RESUMO
The evolution of T cell molecular signatures in the distal lung of patients with severe pneumonia is understudied. Here, we analyzed T cell subsets in longitudinal bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia, including unvaccinated patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or with respiratory failure not linked to pneumonia. In patients with SARS-CoV-2 pneumonia, activation of interferon signaling pathways, low activation of the NF-κB pathway and preferential targeting of spike and nucleocapsid proteins early after intubation were associated with favorable outcomes, whereas loss of interferon signaling, activation of NF-κB-driven programs and specificity for the ORF1ab complex late in disease were associated with mortality. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize individuals who recover, whereas responses against nonstructural proteins and activation of NF-κB are associated with poor outcomes.
RESUMO
Some patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe pneumonia and acute respiratory distress syndrome1 (ARDS). Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from that in other types of pneumonia2. Here we investigate SARS-CoV-2 pathobiology by characterizing the immune response in the alveoli of patients infected with the virus. We collected bronchoalveolar lavage fluid samples from 88 patients with SARS-CoV-2-induced respiratory failure and 211 patients with known or suspected pneumonia from other pathogens, and analysed them using flow cytometry and bulk transcriptomic profiling. We performed single-cell RNA sequencing on 10 bronchoalveolar lavage fluid samples collected from patients with severe coronavirus disease 2019 (COVID-19) within 48 h of intubation. In the majority of patients with SARS-CoV-2 infection, the alveolar space was persistently enriched in T cells and monocytes. Bulk and single-cell transcriptomic profiling suggested that SARS-CoV-2 infects alveolar macrophages, which in turn respond by producing T cell chemoattractants. These T cells produce interferon-γ to induce inflammatory cytokine release from alveolar macrophages and further promote T cell activation. Collectively, our results suggest that SARS-CoV-2 causes a slowly unfolding, spatially limited alveolitis in which alveolar macrophages containing SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation.
Assuntos
COVID-19/imunologia , COVID-19/virologia , Macrófagos Alveolares/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , SARS-CoV-2/patogenicidade , Linfócitos T/imunologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , COVID-19/genética , Estudos de Coortes , Humanos , Interferon gama/imunologia , Interferons/imunologia , Interferons/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Pneumonia Viral/genética , RNA-Seq , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Análise de Célula Única , Linfócitos T/metabolismo , Fatores de TempoRESUMO
The mitochondrial electron transport chain (ETC) is necessary for tumour growth1-6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies7-9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)12, which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.
Assuntos
Mitocôndrias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Ubiquinona/análogos & derivados , Animais , Linhagem Celular Tumoral , Proliferação de Células , Ciona intestinalis/enzimologia , Ciclo do Ácido Cítrico , Citosol/metabolismo , Di-Hidro-Orotato Desidrogenase , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/deficiência , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Levilactobacillus brevis/enzimologia , Masculino , Camundongos , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Neoplasias/enzimologia , Fosforilação Oxidativa , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ubiquinona/metabolismoRESUMO
Pulmonary fibrosis is a relentlessly progressive and often fatal disease with a paucity of available therapies. Genetic evidence implicates disordered epithelial repair, which is normally achieved by the differentiation of small cuboidal alveolar type 2 (AT2) cells into large, flattened alveolar type 1 (AT1) cells as an initiating event in pulmonary fibrosis pathogenesis. Using models of pulmonary fibrosis in young adult and old mice and a model of adult alveologenesis after pneumonectomy, we show that administration of ISRIB, a small molecule that restores protein translation by EIF2B during activation of the integrated stress response (ISR), accelerated the differentiation of AT2 into AT1 cells. Accelerated epithelial repair reduced the recruitment of profibrotic monocyte-derived alveolar macrophages and ameliorated lung fibrosis. These findings suggest a dysfunctional role for the ISR in regeneration of the alveolar epithelium after injury with implications for therapy.
Assuntos
Acetamidas/farmacologia , Células Epiteliais Alveolares/efeitos dos fármacos , Cicloexilaminas/farmacologia , Proteostase/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Acetamidas/uso terapêutico , Fatores Etários , Células Epiteliais Alveolares/citologia , Animais , Amianto , Bleomicina , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Cicloexilaminas/uso terapêutico , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteostase/fisiologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Estresse Fisiológico/efeitos dos fármacosRESUMO
Pathogen clearance and resolution of inflammation in patients with pneumonia require an effective local T cell response. Nevertheless, local T cell activation may drive lung injury, particularly during prolonged episodes of respiratory failure characteristic of severe SARS-CoV-2 pneumonia. While T cell responses in the peripheral blood are well described, the evolution of T cell phenotypes and molecular signatures in the distal lung of patients with severe pneumonia caused by SARS-CoV-2 or other pathogens is understudied. Accordingly, we serially obtained 432 bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia and respiratory failure, including 74 unvaccinated patients with COVID-19, and performed flow cytometry, transcriptional, and T cell receptor profiling on sorted CD8+ and CD4+ T cell subsets. In patients with COVID-19 but not pneumonia secondary to other pathogens, we found that early and persistent enrichment in CD8+ and CD4+ T cell subsets correlated with survival to hospital discharge. Activation of interferon signaling pathways early after intubation for COVID-19 was associated with favorable outcomes, while activation of NF-κB-driven programs late in disease was associated with poor outcomes. Patients with SARS-CoV-2 pneumonia whose alveolar T cells preferentially targeted the Spike and Nucleocapsid proteins tended to experience more favorable outcomes than patients whose T cells predominantly targeted the ORF1ab polyprotein complex. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize patients who recover, yet these responses progress to NF-κB activation against non-structural proteins in patients who go on to experience poor clinical outcomes.
RESUMO
Some patients infected with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) develop severe pneumonia and the acute respiratory distress syndrome (ARDS) [1]. Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from other types of pneumonia [2]. We collected bronchoalveolar lavage fluid samples from 86 patients with SARS-CoV-2-induced respiratory failure and 252 patients with known or suspected pneumonia from other pathogens and subjected them to flow cytometry and bulk transcriptomic profiling. We performed single cell RNA-Seq in 5 bronchoalveolar lavage fluid samples collected from patients with severe COVID-19 within 48 hours of intubation. In the majority of patients with SARS-CoV-2 infection at the onset of mechanical ventilation, the alveolar space is persistently enriched in alveolar macrophages and T cells without neutrophilia. Bulk and single cell transcriptomic profiling suggest SARS-CoV-2 infects alveolar macrophages that respond by recruiting T cells. These T cells release interferon-gamma to induce inflammatory cytokine release from alveolar macrophages and further promote T cell recruitment. Our results suggest SARS-CoV-2 causes a slowly unfolding, spatially-limited alveolitis in which alveolar macrophages harboring SARS-CoV-2 transcripts and T cells form a positive feedback loop that drives progressive alveolar inflammation. This manuscript is accompanied by an online resource: https://www.nupulmonary.org/covid-19/. ONE SENTENCE SUMMARY: SARS-CoV-2-infected alveolar macrophages form positive feedback loops with T cells in patients with severe COVID-19.