RESUMEN
For the treatment of severe COVID-19, supplementation with human plasma-purified α-1 antitrypsin (AAT) to patients is currently considered. AAT inhibits host proteases that facilitate viral entry and possesses broad anti-inflammatory and immunomodulatory activities. Researchers have demonstrated that an interaction between SARS-CoV-2 spike protein (S) and lipopolysaccharides (LPS) enhances pro-inflammatory responses in vitro and in vivo. Hence, we wanted to understand the potential anti-inflammatory activities of plasma-derived and recombinant AAT (recAAT) in a model of human total peripheral blood mononuclear cells (PBMCs) exposed to a combination of CHO expressed trimeric spike protein and LPS, ex vivo. We confirmed that cytokine production was enhanced in PBMCs within six hours when low levels of LPS were combined with purified spike proteins ("spike"). In the presence of 0.5 mg/mL recAAT, however, LPS/spike-induced TNF-α and IL-1ß mRNA expression and protein release were significantly inhibited (by about 46-50%) relative to LPS/spike alone. Although without statistical significance, recAAT also reduced production of IL-6 and IL-8. Notably, under the same experimental conditions, the plasma-derived AAT preparation Respreeza (used in native and oxidized forms) did not show significant effects. Our findings imply that an early pro-inflammatory activation of human PBMCs is better controlled by the recombinant version of AAT than the human plasma-derived AAT used here. Considering the increasing clinical interest in AAT therapy as useful to ameliorate the hyper-inflammation seen during COVID-19 infection, different AAT preparations require careful evaluation.
Asunto(s)
Antiinflamatorios/farmacología , Leucocitos Mononucleares/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , alfa 1-Antitripsina/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/inmunología , Células CHO , COVID-19/terapia , Células Cultivadas , Cricetulus , Citocinas/metabolismo , Humanos , Inflamación/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/inmunología , Lipopolisacáridos/toxicidad , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , alfa 1-Antitripsina/química , alfa 1-Antitripsina/inmunologíaRESUMEN
Acute respiratory distress syndrome is characterized by hypoxemia, altered alveolar-capillary permeability, and neutrophil-dominated inflammatory pulmonary edema. Despite decades of research, an effective drug therapy for acute respiratory distress syndrome remains elusive. The ideal pharmacotherapy for acute respiratory distress syndrome should demonstrate antiprotease activity and target injurious inflammatory pathways while maintaining host defense against infection. Furthermore, a drug with a reputable safety profile, low possibility of off-target effects, and well-known pharmacokinetics would be desirable. The endogenous 52-kd serine protease α1-antitrypsin has the potential to be a novel treatment option for acute respiratory distress syndrome. The main function of α1-antitrypsin is as an antiprotease, targeting neutrophil elastase in particular. However, studies have also highlighted the role of α1-antitrypsin in the modulation of inflammation and bacterial clearance. In light of the current SARS-CoV-2 pandemic, the identification of a treatment for acute respiratory distress syndrome is even more pertinent, and α1-antitrypsin has been implicated in the inflammatory response to SARS-CoV-2 infection.
Asunto(s)
Neutrófilos/efectos de los fármacos , Proteínas Inhibidoras de Proteinasas Secretoras/administración & dosificación , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , alfa 1-Antitripsina/administración & dosificación , Animales , COVID-19/enzimología , COVID-19/inmunología , Humanos , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/inmunología , Pulmón/efectos de los fármacos , Pulmón/enzimología , Pulmón/inmunología , Neutrófilos/enzimología , Neutrófilos/inmunología , Proteínas Inhibidoras de Proteinasas Secretoras/inmunología , Síndrome de Dificultad Respiratoria/enzimología , Síndrome de Dificultad Respiratoria/inmunología , alfa 1-Antitripsina/inmunología , Tratamiento Farmacológico de COVID-19RESUMEN
The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359-394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1-0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Péptidos/inmunología , alfa 1-Antitripsina/inmunología , Secuencia de Aminoácidos , Especificidad de Anticuerpos/inmunología , Trampas Extracelulares , Humanos , Lipopolisacáridos/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Péptidos/sangre , Péptidos/química , Desnaturalización ProteicaRESUMEN
Based on previous results demonstrating that complexes of a mutant α1-antitrypsin with the heat shock proteins (HSP)70 and glucose-regulated protein94 (Grp94) circulate in the blood of patients with type 1 diabetes, we raised the hypothesis that these complexes could represent the primary antigen capable of triggering the autoimmune reactions leading to overt diabetes. As a first approach to this issue, we searched whether A1AT and HSPs had a sequence similarity to major islet antigen proteins so as to identify among the similar sequences those with potential relevance for the pathogenesis of diabetes. A thorough in silico analysis was performed to establish the score of similarity of the human proteins: A1AT, pro-insulin (INS), GAD65, IAPP, IA-2, ICA69, Grp94, HSP70 and HSP60. The sequences of A1AT and HSPs with the highest score of similarity to the islet peptides reported in the literature as the main autoantigens in human diabetes were recorded. At variance with other HSPs, also including HSP90 and Grp78, Grp94 contained the highest number and the longest sequences with structural similarity to A1AT and to well-known immunogenic peptides/epitopes of INS, GAD65, and IA-2. The similarity of A1AT with Grp94 and that of Grp94 with INS also suggested a functional relationship among the proteins. Specific sequences were identified in A1AT, Grp94 and HSP70, with the highest score of cross-similarity to a pattern of eight different islet protein epitopes. The similarity also involved recently discovered autoantigens in type 1 diabetes such as a hybrid peptides of insulin and the defective ribosomal insulin gene product. The significant similarity displayed by specific sequences of Grp94 and A1AT to the islet peptides considered main antigens in human diabetes, is a strong indication for testing these sequences as new peptides of immunogenic relevance in diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Proteínas HSP70 de Choque Térmico/genética , Glicoproteínas de Membrana/genética , alfa 1-Antitripsina/genética , Antígenos/genética , Antígenos/inmunología , Simulación por Computador , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Chaperón BiP del Retículo Endoplásmico , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/inmunología , Humanos , Insulina/metabolismo , Glicoproteínas de Membrana/inmunología , Chaperonas Moleculares/genética , Chaperonas Moleculares/inmunología , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , alfa 1-Antitripsina/química , alfa 1-Antitripsina/inmunologíaRESUMEN
Alpha-1 antitrypsin (AAT) is a major inhibitor of serine proteases in mammals. Therefore, its deficiency leads to protease-antiprotease imbalance and a risk for developing lung emphysema. Although therapy with human plasma-purified AAT attenuates AAT deficiency-related emphysema, its impact on lung antibacterial immunity is poorly defined. Here, we examined the effect of AAT therapy on lung protective immunity in AAT-deficient (KO) mice challenged with Streptococcus pneumoniae. AAT-KO mice were highly susceptible to S. pneumoniae, as determined by severe lobar pneumonia and early mortality. Mechanistically, we found that neutrophil-derived elastase (NE) degraded the opsonophagocytically important collectins, surfactant protein A (SP-A) and D (SP-D), which was accompanied by significantly impaired lung bacterial clearance in S. pneumoniae-infected AAT-KO mice. Treatment of S. pneumoniae-infected AAT-KO mice with human AAT protected SP-A and SP-D from NE-mediated degradation and corrected the pulmonary pathology observed in these mice. Likewise, treatment with Sivelestat, a specific inhibitor of NE, also protected collectins from degradation and significantly decreased bacterial loads in S. pneumoniae-infected AAT-KO mice. Our findings show that NE is responsible for the degradation of lung SP-A and SP-D in AAT-KO mice affecting lung protective immunity in AAT deficiency.
Asunto(s)
Pulmón/inmunología , Pulmón/microbiología , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/patogenicidad , Deficiencia de alfa 1-Antitripsina/inmunología , Animales , Femenino , Humanos , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía Neumocócica/tratamiento farmacológico , Neumonía Neumocócica/etiología , Neumonía Neumocócica/inmunología , Enfisema Pulmonar/etiología , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/inmunología , alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/farmacocinética , alfa 1-Antitripsina/uso terapéutico , Deficiencia de alfa 1-Antitripsina/complicaciones , Deficiencia de alfa 1-Antitripsina/genéticaRESUMEN
BACKGROUND: Asthma is considered an incurable disease, although many advances have been made in asthma treatments in recent years. Therefore, elucidating the pathological mechanisms and seeking novel and effective therapeutic strategies for asthma are urgently needed. METHODS: Airway resistance was measured by whole-body plethysmography. H&E staining was used to observe the morphological changes in the lung. Oxidative stress was assessed by measuring the levels of MDA, CAT and SOD. Gene expression was analysed by western blotting and RT-qPCR. ELISA was used to analyse the concentrations of IL-4, IL-5 and IFN-γ. RESULTS: In the present study, we successfully established in vivo and in vitro asthma models. OVA administration led to elevated lung resistance, cell counts in BALF, and cytokine secretion, impaired airway structure and enhanced oxidative stress and autophagy in a mouse model of asthma, while IL-13 induced inflammation, oxidative stress and autophagy in BEAS-2B cells. A1AT reduced lung resistance and cell counts in BALF and suppressed inflammation, oxidative stress and autophagy in a mouse model of asthma and IL-13-induced BEAS-2B cells. Mechanistic investigations revealed that autophagy activation compromised the protective effect of A1AT on IL-13-induced BEAS-2B cells. Further mechanistic studies revealed that A1AT alleviated inflammation and oxidative stress by inhibiting autophagy in the context of asthma. CONCLUSION: We demonstrated that A1AT could alleviate inflammation and oxidative stress by suppressing autophagy in the context of asthma and thus ameliorate asthma. Our study revealed novel pathological mechanisms and provided novel potential therapeutic targets for asthma treatment.
Asunto(s)
Asma/tratamiento farmacológico , Autofagia/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , alfa 1-Antitripsina/farmacología , Animales , Asma/inmunología , Asma/patología , Autofagia/inmunología , Línea Celular , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Ratones , Ratones Endogámicos BALB C , Estrés Oxidativo/inmunología , alfa 1-Antitripsina/inmunologíaRESUMEN
Alpha 1 antitrypsin deficiency (AATD) is an autosomal co-dominant disorder characterized by a low level of circulating AAT, which significantly reduces protection for the lower airways against proteolytic burden caused by neutrophils. Neutrophils, which are terminally differentiated innate immune cells and play a critical role to clear pathogens, accumulate excessively in the lung of AATD individuals. The neutrophil burden in AATD individuals increases the risk for early-onset destructive lung diseases by producing neutrophil products such as reactive oxygen radicals and various proteases. The level of AAT in AATD individuals is not sufficient to inhibit the activity of neutrophil chemotactic factors such as CXCL-8 and LTB4, which could lead to alveolar neutrophil accumulation in AATD individuals. However, as neutrophils have a short lifespan, and apoptotic neutrophils are rapidly cleared by alveolar macrophages that outnumber the apoptotic neutrophils in the pulmonary alveolus, the increased chemotaxis activity does not fully explain the persistent neutrophil accumulation and the resulting chronic inflammation in AATD individuals. Here, we propose that the ability of alveolar macrophages to clear apoptotic neutrophils is impaired in AATD individuals and it could be the main driver to cause neutrophil accumulation in their lung. This study demonstrates that Z-AAT variant significantly increases the expression of pro-inflammatory cytokines including CXCL-8, CXCL1, LTB4, and TNFα in LPS-treated macrophages. These cytokines play a central role in neutrophil recruitment to the lung and in clearance of apoptotic neutrophils by macrophages. Our result shows that LPS treatment significantly reduces the efferocytosis ability of macrophages with the Z-AAT allele by inducing TNFα expression. We incubated monocyte-derived macrophages (MDMs) with apoptotic neutrophils and found that after 3 h of co-incubation, the expression level of CXCL-8 is reduced in M-MDMs but increased in Z-MDMs. This result shows that the expression of inflammatory cytokines could be increased by impaired efferocytosis. It indicates that the efferocytosis ability of macrophages plays an important role in regulating cytokine expression and resolving inflammation. Findings from this study would help us better understand the multifaceted effect of AAT on regulating neutrophil balance in the lung and the underlying mechanisms.
Asunto(s)
Apoptosis/inmunología , Macrófagos/inmunología , Neutrófilos/inmunología , Fagocitosis/inmunología , Deficiencia de alfa 1-Antitripsina/inmunología , Quimiotaxis de Leucocito , Citocinas/metabolismo , Genotipo , Humanos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Neutrófilos/metabolismo , Fagocitosis/efectos de los fármacos , Fagocitosis/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/inmunología , Deficiencia de alfa 1-Antitripsina/genéticaRESUMEN
Rationale: Coronavirus disease (COVID-19) is a global threat to health. Its inflammatory characteristics are incompletely understood.Objectives: To define the cytokine profile of COVID-19 and to identify evidence of immunometabolic alterations in those with severe illness.Methods: Levels of IL-1ß, IL-6, IL-8, IL-10, and sTNFR1 (soluble tumor necrosis factor receptor 1) were assessed in plasma from healthy volunteers, hospitalized but stable patients with COVID-19 (COVIDstable patients), patients with COVID-19 requiring ICU admission (COVIDICU patients), and patients with severe community-acquired pneumonia requiring ICU support (CAPICU patients). Immunometabolic markers were measured in circulating neutrophils from patients with severe COVID-19. The acute phase response of AAT (alpha-1 antitrypsin) to COVID-19 was also evaluated.Measurements and Main Results: IL-1ß, IL-6, IL-8, and sTNFR1 were all increased in patients with COVID-19. COVIDICU patients could be clearly differentiated from COVIDstable patients, and demonstrated higher levels of IL-1ß, IL-6, and sTNFR1 but lower IL-10 than CAPICU patients. COVID-19 neutrophils displayed altered immunometabolism, with increased cytosolic PKM2 (pyruvate kinase M2), phosphorylated PKM2, HIF-1α (hypoxia-inducible factor-1α), and lactate. The production and sialylation of AAT increased in COVID-19, but this antiinflammatory response was overwhelmed in severe illness, with the IL-6:AAT ratio markedly higher in patients requiring ICU admission (P < 0.0001). In critically unwell patients with COVID-19, increases in IL-6:AAT predicted prolonged ICU stay and mortality, whereas improvement in IL-6:AAT was associated with clinical resolution (P < 0.0001).Conclusions: The COVID-19 cytokinemia is distinct from that of other types of pneumonia, leading to organ failure and ICU need. Neutrophils undergo immunometabolic reprogramming in severe COVID-19 illness. Cytokine ratios may predict outcomes in this population.
Asunto(s)
Reacción de Fase Aguda/inmunología , Proteínas Portadoras/metabolismo , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/metabolismo , Citocinas/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ácido Láctico/metabolismo , Proteínas de la Membrana/metabolismo , Neumonía Viral/inmunología , Neumonía Viral/metabolismo , Hormonas Tiroideas/metabolismo , alfa 1-Antitripsina/inmunología , Reacción de Fase Aguda/metabolismo , Adulto , Anciano , Betacoronavirus , Western Blotting , COVID-19 , Estudios de Casos y Controles , Infecciones Comunitarias Adquiridas/inmunología , Infecciones Comunitarias Adquiridas/metabolismo , Infecciones por Coronavirus/mortalidad , Infecciones por Coronavirus/fisiopatología , Enfermedad Crítica , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Hospitalización , Humanos , Unidades de Cuidados Intensivos , Interleucina-10/inmunología , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Interleucina-8/inmunología , Tiempo de Internación , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Neutrófilos/metabolismo , Pandemias , Fosforilación , Neumonía/inmunología , Neumonía/metabolismo , Neumonía Viral/mortalidad , Neumonía Viral/fisiopatología , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , SARS-CoV-2 , Índice de Severidad de la Enfermedad , alfa 1-Antitripsina/metabolismo , Proteínas de Unión a Hormona TiroideRESUMEN
Rheumatoid arthritis (RA) is a chronic autoimmune disease and rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) are the most frequently detected autoantibodies (autoAbs). To date, more than 20% of RA cases are still defined as seronegative forms (seronegative RA, SN-RA). The aim of this study was to identify new antigenic targets of autoAbs in RA patients, which can also be recognized in SN-RA. Using a proteomic approach, we tested sera from SN-RA patients by analyzing synovial fluid (SF) proteins from these patients. Sera from SN-RA patients revealed a strong reactive spot, corresponding to alpha 1 antitrypsin (A1AT). Reverse-phase nanoliquid chromatography and tandem mass spectrometry (Matrix Assisted Laser Desorption/Ionization-Time Of Flight, MALDI-TOF/TOF) confirmed the presence of A1AT in SF and showed that homocysteinylation was one of the post-translational modifications of A1AT. Homocysteinylated (Hcy)-A1AT immunoprecipitated from SN-RA patients' SFs and in vitro modified Hcy-A1AT were used as antigens by Enzyme-Linked ImmunoSorbent Assay (ELISA) to test the presence of specific autoAbs in sera from 111 SN-RA patients, 132 seropositive (SP)-RA patients, and from 95 patients with psoriatic arthritis, 40 patients with osteoarthritis, and 41 healthy subjects as control populations. We observed that a large portion of SN-RA patients (75.7%), and also most of SP-RA patients' sera (87.1%) displayed anti-Hcy-A1AT autoAbs (anti-HATA). Native A1AT was targeted at a lower rate by SP-RA patients autoAbs, while virtually no SN-RA patients' sera showed the presence of anti-native A1AT autoAbs. In conclusion, anti-HATA can be considered potential biomarkers for RA, also in the SN forms. The discovery of novel autoAbs targeting specific autoantigens can represent higher clinic significance for all RA patients' population.
Asunto(s)
Artritis Reumatoide/diagnóstico , Autoanticuerpos/sangre , Autoantígenos/inmunología , alfa 1-Antitripsina/inmunología , Anciano , Artritis Reumatoide/sangre , Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Autoanticuerpos/metabolismo , Autoantígenos/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Voluntarios Sanos , Homocisteína/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Procesamiento Proteico-Postraduccional , Pruebas Serológicas , alfa 1-Antitripsina/metabolismoRESUMEN
Multiple analytical and preclinical studies were performed to compare the biochemical characteristics, pharmacokinetics (PK), safety and neoantigenicity of a new 5% liquid formulation of Alpha-1 Proteinase Inhibitor (Liquid A1PI, Prolastin®-C Liquid) with the lyophilized version (Lyophilized A1PI, Prolastin®-C). Liquid A1PI and Lyophilized A1PI had similar average mass (~52 kDa), and both forms exhibited glycoform patterns consistent with the known banding pattern of A1PI (dominated by the M6 and M4 bands, including deconvoluted masses). Both Liquid A1PI and Lyophilized A1PI yielded average percent purity values ranging from 96% to 99% and had active content ranging from 53⯠mg/mL to 59 â¯mg/mL. The PK profile of Liquid A1PI was similar to Lyophilized A1PI. Safety assessments in rabbits showed good tolerability and no test article-related changes in mortality, clinical signs, clinical pathology, body weight, food consumption, or urinalysis parameters. Following immunodepletion of antibodies that recognize Lyophilized A1PI, there were no significant differences in the anti-drug titers among animals immunized with Lyophilized A1PI and Liquid A1PI (pâ¯>â¯0.05), indicating that no antibodies to neoantigens were generated. Liquid A1PI and Lyophilized A1PI have similar profiles with respect to biochemical characteristics, PK, safety and neoantigenicity.
Asunto(s)
Deficiencia de alfa 1-Antitripsina/tratamiento farmacológico , alfa 1-Antitripsina , Animales , Anticuerpos/sangre , Anticuerpos/inmunología , Liofilización , Humanos , Conejos , alfa 1-Antitripsina/efectos adversos , alfa 1-Antitripsina/inmunología , alfa 1-Antitripsina/farmacocinética , alfa 1-Antitripsina/farmacología , Deficiencia de alfa 1-Antitripsina/sangre , Deficiencia de alfa 1-Antitripsina/inmunologíaRESUMEN
Rationale: The association between non-tuberculous mycobacterial lung disease and alpha-1-antitrypsin (AAT) deficiency is likely due, in part, to underlying emphysema or bronchiectasis. But there is increasing evidence that AAT itself enhances host immunity against microbial pathogens and thus deficiency could compromise host protection. Objectives: The goal of this project is to determine if AAT could augment macrophage activity against non-tuberculous mycobacteria. Methods: We compared the ability of monocyte-derived macrophages cultured in autologous plasma that were obtained immediately before and soon after AAT infusion-given to individuals with AAT deficiency-to control an ex vivo Mycobacterium intracellulare infection. Measurements and Main Results: We found that compared to pre-AAT infused monocyte-derived macrophages plus plasma, macrophages, and contemporaneous plasma obtained after a session of AAT infusion were significantly better able to control M. intracellulare infection; the reduced bacterial burden was linked with greater phagosome-lysosome fusion and increased autophagosome formation/maturation, the latter due to AAT inhibition of both M. intracellulare-induced nuclear factor-kappa B activation and A20 expression. While there was a modest increase in apoptosis in the M. intracellulare-infected post-AAT infused macrophages and plasma, inhibiting caspase-3 in THP-1 cells, monocyte-derived macrophages, and alveolar macrophages unexpectedly reduced the M. intracellulare burden, indicating that apoptosis impairs macrophage control of M. intracellulare and that the host protective effects of AAT occurred despite inducing apoptosis. Conclusion: AAT augments macrophage control of M. intracellulare infection through enhancing phagosome-lysosome fusion and autophagy.
Asunto(s)
Macrófagos Alveolares/inmunología , Complejo Mycobacterium avium/inmunología , Infección por Mycobacterium avium-intracellulare/inmunología , Deficiencia de alfa 1-Antitripsina/inmunología , alfa 1-Antitripsina/inmunología , Autofagia/inmunología , Bronquiectasia/etiología , Enfisema/etiología , Humanos , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/microbiología , Activación de Macrófagos/inmunología , Fagosomas/inmunología , Factor de Transcripción ReIA/metabolismo , Deficiencia de alfa 1-Antitripsina/patologíaRESUMEN
Although wasting marmoset syndrome (WMS) is one of the biggest problems facing captive marmoset colonies, the mechanisms underlying its pathogenesis remain unclear. In our clinical experience, it is difficult to cure WMS-affected marmosets with severe hypoalbuminemia. Thus, the mechanisms underlying hypoalbuminemia in WMS must be understood. In the present study, we investigated whether intestinal protein loss, a known reason for hypoalbuminemia, occurs in this disease. Fecal α1-proteinase inhibitor (α1-PI, also known as α1-antitrypsin) has been used to diagnose intestinal protein loss in other species. To develop an assay system for this protein, marmoset α1-PI was purified from plasma and antibodies against it were developed using the purified protein. Using the antibodies, a sandwich enzyme-linked immunosorbent assay (ELISA) to measure marmoset α1-PI was developed, and its detection sensitivity for fecal samples was â¼20-fold higher than that of a commercial kit for human α1-PI. From this ELISA, the reference intervals for serum and feces of healthy marmosets were 0.87-1.85 mg/ml and 0.53-395.58 µg/g, respectively. The average concentrations of α1-PI in serum and feces of seven WMS-affected marmosets were 1.17 mg/ml and 1357.58 µg/g, respectively. Although there were no significant differences in the serum concentrations between healthy and WMS-affected marmosets, the fecal concentrations were significantly higher in WMS-affected marmosets than in healthy individuals, suggesting that intestinal protein loss occurs in WMS. Intestinal protein loss of WMS-affected marmosets was significantly attenuated with treatment, suggesting that it is one of the mechanisms involved in the hypoalbuminemia observed in WMS.
Asunto(s)
Callithrix/sangre , Hipoalbuminemia/sangre , Síndrome Debilitante/sangre , alfa 1-Antitripsina/sangre , Animales , Anticuerpos/farmacología , Ensayo de Inmunoadsorción Enzimática , Heces/química , Humanos , Hipoalbuminemia/patología , Intestinos/patología , Síndrome Debilitante/tratamiento farmacológico , Síndrome Debilitante/patología , Síndrome Debilitante/veterinaria , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/inmunologíaRESUMEN
Background: Human α1-antitrypsin (hAAT) is a circulating anti-inflammatory serine-protease inhibitor that rises during acute phase responses. in vivo, hAAT reduces bacterial load, without directly inhibiting bacterial growth. In conditions of excess nitric-oxide (NO), hAAT undergoes S-nitrosylation (S-NO-hAAT) and gains antibacterial capacity. The impact of S-NO-hAAT on immune cells has yet to be explored. Aim: Study the effects of S-NO-hAAT on immune cells during bacterial infection. Methods: Clinical-grade hAAT was S-nitrosylated and then compared to unmodified hAAT, functionally, and structurally. Intracellular bacterial clearance by THP-1 macrophages was assessed using live Salmonella typhi. Murine peritoneal macrophages were examined, and signaling pathways were evaluated. S-NO-hAAT was also investigated after blocking free mambranal cysteine residues on cells. Results: S-NO-hAAT (27.5 uM) enhances intracellular bacteria elimination by immunocytes (up to 1-log reduction). S-NO-hAAT causes resting macrophages to exhibit a pro-inflammatory and antibacterial phenotype, including release of inflammatory cytokines and induction of inducible nitric oxide synthase (iNOS) and TLR2. These pro-inflammatory effects are dependent upon cell surface thiols and activation of MAPK pathways. Conclusions: hAAT duality appears to be context-specific, involving S-nitrosylation in a nitric oxide rich environment. Our results suggest that S-nitrosylation facilitates the antibacterial activity of hAAT by promoting its ability to activate innate immune cells. This pro-inflammatory effect may involve transferring of nitric oxide from S-NO-hAAT to a free cysteine residue on cellular targets.
Asunto(s)
Inmunidad Innata , Macrófagos Peritoneales/inmunología , Óxido Nítrico/inmunología , Salmonella typhi/inmunología , alfa 1-Antitripsina/inmunología , Animales , Femenino , Macrófagos Peritoneales/microbiología , Ratones , alfa 1-Antitripsina/genéticaRESUMEN
Alpha-1 antitrypsin (AAT) is an acute phase protein that possesses immune-regulatory and anti-inflammatory functions independent of antiprotease activity. AAT deficiency (AATD) is associated with early-onset emphysema and chronic obstructive pulmonary disease. Of interest are the AATD nonsense mutations (termed null or Q0), the majority of which arise from premature termination codons in the mRNA coding region. We have recently demonstrated that plasma from an AATD patient homozygous for the Null Bolton allele (Q0bolton ) contains AAT protein of truncated size. Although the potential to alleviate the phenotypic consequences of AATD by increasing levels of truncated protein holds therapeutic promise, protein functionality is key. The goal of this study was to evaluate the structural features and anti-inflammatory capacity of Q0bolton-AAT. A low-abundance, truncated AAT protein was confirmed in plasma of a Q0bolton-AATD patient and was secreted by patient-derived induced pluripotent stem cell-hepatic cells. Functional assays confirmed the ability of purified Q0bolton-AAT protein to bind neutrophil elastase and to inhibit protease activity. Q0bolton-AAT bound IL-8 and leukotriene B4, comparable to healthy control M-AAT, and significantly decreased leukotriene B4-induced neutrophil adhesion (p = 0.04). Through a mechanism involving increased mRNA stability (p = 0.007), ataluren treatment of HEK-293 significantly increased Q0bolton-AAT mRNA expression (p = 0.03) and Q0bolton-AAT truncated protein secretion (p = 0.04). Results support the rationale for treatment with pharmacological agents that augment levels of functional Q0bolton-AAT protein, thus offering a potential therapeutic option for AATD patients with rare mutations of similar theratype.
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Alelos , Codón sin Sentido , Deficiencia de alfa 1-Antitripsina , alfa 1-Antitripsina , Adulto , Femenino , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Hígado/inmunología , Hígado/metabolismo , Masculino , alfa 1-Antitripsina/sangre , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/inmunología , Deficiencia de alfa 1-Antitripsina/sangre , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/inmunologíaRESUMEN
Studies to identify novel immune-regulatory functions of active vitamin D (1,25(OH)2D3) in human CD4+ T cells revealed that 1,25(OH)2D3 potently induced expression of the gene SERPINA1, encoding the anti-protease α-1-antitrypsin. We confirmed α-1-antitrypsin protein expression by 1,25(OH)2D3-treated CD4+ T cells, but not in CD8+ T cells or monocytes. α-1-Antitrypsin promotes anti-inflammatory IL-10 synthesis in other immune cell populations. We therefore investigated its immune-regulatory effects in CD4+ T cells. Plasma-derived α-1-antitrypsin drove IL-10 synthesis by CD4+ T cells, which was not dependent on anti-protease activity, but appeared to require a serum-binding factor, since this could not be achieved with recombinant protein. α-1-Antitrypsin is reported to bind complement components, which regulate T cell function. A role for this interaction was therefore probed. Plasma-derived, but not recombinant α-1-antitrypsin contained C3a. Surface Plasmon Resonance and Microscale Thermophoresis demonstrated α-1-antitrypsin binding to C3a. Addition of C3a to CD4+ T cells cultured with recombinant α-1-antitrypsin restored induction of IL-10, whereas neutralisation of C3a abrogated IL-10 induced by plasma-derived α-1-antitrypsin. To interrogate an endogenous role for the α-1-antitrypsin-C3a axis in 1,25(OH)2D3-driven CD4+ T cell IL-10 synthesis, we treated cells from healthy or α-1-antitrypsin-deficient individuals (which transcribe SERPINA1 but do not secrete protein) with 1,25(OH)2D3. A significant correlation was identified between SERPINA1 and IL10 gene expression in healthy donor CD4+ T cells, which was absent in cells from α-1-antitrypsin-deficient individuals. Therefore, α-1-antitrypsin is required for 1,25(OH)2D3-induced IL-10 expression in CD4+ T cells, interacting with C3a to drive IL-10 expression.
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Linfocitos T CD4-Positivos/efectos de los fármacos , Calcitriol/farmacología , Interleucina-10/inmunología , Vitaminas/farmacología , alfa 1-Antitripsina/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Humanos , Factores Inmunológicos/farmacologíaRESUMEN
While interleukin (IL)-1ß is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1ß secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1ß is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1ß. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1ß regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1ß from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1ß release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1ß release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2ß, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1ß release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.
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Inflamasomas/inmunología , Interleucina-1beta/inmunología , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , alfa 1-Antitripsina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antígenos CD36/metabolismo , Humanos , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Leucocitos Mononucleares , Cultivo Primario de Células , Ratas , Receptores Purinérgicos P2X7/metabolismo , Células U937 , alfa 1-Antitripsina/inmunologíaRESUMEN
Introduction: Human α1-antitrypsin (hAAT) is a 394-amino acid long anti-inflammatory, neutrophil elastase inhibitor, which binds elastase via a sequence-specific molecular protrusion (reactive center loop, RCL; positions 357-366). hAAT formulations that lack protease inhibition were shown to maintain their anti-inflammatory activities, suggesting that some attributes of the molecule may reside in extra-RCL segments. Here, we compare the protease-inhibitory and anti-inflammatory profiles of an extra-RCL mutation (cys232pro) and two intra-RCL mutations (pro357cys, pro357ala), to naïve [wild-type (WT)] recombinant hAAT, in vitro, and in vivo. Methods: His-tag recombinant point-mutated hAAT constructs were expressed in HEK-293F cells. Purified proteins were evaluated for elastase inhibition, and their anti-inflammatory activities were assessed using several cell-types: RAW264.7 cells, mouse bone marrow-derived macrophages, and primary peritoneal macrophages. The pharmacokinetics of the recombinant variants and their effect on LPS-induced peritonitis were determined in vivo. Results: Compared to WT and to RCL-mutated hAAT variants, cys232pro exhibited superior anti-inflammatory activities, as well as a longer circulating half-life, despite all three mutated forms of hAAT lacking anti-elastase activity. TNFα expression and its proteolytic membranal shedding were differently affected by the variants; specifically, cys232pro and pro357cys altered supernatant and serum TNFα dynamics without suppressing transcription or shedding. Conclusion: Our data suggest that the anti-inflammatory profile of hAAT extends beyond direct RCL regions. Such regions might be relevant for the elaboration of hAAT formulations, as well as hAAT-based drugs, with enhanced anti-inflammatory attributes.
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alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/inmunología , Animales , Sitios de Unión , Células HEK293 , Humanos , Elastasa de Leucocito/inmunología , Elastasa de Leucocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Peritonitis , Mutación Puntual , Conformación Proteica , Células RAW 264.7RESUMEN
BACKGROUND: Human kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine proteases. KLK6 is synthesized as a preproenzyme, mainly in tissues of the central nervous system (CNS), and secreted as an inactive precursor. Serum KLK6 is a biomarker of unfavorable prognosis for ovarian cancer, but its sensitivity for early detection is relatively low. Differential glycosylation of KLK6 has been identified in ascites fluid obtained from ovarian cancer patients, suggesting the presence of unique KLK6 isoforms in biological samples. METHODS: In the present study, we applied a two-step enrichment approach for KLK6 in ovarian cancer ascites, followed by mice immunization and production of monoclonal antibodies. Immunoaffinity techniques coupled to mass spectrometric methods were employed for hybridoma screening and target antigen identification. RESULTS: We found that the main target of the newly-generated monoclonal antibodies target was the serine protease inhibitor α1-antitrypsin (A1AT). Additional experiments confirmed that A1AT is the main inhibitor of KLK6 in biological fluids. One new antibody (24ED138) was chosen to build a hybrid assay for the accurate quantification of the A1AT-KLK6 complex in biological samples. The aforementioned assay was evaluated with serum samples collected from patients with ovarian cancer (n=24) and normal donors (n=16) and showed slight improvement in sensitivity (~12%) compared to the standard in-house KLK6 assay. CONCLUSIONS: We conclude that KLK6 is present in biological fluids either as free form, or bound to A1AT, and the bound form performs better than total KLK6 as a biomarker of ovarian carcinoma.
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Líquidos Corporales/química , Calicreínas/sangre , Neoplasias Ováricas/diagnóstico , alfa 1-Antitripsina/sangre , Animales , Anticuerpos Monoclonales/inmunología , Biomarcadores/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Calicreínas/inmunología , Ratones , Ratones Endogámicos BALB C , Neoplasias Ováricas/sangre , Neoplasias Ováricas/inmunología , alfa 1-Antitripsina/inmunologíaRESUMEN
In 2011 a novel autoantibody system, anti-carbamylated protein (anti-CarP) antibodies, was described in rheumatoid arthritis (RA) patients. Anti-CarP antibody positivity associates with a more severe disease course, is observed years before disease onset, and may predict the development of RA in arthralgia patients. Although many clinical observations have been carried out, information on the antigenic targets of anti-CarP antibodies is limited. Most studies on anti-CarP antibodies utilize an ELISA-based assay with carbamylated fetal calf serum (Ca-FCS) as antigen, a complex mixture of proteins. Therefore, we analysed the molecular identity of proteins within Ca-FCS that are recognized by anti-CarP antibodies. Ca-FCS was fractionated using ion exchange chromatography, selecting one of the fractions for further investigation. Using mass-spectrometry, carbamylated alpha-1-antitrypsin (Ca-A1AT) was identified as a potential antigenic target of anti-CarP antibodies in RA patients. A1AT contains several lysines on the protein surface that can readily be carbamylated. A large proportion of the RA patients harbour antibodies that bind human Ca-A1AT in ELISA, indicating that Ca-A1AT is indeed an autoantigen for anti-CarP antibodies. Next to the Ca-A1AT protein, several homocitrulline-containing peptides of A1AT were recognized by RA sera. Moreover, we identified a carbamylated peptide of A1AT in the synovial fluid of an RA patient using mass spectrometry. We conclude that Ca-A1AT is not only a target of anti-CarP antibodies but is also present in the synovial compartment, suggesting that Ca-A1AT recognized by anti-CarP antibodies in the joint may contribute to synovial inflammation in anti-CarP-positive RA.
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Artralgia/inmunología , Artritis Reumatoide/inmunología , Autoantígenos/inmunología , Membrana Sinovial/inmunología , alfa 1-Antitripsina/inmunología , Autoanticuerpos/metabolismo , Autoantígenos/aislamiento & purificación , Cromatografía por Intercambio Iónico , Citrulina/análogos & derivados , Citrulina/inmunología , Citrulina/aislamiento & purificación , Biología Computacional , Ensayo de Inmunoadsorción Enzimática , Humanos , Espectrometría de Masas , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Conformación Proteica , Procesamiento Proteico-Postraduccional , alfa 1-Antitripsina/química , alfa 1-Antitripsina/aislamiento & purificaciónRESUMEN
Adeno-associated virus (AAV)-mediated gene therapy is currently being pursued as a treatment for the monogenic disorder α-1-antitrypsin (AAT) deficiency. Results from phase I and II studies have shown relatively stable and dose-dependent increases in transgene-derived wild-type AAT after local intramuscular vector administration. In this report we describe the appearance of transgene-specific T-cell responses in two subjects that were part of the phase II trial. The patient with the more robust T-cell response, which was associated with a reduction in transgene expression, was characterized more thoroughly in this study. We learned that the AAT-specific T cells in this patient were cytolytic in phenotype, mapped to a peptide in the endogenous mutant AAT protein that contained a common polymorphism not incorporated into the transgene, and were restricted by a rare HLA class I C alleles present only in this patient. These human studies illustrate the genetic influence of the endogenous gene and HLA haplotype on the outcome of gene therapy.