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The role of innate immune cells in tissue inflammation in spondyloarthritis

Noordenbos, T.

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Citation for published version (APA):Noordenbos, T. (2017). The role of innate immune cells in tissue inflammation in spondyloarthritis.

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Download date: 26 Dec 2019

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1 Amsterdam Rheumatology and immunology Center and Department of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, The Netherlands,

2 Department of Experimental Immunology, Academic Medical Center/University of Amsterdam, The Netherlands, 3 Department of pathology, Academic Medical Center/University of Amsterdam, The Netherlands,

4 Tytgat Institute for Liver and Intestinal Research, University of Amsterdam, The Netherlands, 5 Hospital Clinic de Barcelona and Institut d’Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain

EXPRESSION OF IL‐17A BY MAST CELLS IN CHRONIC TISSUE INFLAMMATION: A HISTOLOGICAL STUDY

Troy Noordenbos1,2, Iris Blijdorp1,2, Carmen Ambarus3, Sijia Chen1,2, Esther Vogels4, Anja te Velde4, Mercé Alsina5, Juan Canete5, Dominique Baeten1,2, Nataliya Yeremenko1,2

Submitted for publication

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ABSTRACTThe IL-23/IL-17-mediated diseases, psoriasis (PsO) and inflammatory bowel diseases (IBD) are closely related to spondyloarthritis (SpA) and a partial overlapping mechanism is expected. Since we have recently suggested the role of the IL-17-positive mast cell in the pathophysiology of SpA, we now ask if mast cells are important for PsO and IBD. First we validate the specificity of immunostaining and show that the in situ protein detection method used by us and others is genuine and we indicate that the detection of IL-17A is not a result of extracellular binding, yet is originated from intracellular presence. Then we apply immunostainings to examine presence of IL-17A-positive mast cells in inflamed PsO skin and IBD colon. We observed that although upon inflammation the numbers of IL-17A-positive mast cells increase, they were similar between PsO and control conditions, indicating that this phenomenon, other than in spondyloarthritis, is not specific for PsO. Strikingly, while the increase in total mast cells was paralleled by the increase in IL-17A-containing mast cells in the dermis, the colonic submucosa and the synovial tissue, it was not seen in the lamina propria, where number of IL-17A-containing mast cells trends to decrease compared to non-inflamed lamina propria. The observed heterogeneity in the IL-17A-positive mast cells in various locations may indicate a functional difference in the working of the IL-23/IL-17 axis in the different tissues. Our study indicates that IL-17-positive mast cells may not play the same role in SpA as in PsO and IBD.

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INTRODUCTION Recent advances in genetics, preclinical models, translational research, and clinical trials revealed a central role for the IL-23/IL-17 axis in the pathogenesis of spondyloarthritis (SpA) [1–4]. SpA is phenotypically characterized by inflammation of the axial and peripheral skeleton as well as by extra-articular manifestations including skin and gut inflammation. There are three lines of evidence suggesting that inflammation in these different target tissues is driven, at least partially, by similar pathogenic mechanisms. First, there is a significant clinical overlap between SpA, psoriasis (PsO), and inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), as patients with SpA have concomitant PsO in up to 30% and concomitant bowel inflammation in up to 8% of the cases [5]. Moreover, more than half of the SpA patients have subclinical signs of inflammation in the gut [6]. Second, all these conditions are genetically associated with SNPs in the IL-23/IL-17 pathway [7] and at least some of these SNPs, including the protective R381Q gene variant in the IL-23R locus, have been functionally linked to IL-23-induced IL-17 responses [8]. And third, therapeutic targeting of cytokines such as TNF and the IL-23/IL-12 p40 subunit is not only effective in SpA but also in PsO and CD [9–11].

Although these data support the concept that the different disease manifestations of SpA, including the extra-articular manifestations such as gut and skin inflammation, are driven by similar pathogenic pathways, there is also emerging evidence that the functional outcome of these pathways is highly dependent on the immunological context of the local environment [12]. Prototypical examples are the striking differential effects of monoclonal anti-TNF antibodies versus the soluble decoy receptor etanercept in CD but not in SpA or PsO [13,14] and the fact that anti-IL17A monoclonal antibodies are therapeutically effective in SpA and PsO [3,15–17] but not in CD [18]. Thus a better understanding of how these pathogenic cytokine pathways function in the target tissues of human diseases remains warranted to design optimal therapeutic interventions.

Focusing on the role of the IL-23/IL-17 pathway in human SpA, a series of important questions remain unanswered. In contrast to the elegant experimental work deciphering this axis in rodents [19–21], it remains for example unclear which mechanisms exactly drive human TH17 polarization, what kind of cell types besides TH17 cells contribute to IL-17 production, and which downstream cytokines besides IL-17A (with particular attention to IL-17F and IL-22) drive pathology in the context of SpA. As to the IL-17A-producing cell types, several studies have suggested that not only TH17 cells but also Tc17 [22], γδ T cells [23], and group 3 innate lymphoid cells [24,25] can produce IL-17A in SpA-related conditions. Assessing directly the affected target tissues, we reported previously that mast cells are the major IL-17A-positive cell population in peripheral synovitis in SpA and that ex vivo and in vivo targeting of mast cells using tyrosine kinase inhibitors improves synovial inflammation [26,27]. As IL-17A-positive mast cells have also been reported in many other inflammatory conditions [28–30], we aimed in the present study to determine if mast cells are also a major IL-17-positive population in skin and gut tissues that can be affected by SpA and if their presence in these tissues is constitutive or related to inflammation.

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METHODS Patient material Human synovial biopsies were obtained by small bore arthroscopy, as described before [31]. SpA patients (N=10) were fulfilling the ASAS pSpA criteria [32] and RA-patient (N=10) were fulfilling the American College of Rheumatology/European League Against Rheumatism 2010 criteria for RA [33]. Gut biopsies were endoscopically or surgically obtained from CD-patients (N=3), whose diagnosis was confirmed by radiology, endoscopy, and/or histopathology [11], and CU-patients (N=3), fulfilling ECCO criteria [34]. Skin biopsies were obtained from patients diagnosed with PsO (N=10), and controls from patients with non-psoriatic conditions (N=10) such as follicular eczema, basal cell carcinoma, prurigo, scleroderma and dermatitis medicamentosa. Normal tonsil (n=5), skin (n=5) and colon (n=7) were obtained as rest material after surgery, as approved by the Medical Ethics Committee of Slotervaart Hospital and Reade.

Western blotThe following antibodies were used: goat-anti-IL-17A polyclonal (AF-317-NA, R&D Systems), mouse-anti-IL-17A clone 41809 (MAB317, R&D), and mouse-anti-IL-17A clone A7A (ab134782, Abcam). Protein lysates were prepared from 1) normal human PMBC’s that were stimulated for 6 hours with PMA/ionomycin in the presence of secretion inhibitor brefeldin; 2) mast cells isolated from human pediatric tonsillectomies by flow sorting (FACS-ARIA, BD); 3) LAD2 human mast cell line [35]; and 4) primary human fibroblasts-like synoviocytes in standard culture conditions [36,37]. Commercially available recombinant IL-17A protein, homodimer 317-ILB, and heterodimer 5194-IL (R&D) were used as controls. Cells were lysed and total proteins were reduced, denatured, separated by electrophoresis and transferred onto PVDF membranes. Membranes were washed in TBS (pH 8.0) containing 0.05% Tween-20 (TBST), blocked with milk, and incubated with primary antibodies. IRDye800-, IRDye680- (LI-COR Biosciences) or HRP-conjugated secondary antibodies (DAKO) were used for visualization of proteins on the Odyssey (LI-COR Biosciences) or after addition of substrate (Lumi-Light, Roche) on the LAS4000 (GE-Healthcare).

ImagingFive-micrometer sections of paraffin-embedded synovial, gut, skin, or tonsil tissue were deparaffinized and rehydrated. After antigen retrieval by heating in 0.5 M citrate buffer (pH 6) in pressure cooker, sections were blocked with 10% donkey serum and stained with mouse-anti-mast cell tryptase (clone AA1; Abcam) or mouse-anti-CD15 (clone C3D-1; Dako) and goat-anti-IL-17A (polyclonal AF-317-NA; R&D Systems), followed by incubation with AF488 donkey anti-goat and AF555 donkey-anti-mouse antibodies. Acquisition was performed on a Leica DMR, equipped with fluorescent filter boxes. Stainings were quantified by manual counting of positive cells in five high-power fields with a 20x objective by three independent observers (TN, IB and NY).

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MicroarrayLAD2 cells were stimulated with IL-17A [5ug/ml] in culture medium and RNA was collected at 0 hours, 2 hours and 8 hours after stimulation. Extended technical information was described before [38]. In short, the Agilent platform and reagents were used. Data were extracted using Agilent Feature Extraction software (version 10.1; Agilent) and analyzed using the Limma package (30) from the Bioconductor project (www.bioconductor.org). We performed background correction and we applied between-array normalization using scale normalization. The normalized data were then fitted within a linear model, and Limma was used to moderate the standard errors of the estimated log ratios. The expression data of 2 hour or 8 hours were then compared to 0 hours using T statistics. The false discovery rate was set at 0.01, and probes with P values less than 0.001, as adjusted for multiple hypothesis testing, were judged as differentially regulated.

RESULTSSpecificity of IL-17A immunostaining of human mast cellsMultiple histological studies have reported marked staining for IL-17A in mast cells and neutrophils in a variety of different tissues [28,30,39,40]. These findings gave rise to debate, since murine mechanistic studies failed to demonstrate IL-17A production by mast cells and production of IL-17A by neutrophils is conflictingly reported [41–44]. As we previously reported that mast cells constituted the major IL-17A-positive cell population in SpA synovitis [39], we aimed to confirm that IL-17A protein is actually present in human mast cells. We first tested the specificity of various commercially available anti-IL-17A antibodies on a panel of lysates by Western blot. Recombinant IL-17A and the lysate of PMA/ionomycin-stimulated healthy donor PBMC’s (containing native IL-17A) were used as positive control. As negative control, we used the LAD2 mast cell line [45] and fibroblast-like synoviocytes (FLS) as these cells have no detectable IL-17A mRNA, both in normal condition and after stimulation with PMA/ionomycin, and IL-17A is not detectable by ELISA in their culture supernatants (data not shown). The polyclonal anti-IL-17A antibody AF-371-NA used in previous histological studies detected a band in both positive controls with no signal in the negative controls. Both monoclonal antibodies, MAB371 and A7A, detected a band in positive controls, but also in the negative control lysates (Fig. 1A). Collectively, these data confirm the specificity of the polyclonal antibody AF-371-NA for IL-17A (excluding potential 10% cross-reactivity with IL-17F as described in the manufacturer’s datasheet), used in previous histological studies. Furthermore Reich et al performed a protein screening array (2960 human protein) with AF-371-NA and observed no binding for other proteins than human IL-17A and IL-17F [40].

Absence of membrane receptor-bound IL-17A on human mast cellsHaving confirmed the specificity of the anti-IL-17A antibodies, we next explored the possibility that positive immunostaining of mast cells may be due to IL-17 protein bound to its receptors on the surface of mast cells rather than by protein localized in the cell. IL-17A homodimers and IL-17A/IL-17F heterodimers canonically bind to the IL-17RA/IL-17RC complex [46]. In IL-

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1.rhIL17A 2.recIL17A/F 3.�a��e �.LA�2 5.FLS

Mouse monoclonal αIL17 (R&D [41809])

Mouse monoclonal αIL17 (Abcam [7a7])

Goat polyclonal αIL17 (R&D)

�osi��e controls �e�a��e controlsA

rhIL17ATmem

pma/ionobrefeldin

Tonsil mastcells

α-tubulin

stripping

B

Goat polyclonal αIL17 (R&D)

Mouse monoclonal αIL17 (R&D [41809])

Figure 1. Specificity of mouse monoclonal antibodies #41809 and 7a7 is poor. Mast cells contain Il-17A protein that is detected by both, goat polyclonal and mouse monoclonal antibodies (A) Western blot. Positive controls: 1, recombinant IL-17A homodimer, 2, recombinant IL-17A/F heterodimer; 3, lysate of stimulated PBMC (PMA/ionomycin and brefeldin treated). Negative controls: 4, lysate of steady-state culture LAD2 cell line and 5, fibroblast like synoviocytes (FLS) (passage>5). (B) Detection with goat polyclonal antibody of recombinant IL-17A, native variants of IL-17 in stimulated T memory cells treated with brefelding and in purified tonsillar mast cells. Membrane was stripped and reprobed with the mouse monoclonal antibody.

17A-positive neutrophils in tissue an autocrine loop was found between IL-17A and IL-17RC, indicating a role for receptor binding [44]. A similar mechanism may be present in mast cells. LAD2 mast cell line expresses high levels of both mRNA and protein for IL-17RA, however these cells do not express IL-17RC [47] and thus the heterodimeric IL-17RA/IL-17RC complex required for binding of IL-17 is not present. Similar data was obtained on the mRNA level for the mast cell line HMC-1 [48,49].

In order to further confirm that IL-17A is not able to bind its canonical IL-17RA/IL-17RC pair on the membrane of mast cells, we incubated mast cells with IL-17A in vitro and assessed gene transcription activation by microarray. It has been described that in a variety of cell types, including synovial fibroblasts, binding of IL-17A to the IL-17RA/IL-17RC complex induces expression of a set of responsive genes, including BMP6, CXCL1, CXCL2, CXCL3, CCL20, IL-6, IL-8, and PGE2 [50]. Gene expression analysis of the human mast cell line LAD2 stimulated for 2 hours with IL-17A revealed that no single gene was differentially expressed as compared to the unstimulated control. Incubation of mast cells with IL-17A for 8 hours induced expression of 101 genes, including 85 upregulated and 16 downregulated with more than 2-fold change as compared to unstimulated control (Table 1a and 1b). Strikingly,

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only two out of 19 genes previously described to be upregulated upon canonical IL-17A signaling [50,51] were significantly upregulated in our experiments, CEBPB and NFKB2, with minimal fold changes (1,42 and 1,35, respectively) (Table 1c). In line with the low IL-17RC expression, these findings argue against interaction of IL-17A with its canonical receptor pair on the surface of human mast cells.

To further establish that in situ detected IL-17A is not merely bound to IL-17RA or other protein complexes on the cell surface, we performed a Western blot analysis of primary tonsillar mast cells. By transforming the tonsil tissue biopsies into cell suspension, we were able to carefully treat the cells with acid washing in order to disrupt protein interactions with molecules on the plasma membrane. Hence, the prepared cellular lysate did not contain extracellular membrane bound proteins. Both the goat polyclonal antibody and the mouse monoclonal antibody described above detected a clear band of the expected molecular weight of IL-17A (Fig. 1B). Additionally, the presence of IL-17A in intracellular excretory vessels was confirmed by high resolution confocal microscopy and electron microscopy [30,47]. Collectively, these data support the concept that IL-17A protein is localized inside human mast cells rather than bound to the IL-17A receptors on the cell surface.

IL-17-positive mast cells are present in the inflamed PsO skin and IBD gut Having reported the abundant presence of IL-17A-positive mast cells in SpA synovitis [39] and confirmed the specificity of these immunostainings, we next assessed if IL-17A mast cells were present in affected skin from PsO patients and in inflamed colon biopsies of patients with IBD as proxy for the SpA-specific extra-articular manifestations. Similar to the findings in SpA synovitis (Fig. 2A), co-localization of cellular IL-17 staining with mast cell tryptase staining was seen in the dermis, but not the epidermis, of PsO skin (Fig. 2A), and the lamina propria (Fig. 2B) as well as the submucosa (Fig. 2C) of inflamed colon from of IBD patients, where no differences between UC and CD were observed. Besides mast cells, IL-17A protein expression in situ was also detected in CD15-expressing neutrophils (Fig. 2D), although in skin the presence of neutrophils was restricted to the epidermis, Munro’s microabcesses and spongiform pustules of Kogoj, and not found in the dermis. Mast cells and neutrophils constituted almost all IL-17A-expressing cells in all conditions whereas IL-17A-positive CD3-positive or IL-17A-positive CD4-positive cells were virtually not detected in any of the analyzed tissues (Fig. 2E). Collectively, these data indicate that mast cells are major source of IL-17A in inflamed dermis, colonic lamina propria and submucosa in the context of SpA-related diseases.

No specific accumulation of IL-17-positive mast cells in skin inflammation related to PsO or control diseases As we previously observed that the total number of mast cells as well as the proportion of IL-17A-positive mast cells was increased in SpA synovitis compared to rheumatoid arthritis (RA) synovitis as a control [26], we next assessed if a similar disease-related increase was seen in skin and gut. In contrast to the situation in SpA versus RA synovitis, the absolute and relative numbers of IL-17A-positive mast cells were similar between the dermis of PsO patients and

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Figure 2. IL-17A-positive mast cells are found in PsO skin and IBD colon. Immunofluorescence stainings show IL-17-containing mast cells in the (A) skin and (B) colon. (C) The overview picture of a colon sample depicts the difference between lamina propria (left side) and submucosa (right side). Mast cells in the lamina propria are mainly IL-17-negative. (D) Picture shows the presence of IL-17-positive neutrophils in a colon sample of a patient with CD. (E) CD3 expressing T cells were not detected among IL-17A-expressing cells, in a colon sample of a patient with CD. (F) Appearance of IL-17A-positive mast cells confirmed in tonsil. The signal for IL-17A is shown in green in all pictures, the signal for mast cell tryptase is shown in red in picture A-C and F, the signal for CD15 is shown in red in picture D and the signal for CD3 is shown in red in picture E. In picture E no colocalization between IL-17A and CD3 was observed, orange spots are auto-fluorescent erythrocytes.

various non-psoriatic types of skin diseases (Fig. 3). Unexpectedly, IL-17A-positive mast cells were also detected in the dermis of normal skin, indicating their physiologic presence in barrier tissues. Confirming this observation, IL-17A-positive mast cells were also found in the lamina propria and submucosa of normal colon and in tonsils (Fig. 2F). The normal synovial tissue is paucicellular and mast cells are hardly detected [52], and was thus not analyzed.

The number of IL-17A-positive mast cells per 5 high power fields, represented as median and interquartile range, were significantly increased in PsO dermis, 174,0 (142,8-237,0) versus non-inflamed healthy dermis 54,3 (47,9-58,9), p=0,003 and IBD colonic submucosa 298,3 (252,1-337,2) versus normal colonic submucosa 80,2 (59,4-133) p=0,02. Similarly the number of total mast cells was also significantly increased between PsO dermis 246,0 (186,5-375,0) and normal dermis 71,0 (60,8-73,9), p=0,003 and between IBD colonic submucosa 403,6 (336,0-476,9) and normal colonic submucosa 212,5 (101,8-462,5) p=0,02.

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Also in IBD lamina propria the total number of mast cells showed a clear trend towards increase as compared to healthy lamina propria. However, in sharp contrast to the dermis and the colonic submucosa, the trend for the number of IL-17-positive mast cells was reversed in the lamina propria (Fig. 3). Accordingly, the percentage of IL-17A-positive mast cells was significantly lower in IBD 13%(6-31%) versus healthy lamina propria 45%(31-73) p=0,02, where no difference was observed in the percentages of IL-17A-positive mast cells between IBD 89%(82-92) and healthy submucosa 82%(79-90) (Fig. 3).

DISCUSSIONWe recently reported the specific increase of IL-17A-positive mast cells in the actively inflamed synovial tissue of patients with SpA as compared to RA [26]. IL-17A is a crucial cytokine in SpA and IL-17A-containing mast cells were reported in many studies [40,53], yet the reliability and relevance of detection of IL-17A-containing mast cells has been questioned on several grounds. As to the reliability, there are potential concerns about the specificity of the detection tools as these cells can only be identified by immunostaining of paraffin embedded tissue biopsies with only one commercially available antibody [54]. Here, we confirmed the genuine presence of IL-17A protein in primary human tissue mast cells by Western blot analysis. Testing several commercially available antibodies, we confirmed that the commonly used affinity purified goat polyclonal antibody AF-371-NA is specific and sensitive for IL-17A detection. Additionally, we indicated that the IL-17A protein detected by immunostainings is localized inside the mast cell rather than merely bound to

Figure 3. IL-17A-positive mast cells in target tissue of chronic inflammatory arthritis, skin disease and IBD IL-17A-positive mast cells are present in SpA, PsO and IBD target tissue, but also in normal skin and colon. Except in the lamina propria, these cells accumulated upon inflammation. And while SpA synovium was enriched for IL-17A-positive mast cells over the control disease RA, PsO skin contained similar numbers to control conditions. The epidermis did not contain any mast cells and was not displayed in this figure. Full bars represent total mast cells, open bars IL-17-positive mast cells. Data is shown as median with interquartile range. * P<0,05.

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the cell membrane via a specific receptor by demonstrating: a) the absence of IL-17RC, one subunit of the canonical heterodimeric receptor complex for IL-17A binding, on mast cells, b) the absence of IL-17A-induced gene transcription in mast cells stimulated with IL-17A, and c) the presence of IL-17A as assessed by Western blot in lysates of ex vivo acid-treated cells, ruling out binding of IL-17A to cell membrane receptors other than IL-17RA-IL17RC. Collectively, these data do not only confirm the genuine presence of IL-17A protein in human tissue mast cells but also rule out an autocrine IL-17A-IL-RC loop as recently described for neutrophils [44]. It is important to note, however, that our results do not formally imply that IL-17A is also produced by mast cells themselves. Indeed, both murine genetic reporter models and studies with human mast cells isolated from tissue suggested that mast cells cannot produce IL-17A [42,47,55]. The discrepancy between these reports and our current findings is explained by the fact that we recently demonstrated that human tissue mast cells are able to capture exogenous IL-17A from the milieu [47].

As to the relevance of IL-17A-positive mast cells versus other potential sources of IL-17A for chronic tissue inflammation, we recently demonstrated in a proof-of-concept trial that nilotinib, a c-kit inhibitor inducing apoptosis of mast cells, was clinically effective in patients with peripheral SpA [27]. It should be noted that c-kit is also expressed by other cells, including innate lymphoid cells, and that nilotinib also targets a few other tyrosine kinases. It can thus not be formally concluded that the therapeutic benefit of nilotinib observed in this trial was directly related to mast cell targeting. To further explore the potential relevance of IL-17A-positive mast cells for chronic tissue inflammation, we extended our ex vivo tissue analyses in this study. Since SpA fits together with PsO and IBD into a cluster of immune-mediated inflammatory diseases that are strongly related to the IL-23/IL-17 axis, at least partially overlapping cellular and molecular mechanisms are expected to be involved in pathophysiology of these diseases. We therefore hypothesized that accumulation of IL-17A-positive mast cells would not only be a specific features of SpA synovitis, but also of PsO skin and IBD gut. As expected IL-17A-positive mast cells were present in the dermis of PsO patients and in the colon of IBD patients, similar as we reported for SpA synovium previously [26]. Surprisingly, we also detected IL-17A-positive mast cells in healthy skin and colon. As mast cells are known to play a role in host defense in barrier tissues they are present under homeostatic conditions [56,57]. The obtained data suggest that the mast cell/IL-17A axis may also be relevant for barrier defense, however this hypothesis requires further study. So far we have no information whether synovial mast cells are also positive for IL-17A as in normal synovial tissue mast cells extremely scarce [52].

Although already present in normal barrier tissues like skin and colon, the accumulation of mast cells is strongly increased upon inflammation. Remarkably, this increase in total mast cells in the presence of inflammation is correlated with an increase in the IL-17A-positive mast cells with the exception of IBD lamina propria (Fig. 3). It remains to be identified which mechanisms are responsible for the increase in IL-17-positive mast cells. It is possible that the capacity of mast cells to take up IL-17A upon inflammation is increased, or alternatively, more IL-17A is available in the inflamed tissue so that there more mast cells become positive. In the lamina propria of IBD, however, we observed a significant decrease in the fraction of IL-

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17A-positive mast cells over total mast cells. This may indicate several scenarios: a) the lamina propria is infiltrated by a distinct mast cell subset with lower capacity to accumulate IL-17A, b) the available IL-17A for uptake by mast cells is decreased in the lamina propria, or c) mast cells in the lamina propria actively release IL-17A and thus appear empty. Recently it was proposed that IL-17A in the bowel, other than in the skin or the joint, may have a protective role, next to the role as master driver of tissue inflammation [58,59]. The paradoxical worsening of CD upon clinical inhibition of IL-17RA may be explained by such a protective role of IL-17A signaling [60]. Supporting this hypothesis it has been shown in rodents that the integrity of the epithelial barrier partially depends on IL-17 [59].

Surprisingly, mast cells and neutrophils comprise most of the IL-17A-expressing cells in all the analyzed tissues. The canonical producers, IL-23R and RORC expressing T cells and ILCs, are not found to express IL-17A in situ using an immunostaining approach. It remains to be formally determined if this finding indicates that these canonical IL-17A producers are less relevant to these types of tissue inflammation than previously appreciated or, alternatively, that T cells and ILCs immediately secrete IL17A and that therefore immunostaining is not the appropriate technique to detect this production. Analysis of single cells isolated directly from tissues, by qPCR, flow cytometry, or ELISPOT is required to fully address this question and thereby allow a better interpretation of the relevance of IL-17A-positive mast cells for chronic tissue inflammation. These approaches may also allow to assess other, IL-17A-related cytokines, including IL-17F and IL-22, that may influence chronic tissue inflammation [19,20,61,62].

In conclusion, this study demonstrated that human tissue mast cells genuinely contain IL-17A protein, albeit they may not produce this cytokines themselves, and that such IL-17A-containing mast cells are not only found in SpA synovitis but also in psoriatic skin and IBD gut. However, the accumulation of the IL-17A positive mast cells in skin is not disease-specific. Better understanding of the role of IL-17A-positive cells in physiology and pathology will require analysis of the mechanisms of IL-17A uptake and release by mast cells, identification of IL-17A-producing cells in these tissues, and the role of IL-17A-related cytokines such as IL-17F and IL-22 in the different types of chronic tissue inflammation.

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Table S1a. Genes up-regulated in mast cells 8 hours after incubation with IL-17A.

Gene symbol Gene name Fold change

RNVU1-18 RNA, variant U1 small nuclear 18 6.94CREM cAMP responsive element modulator 6.03HSPA1B heat shock 70kDa protein 1B 5.53CCL4L2 chemokine (C-C motif ) ligand 4-like 2 4.54HILPDA hypoxia inducible lipid droplet-associated 4.35GZMB granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1 4.21SLC25A19 solute carrier family 25 (mitochondrial thiamine pyrophosphate carrier) 3.66EZR ezrin 3.63LOH12CR1 loss of heterozygosity, 12, chromosomal region 1 3.60ANKRD37 ankyrin repeat domain 37 3.52SYAP1 synapse associated protein 1 3.46NKD2 naked cuticle homolog 2 (Drosophila) 3.44ADM adrenomedullin 3.43CTH cystathionase (cystathionine gamma-lyase) 3.24HES4 hes family bHLH transcription factor 4 3.21RNU11 RNA, U11 small nuclear 3.18CXCR4 chemokine (C-X-C motif ) receptor 4 3.11NDEL1 nudE neurodevelopment protein 1-like 1 3.10SLC19A2 solute carrier family 19 (thiamine transporter) 3.06MOB3B MOB kinase activator 3B 3.06SAP30 Sin3A-associated protein, 30kDa 2.98ZNF184 zinc finger protein 184 2.94PLIN2 perilipin 2 2.92APBB3 amyloid beta (A4) precursor protein-binding, family B, member 3 2.91CDK11B cyclin-dependent kinase 11B 2.90AVPI1 arginine vasopressin-induced 1 2.79EIF2AK3 eukaryotic translation initiation factor 2-alpha kinase 3 2.77JMY junction mediating and regulatory protein, p53 cofactor 2.76IRX4 iroquois homeobox 4 (IRX4), transcript variant 5 2.75ADCYAP1 adenylate cyclase activating polypeptide 1 (pituitary) 2.74RANBP2 RAN binding protein 2 2.68CDK11A cyclin-dependent kinase 11A 2.67C9orf72 chromosome 9 open reading frame 72 2.65BCL11A B-cell CLL/lymphoma 11A (zinc finger protein) 2.64HSPA1A heat shock 70kDa protein 1A 2.61MFSD12 major facilitator superfamily domain containing 12 2.58RBKS ribokinase (RBKS), transcript variant 1 2.58SUPV3L1 suppressor of var1, 3-like 1 (S. cerevisiae) 2.57B4GALT5 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 5 2.57CASP9 caspase 9, apoptosis-related cysteine peptidase 2.55MEDAG mesenteric estrogen-dependent adipogenesis 2.54ZBTB1 zinc finger and BTB domain containing 1 2.54

SUPPLEMENTARY MATERIALS

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Table S1a. (continued).


SNHG15 small nucleolar RNA host gene 15 (non-protein coding) 2.51FAM115C family with sequence similarity 115, member C 2.50NR4A2 nuclear receptor subfamily 4, group A, member 2 2.47ZNF331 zinc finger protein 331 2.47FOSL2 FOS-like antigen 2 2.45SLC7A5 solute carrier family 7 (amino acid transporter light chain, L system), member 5 2.44SIK1 salt-inducible kinase 1 2.42FAM177A1 family with sequence similarity 177, member A1 2.41EHD1 EH-domain containing 1 (EHD1), transcript variant 2 2.38SELK selenoprotein K 2.37HOXA5 homeobox A5 2.36SH3TC1 SH3 domain and tetratricopeptide repeats 1 2.35NR4A3 nuclear receptor subfamily 4, group A, member 3 2.31SPAG4 sperm associated antigen 4 2.31ERO1L ERO1-like (S. cerevisiae) 2.30TUBA4A tubulin, alpha 4a 2.30SNORD3B-1 small nucleolar RNA, C/D box 3B-1 2.28FBXL14 F-box and leucine-rich repeat protein 14 2.26PKIG protein kinase (cAMP-dependent, catalytic) inhibitor gamma 2.25UCN2 urocortin 2 2.21HS3ST2 heparan sulfate (glucosamine) 3-O-sulfotransferase 2 2.21BNIP3 BCL2/adenovirus E1B 19kDa interacting protein 3 2.19LGALS12 lectin, galactoside-binding, soluble, 12 2.16AGAP3 ArfGAP with GTPase domain, ankyrin repeat and PH domain 3 2.16CCNH cyclin h 2.15RFX3 regulatory factor X, 3 (influences HLA class II expression) 2.13MXI1 MAX interactor 1, dimerization protein 2.12PBX4 pre-B-cell leukemia homeobox 4 2.12GABARAPL1 GABA(A) receptor-associated protein like 1 2.11PHF20 PHD finger protein 20 2.09PLAUR plasminogen activator, urokinase receptor 2.08HK2 hexokinase 2 2.07GALC galactosylceramidase 2.05RGS2 regulator of G-protein signaling 2 2,04SEMA6A sema domain, transmembrane domain (TM), and cytoplasmic domain,

(semaphorin) 6A 2.03

TOB2 transducer of ERBB2 2.03FAM13A family with sequence similarity 13, member A 2.02KIF27 kinesin family member 27 2.02RELT RELT tumor necrosis factor receptor 2.01SEMA4D sema domain, immunoglobulin domain (Ig), transmembrane domain (TM)

and short cytoplasmic domain, (semaphorin) 4D2.01

XLOC_014512 BROAD Institute lincRNA 2,00RGPD6 RANBP2-like and GRIP domain containing 6 2.00

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Table S1b. Genes down-regulated in mast cells 8 hours after incubation with IL-17A.


EGR1 early growth response 1 -3.02SNAR-G1 small ILF3/NF90-associated RNA G1 -2.92SNAR-H small ILF3/NF90-associated RNA H -2.89EGR2 early growth response 2 -2.87SNAR-D small ILF3/NF90-associated RNA D -2.72SNAR-G2 small ILF3/NF90-associated RNA G2 -2.60SNAR-B2 small ILF3/NF90-associated RNA B2 -2.60SNAR-F small ILF3/NF90-associated RNA F -2.59EGR3 early growth response 3 -2.42LRRC70 leucine rich repeat containing 70 -2.39SPRED1 sprouty-related, EVH1 domain containing 1 -2.31SNAR-A3 small ILF3/NF90-associated RNA A3 -2.26FASLG Fas ligand (TNF superfamily, member 6) -2.23SNORD3B-1 small nucleolar RNA, C/D box 3B-1 -2.17ALPK3 alpha-kinase 3 -2.03SGMS1 sphingomyelin synthase 1 -2.00

Table S1c. Genes encoding proteins involved in IL-17A signaling pathway.

Gene symbol Gene name Fold change p-value

CEBPB CCAAT/enhancer binding protein 1.43 0,0002NFKB2 nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 1.35 0,0006LCN2 lipocalin 2 1,30 0,0038MAPK1 mitogen-activated protein kinase 1 1,24 0,0092CCL7 chemokine (C-C motif ) ligand 7 1,09 0,2762CXCL5 chemokine (C-X-C motif ) ligand 5 1,07 0,4257JUN jun proto-oncogene 1,07 0,5180IL17RA interleukin 17 receptor A 1,04 0,5572FOS FBJ murine osteosarcoma viral oncogene homolog 1,04 0,6933CEBPD CCAAT/enhancer binding protein -0,99 0,9404IL17RC interleukin 17 receptor C -0,98 0,7935IL6 interleukin 6 -0,97 0,6783GSK3B glycogen synthase kinase 3 beta -0,94 0,4118MAP3K7CL MAP3K7 C-terminal like -0,91 0,2341MMP1 matrix metallopeptidase 1 -0,91 0,2826CCL2 chemokine (C-C motif ) ligand 2 -0,81 0,0263IKBKE inhibitor of kappa light polypeptide gene enhancer in B-cells,

kinase epsilon -0,72 0,0004

NFKBIA nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha

-0,71 0,0001

NFKBID nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, delta

-0,61 0,00005

uva-dare (digital academic repository) the role of innate ... · il-17 pathway [7] and at least...

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