IL-25 exacerbates autoimmune aortitis in IL-1 receptor antagonist-deficient mice

IL-25, a member of the IL-17 family of cytokines, is known to enhance type 2 immune responses, but suppress type 3 (IL-17A)-mediated immune responses. Mice deficient in IL-1 receptor antagonist (Il1rn−/− mice) have excessive IL-1 signaling, resulting in spontaneous development of IL-1–, TNF– and IL-17A–dependent aortitis. We found that expression of II25 mRNA was increased in the aortae of Il1rn−/− mice, suggesting that IL-25 may suppress development of IL-1–, TNF– and IL-17A–dependent aortitis in Il1rn−/− mice by inhibiting type 3-mediated immune responses. However, we unexpectedly found that Il25−/−Il1rn−/− mice showed attenuated development of aortitis, accompanied by reduced accumulation of inflammatory cells such as dendritic cells, macrophages and neutrophils and reduced mRNA expression of Il17a and Tnfa—but not Il4 or Il13—in local lesions compared with Il1rn−/− mice. Tissue–, but not immune cell–, derived IL-25 was crucial for development of aortitis. IL-25 enhanced IL-1β and TNF production by IL-25 receptor–expressing dendritic cells and macrophages, respectively, at inflammatory sites of aortae of Il1rn−/− mice, contributing to exacerbation of development of IL-1–, TNF– and IL-17A–dependent aortitis in those mice. Our findings suggest that neutralization of IL-25 may be a potential therapeutic target for aortitis.

of Anakinra (recombinant human IL-1RN) in GCA patients found that its blockade of IL-1 activity was effective for treatment of the disease 24 .
Using that same Il1rn −/− mouse model, we found that the expression level of IL-25 (also called IL-17E), which is a member of the IL-17 family of cytokines, was increased in the aorta. IL-25 binds to IL-25 receptor (IL-25R), which consists of IL-17RA and IL-17RB. IL-25 is preferentially produced by epithelial cells and such immune cells as macrophages, mast cells, basophils, eosinophils and T cells 25,26 . IL-25 induces production of type 2 cytokines by various types of cells, including Th2 cells, Th9 cells, invariant NKT cells and group 2 innate lymphoid cells 27,28 . Those cytokines are involved in such type 2 immune responses as protection from nematode infection and development of allergic disorders [26][27][28][29] . In addition, IL-25 plays dual roles in type 3 immune responses: it can suppress IL-17Amediated autoimmune diseases 25,30,31 , but it enhances IL-17A-mediated dermatitis 32,33 . However, the role of IL-25 in IL-1-, TNF-and IL-17A-mediated aortitis in Il1rn −/− mice has been unclear. Here, we demonstrate that IL-25 plays a facilitative role in the development of IL-1-, TNF-and IL-17A-mediated aortitis in Il1rn −/− mice.
IL-25 enhances type 3-related cytokines, but not type 2 cytokines, in aortitis. In the aortae of Il1rn −/− mice, the expression level of Ifng mRNA was comparable-but the levels of Il4 and Il13 mRNA were lowercompared with in wild-type mice (Fig. 5). Regarding type 3 and type 3-related cytokines such as IL-17A, IL-6, IL-23p19 and TNF, the expression levels of Il17a and Tnfa mRNA were significantly higher, while those of Il6 and Il23p19 mRNA were comparable, in the aortae of Il1rn −/− mice compared with wild-type mice (Fig. 4a). Although IL-25 is known to induce production of type 2 cytokines by various types of cells 27,28 , the expression levels of Il4 and Il13 mRNA were comparable in the aortae of Il25 −/− Il1rn −/− mice and Il1rn −/− mice (Fig. 5), suggesting that IL-25 is not crucial for induction of such type 2 cytokines in the lesions of aortitis. In addition, the expression levels of Ifng, Il6 and Il23p19 mRNA were comparable in the aortae of Il1rn −/− mice and Il25 −/− Il1rn −/− mice (Fig. 5). On the other hand, the expression levels of Il6, Il17a and Tnfa mRNA were significantly reduced in Il25 −/− Il1rn −/− mice compared with Il1rn −/− mice (Fig. 5). IL-25 was reported to inhibit IL-13-dependent Th17 cell differentiation, thereby contributing to suppression of Th17-mediated autoimmune diseases 25,30 . On the other hand, our results suggest that IL-25 may enhance production of IL-6, IL-17A and TNF, but not IL-23, thereby contributing to development of IL-1-, TNF-and IL-17A-dependent aortitis in Il1rn −/− mice.

IL-25 enhances production of IL-1β by dendritic cells and TNF by macrophages.
We next investigated the roles of IL-25 in production of IL-1β by DCs, which were shown to produce IL-1β and to express IL-17RB in the aortae of Il1rn −/− mice (Figs. 3b and 6), and in production of TNF by M2 macrophages, which express IL-17RB and produce TNF in response to IL-25 in humans 34 . Therefore, we isolated single cells from the aortae of Il1rn −/− mice and cultured them in the presence and absence of IL-25. Immunohistochemical analysis showed increased expression of IL-1β in CD11c + DCs and of TNF in Mac2 + macrophages derived from the aortae of Il1rn −/− mice after IL-25 stimulation (Fig. 7b-e). These observations suggest that non-immune cell-derived IL-25 is crucial for production of IL-1β by IL-17RB + CD11c + DCs and production of TNF by IL-17RB + Mac2 + macrophages, and that it contributes to development of aortitis in Il1rn −/− mice.

Discussion
Il1rn −/− mice spontaneously develop aortitis resembling such LVV as TAK and GCA; that is, infiltration of inflammatory cells into all aortic layers, necrosis of vascular smooth muscle cells, disruption of elastic fibers, and vascular remodeling, including stenosis of the vascular lumen and aortic dilatation 1 (Fig. 1a). It was reported that the development of aortitis was diminished in Since IL-1 and/or TNF can activate Th17 cells 35 , it is thought that IL-1-and/or TNF-induced T cell-derived IL-17 may be involved in development of aortitis in Il1rn −/− mice. In the present study, we demonstrated that IL-25 is crucial for development of IL-1-, TNF-and IL-17-mediated aortitis in Il1rn −/− mice.
We found that expression of Il25 mRNA was significantly increased in the aortae of Il1rn −/− mice compared with wild-type mice (Fig. 4a). It has been reported that the source of IL-25 is resident microglia and/or brain capillary endothelial cells in EAE and/or MS 30,36 . In addition, IL-25 was expressed in lung and/or nasal epithelial cells of mice and/or humans [37][38][39] , in B cells, smooth muscle cells and endothelial cells in atherosclerotic arteries of humans 40 , in keratinocytes of patients with atopic dermatitis 41 , and in subepithelial macrophage-like cells and epithelial cells in the human colon 42 . We previously reported that IL-25 is crucial for development of allergic contact dermatitis as well as asthma in mice 32,38 . Although IL-25 proteins were detectable in the lung during asthma 38 , they were hardly detectable in the skin during allergic contact dermatitis 32 . Like in allergic contact dermatitis, IL-25 proteins were hardly detectable in aortae of Il1rn −/− mice (12 weeks) by immunofluorescence using anti-IL-25 Ab (data not shown). In addition to such non-immune cells as epithelial cells, endothelial cells, fibroblasts and tuft cells, immune cells such as mast cells, eosinophils and alveolar macrophages (M2) are known to be sources of IL-25 [43][44][45] . Although there was macrophage infiltration in the aortitis of Il1rn −/− mice (Fig. 1c), CD206 + M2 macrophages were not observed among them (Fig. 6). Infiltrating granulocytes in the aortitis of Il1rn −/− mice were overwhelmingly neutrophils (Fig. 1c), not eosinophils (data not shown). Mast cells were observed in the aortitis of Il1rn −/− mice, but their number was relatively small compared with macrophages and  www.nature.com/scientificreports www.nature.com/scientificreports/ neutrophils (Figs. 1c and 4e). Regarding this, using bone marrow cell transfer analysis, we showed that IL-25 derived from non-hematopoietic cells rather than immune cells was crucial for development of IL-1-, TNF-and IL-17-mediated aortitis in Il1rn −/− mice. Since the levels of IL-25 proteins in bronchoalveolar lavages from mice with ILC2-dependent innate-type airway inflammation and the levels of Il25 mRNA in the skin of mice with imiquimod-induced psoriatic dermatitis peaked at one hour after antigen challenge 46,47 , IL-25 production in the onset phase of aortitis in Il1rn −/− mice (i.e., 4 weeks) may be important for development of the disease.  As shown in Fig. 6, we demonstrated that type 2 immune cells (CD3 + T cells, including Th2 cells, and CD206 + M2-macrophages) did not express IL-25 receptor (IL-17RB). Thus, it seemed that IL-25 cannot activate such immune cells to induce type 2 immune responses in the aortae of Il1rn −/− mice. On the other hand, in the local lesions of aortitis in Il1rn −/− mice, we identified CD11c + DCs, Mac2 + macrophages, γδ T cells and B cells as IL-25 receptor (IL-17RB)-expressing cells (Fig. 6). We cultured GM-CSF-induced and GM-CSF plus IL-4-induced BM cell-derived DCs (BMDCs) and TNF-and LPS-treated BMDCs in the presence of IL-25 in vitro. However, we found that IL-25 did not induce production of IL-1β or TNF by those cells (data not shown), because Il17rb mRNA was hardly detected by qPCR (data not shown). In addition, IL-17RB was expressed on M2-but not M0 or M1-macrophages derived from human PBMCs, and the M2 macrophages produced TNF, IL-6 and certain chemokines in response to IL-25 34 . We generated M2 and M0 macrophages from BM cells in the presence of rmM-CSF and rmIL-4, respectively, and then stimulated them in vitro with IL-25. However, we found that IL-25 did not induce TNF production by either of those macrophages because their expression of Il17rb mRNA was below the limit of detection by qPCR (data not shown). Consistent with this, IL-17RB was not expressed on CD206 + M2 macrophages or CD86 + M1 macrophages from the aortae of Il1rn −/− mice (Fig. 6). Thus, IL-17RB + Mac2 + macrophages seen in the aortae of Il1rn −/− mice were a distinct population from the CD206 + M2 and CD86 + M1 macrophages. Likewise, γδ T cells purified from the peritoneal cavity of naïve wild-type mice did not produce IL-17A in response to IL-25, since expression of Il17rb mRNA was below the limit of detection by qPCR (data not shown). Further study is needed to elucidate the contribution of IL-17RB + γδ T cells in local lesions to development of aortitis in Il1rn −/− mice. Since no CD3 + T cells, including Th2 cells, in the aortae expressed IL-25R, Th2 cells apparently do not produce IL-13 in the aortae of Il1rn −/− mice. On the other hand, IL-25R-expressing DCs and macrophages can produce IL-1β and TNF, respectively, in response to IL-25 (Fig. 7). DC-derived IL-1β and macrophage-derived TNF are thought to subsequently activate Th17 cells to induce IL-17, contributing to development of aortitis in Il1rn −/− mice. Therefore, the function of IL-25 is reflected by the types of IL-25R-expressing cells in the local site.

Number of cells (cells/aorta)
As shown in Fig. 4b, the development of aortitis was attenuated, but not completely abrogated, in Il25 −/− Il1rn −/− mice compared with Il1rn −/− mice. Recently, it was reported that IL-17B, which (like IL-25) is a ligand for IL-17RB, is crucial for development of bleomycin-induced pulmonary fibrosis in mice by promoting type 3 immune responses in mice 49 . Therefore, IL-17B may have a compensatory role in induction of type 3 immune responses in the aortitis of Il25 −/− Il1rn −/− mice.
In the present study, we identified the steps of a new function of IL-25: 1. Non-immune cell-derived IL-25 can induce production of IL-1β and TNF by IL-17RB-expressing DCs and macrophages. 2. DC-derived IL-1β and macrophage-derived TNF can activate Th17 cells to produce IL-17A. And 3. Th17 cell-derived IL-17A contributes to development of aortitis in Il1rn −/− mice. Our findings improve our understanding of the molecular mechanisms involved in development of aortitis and suggest that neutralization of IL-25 may be a potential therapeutic target for aortitis.