Biology subjects

Elks, P. M.

Publications and source records attributed to Elks, P. M..

13 recordsLinked to original sources

bioRxiv2025

During an inflammatory response, zebrafish tnfa and tnfb are expressed by different cell types and have distinct expression kinetics

Tumour necrosis factor (TNF, TNFSF2) is a key orchestrator of inflammation. Though mammalian TNF biology is well studied, the pleotropic nature of this cytokine during inflammation or infection in other vertebrates remains elusive. Interestingly, zebrafish possess two homologues of mammalian TNF, Tnfa and Tnfb. Previous studies have predominantly focused on the role of tnfa during infection or inflammation, while tnfb may also have important roles, yet has remained largely unexplored. Here, we generated and characterized two transgenic reporter lines, Tg(-3.2tnfb:eGFP-F)ump21Tg and Tg(-6.2tnfb:mCherry-F)ump22Tg, marking tnfb expression in zebrafish. By combining our tnfb reporters with other available reporter lines, with high-resolution in vivo microscopy, and with public scRNAseq datasets, we define, in a cell type-specific manner, the expression kinetics of tnfb compared to those of tnfa and il1b. We report on constitutive tnfb expression in neuromast mantle cells from 32 hours post-fertilization onwards, and inducible tnfb expression in subpopulations of macrophages and neutrophils after caudal fin fold amputation and E. coli injection. After amputation, tnfb kinetics of expression are cell-dependent, with expression in macrophages detected prior to that in neutrophils. When analysing the kinetics of expression of tnfb, tnfa and il1b after wounding, our data indicate that, at least in macrophages, a sequential expression occurs, with tnfb being expressed first, followed by tnfa and il1b. Combined, these data show that tnfb has distinct characteristics from tnfa, indicative of a differential role of these cytokines. Our new tnfb reporter lines, set the stage for future studies in which both TNF homologs can be investigated placing zebrafish as an especially suited model to dissect the pleiotropic nature of Tnf in non-mammalian vertebrates.

immunology↗

bioRxiv2025

Telomerase reverse transcriptase is required for resistance to mycobacterial infection

Age is an important risk factor for infections such as tuberculosis (TB). Telomerase is expressed in immune cells yet leukocyte telomere length declines during ageing suggesting an age-dependent loss of telomerase activity in the immune system. Leukocyte telomere length has been correlated with worse outcomes in TB patients, however the mechanisms linking telomere biology to TB susceptibility and response to therapy are unexplored. Here we use the zebrafish-Mycobacterium marinum model to investigate the role of telomerase in TB resistance. We find depletion and inhibition of Tert, the catalytic subunit of telomerase, increases bacterial burden in zebrafish embryos. The Tert depletion infection susceptibility phenotype could not be rescued by p53 or STING depletion suggesting a non-canonical role for Tert in controlling mycobacterial infection. Consistent with a previously described role for Tert in developmental hematopoiesis, we find Tert is necessary for demand-driven emergency myelopoiesis to support containment of extended mycobacterial infection. Our findings establish a previously undescribed role for host telomerase in supporting infection demand-driven hematopoiesis to control infection.

immunology↗

bioRxiv2024

Exploring the role of E. faecalis Enterococcal Polysaccharide Antigen (EPA) and lipoproteins in evasion of phagocytosis

Enterococcus faecalis is an opportunistic pathogen frequently causing nosocomial infections. The virulence of this organism is underpinned by its capacity to evade phagocytosis, allowing dissemination in the host. Immune evasion requires a surface polysaccharide produced by all enterococci, known as the Enterococcal Polysaccharide Antigen (EPA). EPA consists of a cell wall-anchored rhamnose backbone substituted by strain-specific polysaccharides called "decorations", essential for the biological activity of this polymer. However, the structural determinants required for innate immune evasion remain unknown, partly due to a lack of suitable validated assays. Here, we describe a quantitative, in vitro assay to investigate how EPA decorations alter phagocytosis. Using the E. faecalis model strain OG1RF, we demonstrate that a mutant with a deletion of the locus encoding EPA decorations can be used as a platform strain to express heterologous decorations, thereby providing an experimental system to investigate the inhibition of phagocytosis by strain-specific decorations. We show that the aggregation of cells lacking decorations is increasing phagocytosis and that this process does not involve the recognition of lipoproteins by macrophages. Collectively, our work provides novel insights into innate immune evasion by enterococci and paves the way for further studies to explore the structure/function relationship of EPA decorations.

microbiology↗

bioRxiv2023

Candida albicans suppression of neutrophil reactive nitrogen species is rescued by host Hif-1alpha in vivo

Candida albicans is a human commensal that can cause life-threatening invasive infection in immunocompromised individuals. Human immunity to C. albicans infection is thought to be largely dependent on neutrophil reactive oxygen and nitrogen species (ROS/RNS) generation by neutrophils. Despite this, our understanding of innate immune killing and escape by C. albicans is primarily studied in macrophages and the precise mechanisms of evasion are unclear in neutrophils. Here we sought to determine the importance of neutrophil reactive nitrogen species (RNS) production during C. albicans infection in vivo. Using a zebrafish model, we found that C. albicans rapidly downregulated neutrophil RNS below basal levels during the first day post infection, a time at which neutrophil RNS is upregulated in bacterial infections as an important host-defense mechanism, indicating fungal evasion of host neutrophils. We confirmed downregulation of RNS in human primary neutrophils and with clinical Candida spp. isolates, including emerging human pathogens C. auris and C. glabrata. Using a zebrafish arginase 2 transgenic line and a C. albicans car1{Delta} mutant, we show that both host and fungal arginase contribute the reduction in neutrophil RNS. Despite pathogen downregulation, upregulation of neutrophil RNS via Hypoxia inducible factor (Hif)-1 stabilisation, was sufficient to improve C. albicans infection survival, dependent on the presence of neutrophils and Nitric oxide synthase 2 (Nos2). Finally, restoration of neutrophil RNS, via Hif-1 stabilisation, was synergistic with clinically relevant antifungal treatment, increasing survival and clearance of C. albicans infections. Together, these data demonstrate that restoration of the neutrophil RNS response in C. albicans infection improves infection outcomes, highlighting the potential of targeting Hif-1 and RNS in host directed therapies against fungal infections.

immunology↗

bioRxiv2023

Tribbles1 and Cop1 cooperate to protect the host during in vivo mycobacterial infection

Tuberculosis is a major global health problem and is one of the top 10 causes of death worldwide. There is a pressing need for new treatments that circumvent emerging antibiotic resistance. Mycobacterium tuberculosis parasitises macrophages, reprogramming them to establish a niche in which to proliferate, therefore macrophage manipulation is a potential host-directed therapy if druggable molecular targets could be identified. The pseudokinase Tribbles1 (Trib1) regulates multiple innate immune processes and inflammatory profiles making it a potential drug target in infections. Trib1 controls macrophage function, cytokine production and macrophage polarisation. Despite wide-ranging effects on leukocyte biology, data exploring the roles of Tribbles in infection in vivo are limited. Here, we identify that human Tribbles 1 is expressed in monocytes and is upregulated at the transcript level after stimulation with mycobacterial antigen. To investigate the mechanistic roles of Tribbles in the host response to mycobacteria in vivo, we used a zebrafish Mycobacterium marinum (Mm) infection tuberculosis model. Zebrafish Tribbles family members were characterised and shown to have substantial mRNA and protein sequence homology to their human orthologues. trib1 overexpression was host-protective against Mm infection, reducing burden by approximately 50%. Conversely, trib1 knockdown exhibited increased infection. Mechanistically, trib1 overexpression significantly increased the levels of pro-inflammatory factors il-1{beta} and nitric oxide. The host-protective effect of trib1 was found to be dependent on the E3 ubiquitin kinase Cop1. These findings highlight the importance of Trib1 and Cop1 as immune regulators during infection in vivo and suggest that enhancing macrophage TRIB1 levels may provide a tractable therapeutic intervention to improve bacterial infection outcomes in tuberculosis.

immunology↗

bioRxiv2022

An arginase 2 promoter transgenic illuminates anti-inflammatory signalling in zebrafish

The innate immune response to inflammatory stimuli must be finely balanced to produce an appropriate pro-inflammatory response while allowing a subsequent return to homeostasis. In recent years, in vivo transgenic zebrafish models have shed light on the temporal regulation of the pro-inflammatory innate response to immune challenges. However, until now, there have been no zebrafish transgenic models of anti-inflammatory signalling. We compared existing expression data of arginase genes in zebrafish neutrophils and macrophages, strong candidates for an anti-inflammatory marker, and identified that arginase 2 is the most highly expressed Arginase in zebrafish immune cells. We developed an arginase 2 (arg2) bacterial artificial chromosome (BAC) transgenic line, TgBAC(arg2:eGFP)sh571, driving GFP expression under the control of the arg2 promoter. We show that, under resting conditions, arg2:GFP is expressed in ionocytes, matching the in situ hybridisation pattern. Upon immune challenge by injury, bacterial and fungal insults, arg2:GFP is predominantly expressed in neutrophils at early timepoints post-insult. Later in infections, arg2:GFP is expressed in cells associated with foci of infection (including neutrophils and macrophages), alongside liver expression. Our data indicate that arginase 2 is predominantly expressed in neutrophils after immune challenge and suggest that anti-inflammatory signals coincide with pro-inflammatory signals during early wound and infection responses.

immunology↗

bioRxiv2019

Hif-1alpha stabilisation is protective against infection in a zebrafish model of co-morbidity

Multi-drug resistant tuberculosis is a worldwide problem and there is an urgent need for host-derived therapeutic targets, circumventing emerging drug resistance. We have previously shown that hypoxia inducible-1 (Hif-1) stabilisation helps the host to clear mycobacterial infection via neutrophil activation. However, Hif-1 stabilisation has also been implicated in chronic inflammatory diseases caused by prolonged neutrophilic inflammation. Comorbid infection and inflammation can be found together in disease settings, so it is unclear as to whether Hif-1 stabilisation would be beneficial in a holistic disease setting. Here, we set out to understand the effects of Hif-1 on neutrophil behaviour in disease-relevant settings by combining two well-characterised in vivo zebrafish models: TB infection (Mycobacterium marinum infection) and wounding (tailfin transection). We demonstrate during systemic infection, that wounding leads to increased infection burden, but the protective effect of Hif-1 stabilisation remains. A local Mm infection near to the tailfin wound site caused neutrophil migration between sites that was reduced by Hif-1 stabilisation. Our data indicate that the protective effect of Hif-1 against Mm is maintained in the presence of inflammation, highlighting its potential as a host-derived target against TB infection in a disease relevant setting.

immunology↗

bioRxiv2019

Analysis tools to quantify dissemination of pathology in zebrafish larvae

We used the zebrafish larval Mycobacterium marinum infection model of tuberculosis to develop image analysis software called QuantiFish, to enable rapid, objective quantitation of bacterial load and dissemination. Our analysis tools provide novel outcome measures of infection; the proportional distribution of bacteria between sites and their spatial distribution throughout the host. We anticipate the broader application of these parameters to other zebrafish models of infection and metastatic cancer.

microbiology↗

bioRxiv2019

The CXCL12/CXCR4 signalling axis retains neutrophils at inflammatory sites in zebrafish

The inappropriate retention of neutrophils in the lung is a major driver of the excessive tissue damage characteristic of respiratory inflammatory diseases including COPD, ARDS and cystic fibrosis. The molecular programmes which orchestrate neutrophil recruitment to inflammatory sites through chemotactic guidance have been well studied. However, how neutrophil sensitivity to these cues is modulated during inflammation resolution is not understood. The identification of neutrophil reverse migration as a mechanism of inflammation resolution and the ability to modulate this therapeutically has identified a new target to treat inflammatory disease. Here we investigate the role of the CXCL12/CXCR4 signalling axis in modulating neutrophil retention at inflammatory sites. We used an in vivo tissue injury model to study inflammation using transgenic zebrafish larvae. Expression of cxcl12a and cxcr4b during the tissue damage response was assessed using in situ hybridisation and analysis of RNA sequencing data. CRISPR/Cas9 was used to knockdown cxcl12a and cxcr4b in zebrafish larvae. The CXCR4 antagonist AMD3100 was used to block the Cxcl12/Cxcr4 signalling axis pharmacologically. We identified that cxcr4b and cxcl12a are expressed at the wound site in zebrafish larvae during the inflammatory response. Following tail-fin transection, removal of neutrophils from inflammatory sites is significantly increased in cxcr4b and cxcl12a CRISPR knockdown larvae. Pharmacological inhibition of the Cxcl12/Cxcr4 signalling axis accelerates inflammation resolution, an effect caused by an increase in neutrophil reverse migration. The findings of this study suggest that CXCR4/CXCL12 signalling may play an important role in neutrophil retention at inflammatory sites, identifying a potential new target for the therapeutic removal of neutrophils from the lung in chronic inflammatory disease.

immunology↗

bioRxiv2019

Hif-1α stabilisation polarises macrophages via cyclooxygenase/prostaglandin E2 in vivo

Macrophage subtypes are poorly characterised in disease systems in vivo. The initial innate immune response to injury and infectious stimuli through M1 polarisation is important for the outcome of disease. Appropriate macrophage polarisation requires complex coordination of local microenvironmental cues and cytokine signalling to influence immune cell phenotypes. If the molecular mechanisms behind macrophage polarisation were better understood then macrophages could be pharmacologically tuned to better deal with bacterial infections, for example tuberculosis. Here, using zebrafish tnfa:GFP transgenic lines as in vivo readouts of M1 macrophages, we show that hypoxia and stabilisation of Hif-1 polarises macrophages to a tnfa expressing phenotype. We demonstrate a novel mechanism of Hif-1 mediated macrophage tnfa upregulation via a cyclooxygenase/prostaglandin E2 axis, a mechanism that is conserved in human primary macrophages. These findings uncover a novel macrophage HIF/COX/TNF axis that links microenvironmental cues to macrophage phenotype that may have implications in inflammation, infection and cancer, where hypoxia is a common microenvironmental feature and where cyclooxygenase and Tnfa are major mechanistic players.

immunology↗

bioRxiv2019

Non-apoptotic pioneer neutrophils initiate an endogenous swarming response in a zebrafish tissue injury model

Neutrophils are rapidly recruited to inflammatory sites where they coordinate their migration to form clusters, a process termed neutrophil swarming. The factors which modulate neutrophil swarming during its early stages are not fully understood, requiring the development of new in vivo models. Using transgenic zebrafish larvae to study endogenous neutrophil migration in a tissue damage model, we demonstrate that neutrophil swarming is a conserved process in zebrafish immunity, sharing essential features with mammalian systems. We show that neutrophil swarms initially develop around a pioneer neutrophil, in a three-phase sequence of events. By adopting a high-resolution confocal microscopy approach, we observed the release of cell fragments by early swarming neutrophils. We developed a neutrophil specific histone H2A transgenic reporter line TgBAC(mpx:GFP)i114;Tg(lyz:H2A-mCherry)sh530 to study neutrophil extracellular traps (NETs), and found that endogenous neutrophils recruited to sites of tissue damage released NETs at the start of the swarming process. The optical transparency achieved using the zebrafish model has provided some of the highest resolution imaging of NET release in vivo to date. Using a combination of transgenic reporter lines and DNA intercalating agents, we demonstrate that pioneer neutrophils release extracellular traps during the swarming response, suggesting that cell death signalling via NETosis might be important in driving the swarming response.

immunology↗

bioRxiv2018

Macrophage Il-1beta protects against mycobacterial infection downstream of Hif-1alpha in zebrafish

Drug resistant mycobacteria are a rising problem worldwide. There is an urgent need to understand the immune response to TB to identify host targets that, if targeted therapeutically, could be used to tackle these currently untreatable infections. Here, we use an Il-1{beta} fluorescent transgenic line to show that there is an early innate immune pro-inflammatory response to well-established zebrafish models of inflammation and Mycobacterium marinum (Mm) infection. We demonstrate that host-derived hypoxia signalling, mediated by the Hif-1 transcription factor, can prime macrophages with increased levels of Il-1{beta} in the absence of infection, upregulating neutrophil antimicrobial nitric oxide production, leading to greater protection against infection. Our data link Hif-1 to proinflammatory macrophage Il-1{beta} transcription in vivo during early mycobacterial infection and importantly highlight a host protective mechanism, via antimicrobial nitric oxide, that decreases disease outcomes and that could be targeted therapeutically to stimulate the innate immune response to better deal with infections.

immunology↗

bioRxiv2017

Polymersomes Targeting Mononuclear Phagocytes

Mononuclear phagocytes such as monocytes, tissue-specific macrophages and dendritic cells are primary actors in both innate and adaptive immunity, as well as tissue homoeostasis. They have key roles in a range of physiological and pathological processes, so any strategy targeting these cells will have wide-ranging impact. These phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favouring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis, which is the most pandemic and one of the deadliest disease with one third of the worlds population infected, and 1.8 million deaths worldwide in 2015. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to both circulating monocytes and resident macrophages, using pH sensitive nanoscopic polymersomes made of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-co-poly(2-(di-isopropylamino)ethyl methacrylate) (PMPC-PDPA). Polymersome selectivity to mononuclear phagocytes is demonstrated and ascribed to the polymerised phosphorylcholine motifs affinity toward scavenger receptors. Finally, we demonstrate the successful exploitation of this targeting for the effective eradication of intracellular bacteria that cause tuberculosis Mycobacterium tuberculosis as well as other intracellular parasites including the Mycobacterium bovis, Mycobacterium marinum and the most common bacteria associated with antibiotic resistance, the Staphylococcus aureus.

immunology↗