In this study, we observed that although the proportion of ILC3s in the peripheral blood circulation of tendinopathy patients was not significantly different from that of healthy controls, the expression levels of surface molecules characteristic of ILC3s (CD45, ICOS, IL-23R), related inflammatory factors (IL-17A and IL-22), and their characteristic transcription factors (RORC) tended to be upregulated in diseased tendon tissues.
Soft tissue lesions of the shoulder such as tendinopathy cause pain, loss of function, joint failure, and the development of secondary osteoarthritis, resulting in a huge social and economic burden. Identification of key immune cell populations that act as master regulators in this inflammatory process will advance our understanding of its pathogenic mechanisms. Accumulating evidence supports the contribution of inflammation in the development of tendinopathy. Recent studies have highlighted the importance of the innate immune response during the persistence of inflammation. As a newly discovered class of innate immune cells, the role of ILC3s in the development of tendinopathy remains to be examined.
Researchers have found that ILC3s are involved in promoting the development of rheumatoid arthritis. IL-17A secreted by Th17 cells is also a characteristic cytokine of ILC3s, and when IL-17A stimulates tenocytes, tenocytes produce large amounts of TNF-α, MIP-1α, IL-6, IL-8 and MCP-1, and tenocyte apoptosis is increased. Meanwhile, ILC3s have been found to be present in tendon soft tissues.
We collected peripheral blood and tendon tissues from patients who met the diagnostic criteria for tendinopathy and control patients and then examined the infiltration of inflammatory cells in tendon tissues from these tendinopathy patients and control patients (Fig. 1) and the proportion of ILC3s in the peripheral blood circulation (Fig. 2A). Interestingly, although the proportion of ILC3s in the peripheral blood circulation of tendinopathy patients was not significantly different from that of control patients (Fig. 2B-D), HampE sections showed that more inflammatory cells were infiltrated in the tendon tissue of tendinopathy patients (Fig. 1). Considering that tendinopathy is a local inflammatory response, and previous inflammation suggested that the proportion of ILC3s in tendon soft tissue was higher than its proportion in the peripheral blood, we then examined the relevant surface markers of ILC3s in tendon tissue. ILC is defined by characteristic antigens expressed on its surface, characteristic cytokines secreted by it, and specific transcription factors. For example, human ILC1s express CD56 to secrete IFN-γ, and their transcription factor is T-beta ; ILC2s express CRTH2 to secrete IL-13, and their transcription factor is RORA ; and ILC3s express IL-23R, secrete IL-17A, and their transcription factor is RORC . As shown in Fig. 3A and B, the expression of CD45, a common leukocyte differentiation marker, was upregulated; additionally, the expression of IL-23R, a surface marker of ILC3s, was also upregulated relative to tendon tissues from control patients. Furthermore, as an activation marker of leukocytes, the expression of ICOS in tendon tissues from tendinopathy patients also tended to be consistent with that of IL-23R (Fig. 3C). The expression of IL-23, a cytokine that activates ILC3s, was also upregulated in tendon tissues from patients with tendinopathy (Fig. 3D).
The flow staining protocol for ILC3s consists of a variety of protocols. In this study, our staining protocol defines ILC3s as Lin−IL-2R+IL-23R+, which is commonly used in the flow cytometry detection of ILC3s. These Lin−IL-2R+IL-23R+cells may include two populations of cells, namely, NKp44+ ILC3s and NKp44− ILC3s; however, according to previous studies, NKp44+ mainly secretes IL-17A, and NKp44− ILC3s mainly secretes IL-22. Therefore, we then examined the expression of these two cytokines. As shown in Fig. 4A and B, the expression levels of IL-17A and IL-22 were upregulated in the tendon tissue of patients with tendinopathy, suggesting that both NKp44+ ILC3s and NKp44− ILC3s were upregulated in the tendon tissue of patients with tendinopathy. As a characteristic transcription factor of ILC3s, RORC also maintained a consistent trend of upregulation with IL-17A and IL-22 (Fig. 4C).
Based on these results, we propose that ILC3s are potential immune factors for tendon sheath lesions. A better insight into the mechanisms by which ILC3s are involved in lesion development may advance the development of cell-targeted therapeutic modalities for early tendon disorders in humans.