Biology subjects

Keely, S.

Publications and source records attributed to Keely, S..

7 recordsLinked to original sources

bioRxiv2025

A microbiome meta-transcriptomics pipeline identifies a novel human neutrophil elastase inhibitor that protects the colonic epithelial barrier

Inflammatory Bowel Diseases (IBD) are lifelong conditions. Current therapeutic approaches target inflammatory signalling rather than improving barrier permeability or repair. The gut microbiome provides an exciting opportunity for novel drug discovery to leverage its role in healthy gut homeostasis. There is a clear need to identify bioactive molecules within the microbiota that could protect the intestinal barrier. Our group has developed a systematic pipeline using metatranscriptomic data to identify, produce, purify, and test microbial proteins in IBD, pinpointing multiple novel microbiota-derived proteins linked to disease activity. We identified a new microbiota protein (BMG-1), that specifically inhibits human neutrophil elastase, a pathogenic protease in IBD. This protease inhibition allows protection of the intestinal epithelial barrier from permeability and promotes epithelial healing. BMG-1 also reduces colon damage in a mouse model of colitis. These findings demonstrate the gut microbiota can specifically regulate the balance of protease/anti-protease activity in the colon, and this represents a novel therapeutic strategy for IBD.

microbiology↗

bioRxiv2025

Microbiota Modulation Induces Elevated Duodenal Eosinophils Upon Gluten Exposure in Mice: Implications for Non-Coeliac Gluten Sensitivity

A growing proportion of the non-celiac population experience adverse symptoms to gluten. The pathogenesis of non-coeliac gluten sensitivity (NCGS) is unclear, but elevated duodenal eosinophils and altered mucosa-associated microbiota (MAM) populations have been reported. Given the microbiomes role in gluten digestion and its susceptibility to antibiotics, we hypothesised that altering the microbiome with antibiotics would modify immune responses to gluten in mice. BALB/C mice consuming gluten-free chow received amoxicillin/clavulanate (5mg/kg) or PBS-vehicle daily for 5 days. Mice were then treated with a 3mg wheat-gluten suspension, or vehicle, on days 4 and 5 before sacrifice on day 7. Duodenal immune cells were analysed by histology and flow cytometry, while the duodenal MAM and faecal microbiome were characterised via 16S rRNA and shotgun metagenomic sequencing, respectively. Antibiotic treatment followed by gluten reintroduction significantly reduced Staphylococcus in the duodenal MAM, enriched Bacteroides in faeces, and resulted in altered microbial carbohydrate and lipid metabolism, compared to vehicle controls. Treatment with antibiotics and gluten also increased duodenal eosinophils which positively correlated with the genus Blautia. Flow cytometry revealed that antibiotics and gluten treatment resulted in a greater proportion of active eosinophils and epithelial {gamma}{delta} T-cells, compared to vehicle control mice. This study demonstrated that modulating the microbiome with antibiotics was sufficient to alter the immune response to gluten in mice. These findings suggest that the microbiome may determine the capacity for gluten to induce an immune response and offers a valuable insight into potential mechanisms underlying NCGS. New & NoteworthyA mouse model examined how microbial modulation affects immune responses to gluten. Antibiotic treatment followed by gluten reintroduction reduced duodenal Staphylococcus and altered microbial carbohydrate and lipid metabolism pathways in the faecal microbiome. Antibiotics and gluten treatment resulted in increased abundance and activation of duodenal eosinophils, and elevated {gamma}{delta} T-cells in the duodenal epithelium. These findings highlight the role the microbiome plays in gluten-induced immune responses, providing insights into mechanisms behind non-coeliac gluten sensitivity.

immunology↗

bioRxiv2025

Altered Duodenal Mucosa-Associated Microbiota and Immune Profiles in Functional Dyspepsia: A Study of Host-Microbiome Homeostasis

Recent work suggests an altered duodenal mucosa-associated microbiota (MAM) in patients with functional dyspepsia (FD) when compared to outpatient controls, these differences may reflect alterations in host-microbiome homeostasis. Given that specific mucosal immune signatures have been identified in FD, we hypothesised that these changes would be associated with specific microbial changes. We aimed to profile the duodenal MAM to identify microbes associated with known changes in FD mucosal and peripheral immune homeostasis. Duodenal biopsies were collected from 11 asymptomatic outpatient controls and 17 FD patients. Separate biopsies were collected for MAM 16S rRNA amplicon sequencing, histological evaluation, and mucosal lamina propria mononuclear cell (LPMC) isolation. Where available peripheral blood mononuclear cells (PBMC) were isolated from whole blood. PBMC and LPMC populations were analysed for CD4 and CD8 T cell populations by flow cytometry. Initial comparisons of histological and immune measures revealed significant differences between FD and outpatient controls, with decreased villi goblet cells and increased LPMC CD4 Central Memory, LPMC CD8, and PBMC CD4+ Central Memory Th17 in FD compared to controls. While microbiome profiles varied between groups, specific associations with the histological and immunological scoring found that in controls, villi goblet cells correlated positively with Massilia and negatively with Exiguobacterium, while FD patients showed no significant correlations. Additionally, controls exhibited a negative correlation between LPMC CD4 Central Memory and Veillonella, with FD patients showing no significant correlation. Notably, FD patients demonstrated a significant negative correlation between LPMC CD8 and Sulfophobococcus, and a positive correlation between PBMC CD4+ Central Memory Th17 and both Gemella and Fusobacterium. ImportanceWhile several papers have reported the alteration in FD duodenal MAM, study numbers are low and consensus on specific biomarker signatures within the microbiome have not been reached. Our findings contribute to this growing body of evidence, indicating that patients with FD exhibit distinct alterations in duodenal MAM and immune profiles compared to outpatient controls. Furthermore, the immune-microbiome associations present in control populations were absent in FD patients suggesting a loss of host-microbiome homeostasis that may contribute to FD pathophysiology. Our work highlights potential microbial biomarkers which may be a consequence or driver of the mucosal micro-inflammation state which is a characteristic of FD. Future work is required to validate whether these specific microbes are responsible for driving inappropriate host immune responses to inform treatment strategies and assist with disorder diagnosis.

microbiology↗

bioRxiv2024

A rational approach for the targeted discovery and characterisation of microbiome-derived therapeutics

The human gut microbiome is intrinsically involved in health and disease, representing a wealth of untapped therapeutic potential. Here, we demonstrate the utility and potential of a metagenome guided, large cohort-based approach for the rational selection of live biotherapeutics from the human gut. We applied this approach to Inflammatory Bowel Disease (IBD), identifying several lead candidates that were significantly depleted in individuals with IBD compared to healthy controls. Their therapeutic potential was assessed in preclinical models of IBD where they improved markers of disease pathology by reducing inflammation and promoting mucosal healing and wound repair. All leads had excellent safety profiles in silico and in vitro, and several additionally presented favourable manufacturing properties, supporting their progression into clinical trials. We believe that this rational approach will be generalisable to any disease state with underlying microbiome aetiology and will expedite the development of novel microbiome-derived therapeutics to improve human health.

microbiology↗

bioRxiv2024

GSDMD pore formation regulates caspase-4 cleavage to limit IL-18 production in the intestinal epithelium

Epithelial inflammasomes induce pyroptosis and release cytokines to defend against cytosolic pathogens. However, pyroptosis in epithelial barriers must be carefully regulated to facilitate elimination of infected cells while limiting widespread pyroptosis to preserve the single cell barrier. How epithelial cells achieve this is unknown. In this study, we describe a novel epithelial caspase regulation mechanism. By examining caspase-4 activation in human epithelial cells, we discovered that GSDMD pore formation serves as a signal to terminate caspase-4 activity thus facilitating epithelial cell expulsion while controlling cytokine secretion. Inhibition of epithelial pyroptosis led to IL-18 hyperproduction, likely as a mechanism to combat increased pathogen burden and initiate a wider immune response. Moreover, we demonstrate that full-length, rather than cleaved caspase-4 is active against IL-18 and propose that GSDMD pore formation facilitates cleavage of caspase-4 to terminate its catalytic activity. By comparing human cells and murine epithelial organoids to immune cells, we show that GSDMD pore mediated inhibition of caspase activity is largely specific to epithelial cells. Overall, these studies characterise a novel, epithelial-specific negative feedback loop that modulates inflammasome activity and challenge the dogma that autocatalytic caspase cleavage is required for caspase activity against substrates. Graphical AbstractIn intestinal epithelial cells, caspase activation simultaneously leads to GSDMD pore formation and IL-18 release. GSDMD pore formation provides a signal to terminate caspase activity and limit cytokine production. In GSDMD deficient cells, lack of an inhibition signal leads to caspase mediated IL-18 hyperproduction. Upon cell death this leads to release of massive amounts of IL-18. Created with BioRender.com O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/578487v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@46f768org.highwire.dtl.DTLVardef@11cf918org.highwire.dtl.DTLVardef@125be35org.highwire.dtl.DTLVardef@ea9ead_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Hypoxia alters the effects of hypomethylating agents in acute myeloid leukaemia cells.

BackgroundAcute myeloid leukaemia (AML) is a deadly haematological malignancy that originates from mutated myeloid progenitor cells that lie quiescent in the hypoxic bone marrow. Elderly patients who cannot tolerate standard chemotherapies are administered low-dose hypomethylating agents (HMA) which act in a replication-dependent manner to reprogram the epigenome. Relapse is common following HMA treatment and may arise from quiescent leukaemia cells in the hypoxic bone marrow. Therefore, the effects of hypoxia on HMA efficacy may influence AML progression. ResultsAML cell lines (MOLM-13, MV-4-11, HL-60) were treated with decitabine (100nM) or azacitidine (500-2000nM) in normoxic (21% O2) and hypoxic (1% O2) conditions. Exposure to hypoxia significantly reduced AML cell growth across all cell lines, with no additional effects observed upon HMA treatment. This was associated with distinct effects on DNA methylation. The extent of hypomethylation induced by AZA treatment was reduced in hypoxia, whereas DAC-induced hypomethylation was maintained in low oxygen conditions. Transcriptional response to HMA treatment were also altered in hypoxia, with HMAs failing to up-regulate antigen presentation pathways in hypoxia. In particular, human leukocyte antigens (HLAs) such as HLA-DR were increased upon HMA treatment in normoxia, but not hypoxia. ConclusionOur results suggest that HMA-induced antigen presentation may be impaired in hypoxic tissues such as the bone marrow. This study highlights the need to consider microenvironmental factors when designing co-treatment strategies to improve HMA therapeutic efficacy.

cancer biology↗

bioRxiv2023

Fecal microbial transfer and complex carbohydrates mediate protection against COPD

ObjectiveChronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development of effective therapies. The gastrointestinal microbiome has been implicated in chronic lung diseases via the gut-lung axis, but its role is unclear. DesignUsing an in vivo mouse model of cigarette smoke-induced COPD and fecal microbial transfer (FMT), we characterized the fecal microbiota using metagenomics, proteomics and metabolomics. Findings were correlated with airway and systemic inflammation, lung and gut histopathology, and lung function. Complex carbohydrates were assessed in mice using a high resistant starch diet, and in sixteen COPD patients using a randomized, double-blind, placebo-controlled pilot study of inulin supplementation. ResultsFMT alleviated hallmark features of COPD (inflammation, alveolar destruction, impaired lung function), gastrointestinal pathology and systemic immune changes. Protective effects were additive to smoking cessation. Disease features correlated with the relative abundance of Muribaculaceae, Desulfovibrionaceae and Lachnospiraceae family members. Proteomics and metabolomics identified downregulation of glucose and starch metabolism in cigarette smoke-associated microbiota, and supplementation of mice or human patients with complex carbohydrates improved disease outcomes. ConclusionThe gut microbiome contributes to COPD pathogenesis and can be targeted therapeutically. What is already known on this topicO_LIChanges in gut microbiota are associated with COPD but the underlying host and microbial mechanisms are unclear, limiting the therapeutic applications. C_LI What this study addsO_LIMicrobiome composition and metabolism is reproducibly correlated with lung and gastrointestinal pathology in experimental COPD. C_LIO_LIMicrobiome modifying interventions effectively alleviate disease, including protective effects supplementing smoking cessation. C_LIO_LINutritional interventions targeting the microbiome in COPD patients demonstrate efficacy in a small pilot study. C_LI How this study might affect research, practice or policyO_LIMicrobiome-targeting therapeutics and nutritional interventions may be developed for COPD, including as supplements to smoking cessation. C_LI

physiology↗