Biology subjects

Singanayagam, A.

Publications and source records attributed to Singanayagam, A..

8 recordsLinked to original sources

bioRxiv2025

Neutrophil and NET-driven pulmonary microvascular injury following myocardial injury: attenuation by S100A8/A9 inhibition

Myocardial infarction (MI) triggers not only local cardiac damage but also a systemic inflammatory response that extends to remote organs. The pulmonary microcirculation, by virtue of its dense capillary network and direct anatomical proximity to the heart, is particularly vulnerable. Neutrophils and their effector mechanisms, including neutrophil extracellular traps (NETs) and the alarmin S100A8/A9, have been implicated in adverse cardiovascular outcomes. However, their role in remote damage post-MI remains unclear. Using intravital in vivo imaging in murine MI models and analysis of human lung tissues, we show that MI induces rapid pulmonary neutrophil and platelet recruitment, formation of platelet-neutrophil aggregates within capillaries, and endothelial activation. These changes are accompanied by NET release, fibrin deposition, and microvascular obstruction, leading to impaired vascular perfusion and necrosis. These pulmonary disturbances closely parallel those in the infarcted myocardium and exceed responses observed in other organs such as the kidney and liver, highlighting the lung as a vulnerable target organ. Increased neutrophil recruitment was associated with marked upregulation of the neutrophil-derived, NET-associated alarmin S100A8/A9 in mouse and human lungs, where it co-localised with infiltrating neutrophils, NETs, and platelet aggregates. Additionally, we show that short-term pharmacological inhibition of S100A8/A9 with ABR-238901 significantly attenuated pulmonary neutrophil infiltration, reduced NETosis and fibrin deposition, and restored capillary perfusion while rebalancing the pulmonary immune landscape. Together, these findings identify the lung as a principal site of remote thrombo-inflammatory injury after MI and implicate S100A8/A9, a neutrophil-derived, NET-associated alarmin, as a mechanistic driver of pulmonary microvascular dysfunction. We propose that targeting this pathway could provide dual protection for both cardiac and pulmonary microcirculations in the acute phase of myocardial injury. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/675647v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@289725org.highwire.dtl.DTLVardef@db2011org.highwire.dtl.DTLVardef@16790dorg.highwire.dtl.DTLVardef@1655b17_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Targeting the expansion of myeloid-derived suppressor cells in liver cirrhosis

Background and aimsPreviously, we identified immune-suppressive circulating monocytic myeloid-derived suppressor cells (M-MDSC) in patients with cirrhosis and liver failure, which increased with disease severity and were associated with infections and mortality. Impaired immune responses and M-MDSC expansion were reversed by ex vivo polyinosinic:polycytidylic acid (poly(I:C)) treatment. Here, we aimed to investigate hepatic MDSC subsets in liver biopsies of cirrhotic patients and identify MDSC subsets in murine models to assess the safety and efficacy of poly(I:C) in vivo. Methods22 cirrhotic patients and 4 controls were clinically characterised. MDSC were identified in liver biopsies (immunofluorescence) and in the circulation (flow cytometry). M- MDSC phenotype and function following poly(I:C) stimulation were assessed ex vivo. Carbon tetrachloride-based murine models of liver fibrosis were used. Poly(I:C) was administered therapeutically. MDSC biology was investigated with flow cytometry, immunofluorescence and T-cell proliferation assay. Hepatic histopathology, transcriptomics (BulkRNAseq) and serum markers were assessed. ResultsBesides circulating M-MDSC, hepatic CD14+CD84+M-MDSC and CD15+CD84+ polymorphonuclear-MDSC expanded in cirrhotic patients and indicated disease severity, infections and poor survival. Poly(I:C) treatment reversed phenotype and function of circulating M-MDSC ex vivo. Circulating and hepatic MDSC expanded in our murine models of liver fibrosis and suppressed T-cell proliferation. Lipopolysaccharide and E.coli challenge exacerbated hepatic MDSC and fibrosis compared to CCl4 controls. Poly(I:C) therapy reduced MDSC expansion in fibrotic mice with bacterial infection and CCl4-induced fibrosis. ConclusionHepatic MDSC expanded in cirrhotic patients and were linked with disease severity and poor prognosis. Poly(I:C) reversed frequency and function of M-MDSC ex vivo. Poly(I:C) therapy reversed MDSC expansion and fibrosis in a murine model of liver fibrosis with infection. Thus, we highlighted poly(I:C) as a potential immunotherapy for the treatment of immuneparesis in cirrhosis.

immunology↗

bioRxiv2021

Ultrastructural insight into SARS-CoV-2 attachment, entry and budding in human airway epithelium

Ultrastructural studies of SARS-CoV-2 infected cells are crucial to better understand the mechanisms of viral entry and budding within host cells. Many studies are limited by the lack of access to appropriate cellular models. As the airway epithelium is the primary site of infection it is essential to study SARS-CoV-2 infection of these cells. Here, we examined human airway epithelium, grown as highly differentiated air-liquid interface cultures and infected with three different isolates of SARS-CoV-2 including the B.1.1.7 variant (Variant of Concern 202012/01) by transmission electron microscopy and tomography. For all isolates, the virus infected ciliated but not goblet epithelial cells. Two key SARS-CoV-2 entry molecules, ACE2 and TMPRSS2, were found to be localised to the plasma membrane including microvilli but excluded from cilia. Consistent with these observations, extracellular virions were frequently seen associated with microvilli and the apical plasma membrane but rarely with ciliary membranes. Profiles indicative of viral fusion at the apical plasma membrane demonstrate that the plasma membrane is one site of entry where direct fusion releasing the nucleoprotein-encapsidated genome occurs. Intact intracellular virions were found within ciliated cells in compartments with a single membrane bearing S glycoprotein. Profiles strongly suggesting viral budding from the membrane was observed in these compartments and this may explain how virions gain their S glycoprotein containing envelope.

microbiology↗

bioRxiv2021

Increased transmission of SARS-CoV-2 lineage B.1.1.7 (VOC 2020212/01) is not accounted for by a replicative advantage in primary airway cells or antibody escape

Lineage B.1.1.7 (Variant of Concern 202012/01) is a new SARS-CoV-2 variant which was first sequenced in the UK in September 2020 before becoming the majority strain in the UK and spreading worldwide. The rapid spread of the B.1.1.7 variant results from increased transmissibility but the virological characteristics which underpin this advantage over other circulating strains remain unknown. Here, we demonstrate that there is no difference in viral replication between B.1.1.7 and other contemporaneous SARS-CoV-2 strains in primary human airway epithelial (HAE) cells. However, B.1.1.7 replication is disadvantaged in Vero cells potentially due to increased furin-mediated cleavage of its spike protein as a result of a P681H mutation directly adjacent to the S1/S2 cleavage site. In addition, we show that B.1.1.7 does not escape neutralisation by convalescent or post-vaccination sera. Thus, increased transmission of B.1.1.7 is not caused by increased replication, as measured on HAE cells, or escape from serological immunity.

microbiology↗

bioRxiv2020

Inhaled corticosteroids downregulate the SARS-CoV-2 receptor ACE2 in COPD through suppression of type I interferon

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is a new rapidly spreading infectious disease. Early reports of hospitalised COVID-19 cases have shown relatively low frequency of chronic lung diseases such as chronic obstructive pulmonary disease (COPD) but increased risk of adverse outcome. The mechanisms of altered susceptibility to viral acquisition and/or severe disease in at-risk groups are poorly understood. Inhaled corticosteroids (ICS) are widely used in the treatment of COPD but the extent to which these therapies protect or expose patients with a COPD to risk of increased COVID-19 severity is unknown. Here, using a combination of human and animal in vitro and in vivo disease models, we show that ICS administration attenuates pulmonary expression of the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme (ACE)-2. This effect was mechanistically driven by suppression of type I interferon as exogenous interferon-{beta} reversed ACE2 downregulation by ICS. Mice deficient in the type I interferon-/{beta} receptor (Ifnar1-/-) also had reduced expression of ACE2. Collectively, these data suggest that use of ICS therapies in COPD reduces expression of the SARS-CoV-2 entry receptor ACE2 and this effect may thus contribute to altered susceptibility to COVID-19 in patients with COPD.

immunology↗

bioRxiv2019

MUC5AC drives COPD exacerbation severity through amplification of virus-induced airway inflammation

The respiratory tract surface is protected from inhaled pathogens by a secreted layer of mucus that is rich in mucin glycoproteins. Disrupted mucus production is a cardinal feature of chronic respiratory diseases but how this alteration affect interactions between mucins and pathogens is complex and poorly understood. Here, we identify a central and unexpected role for the major airway mucin MUC5AC in pathogenesis of virus-induced exacerbations of chronic obstructive pulmonary disease (COPD). Virus induction of MUC5AC is augmented in COPD compared to healthy subjects, is enhanced in frequent exacerbators and correlates with inflammation, symptom severity and secondary bacterial infection during exacerbation. MUC5AC is functionally related to inflammation as MUC5AC-deficient (Muc5ac-/-) mice had attenuated rhinovirus-induced airway inflammation whilst exogenous MUC5AC glycoprotein administration augmented virus-induced inflammatory responses and bacterial load. Mechanistically, MUC5AC-augmentation of rhinovirus-induced inflammation occurred through release of extracellular adenosine triphosphate (ATP). Therapeutic suppression of virus-induced MUC5AC release using an epidermal growth factor receptor (EGFR) inhibitor ameliorated exaggerated pro-inflammatory responses in a mouse COPD exacerbation model. Collectively, these studies demonstrate previously unrecognised pro-inflammatory effects of MUC5AC during infection and thus highlight a key unforeseen role in driving COPD exacerbation severity.

immunology↗

bioRxiv2019

Anti-microbial immunity is impaired in COPD patients with frequent exacerbations

BackgroundPatients with frequent exacerbations represent a chronic obstructive pulmonary disease (COPD) sub-group requiring better treatment options. The aim of this study was to determine the innate immune mechanisms that underlie susceptibility to frequent exacerbations in COPD.\n\nMethodsWe measured sputum expression of immune mediators and bacterial loads in samples from patients with COPD at stable state and during virus-associated exacerbations. Ex vivo immune responses to rhinovirus infection in differentiated bronchial epithelial cells (BECs) sampled from patients with COPD were additionally evaluated. Patients were stratified as frequent exacerbators ([≥]2 exacerbations in the preceding year) or infrequent exacerbators (<2 exacerbations in the preceding year) with comparisons made between these groups.\n\nResultsFrequent exacerbators had reduced sputum cell mRNA expression of the anti-viral immune mediators type I and III interferons and reduced interferon-stimulated gene (ISG) expression when clinically stable and during virus-associated exacerbation. RV-induction of interferon and ISGs ex vivo was also impaired in differentiated BECs from frequent exacerbators. Frequent exacerbators also had reduced sputum levels of the anti-microbial peptide mannose-binding lectin (MBL)-2 with an associated increase in sputum bacterial loads at 2 weeks following virus-associated exacerbation onset. MBL-2 levels correlated negatively with bacterial loads during exacerbation.\n\nConclusionThese data implicate deficient airway innate immunity in the increased propensity to exacerbations observed in some patients with COPD. Therapeutic approaches to boost innate antimicrobial immunity in the lung could be a viable strategy for prevention/treatment of frequent exacerbations.

immunology↗

bioRxiv2019

Use of a pandemic H1N1 strain with updated haemagglutinin and neuraminidase results in increased nasopharyngeal shedding and improved immunogenicity to Russian-backbone live attenuated influenza vaccine among children aged 2 - 4 years old: an open label, prospective, observational, phase 4 study in The Gambia.

BackgroundPoor efficacy and effectiveness of the pandemic H1N1 (pH1N1) component in intranasal live attenuated influenza vaccine (LAIV) has been demonstrated in several studies. The reasons for this are unclear, but may be due to impaired replicative fitness of pH1N1 A/California/07/2009-like (Cal09) LAIV strains. MethodsIn an open-label, prospective, observational, phase 4 study, we evaluated the impact of updating the pH1N1 component in the Russian-backbone trivalent LAIV from Cal09 in 2016-17 (n=118) to an A/Michigan/45/2015-like strain (A/17/New York/15/5364, NY15) in 2017-18 (n=126), on shedding and immunogenicity in Gambian children aged 2-4 years old. The study was nested within a larger randomised controlled trial investigating LAIV-microbiome interactions (ClinicalTrials.gov NCT02972957). FindingsCal09 showed impaired nasopharyngeal shedding compared to H3N2 and influenza B, along with sub-optimal serum antibody and T-cell responses. Following the switch to NY15, a significant increase in pH1N1 shedding was seen, along with improvements in seroconversion and influenza-specific CD4+ T-cell responses. Viral kinetics in vitro mirrored these findings, with NY15 showing greater replicative ability than Cal09 in human nasal epithelial cells. Persistent shedding to day 7 was independently associated with both seroconversion and CD4+ T cell response in multivariable logistic regression. InterpretationOur results suggest that the pH1N1 component switch in LAIV may have overcome problems in prior formulations. LAIV effectiveness against pH1N1 should therefore improve in upcoming influenza seasons. Our data also highlight the importance of evaluating replicative fitness, in addition to antigenicity, when selecting annual LAIV components and design of potentially more effective vaccines. FundingThe Wellcome Trust.

clinical trials↗