RESUMO
Smoking is a leading risk factor of chronic obstructive pulmonary disease (COPD), that is characterized by chronic lung inflammation, tissue remodeling and emphysema. Although inflammation is critical to COPD pathogenesis, the cellular and molecular basis underlying smoking-induced lung inflammation and pathology remains unclear. Using murine smoke models and single-cell RNA-sequencing, we show that smoking establishes a self-amplifying inflammatory loop characterized by an influx of molecularly heterogeneous neutrophil subsets and excessive recruitment of monocyte-derived alveolar macrophages (MoAM). In contrast to tissue-resident AM, MoAM are absent in homeostasis and characterized by a pro-inflammatory gene signature. Moreover, MoAM represent 46% of AM in emphysematous mice and express markers causally linked to emphysema. We also demonstrate the presence of pro-inflammatory and tissue remodeling associated MoAM orthologs in humans that are significantly increased in emphysematous COPD patients. Inhibition of the IRAK4 kinase depletes a rare inflammatory neutrophil subset, diminishes MoAM recruitment, and alleviates inflammation in the lung of cigarette smoke-exposed mice. This study extends our understanding of the molecular signaling circuits and cellular dynamics in smoking-induced lung inflammation and pathology, highlights the functional consequence of monocyte and neutrophil recruitment, identifies MoAM as key drivers of the inflammatory process, and supports their contribution to pathological tissue remodeling.
Assuntos
Enfisema , Pneumonia , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Humanos , Camundongos , Animais , Macrófagos Alveolares/patologia , Monócitos/patologia , Pneumonia/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/patologia , Inflamação/patologia , Enfisema/patologiaRESUMO
Profibrotic and prohomeostatic macrophage phenotypes remain ill-defined, both in vivo and in vitro, impeding the successful development of drugs that reprogram macrophages as an attractive therapeutic approach to manage fibrotic disease. The goal of this study was to reveal profibrotic and prohomeostatic macrophage phenotypes that could guide the design of new therapeutic approaches targeting macrophages to treat fibrotic disease. This study used nintedanib, a broad kinase inhibitor approved for idiopathic pulmonary fibrosis, to dissect lung macrophage phenotypes during fibrosis-linked inflammation by combining in vivo and in vitro bulk and single-cell RNA-sequencing approaches. In the bleomycin model, nintedanib drove the expression of IL-4/IL-13-associated genes important for tissue regeneration and repair at early and late time points in lung macrophages. These findings were replicated in vitro in mouse primary bone marrow-derived macrophages exposed to IL-4/IL-13 and nintedanib. In addition, nintedanib promoted the expression of IL-4/IL-13 pathway genes in human macrophages in vitro. The molecular mechanism was connected to inhibition of the colony stimulating factor 1 (CSF1) receptor in both human and mouse macrophages. Moreover, nintedanib counterbalanced the effects of TNF on IL-4/IL-13 in macrophages to promote expression of IL-4/IL-13-regulated tissue repair genes in fibrotic contexts in vivo and in vitro. This study demonstrates that one of nintedanib's antifibrotic mechanisms is to increase IL-4 signaling in macrophages through inhibition of the CSF1 receptor, resulting in the promotion of tissue repair phenotypes.
Assuntos
Fibrose Pulmonar Idiopática , Indóis , Macrófagos , Indóis/farmacologia , Animais , Camundongos , Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Interleucina-4/metabolismo , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismoRESUMO
Despite its high prevalence, the cellular and molecular mechanisms of chronic obstructive pulmonary disease (COPD) are far from being understood. Here, we determine disease-related changes in cellular and molecular compositions within the alveolar space and peripheral blood of a cohort of COPD patients and controls. Myeloid cells were the largest cellular compartment in the alveolar space with invading monocytes and proliferating macrophages elevated in COPD. Modeling cell-to-cell communication, signaling pathway usage, and transcription factor binding predicts TGF-ß1 to be a major upstream regulator of transcriptional changes in alveolar macrophages of COPD patients. Functionally, macrophages in COPD showed reduced antigen presentation capacity, accumulation of cholesteryl ester, reduced cellular chemotaxis, and mitochondrial dysfunction, reminiscent of impaired immune activation.
Assuntos
Macrófagos Alveolares , Doença Pulmonar Obstrutiva Crônica , Quimiotaxia/fisiologia , Humanos , Macrófagos/metabolismo , Monócitos/metabolismoRESUMO
BACKGROUND: Palmoplantar pustulosis (PPP) is a severe inflammatory skin disorder characterized by eruptions of painful, neutrophil-filled pustules on the palms and soles. Although PPP has a profound effect on quality of life, it remains poorly understood and notoriously difficult to treat. OBJECTIVE: We sought to investigate the immune pathways that underlie the pathogenesis of PPP. METHODS: We applied bulk and single-cell RNA sequencing (RNA-Seq) methods to the analysis of skin biopsy samples and peripheral blood mononuclear cells. We validated our results by flow cytometry and immune fluorescence microscopy RESULTS: Bulk RNA-Seq of patient skin detected an unexpected signature of T-cell activation, with a significant overexpression of several TH2 genes typically upregulated in atopic dermatitis. To further explore these findings, we carried out single-cell RNA-Seq in peripheral blood mononuclear cells of healthy and affected individuals. Memory CD4+ T cells of PPP patients were skewed toward a TH17 phenotype, a phenomenon that was particularly significant among cutaneous lymphocyte-associated antigen-positive skin-homing cells. We also identified a subset of memory CD4+ T cells that expressed both TH17 (KLRB1/CD161) and TH2 (GATA3) markers, with pseudotime analysis suggesting that the population was the result of TH17 to TH2 plasticity. Interestingly, the GATA3+/CD161+ cells were overrepresented among the peripheral blood mononuclear cells of affected individuals, both in the single-cell RNA-Seq data set and in independent flow cytometry experiments. Dual-positive cells were also detected in patient skin by immune fluorescence microscopy. CONCLUSIONS: PPP is associated with complex T-cell activation patterns and may explain why biologic drugs that target individual T helper cell populations have shown limited therapeutic efficacy.
Assuntos
Produtos Biológicos , Psoríase , Dermatopatias Vesiculobolhosas , Plasticidade Celular , Doença Crônica , Humanos , Leucócitos Mononucleares/patologia , Qualidade de Vida , Análise de Célula ÚnicaRESUMO
Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and causes remodeling of the small airways. However, the exact smoke-induced effects on the different types of small airway epithelial cells (SAECs) are poorly understood. Here, using air-liquid interface (ALI) cultures, single-cell RNA-sequencing reveals previously unrecognized transcriptional heterogeneity within the small airway epithelium and cell type-specific effects upon acute and chronic cigarette smoke exposure. Smoke triggers detoxification and inflammatory responses and aberrantly activates and alters basal cell differentiation. This results in an increase of inflammatory basal-to-secretory cell intermediates and, particularly after chronic smoke exposure, a massive expansion of a rare inflammatory and squamous metaplasia associated KRT6A+ basal cell state and an altered secretory cell landscape. ALI cultures originating from healthy non-smokers and COPD smokers show similar responses to cigarette smoke exposure, although an increased pro-inflammatory profile is conserved in the latter. Taken together, the in vitro models provide high-resolution insights into the smoke-induced remodeling of the small airways resembling the pathological processes in COPD airways. The data may also help to better understand other lung diseases including COVID-19, as the data reflect the smoke-dependent variable induction of SARS-CoV-2 entry factors across SAEC populations.
Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Células Epiteliais Alveolares/efeitos dos fármacos , Fumar Cigarros/efeitos adversos , Células Epiteliais/metabolismo , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Fumar Cigarros/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Neoplasia de Células Basais/metabolismo , Cultura Primária de Células , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Fumaça , Fumar/efeitos adversos , Fumar/metabolismoRESUMO
IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we combined in vivo and in vitro approaches using primary mouse and human cells, as well as, acute and chronic mouse models of lung inflammation to provide mechanistic insight into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. IL-36 receptor deficient mice exposed to cigarette smoke or cigarette smoke and H1N1 influenza virus had attenuated lung inflammation compared with wild-type controls. We identified neutrophils as a source of IL-36 and show that IL-36 is a key upstream amplifier of lung inflammation by promoting activation of neutrophils, macrophages and fibroblasts through cooperation with GM-CSF and the viral mimic poly(I:C). Our data implicate IL-36, independent of other IL-1 family members, as a key upstream amplifier of neutrophilic lung inflammation, providing a rationale for targeting IL-36 to improve treatment of a variety of neutrophilic lung diseases.
Assuntos
Interleucina-1/metabolismo , Pulmão/metabolismo , Ativação de Neutrófilo , Neutrófilos/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Pneumonia Viral/metabolismo , Receptores de Interleucina-1/metabolismo , Animais , Células Cultivadas , Fumar Cigarros , Modelos Animais de Doenças , Feminino , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Interleucina-1/genética , Pulmão/imunologia , Pulmão/virologia , Ativação de Macrófagos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/virologia , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Pneumonia Viral/genética , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Receptores de Interleucina-1/genética , Transdução de SinaisRESUMO
INTRODUCTION: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are being evaluated for their use in pharmacological and toxicological testing, particularly for electrophysiological side effects. However, little is known about the composition of the commercially available iCell cardiomyocyte (Fuijifilm Cellular Dynamics) cultures and the transcriptomic phenotype of individual cells. METHODS: We characterized iCell cardiomyocytes (assumed to be a mixture of nodal-, atrial-, and ventricular-like cardiomyocytes together with potential residual non-myocytes) using bulk RNA-sequencing, followed by investigation of cellular heterogeneity using two different single-cell RNA-sequencing platforms. RESULTS: Bulk RNA-sequencing identified key cardiac markers (TNNT2, MYL7) as well as fibroblast associated genes (P4HB, VIM), and cardiac ion channels in the iCell cardiomyocyte culture. High-resolution single cell RNA-sequencing demonstrated that both, cardiac and fibroblast-related genes were co-expressed throughout the cell population. This approach resolved two cell clusters within iCell cardiomyocytes. Interestingly, these clusters could not be associated with known cardiac subtypes. However, transcripts of ion channels potentially useful as functional markers for cardiac subtypes were below the detection limits of the single-cell approaches used. Instead, one cluster (10.8% of the cells) is defined by co-expression of cardiac and cell cycle-related genes (e.g. TOP2A). Incorporation of bromodeoxyuridine further confirmed the capability of iCell cardiomyocytes to enter cell cycle. DISCUSSION: The co-expression of cardiac related genes with cell cycle or fibroblast related genes may be interpreted either as aberrant or as an immature feature. However, this excludes the presence of a non-cardiomyocyte sub-population and indicates that some cardiomyocytes themselves enter cell cycle.
Assuntos
Miócitos Cardíacos/fisiologia , RNA-Seq/métodos , Análise de Célula Única/métodos , Biomarcadores/análise , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular , Separação Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Transcriptoma/fisiologiaRESUMO
BACKGROUND: Short non-coding microRNAs (miRNAs) are involved in various cellular processes during disease progression of Crohn's disease (CD) and remarkably stable in feces, which make them attractive biomarker candidates for reflecting intestinal inflammatory processes. Here we investigated the potential of fecal miRNAs as noninvasive and translational CD biomarkers. METHODS: MiRNAs were screened in feces of 52 patients with CD and 15 healthy controls using RNA sequencing and the results were confirmed by PCR. The relationship between fecal miRNA levels and the clinical CD activity index (CDAI) or CD endoscopic index of severity (CDEIS) was explored, respectively. Additionally, fecal miRNAs were investigated in dextran sodium sulfate, adoptive T-cell transfer, and Helicobacter typhlonius/stress-induced murine colitis models using the NanoString platform. RESULTS: Nine miRNAs (miR-15a-5p, miR-16-5p, miR-128-3p, miR-142-5p, miR-24-3p, miR-27a-3p, miR-223-3p, miR-223-5p, and miR-3074-5p) were significantly (adj. P < 0.05, >3-fold) increased whereas 8 miRNAs (miR-10a-5p, miR-10b-5p, miR-141-3p, miR-192-5p, miR-200a-3p, miR-375, miR-378a-3p, and let-7g-5p) were significantly decreased in CD. MiR-192-5p, miR-375, and miR-141-3p correlated (P < 0.05) with both CDAI and CDEIS whereas miR-15a-5p correlated only with CDEIS. Deregulated expression of miR-223-3p, miR-16-5p, miR-15a-5p, miR-24-3p, and miR-200a-3p was also observed in murine models. The identified altered fecal miRNA levels reflect pathophysiological mechanisms in CD, such as Th1 and Th17 inflammation, autophagy, and fibrotic processes. CONCLUSIONS: Our translational study assessed global fecal miRNA changes of patients with CD and relevant preclinical models. These fecal miRNAs show promise as translational and clinically useful noninvasive biomarkers for mechanistic investigation of intestinal pathophysiology, including monitoring of disease progression.
RESUMO
Cigarette smoke (CS) is the leading risk factor to develop COPD. Therefore, the pathologic effects of whole CS on the differentiation of primary small airway epithelial cells (SAEC) were investigated, using cells from three healthy donors and three COPD patients, cultured under ALI (air-liquid interface) conditions. The analysis of the epithelial physiology demonstrated that CS impaired barrier formation and reduced cilia beat activity. Although, COPD-derived ALI cultures preserved some features known from COPD patients, CS-induced effects were similarly pronounced in ALI cultures from patients compared to healthy controls. RNA sequencing analyses revealed the deregulation of marker genes for basal and secretory cells upon CS exposure. The comparison between gene signatures obtained from the in vitro model (CS vs. air) with a published data set from human epithelial brushes (smoker vs. non-smoker) revealed a high degree of similarity between deregulated genes and pathways induced by CS. Taken together, whole cigarette smoke alters the differentiation of small airway basal cells in vitro. The established model showed a good translatability to the situation in vivo. Thus, the model can help to identify and test novel therapeutic approaches to restore the impaired epithelial repair mechanisms in COPD, which is still a high medical need.
Assuntos
Bronquíolos/patologia , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Fumaça/efeitos adversos , Produtos do Tabaco/toxicidade , Adulto , Idoso , Bronquíolos/citologia , Bronquíolos/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Doença Pulmonar Obstrutiva Crônica/etiologia , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/patologia , Fumar/efeitos adversosRESUMO
Combining single-cell RNA sequencing (scRNA-seq) with upstream cell preservation procedures such as cryopreservation or methanol fixation has recently become more common. By separating cell handling and preparation, from downstream library generation, scRNA-seq workflows are more flexible and manageable. However, the inherent transcriptomic changes associated with cell preservation and how they may bias further downstream analysis remain unknown. Here, we present a side-by-side droplet-based scRNA-seq analysis, comparing the gold standard - fresh cells - to three different cell preservation workflows: dimethyl sulfoxide based cryopreservation, methanol fixation and CellCover reagent. Cryopreservation proved to be the most robust protocol, maximizing both cell integrity and low background ambient RNA. Importantly, gene expression profiles from fresh cells correlated most with those of cryopreserved cells. Such similarities were consistently observed across the tested cell lines (R ≥ 0.97), monocyte-derived macrophages (R = 0.97) and immune cells (R = 0.99). In contrast, both methanol fixation and CellCover preservation showed an increased ambient RNA background and an overall lower gene expression correlation to fresh cells. Thus, our results demonstrate the superiority of cryopreservation over other cell preservation methods. We expect our comparative study to provide single-cell omics researchers invaluable support when integrating cell preservation into their scRNA-seq studies.