IL-6, IL-17 and Stat3 are required for auto-inflammatory syndrome development in mouse

Auto-inflammatory syndrome, a condition clinically distinct from rheumatoid arthritis, is characterized by systemic inflammation in tissues such as major joints, skin, and internal organs. Autonomous innate-immune activation is thought to promote this inflammation, but underlying pathological mechanisms have not been clarified nor are treatment strategies established. Here, we newly established a mouse model in which IL-1 signaling is conditionally activated in adult mice (hIL-1 cTg) and observed phenotypes similar to those seen in auto-inflammatory syndrome patients. In serum of hIL-1 cTg mice, IL-6 and IL-17 levels significantly increased, and signal transducer and activator of transcription 3 (Stat3) was activated in joints. When we crossed hIL-1 cTg with either IL-6- or IL-17-deficient mice or with Stat3 conditional knockout mice, phenotypes seen in hIL-1 cTg mice were significantly ameliorated. Thus, IL-6, IL-17 and Stat3 all represent potential therapeutic targets for this syndrome.

Histological arthritis score. Ankle joints were removed from control and hIL-1 cTg mice three weeks after PolyI-PolyC injection, and each sample was stained with Safranin O. Safranin O-positive areas were scored as previously described 29 . Articular cartilage damage was assessed in sagittal sections of ankle joints and graded according to a modified Mankin histologic score for the talus articular side 30 . A total modified Mankin score representing the overall state of cartilage in the joint was calculated for each set of experiments.
Three-dimensional microcomputed tomography (Micro-CT) analysis. Changes in bony microstructure around the ankles were assessed in mice three weeks after PolyI-PolyC injection using micro-CT with an X-ray micro-CT system (CosmoScan GX; Rigaku Corporation, Tokyo, Japan). Eroded areas as a proportion of total cortical bone area were calculated at ankle joints.
Histopathological and fluorescent immunohistochemical analysis of joints. Ankle joints were removed from control and hIL-1 cTg mice three weeks after PolyI-PolyC injection, fixed in 10% neutral-buffered formalin and embedded in paraffin, and then tissue blocks were cut into 4-μm sections. Ankles were decalcified in 10% EDTA, pH7.4, before embedding. Hematoxylin and eosin (H&E) or safranin-O staining was performed according to standard procedures. For each fluorescent immunohistochemistry assay, sections were subjected to microwave treatment for 10 min in 10 mM citrate buffer solution (pH 6.0) for antigen retrieval. After blocking with 3% BSA in PBS for 1 h, sections were stained for 6 h with rabbit anti-mouse pSTAT3 (1:100 dilution; Cell Signaling Techniques, Inc.), mouse anti-mouse pSTAT3 (1:100 dilution; Cell Signaling Techniques, Inc.), rabbit anti-mouse IL-6 (1:100 dilution; Abcam), rabbit anti-mouse CD45 (1:100 dilution; Abcam), rabbit anti-mouse  showing construct used to construct inducible human IL-1α (hIL-1α) conditional transgenic mice. The chick actin promoter (CAG) was linked to a neomycin resistance gene (Neo) and a poly A sequence, and both were flanked by floxP sites (triangles). The hIL-1α gene was then inserted downstream of those sequences to yield hIL-1α transgenic construct. The construct was injected into C57/Bl6 mouse embryo to become hIL-1α transgenic mice, which were crossed with Mx Cre transgenic mice to establish inducible hIL-1α conditional transgenic mice (hIL-1α cTg). PolyI-PolyC injection of these mice activated Cre expression via the Mx promoter, excising the Neo-Poly A sequence and enabling hIL-1α expression driven by the CAG promoter. (b-g) PolyI-PolyC was injected into eight-week-old control (Ctl) or hIL-1α cTg (cTg) mice, and three weeks after injection, peripheral blood mononuclear cells (left panel) and sera (right panel) were isolated, and hIL-1α protein levels determined by ELISA (b). Data in (b) represent mean human IL-1α (pg/ml) ± SD (left, n = 3 for Ctl; n = 5 for cTg; * P < 0.05; right, n = 3 for Ctl; n = 3 for cTg). Ankle thickness was measured at indicated time points (c). Ankle thickness of cTg mice not treated with PolyI-PolyC is shown in (c). Ankle thickness is shown as mean thickness ± SD (c) (n = 3 for controls treated with PolyI-PolyC, n = 3 for cTg mice treated with PolyI-PolyC, and n = 3 cTg mice not treated with PolyI-PolyC, ** P < 0.01). Ankle tissue specimens from Ctl or cTg mice were stained with hematoxylin eosin (HE, left upper panels) or safranin O and methyl green (left lower panels) three weeks after PolyI-PolyC injection when mice were 11 weeks old (d), the safranin O-positive articular cartilage area was scored (d, right panel) (n = 3 for control; n = 3 for cTg mice; * P < 0.05) and Mankin scores were evaluated (e) (n = 3 for control; n = 3 for cTg mice; *** P < 0.001). Bar, 100 µm. Bone destruction at ankle joints in Ctl or cTg mice was evaluated by using micro-CT analysis three weeks after PolyI-PolyC injection when mice were 11 weeks old (f). White blood cell (WBC), hemoglobin (Hb) or platelet (Plt) counts were also evaluated (g). Data in (g) represent mean WBC, Hb or Plt counts in peripheral blood ± SD (n = 7 for control; n = 3 for cTg mice; * P < 0.05, *** P < 0.001). Flow cytometeric analysis and sorting. Antibodies purchased from BioLegend and eBioscience were diluted 1:400. For IL-17A intracellular staining, cells were stimulated 4 h in complete medium with PMA (50 ng ml −1 ) and ionomycin (1000 ng ml −1 ; both from Sigma-Aldrich) in the presence of brefeldin A (eBioscience). Surface staining was then performed in the presence of Fc-blocking antibodies (2.4G2), followed by intracellular staining with anti-IL-17A antibodies in IC fixation buffer (#00-8222-49,eBioscience), according to the manufacturer's instructions. We performed flow cytometry acquisition on a FACS Canto II cytometer (BD Biosciences, San Jose, CA, USA) and analysed data using FlowJo software (Tree Star, Ashland, OR, USA). We sorted mouse CD3-positive, B220-positive, Gr-1-negative/CD11b-positive, Gr-1-positive/CD11b-positive and CD45-ngetaive cells using a FACS Aria II system (BD Biosciences).
Antibodies for flow cytometry. Realtime PCR analysis. Total RNAs were isolated from sorted cells using TRIzol reagent (Invitrogen Corp.), and cDNA was generated using oligo(dT) primers and reverse transcriptase (Wako Pure Chemicals Industries). Quantitative realtime PCR was performed using SYBR Premix ExTaq II reagent and a DICE Thermal cycler (Takara Bio Inc.), according to the manufacturer's instructions. β-actin (Actb) expression was analyzed as an internal control. Primers for realtime PCR were as follows.
Study approval. Mice protocols were approved by that committee, and all experiments were carried out based on committee guidelines.

Results
Establishment of IL-1 cTg mice. First, we created a conditional IL-1 transgenic construct in which the chick actin promoter (CAG) was linked to a neomycin (Neo)-poly A sequence flanked by flox sites and followed by the human IL-1α (hIL-1α) sequence ( Fig. 1a). At steady state, the CAG promoter activity promotes Neo expression terminated by the poly A sequence, blocking induction of hIL-1α expression (Fig. 1a). We injected that construct into C57BL/6 mouse embryos to establish an hIL-1α transgenic line in this strain. We then crossed hIL-1α transgenic mice with Mx Cre mice to yield Mx Cre/hIL-1α transgenic mice, hereafter called hIL-1α conditional transgenic mice (hIL-1α cTg) (Fig. 1a). In hIL-1α cTg mice, injection of PolyI-PolyC activated the Mx promoter and excised the Neo-poly A sequence, enabling hIL-1α expression (Fig. 1a).
For all experiments, we injected PolyI-PolyC into eight-week-old hIL-1α cTg or control (Ctl) mice. Three weeks later, we isolated peripheral blood mononuclear cells and determined intracellular hIL-1 levels by ELISA (Fig. 1b). As shown in Fig. 1b, hIL-1α protein was specifically detected in cells or sera from hIL-1α cTg mice, indicating successful establishment of the Tg line. In these conditions, we detected major joint dominant arthritis development in 100% of hIL-1α cTg mice but did not observe arthritis in control mice before or after PolyI-PolyC injection. When we evaluated ankle joint swelling in hIL-1α cTg mice based on ankle thickness, we found that thickness increased significantly in hIL-1α cTg mice after PolyI-PolyC injection (Fig. 1c). Ankle thickness was unchanged in hIL-1α cTg mice in the absence of PolyI-PolyC injection (Fig. 1c). Hematoxylin and eosin (H&E) staining of joint tissue indicated joint destruction and synovitis in hIL-1α cTg mice by three weeks after PolyI-PolyC injection (Fig. 1d). Safranin-O staining of joint tissues also showed synovitis development and loss of articular cartilage in ankle joints of hIL-1α cTg mice (Fig. 1d). As a result, the Mankin score, as determined by histological examination of articular cartilage damage, was significantly higher in hIL-1α cTg than in control mice (Fig. 1e). Micro CT analysis demonstrated severe joint destruction in hIL-1α cTg relative to control mice (Fig. 1f). Peripheral blood tests showed significantly elevated white blood cell (WBC) and platelet (Plt) counts, and hIL-1α cTg mice showed anemia, as evidenced by significantly lower hemoglobin (Hb) levels (Fig. 1g).
We also detected significantly larger spleen, splenomegaly, in hIL-1α cTg compared with control mice (Fig. S1a). Hepatitis and dermatitis were also observed in hIL-1α cTg mice, although hIL-1α cTg mice did not exhibit fever (Fig. S1b,c). hIL-1α cTg mice exhibited thinner and thicker respective subcutaneous fatty and dermal layers than did control mice (Fig. S1d). We also detected infiltration of inflammatory cells into dermal tissues in hIL-1α cTg mice (Fig. S1d) and identified those cells as Myeloperoxigenase (MPO)-positive neutrophils (Fig. S1e). hIL-1α cTg also showed lower body temperature than did control mice (Fig. S1f).

IL-17 functions in arthritis development in hIL-1α cTg mice.
We next undertook a similar cross of hIL-1α cTg mice with IL-17A/F-deficient (IL-17 KO) mice to evaluate potential involvement of the cytokine IL-17 in hIL-1α cTg phenotypes. hIL-1α cTg/IL-17 KO mice showed improvement similar to that shown by hIL-1α cTg/IL-6 mice in terms of arthritis development (Fig. 4). Increased ankle thickness in hIL-1α cTg mice was significantly inhibited in hIL-1α cTg/IL-17 KO mice (Fig. 4a). Synovitis and articular cartilage loss was also inhibited by IL-17A/F-deficiency in hIL-1α cTg mice (Fig. 4b).
Although not significant, increases in the Mankin score seen in hIL-1α cTg mice were diminished in hIL-1α cTg/IL-17 KO mice (Fig. 4c). Bone erosion was also inhibited (Fig. 4d), and elevated platelet counts were significantly inhibited in hIL-1α cTg/IL-17 KO relative to hIL-1α cTg mice (Fig. 4e). Moreover, although not significant, both elevation in WBC and reduction in hemoglobin levels appeared to be partially rescued in IL-17-deficient hIL-1α cTg mice (Fig. 4e).

Discussion
Auto-inflammatory syndrome(s) are marked by systemic inflammation; those syndromes include diseases such as TRAPS, FMF and AOSD, most of them rare diseases. Patients with these syndromes exhibit major joint dominant arthritis and other systemic inflammatory symptoms, and thus are clinically distinct from RA patients. At present, treatment protocols for RA include administration of disease-modifying anti-rheumatic drugs (DMARDs) followed by biologics. However, since pathological mechanisms underlying auto-inflammatory syndromes are not fully characterized, and the diseases are rare, standard protocols to treat these conditions have not been established. It is currently thought that activation of inflammatory cytokine expression underlies these syndromes 32,33 .
Here, we show that mice in which IL-1 signaling is upregulated after birth exhibit symptoms similar to those seen in auto-inflammatory syndrome patients, such as major joint dominant arthritis and splenomegaly. Indeed, administration of CD4-depleting antibody, which effectively inhibits arthritis development in an RA model 34 , to hIL-1α cTg mice did not inhibit arthritis development (Fig. S3). This outcome is likely due to the fact that IL-17 and IL-6 are also expressed in joints of cTg mice (Fig. S4). IL-6 and IL-17 expression in cTg mouse joints was Stat3-dependent, as expression of both decreased in joint tissue of cTg/Stat3 cKO animals (Fig. S4). We demonstrate that, although phenotypes were induced by activated IL-1 signaling, targeting of either IL-6, IL-17 or Stat3 ameliorated those symptoms, suggesting that all of these factors warrant attention as potential therapeutic targets for the syndrome. Indeed, bone erosion was significantly induced by hIL-1α in cTg mice, but that phenotype was significantly blocked in mice lacking either IL-17 or Stat3 (Fig. S5). Pathological mechanisms underlying RA versus auto-inflammatory syndromes differ in the following ways. Elevated TNFα and/or IL-6 levels promote minor joint dominant arthritis, and activation of T cells such as TH17 cells is required for RA pathogenesis [35][36][37] . Stat3 is also reportedly required for arthritis development in RA [37][38][39] . By contrast, inflammasome activation followed by expression of inflammatory cytokines by macrophages 40 reportedly promotes pathogenesis of auto-inflammatory syndromes [41][42][43] . However, inflammasome-independent auto-inflammatory syndromes have also been reported 44,45 ; thus not all mechanisms underlying auto-inflammatory syndromes have been defined 46 . Inflammasomes were activated in auto-inflammatory syndrome patients, who also exhibited conversion of pro-IL-1β to an active form to secret by caspase 1 activity 47,48 . Inflammasomes are reportedly activated by activities such as macrophage activation 49,50 ; however, molecular mechanisms underlying these processes remain unclear. In our model, serum IL-1β levels increased in hIL-1α cTg mice (Fig. S6), suggesting that IL-1α stimulates inflammasome activation, and our model recapitulates, at least in part, human auto-inflammatory syndrome. Here, we show that activation of IL-1 signaling promotes major joint dominant arthritis, and that upregulation of either of IL-6, IL-17 or Stat3, which also function in RA, was required for arthritic phenotypes seen in hIL-1 cTg mice. At present, we do not know how activated IL-1 signals trigger IL-6 and IL-17 expression and Stat3 activation. Previously, we demonstrated that IL-1 signals promote IL-6 expression followed by activation of a Stat3-dependent auto-amplification loop responsible for IL-6 production 38 . IL-6 is known to promote IL-17 expression 51 . Furthermore, the IL-17-triggered positive feedback loop between IL-17 and IL-6 and its downstream effector Stat3 reportedly promotes arthritis development in gp130 mutant F759 mice 31 . In our mouse model, serum IL-17 and IL-6 levels were significantly elevated, Stat3 was activated in joints following IL-1 induction, and arthritis development was significantly blocked by deletion of IL-17, IL-6 or Stat3, suggesting that the IL-17-mediated positive feedback loop of inflammatory cytokines is induced by IL-1. Why arthritis occurs in minor versus major joints is a subject for future investigation.
Animal models have helped investigators understand the pathogenesis of many diseases. Both RA and auto-inflammatory syndromes develop after birth, and adult-onset animal models relevant to RA, such as CIA and AIA, are available; moreover, the function of IL-17 and Stat3 in RA development has been demonstrated using animal models 22,29,35,38 . By contrast, there are currently no adult-onset animal models available to study auto-inflammatory syndromes. Here, we established an adult-onset auto-inflammatory syndrome model using the Cre-loxP system to activate IL-1 signaling, which promoted systemic inflammation in adults as evidenced by inflammasome activation. We were able to establish this model on a C57BL/6 background, a strain considered resistant to arthritis development. We then crossed this model with C57BL/6 mice harboring various mutations in genes encoding cytokines and a transcription factor in order to identify potential effectors of IL-1 signaling. That analysis identified 3 factors, IL-6, IL-17, and Stat3, that may serve as therapeutic targets in treatment of auto-inflammatory syndromes. IL-6 is reportedly a therapeutic target in several auto-inflammatory diseases [52][53][54] ; however, IL-17 and Stat3 have not been previously identified as therapeutic targets in these conditions. Among auto-inflammatory syndromes, AOSD shows greatest similarity to our hIL-1 cTg mice, although our mice do not exhibit all phenotypes seen in patients with AOSD. Still's disease is also an arthritis disease first described by Still in 1897 55 and currently known as systemic juvenile idiopathic arthritis 56 . In 1971, Bywaters described 14 children with pediatric Still's disease resembling AOSD 57 . AOSD is diagnosed by 5 criteria, which must include at least two major ones. Major criteria include: arthralgia for more than two weeks, intermittent fever for longer than a week, typical rash, and a WBC greater than 10,000. Minor criteria include: sore throat, lymphadenopathy and/or splenomegaly, abnormal liver function tests (LFT), and anti-nuclear antibody (ANA)and rheumatoid factor (RF)-negative status. Exclusion criteria include infection, malignancies and rheumatic diseases. A limitation of our study is that differences exist between human auto inflammatory diseases and our models. For example, we did not detect fever in our mouse models (Fig. S1f). Also, serum ferritin levels are reportedly frequently elevated in AOSD patients 58 , an effect we also did not observe in our models. We cannot yet Figure 5. Stat3 deletion rescues hIL-1α cTg phenotypes. hIL-1α cTg (cTg) mice were crossed with Stat3 floxed (Stat3 cKO) mice to yield cTg/Stat3 cKO mice. PolyI-PolyC was injected into eight-week-old Ctl, cTg or cTg/Stat3 cKO mice, and ankle thickness evaluated at indicated time points (a). Data represent mean ankle thickness ± SD (n = 3 for control; n = 3 for cTg mice; n = 5 for cTg/Stat3 cKO; ** P < 0.01, * P < 0.05, NS not significant, cTg vs cTg/Stat3 cKO). Three weeks after PolyI-PolyC injection, ankle joints (b-d) and peripheral blood (e) were collected. Ankle tissue specimens from all three genotypes were stained with HE (upper panels) or safranin O and methyl green (lower panels) (b), and the safranin O-positive articular cartilage area (b) and Mankin scores were evaluated (c). Data in (b,c) represent mean safranin O-positive area (b) (n = 3 for control; n = 3 for cTg mice; n = 4 for cTg + Stat3 cKO mice; * P < 0.05, ** P < 0.01) or Mankin score (c) ± SD (n = 5 for control; n = 3 for cTg mice; n = 3 for cTg + Stat3 cKO mice; ** P < 0.01, *** P < 0.001). Bar, 100 µm. Destruction of ankle bone from Ctl, cTg or cTg + Stat3 cKO mice was evaluated using micro-CT (d). White blood cell (WBC), hemoglobin (Hb) and platelet (Plt) counts in peripheral blood were scored (e). Data represent mean WBC, Hb or Plt ± SD (n = 5 for control; n = 4 for cTg mice; n = 5 for cTg/Stat3 cKO; *** P < 0.001, ** P < 0.01, * P < 0.05, NS not significant). explain differences between the human syndrome and phenotypes seen in animal models. Nonetheless, patients with AOSD have been treated with non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, methotrexate and cyclosporine, and more recently with anti-IL-1 receptor or anti-IL-6 receptor antibodies 33,59 ; however, consistent strategies useful to treat AOSD are not yet established. Our study provides valuable information relevant to possible therapeutic options for auto-inflammatory syndromes.