Biology subjects

Wright, H. L.

Publications and source records attributed to Wright, H. L..

4 recordsLinked to original sources

bioRxiv2025

Cytokine-Induced Transcriptional Changes in Human Neutrophils Reveal Immune Regulatory Plasticity

Neutrophils are the major cellular constituent of blood leukocytes and play a central role in the inflammatory response, producing an array of destructive molecules and antimicrobial proteases that characterise the cells as front-line defenders of the innate immune system, crucial to host defence. It is now appreciated that neutrophils produce and respond to a variety of inflammatory signals and are able to regulate both the innate and adaptive immune responses. However, the mechanisms by which neutrophils respond to different inflammatory signals to regulate their own function and the functions of other immune cells are incompletely defined. In this study, we performed RNA sequencing with bioinformatics analysis of healthy human neutrophils exposed for 1h to a range of pro-inflammatory cytokines. GM-CSF and TNF induced significant changes in expression in the most transcripts including activation of genes regulating apoptosis and genes encoding cytokines and chemokines that can drive the differentiation and activation of CD4 T cells. Stimulation of neutrophils with G-CSF, IFN, IFN{gamma}, IL-1{beta}, or IL-8 resulted in expression of discrete gene sets and differential activation of signalling pathways including changes in cell adhesion and migration, immune receptor expression, apoptosis, and production of pro-inflammatory prostaglandins. This work defines the differential gene expression patterns in neutrophils exposed to different regulatory cytokines. This is important in both increasing our understanding of the role of neutrophils in driving innate and adaptive immune responses and, importantly, for deconvoluting the neutrophil gene expression signatures observed in inflammatory diseases.

immunology↗

bioRxiv2022

LAT1 enables T cell activation under inflammatory conditions: a new therapeutic target for rheumatoid arthritis

ObjectiveTo assess the L-type amino acid transporter-1 (LAT1) as a possible therapeutic target for rheumatoid arthritis (RA). MethodsSynovial LAT1 expression was monitored by immunohistochemistry and transcriptomic datasets. The contribution of LAT1 to gene expression and immune synapse formation was assessed by RNA-sequencing and total internal reflection fluorescent (TIRF) microscopy, respectively. Mouse models of RA were used to assess the impact of therapeutic targeting of LAT1. ResultsLAT1 was strongly expressed by CD4+ T cells in the synovial membrane of patients with active RA and the level of expression correlated with levels of ESR and CRP as well as DAS-28 scores. Deletion of LAT1 in murine CD4+ T cells inhibited the development of experimental arthritis and prevented the differentiation of CD4+ T cells expressing IFN-{gamma} and TNF-, without affecting regulatory T cells. LAT1 deficient CD4+ T cells demonstrated reduced transcription of genes associated with TCR/CD28 signalling, including Akt1, Akt2, Nfatc2, Nfkb1 and Nfkb2. Functional studies using TIRF microscopy revealed a significant impairment of immune synapse formation with reduced recruitment of CD3{zeta} and phospho-tyrosine signalling molecules in LAT1 deficient CD4+ T cells from the inflamed joints but not the draining lymph nodes of arthritic mice. Finally, it was shown that a small molecule LAT1 inhibitor, currently undergoing clinical trials in man, was highly effective in treating experimental arthritis in mice. ConclusionsIt was concluded that LAT1 plays a critical role in activation of pathogenic T cell subsets under inflammatory conditions and represents a promising new therapeutic target for RA. Key MessagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LILAT1 is an amino acid transporter that has previously been shown to play a role in T cell activation. C_LI What does this study add?O_LILAT1 is expressed by synovial T cells in human rheumatoid arthritis and the level of expression correlates with disease severity. C_LIO_LILAT1 expression by T cells is necessary for development of severe arthritis in animal models. C_LIO_LILAT1 is required for immune synapse formation and activation of pathogenic CD4+ T cell subsets in the inflamed joint, but not the lymph nodes. C_LIO_LIA small molecular weight LAT1 inhibitor, currently in clinical trials for cancer, is highly effective in animal models of rheumatoid arthritis. C_LI How might this impact on clinical practice of future developments?O_LIThe context-specific nature of LAT1 involvement in T cell activation positions it as an ideal therapeutic target to distinguish between pathogenic and protective T cell responses and this study provides the scientific rationale for clinical evaluation of LAT1 inhibitors in the treatment of rheumatoid arthritis. C_LI

immunology↗

bioRxiv2021

An improved culture protocol for the differentiation and survival of human promyelocytic leukaemia PLB-985 cell-line into mature neutrophil-like granulocytes

Circulating blood neutrophils are short-lived, lack proliferation capacity and cannot be transfected in vitro to express exogenous genes or proteins. These properties have made the ex vivo genetic manipulation of neutrophils challenging and hindered biochemical and molecular studies investigating the function of specific genes and proteins. Improved methodology for differentiating cell lines into mature neutrophil-like phenotypes, with similar morphological and functional properties to blood neutrophils would, therefore, be an important tool to probe the molecular properties of mature cells. The PLB-985 cell line was cultured in RPMI-1640 medium supplemented foetal calf serum (FCS) and penicillin/streptomycin. For induction of differentiation into neutrophil-like cells, the medium was supplemented with sodium pyruvate, N, N-dimethylformamide (DMF) and all-trans retinoic acid (ATRA), FCS and penicillin/streptomycin. The cytokines G-CSF and GM-CSF were used to enhance differentiation, prolong viability and delay the progression of the differentiated cells into apoptosis. The modified culture protocol and conditions induced PLB-985 cells to differentiate into mature, neutrophil-like granulocytes that resembled the morphology of mature blood neutrophils as evident by acquisition of a multi-lobed nucleus and granulated cytoplasm. These modified culture conditions resulted in enhanced differentiation into neutrophil-like cells and the apoptosis of these differentiated cells was delayed by supplementation with cytokines. This experimental system should be useful for studies probing the function of specific genes and proteins in human neutrophils.

immunology↗

bioRxiv2020

Type I Interferon regulates cytokine-delayed neutrophil apoptosis, reactive oxygen species production and chemokine expression via activation of p38 MAPK

ABSTRACTInterferons (IFNs) are key regulators of a number of inflammatory conditions in which neutrophils play an important role in pathology, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), where Type I IFNs are implicated in disease pathology. However, IFNs are usually generated in vivo together with other cytokines that also have immunoregulatory functions but such interactions are poorly-defined experimentally. We measured the effects of Type-I IFN (IFNα), elevated in both RA and SLE, on the functions of healthy neutrophils incubated in vitro in the absence and presence of pro-inflammatory cytokines typically elevated in inflammatory diseases (TNFα, GM-CSF). IFNα alone had no effect on neutrophil apoptosis, however it did abrogate the anti-apoptotic effect of GM-CSF (18h, p< 0.01). The enhanced stabilty of the anti-apoptotic protein Mcl-1 and delayed activation of caspase activation normally regulated by GM-CSF were blocked by IFNα: this effect was mediated, in part, by activation of p38 MAPK, increased turnover of the anti-apoptotic protein Mcl-1 and cleavage of caspases. IFNα alone also primed ROS production alone and maintained the transient priming effect of TNF for up to 4h: it also down-regulated GM-CSF and TNFα-activated expression of CXCL1, CXCL2, CXCL3, CXCL8, CCL3 and CCL4, but in contrast increased the expression of CXCL10. These novel data identify complex regulatory signalling networks in which Type I IFNs profoundly alter the response of neutrophils to inflammatory cytokines. This is likely to have important consequences in vivo and may explain the complexity and heterogeneity of inflammatory diseases such as RA, in which multiple cytokine cascades have been activated.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗