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1.
Nat Immunol ; 23(1): 23-32, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34937933

RESUMO

Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates1-6. Here, we characterized nasal and systemic immune cells in individuals with COVID-19 who were hospitalized or convalescent and compared the immune cells to those seen in healthy donors. We observed increased nasal granulocytes, monocytes, CD11c+ natural killer (NK) cells and CD4+ T effector cells during acute COVID-19. The mucosal proinflammatory populations positively associated with peripheral blood human leukocyte antigen (HLA)-DRlow monocytes, CD38+PD1+CD4+ T effector (Teff) cells and plasmablasts. However, there was no general lymphopenia in nasal mucosa, unlike in peripheral blood. Moreover, nasal neutrophils negatively associated with oxygen saturation levels in blood. Following convalescence, nasal immune cells mostly normalized, except for CD127+ granulocytes and CD38+CD8+ tissue-resident memory T cells (TRM). SARS-CoV-2-specific CD8+ T cells persisted at least 2 months after viral clearance in the nasal mucosa, indicating that COVID-19 has both transient and long-term effects on upper respiratory tract immune responses.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Nasofaringe/imunologia , Nariz/citologia , Mucosa Respiratória/imunologia , SARS-CoV-2/imunologia , Anticorpos Antivirais/sangue , COVID-19/imunologia , COVID-19/patologia , Granulócitos/imunologia , Antígenos HLA-DR/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Células T de Memória/imunologia , Monócitos/imunologia , Nasofaringe/citologia , Nasofaringe/virologia , Neutrófilos/imunologia , Nariz/imunologia , Nariz/virologia , Estudos Prospectivos , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia
2.
Cell ; 175(6): 1634-1650.e17, 2018 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-30433869

RESUMO

Innate immune memory is an emerging area of research. However, innate immune memory at major mucosal sites remains poorly understood. Here, we show that respiratory viral infection induces long-lasting memory alveolar macrophages (AMs). Memory AMs are programed to express high MHC II, a defense-ready gene signature, and increased glycolytic metabolism, and produce, upon re-stimulation, neutrophil chemokines. Using a multitude of approaches, we reveal that the priming, but not maintenance, of memory AMs requires the help from effector CD8 T cells. T cells jump-start this process via IFN-γ production. We further find that formation and maintenance of memory AMs are independent of monocytes or bone marrow progenitors. Finally, we demonstrate that memory AMs are poised for robust trained immunity against bacterial infection in the lung via rapid induction of chemokines and neutrophilia. Our study thus establishes a new paradigm of immunological memory formation whereby adaptive T-lymphocytes render innate memory of mucosal-associated macrophages.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunidade Inata , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Memória Imunológica , Pulmão/citologia , Macrófagos Alveolares/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Monócitos/citologia , Monócitos/imunologia , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Linfócitos T Auxiliares-Indutores/citologia
3.
Nature ; 629(8013): 869-877, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38693267

RESUMO

Airway hillocks are stratified epithelial structures of unknown function1. Hillocks persist for months and have a unique population of basal stem cells that express genes associated with barrier function and cell adhesion. Hillock basal stem cells continually replenish overlying squamous barrier cells. They exhibit dramatically higher turnover than the abundant, largely quiescent classic pseudostratified airway epithelium. Hillocks resist a remarkably broad spectrum of injuries, including toxins, infection, acid and physical injury because hillock squamous cells shield underlying hillock basal stem cells from injury. Hillock basal stem cells are capable of massive clonal expansion that is sufficient to resurface denuded airway, and eventually regenerate normal airway epithelium with each of its six component cell types. Hillock basal stem cells preferentially stratify and keratinize in the setting of retinoic acid signalling inhibition, a known cause of squamous metaplasia2,3. Here we show that mouse hillock expansion is the cause of vitamin A deficiency-induced squamous metaplasia. Finally, we identify human hillocks whose basal stem cells generate functional squamous barrier structures in culture. The existence of hillocks reframes our understanding of airway epithelial regeneration. Furthermore, we show that hillocks are one origin of 'squamous metaplasia', which is long thought to be a precursor of lung cancer.


Assuntos
Plasticidade Celular , Células Epiteliais , Regeneração , Mucosa Respiratória , Células-Tronco , Animais , Feminino , Humanos , Masculino , Camundongos , Células Epiteliais/citologia , Células Epiteliais/patologia , Metaplasia/etiologia , Metaplasia/patologia , Mucosa Respiratória/citologia , Mucosa Respiratória/lesões , Mucosa Respiratória/patologia , Células-Tronco/citologia , Tretinoína/metabolismo , Tretinoína/farmacologia , Vitamina A/metabolismo , Vitamina A/farmacologia , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos C57BL
4.
Nat Immunol ; 17(4): 441-50, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26855029

RESUMO

Epithelial tissues continually undergo apoptosis. Commensal organisms that inhabit the epithelium influence tissue homeostasis, in which regulatory T cells (Treg cells) have a central role. However, the physiological importance of epithelial cell apoptosis and how the number of Treg cells is regulated are both incompletely understood. Here we found that apoptotic epithelial cells negatively regulated the commensal-stimulated proliferation of Treg cells. Gut commensals stimulated CX3CR1(+)CD103(-)CD11b(+) dendritic cells (DCs) to produce interferon-ß (IFN-ß), which augmented the proliferation of Treg cells in the intestine. Conversely, phosphatidylserine exposed on apoptotic epithelial cells suppressed IFN-ß production by the DCs via inhibitory signaling mediated by the cell-surface glycoprotein CD300a and thus suppressed Treg cell proliferation. Our findings reveal a regulatory role for apoptotic epithelial cells in maintaining the number of Treg cell and tissue homeostasis.


Assuntos
Apoptose/imunologia , Epiderme/imunologia , Células Epiteliais/imunologia , Microbioma Gastrointestinal/imunologia , Interferon beta/imunologia , Mucosa Intestinal/imunologia , Mucosa Respiratória/imunologia , Linfócitos T Reguladores/imunologia , Alérgenos/toxicidade , Animais , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Colo/citologia , Colo/imunologia , Células Dendríticas/imunologia , Dermatite Alérgica de Contato/etiologia , Dermatite Alérgica de Contato/imunologia , Dermatite Alérgica de Contato/patologia , Sulfato de Dextrana/toxicidade , Células Epidérmicas , Citometria de Fluxo , Imuno-Histoquímica , Mucosa Intestinal/citologia , Células de Langerhans/imunologia , Pulmão/citologia , Pulmão/imunologia , Camundongos , Camundongos Knockout , Ovalbumina/toxicidade , Reação em Cadeia da Polimerase em Tempo Real , Receptores Imunológicos/genética , Mucosa Respiratória/citologia , Infecções por Salmonella/imunologia , Salmonella typhimurium
5.
Genes Dev ; 34(15-16): 1017-1032, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32747478

RESUMO

As one of the most common forms of cancer, lung cancers present as a collection of different histological subtypes. These subtypes are characterized by distinct sets of driver mutations and phenotypic appearance, and they often show varying degrees of heterogenicity, aggressiveness, and response/resistance to therapy. Intriguingly, lung cancers are also capable of showing features of multiple subtypes or converting from one subtype to another. The intertumoral and intratumoral heterogeneity of lung cancers as well as incidences of subtype transdifferentiation raise the question of to what extent the tumor characteristics are dictated by the cell of origin rather than the acquired driver lesions. We provide here an overview of the studies in experimental mouse models that try to address this question. These studies convincingly show that both the cell of origin and the genetic driver lesions play a critical role in shaping the phenotypes of lung tumors. However, they also illustrate that there is far from a direct one-to-one relationship between the cell of origin and the cancer subtype, as most epithelial cells can be reprogrammed toward diverse lung cancer fates when exposed to the appropriate set of driver mutations.


Assuntos
Neoplasias Pulmonares/etiologia , Adenocarcinoma/etiologia , Animais , Carcinoma de Células Escamosas/etiologia , Modelos Animais de Doenças , Células Epiteliais , Neoplasias Pulmonares/genética , Camundongos , Mucosa Respiratória/citologia , Carcinoma de Pequenas Células do Pulmão/etiologia
6.
Nat Immunol ; 16(1): 27-35, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25521682

RESUMO

The epithelial surfaces of the lungs are in direct contact with the environment and are subjected to dynamic physical forces as airway tubes and alveoli are stretched and compressed during ventilation. Mucociliary clearance in conducting airways, reduction of surface tension in the alveoli, and maintenance of near sterility have been accommodated by the evolution of a multi-tiered innate host-defense system. The biophysical nature of pulmonary host defenses are integrated with the ability of respiratory epithelial cells to respond to and 'instruct' the professional immune system to protect the lungs from infection and injury.


Assuntos
Imunidade Inata/imunologia , Pulmão/imunologia , Mucosa Respiratória/imunologia , Animais , Homeostase/imunologia , Humanos , Pulmão/citologia , Mucinas/imunologia , Mucosa Respiratória/citologia , Transdução de Sinais/imunologia
7.
Nat Immunol ; 16(1): 36-44, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25521683

RESUMO

Gas exchange is the vital function of the lungs. It occurs in the alveoli, where oxygen and carbon dioxide diffuse across the alveolar epithelium and the capillary endothelium surrounding the alveoli, separated only by a fused basement membrane 0.2-0.5 µm in thickness. This tenuous barrier is exposed to dangerous or innocuous particles, toxins, allergens and infectious agents inhaled with the air or carried in the blood. The lung immune system has evolved to ward off pathogens and restrain inflammation-mediated damage to maintain gas exchange. Lung-resident macrophages and dendritic cells are located in close proximity to the epithelial surface of the respiratory system and the capillaries to sample and examine the air-borne and blood-borne material. In communication with alveolar epithelial cells, they set the threshold and the quality of the immune response.


Assuntos
Células Dendríticas/imunologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Alvéolos Pulmonares/imunologia , Mucosa Respiratória/imunologia , Animais , Citocinas/imunologia , Células Dendríticas/citologia , Humanos , Pulmão/citologia , Macrófagos Alveolares/citologia , Alvéolos Pulmonares/citologia , Mucosa Respiratória/citologia , Infecções Respiratórias/imunologia
8.
Immunity ; 49(2): 275-287.e5, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30054206

RESUMO

Airway epithelial cells (AECs) secrete innate immune cytokines that regulate adaptive immune effector cells. In allergen-sensitized humans and mice, the airway and alveolar microenvironment is enriched with colony stimulating factor-1 (CSF1) in response to allergen exposure. In this study we found that AEC-derived CSF1 had a critical role in the production of allergen reactive-IgE production. Furthermore, spatiotemporally secreted CSF1 regulated the recruitment of alveolar dendritic cells (DCs) and enhanced the migration of conventional DC2s (cDC2s) to the draining lymph node in an interferon regulatory factor 4 (IRF4)-dependent manner. CSF1 selectively upregulated the expression of the chemokine receptor CCR7 on the CSF1R+ cDC2, but not the cDC1, population in response to allergen stimuli. Our data describe the functional specification of CSF1-dependent DC subsets that link the innate and adaptive immune responses in T helper 2 (Th2) cell-mediated allergic lung inflammation.


Assuntos
Alérgenos/imunologia , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Macrófagos/imunologia , Receptores CCR7/biossíntese , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Animais , Linhagem Celular , Movimento Celular/imunologia , Células Dendríticas/classificação , Células Epiteliais/citologia , Células Epiteliais/imunologia , Humanos , Imunidade Inata/imunologia , Imunoglobulina E/imunologia , Fatores Reguladores de Interferon/imunologia , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células RAW 264.7 , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Células Th2/imunologia , Regulação para Cima/imunologia
9.
Proc Natl Acad Sci U S A ; 121(31): e2320303121, 2024 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-39008691

RESUMO

Influenza viruses pose a significant burden on global human health. Influenza has a broad cellular tropism in the airway, but how infection of different epithelial cell types impacts replication kinetics and burden in the airways is not fully understood. Using primary human airway cultures, which recapitulate the diverse epithelial cell landscape of the human airways, we investigated the impact of cell type composition on virus tropism and replication kinetics. Cultures were highly diverse across multiple donors and 30 independent differentiation conditions and supported a range of influenza replication. Although many cell types were susceptible to influenza, ciliated and secretory cells were predominantly infected. Despite the strong tropism preference for secretory and ciliated cells, which consistently make up 75% or more of infected cells, only ciliated cells were associated with increased virus production. Surprisingly, infected secretory cells were associated with overall reduced virus output. The disparate response and contribution to influenza virus production could be due to different pro- and antiviral interferon-stimulated gene signatures between ciliated and secretory populations, which were interrogated with single-cell RNA sequencing. These data highlight the heterogeneous outcomes of influenza virus infections in the complex cellular environment of the human airway and the disparate impacts of infected cell identity on multiround burst size, even among preferentially infected cell types.


Assuntos
Células Epiteliais , Influenza Humana , Tropismo Viral , Replicação Viral , Humanos , Influenza Humana/virologia , Replicação Viral/fisiologia , Células Epiteliais/virologia , Células Epiteliais/metabolismo , Cílios/virologia , Cílios/metabolismo , Células Cultivadas , Mucosa Respiratória/virologia , Mucosa Respiratória/citologia
10.
Nature ; 578(7794): 266-272, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31996850

RESUMO

Tobacco smoking causes lung cancer1-3, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the genome of lung cancer cells are well-documented6-10, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0-6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.


Assuntos
Brônquios/metabolismo , Mutagênese , Mutação/genética , Mucosa Respiratória/metabolismo , Fumar Tabaco/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Brônquios/citologia , Brônquios/patologia , Criança , Células Clonais/citologia , Células Clonais/metabolismo , Análise Mutacional de DNA , Feminino , Humanos , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , Fumantes , Telômero/genética , Telômero/metabolismo , Fumar Tabaco/efeitos adversos , Fumar Tabaco/patologia , Adulto Jovem
11.
Nature ; 585(7826): 584-587, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32698191

RESUMO

Coronavirus disease 2019 (COVID-19) has rapidly become a global pandemic and no antiviral drug or vaccine is yet available for the treatment of this disease1-3. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro. Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes COVID-19-worldwide but there is no definitive evidence that HCQ is effective for treating COVID-194-7. Here we evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in African green monkey kidney cells (Vero E6) but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to a placebo treatment, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor the combination of HCQ and AZTH showed a significant effect on viral load in any of the analysed tissues. When the drug was used as a pre-exposure prophylaxis treatment, HCQ did not confer protection against infection with SARS-CoV-2. Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral drug for the treatment of COVID-19 in humans.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Hidroxicloroquina/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Animais , Azitromicina/farmacologia , Azitromicina/uso terapêutico , COVID-19 , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/fisiopatologia , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Humanos , Hidroxicloroquina/farmacocinética , Hidroxicloroquina/farmacologia , Técnicas In Vitro , Cinética , Macaca fascicularis , Masculino , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/fisiopatologia , Profilaxia Pré-Exposição , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/virologia , SARS-CoV-2 , Fatores de Tempo , Falha de Tratamento , Células Vero , Carga Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19
12.
EMBO J ; 40(15): e107826, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34101213

RESUMO

SARS-CoV-2 infection causes broad-spectrum immunopathological disease, exacerbated by inflammatory co-morbidities. A better understanding of mechanisms underpinning virus-associated inflammation is required to develop effective therapeutics. Here, we discover that SARS-CoV-2 replicates rapidly in lung epithelial cells despite triggering a robust innate immune response through the activation of cytoplasmic RNA sensors RIG-I and MDA5. The inflammatory mediators produced during epithelial cell infection can stimulate primary human macrophages to enhance cytokine production and drive cellular activation. Critically, this can be limited by abrogating RNA sensing or by inhibiting downstream signalling pathways. SARS-CoV-2 further exacerbates the local inflammatory environment when macrophages or epithelial cells are primed with exogenous inflammatory stimuli. We propose that RNA sensing of SARS-CoV-2 in lung epithelium is a key driver of inflammation, the extent of which is influenced by the inflammatory state of the local environment, and that specific inhibition of innate immune pathways may beneficially mitigate inflammation-associated COVID-19.


Assuntos
COVID-19/imunologia , Proteína DEAD-box 58/imunologia , Células Epiteliais/imunologia , Helicase IFIH1 Induzida por Interferon/imunologia , Macrófagos/imunologia , RNA Viral/imunologia , Receptores Imunológicos/imunologia , SARS-CoV-2 , COVID-19/genética , COVID-19/virologia , Linhagem Celular , Citocinas/genética , Citocinas/imunologia , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Inflamação/genética , Inflamação/imunologia , Inflamação/virologia , Janus Quinases/imunologia , Pulmão/citologia , Pulmão/imunologia , Pulmão/virologia , Ativação de Macrófagos , NF-kappa B/imunologia , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologia , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Fatores de Transcrição STAT/imunologia , Replicação Viral
13.
J Virol ; 98(3): e0157623, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38323814

RESUMO

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.


Assuntos
Adenovírus Humanos , Proteínas do Capsídeo , Lactoferrina , Receptores Virais , Internalização do Vírus , Humanos , Infecções por Adenovirus Humanos/metabolismo , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/química , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Adenovírus Humanos/ultraestrutura , Sítios de Ligação/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Lactoferrina/química , Lactoferrina/genética , Lactoferrina/metabolismo , Lactoferrina/ultraestrutura , Modelos Biológicos , Mutação , Ligação Proteica , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Receptores Virais/ultraestrutura , Solubilidade , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia
14.
Am J Respir Crit Care Med ; 210(6): 788-800, 2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-38573173

RESUMO

Rationale: Pulmonary ionocytes are a newly discovered airway epithelial cell type proposed to be a major contributor to cystic fibrosis (CF) lung disease based on observations they express the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel at a higher level than any other cell type in the airway epithelia. Moreover, genetically manipulated experimental models that lack ionocytes develop NaCl transport abnormalities and airway surface liquid (ASL) dehydration consistent with CF. However, no direct evidence indicates ionocytes engage in NaCl transport or contribute to ASL formation, questioning the relevance of ionocytes to CF lung disease. Objectives: To determine the ion transport properties of pulmonary ionocytes and club cells in genetically intact healthy and CF airway epithelia. Methods: We measured ion transport at the single-cell level using a self-referencing ion-selective microelectrode technique in primary human bronchial epithelial cell culture. Measurements and Main Results: cAMP-stimulated non-CF ionocytes do not secrete Na+ or Cl- into the ASL, but rather modulate its pH by secreting bicarbonate via CFTR-linked Cl-/bicarbonate exchange. Non-CF club cells secrete Na+ and Cl- to the lumen side after cAMP stimulation. CF ionocytes and club cells do not transport ions in response to cAMP stimulation, but incubation with CFTR modulators elexacaftor/tezacaftor/ivacaftor restores transport properties. Conclusions: We conclude that ionocytes do not contribute to ASL formation but regulate ASL pH. Club cells secrete the bulk of airway fluid. In CF, abnormal ionocyte and club cell function results in acidic and dehydrated ASL, causing reduced antimicrobial properties and mucociliary clearance.


Assuntos
Brônquios , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Células Epiteliais , Humanos , Células Epiteliais/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Concentração de Íons de Hidrogênio , Brônquios/citologia , Transporte de Íons/fisiologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/citologia , Células Cultivadas , Sódio/metabolismo , Cloretos/metabolismo , AMP Cíclico/metabolismo , Aminofenóis/farmacologia
15.
Biochem J ; 481(14): 959-980, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-38941070

RESUMO

While IκB-kinase-ε (IKKε) induces immunomodulatory genes following viral stimuli, its up-regulation by inflammatory cytokines remains under-explored. Since airway epithelial cells respond to airborne insults and potentiate inflammation, IKKε expression was characterized in pulmonary epithelial cell lines (A549, BEAS-2B) and primary human bronchial epithelial cells grown as submersion or differentiated air-liquid interface cultures. IKKε expression was up-regulated by the pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNFα). Thus, mechanistic interrogations in A549 cells were used to demonstrate the NF-κB dependence of cytokine-induced IKKε. Furthermore, chromatin immunoprecipitation in A549 and BEAS-2B cells revealed robust recruitment of the NF-κB subunit, p65, to one 5' and two intronic regions within the IKKε locus (IKBKE). In addition, IL-1ß and TNFα induced strong RNA polymerase 2 recruitment to the 5' region, the first intron, and the transcription start site. Stable transfection of the p65-binding regions into A549 cells revealed IL-1ß- and TNFα-inducible reporter activity that required NF-κB, but was not repressed by glucocorticoid. While critical NF-κB motifs were identified in the 5' and downstream intronic regions, the first intronic region did not contain functional NF-κB motifs. Thus, IL-1ß- and TNFα-induced IKKε expression involves three NF-κB-binding regions, containing multiple functional NF-κB motifs, and potentially other mechanisms of p65 binding through non-classical NF-κB binding motifs. By enhancing IKKε expression, IL-1ß may prime, or potentiate, responses to alternative stimuli, as modelled by IKKε phosphorylation induced by phorbol 12-myristate 13-acetate. However, since IKKε expression was only partially repressed by glucocorticoid, IKKε-dependent responses could contribute to glucocorticoid-resistant disease.


Assuntos
Células Epiteliais , Quinase I-kappa B , Humanos , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células A549 , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/genética , Interleucina-1beta/farmacologia , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , NF-kappa B/metabolismo , NF-kappa B/genética , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Pulmão/metabolismo , Pulmão/citologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/citologia , Regulação da Expressão Gênica/efeitos dos fármacos
16.
Am J Respir Cell Mol Biol ; 71(3): 267-281, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38843491

RESUMO

The human airway mucociliary epithelium can be recapitulated in vitro using primary cells cultured in an air-liquid interface (ALI), a reliable surrogate to perform pathophysiological studies. As tremendous variations exist among media used for ALI-cultured human airway epithelial cells, the aim of our study was to evaluate the impact of several media (BEGM, PneumaCult, Half & Half, and Clancy) on cell type distribution using single-cell RNA sequencing and imaging. Our work revealed the impact of these media on cell composition, gene expression profile, cell signaling, and epithelial morphology. We found higher proportions of multiciliated cells in PneumaCult-ALI and Half & Half, stronger EGF signaling from basal cells in BEGM-ALI, differential expression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry factor ACE2, and distinct secretome transcripts depending on the media used. We also established that proliferation in PneumaCult-Ex Plus favored secretory cell fate, showing the key influence of proliferation media on late differentiation epithelial characteristics. Altogether, our data offer a comprehensive repertoire for evaluating the effects of culture conditions on airway epithelial differentiation and will aid in choosing the most relevant medium according to the processes to be investigated, such as cilia, mucus biology, or viral infection. We detail useful parameters that should be explored to document airway epithelial cell fate and morphology.


Assuntos
Diferenciação Celular , Proliferação de Células , Células Epiteliais , Mucosa Respiratória , Humanos , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/citologia , Regeneração , Células Cultivadas , SARS-CoV-2 , COVID-19/virologia , COVID-19/patologia , COVID-19/metabolismo , Técnicas de Cultura de Células/métodos , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Meios de Cultura
17.
Am J Physiol Lung Cell Mol Physiol ; 327(4): L587-L599, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39137525

RESUMO

Basal cells are adult stem cells in the airway epithelium and regenerate differentiated cell populations, including the mucosecretory and ciliated cells that enact mucociliary clearance. Human basal cells can proliferate and produce differentiated epithelium in vitro. However, studies of airway epithelial differentiation mostly rely on immunohistochemical or immunofluorescence-based staining approaches, meaning that a dynamic approach is lacking, and quantitative data are limited. Here, we use a lentiviral reporter gene approach to transduce primary human basal cells with bioluminescence reporter constructs to monitor airway epithelial differentiation longitudinally. We generated three constructs driven by promoter sequences from the TP63, MUC5AC, and FOXJ1 genes to quantitatively assess basal cell, mucosecretory cell, and ciliated cell abundance, respectively. We validated these constructs by tracking differentiation of basal cells in air-liquid interface and organoid ("bronchosphere") cultures. Transduced cells also responded appropriately to stimulation with interleukin 13 (IL-13; to increase mucosecretory differentiation and mucus production) and IL-6 (to increase ciliated cell differentiation). These constructs represent a new tool for monitoring airway epithelial cell differentiation in primary epithelial and/or induced pluripotent stem cell (iPSC)-derived cell cultures.NEW & NOTEWORTHY Orr et al. generated and validated new lentiviral vectors to monitor the differentiation of airway basal cells, goblet cells, or multiciliated cells using bioluminescence.


Assuntos
Diferenciação Celular , Células Epiteliais , Lentivirus , Humanos , Lentivirus/genética , Células Epiteliais/metabolismo , Células Epiteliais/citologia , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Mucina-5AC/metabolismo , Mucina-5AC/genética , Medições Luminescentes/métodos , Células Cultivadas , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Interleucina-13/metabolismo , Interleucina-13/farmacologia , Interleucina-6/metabolismo , Interleucina-6/genética , Genes Reporter , Fatores de Transcrição , Proteínas Supressoras de Tumor
18.
Am J Hum Genet ; 108(7): 1318-1329, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34077761

RESUMO

TP73 belongs to the TP53 family of transcription factors and has therefore been well studied in cancer research. Studies in mice, however, have revealed non-oncogenic activities related to multiciliogenesis. Utilizing whole-exome sequencing analysis in a cohort of individuals with a mucociliary clearance disorder and cortical malformation, we identified homozygous loss-of-function variants in TP73 in seven individuals from five unrelated families. All affected individuals exhibit a chronic airway disease as well as a brain malformation consistent with lissencephaly. We performed high-speed video microscopy, immunofluorescence analyses, and transmission electron microscopy in respiratory epithelial cells after spheroid or air liquid interface culture to analyze ciliary function, ciliary length, and number of multiciliated cells (MCCs). The respiratory epithelial cells studied display reduced ciliary length and basal bodies mislocalized within the cytoplasm. The number of MCCs is severely reduced, consistent with a reduced number of cells expressing the transcription factors crucial for multiciliogenesis (FOXJ1, RFX2). Our data demonstrate that autosomal-recessive deleterious variants in the TP53 family member TP73 cause a mucociliary clearance disorder due to a defect in MCC differentiation.


Assuntos
Lisencefalia/genética , Depuração Mucociliar/genética , Mucosa Respiratória/metabolismo , Proteína Tumoral p73/genética , Diferenciação Celular/genética , Células Cultivadas , Ciliopatias/genética , Genes Recessivos , Homozigoto , Humanos , Mutação com Perda de Função , Microscopia de Vídeo , Mucosa Respiratória/citologia , Mucosa Respiratória/ultraestrutura , Sequenciamento do Exoma
19.
Respir Res ; 25(1): 180, 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38664797

RESUMO

BACKGROUND: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium. METHODS: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties. RESULTS: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells. CONCLUSION: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.


Assuntos
Células-Tronco Pluripotentes Induzidas , Mucosa Respiratória , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Organoides/metabolismo
20.
J Immunol ; 208(2): 407-419, 2022 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-34965963

RESUMO

Tuberculosis consistently causes more deaths worldwide annually than any other single pathogen, making new effective vaccines an urgent priority for global public health. Among potential adjuvants, STING-activating cyclic dinucleotides (CDNs) uniquely stimulate a cytosolic sensing pathway activated only by pathogens. Recently, we demonstrated that a CDN-adjuvanted protein subunit vaccine robustly protects against tuberculosis infection in mice. In this study, we delineate the mechanistic basis underlying the efficacy of CDN vaccines for tuberculosis. CDN vaccines elicit CD4 T cells that home to lung parenchyma and penetrate into macrophage lesions in the lung. Although CDNs, like other mucosal vaccines, generate B cell-containing lymphoid structures in the lungs, protection is independent of B cells. Mucosal vaccination with a CDN vaccine induces Th1, Th17, and Th1-Th17 cells, and protection is dependent upon both IL-17 and IFN-γ. Single-cell RNA sequencing experiments reveal that vaccination enhances a metabolic state in Th17 cells reflective of activated effector function and implicate expression of Tnfsf8 (CD153) in vaccine-induced protection. Finally, we demonstrate that simply eliciting Th17 cells via mucosal vaccination with any adjuvant is not sufficient for protection. A vaccine adjuvanted with deacylated monophosphoryl lipid A (MPLA) failed to protect against tuberculosis infection when delivered mucosally, despite eliciting Th17 cells, highlighting the unique promise of CDNs as adjuvants for tuberculosis vaccines.


Assuntos
Adjuvantes Imunológicos/farmacologia , Interleucina-17/imunologia , Mycobacterium tuberculosis/imunologia , Células Th17/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/prevenção & controle , Animais , Ligante CD30/metabolismo , Interferon gama/imunologia , Pulmão/citologia , Pulmão/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Tuberculose Pulmonar/imunologia , Vacinação
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