Background/Purpose: Ankylosing 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.

To determine if human entheseal tissue harbours a population of MSCs and to investigate the effect of spondyloarthritis associated pro-inflammatory cytokines on MSC osteogenesis and adipogenesis.

Methods: Samples from healthy spinous process and interspinous ligament (10 male, 10 female, median age = 49) were divided into entheseal soft tissue (EST) and peri-entheseal bone (PEB) (1) and enzymatically digested. 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 assessed 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 and adipogenic cultures were also supplemented with IL-17A (50ng/ml), IL-22 (10ng/ml) or TNF-α (1ng/ml) to determine the effect of these cytokines on adipogenesis and osteogenesis.

Results: As a proportion of total cellularity EST developed approximately 5 fold more CFU-F than matched PEB (p< 0.0001). Cultured cells were overwhelmingly positive for expression of MSC markers CD73, CD90, CD105 (median 98.66% range: 87.69-98.7%) and negative for CD14, CD19, CD45 and HLA-DR (median 0.34% 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 2.75-fold greater osteogenic (p< 0.05) and -4.4-fold adipogenic (p< 0.05) potential than matched EST MSCs. Calcium accumulation was significantly lower for PEB MSCs when exposed to any cytokine (p< 0.05), though addition of IL-17A to EST MSCs significantly increased calcium accumulation by 1.5-fold (p< 0.05). Addition of IL-17A or TNF-α significantly decreased lipid accumulation for EST MSCs (p< 0.05).

Conclusion: Both 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. IL-17A is capable of enhancing osteogenesis and impairing lipid accumulation in entheseal soft tissue MSCs. These findings are potentially important in explaining the altered bone and fat phenotype observed in AS as the aberrant new bone formation arises in this tissue.

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.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/il-17a-induces-distinct-functional-differences-between-two-novel-mesenchymal-stem-cell-populations-identified-at-the-human-enthesis/