Biology subjects

Fleming, I.

Publications and source records attributed to Fleming, I..

24 records · Page 2Linked to original sources

bioRxiv2023

Pro-inflammatory feedback loops define immune responses to pathogenic lentivirus infection

HIV causes chronic inflammation and AIDS in humans, though the rate of disease progression varies between individuals. Similarly, simian lentiviruses vary in their pathogenicity based on characteristics of both the host (simian species) and virus strain. Here, we profile immune responses in pig-tailed macaques infected with variants of SIV that differ in virulence to understand the immune mechanisms underlying lentiviral pathogenicity. Compared to a minimally pathogenic lentiviral variant, infection with a highly pathogenic variant results in a more delayed, broad, and sustained activation of inflammatory pathways, including an extensive global interferon signature. Conversely, individual cells infected with highly pathogenic lentivirus upregulated fewer interferon-stimulated genes at a lower magnitude, indicating that highly pathogenic lentivirus has evolved to partially escape from interferon responses. Further, we identified distinct gene co-expression patterns and cell-cell communication pathways that implicate CXCL10 and CXCL16 as important molecular drivers of inflammatory pathways specifically in response to highly pathogenic lentivirus infection. Immune responses to highly pathogenic lentivirus infection are characterized by amplifying regulatory circuits of pro-inflammatory cytokines with dense longitudinal connectivity. Our work presents a model of lentiviral pathogenicity where failures in early viral control mechanisms lead to delayed, sustained, and amplifying pro-inflammatory circuits, which has implications for other viral infections with highly variable disease courses.

immunology↗

bioRxiv2023

Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

Myocarditis is a challenging inflammatory disease of the heart and better understanding of its etiology is needed to develop specific drug therapies. Epoxyeicosatrienoic acids (EETs), active molecules synthesized by cytochrome P450 enzymes from arachidonic acids and hydrolyzed to less active dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH), have been attributed anti-inflammatory activity. Here, we investigated whether EETs have immunomodulatory activity and exert protective effects on coxsackie B3 virus (CVB3)-induced myocarditis. Viral infection altered eicosanoid epoxide and diol levels in both patients with myocarditis and, in the murine heart, and correlated with the increased expression and activity of the sEH after CVB3 infection. Administration of a sEH inhibitor prevented CVB3-induced cardiac dysfunction and inflammatory infiltration. Importantly, EET/sEH inhibitor treatment attenuated vial infection/ improved viral resistance by activating type I interferon (IFN) signaling. At the molecular level, EETs enhanced the interaction between glycogen synthase kinase 3{beta} (GSK3{beta}) and TANK-binding kinase 1 (TBK1) to promote IFN-{beta} production. Our findings revealed that EETs and sEH inhibitors prevent the progress of CVB3-induced myocarditis, particularly by promoting viral resistance by increasing IFN production.

immunology↗

bioRxiv2022

A novel role for cystathionine gamma lyase in the control of p53: impact on endothelial senescence and metabolic reprograming

AimsAdvanced age is unequivocally linked to the development of cardiovascular disease, however, the mechanisms leading to loss of endothelial cell regenerative capacity during aging remain poorly understood. Here we aimed to investigate novel mechanisms involved in endothelial cell senescence, that impact on endothelial cell transcription and the vascular repair response upon injury Methods and resultsRNA sequencing of a unique collection of native endothelial cells from young and aged individuals, showed that aging (20 vs. 80 years) is characterized by p53- mediated reprogramming to promote the expression of senescence-associate genes. Molecular analysis revelead that p53 accumulated and acetylated in the nucleus of aged human endothelial cells to suppress glycolysis. Metabolic flux analysis identified an associated reduction in glucose uptake and ATP availability that inhibited the assembly of the telomerase complex, which was essential for proliferation. Nuclear translocation of p53 in aged endothelial cells was attributed to the loss of the vasoprotective enzyme, cystathionine {gamma}-lyase (CSE), which physically anchored p53 in the cytosol. In mice, loss of endothelial cell CSE activated p53 and arrested vascular repair upon injury, while the AAV9 mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in human native aged endothelial cells maintained low p53 activity and re-activated telomerase to revert endothelial cell senescence. ConclusionOur data identified the interaction between CSE and p53 as a promising target to preserve vascular regeneration during aging. Key QuestionTo identify the mechanisms that regulate endothelial cell senescence under native conditions and their impact on vascular repair in aging. Key FindingLack of a physical interaction between CSE and p53 metabolically reprogrammes endothelial cells to reduce telomerase activity and halt endothelial cell regeneration. Take home messageInterventions to increase CSE expression represent a novel therapy against p53-induced endothelial cell cycle arrest and senescense Translational perspectiveEndothelial rejuvenation strategies could serve as promising therapies against age-related cardiovascular diseases. By investigating human native endothelial cells from young and aged individuals, we identified that the age-related nuclear accumulation of p53 reprograms endothelial cell metabolism, regulates telomerase activity and inhibits endothelial cell regeneration. Nuclear localization of p53 resulted from a loss of its interaction with the cysteine catabolizing enzyme cystathionine {gamma}-lyase in the cytoplasm. Enhancing the physical interaction of p53 with CSE by gene therapy could revert endothelial cell senescence and activate endothelial reparative responses.

cell biology↗

bioRxiv2021

Clinical implementation of single-cell RNA sequencing using liver fine needle aspirate tissue sampling and centralized processing captures compartment specific immuno-diversity

Blood samples are frequently collected in human studies of the immune system but poorly represent tissue-resident immunity. Understanding the immunopathogenesis of tissue-restricted diseases, such as chronic hepatitis B, necessitates direct investigation of local immune responses. We developed a workflow that enables frequent, minimally invasive collection of liver fine-needle aspirates in multi-site international studies and centralized single-cell RNA sequencing data generation using the Seq-Well S3 picowell-based technology. All immunological cell types were captured, including liver macrophages, and showed distinct compartmentalization and transcriptional profiles, providing a systematic assessment of the capabilities and limitations of peripheral blood samples when investigating tissue-restricted diseases. The ability to electively sample the liver of chronic viral hepatitis patients and generate high-resolution data will enable multi-site clinical studies to power fundamental and therapeutic discovery.

immunology↗

bioRxiv2021

Single-cell profiling of environmental enteropathy reveals signatures of epithelial remodeling and immune activation in severe disease

Environmental enteropathy (EE) is a subclinical condition of the small intestine that is highly prevalent in low- and middle-income countries. It is thought to be a key contributing factor to childhood malnutrition, growth-stunting, and diminished oral vaccine responses. While EE has been shown to be the by-product of recurrent enteric infection, its full pathophysiology remains unclear. Here, we mapped the cellular and molecular correlates of EE severity by performing high-throughput single-cell RNA-sequencing on 33 small intestinal biopsies from 11 adults with EE from Lusaka, Zambia (8 HIV-negative, 3 HIV-positive), 6 adults without EE in Boston, USA, and 2 adults from Durban, South Africa, which we complemented with published data from 3 additional South African adults from the same clinical site. By using these data to reanalyze previously-defined bulk-transcriptomic signatures of reduced villus height and decreased plasma LPS levels in EE, we found that these signatures may be driven by an increased abundance of surface mucosal cells - a gastric-like subset previously implicated in epithelial repair in the gastrointestinal tract. In addition, we identified several cell subsets whose fractional abundances associate with histologically determined EE severity, small intestinal region, and HIV infection. Furthermore, by comparing distal duodenal EE samples with those from three control cohorts, we identified dysregulated WNT and MAPK signaling in the EE epithelium and a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells but with increased pro-inflammatory cytokine expression in the EE cohort. Altogether, our work illuminates epithelial and immune correlates of EE and provides new molecular targets for intervention. One Sentence SummaryUsing single-cell RNA-sequencing, we characterize the pathophysiology of environmental enteropathy (EE) - a highly prevalent condition of the small intestine that is thought to be a primary cause of global growth-stunting cases and a key contributing factor to childhood malnutrition and diminished oral vaccine responses - to derive insights into the epithelial and immune correlates of disease severity, suggesting new therapeutic targets for future investigation.

genomics↗

bioRxiv2020

Leukocyte dynamics after intracerebral hemorrhage in a living patient reveal rapid adaptations to tissue milieu

Intracerebral hemorrhage (ICH) is a devastating form of stroke with a high mortality rate and few treatment options. Discovery of therapeutic interventions has been slow given the challenges associated with studying acute injury, particularly over time, in the human brain. Inflammation induced by exposure of brain tissue to blood appears to be a major part of brain tissue injury. Here we longitudinally profiled blood and cerebral hematoma effluent from a patient enrolled in the Minimally Invasive Surgery with Thrombolysis in Intracerebral Haemorrhage Evacuation (MISTIEIII) trial, offering a rare window into the local and systemic immune responses to acute brain injury. Using single-cell RNA-sequencing, we characterized the local cellular response during ICH in the brain of a living patient at single-cell resolution for the first time. Our analysis revealed rapid shifts in the activation states of myeloid and T cells in the brain over time, suggesting that leukocyte responses are dynamically reshaped by the hematoma microenvironment. Interestingly, the patient had an asymptomatic re-bleed (second local exposure to blood) that our transcriptional data indicated occurred more than 30 hours prior to detection by CT scan. This case highlights the rapid immune dynamics in the brain after ICH and suggests that sensitive methods like scRNA-seq can inform our understanding of complex intracerebral events.

immunology↗