Introduction

Spondyloarthritis (SpA) includes a family of diseases that share a common genetic predisposition and several clinical manifestations. These diseases include ankylosing spondylitis (AS), reactive arthritis (ReA), psoriatic arthritis (PsA) and inflammatory bowel disease (IBD)-related arthritis. The enthesis represents the insertion point where tendons or ligaments attach to the bone and inflammation of this anatomic site (enthesitis) is a cardinal feature of SpA.

Neutrophils are innate immune cells implicated in the pathogenesis of psoriasis, PsA, uveitis and IBD [1,2,3,4,5]. They are prominent in other diseases that also fall within the SpA spectrum, including synovitis, acne, pustulosis, hyperostosis and osteitis (SAPHO) and chronic recurrent multifocal osteomyelitis (CRMO) [6, 7]. Neutrophils are also present in the sacroiliac joints of patients with early sacroiliitis and suspected AS [33].

Fig. 1
figure 1

Early inflammatory infiltrates at entheses show abundant neutrophils. A Clinical peripheral inflammation scores recorded weekly over a period of 11 weeks post intraperitoneal curdlan injection (n = 6 SKG, n = 4 BALB/c controls). B Prominent inflammatory infiltrates (red arrowheads) in female SKG mice (6 weeks post curdlan injection) are seen in H&E-stained sections (×4 magnification) at the spine base (SB), a region of high mobility. Lesser infiltrates are present in other regions of the spine, including the thoracolumbar (TL), lumbosacral (LS) and mid tail (MT) sites. C, D Immunohistochemistry (IHC) demonstrating abundant neutrophils, as stained by anti-MPO antibodies, present in SKG spine base enthesis (C) and at ankle enthesis (D). Black arrow: vertebral body. Red arrowhead: intervertebral disc. Black arrowhead: site of insertion of a tendon. Characteristic neutrophil trilobed nuclei can be seen in enlarged insets (bottom right)

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Multi-lobed nuclei of neutrophils were easily visualized in tissues at spine and ankle entheseal sites, and these cells stained positive for myeloperoxidase (MPO), a marker for neutrophils (Fig. 1C, D). Consistent with prior studies, gene array analysis from tissues at both ankle and spine inflamed entheseal sites revealed that regulators of pro-inflammatory cytokine genes including TNF, IL-1β, IL-6, IL-17, IL-23 and IL-12 were activated (Supplementary Table 2). In order to further evaluate the type of early cellular infiltration and gene expression patterns at spine and ankle entheseal sites, inflamed entheseal tissue from the axial skeleton and ankles of SKG and control BALB/c mice (n = 3 per group) was microdissected via laser capture microscopy 3 weeks post curdlan administration (Fig. 2A and Supplementary Figure 1).

Fig. 2
figure 2

S100A8/A9 and other neutrophil-related genes are the most highly expressed at SKG spine enthesis. A Inflamed spine base enthesis (red square) microdissected via laser capture microscopy (H&E, upper panel) and in unstained paraffin sections before (left lower panel) and after (right lower panel) microdissection, ×4 magnification. B Top twenty most highly expressed genes in spine base entheses in Affymetrix gene array. S100A8 and S100A9, components of calprotectin, and other neutrophil-related genes are highlighted in red. C H&E stain of inflammatory infiltrate at axial spine enthesis in control BALB/c and SKG mice (red square), ×4 magnification, and IHC for S100A8 (×20 magnification) confirming protein expression of S100A8 at this site. D, E Murine neutrophil signature score (D) and heatmap (E) show that the neutrophil signature is significantly prominent in spine base inflamed enthesis from SKG compared to BALB/c control mice at entheseal sites (n= 3 per group). Granulocyte signature of resting granulocytes from human peripheral blood was used as a reference in D [31]

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Neutrophil-associated genes and pathways are highly upregulated at axial and peripheral enthesis sites during early inflammation in SKG mice

The early inflamed entheseal tissue obtained by laser capture from SKG and BALB/c control spine entheseal sites (n= 3 per group) was subjected to Affymetrix gene array analysis. A cutoff of greater than 2-fold change in gene expression compared to control was deemed significant. The most highly upregulated genes at both axial (Fig. 2B) and peripheral entheseal sites (Supplementary Table 3) in SKG mice were S100A8 and S100A9, the protein products of which form the heterodimer alarmin calprotectin. This alarmin is highly expressed intracellularly by neutrophils and is released extracellularly upon their activation. Protein expression of S100A8 was confirmed through IHC (Fig. 2C). Activation of upstream regulators of MYD88 and TLR4 (the endogenous receptor for S100A8/A9 agonists) was also found, in line with previously published data highlighting these genes in S100A8/A9 signaling (Supplementary Table 2) [34, 35].

The strong presence of neutrophils within entheseal infiltrates was supported by the top twenty perturbed pathways in the signaling pathway impact analysis (SPIA) [30], where multiple pathways related to neutrophil function, survival and differentiation were activated, highlighted in red in Table 1. Furthermore, the presence of neutrophils was confirmed through the neutrophil gene signature scores and heat map (Fig. 2D and E) using a previously validated neutrophil gene signature [32]. In addition, a significant fold-change expression of several neutrophil-associated genes was noted in the top 20 expressed genes at spine axial enthesis, highlighted in red in Fig. 2B. Notable among these genes are C-type lectin domain family 7 member A (Clec7a, Dectin-1), typically expressed by myeloid cells (neutrophils, macrophages and dendritic cells) [36], Cathepsin S (Ctss), known to activate IL-36 and drive neutrophil biology in psoriasis [37], C-type lectin domain family 4 member D (Clec4d), implicated in the recruitment of neutrophils at sites of inflammation [38], and Cytochrome B-245 Beta Chain (Cybb), encoding a subunit of Nox2, an important gene in reactive oxygen species production by neutrophils [39, 40].

Table 1 Activated pathways related to neutrophil function, survival and differentiation at axial enthesis from SKG mice
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Neutrophils are present at healthy human entheses and express IL-23 upon stimulation with fungal adjuvant

To confirm the presence of neutrophils in the human enthesis, tissue sections from human spinal enthesis samples (Supplementary Figure 2) were stained for MPO. MPO expressing cells were present in the peri-entheseal bone marrow (Fig. 3A) but were not detectable in the enthesis soft tissues. However, neutrophils showed margination in the blood vessels found within entheseal soft tissue (Fig. 3B), suggesting that these neutrophils are activated. Following enzymatic digestion of the non-inflamed human peri-entheseal bone and enthesis soft tissue, flow cytometry also confirmed the presence of neutrophils (CD45+ CD66b+ CD16+) (Fig. 3C) in peri-entheseal bone marrow but not in entheseal soft tissue (data not shown). It has previously been shown in mice that intraperitoneal fungal adjuvants rapidly spread to the joints and tibial bone marrow [41].

Fig. 3
figure 3

Neutrophils at healthy human axial enthesis are capable of producing IL-23 upon fungal adjuvant stimulation. A IHC for MPO, human non-inflamed axial enthesis: MPO-expressing neutrophils are present in peri-entheseal bone (3 samples examined). Scale bar (right) represents 100 μm. B MPO-expressing neutrophils are also present in entheseal soft tissue, where they are localized to the blood vessels and are seen marginating (3 examined). Scale bar (right) represents 100 μm. C Digestion of the peri-entheseal bone with subsequent cell sorting also shows that neutrophils are present at/near human axial enthesis (CD45, all leukocytes; CD66b, granulocyte marker; CD16, neutrophil specific marker) (n = 3 samples). D Axial entheseal neutrophils (n = 4 samples) were stimulated with the fungal adjuvant zymosan (0.5mg/ml), zymosan plus IFNγ (20ng/ml) and IFNγ alone for 48 h. IL-23 was measured by a sandwich ELISA for p40 and p19. Paired t test (**p < 0.01)

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The SKG model is an IL-23-dependent model accelerated by fungal adjuvant that demonstrates arthritis and enthesitis, including early neutrophilic inflammation. Thus, we tested the ability of human entheseal neutrophils to secrete IL-23, as IL-23 secreting cells could initiate inflammation in SpA through activation at these sites by fungal and other adjuvants. When stimulated with the fungal adjuvant zymosan, enthesis-derived neutrophils significantly upregulated secretion of IL-23 (Fig. 3D), a finding enhanced by the co-administration of IFNγ, but not by administration of IFNγ alone. We also tested isolated blood neutrophils stimulated with two different fungal adjuvants (0.5mg/ml) for production of IL-23, as measured by ELISA (n=3). Mannan and zymosan (Supplementary Figure 3) show similar IL-23 induction capacity in neutrophils, while curdlan shows stronger induction capacity.

Stromal cells from healthy human entheses produce proinflammatory chemokines upon fungal adjuvant administration

Activation of stromal cells from entheseal sites is thought to play a role in the initiation of enthesitis [42]. When stimulated with the fungal adjuvant mannan, entheseal stromal cells significantly upregulated secretion of the neutrophil chemoattractant IL-8 (Fig. 4A). Mannan stimulation also resulted in the significant upregulation of other disease-relevant chemokines and cytokines including IL-6 (involved in promoting the IL-23/IL-17 axis), CCL20 (chemoattractant for IL-17-expressing cells) and CCL2 (chemoattractant for monocytes) (Fig. 4B–D). Adhesion molecules, whose upregulation is required for leukocyte recruitment, were also upregulated by mannan stimulation, including ICAM-1 (Fig. 4E) and VCAM-1 (Fig. 4F).

Fig. 4
figure 4

Human entheseal stromal cells produce chemokines and adhesion molecules important for leukocyte recruitment. Stromal cells were isolated from human entheseal sites and stimulated with the fungal adjuvant mannan (1 mg/ml) for 24 h. A–D Supernatant was analyzed for IL-8, IL-6, CCL2 and CCL20 protein (n = 3). E, F Expression of the adhesion molecules ICAM-1 and VCAM-1 was analyzed by FACS, and median fluorescence intensity was quantified (n = 3). Paired t test (*p < 0.05, **p < 0.01)

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Discussion

We report that neutrophils are present in both non-inflamed human and diseased murine entheses. Since neutrophils are short-lived and thus difficult to manipulate and study in human samples, we have used the SKG mouse as a model of SpA. We confirm a key early role for neutrophils in the SKG enthesitis and for the first time confirm the presence of human enthesis neutrophils that have the capability to produce IL-23.

We demonstrate the presence of neutrophils by H&E staining and expression of MPO and by noting the presence of a neutrophil gene signature and upregulation of neutrophil-associated pathways amidst the 20 most activated pathways in IPA analysis. In the early entheseal inflammatory infiltrates in the spine, we find a highly upregulated gene and protein expression of the alarmins S100A8 and S100A9, components of calprotectin, a myeloid lineage factor. Calprotectin has been shown to be upregulated in the psoriatic skin and has been implicated in disease pathogenesis [43]. Calprotectin is also a biomarker for several inflammatory diseases, most notably inflammatory bowel disease, and is a marker of radiographic progression in SpA [44]. Furthermore, S100A8 and S100A9 are products of neutrophils and monocytes and are known to modulate the early stromal microenvironment during the development of tendinopathy and to influence the composition of the inflammatory cell infiltrate [45].

Our integrated evaluation of murine and human tissue supports an important role of neutrophils in the earliest stages of SpA. We report that neutrophils isolated from non-inflamed human entheses are capable of producing IL-23 in response to stimulation with the fungal adjuvant zymosan. Zymosan activates TRL2 and Dectin-1 pathways, and in line with our observations, it has been reported that IL-23 is also produced by peripheral blood neutrophils using TLR agonists [24]. In addition IL-23 producing CD14+ monocytes have been found in human entheses [46]. Until recently, IL-23 was thought to be largely produced by professional antigen presenting cells such as macrophages and dendritic cells. However, Kvedaraite et al. have reported that in IBD, the main source of IL-23 is tissue infiltrating neutrophils [22]. In our study, we also show that human entheseal stromal cells can produce chemokines associated with neutrophil chemotaxis following fungal adjuvant stimulation. It has been previously demonstrated that through an autocrine effect of S100A8/A9 on neutrophils, there is promotion of their slow-rolling and adhesion in the blood vessel walls [47]. Interestingly, in non-inflamed human entheseal tissue, we observe neutrophils lining blood vessels. We hypothesize that once present at entheseal sites, activated neutrophils could initially promote further neutrophil infiltration and contribute to the propagation of inflammation when normal tissue reparative processes are dysregulated. This hypothesis needs to be validated through additional work.

Limitations of this study include the fact that other myeloid cells may play a role in entheseal pathology in the SKG model and are likely contributors in early inflammation. We hypothesize that there is an upregulation of IL-8 in local fibroblasts, with subsequent recruitment of neutrophils, but we do not exclude a role for monocytes/macrophages or other cell types in early disease pathogenesis. Instead, we highlight the presence of neutrophils, an underappreciated cell in entheseal pathogenesis. We have not isolated neutrophils from SKG mice to directly show production of IL-23 upon fungal stimulation (as was done with human peri-entheseal bone neutrophils). However, we infer this association from the combination of gene array analysis and histologic data, where a prominent neutrophilic infiltrate is present at early timepoints in inflammation. Finally, we note that although IL-23 inhibition has been shown to be ineffective in treating axial SpA patients with established disease [48, 49], it is still likely that it plays a role as an important contributing cytokine in the development of enthesitis and possibly also as a key contributor in early SpA pathogenesis.

Conclusion

The SpA group of diseases are strongly linked to the IL-23/17 cytokine axis, and are linked to neutrophil biology including maturation, marrow egression, tissue infiltration and activation. Furthermore, the target organs in SpA, including the skin, eye and gut, as well as more rare skeletal diseases including SAPHO, show significant macroscopic or microscopic neutrophil infiltration [39]. Our data suggest that neutrophils play an important role in the earliest enthesitis lesions in the SKG mouse model of SpA (an IL-23/17 dependent model), and we demonstrate that the normal human enthesis contains neutrophils that, when stimulated, can produce IL-23. These findings suggest that this innate immune cell type might play a key role in disease pathogenesis.