Biology subjects

Bowness, P.

Publications and source records attributed to Bowness, P..

10 recordsLinked to original sources

bioRxiv2025

Inflammatory disease microbiomes share a functional pathogenicity predicted by C-reactive protein

We examine disease-specific and cross-disease functions of the human gut microbiome by colonizing germ-free mice, at risk for inflammatory arthritis, colitis, or neuroinflammation, with over 100 human fecal microbiomes from subjects with rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, ulcerative colitis, Crohns disease, or colorectal cancer. We find common inflammatory phenotypes driven by microbiomes from individuals with intestinal inflammation or inflammatory arthritis, as well as distinct functions specific to microbiomes from multiple sclerosis patients. Inflammatory disease in mice colonized with human microbiomes correlated with systemic inflammation, measured by C-reactive protein, in the human donors. These cross-disease patterns of human microbiome pathogenicity mirror features of the inflammatory diseases, including therapeutic targets and the presence or absence of systemic inflammation, suggesting shared and disease-specific mechanisms by which the microbiome is shaped and drives pathogenic inflammatory responses.

immunology↗

bioRxiv2024

CD4+ tissue resident memory Th17 cells drive IL-17A-mediated joint pathology in Spondyloarthritis

ObjectivesInterleukin (IL)-17A is a key driver of Spondyloarthritis (SpA) joint pathology. We aimed to identify its cellular source in synovial tissue from patients with two forms of SpA namely Axial SpA (AxSpA) and Psoriatic arthritis (PsA). MethodsSynovial tissue from patients with SpA was profiled using single-cell RNA sequencing (scRNA-seq: AxSpA, n=5 and PsA, n=6) or spatial RNA profiling (PsA, n=4). CellPhoneDB was used to infer cell-cell communication. Tissue resident memory Th17 (TRM17)-like cells were generated in vitro using blood memory CD4+ T cells from SpA patients. An epigenetic inhibitor library, siRNA and clustered regularly interspaced short palindromic repeats (CRISPR) were used to identify epigenetic regulator(s) for TRM17. ResultsscRNA-seq showed that CD4+CXCR6+ TRM17 cells are the predominant spontaneous IL17A producers in SpA synovium. Cell-cell communication and single-cell spatial analysis support the interaction between TRM17 and CLEC10A+ dendritic cells, which were activated in SpA. Both sublining and lining fibroblasts in SpA synovium showed evidence of IL-17A activation. In vitro-generated CD4+ TRM17-like cells phenocopied joint tissue TRM17, producing IL-17A/F upon T cell receptor (TCR) stimulation, which was further enhanced by cytokines. Perturbation of BRD1 inhibited the generation of TRM17-like cells. ConclusionsCD4+ TRM17 cells are the predominant source of IL-17A in SpA synovial tissue. TCR stimulation is essential for the secretion of IL-17A by CD4+TRM17-like cells. The epigenetic regulator BRD1 contributes to the generation of CD4+TRM17. Depleting CD4+TRM17 cells in SpA is thus a therapeutic strategy with potential to induce long-term remission. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSInterleukin (IL)-17A plays a key role in the immunopathogenesis of Spondyloarthritis (SpA), but its cellular source in joint tissue has not been determined previously. The induction and accumulation of CD4+ tissue resident memory Th17 (TRM17) cells following the clearance of pathogens has been described in skin, lung and kidney. Whether CD4+ TRM17 cells also accumulate in the joint and contribute to the pathology of SpA is not clear. What this study addsO_LICD4+ TRM17 cells are present in SpA synovial tissue and are the predominant source of IL17A C_LIO_LICD4+ TRM17 cells in SpA joints express IL17A without any in vitro exogenous stimulation C_LIO_LIT cell receptor (TCR) rather than cytokine stimulation is essential for IL-17A production by CD4+ TRM17-like cells C_LIO_LIThe epigenetic regulator BRD1 contributes to the generation of CD4+ TRM17-like cells. C_LI How this study might affect research, practice or policyOur findings identify CD4+ TRM17 cells as the primary source of IL-17A in SpA synovium, a previously unrecognized role for these cells. Key questions remain: How do CD4+ TRM17 cells relate to IL-17A producers in synovial fluid? What mechanisms induce and maintain them in the joint? How do they interact with other cells to promote arthritis? These questions warrant further investigation. In addition, our data suggest that targeting CD4+ TRM17 cells, the "factory" of IL-17A in SpA synovial tissue, has the potential to induce long-term remission, encouraging future efforts to develop new therapies to deplete CD4+ TRM17 cells in SpA.

immunology↗

bioRxiv2024

AxSpA joint tissue is characterised by HLA-DR+ tissue resident memory (TRM) and killer cell immunoglobulin-like receptor (KIR)+ CD8+ T cell subsets

ObjectiveAxial Spondyloarthritis (AxSpA) is a common inflammatory arthritis with HLA-B*27 as the major genetic risk. Recent discoveries of AxSpA-specific T cell receptor (TCR) motifs and the self and bacterial peptides that they recognize support a pathogenic role of CD8+ T cells. Despite of previous work on synovial fluid, the characteristics of CD8 cells in joint tissue are currently unknown. MethodSynovial tissues from 5 AxSpA patients were used for single cell RNA sequencing (scRNA-seq). Paired TCR sequencing was carried out for 2. The abundance of KIR+CD8+ T cells in the blood from 9 AxSpA patients and 10 healthy controls was measured using flow cytometry. The expression of naive and memory T cell markers (CCR7, CD45RA and CD45RO) were compared between KIR+ and KIR- CD8 cells. ResultsWe observed conventional, TRAV1-2+ mucosal-associated invariant T (MAIT) cell and MKI67+ proliferating cell populations in synovium. Following sub-clustering of conventional CD8+ T cells, HLA-DR+ tissue resident memory (TRM), circulating, KIR+ and FCGR3A+ (encoding CD16) cell subsets were observed. HLA-DR+ TRM and KIR+ cells were clonally expanded and exhibited distinct transcriptional features, enriched for T cell activation pathways and natural killer (NK) cell-mediated cytotoxicity pathway respectively. Lastly, KIR+CD8+ T cells were increased in AxSpA blood and enriched for CD45RA+CCR7- TEMRA cells. ConclusionHere we present the very first transcriptomic profiling of CD8+ T cells in synovium tissue and highlight potential roles of HLA-DR+ TRM and KIR+ cells in AxSpA pathology. This study adds novel insights to the disease mechanisms and offers new therapeutic opportunities.

immunology↗

bioRxiv2023

Trained immunity in human monocyte enhances myeloid-T-cell pathogenic crosstalk in Ankylosing Spondylitis

Recent studies in infectious, cardiovascular and neurodegenerative diseases have established the presence of memory in innate immune cells. This "trained immunity (TI)" leads to an enhanced response to a second challenge. Monocytes in Ankylosing Spondylitis (AS), a common form of inflammatory arthritis, are known to be hyper-responsive to microbial stimulus lipopolysaccharide (LPS). We asked if TI is present in AS monocytes and, if so, how it contributes to disease pathology. Using Single-cell RNA sequencing (scRNA-seq), flow cytometry and enzyme-linked immunosorbent assays (ELISA), we identify a subset of monocytes from AS patients exhibiting features of trained immunity and being hyperresponsive to LPS stimulation. Surprisingly, both trained monocytes in AS and {beta}-glucan-trained monocytes from healthy donors are hyper-responsive to T-cell-induced activation. scRNA-seq of AS synovial mononuclear cells shows enrichment of a monocyte population with these/analogous features. Additionally, T cell-stimulated monocytes act back on T-cells to support Th17 responses (of established pathology in AS). Lastly, using genetic and chemical perturbations we show that ERN1, an AS risk gene enriched in this trained monocyte population, contributes to T-cell-induced monocyte activation. Our data provide strong evidence for the first time for the key role of TI in common human inflammatory arthritis.

molecular biology↗

bioRxiv2021

Disruption of c-MYC binding and chromosomal looping involving genetic variants associated with ankylosing spondylitis upstream of RUNX3 promoter

BackgroundAnkylosing Spondylitis (AS) is a common form of inflammatory spinal arthritis with a complex aetiology and high heritability, involving more than 100 genetic associations. These include several AS-associated single nucleotide polymorphisms (SNPs) upstream of RUNX3, which encodes the multifunctional RUNT-related transcription factor (TF) 3. The lead associated SNP rs6600247 (p= 2.6 x 10-15) lies ~13kb upstream of the RUNX3 promoter adjacent to a c-MYC TF binding-site. The effect of rs6600247 genotype on DNA binding and chromosome looping were investigated by electrophoretic mobility gel shift assays (EMSA), Western blotting-EMSA (WEMSA) and Chromosome Conformation Capture (3C). ResultsInterrogation of ENCODE published data showed open chromatin in the region overlapping rs6600247 in primary human CD14+ monocytes in contrast to Jurkat T cell line or primary T-cells. The rs6600247 AS-risk allele is predicted to specifically disrupt a c-MYC binding-site. Using a 50bp DNA probe spanning rs6600247 there was consistently less binding to the AS-risk "C" allele of both purified c-MYC protein and nuclear extracts (NE) from monocyte-like U937 cells. WEMSA on U937 NE and purified c-MYC protein confirmed these differences (n=2; p<0.05). 3C experiments demonstrated negligible interaction between the region encompassing rs6600247 and the RUNX3 promoter. A stronger interaction frequency was demonstrated between the RUNX3 promoter and the previously characterised AS-associated SNP rs4648889. ConclusionsThe lead SNP rs6600247, located in an enhancer-like region upstream of the RUNX3 promoter, modulates c-MYC binding. However, the region encompassing rs6600247 has rather limited physical interaction with the promoter of RUNX3. In contrast a clear chromatin looping event between the region encompassing rs4648889 and the RUNX3 promoter was observed. These data provide further evidence for complexity in the regulatory elements upstream of the RUNX3 promoter and the involvement of RUNX3 transcriptional regulation in AS.

genomics↗

bioRxiv2021

Single cell analysis of spondyloarthritis regulatory T cells identifies distinct synovial gene expression patterns and clonal fates

Regulatory T cells (Tregs) play an important role in controlling inflammation and limiting autoimmunity, but their phenotypes at inflammatory sites in human disease are poorly understood. We here analyze the single-cell transcriptome of >16,000 Tregs obtained from peripheral blood and synovial fluid of two patients with HLA-B27+ ankylosing spondylitis and three patients with psoriatic arthritis, closely related forms of inflammatory spondyloarthritis. We identify multiple Treg clusters with distinct transcriptomic profiles, including, among others, a regulatory CD8+ subset expressing cytotoxic markers/genes, and a Th17-like RORC+ Treg subset characterized by IL-10 and LAG-3 expression. Synovial Tregs show upregulation of interferon signature and TNF receptor superfamily genes, and marked clonal expansion, consistent with tissue adaptation and antigen contact respectively. Individual synovial Treg clones map to different clusters indicating cell fate divergence. Finally, we demonstrate that LAG-3 directly inhibits IL-12/23 and TNF secretion by patient-derived monocytes, a mechanism with translational potential in SpA. Our detailed characterization of Tregs at an important inflammatory site illustrates the marked specialization of Treg subpopulations.

immunology↗

bioRxiv2021

Ex vivo mass cytometry analysis reveals a profound myeloid proinflammatory signature in psoriatic arthritis synovial fluid

ObjectivesA number of immune populations have been implicated in psoriatic arthritis (PsA) pathogenesis. This study used mass cytometry (CyTOF) combined with transcriptomic analysis to generate a high-dimensional dataset of matched PsA synovial fluid (SF) and blood leucocytes, with the aim of identifying cytokine production ex vivo in unstimulated lymphoid and myeloid cells. MethodsFresh SF and paired blood were either fixed or incubated with protein transport inhibitors for 6 h. Samples were stained with two CyTOF panels: a phenotyping panel and an intracellular panel, including antibodies to both T cell and myeloid cell secreted proteins. Transcriptomic analysis by gene array of key expanded cell populations and single-cell RNA-seq, and ELISA and LEGENDplex analysis of PsA SF were also performed. ResultsWe observed marked changes in the myeloid compartment of PsA SF relative to blood, with expansion of intermediate monocytes, macrophages and dendritic cell populations. Classical monocytes, intermediate monocytes and macrophages spontaneously produced significant levels of the proinflammatory mediators osteopontin and CCL2 in the absence of any in vitro stimulation. By contrast minimal spontaneous cytokine production by T cells was detected. Gene expression analysis showed the genes for osteopontin and CCL2 to be amongst those most highly upregulated by PsA monocytes/macrophages; and both proteins were elevated in PsA SF. ConclusionsUsing multiomic analyses we have generated a comprehensive cellular map of PsA SF and blood to reveal key expanded myeloid proinflammatory modules in PsA of potential pathogenic and therapeutic importance.

immunology↗

bioRxiv2019

Histone H3K27me3 demethylases regulate human Th17 cell development and effector functions by impacting on metabolism

T helper (Th) cells are CD4+ effector T cells that play an instrumental role in immunity by shaping the inflammatory cytokine environment in a variety of physiological and pathological situations. Using a combined chemico-genetic approach we identify histone H3K27 demethylases KDM6A and KDM6B as central regulators of human Th subsets. The prototypic KDM6 inhibitor GSK-J4 increases genome-wide levels of the repressive H3K27me3 chromatin mark and leads to suppression of the key transcription factor ROR{gamma}t during Th17 differentiation, whereas in mature Th17 cells an altered transcriptional program leads to a profound metabolic reprogramming with concomitant suppression of IL-17 cytokine levels and reduced proliferation. Single cell analysis reveals a specific shift from highly inflammatory cell subsets towards a resting state upon demethylase inhibition. The root cause of the observed anti-inflammatory phenotype in stimulated Th17 cells is reduced expression of key metabolic transcription factors, such as PPRC1 and c-myc. Overall, this leads to reduced mitochondrial biogenesis resulting in a metabolic switch with concomitant anti-inflammatory effects. These data are consistent with an opposing effect of GSK-J4 on Th17 T-cell differentiation pathways directly related to proliferation and effector cytokine profiles.

immunology↗

bioRxiv2019

Single-cell sequencing reveals a clonal expansion of pro-inflammatory synovial CD8 T cells expressing tissue homing receptors in psoriatic arthritis

Psoriatic arthritis (PsA) is a debilitating immune-mediated inflammatory arthritis of unknown pathogenesis commonly affecting patients with skin psoriasis. We used three complementary single cell approaches to study leukocytes from PsA joints. Mass cytometry (CyTOF) demonstrated marked (>3 fold) expansion of memory CD8 T cells in the joints compared to matched blood. Further exploration of the memory CD8 compartment using both droplet and plate based single cell RNA sequencing of paired alpha and beta chain T cell receptor sequences identified pronounced CD8 T cell clonal expansions within the joints, strongly suggesting antigen driven expansion. These clonotypes exhibited distinct gene expression profiles including cycling, activation, tissue homing and tissue residency markers. Pseudotime analysis of these clonal CD8 populations identified trajectories in which tissue residency can represent an intermediate developmental state giving rise to activated, cycling and exhausted CD8 populations. Comparing T-cell clonality across patients further revealed specificity convergence of clones against a putative common antigen. We identify chemokine receptor CXCR3 as upregulated in expanded synovial clones, and elevation of two CXCR3 ligands, CXCL9 and CXCL10, in PsA synovial fluid.

immunology↗

bioRxiv2018

The Autoimmune Disease Risk Allele rs6897932 Modulates Monocyte IL7R Surface and Soluble Receptor Levels in a Context-Specific Manner

IL-7 is a key factor in T-cell immunity and IL7R polymorphisms are implicated in autoimmune pathogenesis. IL7R mRNA is induced in stimulated monocytes in a genetically determined manner, yet a role for IL7R in monocyte biology remains unexplored. Here we characterize genetic regulation of IL7R at the protein level across multiple cell subsets and conditions in healthy individuals. We find monocyte surface and soluble IL7R (sIL7R) protein are markedly expressed in response to lipopolysaccharide (LPS). We further demonstrate alleles of rs6897932, a non-synonymous IL7R polymorphism associated with susceptibility to Multiple Sclerosis, Ankylosing Spondylitis and Primary Biliary Cirrhosis, form the key determinant of both surface IL7R and sIL7R in the context of inflammation. No effect of this allele was observed in unstimulated monocytes or across lymphoid subsets. Production of sIL7R by monocytes greatly exceeded that of CD4+ T-cells, and was strongly associated with both rs6897932 genotype and expression of the splicing factor gene DDX39A. Stimulated monocytes were sensitive to exogenous IL-7, which elicits a defined transcriptional signature. Flow cytometry and single-cell sequencing of synovial fluid derived monocytes from patients with spondyloarthritis showed an enlarged subset of IL7R+ monocytes with a unique transcriptional profile that markedly overlaps that induced by IL-7 in-vitro and shows similarity to the previously described Mono4 subset. These data demonstrate disease-associated genetic variants at IL7R specifically impact monocyte surface IL7R and sIL7R following innate immune stimulation, suggesting a previously unappreciated key role for monocytes in IL-7 pathway biology and IL7R-associated diseases.

immunology↗