Biology subjects

Lloyd, C. M.

Publications and source records attributed to Lloyd, C. M..

7 recordsLinked to original sources

bioRxiv2026

Organ on chip model of respiratory vascular interactions under COPD relevant oxidative stress

Oxidative stress-induced airway injury contributes to chronic obstructive pulmonary disease (COPD). Cardiovascular complications increase COPD morbidity and mortality, but mechanistic links between airway injury and vascular dysfunction remain unclear, largely due to limitations of in vitro models that fail to replicate the multicellular lung environment. We developed REVAS, a modular organ-on-chip platform to study human respiratory-vascular cell-cell interactions at baseline and under oxidative stress conditions. REVAS consists of two respiratory chips hosting airway epithelium and microvascular endothelium, and a vascular chip hosting pulmonary artery endothelial cells co-cultured with vascular support cells, including smooth muscle cells, pericytes and fibroblasts. We studied effects of vascular support and respiratory cells on vascular endothelial phenotype at baseline and under H2O2-induced epithelial oxidative stress using functional assays, proteomic and transcriptomic analyses. Multicellular environment enhanced vascular endothelial barrier function and promoted respiratory and vascular cell differentiation at baseline. Mural cells altered endothelial cell-matrix interactions, metabolism and cytoskeletal remodelling, while respiratory cells promoted endothelial aerobic respiration and quiescent phenotype. Epithelial oxidative stress triggered inflammatory gene expression across all respiratory and vascular cells alongside apoptotic, reparative and pro-angiogenic signalling in endothelial and mural cells, accompanied by increased release of COPD-relevant cytokines and chemokines, including IL-6, TNF-/{beta}, IL-8, CCL5, CXCL9, PDGF, TGF-{beta}. Comparative analyses with COPD endothelial datasets confirmed that REVAS recapitulates key features of disease-associated endothelial dysfunction. These findings demonstrate that airway epithelial injury drives downstream vascular responses linked to inflammation and vascular remodelling, establishing REVAS as a human-relevant platform for mechanistic and therapeutic evaluation of cell-cell interactions in COPD and related lung diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/730087v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@e29fd8org.highwire.dtl.DTLVardef@6c3d22org.highwire.dtl.DTLVardef@21a53forg.highwire.dtl.DTLVardef@e7f432_HPS_FORMAT_FIGEXP M_FIG C_FIG REVAS: a microfluidic platform developed to model multicellular interactions between airway epithelium and pulmonary vasculature under basal and oxidative stress. COPD: Chronic Obstructive Pulmonary Disease; EMT: endothelial-to-mesenchymal transition; HsEpCs: human small airway epithelial cells; HPMVECs: human pulmonary microvascular endothelial cells; HPAECs: human pulmonary artery endothelial cells; HPASMCs: human pulmonary artery smooth mucle cells; HPFs: human pulmonary fibcroblasts; HPCs: human pericytes.

bioengineering↗

bioRxiv2025

Differentiation of human T follicular helper cells in vitro requires co-operation between STAT3 and SMAD signaling cytokines that is restrained by IL-2

T follicular helper cells (TFH) are a CD4 T cell subset required for effective humoral immunity; promoting antibody responses to vaccines and natural infections. The mechanisms by which human TFH differentiate are not fully understood, and given the anatomical localization to the secondary lymphoid tissues, and asynchronous nature of in vivo TFH populations, studying TFH differentiation in vitro represents an attractive alternative, through which the mechanisms which govern TFH differentiation can be explored. Here we demonstrate reliable differentiation of in vitro TFH from naive human CD4 T cells in a STAT3 dependent fashion. Cooperation between STAT3 and SMAD signaling cytokines led to expression of the lineage defining transcriptional regulator BCL6, multiple downstream proteins required for TFH function and localization (including PD1 and CXCR5), and potent IL-21 production capacity by these in vitro TFH. Critically we found that autocrine production of IL-2 acted as an intrinsic brake on TFH differentiation in vitro; promoting proliferation but limiting the acquisition of a TFH phenotype resembling that found in germinal centers in vivo; neutralization of IL-2 resulting in the emergence of this phenotype. Overall, we show that human TFH expansion, differentiation and function relies on careful regulation of pro- and anti-TFH cytokine signaling.

immunology↗

bioRxiv2023

Adventitial mast cell activation disrupts endothelial cell/pericyte interactions during early life allergic asthma

Allergic asthma generally starts during early life and is linked to substantial tissue remodelling and lung dysfunction. Although angiogenesis is a feature of the disrupted airway, the impact of allergic asthma on the pulmonary microcirculation during early life is unknown. Here, using quantitative imaging in precision-cut lung slices (PCLS), we report that exposure of neonatal mice to house dust mite (HDM) extract disrupts endothelial cell/pericyte interactions in adventitial areas. Central to the blood vessel structure, the loss of pericyte coverage was driven by mast cell (MCs) proteases, such as tryptase, that can induce pericyte retraction and loss of the critical adhesion molecule N-Cadherin. Furthermore, spatial transcriptomics of paediatric asthmatic endobronchial biopsies suggests intense vascular stress and remodelling linked with increased expression of MC activation pathways in regions enriched in blood vessels. These data provide previously unappreciated insights into the pathophysiology of allergic asthma with potential long-term vascular defects.

immunology↗

bioRxiv2022

Lung extracellular matrix modulates KRT5+ basal cell activity in pulmonary fibrosis

Aberrant expansion of KRT5+ basal cells in the distal lung accompanies progressive alveolar epithelial cell loss and tissue remodelling during fibrogenesis in idiopathic pulmonary fibrosis (IPF). The mechanisms determining activity of KRT5+ cells in IPF have not been delineated. Here, we reveal a potential mechanism by which KRT5+ cells migrate within the fibrotic lung, navigating regional differences in collagen topography. In vitro, KRT5+ cell migratory characteristics and expression of remodelling genes are modulated by extracellular matrix (ECM) composition and organisation. Mass spectrometry-based proteomics revealed compositional differences in ECM components secreted by primary human lung fibroblasts (HLF) from IPF patients compared to controls. Over-expression of ECM glycoprotein, Secreted Protein Acidic and Cysteine Rich (SPARC) in the IPF HLF matrix restricts KRT5+ cell migration in vitro. Together, our findings demonstrate how changes to the ECM in IPF directly influence KRT5+ cell behaviour and function contributing to remodelling events in the fibrotic niche.

cell biology↗

bioRxiv2020

Epigenetic alterations underlie airway macrophage differentiation and phenotype during lung fibrosis

Airway macrophages (AMs) are key regulators of the lung environment and are implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a fatal respiratory disease with no cure. However, the epigenetics of AMs development and function in IPF are limited. Here, we characterised the DNA-methylation (DNAm) profile of AMs from IPF (n=30) and healthy (n=14) donors. Our analysis revealed epigenetic heterogeneity was a key characteristic of IPF AMs. DNAm clock analysis indicated epigenetic alterations in IPF-AMs was not associated with accelerated ageing. In differential DNAm analysis, we identified numerous differentially methylated positions (DMPs, n=11) and regions (DMRs, n=49) between healthy and IPF AMs respectively. DMPs and DMRs encompassed genes involved in lipid (LPCAT1) and glucose (PFKB3) metabolism and importantly, DNAm status was associated with disease severity in IPF. Collectively, our data identify that profound changes in the epigenome underpin the development and function of AMs in the IPF lung.

immunology↗

bioRxiv2020

Comparative primary paediatric nasal epithelial cell culture differentiation and RSV-induced cytopathogenesis following culture in two commercial media.

The culture of differentiated human airway epithelial cells allows the study of pathogen-host interactions and innate immune responses in a physiologically relevant in vitro model. As the use of primary cell culture has gained popularity the availability of the reagents needed to generate these cultures has increased. In this study we assessed two different media, Promocell and PneumaCult, during the differentiation and maintenance of well-differentiated primary nasal epithelial cell cultures (WD-PNECs). We compared and contrasted the consequences of these media on WD-PNEC morphological and physiological characteristics and their responses to respiratory syncytial virus (RSV) infection. We found that cultures generated using PneumaCult resulted in greater total numbers of smaller, tightly packed, pseudostratified cells. However, cultures from both media resulted in similar proportions of ciliated and goblet cells. There were no differences in RSV growth kinetics, although more ciliated cells were infected in the PneumaCult cultures. There was also significantly more IL-29/IFN{lambda}1 secreted from PneumaCult compared to Promocell cultures. In conclusion, the type of medium used for the differentiation of primary human airway epithelial cells impacts experimental results.

cell biology↗

bioRxiv2018

Therapeutic ICOS blockade reduces T follicular helper cells and improves allergic airway disease

Allergic asthma is a disease of chronic airway inflammation and remodelling, characterised by a dysregulated type 2 response and allergen-specific IgE. T follicular helper cells (TFH) are critical to antibody production and have recently been implicated in allergic airway disease (AAD) pathogenesis. The role of TFH in established disease and the therapeutic potential of targeting them are however not fully understood. Using two aeroallergen driven murine models of chronic AAD, TFH were first identified in the lung draining lymph nodes but with prolonged exposure were present in the lung itself. Sustained allergen exposure led to the accumulation of TFH, and concomitant development of germinal centre B cells. Blockade of Inducible T cell co-stimulator (ICOS) signalling during established AAD depleted TFH without adversely affecting the differentiation of other CD4+ T cell subsets. This resulted in impaired germinal centre responses, reduced allergen specific IgE and ameliorated inflammation and airway hyper-responsiveness, including reduced pulmonary IL-13. TFH did not however appear to produce IL-13 directly, suggesting they indirectly promote type-2 inflammation in the lungs. These data show that TFH play a pivotal role in the regulation of AAD and that targeting the ICOS-L pathway could represent a novel therapeutic approach in this disease.

immunology↗