Biology subjects

Renshaw, S.

Publications and source records attributed to Renshaw, S..

10 recordsLinked to original sources

bioRxiv2025

Verapamil limits inflammation by restoring VGCC-driven epithelial Ca2+ in models of cystic fibrosis

RationaleIn cystic fibrosis (CF), caused by mutations in the CFTR gene, excessive neutrophilic inflammation drives lung damage and premature mortality. Current anti-inflammatory therapies have limited efficacy, partly due to an incomplete understanding of the mechanisms underlying CF-associated inflammation. Increased epithelial Ca{superscript 2} signaling has been implicated, but how CFTR dysfunction perturbs the CFTR/Ca{superscript 2} axis to promote inflammation remains unclear. ObjectivesUsing Cftr-depleted zebrafish and human approaches, we sought to i) define how CFTR dysfunction alters epithelial Ca{superscript 2} signaling and promotes inflammation, and ii) assess whether Ca{superscript 2} channel antagonists can attenuate inflammatory damage in CF. Methods and ResultsWe generated a transgenic zebrafish line expressing the Ca{superscript 2} reporter GCaMP6 under an epithelial promoter to dynamically map epithelial Ca{superscript 2} activity in vivo. Cftr-depleted zebrafish exhibited exaggerated epithelial Ca{superscript 2} elevations following injury compared with wild-type animals. Genetic or pharmacological inhibition of L-type voltage-gated Ca{superscript 2} channels (VGCCs) normalized epithelial Ca{superscript 2} responses and reduced both oxidative stress and neutrophil recruitment. Among FDA-approved VGCCs antagonists tested, only verapamil promoted resolution of neutrophilic inflammation and improved tissue repair in CF zebrafish. Mechanistically, verapamil reduced aberrant epithelial Ca{superscript 2} fluxes, ROS generation and NF-{kappa}B activation. These effects were confirmed in the human CF epithelial cell CFBE41o-, where verapamil reduced Ca2+ and oxidative stress. ConclusionsThese findings establish dysregulated Ca{superscript 2} signaling via L-type VGCCs as a pathogenic driver of inflammation in CF and identify verapamil as a promising therapeutic candidate. By restoring Ca{superscript 2} and oxidative homeostasis, verapamil alleviates inflammatory damage, supporting its repurposing as a candidate anti-inflammatory therapy in CF.

immunology↗

bioRxiv2024

Curcumin-mediated NRF2 induction limits inflammatory damage in preclinical models of cystic fibrosis

Overactive inflammation is directly correlated with airway damage and early death in individuals with cystic fibrosis (CF), a genetic disorder caused by mutation in the CFTR gene. Reducing the impact of inflammatory damage is therefore a major concern in CF. Several studies indicate that a decrease in the nuclear factor erythroid 2-related factor-2 (NRF2) signaling in people with CF may hamper their ability to alleviate oxidative stress and inflammation, although the role of NRF2 in CF inflammatory damage has not been determined. Therefore, we examined whether the phytochemical curcumin, an activator of NRF2, might provide a beneficial effect in the context of CF. Herein, combining Cftr-depleted zebrafish larvae as innovative biomedical model with CF patient-derived airway organoids (AOs), we sought to understand how NRF2 dysfunction leads to abnormal inflammatory status and impaired tissue remodeling, and determine the effects of curcumin in reducing inflammation and tissue damage in CF. We demonstrate that NFR2 is instrumental in efficiently regulating inflammatory and repair processes in vivo, thereby preventing acute neutrophilic inflammation and tissue damage. Importantly, curcumin treatment restores NRF2 activity in both CF zebrafish and AOs. Curcumin reduces neutrophilic inflammation in CF context, by rebalancing the production of epithelial ROS and pro-inflammatory cytokines. Furthermore, curcumin alleviates CF-associated tissue remodeling and allows tissue repair to occur. Our findings demonstrate that curcumin reduces inflammatory damage by restoring normal NRF2 activity, since disruption of Nrf2 pathway abrogated the effect of treatment in CF zebrafish. This work highlights the protective role of NRF2 in limiting inflammation and injury, and show that therapeutic strategies to normalize NRF2 activity using curcumin might simultaneously reduce inflammation and enhance tissue repair, and thus prevent infectious and inflammatory lung damage in CF.

immunology↗

bioRxiv2024

Pulses of Class I PI3kinase activity identify the release and recapture of prey from neutrophil phagosomes

Class I PI3kinases coordinate the delivery of microbicidal effectors to the phagosome by forming the phosphoinositide lipid second messenger, phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3). However, the dynamics of PIP3 in neutrophils during a bacterial infection are unknown. We have therefore developed an in vivo, live zebrafish infection model that enables visualisation of dynamic changes in Class 1 PI3kinases (PI3K) signalling on neutrophil phagosomes in real-time. We have identified that on approximately 12% of neutrophil phagosomes PHAkt-eGFP, a reporter for Class 1 PI3K signalling, re-recruits in pulsatile bursts. This phenomenon occurred on phagosomes containing structurally and morphologically distinct prey, including Staphylococcus aureus and Mycobacterium abscessus, and was dependent on the activity of the Class 1 PI3K isoform, PI3kinase {gamma}. Detailed imaging suggested that pulsing phagosomes represent neutrophils transiently reopening and reclosing phagosomes. This finding challenges the concept that phagosomes remain closed after prey engulfment and we propose that neutrophils occasionally use this alternative pathway of phagosome maturation to release phagosome contents and/or to restart phagosome maturation if digestion has stalled.

cell biology↗

bioRxiv2023

Determining the importance of the stringent response for methicillin-resistant Staphylococcus aureus virulence using a zebrafish model of infection

Staphylococcus aureus is a bacterial pathogen that poses a major threat to human health. The ability of this bacterium to adapt to stresses encountered in the host is essential for disease. The stringent response is a signalling pathway utilised by all bacteria to alarm cells when stressed, and has been linked to the virulence of a number of species. This signalling pathway is controlled by the nucleotide alarmones guanosine tetra-(ppGpp) and pentaphosphate (pppGpp: collectively termed (p)ppGpp), produced in S. aureus by three synthetase enzymes: Rel, RelP and RelQ. Here, we used a triple (p)ppGpp synthetase mutant ((p)ppGpp0) to examine the importance of this signalling network for the survival and virulence of S. aureus in vivo. Using an established zebrafish larval infection model, we observed that infection with (p)ppGpp0 resulted in attenuated virulence, which was not due to a reduced ability of the mutant to replicate in vivo. Of the three (p)ppGpp synthetases, Rel was established as key during infection, but roles for RelP and RelQ were also observed. Zebrafish myeloid cell depletion restored the virulence of (p)ppGpp0 during systemic infection, indicating that (p)ppGpp is important for survival within host phagocytes. Primary macrophages infection studies, followed by in vitro tolerance assays to key innate immune effectors, demonstrated that (p)ppGpp0 was more susceptible to stressors found within the intracellular macrophage environment, with roles for all three synthetases implicated. Lastly, the absence of CodY, a transcription factor linked to the stringent response, significantly increased the tolerance of S. aureus to phagolysosomal-like stressors in vitro, but had no impact in vivo. Taken together, these results define the importance of the stringent response for S. aureus infection, revealing that (p)ppGpp produced by all three synthetases is required for bacterial survival within the host environment by mediating adaptation to the phagolysosome.

microbiology↗

bioRxiv2023

Analysis of the Distal Urinary Tract in Larval and Adult Zebrafish Reveals Unrecognized Homology to the Human System

Little is known about the distal excretory component of the urinary tract in Danio rerio (zebrafish). This component is affected by many human diseases and disorders of development. Here, we undertook multi-level analyses to determine the structure and composition of the distal urinary tract in the zebrafish. In silico searches identified uroplakin 1a, 2-like and 3-like genes in the zebrafish genome (orthologues to genes that encode proteins that characterise the urothelium in humans). In situ hybridization demonstrated uroplakin-1a expression in the zebrafish pronephros and cloaca from 96 hours post-fertilisation. H&E sectioning of adult zebrafish demonstrated two mesonephric ducts uniting into a urinary bladder which leads to a distinct urethral opening. Immunohistochemistry identified Uroplakin 1a and 2 expression within the urothelial luminal surface and Gata3 expression in cell layers that match human urothelial expression. Fluorescent dye injections demonstrated urinary bladder function, including urine storage, intermittent micturition, and a urethral orifice separate from the larger anal canal and rectum. Our findings reveal homology between the urinary tracts of zebrafish and humans and offer the former as a model system to study disease. Summary StatementThe excretory components of the distal urinary tract in larval and adult zebrafish have been incompletely evaluated. Here, we demonstrate close homology in urinary tract anatomy between zebrafish and human, including the presence of a distinct adult urinary bladder capable of periodic micturition

evolutionary biology↗

bioRxiv2022

A p21-GFP zebrafish model of senescence for rapid testing of senolytics in vivo.

Senescence drives the onset and severity of multiple ageing-associated diseases as well as frailty. As a result, there has been an increased interest in mechanistic studies and in the search for compounds targeting senescent cells, known as senolytics. Mammalian models are commonly used to test senolytics and generate functional and toxicity data at the level of organs and systems, yet this is expensive and time consuming. Zebrafish share high homology in genes associated with human ageing and disease. They can be genetically-modified relatively easily. In larvae, most organs develop within 5 days of fertilisation and are transparent, which allows tracking of fluorescent cells in vivo in real time, testing drug off-target toxicity and assessment of cellular and phenotypic changes. Here, we have generated a transgenic zebrafish line that expresses green fluorescent protein (GFP) under the promoter of a key senescence marker, p21. We show an increase in p21:GFP+ cells in larvae following exposure to ionising radiation and with natural ageing. p21:GFP+ cells display other markers of senescence, including senescence-associated {beta}-galactosidase and IL6. The observed increase in senescent cells following irradiation is associated with a reduction in the thickness of muscle fibres and mobility, two important ageing phenotypes. We also show that quercetin and dasatinib, two senolytics currently in clinical trials, reduce the number of p21:GFP+ cells, in a rapid 5-day assay. This model provides an important tool to study senescence in a living organism, allowing the rapid selection of senolytics before moving to more expensive and time-consuming mammalian systems.

pharmacology and toxicology↗

bioRxiv2020

Blood vessel occlusion by Cryptococcus neoformans is a mechanism for hemorrhagic dissemination of infection

Meningitis caused by infectious pathogens are associated with vessel damage and infarct formation, however the physiological cause is unknown. Cryptococcus neoformans, is a human fungal pathogen and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of cases, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model for in vivo live imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on there size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a single or small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel stifness via cell junctions and found increased stiffness resulted in increased dissemination. Therefore, global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction more generally in infective meningitis.

pathology↗

bioRxiv2019

Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation

Neutrophilic inflammation with prolonged neutrophil survival is common to many inflammatory conditions, including chronic obstructive pulmonary disease (COPD). There are few specific therapies that reverse neutrophilic inflammation, but uncovering mechanisms regulating neutrophil survival is likely to identify novel therapeutic targets. Screening of 367 kinase inhibitors in human neutrophils and a zebrafish tail fin injury model identified ErbBs as common targets of compounds that accelerated inflammation resolution. The ErbB inhibitors gefitinib, CP-724714, erbstatin and tyrphostin AG825 significantly accelerated apoptosis of human neutrophils, including neutrophils from people with COPD. Neutrophil apoptosis was also increased in Tyrphostin AG825 treated-zebrafish in vivo. Tyrphostin AG825 decreased peritoneal inflammation in zymosan-treated mice, and increased lung neutrophil apoptosis and macrophage efferocytosis in a murine acute lung injury model. Tyrphostin AG825 and knockdown of egfra and erbb2 by CRISPR/Cas9 reduced inflammation in zebrafish. Our work shows that inhibitors of ErbB kinases have therapeutic potential in neutrophilic inflammatory disease.

immunology↗

bioRxiv2017

Dissemination of Cryptococcus neoformans via localised proliferation and blockage of blood vessels

Cryptococcus neoformans is an opportunistic fungal pathogen that can cause life-threatening cryptoccocal meningitis, predominantly within immunocompromised individuals. Cortical infarcts are observed in as many as 30% of cryptococcal meningitis cases, being particularly common in severe infection. Limited clinical case studies suggest infarcts are secondary to vasculitis and blood vessel damage caused by cryptococcal infection. However, the cause of infarcts in cryptococcal infection has not been determined. To examine potential causes of vascular damage and cryptococcal dissemination in cryptococcal infection, the zebrafish C. neoformans infection model was used. We demonstrate that spread of cryptococci from the vasculature occurs at sites where cryptococci grow within the blood vessels, originating from a single or small number of cryptococci. We find that cryptococcal cells become trapped within the vasculature and can proliferate there resulting in vasodilation. Localised cryptococcal growth in the vasculature is also associated with sites of dissemination - in some cases simultaneously with a loss of blood vessel integrity. Using a cell-cell junction protein reporter (VE-cadherin) we identified sites dissemination associated with both intact blood vessels and where vessel rupture occurred. Thus, we have identified a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of the vascular damage and cortical infarction observed in cryptococcal meningitis.\n\nAuthor summaryHuman infection by the fungal pathogen, Cryptococcus neoformans, can lead to life-threatening cryptococcal meningitis. In severe cases of cryptococcal meningitis, a lack of blood supply can cause tissue death and a resulting area of dead tissue (infarct) in the brain. Although vasculature inflammation in known to occur in cryptococcal meningitis, the cause of infarcts in unknown. Using a zebrafish model of cryptococcal infection, the growth and dissemination of fungal cells was observed over time. We show that cryptococcal cells become trapped and proliferate in the vasculature, resulting in cryptococcoma that damage the blood vessels. We propose that vessel damage results from increased blood pressure caused by cryptococci blocking blood vessels suggesting that the vascular damage that ensues on cryptococcoma formation may in turn be a cause of infarct formation seen in cryptococcal meningitis.

microbiology↗

bioRxiv2017

Fungal derived 15-keto-prostaglandin E2 and host proliferator-activated receptor gamma (PPAR-γ) promote C. neoformans growth during infection.

Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants{Delta} plb1 and{Delta} lac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-{gamma}. C. neoformans infection of macrophages activates host PPAR-{gamma} and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-{gamma} in cryptococcosis. Author SummaryCryptococcus neoformans is an opportunistic fungal pathogen that is responsible for significant numbers of deaths in the immunocompromised population worldwide. Here we address whether eicosanoids produced by C. neoformans manipulate host innate immune cells during infection. Cryptococcus neoformans produces several eicosanoids that are notable for their similarity to vertebrate eicosanoids, it is therefore possible that fungal-derived eicosanoids may provoke physiological effects in the host. Using a combination of in vitro and in vivo infection models we identify a specific eicosanoid species - prostaglandin E2 - that is required by C. neoformans for growth during infection. We subsequently show that prostaglandin E2 must be converted to 15-keto-prostaglandin E2 within the host before it has these effects. Furthermore, we find that prostaglandin E2/15-keto-prostaglandin E2 mediated virulence is via activation of host PPAR-{gamma} - an intracellular eicosanoid receptor known to interact with 15-keto-PGE2.

microbiology↗