of atherosclerotic plaques. To characterize the dynamic behavior of monocytes and macrophages in atherosclerotic plaques over time, we developed a new model of cervical aortic arch transplantation in mice that is amenable to intravital imaging. APPROACH AND RESULTS: Vascularized aortic arch grafts were transplanted heterotropically to the right carotid arteries of recipient mice using microsurgical suture techniques. To image immune cells in atherosclerotic lesions during regression, plaque-bearing aortic arch grafts from B6 ApoE-deficient donors were transplanted into syngeneic CX3CR1 GFP reporter mice. Grafts were evaluated histologically, and monocytic cells in atherosclerotic plaques in ApoE-deficient grafts were imaged intravitally by 2-photon microscopy in serial fashion. In complementary experiments, CCR2+ cells in plaques were serially imaged by positron emission tomography using specific molecular probes. Plaques in ApoE-deficient grafts underwent regression after transplantation into normolipidemic hosts. Intravital imaging revealed clusters of largely immotile CX3CR1+ monocytes/macrophages in regressing plaques that had been recruited from the periphery. We observed a progressive decrease in CX3CR1+ monocytic cells in regressing plaques and a decrease in CCR2+ positron emission tomography signal during 4 months. CONCLUSIONS: Cervical transplantation of atherosclerotic mouse aortic arches represents a novel experimental tool to investigate cellular mechanisms that contribute to the remodeling of atherosclerotic plaques.
Assuntos
Aorta Torácica/diagnóstico por imagem , Aorta Torácica/patologia , Doenças da Aorta/diagnóstico por imagem , Doenças da Aorta/patologia , Aterosclerose/diagnóstico por imagem , Aterosclerose/patologia , Microscopia Intravital/métodos , Microscopia de Fluorescência por Excitação Multifotônica , Monócitos/patologia , Placa Aterosclerótica , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Animais , Aorta Torácica/metabolismo , Aorta Torácica/transplante , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Modelos Animais de Doenças , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout para ApoE , Monócitos/metabolismo , Receptores CCR2/metabolismo , Fatores de Tempo , Proteína Vermelha FluorescenteRESUMO
Motile cilia are essential for clearance of particulates and pathogens from airways. For effective transport, ciliary motor proteins and axonemal structures interact to generate the rhythmic, propulsive bending, but the mechanisms that produce a dynamic waveform remain incompletely understood. Biomechanical measures of human ciliary motion and their relationships to ciliary assembly are needed to illuminate the biophysics of normal ciliary function and to quantify dysfunction in ciliopathies. To these ends, we analyzed ciliary motion by high-speed video microscopy of ciliated cells sampled from human lung airways compared with primary culture cells that undergo ciliogenesis in vitro. Quantitative assessment of waveform parameters showed variations in waveform shape between individual cilia; however, general trends in waveform parameters emerged, associated with progression of cilia length and stage of differentiation. When cilia emerged from cultured cells, beat frequency was initially elevated, then fell and remained stable as cilia lengthened. In contrast, the average bending amplitude and the ability to generate force gradually increased and eventually approached values observed in ex vivo samples. Dynein arm motor proteins DNAH5, DNAH9, DNAH11, and DNAH6 were localized within specific regions of the axoneme in the ex vivo cells; however, distinct stages of in vitro waveform development identified by biomechanical features were associated with the progressive movement of dyneins to the appropriate proximal or distal sections of the cilium. These observations suggest that the stepwise variation in waveform development during ciliogenesis is dependent on cilia length and potentially on outer dynein arm assembly.
Assuntos
Diferenciação Celular , Cílios/metabolismo , Pulmão/citologia , Axonema/metabolismo , Dineínas/metabolismo , Humanos , OrganogêneseRESUMO
To expand the range of functional polymer materials to include fully hydrolytically degradable systems that bear bioinspired phosphorus-containing linkages both along the backbone and as cationic side chain moieties for packaging and delivery of nucleic acids, phosphonium-functionalized polyphosphoester- block-poly(l-lactide) copolymers of various compositions were synthesized, fully characterized, and their self-assembly into nanoparticles were studied. First, an alkyne-functionalized polyphosphoester- block-poly(l-lactide) copolymer was synthesized via a one pot sequential ring opening polymerization of an alkyne-functionalized phospholane monomer, followed by the addition of l-lactide to grow the second block. Second, the alkynyl side groups of the polyphosphoester block were functionalized via photoinitiated thiol-yne radical addition of a phosphonium-functionalized free thiol. The polymers of varying phosphonium substitution degrees were self-assembled in aqueous buffers to afford formation of well-defined core-shell assemblies with an average size ranging between 30 and 50 nm, as determined by dynamic light scattering. Intracellular delivery of the nanoparticles and their effects on cell viability and capability at enhancing transfection efficiency of nucleic acids (e.g., siRNA) were investigated. Cell viability assays demonstrated limited toxicity of the assembly to RAW 264.7 mouse macrophages, except at high polymer concentrations, where the polymer of high degree of phosphonium functionalization induced relatively higher cytotoxicity. Transfection efficiency was strongly affected by the phosphonium-to-phosphate (P+/P-) ratios of the polymers and siRNA, respectively. The AllStars Hs Cell Death siRNA complexed to the various copolymers at a P+/P- ratio of 10:1 induced comparable cell death to Lipofectamine. These fully degradable nanoparticles might provide biocompatible nanocarriers for therapeutic nucleic acid delivery.
Assuntos
Técnicas de Transferência de Genes , Nanopartículas/química , Compostos Organofosforados/química , Polímeros/química , Alcinos/química , Animais , Dioxanos/química , Macrófagos/efeitos dos fármacos , Camundongos , Nanopartículas/administração & dosagem , Fósforo/química , Polímeros/administração & dosagem , Células RAW 264.7 , Compostos de Sulfidrila/químicaRESUMO
Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of ß-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential ß-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific ß-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/ß-catenin but not CBP/ß-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/ß-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/ß-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/ß-catenin interactions in a PKC-dependent manner, likely involving PKCζ. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting ß-catenin to modulate adult progenitor cell behavior in disease.
Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular , Proteína p300 Associada a E1A/fisiologia , Proteína Quinase C/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/fisiologia , Células Epiteliais Alveolares/fisiologia , Animais , Aquaporina 5/genética , Aquaporina 5/metabolismo , Linhagem Celular , Impedância Elétrica , Expressão Gênica , Camundongos , Camundongos Knockout , Fosforilação , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Ratos , Via de Sinalização Wnt , Proteína Wnt-5aRESUMO
Females have a more severe clinical course than males in terms of several inflammatory lung conditions. Notably, females with cystic fibrosis (CF) suffer worse outcomes, particularly in the setting of Pseudomonas aeruginosa infection. Sex hormones have been implicated in experimental and clinical studies; however, immune mechanisms responsible for this sex-based disparity are unknown and the specific sex hormone target for therapeutic manipulation has not been identified. The objective of this study was to assess mechanisms behind the impact of female sex hormones on host immune responses to P. aeruginosa We used wild-type and CF mice, which we hormone manipulated, inoculated with P. aeruginosa, and then examined for outcomes and inflammatory responses. Neutrophils isolated from mice and human subjects were tested for responses to P. aeruginosa We found that female mice inoculated with P. aeruginosa died earlier and showed slower bacterial clearance than males (P < 0.0001). Ovariectomized females supplemented with 17ß-estradiol succumbed to P. aeruginosa challenge earlier than progesterone- or vehicle-supplemented mice (P = 0.0003). 17ß-Estradiol-treated ovariectomized female mice demonstrated increased lung levels of inflammatory cytokines, and when rendered neutropenic the mortality difference was abrogated. Neutrophils treated with 17ß-estradiol demonstrated an enhanced oxidative burst but decreased P. aeruginosa killing and earlier cell necrosis. The estrogen receptor (ER) antagonist ICI 182,780 improved survival in female mice infected with P. aeruginosa and restored neutrophil function. We concluded that ER antagonism rescues estrogen-mediated neutrophil dysfunction and improves survival in response to P. aeruginosa ER-mediated processes may explain the sex-based mortality gap in CF and other inflammatory lung illnesses, and the ER blockade represents a rational therapeutic strategy.
Assuntos
Estradiol/farmacologia , Imunidade Inata/efeitos dos fármacos , Neutrófilos/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Receptores de Estrogênio/antagonistas & inibidores , Infecções Respiratórias/imunologia , Animais , Fibrose Cística/microbiologia , Estradiol/administração & dosagem , Estrogênios/administração & dosagem , Estrogênios/sangue , Estrogênios/farmacologia , Feminino , Humanos , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Necrose , Neutropenia/imunologia , Neutropenia/microbiologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/microbiologia , Ovariectomia , Progesterona/administração & dosagem , Progesterona/sangue , Infecções por Pseudomonas/microbiologia , Explosão Respiratória , Infecções Respiratórias/microbiologiaRESUMO
Purpose To characterize a chemokine receptor type 2 (CCR2)-binding peptide adapted for use as a positron emission tomography (PET) radiotracer for noninvasive detection of lung inflammation in a mouse model of lung injury and in human tissues from subjects with lung disease. Materials and Methods The study was approved by institutional animal and human studies committees. Informed consent was obtained from patients. A 7-amino acid CCR2 binding peptide (extracellular loop 1 inverso [ECL1i]) was conjugated to tetraazacyclododecane tetraacetic acid (DOTA) and labeled with copper 64 (64Cu) or fluorescent dye. Lung inflammation was induced with intratracheal administration of lipopolysaccharide (LPS) in wild-type (n = 19) and CCR2-deficient (n = 4) mice, and these mice were compared with wild-type mice given control saline (n = 5) by using PET performed after intravenous injection of 64Cu-DOTA-ECL1i. Lung immune cells and those binding fluorescently labeled ECL1i in vivo were detected with flow cytometry. Lung inflammation in tissue from subjects with nondiseased lungs donated for lung transplantation (n = 11) and those with chronic obstructive pulmonary disease (COPD) who were undergoing lung transplantation (n = 16) was evaluated for CCR2 with immunostaining and autoradiography (n = 6, COPD) with 64Cu-DOTA-ECL1i. Groups were compared with analysis of variance, the Mann-Whitney U test, or the t test. Results Signal on PET images obtained in mouse lungs after injury with LPS was significantly greater than that in the saline control group (mean = 4.43% of injected dose [ID] per gram of tissue vs 0.99% of injected dose per gram of tissue; P < .001). PET signal was significantly diminished with blocking studies using nonradiolabeled ECL1i in excess (mean = 0.63% ID per gram of tissue; P < .001) and in CCR2-deficient mice (mean = 0.39% ID per gram of tissue; P < .001). The ECL1i signal was associated with an elevated level of mouse lung monocytes. COPD lung tissue displayed significantly elevated CCR2 levels compared with nondiseased tissue (median = 12.8% vs 1.2% cells per sample; P = .002), which was detected with 64Cu-DOTA-ECL1i by using autoradiography. Conclusion 64Cu-DOTA-ECL1i is a promising tool for PET-based detection of CCR2-directed inflammation in an animal model and in human tissues as a step toward clinical translation. © RSNA, 2017 Online supplemental material is available for this article.
Assuntos
Pneumonia/diagnóstico por imagem , Pneumonia/imunologia , Tomografia por Emissão de Pósitrons , Receptores CCR2/análise , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Tomografia por Emissão de Pósitrons/métodosRESUMO
Neutrophils are critical mediators of innate immune responses and contribute to tissue injury. However, immune pathways that regulate neutrophil recruitment to injured tissues during noninfectious inflammation remain poorly understood. DAP12 is a cell membrane-associated protein that is expressed in myeloid cells and can either augment or dampen innate inflammatory responses during infections. To elucidate the role of DAP12 in pulmonary ischemia/reperfusion injury (IRI), we took advantage of a clinically relevant mouse model of transplant-mediated lung IRI. This technique allowed us to dissect the importance of DAP12 in tissue-resident cells and those that infiltrate injured tissue from the periphery during noninfectious inflammation. Macrophages in both mouse and human lungs that have been subjected to cold ischemic storage express DAP12. We found that donor, but not recipient, deficiency in DAP12 protected against pulmonary IRI. Analysis of the immune response showed that DAP12 promotes the survival of tissue-resident alveolar macrophages and contributes to local production of neutrophil chemoattractants. Intravital imaging demonstrated a transendothelial migration defect into DAP12-deficient lungs, which can be rescued by local administration of the neutrophil chemokine CXCL2. We have uncovered a previously unrecognized role for DAP12 expression in tissue-resident alveolar macrophages in mediating acute noninfectious tissue injury through regulation of neutrophil trafficking.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Regulação da Expressão Gênica/imunologia , Transplante de Pulmão , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Disfunção Primária do Enxerto/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Quimiocina CXCL2/genética , Quimiocina CXCL2/imunologia , Humanos , Pulmão/patologia , Macrófagos Alveolares/patologia , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/genética , Neutrófilos/patologia , Disfunção Primária do Enxerto/genética , Disfunção Primária do Enxerto/patologiaRESUMO
Ciliopathies are a growing class of disorders caused by abnormal ciliary axonemal structure and function. Our understanding of the complex genetic and functional phenotypes of these conditions has rapidly progressed. Primary ciliary dyskinesia (PCD) remains the sole genetic disorder of motile cilia dysfunction. However, unlike many Mendelian genetic disorders, PCD is not caused by mutations in a single gene or locus, but rather, autosomal recessive mutation in one of many genes that lead to a similar phenotype. The first reported PCD mutations, more than a decade ago, identified genes encoding known structural components of the ciliary axoneme. In recent years, mutations in genes encoding novel cytoplasmic and regulatory proteins have been discovered. These findings have provided new insights into the functions of the motile cilia, and a better understanding of motile cilia disease. Advances in genetic tools will soon allow more precise genetic testing, mandating that clinicians must understand the genetic basis of PCD. Here, we review genetic mutations, their biological impact on cilia structure and function, and the implication of emerging genetic diagnostic tools.