Biology subjects

Thacker, V. V.

Publications and source records attributed to Thacker, V. V..

7 recordsLinked to original sources

bioRxiv2025

Vehiculation and functional delivery of lipophilic therapeutics and antibiotics via pulmonary surfactant in a lung-on-chip model

Pulmonary surfactant forms a dynamic proteolipid thin-film at the alveolar air-liquid interface. It is both a barrier to but also a "last-mile" carrier for inhaled particulate matter to the distal lung. The role of vehiculation in pulmonary delivery of inhaled therapeutics remains poorly understood, as it cannot be easily studied in animal models, and is not recapitulated in liquid cell culture. Here we adapted a thin-film bridge (TFB) delivery method from vehiculation experiments in acellular models to a lung-on-chip (LoC) platform under breathing-like stretch. Using confocal live-imaging, we demonstrate vehiculation and cellular uptake of fluorescently-labelled cargoes such as Tacrolimus or Beclomethasone. We systematically compared TFB vehiculation to existing in vitro delivery approaches. TFB vehiculation promoted sustained retention of functionally effective Tacrolimus, strong co-localization with surfactant lipids, and accumulation in macrophages in human LoCs reconstituted with in vitro-differentiated alveolar macrophage-like cells. This contrasted with the unphysiological dominant uptake of formulations in liquid by alveolar epithelial cells. Finally, in proof-of-concept experiments, TFB-vehiculated bedaquiline effectively supressed subsequent growth of Mycobacterium tuberculosis in a prophylactic manner. Overall, our work demonstrates an approach for studying drug vehiculation in models of the alveolar interface in vitro, and the feasibility of surfactant-containing therapeutic formulations for direct and effective pulmonary delivery.

bioengineering↗

bioRxiv2025

A bioengineered human urothelial organoid model reveals the urine-urothelium interplay in tissue resilience and UPEC recurrence in urinary tract infections

Urine is a dynamic and highly variable biofluid. Urine-urothelium interactions are a critical yet underexplored factor in bladder homeostasis and urinary tract infections (UTIs). Here, we report on a human mini-bladder model that exposes a stratified urothelium to urine of defined composition, and incorporates micturition. Prolonged exposure to high-solute concentration urine weakens tight junctions, dysregulates immune responses, and reduces bladder tissue resilience. This increases susceptibility to colonization of the bladder by uropathogenic E. coli (UPEC) which reduces efficacy of antibiotic therapy. In high-solute concentration urine, Fosfomycin monotherapy - prescribed for uncomplicated UTIs, induces the formation of cell wall-deficient (CWD) UPEC in the urine (as observed in patients with recurrent UTIs) but also within deeper urothelial layers. Tissue-associated CWD UPEC directly contributed to recurrence. Our findings expand the conceptual role for CWD UPEC in UTIs, and demonstrate the power of the mini-bladder platform to capture urine-urothelial microenvironment dynamics that actively shape UTI pathogenesis and antibiotic tolerance.

bioengineering↗

bioRxiv2025

Bioenergetic reprogramming of macrophages reduces drug tolerance in Mycobacterium tuberculosis

Eradication of Mycobacterium tuberculosis (Mtb) requires strategies targeting bacteria inside the host. Mtb exhibits heterogeneity in redox metabolism inside macrophages to evade killing by anti-TB drugs. If and how macrophage physiology correlates with bacterial redox heterogeneity and drug tolerance remains unclear. Using a fluorescent reporter of mycobacterial redox potential, flow sorting, and RNA sequencing of infected macrophages, we characterized transcriptional and metabolic responses of macrophages harboring redox-diverse Mtb populations. We found that macrophages with suppressed glycolysis and elevated oxidative phosphorylation (OXPHOS) correlated with Mtb populations exhibiting reductive stress and drug tolerance. Conversely, macrophages with elevated glycolysis and suppressed OXPHOS displayed higher mitochondrial reactive oxygen species through reverse electron transport, resulting in oxidative stress in Mtb and enhancing drug efficacy. Computational and genetic approaches identified Nrf2 as a key regulator of macrophage bioenergetics driving redox heterogeneity and drug tolerance in Mtb. Redirecting macrophage metabolism from OXPHOS to glycolysis using an FDA- approved antiemetic drug, meclizine, subverted redox heterogeneity and diminished drug tolerance in macrophages and mice. The pharmacological profile of meclizine (Cmax and AUClast) indicated no adverse interactions with first-line anti-TB drugs in mice. Our data demonstrate the feasibility of reprogramming macrophage metabolism to reduce drug tolerance in Mtb infection.

microbiology↗

bioRxiv2021

Dynamic persistence of intracellular bacterial communities of uropathogenic Escherichia coli in a human bladder-chip model of urinary tract infections

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 harbours of bacterial persistence, with significant consequences for non-compliance with antibiotic regimens.

microbiology↗

bioRxiv2020

Invasion into the bladder wall generates solitary subpopulations of uropathogenic Escherichia coli that are protected from killing by antibiotics and neutrophil swarms in an organoid model

Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infections (UTIs) requiring antibiotic therapy. Recurrent infections, which occur in a quarter of treated individuals, may arise from "quiescent intracellular reservoirs" of bacteria that invade deeper layers of the bladder wall following infection and exfoliation of superficial umbrella cells. Here, we present a novel bladder organoid model of UPEC infection that recapitulates the stratified bladder architecture within a small volume suitable for live-cell imaging of host-pathogen dynamics with high spatiotemporal resolution. We confirm that bacteria injected into the organoid lumen rapidly enter superficial cells that resemble umbrella cells and proliferate to generate tightly packed colonies that resemble intracellular bacterial communities (IBCs), a hallmark of UPEC pathogenesis. Unexpectedly, at early stages of infection we detect individual "solitary" bacteria that penetrate deeper layers of the organoid wall, where they evade killing by antibiotics and neutrophils. Volumetric serial block face scanning electron microscopy of infected organoids reveals that solitary bacteria can be found throughout the bladder wall and may be intracellular or pericellular (sandwiched between uroepithelial cells). Unlike bacteria within IBCs, which are coccoid-shaped and non-flagellated, solitary bacteria within the bladder wall are rod-shaped and flagellated. We conclude that early invasion of deeper layers of the bladder wall, independent of IBC formation, results in the establishment of reservoirs of solitary bacteria that resist elimination by antibiotics and the host innate immune response.

microbiology↗

bioRxiv2020

Rapid endothelialitis and vascular inflammation characterise SARS-CoV-2 infection in a human lung-on-chip model

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.

microbiology↗

bioRxiv2020

A lung-on-chip infection model reveals protective and permissive roles of alveolar epithelial cells in tuberculosis

Mycobacterium tuberculosis (Mtb) makes first contact with a host in the alveolar space, an interaction largely inaccessible to experimental observation. We establish a lung-on-chip model for early tuberculosis and use time-lapse imaging to reveal the dynamics of host-Mtb interactions at an air-liquid interface with a spatiotemporal resolution unattainable in animal models. By reconstituting host physiology in a modular manner, we probe the role of pulmonary surfactant secreted by alveolar epithelial cells (AECs) in early infection. This is difficult to study directly in animal models, as surfactant-deficient animals are either non-viable or develop acute lung pathologies. We demonstrate that surfactant deficiency results in rapid and uncontrolled Mtb growth in both macrophages and AECs. 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 1,2 from the bacterial cell surface and consistent with this mechanism of action, we show that attenuation of bacteria lacking the virulence-associated ESX-1 secretion system is independent of surfactant levels. These findings may partly explain why individuals with compromised surfactant function, such as smokers and elderly persons, are at increased risk of developing active tuberculosis.

microbiology↗