Adolescent idiopathic scoliosis (AIS), defined by an age at presentation of 11 to 18 years, has a prevalence of 0.47% and accounts for approximately 90% of all cases of idiopathic scoliosis. Despite decades of research, the exact aetiology of AIS remains unknown. It is becoming evident that it is the result of a complex interplay of genetic, internal, and environmental factors. It has been hypothesized that genetic variants act as the initial trigger that allow epigenetic factors to propagate AIS, which could also explain the wide phenotypic variation in the presentation of the disorder. A better understanding of the underlying aetiological mechanisms could help to establish the diagnosis earlier and allow a more accurate prediction of deformity progression. This, in turn, would prompt imaging and therapeutic intervention at the appropriate time, thereby achieving the best clinical outcome for this group of patients. Cite this article: Bone Joint J 2022;104-B(8):915–921.

Adolescent idiopathic scoliosis (AIS), defined by an age at presentation of 11 to 18 years, has a prevalence of 0.47% and accounts for approximately 90% of all cases of idiopathic scoliosis. Despite decades of research, the exact aetiology of AIS remains unknown. It is becoming evident that it is the result of a complex interplay of genetic, internal, and environmental factors. It has been hypothesized that genetic variants act as the initial trigger that allow epigenetic factors to propagate AIS, which could also explain the wide phenotypic variation in the presentation of the disorder. A better understanding of the underlying aetiological mechanisms could help to establish the diagnosis earlier and allow a more accurate prediction of deformity progression. This, in turn, would prompt imaging and therapeutic intervention at the appropriate time, thereby achieving the best clinical outcome for this group of patients. Cite this article: Bone Joint J 2022;104-B(8):915-921.

Objective Bacterial and viral infectious triggers are linked to spondyloarthritis (SpA) including psoriatic arthritis (PsA) development, likely via dendritic cell activation. We investigated spinal entheseal plasmacytoid dendritic cells (pDCs) toll-like receptor (TLR)-7 and 9 activation and therapeutic modulation, including JAK inhibition. We also investigated if COVID-19 infection, a potent TLR-7 stimulator triggered PsA flares. Methods Normal entheseal pDCs were characterized and stimulated with imiquimod and CpG oligodeoxynucleotides (ODN) to evaluate TNF and IFNα production. NanoString gene expression assay of total pDCs RNA was performed pre- and post- ODN stimulation. Pharmacological inhibition of induced IFNα protein was performed with Tofacitinib and PDE4 inhibition. The impact of SARS-CoV2 viral infection on PsA flares was evaluated. Results CD45+HLA-DR+CD123+CD303+CD11c- entheseal pDCs were more numerous than blood pDCs (1.9 ± 0.8% vs 0.2 ± 0.07% of CD45+ cells, p=0.008) and showed inducible IFNα and TNF protein following ODN/imiquimod stimulation and were the sole entheseal IFNα producers. NanoString data identified 11 significantly upregulated differentially expressed genes (DEGs) including TNF in stimulated pDCs. Canonical pathway analysis revealed activation of dendritic cell maturation, NF-κB signaling, toll-like receptor signaling and JAK/STAT signaling pathways following ODN stimulation. Both tofacitinib and PDE4i strongly attenuated ODN induced IFNα. DAPSA scores elevations occurred in 18 PsA cases with SARS-CoV2 infection (9.7 ± 4 pre-infection and 35.3 ± 7.5 during infection). Conclusion Entheseal pDCs link microbes to TNF/IFNα production. SARS-CoV-2 infection is associated with PsA Flares and JAK inhibition suppressed activated entheseal plasmacytoid dendritic Type-1 interferon responses as pointers towards a novel mechanism of PsA and SpA-related arthropathy.

AIMS:The aim of this study was to determine whether there is an increased prevalence of scoliosis in patients who have suffered from a haematopoietic malignancy in childhood.METHODS:Patients with a history of lymphoma or leukaemia with a current age between 12 and 25 years were identified from the regional paediatric oncology database. The medical records and radiological findings were reviewed, and any spinal deformity identified. The treatment of the malignancy and the spinal deformity, if any, was noted.RESULTS:From a cohort of 346 patients, 19 (5.5%) had radiological evidence of scoliosis, defined as a Cobb angle of > 10°. A total of five patients (1.4% of the total cohort) had a Cobb angle of > 40°, all of whom had corrective surgery. No patient with scoliosis had other pathology as a possible cause of the scoliosis and all had been treated with high doses of steroids for leukaemia, either acute or chronic myeloid, or acute lymphoblastic.CONCLUSION:There is an increased prevalence of idiopathic-like scoliosis and larger curves (Cobb angle of > 40°) associated with childhood leukaemia, which has not been previously reported in the literature. Causative factors may relate to the underlying disease process and/or its treatment. Cite this article: Bone Joint J 2021;103-B(8):1400-1404.

OBJECTIVE:The spondylarthritides (SpA) are intimately linked to new bone formation and IL-17A and TNF pathways. We investigated spinal soft tissue and bone mesenchymal stem cell (MSC) responses to IL-17A and TNF, including their osteogenesis, adipogenesis, and stromal supportive function and ability to support lymphocyte recruitment.METHODS:Normal spinal peri-entheseal bone (PEB) and entheseal soft tissue (EST) were characterized for MSCs by immunophenotypic, osteogenic, chondrogenic, and adipogenic differentiation criteria. Functional and gene transcriptomic analysis was carried out on undifferentiated, adipo- differentiated, and osteo-differentiated MSCs. The enthesis C-C Motif Chemokine Ligand 20-C-C Motif Chemokine Receptor 6 (CCL20-CCR6) axis was investigated at transcript and protein levels to ascertain whether entheseal MSCs influence local immune cell populations.RESULTS:Cultured MSCs from both PEB and EST displayed a tri-lineage differentiation ability. EST MSCs exhibited 4.9-fold greater adipogenesis (p < 0.001) and a 3-fold lower osteogenic capacity (p < 0.05). IL-17A induced greater osteogenesis in PEB MSCs compared to EST MSCs. IL-17A suppressed adipogenic differentiation, with a significant decrease in fatty acid-binding protein 4 (FABP4), peroxisome proliferator-activated receptor gamma (PPARγ), Cell Death Inducing DFFA Like Effector C (CIDEC), and Perilipin-1 (PLIN1). IL-17A significantly increased the CCL20 transcript (p < 0.01) and protein expression (p < 0.001) in MSCs supporting a role in type 17 lymphocyte recruitment.CONCLUSIONS:Normal spinal enthesis harbors resident MSCs with different in vitro functionalities in bone and soft tissue, especially in response to IL-17A, which enhanced osteogenesis and CCL20 production and reduced adipogenesis compared to unstimulated MSCs. This MSC-stromal-enthesis immune system may be a hitherto unappreciated mechanism of "fine tuning" tissue repair responses at the enthesis in health and could be relevant for SpA understanding.

Background: We have previously reported that the normal spinal enthesis has populations of conventional T-cells including CD4+ & CD8+ T-cells that could be induced to produce IL-17A and TNF following anti-CD3/CD28 stimulation. The biology of such cells in health including their normal function and antigen reactivity is completely unknown. The purpose of this work was to define the phenotype, functionality and TCR reactivity of such T-cells in health. Objectives: To investigate whether the T-cells at the normal enthesis were regulatory in nature and to determine the type of regulatory T-cell as Tr1 or FOXP3 regulatory T-cell and to determine T-cell reactivity. Methods: Healthy interspinous ligament and spinous process with matched peripheral blood were harvested from patients undergoing elective spinal surgery (n=20). Entheseal soft tissue (EST) & peri-entheseal bone (PEB) was enzymatically digested and then sorted. Tr1 and Treg phenotypes were investigated using flow cytometry. Analysis of cytokines, growth factors and chemokines was performed by qRT-PCR, ELISA and flow cytometry. TCR sequencing was performed and a search for putative T-cell reactivity was done using TCR3 database. Results: Pro-inflammatory cytokine transcripts including IL-17A, IL-17F, IL-22, IL-23 (p19) & TNF were very low or undetectable in the Enthesis T-cells (Fig 1). Flow cytometry confirmed entheseal T-cells had a Tr1 phenotype (CD4+ LAG3+ CD49b+). Intracellular flow cytometry showed enthesis T-cells had very low FOXP3 expression, when compared to their blood counterparts. Intracellular flow cytometry and gene expression showed high basal expression of growth factors and regulatory proteins such as IL-10 & TGFβ, when compared to blood T-cells. RNA-Seq data, showed 13 potential TCR clonal sequences the most common of which are predicted to be reactive viral infection was CMV present in 8 sequences and Influenza A virus present in 2 sequences. Conclusion: The healthy human enthesis has regulatory T-cells of a Tr1 phenotype rather than a FOXP3 Treg phenotype. Many clones have antigen specificity indicating reactivity to prior infection. These findings suggest that conventional entheseal T-cells have a role in enthesis immune homeostasis. Disclosure of Interests: Hannah Rowe Grant/research support from: Novartis UK Investigator Initiated non-clinical research funding support, Abdulla Watad: None declared, Tobias Russell Grant/research support from: Novartis UK Investigator Initiated non-clinical research funding support, Kassem Sharif: None declared, Darren Newton: None declared, Miriam Wittmann: None declared, Qiao Zhou Grant/research support from: Funded by the PARTNER fellowship program, Almas Khan: None declared, Peter Loughenbury: None declared, Robert Dunsmuir: None declared, Abhay S Rao: None declared, Peter Millner: None declared, Tony Kenna: None declared, Matthew Brown: None declared, Charlie Bridgewood: None declared, Dennis McGonagle Grant/research support from: Janssen Research & Development, LLC

Background: It is well known that viral infections may trigger psoriatic arthritis (PsA), a disease that typically has extensive pre-clinical entheseal abnormalites. Skin resident plasmacytoid dendritic cells (pDCs) produce IFNα that contribute to T cell expansion and the development of experimental psoriasis [1, 2]. IFN pathway SNPs have been reported in both PsA and psoriasis and we previously reported the presence of pDCs at the human enthesis [3]. Objectives: To investigate whether the TLR9 agonist ODN that replicates viral infection activate a wide array of of entheseal derived pDCs molecular cascades including the TNF pathway that might provide a link between viral infection and PsA. Methods: pDCs were sorted from enthesis and blood and stimulated with ODN as previously described (n=16) [3, 4]. IFNα protein pre and post stimulation were detected by ELISA. Intracellular flow cytometry (IFC) of entheseal pDCs was used to detect TNF protein. RNA was extracted post-stimulation. The mRNA were hybridised and tagged by probes then measured on the nCounter platform. Data was analysed using nSolver 4.0. Log2 |fold change| >1 and P-value <0.05 were considered statistically significant. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) of differentially expressed genes (DEGs) were analyzed using DAVID. Protein-protein interaction (PPI) network was drawn by STRING. Results: Stimulated entheseal pDCs showed a strong DEGs pattern pointing towards increased TNF expression. There were 11 genes significantly upregulated including TNF. RIPK3 is involved in TNF signalling pathway. TNF, RIPK3 and ZBP1 are involved in necroptosis. TNF and ITGB2 are involved in IL-4 and IL-13 signaling pathway. TNF, HLA-DOA, ITGB2/TLR7 are involved in virus infection. Together it highlights extremely activated TNF pathway genes. IFN protein was induced in sorted entheseal pDCs following stimulation (n=8). TNF protein was detected by IFC on stimulated entheseal pDCs (CD45+HLA-DR+CD123+CD303+ CD11c-) (n=3). We also compared entheseal and matched peripheral blood pDCs (n=8) following stimulation where no major differences in the TNF pathway were present between groups. The KEGG analysis was mapped in Figure 1. GO analysis showed the most significant change in biological processes was enriched in the positive regulation of DNA binding transcription factor activity. The change in molecular function was mainly enriched in p53 binding. Conclusion: Entheseal pDCs, upon viral molecule stimulation, show several markers of activation. However, TNF pathway genes were highly activiated which provides a novel mechanistic link between viral infection and PsA as reported in epidemiological studies. References: [1]Nestle, F.O., et al., Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production. J Exp Med, 2005. 202 (1): p. 135-43. [2]Christophers, E., et al., The risk of psoriatic arthritis remains constant following initial diagnosis of psoriasis among patients seen in European dermatology clinics. J Eur Acad Dermatol Venereol, 2010. 24 (5): p. 548-54. [3]Zhou, Q., PLASMACYTOID DENDRITIC CELLS IN THE ENTHESIS: PHENOTYPING AND FUNCTION INVESTIGATION. Annals of the Rheumatic Diseases, 2019. 78 . [4]Bridgewood, C., et al., Identification of myeloid cells in the human enthesis as the main source of local IL-23 production. Ann Rheum Dis, 2019. 78 (7): p. 929-933. Disclosure of Interests: Qiao Zhou: None declared, Jayakumar Vadakekolathu: None declared, Kassem Sharif: None declared, Tobias Russell Grant/research support from: Novartis UK Investigator Initiated non-clinical research funding support, Hannah Rowe Grant/research support from: Novartis UK Investigator Initiated non-clinical research funding support, Peter Millner: None declared, Peter Loughenbury: None declared, Abhay S Rao: None declared, Robert Dunsmuir: None declared, Charlie Bridgewood: None declared, Yasser El-Sherbiny: None declared, Dennis McGonagle Grant/research support from: Janssen Research & Development, LLC

Background The human enthesis conventional T cells are poorly characterised. Objectives To study the biology of the conventional T cells in human enthesis. Methods CD4+ and CD8+ T cells were investigated in 25 enthesis samples using immunofluorescence, cytometrically, bulk RNAseq and quantitative real-time PCR following anti-CD3/CD28 bead stimulation to determine interleukin (IL)-17A and tumour necrosis factor (TNF) levels. T-cell receptor (TCR) repertoires were characterised and a search for putative T-cell reactivity was carried out using TCR3 database. The impact of pharmacological antagonism with retinoic acid receptor-related orphan nuclear receptor gamma t inhibitor (RORγti), methotrexate and phosphodiesterase type 4 inhibitor (PDE4i) was investigated. Results Immunofluorescence and cytometry suggested entheseal resident CD4+ and CD8+ T cells with a resident memory phenotype (CD69+/CD45RA-) and tissue residency gene transcripts (higher NR4A1/AhR and lower KLF2/T-bet transcripts). Both CD4+ and CD8+ T cells showed increased expression of immunomodulatory genes including IL-10 and TGF-β compared with peripheral blood T cells with entheseal CD8+ T cells having higher CD103, CD49a and lower SIPR1 transcript that matched CD4+ T cells. Following stimulation, CD4+ T cells produced more TNF than CD8+ T cells and IL-17A was produced exclusively by CD4+ T cells. RNAseq suggested both Cytomegalovirus and influenza A virus entheseal resident T-cell clonotype reactivity. TNF and IL-17A production from CD4+ T cells was effectively inhibited by PDE4i, while RORγti only reduced IL-17A secretion. Conclusions Healthy human entheseal CD4+ and CD8+ T cells exhibit regulatory characteristics and are predicted to exhibit antiviral reactivity with CD8+ T cells expressing higher levels of transcripts suggestive of tissue residency. Inducible IL-17A and TNF production can be robustly inhibited in vitro.

Background: Fat formation in the bone adjacent to the enthesis is an important but poorly characterised intermediate stage in new bone formation that occurs in the spine in AS. We and others have previously reported that IL-17A can increase mesenchymal stem cell (MSC) mediated osteogenesis in normal and AS spinal tissue (1, 2). Objectives: Herein we investigate the impact of IL-17A & TNF on MSC adipogenesis from spinal enthesis tissue. Methods: Samples from healthy spinous process and interspinous ligament (n=14, median age = 53) were separated into the peri-entheseal bone (PEB) and entheseal soft tissue (EST) & enzymatically digested. Minimally passaged (<p3) MSCs were cultured in a complete adipogenic media, with some cultures supplemented with either IL-17A (50ng/ml), TNF (1ng/ml) or IL-17A & TNF for 3 weeks. Adipogeneis was quantitatively assessed by Oil Red O staining at day 21. IL-17A’s effect on adipogeneis was further investigated by RNA extractions at Day 0, 3, 5, 7, 15 & 21 with supporting Oil Red O staining. 48 adipogenic and IL-17A target genes were used to investigate adipogenic progression and IL-17A effects on it over 21 day adipogenic differentiation. Results: EST MSCs have a significantly higher adipogenic potential than matched PEB MSCs (n=14, p<0.001). TNF and IL-17A both cause significant decreases (all p<0.01, n=5) in adipogenesis for both PEB and EST MSCs. EST MSCs produced lipid vesicles by day-3 post-induction, with significant inhibition by IL-17A (p<0.01, n=4) seen from day 15 onwards. IL-17A caused a significant decrease in overall Oil Red O staining, and it changed the morphology of lipid vesicles with a majority of cells consistent with immature pre-adipocytes. This was supported by gene expression data, which indicated significant decreases in transcripts encoding vesicle fusion proteins (CIDEC p<0.05, PLIN1 p<0.01). PLIN1 also aids protection against lipolysis (4). Transcripts associated with osteogenesis (CEBPβ (3)) and MSC stromal support (CXCL12) were significantly upregulated in adipogenically-induced cultures stimulated with IL-17A when compared to control adipogenic media. TNF & IL-17A combination demonstrated that IL-17A drove the vesicle morphology changes, with TNF alone not showing the same vesicle changes. Conclusion: Given the inverse link between MSC mediated osteogeneis & adipogenesis, these findings reveal a role of IL-17A especially on EST MSCs. The rapid formation of adipocytes seen in EST MSCs may be relevant to MRI determined peri-entheseal bone “shiny corners” due to post inflammation fat accumulation. Elevated transcripts associated with pre-adipocytes & undifferentiated MSCs support the idea of plasticity between early osteogenesis & adipogenesis. Downregulation of transcripts for proteins associated with protection against lipolysis allows for the rationalising of the gradual loss of the shiny corners seen in AS preceding subsequent new bone formation. References: [1]RUSSELL, T., A. WATAD, C. BRIDGEWOOD, A. KHAN, A.S. RAO, P. LOUGHENBURY, P. MILNER, R. DUNSMUIR, T. BABOOLAL, E. JONES, R. CUTHBERT and D. MCGONAGLE. IL-17A Induces Distinct Functional Differences Between Two Novel Mesenchymal Stem Cell Populations Identified at the Human Enthesis. Arthritis Rheumatol , 2019, 71 Suppl 10, pp.1-5362. [2]JO, S., S.E. WANG, Y.L. LEE, S. KANG, B. LEE, J. HAN, I.H. SUNG, Y.S. PARK, S.C. BAE and T.H. KIM. IL-17A induces osteoblast differentiation by activating JAK2/STAT3 in ankylosing spondylitis. Arthritis Res Ther , 2018, 20(1), p.115. [3]AHMED, M. and S.L. GAFFEN. IL-17 in obesity and adipogenesis. Cytokine Growth Factor Rev , 2010, 21(6), pp.449-53. [4]HANSEN, J.S., S. DE MARE, H.A. JONES, O. GORANSSON and K. LINDKVIST-PETERSSON. Visualization of lipid directed dynamics of perilipin 1 in human primary adipocytes. Sci Rep , 2017, 7(1), p.15011. Disclosure of Interests: Tobias Russell Grant/research support from: Novartis UK Investigator Initiated non-clinical research funding support, Charlie Bridgewood: None declared, Almas Khan: None declared, Abhay S Rao: None declared, Peter Loughenbury: None declared, Peter Millner: None declared, Robert Dunsmuir: None declared, Ala Altaie: None declared, Elena Jones: None declared, Dennis McGonagle Grant/research support from: Janssen Research & Development, LLC

Background: In patients with spondyloarthritis, the presence of intestinal inflammation and an increase in digestive permeability responsible for bacterial translocation has been described. No data are available on the effect of non-steroidal anti-inflammatory drugs (NSAIDs) on this bacterial translocation in patients with spondyloarthritis. Zonulin and lipopolysaccharide (LPS) have been described as good biomarkers of intestinal permeability and bacterial translocation

ObjectivesMurine models of interleukin (IL)-23-driven spondyloarthritis (SpA) have demonstrated entheseal accumulation of γδT-cells which were responsible for the majority of local IL-17A production. However, IL-23 blockers are ineffective in axial inflammation in man. This study investigated γδT-cell subsets in the normal human enthesis to explore the biology of the IL-23/17 axis.MethodsHuman spinous processes entheseal soft tissue (EST) and peri-entheseal bone (PEB) were harvested during elective orthopaedic procedures. Entheseal γδT-cells were evaluated using immunohistochemistry and isolated and characterised using flow cytometry. RNA was isolated from γδT-cell subsets and analysed by qPCR. Entheseal γδT-cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, anti-CD3/28 or IL-23 and IL-17A production was measured by high-sensitivity ELISA and qPCR.ResultsEntheseal γδT-cells were confirmed immunohistochemically with Vδ1 and Vδ2 subsets that are cytometrically defined. Transcript profiles of both cell populations suggested tissue residency and immunomodulatory status. Entheseal Vδ2 cells expressed high relative abundance of IL-23/17-associated transcripts including IL-23R, RORC and CCR6, whereas the Vδ1 subset almost completely lacked detectable IL-23R transcript. Following PMA stimulation IL-17A was detectable in both Vδ1 and Vδ2 subsets, and following CD3/CD28 stimulation both subsets showed IL-17A and IL-17F transcripts with neither transcript being detectable in the Vδ1 subset following IL-23 stimulation.ConclusionSpinal entheseal Vδ1 and Vδ2 subsets are tissue resident cells with inducible IL-17A production with evidence that the Vδ1 subset does so independently of IL-23R expression.

Background Enthesitis or inflammation of tendon/ligament anchorage points is the cardinal lesion in spondyloarthritis (SpA)[1]. Human SpA is thought to be driven by IL-17 producing T-cells and can be successfully therapeutically targeted with anti-IL-17A and more recently anti-IL-17F[2]. The CCR6-CCL20 axis is thought to be crucial to tissue recruitment of IL-17 producing T-cells into tissues, however it is presently unknown if CCR6+ T-cells reside at the healthy human enthesis[3]. Moreover, the ability of IL-17 to drive CCL20 production and hence to promote further T-cell migration to the enthesis has not been tested. Objectives To determine if enthesis T cells secrete IL-17A and IL-17F. To determine if CCR6+ T-cells reside at the normal human enthesis. To study the effect of both IL-17A and IL-17F on CCL20 induction from entheseal stromal cells. Methods Normal spinous process enthesis was obtained from patients undergoing spinal decompression or surgery for scoliosis correction. Following enzymatic digestion, CD4+ and CD8+ T cells were stimulated with anti CD3/CD28 and IL-17A and IL-17F measured by intracellular FACS. Stromal cells were isolated from both the peri-entheseal bone (PEB) and enthesis soft tissue (ST). Stromal cells were stimulated with combinations of IL-17A or IL-17F and TNF and subsequently CCL20 was measured by ELISA. Normal PEB and ST were also analysed for the presence CCR6+ T-cells by multiparameter FACS including CD4+ and CD8 Results Entheseal CD4+ cells secreted IL-17A and IL-17F following stimulation. When used as single agents IL-17A, IL-17F and TNF were able to induce minimal CCL20 from entheseal stromal cells, but great synergy was reported between IL-17A/TNF and IL-17F/TNF. No synergy was reported for IL-17A/IL-17F. CCR6+ CD4+ and CD8+ cells were also confirmed in the enthesis. Conclusion Resident CD4+ T cells at the enthesis secrete IL-17A and IL-17F following stimulation. The normal human enthesis contains CCR6+ T cell populations, suggesting a CCR6/CCL20 axis is important to entheseal homeostasis. IL-17A/F dramatically synergize with TNF to induce CCL20 from enthesis stromal cells, which would be well placed to mediate further migration of IL-17 producing lymphocytes to entheseal tissues. References [1] Bridgewood, C., et al., Spondyloarthritis: new insights into clinical aspects, translational immunology and therapeutics. Current opinion in rheumatology, 2018. 30(5): p. 526-532. [2] Hall, A.O.H., J.E. Towne, and S.E. Plevy, Get the IL-17F outta here! Nature immunology, 2018: p. 1. [3] Singh, S.P., et al., Human T cells that are able to produce IL-17 express the chemokine receptor CCR6. The Journal of Immunology, 2008. 180(1): p. 214-221. Disclosure of Interests Charlie Bridgewood: None declared, Tobias Russell Grant/research support from: PhD Project is funded by Novartis., Abdulla Watad: None declared, Hannah Rowe: None declared, Qiao Zhou: None declared, Almas Khan: None declared, Peter Loughenbury: None declared, Abhay S Rao: None declared, Peter Millner: None declared, Robert Dunsmuir: None declared, Richard Cuthbert: None declared, Dennis McGonagle Consultant for: Lilly, Novartis UCB, Speakers bureau: Lilly, Novartis UCB

BackgroundMucosal associated invariant T-cells (MAITs) are innate-like T lymphocytes that express a semi-invariant TCR repertoire. They are activated by microbial ligands or cytokines including IL-23 and secrete inflammatory cytokines, including IFNγ and IL-17A. MAIT cells are enriched at mucosal surfaces and have been implicated in the pathogenesis of spondyloarthritis (SpA) (1) and inflammatory bowel disease (IBD). Although the human enthesis is not a mucosal surface it is the primary site of inflammation in SpA which has strong association with IBD.ObjectivesTo investigate if a population of MAITs is present at the normal human enthesis thereby establishing a potential link between gut and joint inflammation.MethodsHealthy interspinous ligament and spinous process were harvested from patients undergoing elective surgery for the correction of mechanical spinal defects. Entheseal soft tissue (EST) and peri-entheseal bone (PEB) were separated and cells were harvested by enzymatic and mechanical digestion respectively. The proportion of cells expressing markers consistent with MAITS (CD45+, CD3+, CD161+, TCRVα7.2+) were measured by flow cytometry in EST, PEB and matched blood. Expression of CD69 and CD45RA were examined for phenotypic analysis. Transcript analysis for IL-23/IL-17 axis and immunomodulatory genes was performed on sorted entheseal MAITs and analysed by TaqMan array.ResultsAs a proportion of total T-cells, MAITs were of approximately 3 fold and 2.5 fold greater abundance in EST and PEB respectively in comparison to matched peripheral blood (both p=0.034). MAITs in entheseal tissue had an overwhelming resident memory phenotype (CD69+, CD45RA-) median 53.2% (range 42.4 – 78.6%) in EST and 54.9% (45.2 - 82.1%) in PEB compared to those from blood 17.7 (6.8 – 69.4). MAITs robustly expressed RORC, CCR6 and IL-23R transcript. Compared to conventional entheseal T-cells, MAITs expressed significantly less TGFβ (6-fold, p>0.001) and significantly more IL-23R (29-fold, p=0.004).ConclusionHealthy human entheseal tissue contains an enriched population of MAITs that strongly express IL-23R transcript at a frequency comparable to that reported in the colon (2). The majority of these cells express a resident memory phenotype suggesting that they are a distinct population residing in entheseal tissue. These observations are potentially relevant to SpA pathogenesis and the observed link between SpA and IBD.References[1] Gracey E, Qaiyum Z, Almaghlouth I, Lawson D, Karki S, Avvaru N, et al. IL-7 primes IL-17 in mucosal-associated invariant T (MAIT) cells, which contribute to the Th17-axis in ankylosing spondylitis. Annals of the rheumatic diseases. 2016:annrheumdis-2015-208902. [2] Salou M, Franciszkiewicz K, Lantz O. MAIT cells in infectious diseases. Current opinion in immunology. 2017;48:7-14.Disclosure of InterestsRichard Cuthbert: None declared, Qiao Zhou: None declared, Abdulla Watad: None declared, Robert Dunsmuir: None declared, Peter Loughenbury: None declared, Almas Khan: None declared, Peter Millner: None declared, Charlie Bridgewood: None declared, Dennis McGonagle Consultant for: Lilly, Novartis UCB, Speakers bureau: Lilly, Novartis UCB

BackgroundAnkylosing spondylitis (AS) is associated with entheseal inflammation and new bone formation. Resident populations of lymphocytes have been identified at the enthesis that on stimulation with IL-23 produce pro-inflammatory cytokines including IL-17A and IL-22 (1) which drive inflammation and may also influence osteogenesis. Surprisingly, enthesis resident mesenchymal stem cells (MSCs) have not been phenotypically or functionally characterised.ObjectivesTo determine if human enthesis including entheseal soft tissue and peri-entheseal bone harbours a population of MSCs. Furthermore, to investigate the effect of spondyloarthritis associated pro-inflammatory cytokines on MSC osteogenesis.MethodsSamples from healthy the spinous process and interspinous ligament (male = 5, female = 10, median age = 19) were divided into entheseal soft tissue (EST) and peri-entheseal bone (PEB) and enzymatically digested (1). MSCs content was assessed using a CFU-F assay. Flow cytometry was used to examine expression of MSCs specific markers in plastic adherent cultures. Following osteogenic, chondrogenic and adipogenic inductions, osteogenesis was qualitatively by alkaline phosphatase and alizarin red staining and quantitatively by measurement of calcium accumulation. Chondrogenesis and adipogenesis were assessed using glycosaminoglycan assay and oil red o staining respectively. Osteogenic cultures were also supplemented with IL-17A (50ng/ml), IL-22 (10ng/ml) or TNF-α (1ng/ml).ResultsAs a proportion of total cellularity EST developed approximately 5 fold more colonies than matched PEB (p<0.0001). Cultured cells were overwhelmingly positive for expression of MSC markers CD73, CD90, CD105 (PEB median 98.66% range: 95.17-98.96%, EST median 98.42% range: 87.69-98.7%) and negative for CD14, CD19, CD45 and HLA-DR (PEB median 0.34% range: 0-2.91%, EST median 1.1% range: 0-2.53%) however some CD34 expression was noted particularly in EST cultures (median 3.74% range: 0-29%). Both populations were capable of tri-lineage differentiation, although PEB MSCs had greater osteogenic (p<0.025) and lower adipogenic (p<0.05) potential than matched EST MSCs. Addition of IL-22 (p<0.01) or IL-17A (p<0.005) caused a significant decrease in the calcium deposited in PEB MSCs but not in EST MSCs.ConclusionBoth the EST and PEB contain cells that meet the ISCT criteria defining MSCs. However, MSCs from these sources are functionally distinct in terms of their differentiation potential and response to inflammatory cytokines. The cytokines tested had a negative influence on osteogenesis in the conditions tested.References[1] CUTHBERT, R.J., E.M. FRAGKAKIS, R. DUNSMUIR, Z. LI, M. COLES, H. MARZO-ORTEGA, P.V. GIANNOUDIS, E. JONES, Y.M. EL-SHERBINY and D. MCGONAGLE. Brief Report: Group 3 Innate Lymphoid Cells in Human Enthesis. Arthritis Rheumatol, 2017, 69(9), pp.1816-1822.Disclosure of InterestsTobias Russell Grant/research support from: PhD Project is funded by Novartis., Abdulla Watad: None declared, Charlie Bridgewood: None declared, Almas Khan: None declared, Peter Millner: None declared, Peter Loughenbury: None declared, Abhay S Rao: None declared, Robert Dunsmuir: None declared, Thomas Baboolal: None declared, Elena Jones: None declared, Dennis McGonagle Consultant for: Lilly, Novartis UCB, Speakers bureau: Lilly, Novartis UCB, Richard Cuthbert: None declared

Background:Plasmacytoid dendritic cells (pDCs) play an important role in linking innate and adaptive immune responses and selectively express Toll-like receptor (TLR) 7 and TLR9 that sense RNA and DNA respectively thus regulating the secretion of type I interferons (IFN) and other inflammatory cytokines such as TNF [1]. pDCs have been directly implicated in psoriasis immunopathology and are activated following anti-viral vaccination, with vaccination also linked to psoriatic arthritis onset [2-3]. Inflammation of the enthesis (enthesitis) is a cardinal spondyloarthritis associated lesion so we hypothesized the presence of enthesis resident pDCsObjectives:To investigate whether the human enthesis contains a resident pDC population and to compare the responses of TLR7/9 agonists on entheseal pDCs function relative to peripheral blood derived pDCs.Methods:Normal interspinous process enthesis and matched peripheral blood (PB) were obtained from patients (n=11) undergoing spinal decompression or scolosis corrective surgery. Cells were isolated from perientheseal bone by mechanical digestion. Cells were stimulated with either TLR7 agonist (imiquimod) or TLR9 agonist (ODN-2216). Flow cytometry was used to phenotype pDCs, and intracellular staining used to measure IFNα and TNF. IFNα from supernatant was also measured by ELISA. Results: pDCs were identified in the human enthesis with a typical phenotype (CD45+HLA-DR+CD123+CD303+CD11c-). By intracellular FACS, following ODN or imiquimod stimulation, IFNα and TNF was detected in entheseal pDCs and also PB. IFNα and TNF induction trended upwards in entheseal pDC when compared with unstimulated pDCs. IFNα was also detected by ELISA following ODN or imiquimod stimulation of entheseal derived pDCs.Conclusion:The human enthesis contains a resident population of pDCs that produce IFNα and TNF following induction with relevant TLR agonists. For the first time, our findings provide a link between viral illness and vaccination and pivotal innate immune cell production of IFNα and TNF.References:[1] Melissa Swiecki and Marco Colonna, The multifaceted biology of plasmacytoid dendritic cells. Nat Rev Immunol. 2015 Aug;15(8):471-85. [2] Gunes AT, Fetil E, Akarsu S, et al., Possible Triggering Effect of Influenza Vaccination on Psoriasis. J Immunol Res. 2015;2015:258430. [3] Nestle FO, Conrad C, Tun-Kyi A, et al., Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production. J Exp Med. 2005 Jul 4;202(1):135-43.Disclosure of Interests:Qiao Zhou: None declared, Richard Cuthbert: None declared, Abdulla Watad: None declared, Tobias Russell Grant/research support from: PhD Project is funded by Novartis., Hannah Rowe: None declared, Almas Khan: None declared, Peter Millner: None declared, Peter Loughenbury: None declared, Abhay S Rao: None declared, Robert Dunsmuir: None declared, Charlie Bridgewood: None declared, Dennis McGonagle Consultant for: Lilly, Novartis UCB, Speakers bureau: Lilly, Novartis UCB

Background Animal models have demonstrated that enthesitis is the primary lesion in experimental spondyloarthritis (SpA). In mice, innate lymphocytes were suggested as the major cytokine producers at the enthesis. Objectives We tested the hypothesis that the human enthesis harbours tissue resident conventional T-cells. We also assessed their ability to express SpA-related cytokines including TNF and IL-17A and if this could be blocked using psoriasis therapeutic agents (methotrexate (MTX), and phosphodiesterase type 4 inhibitor (PDE4i)) and experimental RORγt inhibitors (RORγti). Methods Entheseal spinous process was obtained from patients undergoing elective orthopedic procedures (n=20) and mechanically digested or processed for confocal staining and flow cytometry. CD4+ and CD8+ T-cells were sorted and RNA was isolated and analysed by qPCR. Magnetically isolated cells were stimulated using an anti-CD3/CD2/CD28 bead with and without the presence of MTX, RORγti and PDE4i. Following stimulation IL-17A and TNF were measured by ELISA and intracellular flow cytometry. Results CD4+ and CD8+ T-cells represent 35.7% and 23.7% of T-cells in the enthesis, respectively, with topographic confirmation by anti-CD3 immunofluorescence staining. Entheseal tissue contained a higher proportion of CD4+ and CD8+ T-cells expressing a resident memory phenotype (CD69+/CD45RA-) compared to matched blood. Sorted T-cells from entheseal tissue had a gene expression profile consistent with a tissue resident phenotype and CD4+ and CD8+ T-cells showed increased expression immunomodulatory genes including IL-10 and TGF-β compared to peripheral blood T-cells (p<0.001). Following stimulation CD4+ T-cells produced more TNF than CD8+ T-cells (p<0.05), IL-17A was robustly detected in CD4+ but not CD8+ T-cells. TNF and IL-17A production from CD4+ T-cells was effectively inhibited by PDE4i (p<0.05), while RORγti only reduced IL-17 secretion (p<0.001). MTX had no significant impact on both TNF and IL-17A production in either cell population. This pattern of inhibition was mirrored in TNF secretion from CD8+ T-cells. Conclusion This is the first description of conventional CD4+ and CD8+ enthesis resident T-cells. PDE4i was effective in abrogating induced TNF production and IL-17, whereas RORγti is highly effective for IL-17A production but not TNF. In contrast, MTX had little effect on in vitro enthesitis model cytokine production. These findings may have some practical implications in the treatment of subclinical enthesitis. Abbreviations: ST, entheseal soft tissue; PEB, peri-entheseal bone. Disclosure of Interests Abdulla Watad: None declared, Hannah Rowe: None declared, Charlie Bridgewood: None declared, Tobias Russell Grant/research support from: PhD Project is funded by Novartis., Qiao Zhou: None declared, Almas Khan: None declared, Robert Dunsmuir: None declared, Peter Loughenbury: None declared, Abhay S Rao: None declared, Peter Millner: None declared, Richard Cuthbert: None declared, Dennis McGonagle Consultant for: Lilly, Novartis UCB, Speakers bureau: Lilly, Novartis UCB