Background/Purpose:  In an IL-23 overexpression animal model of spondyloarthropathy (SpA), primary entheseal disease is driven by innate like lymphocytes at peripheral and spinal enthesis with these cells being reported in the enthesis soft tissue-bone interface. We previously described that the normal human entheseal soft tissue (EST) also had a populations of innate lymphoid cells (ILCs) (Cuthbert et al ACR abstract 2015). However, in human SpA the earliest imaging changes may occur in peri-entheseal bone (PEB) with such bone changes, rather than soft tissue changes, being harbingers of eventual joint ankylosis. The purpose of this work was to define PEB ILCs, to examine ILC3 transcriptional profiles and test entheseal responsiveness to IL-23 stimulation in comparison to other ILC3 sources.

Methods:  Human PEB and EST was harvested from normal spinous process in patients undergoing elective spinal orthopaedic procedures. Interspinous EST was dissected from PEB and enzymatically digested. Knee joint synovial fluid cells from active SpA were also collected as a comparator source of ILCs from an inflammatory environment. ILC3s were isolated for RNA analysis by FACS sorting using accepted phenotypic cell surface markers. For cytokine stimulation unsorted PEB digest was incubated for 48 hours in the presence of IL-1β and IL-23. In all cases TaqMan genes expression assays (Applied Biosystems) were used to measure transcripts of interest.

Results:  Bone adjacent to interpinous process attachment sites contained ILCs including ILC3s (6.1×10^-3%). RORγt and IL-23R were significantly increased in PEB ILC3 isolates compared to unsorted mononuclear cells (p=0.032 and p=0.033 respectively). Expression of immunomodulatory transcripts IL-10 and TGFβ were comparable in ILC3s isolated from all tissues. STAT3 expression was elevated in ILC3s isolated from PEB (p=0.048) compared to both EST and synovial fluid ILC3s as was TNFα. IL-17A and IL-22 expression was not detected in EST but both were detected in PEB and synovial fluid ILC3s. IL-1β/IL-23 stimulation of PEB resulted in 47-fold induction of IL-17A, 43-fold induction of IL-17F and 51-fold induction of IL-22 transcript.

Conclusion:  This is the first study to define the presence of type 3 ILCs in normal peri-entheseal bone. ILC3s from this location exhibit occasional IL17 gene transcripts which increased substantially following priming. This work adds to the emerging data on ILCs resident populations in the SpA associated target tissues.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/enumeration-and-preliminary-characterisation-of-peri-entheseal-bone-type-3-innate-lymphoid-cells/