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1.
Nat Microbiol ; 2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38997518

RESUMO

Approved vaccines are effective against severe COVID-19, but broader immunity is needed against new variants and transmission. Therefore, we developed genome-modified live-attenuated vaccines (LAV) by recoding the SARS-CoV-2 genome, including 'one-to-stop' (OTS) codons, disabling Nsp1 translational repression and removing ORF6, 7ab and 8 to boost host immune responses, as well as the spike polybasic cleavage site to optimize the safety profile. The resulting OTS-modified SARS-CoV-2 LAVs, designated as OTS-206 and OTS-228, are genetically stable and can be intranasally administered, while being adjustable and sustainable regarding the level of attenuation. OTS-228 exhibits an optimal safety profile in preclinical animal models, with no side effects or detectable transmission. A single-dose vaccination induces a sterilizing immunity in vivo against homologous WT SARS-CoV-2 challenge infection and a broad protection against Omicron BA.2, BA.5 and XBB.1.5, with reduced transmission. Finally, this promising LAV approach could be applicable to other emerging viruses.

2.
JCI Insight ; 9(7)2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38470488

RESUMO

Studies on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) have highlighted the crucial role of host proteases for viral replication and the immune response. The serine proteases furin and TMPRSS2 and lysosomal cysteine proteases facilitate viral entry by limited proteolytic processing of the spike (S) protein. While neutrophils are recruited to the lungs during COVID-19 pneumonia, little is known about the role of the neutrophil serine proteases (NSPs) cathepsin G (CatG), elastase (NE), and proteinase 3 (PR3) on SARS-CoV-2 entry and replication. Furthermore, the current paradigm is that NSPs may contribute to the pathogenesis of severe COVID-19. Here, we show that these proteases cleaved the S protein at multiple sites and abrogated viral entry and replication in vitro. In mouse models, CatG significantly inhibited viral replication in the lung. Importantly, lung inflammation and pathology were increased in mice deficient in NE and/or CatG. These results reveal that NSPs contribute to innate defenses against SARS-CoV-2 infection via proteolytic inactivation of the S protein and that NE and CatG limit lung inflammation in vivo. We conclude that therapeutic interventions aiming to reduce the activity of NSPs may interfere with viral clearance and inflammation in COVID-19 patients.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Animais , Camundongos , SARS-CoV-2/metabolismo , Neutrófilos/metabolismo , Glicoproteína da Espícula de Coronavírus , Inflamação , Serina Proteases/metabolismo
3.
Sci Signal ; 16(769): eabm0517, 2023 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-36693132

RESUMO

Neutrophil extracellular traps (NETs) are DNA scaffolds coated with granule proteins that are released by neutrophils to ensnare and kill bacteria. NET formation occurs in response to many stimuli through independent molecular pathways. Although NET release has been equated to a form of lytic cell death, live neutrophils can rapidly release antimicrobial NETs. Gasdermin D (GSDMD), which causes pyroptotic death in macrophages, is thought to be required for NET formation by neutrophils. Through experiments with known physiological activators of NET formation and ligands that activate canonical and noncanonical inflammasome signaling pathways, we demonstrated that Gsdmd-deficient mouse neutrophils were as competent as wild-type mouse neutrophils in producing NETs. Furthermore, GSDMD was not cleaved in wild-type neutrophils during NET release in response to inflammatory mediators. We found that activation of both canonical and noncanonical inflammasome signaling pathways resulted in GSDMD cleavage in wild-type neutrophils but was not associated with cell death. Moreover, NET formation as a result of either pathway of inflammasome activation did not require GSDMD. Together, these data suggest that NETs can be formed by viable neutrophils after inflammasome activation and that this function does not require GSDMD.


Assuntos
Gasderminas , Piroptose , Camundongos , Animais , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inflamassomos/genética , Inflamassomos/metabolismo , Morte Celular , Neutrófilos/metabolismo
5.
Viruses ; 16(1)2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-38275939

RESUMO

The 2023 International African Swine Fever Workshop (IASFW) took place in Beijing, China, on 18-20 September 2023. It was jointly organized by the U.S.-China Center for Animal Health (USCCAH) at Kansas State University (KSU) and the Chinese Veterinary Drug Association (CVDA) and sponsored by the United States Department of Agriculture Foreign Agricultural Service (USDA-FAS), Harbin Veterinary Research Institute, and Zoetis Inc. The objective of this workshop was to provide a platform for ASF researchers around the world to unite and share their knowledge and expertise on ASF control and prevention. A total of 24 outstanding ASF research scientists and experts from 10 countries attended this meeting. The workshop included presentations on current ASF research, opportunities for scientific collaboration, and discussions of lessons and experiences learned from China/Asia, Africa, and Europe. This article summarizes the meeting highlights and presents some critical issues that need to be addressed for ASF control and prevention in the future.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Suínos , Animais , Humanos , Febre Suína Africana/prevenção & controle , Febre Suína Africana/epidemiologia , Ásia , China/epidemiologia , África/epidemiologia , Sus scrofa , Surtos de Doenças/veterinária
6.
Nat Commun ; 13(1): 5929, 2022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36207334

RESUMO

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Cricetinae , Furões , Humanos , Melfalan , Camundongos , Fenótipo , RNA Mensageiro , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , gama-Globulinas
7.
PLoS Pathog ; 18(8): e1010522, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-36006954

RESUMO

African Swine Fever virus (ASFV) is a large double-enveloped DNA virus of the Asfarviridae family that causes a lethal hemorrhagic disease in domestic pigs and wild boars. Since 2007, a highly virulent genotype II strain has emerged and spread in Europe and South-East Asia, where millions of animals succumbed to the disease. Field- and laboratory-attenuated strains of ASFV cause highly variable clinical disease severity and survival, and mechanisms remain unclear. We hypothesized that the immunological and hygienic status of pigs is a determinant of ASF disease course. Here we compared the immunological profile at baseline and in response to ASFV infection in specific pathogen-free (SPF) and farm-raised Large White domestic pigs. At steady state, SPF pigs showed lower white blood cell counts and a lower basal inflammatory and antiviral transcriptomic profile compared to farm pigs, associated with profound differences in gut microbiome composition. After inoculation with a highly virulent ASFV genotype II strain (Armenia 2008), severe clinical signs, viremia and pro-inflammatory cytokines appeared sooner in SPF pigs, indicating a reduced capacity to control early virus replication. In contrast, during infection with an attenuated field isolate (Estonia 2014), SPF pigs presented a milder and shorter clinical disease with full recovery, whereas farm pigs presented severe protracted disease with 50% lethality. Interestingly, farm pigs showed higher production of inflammatory cytokines, whereas SPF pigs produced more anti-inflammatory IL-1ra early after infection and presented a stronger expansion of leukocytes in the recovery phase. Altogether, our data indicate that the hygiene-dependent innate immune status has a double-edge sword impact on immune responses in ASF pathogenesis. While the higher baseline innate immune activity helps the host in reducing initial virus replication, it promotes immunopathological cytokine responses, and delays lymphocyte proliferation after infection with an attenuated strain. Such effects should be considered for live vaccine development and vigilance.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Vírus da Febre Suína Africana/genética , Animais , Citocinas , Higiene , Índice de Gravidade de Doença , Sus scrofa , Suínos
8.
NPJ Vaccines ; 7(1): 82, 2022 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-35879345

RESUMO

Immunization with vesicular stomatitis virus (VSV)-vectored COVID-19 vaccine candidates expressing the SARS-CoV-2 spike protein in place of the VSV glycoprotein relies implicitly on expression of the ACE2 receptor at the muscular injection site. Here, we report that such a viral vector vaccine did not induce protective immunity following intramuscular immunization of K18-hACE2 transgenic mice. However, when the viral vector was trans-complemented with the VSV glycoprotein, intramuscular immunization resulted in high titers of spike-specific neutralizing antibodies. The vaccinated animals were fully protected following infection with a lethal dose of SARS-CoV-2-SD614G via the nasal route, and partially protected if challenged with the SARS-CoV-2Delta variant. While dissemination of the challenge virus to the brain was completely inhibited, replication in the lung with consequent lung pathology was not entirely controlled. Thus, intramuscular immunization was clearly enhanced by trans-complementation of the VSV-vectored vaccines by the VSV glycoprotein and led to protection from COVID-19, although not achieving sterilizing immunity.

9.
Nature ; 602(7896): 307-313, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34937050

RESUMO

Emerging variants of concern (VOCs) are driving the COVID-19 pandemic1,2. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3. Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models-the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals.


Assuntos
COVID-19/transmissão , COVID-19/virologia , Mutação , SARS-CoV-2/classificação , SARS-CoV-2/fisiologia , Replicação Viral , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Animais de Laboratório/virologia , COVID-19/veterinária , Cricetinae , Modelos Animais de Doenças , Células Epiteliais/virologia , Feminino , Furões/virologia , Humanos , Masculino , Mesocricetus/virologia , Camundongos , Camundongos Transgênicos , SARS-CoV-2/genética , SARS-CoV-2/crescimento & desenvolvimento , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Virulência/genética
10.
Front Cell Dev Biol ; 9: 630166, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34858967

RESUMO

Mast cells are multifunctional immune cells scattered in tissues near blood vessels and mucosal surfaces where they mediate important reactions against parasites and contribute to the pathogenesis of allergic reactions. Serine proteases released from secretory granules upon mast cell activation contribute to these functions by modulating cytokine activity, platelet activation and proteolytic neutralization of toxins. The forced release of granule proteases into the cytosol of mast cells to induce cell suicide has recently been proposed as a therapeutic approach to reduce mast cell numbers in allergic diseases, but the molecular pathways involved in granule-mediated mast cell suicide are incompletely defined. To identify intrinsic granule proteases that can cause mast cell death, we used mice deficient in cytosolic serine protease inhibitors and their respective target proteases. We found that deficiency in Serpinb1a, Serpinb6a, and Serpinb9a or in their target proteases did not alter the kinetics of apoptosis induced by growth factor deprivation in vitro or the number of peritoneal mast cells in vivo. The serine protease cathepsin G induced marginal cell death upon mast cell granule permeabilization only when its inhibitors Serpinb1a or Serpinb6a were deleted. In contrast, the serine protease granzyme B was essential for driving apoptosis in mast cells. On granule permeabilization, granzyme B was required for caspase-3 processing and cell death. Moreover, cytosolic granzyme B inhibitor Serpinb9a prevented caspase-3 processing and mast cell death in a granzyme B-dependent manner. Together, our findings demonstrate that cytosolic serpins provide an inhibitory shield preventing granule protease-induced mast cell apoptosis, and that the granzyme B-Serpinb9a-caspase-3 axis is critical in mast cell survival and could be targeted in the context of allergic diseases.

11.
PLoS Pathog ; 17(4): e1009529, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33909707

RESUMO

The human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, possibly due to the properties of the immature neonatal pulmonary immune system. Using the newborn lamb, a classical model of human lung development and a translational model of RSV infection, we aimed to explore the role of cell-mediated immunity in RSV disease during early life. Remarkably, in healthy conditions, the developing T cell compartment of the neonatal lung showed major differences to that seen in the mature adult lung. The most striking observation being a high baseline frequency of bronchoalveolar IL-4-producing CD4+ and CD8+ T cells, which declined progressively over developmental age. RSV infection exacerbated this pro-type 2 environment in the bronchoalveolar space, rather than inducing a type 2 response per se. Moreover, regulatory T cell suppressive functions occurred very early to dampen this pro-type 2 environment, rather than shutting them down afterwards, while γδ T cells dropped and failed to produce IL-17. Importantly, RSV disease severity was related to the magnitude of those unconventional bronchoalveolar T cell responses. These findings provide novel insights in the mechanisms of RSV immunopathogenesis in early life, and constitute a major step for the understanding of RSV disease severity.


Assuntos
Pulmão/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções Respiratórias/imunologia , Linfócitos T/patologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/imunologia , Células Cultivadas , Pré-Escolar , Modelos Animais de Doenças , Progressão da Doença , Humanos , Pulmão/crescimento & desenvolvimento , Pulmão/patologia , Pulmão/virologia , Infecções por Vírus Respiratório Sincicial/congênito , Infecções por Vírus Respiratório Sincicial/patologia , Infecções Respiratórias/congênito , Infecções Respiratórias/patologia , Ovinos/crescimento & desenvolvimento , Ovinos/imunologia , Linfócitos T/imunologia , Linfócitos T/fisiologia
12.
Blood ; 137(21): 2958-2969, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33598715

RESUMO

Eosinophils are white blood cells that contribute to the regulation of immunity and are involved in the pathogenesis of numerous inflammatory diseases. In contrast to other cells of the immune system, no information is available regarding the role of autophagy in eosinophil differentiation and functions. To study the autophagic pathway in eosinophils, we generated conditional knockout mice in which Atg5 is deleted within the eosinophil lineage only (designated Atg5eoΔ mice). Eosinophilia was provoked by crossbreeding Atg5eoΔ mice with Il5 (IL-5) overexpressing transgenic mice (designated Atg5eoΔIl5tg mice). Deletion of Atg5 in eosinophils resulted in a dramatic reduction in the number of mature eosinophils in blood and an increase of immature eosinophils in the bone marrow. Atg5-knockout eosinophil precursors exhibited reduced proliferation under both in vitro and in vivo conditions but no increased cell death. Moreover, reduced differentiation of eosinophils in the absence of Atg5 was also observed in mouse and human models of chronic eosinophilic leukemia. Atg5-knockout blood eosinophils exhibited augmented levels of degranulation and bacterial killing in vitro. Moreover, in an experimental in vivo model, we observed that Atg5eoΔ mice achieve better clearance of the local and systemic bacterial infection with Citrobacter rodentium. Evidence for increased degranulation of ATG5low-expressing human eosinophils was also obtained in both tissues and blood. Taken together, mouse and human eosinophil hematopoiesis and effector functions are regulated by ATG5, which controls the amplitude of overall antibacterial eosinophil immune responses.


Assuntos
Proteína 5 Relacionada à Autofagia/fisiologia , Eosinófilos/fisiologia , Mielopoese/fisiologia , Animais , Proteína 5 Relacionada à Autofagia/biossíntese , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Medula Óssea/patologia , Sistemas CRISPR-Cas , Degranulação Celular , Linhagem Celular Tumoral , Células Cultivadas , Citrobacter rodentium , Ensaio de Unidades Formadoras de Colônias , Infecções por Enterobacteriaceae/imunologia , Eosinófilos/citologia , Eosinófilos/imunologia , Humanos , Síndrome Hipereosinofílica/sangue , Síndrome Hipereosinofílica/patologia , Interleucina-5/genética , Contagem de Leucócitos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Fusão Oncogênica/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética
13.
Nature ; 592(7852): 122-127, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33636719

RESUMO

During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.


Assuntos
COVID-19/transmissão , COVID-19/virologia , Mutação , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Replicação Viral/genética , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Brônquios/citologia , Brônquios/virologia , COVID-19/epidemiologia , Linhagem Celular , Células Cultivadas , Cricetinae , Modelos Animais de Doenças , Células Epiteliais/virologia , Feminino , Furões/virologia , Efeito Fundador , Técnicas de Introdução de Genes , Aptidão Genética , Humanos , Masculino , Mesocricetus , Camundongos , Mucosa Nasal/citologia , Mucosa Nasal/virologia , Ligação Proteica , RNA Viral/análise , Receptores de Coronavírus/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade
14.
Front Immunol ; 11: 562587, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33262755

RESUMO

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes. They quickly respond to antigenic stimulation by producing copious amounts of cytokines and chemokines. iNKT precursors differentiate into three subsets iNKT1, iNKT2, and iNKT17 with specific cytokine production signatures. While key transcription factors drive subset differentiation, factors that regulate iNKT subset homeostasis remain incompletely defined. Transcriptomic analyses of thymic iNKT subsets indicate that Serpinb1a is one of the most specific transcripts for iNKT17 cells suggesting that iNKT cell maintenance and function may be regulated by Serpinb1a. Serpinb1a is a major survival factor in neutrophils and prevents cell death in a cell-autonomous manner. It also controls inflammation in models of bacterial and viral infection as well as in LPS-driven inflammation. Here, we examined the iNKT subsets in neutropenic Serpinb1a-/- mice as well as in Serpinb1a-/- mice with normal neutrophil counts due to transgenic re-expression of SERPINB1 in neutrophils. In steady state, we found no significant effect of Serpinb1a-deficiency on the proliferation and numbers of iNKT subsets in thymus, lymph nodes, lung, liver and spleen. Following systemic activation with α-galactosylceramide, the prototypic glycolipid agonist of iNKT cells, we observed similar serum levels of IFN-γ and IL-4 between genotypes. Moreover, splenic dendritic cells showed normal upregulation of maturation markers following iNKT cell activation with α-galactosylceramide. Finally, lung instillation of α-galactosylceramide induced a similar recruitment of neutrophils and production of iNKT-derived cytokines IL-17, IFN-γ, and IL-4 in wild-type and Serpinb1a-/- mice. Taken together, our results indicate that Serpinb1a, while dominantly expressed in iNKT17 cells, is not essential for iNKT cell homeostasis, subset differentiation and cytokine release.


Assuntos
Diferenciação Celular/genética , Citocinas/metabolismo , Ativação Linfocitária/genética , Células T Matadoras Naturais/imunologia , Serpinas/deficiência , Transdução de Sinais/genética , Animais , Células Dendríticas/imunologia , Feminino , Galactosilceramidas/efeitos adversos , Homeostase/genética , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/metabolismo , Fígado/imunologia , Pulmão/imunologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Neutrófilos/imunologia , Serpinas/genética , Transdução de Sinais/efeitos dos fármacos , Baço/imunologia
15.
Microb Genom ; 6(12)2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33295863

RESUMO

Environmental factors, such as cigarette smoking or lung infections, may influence chronic obstructive pulmonary disease (COPD) progression by modifying the respiratory tract microbiome. However, whether the disease itself induces or maintains dysbiosis remains undefined. In this longitudinal study, we investigated the oropharyngeal microbiota composition and disease progression of mice (in cages of 5-10 mice per cage) before, during and up to 3 months after chronic cigarette smoke exposure or exposure to room air for 6 months. Cigarette smoke exposure induced pulmonary emphysema measurable at the end of exposure for 6 months, as well as 3 months following smoke exposure cessation. Using both classical culture methods and 16S rRNA sequencing, we observed that cigarette smoke exposure altered the relative composition of the oropharyngeal microbiota and reduced its diversity (P <0.001). More than 60 taxa were substantially reduced after 6 months of smoke exposure (P <0.001) However, oropharyngeal microbiota disordering was reversed 3 months after smoke exposure cessation and no significant difference was observed compared to age-matched control mice. The effects of lung infection with Streptococcus pneumoniae on established smoke-induced emphysema and on the oropharyngeal microbiota were also evaluated. Inoculation with S. pneumoniae induced lung damage and altered the microbiota composition for a longer time compared to control groups infected but not previously exposed to smoke (P=0.01). Our data demonstrate effects of cigarette smoke and pneumococcus infection leading to altered microbiota and emphysema development. The reversal of the disordering of the microbiota composition, but not lung damage, following smoke exposure cessation and after clearance of infection suggest that changes in lung structure are not sufficient to sustain a disordered microbiota in mice. Whether changes in the airway microbiota contribute to inducing emphysema requires further investigation.


Assuntos
Bactérias/classificação , Disbiose/etiologia , Orofaringe/microbiologia , Infecções Pneumocócicas/microbiologia , Enfisema Pulmonar/genética , RNA Ribossômico 16S/genética , Fumaça/efeitos adversos , Animais , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/isolamento & purificação , DNA Ribossômico/genética , Modelos Animais de Doenças , Progressão da Doença , Disbiose/induzido quimicamente , Disbiose/complicações , Disbiose/microbiologia , Feminino , Estudos Longitudinais , Camundongos , Filogenia , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/microbiologia , RNA Bacteriano/genética , Análise de Sequência de DNA/métodos , Produtos do Tabaco/efeitos adversos
16.
bioRxiv ; 2020 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-33140052

RESUMO

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic 1 . However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro , it provides a real competitive advantage in vivo , particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

18.
J Exp Med ; 216(11): 2669-2687, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31492810

RESUMO

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology (PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)-induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy.


Assuntos
Citosol/metabolismo , NADPH Oxidase 2/metabolismo , NADPH Oxidases/metabolismo , Neutrófilos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Colite/induzido quimicamente , Colite/prevenção & controle , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/genética , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ácido Trinitrobenzenossulfônico
19.
Cell Rep ; 27(12): 3646-3656.e5, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216481

RESUMO

Neutrophil granule serine proteases contribute to immune responses through cleavage of microbial toxins and structural proteins. They induce tissue damage and modulate inflammation if levels exceed their inhibitors. Here, we show that the intracellular protease inhibitors Serpinb1a and Serpinb6a contribute to monocyte and neutrophil survival in steady-state and inflammatory settings by inhibiting cathepsin G (CatG). Importantly, we found that CatG efficiently cleaved gasdermin D (GSDMD) to generate the signature N-terminal domain GSDMD-p30 known to induce pyroptosis. Yet GSDMD deletion did not rescue neutrophil survival in Sb1a.Sb6a-/- mice. Furthermore, Sb1a.Sb6a-/- mice released high levels of pro-inflammatory cytokines upon endotoxin challenge in vivo in a CatG-dependent manner. Canonical inflammasome activation in Sb1a.Sb6a-/- macrophages showed increased IL-1ß release that was dependent on CatG and GSDMD. Together, our findings demonstrate that cytosolic serpins expressed in myeloid cells prevent cell death and regulate inflammatory responses by inhibiting CatG and alternative activation of GSDMD.


Assuntos
Catepsina G/antagonistas & inibidores , Inflamação/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Monócitos/patologia , Neutrófilos/patologia , Proteínas de Ligação a Fosfato/metabolismo , Serpinas/fisiologia , Animais , Apoptose , Endotoxinas/toxicidade , Feminino , Inflamassomos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Necrose , Neutrófilos/metabolismo , Proteínas de Ligação a Fosfato/genética , Piroptose
20.
Int J Mol Sci ; 20(6)2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30901861

RESUMO

Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS) in young adults leading to severe disability. Besides genetic traits, environmental factors contribute to MS pathogenesis. Cigarette smoking increases the risk of MS in an HLA-dependent fashion, but the underlying mechanisms remain unknown. Here, we explored the effect of cigarette smoke exposure on spontaneous and induced models of experimental autoimmune encephalomyelitis (EAE) by evaluating clinical disease and, when relevant, blood leukocytes and histopathology. In the relapsing-remitting (RR) transgenic model in SJL/J mice, we observed very low incidence in both smoke-exposed and control groups. In the optico-spinal encephalomyelitis (OSE) double transgenic model in C57BL/6 mice, the early onset of EAE prevented a meaningful evaluation of the effects of cigarette smoke. In EAE models induced by immunization, daily exposure to cigarette smoke caused a delayed onset of EAE followed by a protracted disease course in SJL/J mice. In contrast, cigarette smoke exposure ameliorated the EAE clinical score in C57BL/6J mice. Our exploratory studies therefore show that genetic background influences the effects of cigarette smoke on autoimmune neuroinflammation. Importantly, our findings expose the challenge of identifying an animal model for studying the influence of cigarette smoke in MS.


Assuntos
Encefalomielite Autoimune Experimental/diagnóstico , Encefalomielite Autoimune Experimental/etiologia , Patrimônio Genético , Fumar/efeitos adversos , Idade de Início , Animais , Biópsia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/metabolismo , Imuno-Histoquímica , Camundongos , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/etiologia , Esclerose Múltipla/metabolismo , Fenótipo , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença , Medula Espinal/metabolismo , Medula Espinal/patologia
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