Effective clearance of Mycobacterium tuberculosis (Mtb) requires targeting drug-tolerant populations within host macrophages. Here, we show that macrophage metabolic states govern redox heterogeneity and drug response in intracellular Mtb. Using a redox-sensitive fluorescent reporter (Mrx1-roGFP2), flow cytometry, and transcriptomics, we found that macrophages with high oxidative phosphorylation (OXPHOS) and low glycolysis harbor reductive, drug-tolerant Mtb, whereas glycolytically active macrophages generate mitochondrial ROS via reverse electron transport, imposing oxidative stress on Mtb and enhancing drug efficacy. Computational and genetic analyses identified NRF2 as a key regulator linking host metabolism to bacterial redox state and drug tolerance. Pharmacological reprogramming of macrophages with the FDA-approved drug meclizine (MEC) shifted metabolism towards glycolysis, suppressed redox heterogeneity, and reduced Mtb drug tolerance in macrophages and mice. MEC exhibited no adverse interactions with frontline anti-TB drugs. These findings demonstrate the therapeutic potential of host metabolic reprogramming to overcome Mtb drug tolerance.

ABSTRACTThe elderly population is at significant risk of developing respiratory diseases, including tuberculosis (TB) caused by the airborneMycobacterium tuberculosis(M.tb). OnceM.tbreaches the alveolar space, it contacts alveolar lining fluid (ALF) which dictates host cell interactions. We previously determined that age-associated dysfunctionality in human ALF soluble innate components lead to acceleratedM.tbgrowth within human alveolar macrophages. Here we determined the impact of human ALF onM.tbinfection of alveolar epithelial cells (ATs), another critical cellular determinant of infection. We observed that E-ALF-exposedM.tbhad significantly increased intracellular growth in ATs compared to adult ALF (A-ALF)-exposed bacteria. Despite this, there were no alterations in AT inflammatory mediators or cell activation. However, exposure to E-ALF altered endosomal trafficking ofM.tb, driving bacterial translocation to both endosomal and cytosolic compartments in ATs. Our results indicate that exposure ofM.tbto E-ALF promotes translocation of bacteria into the AT cytosol as a potential favorable niche for rapid bacterial growth and at the same time dampens AT’s immune responses. Thus, our findings highlight the influence of the elderly lung mucosa onM.tbinfection of ATs, an unexplored contributing factor to the elderly population’s increased susceptibility of developing active TB disease.

Low-grade inflammation is a hallmark of old age and a central driver of ageing-associated impairment and disease1. Multiple factors can contribute to ageing-associated inflammation2; however, the molecular pathways that transduce aberrant inflammatory signalling and their impact in natural ageing remain unclear. Here we show that the cGAS-STING signalling pathway, which mediates immune sensing of DNA3, is a critical driver of chronic inflammation and functional decline during ageing. Blockade of STING suppresses the inflammatory phenotypes of senescent human cells and tissues, attenuates ageing-related inflammation in multiple peripheral organs and the brain in mice, and leads to an improvement in tissue function. Focusing on the ageing brain, we reveal that activation of STING triggers reactive microglial transcriptional states, neurodegeneration and cognitive decline. Cytosolic DNA released from perturbed mitochondria elicits cGAS activity in old microglia, defining a mechanism by which cGAS-STING signalling is engaged in the ageing brain. Single-nucleus RNA-sequencing analysis of microglia and hippocampi of a cGAS gain-of-function mouse model demonstrates that engagement of cGAS in microglia is sufficient to direct ageing-associated transcriptional microglial states leading to bystander cell inflammation, neurotoxicity and impaired memory capacity. Our findings establish the cGAS-STING pathway as a driver of ageing-related inflammation in peripheral organs and the brain, and reveal blockade of cGAS-STING signalling as a potential strategy to halt neurodegenerative processes during old age.

Mycobacterium tuberculosis (Mtb) cultured axenically without detergent forms biofilm-like cords, a clinical identifier of virulence. In lung-on-chip (LoC) and mouse models, cords in alveolar cells contribute to suppression of innate immune signaling via nuclear compression. Thereafter, extracellular cords cause contact-dependent phagocyte death but grow intercellularly between epithelial cells. The absence of these mechanopathological mechanisms explains the greater proportion of alveolar lesions with increased immune infiltration and dissemination defects in cording-deficient Mtb infections. Compression of Mtb lipid monolayers induces a phase transition that enables mechanical energy storage. Agent-based simulations demonstrate that the increased energy storage capacity is sufficient for the formation of cords that maintain structural integrity despite mechanical perturbation. Bacteria in cords remain translationally active despite antibiotic exposure and regrow rapidly upon cessation of treatment. This study provides a conceptual framework for the biophysics and function in tuberculosis infection and therapy of cord architectures independent of mechanisms ascribed to single bacteria.

Dataset corresponding to the LoC studies in the manuscript titled The cGAS-STING pathway drives type I IFN immunopathology in COVID-19. The following data are included: BioEM.zip: Volumetric electron microscopy - representative movies of volumetric scans of fields of view of the vascular face of uninfected control and SARS-CoV-2 infected LoCs. Blender file containing reconstruction of mitochondria. Cleaved-caspase3.zip: Imaris files for analysis of 3D stacks from two- and three-component LoCs immunostained for cleaved-caspase 3. IFN beta.zip: Imaris files for analysis of 3D stacks from two- and three-component LoCs immunostained for IFN beta. PhosphoSTING.zip: Imaris files for analysis of 3D stacks from two component LoCs immunostained for phosphoSTING. Proteomics_R_code.Rmd: Annotated custom scripts in R for the analysis of the proteomics data.

COVID-19, which is caused by infection with SARS-CoV-2, is characterized by lung pathology and extrapulmonary complications 1 , 2 . Type I interferons (IFNs) have an essential role in the pathogenesis of COVID-19 (refs 3 – 5 ). Although rapid induction of type I IFNs limits virus propagation, a sustained increase in the levels of type I IFNs in the late phase of the infection is associated with aberrant inflammation and poor clinical outcome 5 – 17 . Here we show that the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway, which controls immunity to cytosolic DNA, is a critical driver of aberrant type I IFN responses in COVID-19 (ref. 18 ). Profiling COVID-19 skin manifestations, we uncover a STING-dependent type I IFN signature that is primarily mediated by macrophages adjacent to areas of endothelial cell damage. Moreover, cGAS–STING activity was detected in lung samples from patients with COVID-19 with prominent tissue destruction, and was associated with type I IFN responses. A lung-on-chip model revealed that, in addition to macrophages, infection with SARS-CoV-2 activates cGAS–STING signalling in endothelial cells through mitochondrial DNA release, which leads to cell death and type I IFN production. In mice, pharmacological inhibition of STING reduces severe lung inflammation induced by SARS-CoV-2 and improves disease outcome. Collectively, our study establishes a mechanistic basis of pathological type I IFN responses in COVID-19 and reveals a principle for the development of host-directed therapeutics.

Dataset for manuscript posted at biorxiv: https://doi.org/10.1101/2021.01.03.42483 and in revision for eLife. Data corresponding to each main figure and its associated figure supplements are in seperate .zip folders.

Recurrence of uropathogenic Escherichia coli (UPEC) infections has been attributed to reactivation of quiescent intracellular reservoirs (QIRs) in deep layers of the bladder wall. QIRs are thought to arise late during infection following dispersal of bacteria from intracellular bacterial communities (IBCs) in superficial umbrella cells. Here, we track the formation of QIR-like bacteria in a bladder organoid model that recapitulates the stratified uroepithelium within a volume suitable for high-resolution live-cell imaging. Bacteria injected into the organoid lumen enter umbrella-like cells and proliferate to form IBC-like bodies. In parallel, single bacteria penetrate deeper layers of the organoid wall, where they localize within or between uroepithelial cells. These "solitary" bacteria evade killing by antibiotics and neutrophils and are morphologically distinct from bacteria in IBCs. We conclude that bacteria with QIR-like properties may arise at early stages of infection, independent of IBC formation and rupture.

Uropathogenic Escherichia coli (UPEC) proliferate within superficial bladder umbrella cells to form intracellular bacterial communities (IBCs) during early stages of urinary tract infections. However, the dynamic responses of IBCs to host stresses and antibiotic therapy are difficult to assess in situ. We develop a human bladder-chip model wherein umbrella cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively, and bladder filling and voiding mimicked mechanically by application and release of linear strain. Using time-lapse microscopy, we show that rapid recruitment of neutrophils from the vascular channel to sites of infection leads to swarm and neutrophil extracellular trap formation but does not prevent IBC formation. Subsequently, we tracked bacterial growth dynamics in individual IBCs through two cycles of antibiotic administration interspersed with recovery periods which revealed that the elimination of bacteria within IBCs by the antibiotic was delayed, and in some instances, did not occur at all. During the recovery period, rapid proliferation in a significant fraction of IBCs reseeded new foci of infection through bacterial shedding and host cell exfoliation. These insights reinforce a dynamic role for IBCs as harbors of bacterial persistence, with significant consequences for non-compliance with antibiotic regimens.

In the science fiction series Star Trek, first contact between species occurs on a galactic scale; a recurring theme whose consequences are richly developed and explored throughout the series. In infection biology, first contact of a susceptible host with an infectious agent, albeit on a less gargantuan scale, plays no less of a role in determining subsequent outcomes. Nowhere might this be truer than for tuberculosis, an ancient disease and the leading cause of global mortality from any single infectious agent. Caused by the very slow-growing bacterium M. tuberculosis (Mtb), tuberculosis can be triggered by the infection of the deep areas of the lung by even a single bacterium, yet only 5-10% of individuals exposed to the bacterium go on to develop a full infection. This astonishing diversity in outcomes might in part be a consequence of the early interactions of Mtb bacteria with both nonimmune and immune cells at 'first contact' in the lung. However, even in the best animal model, the disease dynamics at this early stage are extremely hard to follow.

Severe cases of SARS-CoV-2 infection are characterized by hypercoagulopathies and systemic endotheliitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of an immune cell-mediated cytokine storm remain unknown. Using a vascularized lung-on-chip model, we find that infection of alveolar epithelial cells leads to limited apical release of virions, consistent with reports of monoculture infection. However, viral RNA and proteins are rapidly detected in underlying endothelial cells, which are themselves refractory to apical infection in monocultures. Although endothelial infection is unproductive, it leads to the formation of cell clusters with low CD31 expression, a progressive loss of barrier integrity and a pro-coagulatory microenvironment. Viral RNA persists in individual cells generating an inflammatory response, which is transient in epithelial cells but persistent in endothelial cells and typified by IL-6 secretion even in the absence of immune cells. Inhibition of IL-6 signalling with tocilizumab reduces but does not prevent loss of barrier integrity. SARS-CoV-2-mediated endothelial cell damage thus occurs independently of cytokine storm.

We establish a murine lung-on-chip infection model and use time-lapse imaging to reveal the dynamics of host-Mycobacterium tuberculosis interactions at an air-liquid interface with a spatiotemporal resolution unattainable in animal models and to probe the direct role of pulmonary surfactant in early infection. Surfactant deficiency results in rapid and uncontrolled bacterial growth in both macrophages and alveolar epithelial cells. In contrast, under normal surfactant levels, a significant fraction of intracellular bacteria are non-growing. The surfactant-deficient phenotype is rescued by exogenous addition of surfactant replacement formulations, which have no effect on bacterial viability in the absence of host cells. Surfactant partially removes virulence-associated lipids and proteins from the bacterial cell surface. Consistent with this mechanism, the attenuation of bacteria lacking the ESX-1 secretion system is independent of surfactant levels. These findings may partly explain why smokers and elderly persons with compromised surfactant function are at increased risk of developing active tuberculosis.

Abstract Mycobacterium tuberculosis (Mtb) is spread via aerosolized droplets and makes ‘first contact’ with a new host in the alveolar space, an interaction largely inaccessible to experimental observation. We establish a lung-on-chip as an infection model for early tuberculosis, where time-lapse imaging at an air-liquid interface reveals the dynamics of early host-Mtb interactions with a spatiotemporal resolution unattainable in animal models. Pulmonary surfactant mediates a non-growing Mtb population in both alveolar epithelial cells (AECs) and macrophages, although AECs are more permissive to Mtb growth. Defective surfactant production leads to uncontrolled rapid Mtb growth in both cell types which can be partially rescued by formulations containing surfactant phospholipids. AECs thus have both host-protective and pathogen-permissive roles during first contact, which may explain the full spectrum of human disease and inform new therapeutic interventions. One Sentence Summary Live imaging in a lung-on-chip model for early tuberculosis reveals pulmonary surfactant significantly attenuates bacterial growth

Description of the sub-folders Name, type of data, corresponding Figure in the manuscript 3D view of the LoC model - .tiff image stack, Figure 1. Bacterial Growth Rate Data - .tiff image stacks, .csv files and MATLAB code to extract the fluorescence intensity over time, Figure 2, Figure 2 - figure supplement 2, Figure 2 - figure supplement 4, Figure 3, Figure 3 - figure supplement 2, Figure 4. AT Characterization - .tiff image stacks and MATLAB code to extract the number and volume of lamellar bodies from the stack of confocal images, Figure 1, Figure 1 - figure supplement 1, Figue 1 - figure supplement 2. AT Infection in LoC model - .tiff image stacks, Figure 2 - figure supplement 1. AT Infection in vivo - .tiff image stacks, Figure 1 - figure supplement 3. Simulations of in vivo infections - .dat files of growth rates in macrophages for the WT and ESX-1 deficient populations and MATLAB code to simulate an infection from this data, Figure 4.

Severe cases of COVID-19 present with hypercoagulopathies and systemic endothelialitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of immune cell mediated cytokine storms is unknown. This is in part because in vitro models are typically epithelial cell monocultures or fail to recapitulate vascular physiology. We use a vascularised lung-on-chip model where, consistent with monoculture reports, low numbers of SARS-CoV-2 virions are released apically from alveolar epithelial cells. However, rapid infection of the underlying endothelial layer leads to the generation of clusters of endothelial cells with low or no CD31 expression, a progressive loss of endothelial barrier integrity, and a pro-coagulatory microenvironment. These morphological changes do not occur if these cells are exposed to the virus apically. Viral RNA persists in individual cells, which generates a response that is skewed towards NF-KB mediated inflammation, is typified by IL-6 secretion even in the absence of immune cells, and is transient in epithelial cells but persistent in endothelial cells. Perfusion with Tocilizumab, an inhibitor of trans IL-6 signalling slows the loss of barrier integrity but does not prevent the formation of endothelial cell clusters with reduced CD31 expression. SARS-CoV-2 mediated endothelial cell damage occurs despite a lack of rapid viral replication, in a cell-type specific manner and independently of immune-cell mediated cytokine storms, whose effect would only exacerbate the damage. ### Competing Interest Statement The authors have declared no competing interest.

We fabricate heterocomponent dimers built from a single 40 nm gold and a single 40 nm silver nanoparticle separated by sub-5 nm gaps. Successful assembly mediated by a specialized DNA origami platform is verified by scanning electron microscopy and energy-dispersive X-ray characterization. Dark-field optical scattering on individual dimers is consistent with computational simulations. Direct plasmonic coupling between each nanoparticle is observed in both experiment and theory only for these small gap sizes, as it requires the silver dipolar mode energy to drop below the energy of the gold interband transitions. A new interparticle-spacing-dependent coupling model for heterodimers is thus required. Such Janus-like nanoparticle constructs available from DNA-mediated assembly provide an effective tool for controlling symmetry breaking in collective plasmon modes.

Plasmonic sensors are extremely promising candidates for label-free single molecule analysis but require exquisite control over the physical arrangement of metallic nanostructures. We employ self-assembly based on the DNA origami technique for accurate positioning of individual 40 nm gold nanoparticles with gaps of 3.3 +/- 1 nm. This is probed through far field scattering measurements on individual dimers. This plasmonic coupling allows us to use surface enhanced Raman scattering (SERS) to detect a small number of dye molecules as well as short single-stranded DNA oligonucleotides in the vicinity of the dimers. This demonstrates that DNA origami is a powerful tool with great potential for a wide variety of biosensing and single-molecule applications.

During bursts in KO, the rate was comparable to WT (4.150.3Hz). WT SANs (n=5) showed a 70.556.1% increase in rate after exposure to the b-adrenergic agonist ISO (10mM), while KO showed no increase in average rate. However, when considering only rate during burst activity in KO, 6 out of 10 KO SANs responded significantly to ISO 10mM (52516% increase). The specific If inhibitor ivabradine (IVA, 9mM) reduced the spontaneous pacemaker rate in both WT (n=6) and KO (n=8) SANs (4459% and 58.956.8% decrease, respectively), and even during the bursts in the KO (36517.9% decrease). Thus, NCX1 KO SANs can generate bursts of pacemaker activity similar to WT. These bursts are responsive to both ISO and IVA, consistent with If-mediated pacemaker activity despite the absence of NCX.

Plasmonic sensors are extremely promising candidates for label-free single-molecule analysis but require exquisite control over the physical arrangement of metallic nanostructures. Here we employ self-assembly based on the DNA origami technique for accurate positioning of individual gold nanoparticles. Our innovative design leads to strong plasmonic coupling between two 40 nm gold nanoparticles reproducibly held with gaps of 3.3 ± 1 nm. This is confirmed through far field scattering measurements on individual dimers which reveal a significant red shift in the plasmonic resonance peaks, consistent with the high dielectric environment due to the surrounding DNA. We use surface-enhanced Raman scattering (SERS) to demonstrate local field enhancements of several orders of magnitude through detection of a small number of dye molecules as well as short single-stranded DNA oligonucleotides. This demonstrates that DNA origami is a powerful tool for the high-yield creation of SERS-active nanoparticle assemblies with reliable sub-5 nm gap sizes.

DNA nanotechnology excels at rationally designing bottom-up structures that can functionally replicate naturally occurring proteins. We describe the design and generation of stable self-assembled DNA-based nanopores that functionally mimic membrane protein pores and insert into lipid bilayers to support transmembrane water flow. The DNA nanopores consist of a bundle of six hexagonally arranged duplexes which are interconnected by cross-overs. The negatively charged nanobarrels carry lipid anchors to facilitate the pores' insertion into the hydrophobic bilayers. The lipid anchors either neutralize localized negative charges on the DNA backbone to create a hydrophobic belt to resemble amphiphilic protein pores, as demonstrated with alkylated phosphorothioate groups (Nano Letters, 2013, 13, 2351). Alternatively, anchoring can be achieved with few, large hydrophobic group such as porphyrin which doubles as fluorophore (Angew Chem, doi anie.201305765, Front Cover). The nanoarchitectures are correctly assembled as confirmed by AFM, SEC, and DLS, and are fully functional as shown by single-channel current recordings. The small membrane-spanning DNA pores merge the fields of nanopores and DNA-nanotechnology and will help open up the design of entirely new molecular devices for applications within single-molecule research and sensing, electric circuits, catalysis, and nanofluidics.