Hussein Al-Mossawi

Activation‐induced KIR3DL2 binding to HLA‐B27 licenses pathogenic T cell differentiation in Spondyloarthritis

In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin‐like receptor 3DL2 (KIR‐3DL2). The aim of this study was to determine the factors that induce KIR‐3DL2 expression, and to characterize the relationship between HLA–B27 and the phenotype and function of KIR‐3DL2–expressing CD4+ T cells in SpA.

Activation‐Induced Killer Cell Immunoglobulin‐like Receptor 3DL2 Binding to HLA–B27 Licenses Pathogenic T Cell Differentiation in Spondyloarthritis

In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin‐like receptor 3DL2 (KIR‐3DL2). The aim of this study was to determine the factors that induce KIR‐3DL2 expression, and to characterize the relationship between HLA–B27 and the phenotype and function of KIR‐3DL2–expressing CD4+ T cells in SpA.

Context-specific regulation of monocyte surface IL7R expression and soluble receptor secretion by a common autoimmune risk allele

IL-7 is a key factor in T-cell immunity and IL7R polymorphisms are implicated in autoimmune pathogenesis. We previously reported an expression quantitative trait locus (eQTL) at rs931555, 59 to IL7R, associated with stimulated monocyte IL7R mRNA expression. Unlike in T-cells, a role for IL7R in monocyte biology is poorly described. Here we detail replication and characterization of this eQTL at protein level across cell subsets and conditions in a separate cohort. We find rs6897932, a non-synonymous IL7R polymorphism associated with susceptibility to Multiple Sclerosis, Ankylosing Spondylitis and Primary Biliary Cirrhosis, is the key determinant of monocyte IL7R surface expression and soluble IL7R (sIL7R) and functions in a context-specific manner. Monocyte surface IL7R is markedly induced by LPS and TNF stimulation under the genotypic regulation of rs6897932, whereas no effect of this allele was observed on CD4+, CD8+ or CD56+ cell surface IL7R or in unstimulated monocytes. LPS-induced monocyte release of sIL7R was strongly associated with both rs6897932 genotype and expression of the splicing factor gene DDX39A. After induction of IL7R expression, human monocytes display a robust and pleiotropic transcriptional response to exogenous IL-7. Monocytes from the synovial fluid of patients with Spondyloarthritis were similarly found to express high levels of surface IL7R. These data demonstrate that disease-associated genetic variants in the IL7R gene critically impact monocyte IL7R and sIL7R expression following innate immune stimulation, suggesting a previously unappreciated key role for monocytes in IL-7 pathway biology and IL7R-associated diseases.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.

DECIPHERING THE IN VITRO THERAPEUTIC POTENTIAL OF JAK INHIBITORS IN ANKYLOSING SPONDYLITIS

Background In Ankylosing Spondylitis (AS) therapeutic options include blocking of TNF and IL-17, but are expensive and limited compared to other rheumatic diseases. In addition there is no convincing evidence that either of these therapies is able to block new bone formation, the hallmark of AS. Small molecule inhibitors of Janus kinases (JAK) are potent inhibitors of other inflammatory diseases including Rheumatoid Arthritis (RA), and inhibit new bone formation in murine Spondyloarthritis1,2. Objectives We evaluated the ability of 4 different JAK family inhibitors to reduce IL-17A production in vitro using blood and synovial fluid samples from patients with AS, Psoriatric Arthritis (PsA) and RA. We choose to study Tofacitinib, as it shows relative specificity to JAK3, Ruxolitinib (JAK1), CEP-33779 (JAK2) and Bayer-18 (TYK2), in order to decipher the most potent inhibitor for this disease. Methods CD4 T cells were isolated from PBMC of AS (n=43), PsA (n=13), RA patients (n=10) and Healthy controls (HC, n=15) and stimulated for 3 days under Th17-promoting conditions in the presence of JAK inhibitors. Cytokine secretion (IL-17A for all samples, IL-17F, IL-22, GM-CSF and IFNγ for a representative subgroup) was assessed using ELISA, measured in pg/m, and expressed as relative to control. Cytotoxicity and proliferation were measured with Annexin V/7-AAD staining and CFSE dilution on Flow cytometry. TYK2 was silenced in primary human CD4 T cells with siRNA and effects were analysed using ELISA and Western Blot. Results All JAK inhibitors tested reduced secretion of IL-17A in CD4 T cells from AS, PsA, RA patients and HC (see Fig. 1 for data on AS and HC). In a small subset of 10 AS patients Tofacitinib showed statistically significant inhibition of IL-17F, IL-22, GM-CSF and IFNγ secretion, and CEP-33779 of IL-22 and GM-CSF secretion. At the same time cytotoxicity and anti-proliferative capacity of the inhibitors was low. No correlation between inhibitory capacity of the different small molecules and clinical parameters was observed. Bayer-18 did not significantly reduce Th17 cytokine secretion, but siRNA-mediated knock down of TYK2 in primary human CD4 T cells inhibited IL-17A, IL-22 and IL-17F secretion, when knock-down efficacy exceeded 70%. Conclusions All JAK inhibitors tested reduce IL-17A secretion from CD4 T cells in vitro. Despite the fact that our study does not address the specificity of the different JAK inhibitors, we find no clear evidence that targeting any one JAK family member is superior in reducing in vitro Th17 responses. Our siRNA knock-down data suggest that TYK2 is still a valuable target for developing new treatments in AS, even though blockade of its kinase activity was not efficacious in reducing IL-17A secretion. One explanation is that the IL-17A-inducing activity of TYK2 was not inhibited by Bayer-18 in our assays or that the scaffold function of TYK2 is more important in this pathway. References Fleischmann R. et al, Placebo-controlled trial of tofacitinib monotherapy in rheumatoid arthritis. N Engl J Med. 2012;367(6). Lories R. et al, Tofacitinib inhibits inflammation and new bone formation in murine Spondyloarthritis but does not adversely inhibit human MSC function [abstract]. Arthritis Rheumatol. 2015;67 (suppl 10). Acknowledgement AH was supported by DFG (Deutsche Forschungsgemeinschaft, HA-7021/1–1) and DS by SIR (Società Italiana di Reumatologia). Disclosure of Interest None declared

A novel innate lymphoid cell delineates childhood autoimmune arthritis

Inflammation in autoimmune disease is mediated by a complex network of interacting cells. Their identity and cross-talk are encoded in messenger RNA (mRNA). Juvenile idiopathic arthritis (JIA), a chronic autoimmune arthritis of childhood, is characterised by synovial inflammation with infiltration of both innate and adaptive immune cells1. Activated T cells play a role in disease2 but the cell types that drive the recruitment and activation of immune cells within the synovium are not known. Here, we utilised droplet-based and full length single cell mRNA sequencing to obtain a quantitative map of the cellular landscape of JIA. We studied 45,715 cells from the synovial fluid of inflamed knee joints and peripheral blood. We identified a population of synovial innate lymphoid cells (ILCs), shared across patients, that exhibited a unique transcriptional profile in comparison to canonical ILC subtypes. Validation at protein-level across a spectrum of autoimmune arthritides revealed that these ILCs are pathologically expanded in a particular type of JIA. Using statistical tools to assess cellular interactions in synovial fluid, ILCs emerged as a central node of communication, expressing the full repertoire of genes required to orchestrate and maintain the inflammatory milieu. Several ILC-mediated signalling pathways may lend themselves as novel therapeutic targets. Together our findings demonstrate a distinct ILC subtype associated with a tissue-specific childhood autoimmune disease.

Personalized medicine — a new reality in psoriatic arthritis?

Some patients with psoriatic arthritis are refractive to one biologic therapy but not to others, and a strategy for selecting the right therapy for each patient is needed. The findings of a new study highlight the potential benefit of stratifying patients by their immunophenotype to select the optimal biologic to use.

02.35 Time of flight mass cytometry (cytof) and rna sequencing interrogation of ‘pathogenic’ gm-csf lymphocytes in human spondyloarthritis

Background Immunological, genetic and therapeutic studies have implicated T lymphocytes in the pathogenesis of Spondyloarthritis (SpA). GM-CSF is emerging as a cytokine that marks out pathogenic T lymphocytes. Inhibition of this cytokine pathway in the SKG mouse model of SpA leads to improvement of joint scores. Methods Matched blood (PBMC) and synovial fluid (SFMC) from patients with SpA was studied ex-vivo using a 40 colour surface and intracellular CyTOF panel. CyTOF findings were validated by flow cytometry in a cohort of patients with SpA, rheumatoid arthritis (RA) controls and healthy donors. CyTOF data was analysed by multi-dimensional viSNE mapping in order to understand the heterogeneity of the synovial fluid inflammatory response. Novel triple cytokine capture was used to purify GM-CSF producing lymphocytes to high purity and RNA sequencing used to interrogate their transcriptional profile. Results CyTOF revealed ex-vivo GM-CSF production from multiple lymphoid cell lineages, with CD4 and CD8 cells the main producers in PBMC and SFMC. GM-CSF production overlaps with multiple other cytokines including IL-17A, IFN- and TNF-. 43% (SEM 3.98) of all GM-CSF producing cells in SpA PBMC were CD4 cells. The percentage of CD4 cells producing GM-CSF was significantly increased in SpA PBMC ex-vivo compared to healthy controls (mean 7.73% vs 4.96% n=38, p<0.005). The mean percentage of GM-CSF-positive CD4 cells from ex-vivo SFMCs was 32.2%, and was significantly higher compared to matched PBMC (n=5, p=0.005). RNA sequencing of capture-sorted GM-CSF-positive human CD4 cells showed a unique transcriptional profile and validated key genes previously shown to drive pathogenicity in mouse models of type-17 immune-mediated disease. Conclusions The synovial fluid of patients with SpA shows an expanded GM-CSF signature. This corresponds with an expansion of GM-CSF producing T lymphocytes in the peripheral blood of patients with SpA compared to controls. Human GM-CSF-positive CD4 cells have a unique ‘pathogenic’ transcriptional profile.

IDENTIFICATION OF CHROMATIN MODIFYING MECHANISMS IN INFLAMMATORY MACROPHAGES IN RHEUMATOID ARTHRITIS

Background Alterations in epigenetic mechanisms have been implicated in the regulation of pro-inflammatory cytokine production, and bromodomain-containing proteins (the BET family) may have a specific role in the transcriptional regulation of inflammatory genes. Our objective was to examine specific epigenetic variation between the responses of macrophages derived from RA patients and healthy volunteers, and those derived from inflammatory synovial fluid (SF). Delineation of any epigenetic differences between different macrophage populations may highlight new therapeutic targets in inflammatory disease. Materials and methods A library of small molecule inhibitors, including BET protein inhibitors, was used to identify key epigenetic regulators of pro-inflammatory cytokine production from macrophages. Macrophages were differentiated from the blood monocytes of normal volunteers and RA patients. Synovial fluid derived macrophages were isolated from RA patients undergoing therapeutic arthrocentesis. Cell culture media was supplemented with a small molecule inhibitor, and lipopolysaccharide subsequently used to stimulate inflammatory cytokine production. Cell viability was confirmed using WST-1 assay, cytokine production was measured using an MSD platform and qPCR to assess gene expression. Each experimental condition was performed in triplicate, alongside positive and negative controls. The study was ethically approved and all samples were obtained with informed consent. Results Inhibitors of BET proteins (JQ1, I-BET151 and PFI-1) were the only class of inhibitor to show consistent down-regulation of pro-inflammatory cytokines in both healthy and RA-patient derived macrophages. BET protein inhibitors significantly suppressed the production of TNFα and IL-6 production from RA blood derived macrophages and IL-6 from corresponding SF-derived macrophages. However, only JQ1 achieved significant inhibition of TNFα production from SF-derived macrophages; it also abrogated LPS-induced cytokine and chemokine mRNA expression in the macrophages of patients with RA. Conclusions This data suggests that the BET proteins are essential regulators of pro-inflammatory cytokine and chemokine production from the macrophages of patients with RA. The differential response to BET inhibition by macrophages derived from blood and synovial fluid of RA patients could indicate alteration in BET protein distribution at pro-inflammatory cytokine promoters, influenced by the chronic inflammatory environment. This study highlights the therapeutic potential of BET inhibitors (particularly JQ1) in RA. Funding acknowledgements NIHR BRU, BBSRC and GlaxoSmithKline