Red blood cells (RBCs) are essential for aerobic respiration through delivery of oxygen to distant tissues. However, RBCs are currently considered immunologically inert, and few, if any, secondary ...functions of RBCs have been identified. Here, we showed that RBCs serve as critical immune sensors through surface expression of the nucleic acid–sensing Toll-like receptor 9 (TLR9). Mammalian RBCs expressed TLR9 on their surface and bound CpG-containing DNA derived from bacteria, plasmodia, and mitochondria. RBC-bound mitochondrial DNA was increased during human and murine sepsis and pneumonia. In vivo, CpG-carrying RBCs drove accelerated erythrophagocytosis and innate immune activation characterized by increased interferon signaling. Erythroid-specific deletion of TLR9 abrogated erythrophagocytosis and decreased local and systemic cytokine production during CpG-induced inflammation and polymicrobial sepsis. Thus, detection and capture of nucleic acid by TLR9-expressing RBCs regulated red cell clearance and inflammatory cytokine production, demonstrating that RBCs function as immune sentinels during pathologic states. Consistent with these findings, RBC-bound mitochondrial DNA was elevated in individuals with viral pneumonia and sepsis secondary to coronavirus disease 2019 (COVID-19) and associated with anemia and severity of disease. These findings uncover a previously unappreciated role of RBCs as critical players in inflammation distinct from their function in gas transport.
After influenza infection, lineage-negative epithelial progenitors (LNEPs) exhibit a binary response to reconstitute epithelial barriers: activating a Notch-dependent ΔNp63/cytokeratin 5 (Krt5) ...remodelling program or differentiating into alveolar type II cells (AEC2s). Here we show that local lung hypoxia, through hypoxia-inducible factor (HIF1α), drives Notch signalling and Krt5
basal-like cell expansion. Single-cell transcriptional profiling of human AEC2s from fibrotic lungs revealed a hypoxic subpopulation with activated Notch, suppressed surfactant protein C (SPC), and transdifferentiation toward a Krt5
basal-like state. Activated murine Krt5
LNEPs and diseased human AEC2s upregulate strikingly similar core pathways underlying migration and squamous metaplasia. While robust, HIF1α-driven metaplasia is ultimately inferior to AEC2 reconstitution in restoring normal lung function. HIF1α deletion or enhanced Wnt/β-catenin activity in Sox2
LNEPs blocks Notch and Krt5 activation, instead promoting rapid AEC2 differentiation and migration and improving the quality of alveolar repair.
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IJS, NUK, SBMB, UL, UM, UPUK
Kass-Gergi and Vaughan discuss how alveolar repairs after viral injury. Sendai virus (SeV) is a murine paramyxovirus that bears significant homology to human parainfluenza virus 1. For years, it has ...been used as a vector for gene therapy in mouse models, but its antigenicity and induction of host immune responses make it an excellent natural model of injury and repair. The paper by Hernandez et al which capitalizes on these observations and use SeV infection to uncover fascinating and largely novel aspects of epithelial repair after respiratory viral injury, highlighting a previously unknown role for alveolar type 1 (AT1) cells in these processes. The authors observe a previously undescribed phenomenon of AT2-less regions in murine lungs in response to injury, begging direct comparison with mouse models of influenza and SARS-CoV-2 infection.
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
Lipid nanoparticles for delivering mRNA therapeutics hold immense promise for the treatment of a wide range of lung-associated diseases. However, the lack of effective methodologies capable of ...identifying the pulmonary delivery profile of chemically distinct lipid libraries poses a significant obstacle to the advancement of mRNA therapeutics. Here we report the implementation of a barcoded high-throughput screening system as a means to identify the lung-targeting efficacy of cationic, degradable lipid-like materials. We combinatorially synthesize 180 cationic, degradable lipids which are initially screened in vitro. We then use barcoding technology to quantify how the selected 96 distinct lipid nanoparticles deliver DNA barcodes in vivo. The top-performing nanoparticle formulation delivering Cas9-based genetic editors exhibits therapeutic potential for antiangiogenic cancer therapy within a lung tumor model in female mice. These data demonstrate that employing high-throughput barcoding technology as a screening tool for identifying nanoparticles with lung tropism holds potential for the development of next-generation extrahepatic delivery platforms.
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.
► Introduction of the epithelial–mesenchymal transition (EMT) concept derived from developmental and cancer biology ► EMT and associated signaling pathways in lung repair and fibrosis ► EMT phenotypes and their relationship to a pluripotent state or “stemness” ► Candidate airway and alveolar progenitor cells responsible for lung regeneration ► Newly appreciated injury models demonstrating regeneration of functional pulmonary structures
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Despite kerogen's importance as the organic backbone for hydrocarbon production from source rocks such as gas shale, the interplay between kerogen's chemistry, morphology and mechanics remains ...unexplored. As the environmental impact of shale gas rises, identifying functional relations between its geochemical, transport, elastic and fracture properties from realistic molecular models of kerogens becomes all the more important. Here, by using a hybrid experimental-simulation method, we propose a panel of realistic molecular models of mature and immature kerogens that provide a detailed picture of kerogen's nanostructure without considering the presence of clays and other minerals in shales. We probe the models' strengths and limitations, and show that they predict essential features amenable to experimental validation, including pore distribution, vibrational density of states and stiffness. We also show that kerogen's maturation, which manifests itself as an increase in the sp(2)/sp(3) hybridization ratio, entails a crossover from plastic-to-brittle rupture mechanisms.
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IJS, KISLJ, NUK, SBMB, UL, UM, UPUK
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