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 (Ga-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). Ga-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. Ga-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 Ga-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 Ga-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.
The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed ...differentiation of human iPSCs into airway basal cells (“iBCs”), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato), we track and purify these cells as they first emerge as developmentally immature NKX2-1GFP+ lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1GFP+/TP63tdTomato+ cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.
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•Directed differentiation of human iPSCs generates airway basal cells (“iBCs”)•iBCs self-renew and display multipotent differentiation in vitro and in vivo•By single-cell RNA-seq, iBCs are highly similar to adult primary airway basal cells•iBCs enable modeling of acquired and genetic airway diseases
Hawkins and colleagues report a directed differentiation protocol enabling the derivation of airway basal cells (“iBCs”) from human iPSCs. iBCs recapitulate hallmark stem cell properties of primary basal cells, including self-renewal and multi-lineage differentiation, thus enabling modeling of airway diseases in vitro and repopulation of tracheal xenografts in vivo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Summary 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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Expansion microscopy enables nanoimaging with conventional microscopes by physically and isotropically magnifying preserved biological specimens embedded in a crosslinked water-swellable hydrogel. ...Current expansion microscopy protocols require prior treatment with reactive anchoring chemicals to link specific labels and biomolecule classes to the gel. We describe a strategy called Magnify, which uses a mechanically sturdy gel that retains nucleic acids, proteins and lipids without the need for a separate anchoring step. Magnify expands biological specimens up to 11 times and facilitates imaging of cells and tissues with effectively around 25-nm resolution using a diffraction-limited objective lens of about 280 nm on conventional optical microscopes or with around 15 nm effective resolution if combined with super-resolution optical fluctuation imaging. We demonstrate Magnify on a broad range of biological specimens, providing insight into nanoscopic subcellular structures, including synaptic proteins from mouse brain, podocyte foot processes in formalin-fixed paraffin-embedded human kidney and defects in cilia and basal bodies in drug-treated human lung organoids.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. ...However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Chronic obstructive lung disease is characterized by persistent abnormalities in epithelial and immune cell function that are driven, at least in part, by infection. Analysis of parainfluenza virus ...infection in mice revealed an unexpected role for innate immune cells in IL-13-dependent chronic lung disease, but the upstream driver for the immune axis in this model and in humans with similar disease was undefined. We demonstrate here that lung levels of IL-33 are selectively increased in postviral mice with chronic obstructive lung disease and in humans with very severe chronic obstructive pulmonary disease (COPD). In the mouse model, IL-33/IL-33 receptor signaling was required for Il13 and mucin gene expression, and Il33 gene expression was localized to a virus-induced subset of airway serous cells and a constitutive subset of alveolar type 2 cells that are both linked conventionally to progenitor function. In humans with COPD, IL33 gene expression was also associated with IL13 and mucin gene expression, and IL33 induction was traceable to a subset of airway basal cells with increased capacities for pluripotency and ATP-regulated release of IL-33. Together, these findings provide a paradigm for the role of the innate immune system in chronic disease based on the influence of long-term epithelial progenitor cells programmed for excess IL-33 production.
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.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Airway epithelial cell biology has been greatly advanced by studies of genetically defined and modified mice; however it is often difficult to isolate, manipulate, and assay epithelial cell-specific ...responses in vivo. In vitro proliferation and differentiation of mouse airway epithelial cells are made possible by a high-fidelity system for primary culture of mouse tracheal epithelial cells described in this chapter. Using this method, epithelial cells purified from mouse tracheas proliferate in growth factor-enriched medium. Subsequent culture in defined medium and the use of the air–liquid interface condition result in the development of well-differentiated epithelia composed of ciliated and non-ciliated cells with characteristics of native airways. Methods are also provided for manipulation of differentiation and analysis of differentiation and gene expression. These approaches allow the assessment of global responses and those of specific cell subpopulations within the airway epithelium.
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FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Background Some investigators find a deficiency in IFN production from airway epithelial cells infected with human rhinovirus in asthma, but whether this abnormality occurs with other respiratory ...viruses is uncertain. Objective To assess the effect of influenza A virus (IAV) and respiratory syncytial virus (RSV) infection on IFN production and viral level in human bronchial epithelial cells (hBECs) from subjects with and without asthma. Methods Primary-culture hBECs from subjects with mild to severe asthma (n = 11) and controls without asthma (hBECs; n = 7) were infected with live or ultraviolet-inactivated IAV (WS/33 strain), RSV (Long strain), or RSV (A/2001/2-20 strain) with multiplicity of infection 0.01 to 1. Levels of virus along with IFN-β and IFN-λ and IFN-stimulated gene expression (tracked by 2′-5′-oligoadenylate synthetase 1 and myxovirus (influenza virus) resistance 1 mRNA) were determined up to 72 hours postinoculation. Results After IAV infection, viral levels were increased 2-fold in hBECs from asthmatic subjects compared with nonasthmatic control subjects ( P < .05) and this increase occurred in concert with increased IFN-λ1 levels and no significant difference in IFNB1 , 2′-5′-oligoadenylate synthetase 1, or myxovirus (influenza virus) resistance 1mRNA levels. After RSV infections, viral levels were not significantly increased in hBECs from asthmatic versus nonasthmatic subjects and the only significant difference between groups was a decrease in IFN-λ levels ( P < .05) that correlated with a decrease in viral titer. All these differences were found only at isolated time points and were not sustained throughout the 72-hour infection period. Conclusions The results indicate that IAV and RSV control and IFN response to these viruses in airway epithelial cells is remarkably similar between subjects with and without asthma.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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.
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