RESUMO
Macrophages are integral components of the innate immune system, playing a dual role in host defense during infection and pathophysiological states. Macrophages contribute to immune responses and aid in combatting various infections, yet their production of abundant proinflammatory cytokines can lead to uncontrolled inflammation and worsened tissue damage. Therefore, reducing macrophage-derived proinflammatory cytokine release represents a promising approach for treating various acute and chronic inflammatory disorders. However, limited macrophage-specific delivery vehicles have hindered the development of macrophage-targeted therapies. In this study, we screened a pool of 112 lipid nanoparticles (LNPs) to identify an optimal LNP formulation for efficient siRNA delivery. Subsequently, by conjugating the macrophage-specific antibody F4/80 to the LNP surface, we constructed MacLNP, an enhanced LNP formulation designed for targeted macrophage delivery. In both in vitro and in vivo experiments, MacLNP demonstrated a significant enhancement in targeting macrophages. Specifically, delivery of siRNA targeting TAK1, a critical kinase upstream of multiple inflammatory pathways, effectively suppressed the phosphorylation/activation of NF-kB. LNP-mediated inhibition of NF-kB, a key upstream regulator in the classic inflammatory signaling pathway, in the murine macrophage cell line RAW264.7 significantly reduced the release of proinflammatory cytokines after stimulation with the viral RNA mimic Poly(I:C). Finally, intranasal administration of MacLNP-encapsulated TAK1 siRNA markedly ameliorated lung injury induced by influenza infection. In conclusion, our findings validate the potential of targeted macrophage interventions in attenuating inflammatory responses, reinforcing the potential of LNP-mediated macrophage targeting to treat pulmonary inflammatory disorders.
Assuntos
Lipossomos , Nanopartículas , Pneumonia Viral , Camundongos , Humanos , Animais , NF-kappa B/metabolismo , Lipídeos/farmacologia , Macrófagos/metabolismo , RNA Interferente Pequeno/metabolismo , Citocinas/metabolismo , Pneumonia Viral/metabolismoRESUMO
Surfactant protein B (SP-B) deficiency is a rare genetic disease that causes fatal respiratory failure within the first year of life. Currently, the only corrective treatment is lung transplantation. Here, we co-transduced the murine lung with adeno-associated virus 6.2FF (AAV6.2FF) vectors encoding a SaCas9-guide RNA nuclease or donor template to mediate insertion of promoterless reporter genes or the (murine) Sftpb gene in frame with the endogenous surfactant protein C (SP-C) gene, without disrupting SP-C expression. Intranasal administration of 3 × 1011 vg donor template and 1 × 1011 vg nuclease consistently edited approximately 6% of lung epithelial cells. Frequency of gene insertion increased in a dose-dependent manner, reaching 20%-25% editing efficiency with the highest donor template and nuclease doses tested. We next evaluated whether this promoterless gene editing platform could extend survival in the conditional SP-B knockout mouse model. Administration of 1 × 1012 vg SP-B-donor template and 5 × 1011 vg nuclease significantly extended median survival (p = 0.0034) from 5 days in the untreated off doxycycline group to 16 days in the donor AAV and nuclease group, with one gene-edited mouse living 243 days off doxycycline. This AAV6.2FF-based gene editing platform has the potential to correct SP-B deficiency, as well as other disorders of alveolar type II cells.
Assuntos
Doxiciclina , Edição de Genes , Camundongos , Animais , Dependovirus/genética , Vetores Genéticos/genética , RNA Guia de Sistemas CRISPR-Cas , Pulmão/metabolismo , Tensoativos/metabolismo , Sistemas CRISPR-CasRESUMO
Tissue damage in the upper and lower airways caused by mechanical abrasion, noxious chemicals, or pathogenic organisms must be followed by rapid restorative processes; otherwise, persistent immunopathology and disease may ensue. This review will discuss evidence for the important role served by trefoil factor (TFF) family members in healthy and diseased airways of humans and rodents. Collectively, these peptides serve to both maintain and restore homeostasis through their regulation of the mucous layer and their control of cell motility, cell differentiation, and immune function in the upper and lower airways. We will also discuss important differences in which trefoil member tracks with homeostasis and disease between humans and mice, which poses a challenge for research in this area. Moreover, we discuss new evidence supporting newly identified receptor binding partners in the leucine-rich repeat and immunoglobulin-like domain-containing NoGo (LINGO) family in mediating the biological effects of TFF proteins in mouse models of epithelial repair and infection. Recent advances in our knowledge regarding TFF peptides suggest that they may be reasonable therapeutic targets in the treatment of upper and lower airway diseases of diverse etiologies. Further work understanding their role in airway homeostasis, repair, and inflammation will benefit from these newly uncovered receptor-ligand interactions.
Assuntos
Fatores Trefoil , Animais , Pulmão/metabolismo , Camundongos , Peptídeos/metabolismo , Proteínas , Fator Trefoil-2RESUMO
The 9th biennial conference titled "Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases" was hosted virtually, due to the ongoing COVID-19 pandemic, in collaboration with the University of Vermont Larner College of Medicine, the National Heart, Lung, and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, and the International Society for Cell & Gene Therapy. The event was held from July 12th through 15th, 2021 with a pre-conference workshop held on July 9th. As in previous years, the objectives remained to review and discuss the status of active research areas involving stem cells (SCs), cellular therapeutics, and bioengineering as they relate to the human lung. Topics included 1) technological advancements in the in situ analysis of lung tissues, 2) new insights into stem cell signaling and plasticity in lung remodeling and regeneration, 3) the impact of extracellular matrix in stem cell regulation and airway engineering in lung regeneration, 4) differentiating and delivering stem cell therapeutics to the lung, 5) regeneration in response to viral infection, and 6) ethical development of cell-based treatments for lung diseases. This selection of topics represents some of the most dynamic and current research areas in lung biology. The virtual workshop included active discussion on state-of-the-art methods relating to the core features of the 2021 conference, including in situ proteomics, lung-on-chip, induced pluripotent stem cell (iPSC)-airway differentiation, and light sheet microscopy. The conference concluded with an open discussion to suggest funding priorities and recommendations for future research directions in basic and translational lung biology.
Assuntos
COVID-19 , Células-Tronco Pluripotentes Induzidas , Bioengenharia , Biologia , COVID-19/terapia , Humanos , Pulmão , PandemiasRESUMO
Chronic obstructive pulmonary disease (COPD) and asthma remain prevalent human lung diseases. Variability in epithelial and inflammatory components that results in pathologic heterogeneity complicates the development of treatments for these diseases. Early childhood infection with parainfluenza virus or respiratory syncytial virus is strongly associated with the development of asthma and COPD later in life, and exacerbations of these diseases correlate with the presence of viral RNA in the lung. Well-characterized animal models of postviral chronic lung diseases are necessary to study the underlying mechanisms of viral-related COPD and asthma and to develop appropriate therapies. In this study, we cross-analyzed chronic lung disease caused by infection with Sendai virus (SeV) or influenza A virus in mice. Differences were observed in lesion composition and inflammatory profiles between SeV- and influenza A virus-induced long-term lung disease. In addition, a primary SeV infection led to worsened pathologic findings on secondary heterologous viral challenge, whereas the reversed infection scheme protected against disease in response to a secondary viral challenge >1 month after the primary infection. These data demonstrate the differential effect of primary viral infections in the susceptibility to disease exacerbation in response to a different secondary viral infection and highlight the usefulness of these viral models as tools to understand the underlying mechanisms that mediate distinct chronic postviral lung diseases.
Assuntos
Asma/patologia , Vírus da Influenza A/fisiologia , Influenza Humana/patologia , Infecções por Paramyxoviridae/patologia , Paramyxoviridae/fisiologia , Doença Pulmonar Obstrutiva Crônica/virologia , Superinfecção/patologia , Animais , Asma/virologia , Doença Crônica , Progressão da Doença , Feminino , Humanos , Influenza Humana/virologia , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Paramyxoviridae/virologia , Superinfecção/virologiaRESUMO
Broadly, tissue regeneration is achieved in two ways: by proliferation of common differentiated cells and/or by deployment of specialized stem/progenitor cells. Which of these pathways applies is both organ- and injury-specific. Current models in the lung posit that epithelial repair can be attributed to cells expressing mature lineage markers. By contrast, here we define the regenerative role of previously uncharacterized, rare lineage-negative epithelial stem/progenitor (LNEP) cells present within normal distal lung. Quiescent LNEPs activate a ΔNp63 (a p63 splice variant) and cytokeratin 5 remodelling program after influenza or bleomycin injury in mice. Activated cells proliferate and migrate widely to occupy heavily injured areas depleted of mature lineages, at which point they differentiate towards mature epithelium. Lineage tracing revealed scant contribution of pre-existing mature epithelial cells in such repair, whereas orthotopic transplantation of LNEPs, isolated by a definitive surface profile identified through single-cell sequencing, directly demonstrated the proliferative capacity and multipotency of this population. LNEPs require Notch signalling to activate the ΔNp63 and cytokeratin 5 program, and subsequent Notch blockade promotes an alveolar cell fate. Persistent Notch signalling after injury led to parenchymal 'micro-honeycombing' (alveolar cysts), indicative of failed regeneration. Lungs from patients with fibrosis show analogous honeycomb cysts with evidence of hyperactive Notch signalling. Our findings indicate that distinct stem/progenitor cell pools repopulate injured tissue depending on the extent of the injury, and the outcomes of regeneration or fibrosis may depend in part on the dynamics of LNEP Notch signalling.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/patologia , Lesão Pulmonar/patologia , Pulmão/citologia , Pulmão/patologia , Reepitelização , Células-Tronco/citologia , Animais , Bleomicina , Linhagem da Célula , Proliferação de Células , Separação Celular , Cistos/metabolismo , Cistos/patologia , Células Epiteliais/metabolismo , Feminino , Humanos , Queratina-5/metabolismo , Pulmão/fisiologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/virologia , Masculino , Camundongos , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Transplante de Células-Tronco , Células-Tronco/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
H1N1 influenza virus infection induces dramatic and permanent alveolar remodeling mediated by p63+ progenitor cell expansion in both mice and some patients with acute respiratory distress syndrome. This persistent lung epithelial dysplasia is accompanied by chronic inflammation, but the driver(s) of this pathology are unknown. This work identified de novo appearance of solitary chemosensory cells (SCCs), as defined by the tuft cell marker doublecortin-like kinase 1, in post-influenza lungs, arising in close proximity with the dysplastic epithelium, whereas uninjured lungs are devoid of SCCs. Interestingly, fate mapping demonstrated that these cells are derived from p63-expressing lineage-negative progenitors, the same cell of origin as the dysplastic epithelium. Direct activation of SCCs with denatonium + succinate increased plasma extravasation specifically in post-influenza virus-injured lungs. Thus we demonstrate the previously unrecognized development and activity of SCCs in the lung following influenza virus infection, implicating SCCs as a central feature of dysplastic remodeling.
Assuntos
Lesão Pulmonar Aguda/patologia , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/patologia , Síndrome do Desconforto Respiratório/patologia , Mucosa Respiratória/patologia , Lesão Pulmonar Aguda/virologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Quinases Semelhantes a Duplacortina , Células Epiteliais/patologia , Feminino , Humanos , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Mucosa Respiratória/virologiaRESUMO
Trefoil factors (TFFs) are small secreted proteins that regulate tissue integrity and repair at mucosal surfaces, particularly in the gastrointestinal tract. However, their relative contribution(s) to controlling baseline lung function or the extent of infection-induced lung injury are unknown issues. With the use of irradiation bone marrow chimeras, we found that TFF2 produced from both hematopoietic- and nonhematopoietic-derived cells is essential for host protection, proliferation of alveolar type 2 cells, and restoration of pulmonary gas exchange after infection with the hookworm parasite Nippostrongylus brasiliensis. In the absence of TFF2, lung epithelia were unable to proliferate and expressed reduced lung mRNA transcript levels for type 2 response-inducing IL-25 and IL-33 after infectious injury. Strikingly, even in the absence of infection or irradiation, TFF2 deficiency compromised lung structure and function, as characterized by distended alveoli and reduced blood oxygen levels relative to wild-type control mice. Taken together, we show a previously unappreciated role for TFF2, produced by either hematopoietic or nonhematopoietic sources, as a pro-proliferative factor for lung epithelial cells under steady-state and infectious injury conditions.
Assuntos
Células Epiteliais/metabolismo , Pulmão/metabolismo , Alvéolos Pulmonares/metabolismo , Infecções por Strongylida/metabolismo , Fator Trefoil-2/metabolismo , Animais , Proliferação de Células , Células Epiteliais/parasitologia , Células Epiteliais/patologia , Pulmão/parasitologia , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Nippostrongylus , Alvéolos Pulmonares/parasitologia , Alvéolos Pulmonares/patologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/patologiaRESUMO
BACKGROUND: Jaagsiekte sheep retrovirus (JSRV) causes a contagious lung cancer in sheep and goats that can be transmitted by aerosols produced by infected animals. Virus entry into cells is initiated by binding of the viral envelope (Env) protein to a specific cell-surface receptor, Hyal2. Unlike almost all other retroviruses, the JSRV Env protein is also a potent oncoprotein and is responsible for lung cancer in animals. Of concern, Hyal2 is a functional receptor for JSRV in humans. RESULTS: We show here that JSRV is fully capable of infecting human cells, as measured by its reverse transcription and persistence in the DNA of cultured human cells. Several studies have indicated a role for JSRV in human lung cancer while other studies dispute these results. To further investigate the role of JSRV in human lung cancer, we used highly-specific mouse monoclonal antibodies and a rabbit polyclonal antiserum against JSRV Env to test for JSRV expression in human lung cancer. JSRV Env expression was undetectable in lung cancers from 128 human subjects, including 73 cases of bronchioalveolar carcinoma (BAC; currently reclassified as lung invasive adenocarcinoma with a predominant lepidic component), a lung cancer with histology similar to that found in JSRV-infected sheep. The BAC samples included 8 JSRV DNA-positive samples from subjects residing in Sardinia, Italy, where sheep farming is prevalent and JSRV is present. We also tested for neutralizing antibodies in sera from 138 Peruvians living in an area where sheep farming is prevalent and JSRV is present, 24 of whom were directly exposed to sheep, and found none. CONCLUSIONS: We conclude that while JSRV can infect human cells, JSRV plays little if any role in human lung cancer.
Assuntos
Adenocarcinoma/patologia , Adenocarcinoma/virologia , Retrovirus Jaagsiekte de Ovinos/isolamento & purificação , Retrovirus Jaagsiekte de Ovinos/patogenicidade , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Criação de Animais Domésticos , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Feminino , Humanos , Imuno-Histoquímica , Itália , Masculino , Microscopia , Pessoa de Meia-Idade , Exposição Ocupacional , Proteínas do Envelope Viral/análiseAssuntos
Betacoronavirus , Infecções por Coronavirus/complicações , Pneumonia Viral/complicações , Circulação Pulmonar/fisiologia , Síndrome do Desconforto Respiratório/etiologia , COVID-19 , Infecções por Coronavirus/epidemiologia , Humanos , Pandemias , Pneumonia Viral/epidemiologia , Síndrome do Desconforto Respiratório/diagnóstico , Síndrome do Desconforto Respiratório/fisiopatologia , SARS-CoV-2RESUMO
Lung epithelial cells use remarkably adaptive sensing and signaling systems to maintain a physiological state supporting gas exchange and minimizing environmental insults. One facet of epithelial adaptability is the reversible acquisition of mesenchymal features, a process termed epithelial-mesenchymal transition (EMT). Although in the adult, permanent and complete EMT appears rare or non-existent, a growing body of evidence implicates a critical role for the activation of EMT signaling in tissue remodeling, including fibrotic lung disease. The specific phenotypes of cells undergoing EMT re-programming during epithelial responses to injury continue to be defined and are reviewed here. Several recent studies implicate epithelial expression of canonical EMT transcription factors, such as Snail and Twist1, with the acquisition of a less differentiated, more proliferative stem-like state, providing an additional link between activation of EMT signaling and tissue repair. In lung airways, proliferating variant clara cells rely upon Snail for effective epithelial repair, and in the breast, cells possessing the greatest regenerative capacity also express Snail2. The ongoing elucidation of signaling underlying epithelial stem/progenitor expansion coincides with recent discoveries implicating regenerative activity in the lung, possibly including de novo regeneration of airway and alveolar units. It remains largely unknown what signals drive organization of epithelial progenitor cells that expand after lung injury, to what degree such organization is ever functionally relevant, and whether the lung regenerative potential recently observed in mouse models extends to humans. Yet these unknowns with clinical potential bring future mechanistic studies of EMT and lung repair directly into the field of regenerative medicine. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.
Assuntos
Transição Epitelial-Mesenquimal , Pulmão , Animais , Diferenciação Celular , Células Epiteliais/metabolismo , Humanos , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Macrophages are key modulators of all inflammatory diseases and essential for their resolution, making macrophage cell therapy a promising strategy for regenerative medicine. However, since macrophages change rapidly in response to microenvironmental cues, their phenotype must be controlled post-administration. We present a tunable biomaterial-based strategy to control macrophages intracellularly via small molecule-releasing microparticles. Poly(lactic-co-glycolic acid) microparticles encapsulating the anti-inflammatory and anti-fibrotic drug dexamethasone were administered to macrophages in vitro, with uptake rates controlled by different loading regimes. Microparticle dose and dexamethasone content directly affected macrophage phenotype and phagocytic capacity, independent of particle content per cell, leading to an overall pro-reparative, anti-inflammatory, anti-fibrotic phenotype with increased phagocytic and ECM degrading functionality. Intracellularly controlled macrophages partially maintained this phenotype in vivo in a murine pulmonary fibrosis model, with more prominent effects in a pro-fibrotic environment compared to pro-inflammatory. These results suggest that intracellular control using biomaterials has the potential to control macrophage phenotype post-administration, which is essential for successful macrophage cell therapy.
Assuntos
Materiais Biocompatíveis , Dexametasona , Macrófagos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Animais , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Materiais Biocompatíveis/química , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Camundongos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Terapia Baseada em Transplante de Células e Tecidos/métodos , Camundongos Endogâmicos C57BL , Inflamação/patologia , Fibrose Pulmonar/terapia , Fibrose Pulmonar/patologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Fagocitose/efeitos dos fármacos , Células RAW 264.7 , Ácido Poliglicólico/química , Ácido Láctico/química , FibroseRESUMO
IκB kinase (IKK)α controls noncanonical NF-κB signaling required for lymphoid organ development. We showed previously that lymph node formation is ablated in IkkαLyve-1 mice constitutively lacking IKKα in lymphatic endothelial cells (LECs). We now reveal that loss of IKKα in LECs leads to the formation of BALT in the lung. Tertiary lymphoid structures appear only in the lungs of IkkαLyve-1 mice and are not present in any other tissues, and these highly organized BALT structures form after birth and in the absence of inflammation. Additionally, we show that IkkαLyve-1 mice challenged with influenza A virus (IAV) exhibit markedly improved survival and reduced weight loss compared with littermate controls. Importantly, we determine that the improved morbidity and mortality of IkkαLyve-1 mice is independent of viral load and rate of clearance because both mice control and clear IAV infection similarly. Instead, we show that IFN-γ levels are decreased, and infiltration of CD8 T cells and monocytes into IkkαLyve-1 lungs is reduced. We conclude that ablating IKKα in LECs promotes BALT formation and reduces the susceptibility of IkkαLyve-1 mice to IAV infection through a decrease in proinflammatory stimuli.
Assuntos
Homeostase , Quinase I-kappa B , Vírus da Influenza A , Pulmão , Infecções por Orthomyxoviridae , Animais , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Camundongos , Pulmão/imunologia , Pulmão/virologia , Pulmão/patologia , Infecções por Orthomyxoviridae/imunologia , Vírus da Influenza A/imunologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Linfócitos T CD8-Positivos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/imunologia , Interferon gama/metabolismoRESUMO
Red blood cells (RBCs) express the nucleic acid-binding toll-like receptor 9 (TLR9) and bind CpG-containing DNA. However, whether human RBCs express other nucleic acid-binding TLRs is unknown. Here we show that human RBCs express the RNA sensor TLR7. TLR7 is present on the red cell membrane and is associated with the RBC membrane protein Band 3. In patients with SARS-CoV2-associated sepsis, TLR7-Band 3 interactions in the RBC membrane are increased when compared with healthy controls. In vitro, RBCs bind synthetic ssRNA and RNA from ssRNA viruses. Thus, RBCs may serve as a previously unrecognized sink for exogenous RNA, expanding the repertoire of non-gas exchanging functions performed by RBCs.
Assuntos
COVID-19 , Eritrócitos , SARS-CoV-2 , Receptor 7 Toll-Like , Humanos , Receptor 7 Toll-Like/metabolismo , Receptor 7 Toll-Like/genética , Eritrócitos/metabolismo , COVID-19/virologia , COVID-19/metabolismo , SARS-CoV-2/metabolismo , Sepse/metabolismo , Sepse/sangue , Sepse/genética , Membrana Eritrocítica/metabolismo , Masculino , RNA/metabolismo , RNA/genética , FemininoRESUMO
Maintenance of the cellular boundary between airway and alveolar compartments during homeostasis and after injury is essential to prohibit pathological plasticity which can reduce respiratory function. Lung injury and disease can induce either functional alveolar epithelial regeneration or dysplastic formation of keratinized epithelium which does not efficiently contribute to gas exchange. Here we show that Sox2 preserves airway cell identity and prevents fate changes into either functional alveolar tissue or pathological keratinization following lung injury. Loss of Sox2 in airway epithelium leads to a loss of airway epithelial identity with a commensurate gain in alveolar and basal cell identity, in part due to activation of Wnt signaling in secretory cells and increased Trp63 expression in intrapulmonary basal-like progenitors. In idiopathic pulmonary fibrosis, loss of SOX2 expression correlates with increased WNT signaling activity in dysplastic keratinized epithelium. SOX2-deficient dysplastic epithelial cells are also observed in COVID-19 damaged lungs. Thus, Sox2 provides a molecular barrier that suppresses airway epithelial plasticity to prevent acquisition of alveolar or basal cell identity after injury and help guide proper epithelial fate and regeneration.
RESUMO
The ionizable lipidoid is a key component of lipid nanoparticles (LNPs). Degradable lipidoids containing extended alkyl branches have received tremendous attention, yet their optimization and investigation are underappreciated. Here, we devise an in situ construction method for the combinatorial synthesis of degradable branched (DB) lipidoids. We find that appending branch tails to inefficacious lipidoids via degradable linkers boosts mRNA delivery efficiency up to three orders of magnitude. Combinatorial screening and systematic investigation of two libraries of DB-lipidoids reveal important structural criteria that govern their in vivo potency. The lead DB-LNP demonstrates robust delivery of mRNA therapeutics and gene editors into the liver. In a diet-induced obese mouse model, we show that repeated administration of DB-LNP encapsulating mRNA encoding human fibroblast growth factor 21 alleviates obesity and fatty liver. Together, we offer a construction strategy for high-throughput and cost-efficient synthesis of DB-lipidoids. This study provides insights into branched lipidoids for efficient mRNA delivery.
Assuntos
Nanopartículas , Animais , Camundongos , Humanos , RNA Mensageiro/genética , Nanopartículas/química , RNA Interferente PequenoRESUMO
Lipid nanoparticles (LNPs) are widely used for mRNA delivery, with cationic lipids greatly affecting biodistribution, cellular uptake, endosomal escape and transfection efficiency. However, the laborious synthesis of cationic lipids limits the discovery of efficacious candidates and slows down scale-up manufacturing. Here we develop a one-pot, tandem multi-component reaction based on the rationally designed amine-thiol-acrylate conjugation, which enables fast (1 h) and facile room-temperature synthesis of amidine-incorporated degradable (AID) lipids. Structure-activity relationship analysis of a combinatorial library of 100 chemically diverse AID-lipids leads to the identification of a tail-like amine-ring-alkyl aniline that generally affords efficacious lipids. Experimental and theoretical studies show that the embedded bulky benzene ring can enhance endosomal escape and mRNA delivery by enabling the lipid to adopt a more conical shape. The lead AID-lipid can not only mediate local delivery of mRNA vaccines and systemic delivery of mRNA therapeutics, but can also alter the tropism of liver-tropic LNPs to selectively deliver gene editors to the lung and mRNA vaccines to the spleen.
RESUMO
Severe lung injury causes basal stem cells to migrate and outcompete alveolar stem cells resulting in dysplastic repair and a loss of gas exchange function. This "stem cell collision" is part of a multistep process that is now revealed to generate an injury-induced tissue niche (iTCH) containing Keratin 5+ epithelial cells and plastic Pdgfra+ mesenchymal cells. Temporal and spatial single cell analysis reveals that iTCHs are governed by mesenchymal proliferation and Notch signaling, which suppresses Wnt and Fgf signaling in iTCHs. Conversely, loss of Notch in iTCHs rewires alveolar signaling patterns to promote euplastic regeneration and gas exchange. The signaling patterns of iTCHs can differentially phenotype fibrotic from degenerative human lung diseases, through apposing flows of FGF and WNT signaling. These data reveal the emergence of an injury and disease associated iTCH in the lung and the ability of using iTCH specific signaling patterns to discriminate human lung disease phenotypes.
RESUMO
Alveolar epithelial type II (AT2) cell dysfunction is implicated in the pathogenesis of familial and sporadic idiopathic pulmonary fibrosis (IPF). We previously described that expression of an AT2 cell exclusive disease-associated protein isoform (SP-CI73T) in murine and patient-specific induced pluripotent stem cell (iPSC)-derived AT2 cells leads to a block in late macroautophagy and promotes time-dependent mitochondrial impairments; however, how a metabolically dysfunctional AT2 cell results in fibrosis remains elusive. Here using murine and human iPSC-derived AT2 cell models expressing SP-CI73T, we characterize the molecular mechanisms governing alterations in AT2 cell metabolism that lead to increased glycolysis, decreased mitochondrial biogenesis, disrupted fatty acid oxidation, accumulation of impaired mitochondria, and diminished AT2 cell progenitor capacity manifesting as reduced AT2 self-renewal and accumulation of transitional epithelial cells. We identify deficient AMP-kinase signaling as a key upstream signaling hub driving disease in these dysfunctional AT2 cells and augment this pathway to restore alveolar epithelial metabolic function, thus successfully alleviating lung fibrosis in vivo.