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Alveolar type 2 (AT2) cells maintain lung health by acting as stem cells and producing pulmonary surfactant1-3. AT2 dysfunction underlies many lung diseases including interstitial lung disease (ILD), in which some inherited forms result from mislocalisation of surfactant protein C (SFTPC) variants4,5. Disease modelling and dissection of mechanisms remains challenging due to complexities in deriving and maintaining AT2 cells ex vivo. Here, we describe the development of expandable adult AT2-like organoids derived from human fetal lung which are phenotypically stable, can differentiate into AT1-like cells and are genetically manipulable. We use these organoids to test key effectors of SFTPC maturation identified in a forward genetic screen including the E3 ligase ITCH, demonstrating that their depletion phenocopies the pathological SFTPC redistribution seen for the SFTPC-I73T variant. In summary, we demonstrate the development of a novel alveolar organoid model and use it to identify effectors of SFTPC maturation necessary for AT2 health.
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Misfolding of secretory proteins in the endoplasmic reticulum (ER) features in many human diseases. In α1-antitrypsin deficiency, the pathogenic Z variant aberrantly assembles into polymers in the hepatocyte ER, leading to cirrhosis. We show that α1-antitrypsin polymers undergo a liquid:solid phase transition, forming a protein matrix that retards mobility of ER proteins by size-dependent molecular filtration. The Z-α1-antitrypsin phase transition is promoted during ER stress by an ATF6-mediated unfolded protein response. Furthermore, the ER chaperone calreticulin promotes Z-α1-antitrypsin solidification and increases protein matrix stiffness. Single-particle tracking reveals that solidification initiates in cells with normal ER morphology, previously assumed to represent a healthy pool. We show that Z-α1-antitrypsin-induced hypersensitivity to ER stress can be explained by immobilization of ER chaperones within the polymer matrix. This previously unidentified mechanism of ER dysfunction provides a template for understanding a diverse group of related proteinopathies and identifies ER chaperones as potential therapeutic targets.
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The sodium-iodide symporter (NIS, SLC5A5) is expressed at the basolateral membrane of the thyroid follicular cell, and facilitates the thyroidal iodide uptake required for thyroid hormone biosynthesis. Biallelic loss-of-function mutations in NIS are a rare cause of dyshormonogenic congenital hypothyroidism. Affected individuals typically exhibit a normally sited, often goitrous thyroid gland, with absent uptake of radioiodine in the thyroid and other NIS-expressing tissues. We report a novel homozygous NIS mutation (c.1067 C>T, p.S356F) in four siblings from a consanguineous Indian kindred, presenting with significant hypothyroidism. Functional characterization of the mutant protein demonstrated impaired plasma membrane localization and cellular iodide transport.
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Hipotiroidismo Congénito/genética , Mutación/genética , Simportadores/genética , Femenino , Humanos , India , Recién NacidoRESUMEN
BACKGROUND: Alveolar epithelial cell dysfunction plays an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), but remains incompletely understood. Some monogenic forms of pulmonary fibrosis are associated with expression of mutant surfactant protein C (SFTPC). The commonest pathogenic mutant, I73T, mislocalises to the alveolar epithelial cell plasma membrane and displays a toxic gain of function. Because the mechanisms explaining the link between this mutant and IPF are incompletely understood, we sought to interrogate SFTPC trafficking in health and disease to understand the functional significance of SFTPC-I73T relocalisation. METHODS: We performed mechanistic analysis of SFTPC trafficking in a cell model that reproduces the in vivo phenotype and validated findings in human primary alveolar organoids. RESULTS: We show that wild-type SFTPC takes an unexpected indirect trafficking route via the plasma membrane and undergoes the first of multiple cleavage events before reaching the multivesicular body (MVB) for further processing. SFTPC-I73T takes this same route, but its progress is retarded both at the cell surface and due to failure of trafficking into the MVB. Unable to undergo onward trafficking, it is recycled to the plasma membrane as a partially cleaved intermediate. CONCLUSION: These data show for the first time that all SFTPC transits the cell surface during normal trafficking, and the I73T mutation accumulates at the cell surface through both retarded trafficking and active recycling. This understanding of normal SFTPC trafficking and how the I73T mutant disturbs it provides novel insight into SFTPC biology in health and disease, and in the contribution of the SFTPC mutant to IPF development.
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Fibrosis Pulmonar Idiopática , Proteína C Asociada a Surfactante Pulmonar/metabolismo , Células Epiteliales Alveolares , Humanos , Fibrosis Pulmonar Idiopática/genética , Mutación , Proteína C Asociada a Surfactante Pulmonar/genética , TensoactivosRESUMEN
Protein misfolding within the endoplasmic reticulum (ER stress) can be a cause or consequence of pulmonary disease. Mutation of proteins restricted to the alveolar type II pneumocyte can lead to inherited forms of pulmonary fibrosis, but even sporadic cases of pulmonary fibrosis appear to be strongly associated with activation of the unfolded protein response and/or the integrated stress response. Inhalation of smoke can impair protein folding and may be an important cause of pulmonary ER stress. Similarly, tissue hypoxia can lead to impaired protein homeostasis (proteostasis). But the mechanisms linking smoke and hypoxia to ER stress are only partially understood. In this review, we will examine the role of ER stress in the pathogenesis of lung disease by focusing on fibrosis, smoke, and hypoxia.
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Asfixia/fisiopatología , Estrés del Retículo Endoplásmico , Retículo Endoplásmico/patología , Hipoxia/fisiopatología , Fibrosis Pulmonar/fisiopatología , Fumar/fisiopatología , Respuesta de Proteína Desplegada , Animales , Retículo Endoplásmico/metabolismo , Humanos , Pliegue de ProteínaRESUMEN
An important function of the endoplasmic reticulum (ER) is to serve as a site of secretory protein folding. When the accumulation of misfolded proteins threatens to disturb luminal homoeostasis, the cell is said to experience ER stress. By contrast, the accumulation of well-folded proteins inside the ER leads to a distinct form of strain called ER overload. The serpins comprise a large family of proteins whose folding has been studied in great detail. Some mutant serpins misfold to cause ER stress, whereas others fold but then polymerise to cause ER overload. We discuss recent advances in the use of dynamic fluorescence imaging to study these phenomena. We also discuss a new technique that we recently published, rotor-based organelle viscosity imaging (ROVI), which promises to shed more light on the biophysical features of ER stress and ER overload.
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Fenómenos Biofísicos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Polimerizacion , alfa 1-Antitripsina/metabolismo , Animales , Humanos , Cuerpos de Inclusión/metabolismo , ViscosidadRESUMEN
BACKGROUND: Developmental pathways must be responsive to the environment. Phosphorylation of eIF2α enables a family of stress-sensing kinases to trigger the integrated stress response (ISR), which has pro-survival and developmental consequences. Bone morphogenetic proteins (BMPs) regulate multiple developmental processes in organisms from insects to mammals. RESULTS: Here we show in Drosophila that GCN2 antagonises BMP signalling through direct effects on translation and indirectly via the transcription factor crc (dATF4). Expression of a constitutively active GCN2 or loss of the eIF2α phosphatase dPPP1R15 impairs developmental BMP signalling in flies. In cells, inhibition of translation by GCN2 blocks downstream BMP signalling. Moreover, loss of d4E-BP, a target of crc, augments BMP signalling in vitro and rescues tissue development in vivo. CONCLUSION: These results identify a novel mechanism by which the ISR modulates BMP signalling during development.
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Proteínas de Drosophila/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Drosophila , Proteínas de Drosophila/genética , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Transducción de Señal/genéticaRESUMEN
α1-Antitrypsin is a serine protease inhibitor produced in the liver that is responsible for the regulation of pulmonary inflammation. The commonest pathogenic gene mutation yields Z-α1-antitrypsin, which has a propensity to self-associate forming polymers that become trapped in inclusions of endoplasmic reticulum (ER). It is unclear whether these inclusions are connected to the main ER network in Z-α1-antitrypsin-expressing cells. Using live cell imaging, we found that despite inclusions containing an immobile matrix of polymeric α1-antitrypsin, small ER resident proteins can diffuse freely within them. Inclusions have many features to suggest they represent fragmented ER, and some are physically separated from the tubular ER network, yet we observed cargo to be transported between them in a cytosol-dependent fashion that is sensitive to N-ethylmaleimide and dependent on Sar1 and sec22B. We conclude that protein recycling occurs between ER inclusions despite their physical separation.-Dickens, J. A., Ordóñez, A., Chambers, J. E., Beckett, A. J., Patel, V., Malzer, E., Dominicus, C. S., Bradley, J., Peden, A. A., Prior, I. A., Lomas, D. A., Marciniak, S. J. The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin.
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Transporte Biológico/fisiología , Retículo Endoplásmico/metabolismo , Hígado/metabolismo , alfa 1-Antitripsina/metabolismo , Animales , Transporte Biológico/genética , Células CHO , Células Cultivadas , Cricetulus , Mutación/genética , alfa 1-Antitripsina/genéticaRESUMEN
Alpha-1 antitrypsin deficiency is predominantly caused by point mutations that alter the protein's folding. These mutations fall into two broad categories: those that destabilize the protein dramatically and lead to its post-translational degradation and those that affect protein structure more subtly to promote protein polymerization within the endoplasmic reticulum (ER). This distinction is important because it determines the cell's response to each mutant. The severely misfolded mutants trigger an unfolded protein response (UPR) that promotes improved protein folding but can kill the cell in the chronic setting. In contrast, mutations that permit polymer formation fail to activate the UPR but instead promote a nuclear factor-κB-mediated ER overload response. The ability of polymers to increase a cell's sensitivity to ER stress likely explains apparent inconsistencies in the alpha-1 antitrypsin-signaling literature that have linked polymers with the UPR. In this review we discuss the use of mutant serpins to dissect each signaling pathway.
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Estrés del Retículo Endoplásmico/genética , Deficiencia de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Retículo Endoplásmico/metabolismo , Humanos , Mutación , FN-kappa B/metabolismo , Transducción de Señal/genética , Respuesta de Proteína Desplegada , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/metabolismoRESUMEN
Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.
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Polimerizacion/efectos de los fármacos , Inhibidores de Proteasas/metabolismo , Anticuerpos de Cadena Única/farmacología , alfa 1-Antitripsina/metabolismo , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Humanos , Immunoblotting , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Cinética , Elastasa de Leucocito/metabolismo , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación , Inhibidores de Proteasas/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/inmunologíaRESUMEN
α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p<10(-14)). We conclude that latent α1-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α1-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α1-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy.
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alfa 1-Antitripsina/química , Anticuerpos Monoclonales/química , Retículo Endoplásmico/metabolismo , Cinética , Polimerizacion , Estructura Secundaria de ProteínaAsunto(s)
Polímeros/química , Deficiencia de alfa 1-Antitripsina/sangre , Alelos , Biomarcadores/sangre , Biopsia , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoprecipitación , Inflamación , Masculino , Persona de Mediana Edad , Fenotipo , Sensibilidad y Especificidad , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/genéticaRESUMEN
Serpins are protease inhibitors whose most stable state is achieved upon transition of a central 5-stranded ß-sheet to a 6-stranded form. Mutations, low pH, denaturants and elevated temperatures promote this transition, which can result in a growing polymer chain of inactive molecules. Different types of polymer are possible, but, experimentally only heat has been shown to generate polymers in vitro consistent with ex vivo pathological specimens. Many mutations that alter the rate of heat-induced polymerization have been described, but interpretation is problematic because discrimination is lacking between the effect of global changes in native stability and specific effects on structural mechanism. We show that the temperature midpoint (Tm) of thermal denaturation reflects the transition of α1-antitrypsin to the polymerization intermediate, and determine the relationship with fixed-temperature polymerization half-times (t0.5) in the presence of stabilizing additives [TMAO (trimethylamine N-oxide), sucrose and sodium sulfate], point mutations and disulfide bonds. Combined with a retrospective analysis of 31 mutants characterized in the literature, the results of the present study show that global changes to native state stability are the predominant basis for the effects of mutations and osmolytes on heat-induced polymerization, summarized by the equation: ln(t0.5,mutant/t0.5,wild-type)=0.34×ΔTm. It is deviations from this relationship that hold key information about the polymerization process.
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Quimotripsina/química , Quimotripsina/genética , Mutación Puntual/genética , Polimerizacion , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Sustitución de Aminoácidos/genética , Animales , Bovinos , Quimotripsina/metabolismo , Dicroismo Circular , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estudios Retrospectivos , Temperatura , alfa 1-Antitripsina/metabolismoRESUMEN
Since its discovery 50 years ago, α1-antitrypsin deficiency has represented a case study in molecular medicine, with careful clinical characterisation guiding genetic, biochemical, biophysical, structural, cellular, and in vivo studies. Here we highlight the milestones in understanding the disease mechanisms and show how they have spurred the development of novel therapeutic strategies. α1-Antitrypsin deficiency is an archetypal conformational disease. Its pathogenesis demonstrates the interplay between protein folding and quality control mechanisms, with aberrant conformational changes causing liver and lung disease through combined loss- and toxic gain-of-function effects. Moreover, α1-antitrypsin exemplifies the ability of diverse proteins to self-associate into a range of morphologically distinct polymers, suggesting a mechanism for protein and cell evolution.
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Deficiencia de alfa 1-Antitripsina/etiología , Deficiencia de alfa 1-Antitripsina/patología , Animales , Retículo Endoplásmico/metabolismo , Humanos , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Proteolisis , Respuesta de Proteína Desplegada , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/terapiaRESUMEN
Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling. However, whether endogenous expression in primary bronchial epithelial cells has similar consequences remains unclear. Moreover, the mechanism of NF-κB activation has not yet been elucidated. Here, we report excessive NF-κB signalling in resting primary bronchial epithelial cells from ZZ patients compared with wild-type (MM) controls, and this appears to be mediated by mitogen-activated protein/extracellular signal-regulated kinase, EGF receptor and ADAM17 activity. Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin. Treatment of ZZ primary bronchial epithelial cells with purified plasma M α1-antitrypsin attenuates this inflammatory response, opening up new therapeutic options to modulate airway inflammation in the lung.
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Células Epiteliales/metabolismo , Sistema de Señalización de MAP Quinasas , Mutación Missense , Transducción de Señal , alfa 1-Antitripsina/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Línea Celular Tumoral , Citocinas/metabolismo , Estrés del Retículo Endoplásmico , Receptores ErbB/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Pulmón/patología , FN-kappa B/metabolismo , Cultivo Primario de Células , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , alfa 1-Antitripsina/biosíntesisRESUMEN
The common severe Z mutation (E342K) of α1-antitrypsin forms intracellular polymers that are associated with liver cirrhosis. The native fold of this protein is well-established and models have been proposed from crystallographic and biophysical data for the stable inter-molecular configuration that terminates the polymerization pathway. Despite these molecular 'snapshots', the details of the transition between monomer and polymer remain only partially understood. We surveyed the RCL (reactive centre loop) of α1-antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P14P12 and P4, but not P10P8 or P2P1', resulted in a decrease in detectable polymer in a cell model that recapitulates the intracellular polymerization of the Z variant, consistent with polymerization from a near-native conformation. We have developed a FRET (Förster resonance energy transfer)-based assay to monitor polymerization in small sample volumes. An in vitro assessment revealed the position-specific effects on the unimolecular and multimolecular phases of polymerization: the P14P12 region self-inserts early during activation, while the interaction between P6P4 and ß-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P6P4, but not P14P12, yield an increase in the overall apparent activation energy of association from ~360 to 550 kJ mol(-1).
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alfa 1-Antitripsina/genética , Animales , Células COS , Chlorocebus aethiops , Humanos , Modelos Moleculares , Mutación , Polimerizacion , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismoRESUMEN
BACKGROUND: There is a need for biomarkers to better characterise individuals with COPD and to aid with the development of therapeutic interventions. A panel of putative blood biomarkers was assessed in a subgroup of the Evaluation of COPD Longitudinally to Identify Surrogate Endpoints (ECLIPSE) cohort. METHODS: Thirty-four blood biomarkers were assessed in 201 subjects with COPD, 37 ex-smoker controls with normal lung function and 37 healthy non-smokers selected from the ECLIPSE cohort. Biomarker repeatability was assessed using baseline and 3-month samples. Intergroup comparisons were made using analysis of variance, repeatability was assessed through Bland-Altman plots, and correlations between biomarkers and clinical characteristics were assessed using Spearman correlation coefficients. RESULTS: Fifteen biomarkers were significantly different in individuals with COPD when compared to former or non-smoker controls. Some biomarkers, including tumor necrosis factor-α and interferon-γ, were measurable in only a minority of subjects whilst others such as C-reactive protein showed wide variability over the 3-month replication period. Fibrinogen was the most repeatable biomarker and exhibited a weak correlation with 6-minute walk distance, exacerbation rate, BODE index and MRC dyspnoea score in COPD subjects. 33% (66/201) of the COPD subjects reported at least 1 exacerbation over the 3 month study with 18% (36/201) reporting the exacerbation within 30 days of the 3-month visit. CRP, fibrinogen interleukin-6 and surfactant protein-D were significantly elevated in those COPD subjects with exacerbations within 30 days of the 3-month visit compared with those individuals that did not exacerbate or whose exacerbations had resolved. CONCLUSIONS: Only a few of the biomarkers assessed may be useful in diagnosis or management of COPD where the diagnosis is based on airflow obstruction (GOLD). Further analysis of more promising biomarkers may reveal utility in subsets of patients. Fibrinogen in particular has emerged as a potentially useful biomarker from this cohort and requires further investigation. TRIAL REGISTRATION: SCO104960, clinicaltrials.gov identifier NCT00292552.
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Citocinas/sangre , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Adulto , Anciano , Biomarcadores/sangre , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Enfermedad Pulmonar Obstructiva Crónica/epidemiología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Estados Unidos/epidemiologíaRESUMEN
Alpha-1-antitrypsin is the most abundant circulating protease inhibitor. It is mainly produced by the liver and secreted into the circulation where it acts to prevent excessive proteolytic damage in the lungs by the enzyme neutrophil elastase. The most common severe deficiency allele is the Z mutation, which causes the protein to self-associate into ordered polymers. These polymers accumulate within hepatocytes to cause liver damage. The resulting lack of circulating α(1)-antitrypsin predisposes the Z homozygote to proteolytic lung damage and emphysema. Other pathways may also contribute to the development of lung disease. In particular, polymers of Z α(1)-antitrypsin can form within the lung where they act as a pro-inflammatory stimulus that may exacerbate protease-mediated lung damage. Researchers recognized in the 1980s that plasma α(1)-antitrypsin levels could be restored by intravenous infusions of purified human protein. Alpha-1-antitrypsin replacement therapy was introduced in 1987 but subsequent clinical trials have produced conflicting results, and to date there remains no widely accepted clinical evidence of the efficacy of α(1)-antitrypsin replacement therapy. This review addresses our current understanding of disease pathogenesis in α(1)-antitrypsin deficiency and questions why this treatment in isolation may not be effective. In particular it discusses the possible role of α(1)-antitrypsin polymers in exacerbating intrapulmonary inflammation and attenuating the efficacy of α(1)-antitrypsin replacement therapy.