Normal human enthesis harbours conventional CD4+ and CD8+ T cells with regulatory features and inducible IL-17A and TNF expression.

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-ß 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.

Evidence that tissue resident human enthesis γδT-cells can produce IL-17A independently of IL-23R transcript expression.

OBJECTIVES: Murine 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. METHODS: Human 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. RESULTS: Entheseal 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. CONCLUSION: Spinal 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.

Identification of myeloid cells in the human enthesis as the main source of local IL-23 production.

OBJECTIVE: We investigated whether the normal human spinal enthesis contained resident myeloid cell populations, capable of producing pivotal proinflammatory cytokines including tumour necrosis factor (TNF) and interleukin (IL)-23 and determined whether these could be modified by PDE4 inhibition. METHODS: Normal human enthesis soft tissue (ST) and adjacent perientheseal bone (PEB) (n=15) were evaluated using immunohistochemistry (IHC), digested for myeloid cell phenotyping, sorted and stimulated with different adjuvants (lipopolysaccharide and mannan). Stimulated enthesis fractions were analysed for inducible production of spondyloarthropathy disease-relevant mediators (IL-23 full protein, TNF, IL-1ß and CCL20). Myeloid populations were also compared with matched blood populations for further mRNA analysis and the effect of PDE4 inhibition was assessed. RESULTS: A myeloid cell population (CD45+ HLADR+ CD14+ CD11c+) phenotype was isolated from both the ST and adjacent PEB and termed 'CD14+ myeloid cells' with tissue localisation confirmed by CD14+ IHC. The CD14- fraction contained a CD123+ HLADR+ CD11c- cell population (plasmacytoid dendritic cells). The CD14+ population was the dominant entheseal producer of IL-23, IL-1ß, TNF and CCL20. IL-23 and TNF from the CD14+ population could be downregulated by a PDE4I and other agents (histamine and 8-Bromo-cAMP) which elevate cAMP. Entheseal CD14+ cells had a broadly similar gene expression profile to the corresponding CD14+ population from matched blood but showed significantly lower CCR2 gene expression. CONCLUSIONS: The human enthesis contains a CD14+ myeloid population that produces most of the inducible IL-23, IL-1ß, TNF and CCL20. This population has similar gene expression profile to the matched blood CD14+ population.

Complications in spinal deformity surgery in the United Kingdom: 5-year results of the annual British Scoliosis Society National Audit of Morbidity and Mortality.

PURPOSE: To provide a 5-year national overview of corrective spinal deformity surgery in the United Kingdom. METHODS: Since 2008, the British Scoliosis Society has collected predefined data on spinal deformity surgeries carried out by its members. Participating units collect and submit annual anonymised data pertaining to the number of deformity surgeries performed, age groups, aetiology (idiopathic versus non-idiopathic), mortality, deep infections and neurological deficit (complete, incomplete without resolution and incomplete with resolution). Overall aetiology proportions and complication rates were calculated, as well as funnel plots with control limits of individual complication rates by cases performed. RESULTS: Between 2008 and 2012, 9,295 corrective spinal deformity procedures were performed. 4,445 (48%) were recorded as idiopathic and 2,917 (31%) as non-idiopathic. There were a total of 339 complications (3.6%). Deep infections occurred in 222 (2.82%), incomplete neurological deficit with resolution in 59 (0.65%), incomplete neurological deficit without resolution in 29 (0.32%), complete neurological deficit in 12 (0.13%) and mortality in 17 (0.19%). CONCLUSION: The complication rates reported in this study compare well with previously published studies. These reported results will hopefully serve to provide a benchmark for units in the UK providing corrective spinal deformity surgery to allow individual units to compare their complication rates against national averages and to provide national complication figures to aid in the consenting process of patients. Use of a spinal deformity registry, such as the British Spine Registry, is required to ensure ongoing service development and optimal healthcare provision.

Adolescent idiopathic scoliosis : a review of aetiological theories of a multifactorial disease.

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.

SARS-CoV-2 Infection Induces Psoriatic Arthritis Flares and Enthesis Resident Plasmacytoid Dendritic Cell Type-1 Interferon Inhibition by JAK Antagonism Offer Novel Spondyloarthritis Pathogenesis Insights.

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.

Vertebral body versus iliac crest bone marrow as a source of multipotential stromal cells: Comparison of processing techniques, tri-lineage differentiation and application on a scaffold for spine fusion.

The potential use of bone progenitors, multipotential stromal cells (MSCs) helping spine fusion is increasing, but convenient MSC sources and effective processing methods are critical factors yet to be optimised. The aim of this study was to test the effect of bone marrow processing on the MSC abundance and to compare the differentiation capabilities of vertebral body-bone marrow (VB-BM) MSCs versus iliac crest-bone marrow (IC-BM) MSCs. We assessed the effect of the red blood cell lysis (ammonium chloride, AC) and density-gradient centrifugation (Lymphoprep™, LMP), on the extracted VB-BM and IC-BM MSC numbers. The MSC abundance (indicated by colony counts and CD45lowCD271high cell numbers), phenotype, proliferation and tri-lineage differentiation of VB-BM MSCs were compared with donor-matched IC-BM MSCs. Importantly, the MSC attachment and osteogenesis were examined when VB-BM and IC-BM samples were loaded on a beta-tricalcium phosphate scaffold. In contrast to LMP, using AC yielded more colonies from IC-BM and VB-BM aspirates (p = 0.0019 & p = 0.0201 respectively). For IC-BM and VB-BM, the colony counts and CD45lowCD271high cell numbers were comparable (p = 0.5186, p = 0.2640 respectively). Furthermore, cultured VB-BM MSCs exhibited the same phenotype, proliferative and adipogenic potential, but a higher osteogenic and chondrogenic capabilities than IC-BM MSCs (p = 0.0010 and p = 0.0005 for calcium and glycosaminoglycan (GAG) levels, respectively). The gene expression data confirmed higher chondrogenesis for VB-BM MSCs than IC-BM MSCs, but osteogenic gene expression levels were comparable. When loaded on Vitoss™, both MSCs showed a similar degree of attachment and survival, but a better osteogenic ability was detected for VB-BM MSCs as measured by alkaline phosphatase activity (p = 0.0386). Collectively, the BM processing using AC had more MSC yield than using LMP. VB-BM MSCs have a comparable phenotype and proliferative capacity, but higher chondrogenesis and osteogenesis with or without using scaffold than donor-matched IC-BM MSCs. Given better accessibility, VB-BM could be an ideal MSC source for spinal bone fusion.

Benefits of the use of blood conservation in scoliosis surgery.

AIM: To investigate whether autologous blood transfusion (ABT) drains and intra-operative cell salvage reduced donor blood transfusion requirements during scoliosis surgery. METHODS: Retrospective data collection on transfusion requirements of patients undergoing scoliosis surgery is between January 2006 and March 2010. There were three distinct phases of transfusion practice over this time: Group A received "traditional treatment" with allogeneic red cell transfusion (ARCT) in response to an intra- or post-operative anaemia (Hb < 8 g/dL or a symptomatic anaemia); Group B received intra-operative cell salvage in addition to "traditional treatment". In group C, ABT wound drains were used together with both intra-operative cell salvage and "traditional treatment". RESULTS: Data from 97 procedures on 77 patients, there was no difference in mean preoperative haemoglobin levels between the groups (A: 13.1 g/dL; B: 13.49 g/dL; C: 13.66 g/dL). Allogeneic red cell transfusion was required for 22 of the 37 procedures (59%) in group A, 17 of 30 (57%) in group B and 16 of 30 (53%) in group C. There was an overall 6% reduction in the proportion of patients requiring an ARCT between groups A and C but this was not statistically significant (χ2 = 0.398). Patients in group C received fewer units (mean 2.19) than group B (mean 2.94) (P = 0.984) and significantly fewer than those in group A (mean 3.82) (P = 0.0322). Mean length of inpatient stay was lower in group C (8.65 d) than in groups B (12.83) or A (12.62). CONCLUSION: When used alongside measures to minimise blood loss during surgery, ABT drains and intra-operative cell salvage leads to a reduced need for donor blood transfusion in patients undergoing scoliosis surgery.

A survey of current controversies in scoliosis surgery in the United Kingdom.

STUDY DESIGN: A 10-point questionnaire was constructed to identify the philosophy of surgeons on various aspects of scoliosis surgery, such as choice of implant, bone graft, autologous blood transfusion, cord monitoring, and computer-assisted surgery. Comparisons were then made with recommendations published in the spinal literature. OBJECTIVE: To determine certain aspects of the current practice of scoliosis surgery in the United Kingdom. SUMMARY OF BACKGROUND DATA: Guidelines for good clinical practice in spinal deformity surgery are available in the United Kingdom but do not cover a number of controversial issues. METHODS: Consultants and fellows attended the 2009 British Scoliosis Society meeting. Fifty questionnaires were completed by 45 consultants and 5 fellows. RESULTS: All pedicle screw constructs favored by 25 of 50, hybrid 24 of 50 (1 undecided). Posterior construct of fewer than 10 levels, 20 of 50 would not cross-link, 11 of 50 used 1, and 19 of 20 used 2 or more. More than 10 levels 17 of 50 considered cross-links unnecessary, 4 of 50 used 1 and 29 of 50 used 2 or more. Eighty-eight percent preferred titanium alloy implants, whereas others used a mixture of stainless steel and cobalt chrome. When using bone graft, respondents used bone substitutes (24), iliac crest graft (14), allograft (12) and demineralized bone matrix (9) in addition to local bone. Ten of 50 would use recombinant bone morphogenetic protein (3 for revision cases only). Thirty-nine of 50 routinely used intraoperative cell salvage and 4 of 50 never used autologous blood. All used cord monitoring: sensory (19 of 50), motor (2 of 50), and combined (29 of 50). None used computer-aided surgery. Twenty-six operated alone, 12 operated in pairs, and 12 varied depending on type of case. CONCLUSION: This survey shows interesting variations in scoliosis surgery in the United Kingdom. It may reflect the conflicting evidence in the literature.