ORCID Profile
0000-0001-6275-2847
Current Organisations
Flinders University
,
University of Adelaide
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Publisher: MDPI AG
Date: 21-01-2022
DOI: 10.3390/JCM11030535
Abstract: Primary Sjögren’s syndrome (SjS) is an inflammatory autoimmune disorder which targets the lacrimal and salivary glands, resulting in glandular dysfunction. Currently, the immune drivers of SjS remain poorly understood and peripheral biomarkers of disease are lacking. The present study therefore sought to investigate the immune cell constituents of the SjS peripheral blood, and to assess the role of the BTLA/HVEM/CD160 co-stimulatory network by characterizing expression within the periphery. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of n = 10 patients with SjS and n = 10 age- and sex-matched healthy control donors. Cells were ided and stained with three panels of antibodies, allowing assessment of T, B, and myeloid cell subsets, and measurement of BTLA, HVEM, and CD160 surface expression by flow cytometry. We identified distinct alterations in proportions of peripheral T, B, and myeloid cell types in SjS compared with healthy controls. Expression of BTLA/CD160/HVEM and frequency of BTLA/CD160/HVEM-expressing cells were significantly altered in peripheral SjS lymphocytes. The proportion of T cells co-expressing BTLA/HVEM and CD160/HVEM were significantly reduced in SjS. We found decreased BTLA and HVEM levels on peripheral B and T cells of SjS patients, and decreased BTLA/HVEM and CD160/HVEM co-expression, demonstrating dysregulation of the BTLA/HVEM axis in the peripheral blood of SjS patients. These results indicate the potential of targeting the BTLA-HVEM axis for the treatment of SjS.
Publisher: Elsevier BV
Date: 10-2018
DOI: 10.1016/J.JIM.2018.06.019
Abstract: Macrophage cell lines are a useful model to explore the properties of primary macrophages. However, a major limitation in the use of these cells is that when they are differentiated, they become adherent and hence present with the same limitation as natural macrophages. The cells need to be detached and are often subjected to detachment techniques such as detachment buffers containing proteolytic enzymes or scraping with a rubber 'policeman'. These steps are time-consuming, reduce cell yields as well as cell viability and function. We have therefore investigated the possibility of differentiating the human macrophage THP-1 cell line in polystyrene FACS tubes to enable cells to be directly used for investigations by flow cytometry. Here we demonstrate that when the human macrophage cell line THP-1 are cultured in FACS tubes with phorbol myristate acetate added, they undergo differentiation into macrophages, assessed morphologically and by autofluorescence expression, in a similar manner to those cultured in tissue culture dishes. The cells can be readily washed and adjusted in concentration by centrifugation in the same tubes and can be directly tested for expression of cell surface markers and function by flow cytometry. This avoids the use of either detachment reagents or physical cell scraping. Consequently, we showed that the tube culture method results in increased cell yield and viability compared to those subjected to detachment procedures. The tube method generated functional macrophages which expressed the complement receptors, CR3 and CR4, and effectively phagocytosed complement opsonised Staphylococcus aureus via these receptors.
Publisher: Informa UK Limited
Date: 07-01-2020
Publisher: Research Square Platform LLC
Date: 07-06-2023
DOI: 10.21203/RS.3.RS-3001427/V1
Abstract: New-onset HLA-DR-associated anti-citrullinated protein autoantibody (ACPA) + rheumatoid arthritis (RA) synovial tissue (ST) contains highly-expanded TCR-αβ clonotypes, some viral-antigen-reactive. However, it is unknown how viral-specific T-cells sustain ACPA + RA. We studied paired peripheral blood (PB) and ST TCR repertoires in drug-naïve ACPA + HLA-DRB1*04:01 + diffuse-myeloid RA ST pathology. To model effects of viral infection, we induced ovalbumin antigen-induced arthritis (AIA) in mice with latent murine-cytomegalovirus or recovered from acute lymphocytic-choriomeningitis virus. We show that most clonally-expanded CD8 + T cells had polyfunctional TNF + IFNγ + cytotoxic T-lymphocyte (CTL) signatures. Transcriptomic profiles of ST CD4 + T-cell clonotypes were Th2-like and IL-6-signaled central memory-like. CMV-specific tetramers confirmed in-silico prediction of viral-epitope recognition by CTL. Perivascular GZMB + CD8 + T cells co-localized with CD4 + T-cells, dendritic cells, fibroblasts and IL-6. After viral infection, AIA severity increased with enhanced viral and ovalbumin-specific TNF + IFN-γ + T-cell cytokines, suggesting that in the diffuse-myeloid rheumatoid-synovial perivascular niche, polyfunctional bystander-CTL reinforce myeloid- and CD4 + T-cell activation.
Publisher: Springer Science and Business Media LLC
Date: 22-06-2017
DOI: 10.1038/S41598-017-04325-0
Abstract: Complement Receptor Immunoglobulin (CRIg), selectively expressed by macrophages, plays an important role in innate immunity by promoting phagocytosis of bacteria. Thus modulation of CRIg on macrophages by cytokines can be an important mechanism by which cytokines regulate anti-microbial immunity. The effects of the cytokines, tumor necrosis factor, transforming growth factor-β1, interferon-γ, interleukin (IL)-4, IL-13, IL-10, IL-1β, IL-6, lymphotoxin-α, macrophage-colony stimulating factor (M-CSF) and GM-CSF on CRIg expression were examined in human macrophages. We demonstrated that cytokines regulated the CRIg expression on macrophages during their development from monocytes in culture at the transcriptional level using qPCR and protein by Western blotting. Both CRIg spliced forms (Long and Short), were similarly regulated by cytokines. Direct addition of cytokines to matured CRIg+ macrophages also changed CRIg mRNA expression, suggesting that cytokines control macrophage function via CRIg, at two checkpoints. Interestingly the classical complement receptors, CR3 and CR4 were differentially regulated by cytokines. The changes in CRIg but not CR3/CR4 mRNA expression correlated with ability to phagocytose Candida albicans by macrophages. These findings suggest that CRIg is likely to be a control point in infection and immunity through which cytokines can mediate their effects, and is differentially regulated from CR3 and CR4 by cytokines.
Publisher: BMJ
Date: 10-2022
DOI: 10.1136/RMDOPEN-2022-002563
Abstract: Programmed cell death protein 1 (PD-1)-expressing T cells are implicated in the pathogenesis of autoimmune inflammatory diseases such as rheumatoid arthritis. A subset of CXCR5 − T cells, termed T peripheral helper (Tph) cells, which drive B cell differentiation, have been identified in ectopic lymphoid structures in established rheumatoid arthritis synovial tissue. Here, we aimed to characterise these in treatment-naïve, early rheumatoid arthritis to determine whether these cells accumulate prior to fully established disease. Fresh dissociated tissue and peripheral blood mononuclear cell (PBMC) suspensions were stained with Zombie UV, followed by anti-CD45RO, PD-1, CD3, ICOS, CD8, CD4, CD20, CXCR5, TIGIT and CD38 antibodies prior to analysis. For histology, rheumatoid arthritis synovial sections were prepared for Opal multispectral immunofluorescence with anti-CD45RO, CD20, PD-1 and CXCR5 antibodies. Images were acquired on the Perkin Elmer Vectra V.3.0 imaging system and analysed using InForm Advanced Image Analysis software. Flow cytometry revealed T cell infiltration in the rheumatoid arthritis synovium with differential expression of PD-1, CD45RO, ICOS, TIGIT and CD38. We observed a higher frequency of PD1 hi CXCR5 − Tph in rheumatoid arthritis synovial tissue and PBMCs versus controls, and no significant difference in T follicular helper cell frequency. Microscopy identified a 10-fold increase of Tph cells in early rheumatoid arthritis synovial follicular and diffuse regions, and identified Tph adjacent to germinal centre B cells. These data demonstrate that PD-1 hi Tph cells are present in early rheumatoid arthritis, but not osteoarthritis synovium, and therefore may provide a target for treatment of patients with early rheumatoid arthritis.
Publisher: Frontiers Media SA
Date: 10-12-2019
Publisher: Frontiers Media SA
Date: 04-03-2022
DOI: 10.3389/FIMMU.2022.840510
Abstract: The phagocytosis-promoting complement receptor, Complement Receptor Immunoglobulin (CRIg), is exclusively expressed on macrophages. It has been demonstrated that expression in macrophages could be modulated by inflammatory mediators, including cytokines. This raised the possibility that a major phagocyte, the neutrophil, may also express CRIg following activation with inflammatory mediators. Here we show that resting peripheral blood neutrophil lysates subjected to protein analysis by Western blot revealed a 35 kDa CRIg isoform, consistent with the expression of CRIg mRNA by RT-PCR. By flow cytometry, CRIg was detected intracellularly and in very minor amounts on the cell surface. Interestingly, expression on the cell surface was significantly increased to functional levels after activation with inflammatory mediators/neutrophil activators N-Formylmethionine-leucyl-phenylalanine, tumor necrosis factor (TNF), Granulocyte-Macrophage Colony stimulating Factor (GM-CSF), bacterial lipopolysaccharide, leukotriene B4 and phorbol myristate acetate. The increase in expression required p38 MAP kinase and protein kinase C activation, as well as intracellular calcium. Neutrophils which were defective in actin microfilament reorganization due to a mutation in ARPC1B or inhibition of its upstream regulator, Rac2 lose their ability to upregulate CRIg expression. Inhibition of another small GTPase, Rab27a, with pharmacological inhibitors prevented the increase in CRIg expression, suggesting a requirement for the actin cytoskeleton and exocytosis. Engagement of CRIg on TNF-primed neutrophils with an anti-CRIg monoclonal antibody increased the release of superoxide and promoted the activation of p38 but not ERK1/ERK2 or JNK MAP kinases. The TNF-induced increase in killing of Staphylococcus aureus was blocked by the anti-CRIg antibody. Adding to the anti-microbial role of CRIg, it was found that GM-CSF priming lead to the release of neutrophil extracellular traps. Interestingly in contrast to the above mediators the anti-inflammatory cytokine IL-10 caused a decrease in basal expression and GM-CSF induced increase in CRIg expression. The data demonstrate that neutrophils also express CRIg which is regulated by inflammatory mediators and cytokines. The findings show that the neutrophil antimicrobial function involving CRIg requires priming as a means of arming the cell strategically with microbial invasion of tissues and the bloodstream.
Publisher: IntechOpen
Date: 24-04-2019
Publisher: Springer Science and Business Media LLC
Date: 17-02-2023
DOI: 10.1038/S41598-023-29971-5
Abstract: Programmed cell death protein 1 (PD-1)-expressing T cells are expanded in in iduals with established rheumatoid arthritis (RA). However, little is known about their functional role in the pathogenesis of early RA. To address this, we investigated the transcriptomic profiles of circulating CD4 + and CD8 + PD-1 + lymphocytes from patients with early RA (n = 5) using fluorescence activated cell sorting in conjunction with total RNA sequencing. Additionally, we assessed for alterations in CD4 + PD-1 + gene signatures in previously published synovial tissue (ST) biopsy data (n = 19) (GSE89408, GSE97165) before and after six-months of triple disease modifying anti-rheumatic drug (tDMARD) treatment. Comparisons of gene signatures between CD4 + PD-1 + vs. PD-1 − cells identified significant upregulation of genes including CXCL13 and MAF , and in pathways including Th1 and Th2, cross talk between dendritic cells and NK cells, B cell development and antigen presentation. Gene signatures from early RA ST before and after six-month tDMARD treatment revealed downregulation of the CD4 + PD-1 + signatures following treatment, identifying a mechanism through which tDMARDs exert their effect by influencing T cell populations. Furthermore, we identify factors associated with B cell help that are enhanced in the ST compared with PBMCs, highlighting their importance in driving synovial inflammation.
Publisher: SMW Supporting Association
Date: 05-04-2016
Publisher: Springer Science and Business Media LLC
Date: 25-03-2021
DOI: 10.1038/S42003-021-01943-3
Abstract: Vitamin D deficiency remains a global concern. This ‘sunshine’ vitamin is converted through a multistep process to active 1,25-dihydroxyvitamin D 3 (1,25D), the final step of which can occur in macrophages. Here we demonstrate a role for vitamin D in innate immunity. The expression of the complement receptor immunoglobulin (CRIg), which plays an important role in innate immunity, is upregulated by 1,25D in human macrophages. Monocytes cultured in 1,25D differentiated into macrophages displaying increased CRIg mRNA, protein and cell surface expression but not in classical complement receptors, CR3 and CR4. This was associated with increases in phagocytosis of complement opsonised Staphylococcus aureus and Candida albicans . Treating macrophages with 1,25D for 24 h also increases CRIg expression. While treating macrophages with 25-hydroxyvitamin D 3 does not increase CRIg expression, added together with the toll like receptor 2 agonist, triacylated lipopeptide, Pam3CSK4, which promotes the conversion of 25-hydroxyvitamin D 3 to 1,25D, leads to an increase in CRIg expression and increases in CYP27B1 mRNA. These findings suggest that macrophages harbour a vitamin D-primed innate defence mechanism, involving CRIg.
No related grants have been discovered for Annabelle Small.