Anti-Arthritic Effect of TLR4 Inhibitor TAK-242 Through the Inhibition of Th1 and Th17 Cell Proliferation in a Curdlan-Induced Spondyloarthritis SKG Mouse Model

Background: Toll-like receptor 4 (TLR4) is involved in the pathogenesis of arthritic diseases. The TLR4 inhibitor TAK-242 has shown potential as a possible treatment for rheumatoid arthritis in an adjuvant-induced arthritis rat model. However, further investigation of the anti-arthritis effect of TAK-242 in other animal models of arthritis disease is required. Objective: This study was conducted to examine the anti-arthritic effects and mechanisms of TAK-242 on curdlan-induced spondyloarthritis in SKG mice. Methods: Curdlan-injected SKG ZAP-70 W163C mice were intraperitoneally administered with TAK-242 (3 mg/kg) every other day or anti-IL-23 antibody (100 μg) weekly for 9 weeks. The therapeutic effect of TAK-242 was evaluated in the SKG mice by measuring the inammation level of foot, ileum, lung and tail by histology, the levels of proinammatory cytokines in serum, bone mineral density (BMD) in femur and spine, and CD4+T cell subtypes Th1 and Th17 in spleen. Results: TAK-242 treatment signicantly reduced clinical score, paw width, and colon length in SKG mice. Consistent with the therapeutic effect, TAK-242 also reduced peripheral arthritis and IL-17 expression in the foot and ileum tissues. It also inhibited inammation of the lungs, ileum, paws, and tail and reduced serum levels of pro-inammatory cytokines such as IL-6 and TNF-α. TAK-242 recovered the reduced bone density in the femur but not in the spine. Treatment with TAK-242 also suppressed the populations of Th1 and Th17 cells in the spleen of SKG mice. Conclusions: TAK-242 may show benecial anti-arthritis effects by inhibiting the proliferation of proinammatory Th1 and Th17 cells. Further research is needed for TAK-242 to be repositioned as a treatment for arthritis disease.


Introduction
Chronic in ammation in autoimmune diseases such as spinal arthritis (SpA) and rheumatoid arthritis (RA) causes damage to tissues and cells, resulting in the release of molecules such as damage associated molecular patterns (DAMPs) into nearby tissues. DAMPs further activate the in ammatory response through the signaling pathway regulated by toll-like receptors (TLRs) that are expressed in tissues or immune cells [1]. TLRs are expressed in most cells, which play an important role as the front line of innate immunity. Ten TLRs have been identi ed in humans, and the roles of TLR1-9 have been determined [2]. TLR4 triggers immune responses by recognizing invading organisms as pathogenassociated molecular patterns (PAMPs) [3]. TLR4 also recognizes various molecules, such as RNA or DNA released from damaged cells, to initiate immune responses as DAMPs. All the TLR-mediated in ammatory responses remove harmful stimuli or repair damaged tissues. However, under chronic in ammatory conditions, persistent innate damage signals could lead to harmful in ammation, resulting in more destruction than the original injury [4]. Thus, inappropriate TLR activation has been suggested to be involved in the chronic and persistent in ammation of RA [5]. In our previous study, we found that the TLR4 inhibitor TAK-242 (resatorvid) signi cantly ameliorated in ammatory symptoms in joint tissues in an adjuvant-induced arthritis (AIA) rat model [6]. Although TAK-242 was initially developed for sepsis treatment [7], it has the potential to be applied for treatment of other in ammatory or non-in ammatory diseases such as cancer and hyperalgesia through drug repositioning [8][9][10].
Spondyloarthritis (SpA) refers to several clinical symptoms characterized as psoriatic arthritis, enteropathic arthritis, reactive arthritis, a subgroup of juvenile idiopathic arthritis, and ankylosing spondylitis (the prototypic and best studied subtype) [11]. The disease subtypes all show joint in ammation (peripheral and axial skeleton); skin, gut and eye manifestations; and the absence of diagnostic autoantibodies (seronegative) [12]. The pathogenesis of SpA also involves genetic polymorphisms and overexpression of TLRs. For example, TLR4 gene polymorphism may be related to the development of psoriatic arthritis [13]. TLR4 expression was increased in patients with ankylosing spondylitis [14]. The IL-23/IL-17 immune axis also plays an important role in the pathogenesis of SpA. Thus, IL-23 and IL-17 cytokines have been used as therapeutic targets by many researchers [15]. Several therapeutic antibodies against IL-23 and IL-17 such as ustekinumab, secukinumab, ixekizumab and brodalumal have been clinically used for the treatment of SpA patients [16][17][18] and for the ankylosing spondylitis and/or psoriatic arthritis subforms of SpA, with promising clinical e cacy.
The SKG mouse has a mutation in the gene encoding the SH2 domain of ZAP-70, a key signal transduction molecule in T cells. The mutation changes the thresholds of T cells to thymic selection, leading to the positive selection of otherwise negatively selected autoimmune T cells [19]. Curdlan is a water-insoluble linear β-1,3-glucan, a high-molecular-weight polymer of glucose. The injection of curdlan into the SKG mouse results in the development of SpA-like symptoms, including chronic in ammation of the peripheral joint, spine, skin, ileum and colon. These features resemble the characteristics of human SpA [20]. The mechanisms by which curdlan induces arthritis drives preferential differentiation and expansion of Th17 cells or dendritic cells (DCs) through TLR and dectin-1 [21,22].
In this study, we examined whether TAK-242, a TLR4 inhibitor, has an anti-arthritic effect in the curdlaninduced spondyloarthritis SKG mouse model [23]. We showed that TAK-242 suppressed SpA in curdlaninjected SKG mice through inhibition of the proliferation of Th1 and Th17 cells. These ndings suggest that TAK-242 has the potential to be developed as a treatment for SpA.

Materials And Methods
Induction of SpA in SKG mice and TAK-242 treatment Female SKG mice were obtained from CLEA-Japan, Inc. The mice were maintained under speci c pathogen-free conditions. SpA was induced in mice between 8 and 10 weeks of age by intraperitoneally (I.P) injecting 3 mg curdlan (Wako Chemicals, Osaka, Japan). Mice received either anti-IL-23 antibody or TAK-242 injection. Mice were I.P injected with 100 μg of anti-mouse anti-IL-23(P19) monoclonal antibody (BioXcell, Seoul, Korea) 1 day before curdlan injection and weekly until mice were killed. In another group of mice, TAK-242 (3 mg/kg) was given I.P 1 day before curdlan injection and three times per week. All mice were handled according to protocols that were approved by the Committee on Animals of Kyung Hee University Hospital at Gangdong.

Clinical and histologic scoring
Clinical scores were monitored weekly and scored as follows: 0, no joint swelling or redness of nger, wrist and ankle joint; 0.1, swelling or redness of one nger joint; 0.5, mild swelling or redness of wrist and ankle; and 1.0, severe swelling or redness of the wrist and ankle. Mice were monitored by the same observer who was blinded with regard to treatment. Calipers were used to measure the wrist and ankle width. Scores for the affected joints of 4 legs were summed; the maximum score was 6. The hematoxylin and eosin (H&E)-stained sections of the ileum, tail, lung and peripheral joint were scored as described previously [24]: 0 = no in ammation, 1 = few in ltrating immune cells, 2 = 1-2 small patches in ammation, 3 = in ammation and reduced joint space throughout the ankle joint, and 4 = in ammation in > 70% of the tissue.

Histology
At the experimental endpoint of 9 weeks, the peripheral joint, lung, tail and ileum from control and treated mice were xed in 10% formalin (Sigma-Aldrich, St. Louis, MO, USA) and embedded in para n. Tissue specimens of joint and ileum were sectioned at 7 µm and 4 µm thickness, respectively. The sections were dewaxed using xylene (Duksan General Science, Seoul, Korea), dehydrated using an alcohol gradient, and stained with H&E (Cancer Diagnostics Inc. Durham, NC, USA). For immunohistochemistry, sections were depara nized, hydrated and incubated in 20 µg/mL proteinase K (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 37℃. Endogenous peroxidases were quenched in 3% hydrogen peroxide solution (Qiagen, Hilden, Germany) in methanol for 10 min. After washing with phosphate buffered saline (PBS), permeabilization with 0.4% Triton X-100 in PBS for 10 min and blocking with 1% bovine serum albumin (BSA) for 1 h at room temperature, the slides were incubated with primary antibody against IL-17A (Santa Cruz Biotechnology Inc., Dallas, TX, USA) at a 1:100 dilution for overnight at 4℃. Sections were then washed with PBS and incubated with horse anti-mouse/rabbit IgG antibody (H+L) (Universal, biotinylated, R.T.U [Vector Laboratories Inc., Burlingame, CA, USA]) for 30 min at room temperature. Next, 3,3'diaminobenzidine peroxidase substrate solution was added, and dehydration, clearing and mounting was conducted by routine procedures. Samples were observed under a microscope and photographs were taken. Sections were analyzed, and in ammation was scored blindly by at least 3 independent researchers (CHM, HMD and LKM).

Micro-computed tomography (CT) imaging
The bodies of mice were stored in 4% formalin. The mice were washed twice with PBS and imaged at a resolution of 100 μm with a NanoSPECT/CT (Bioscan Inc., Santa Barbara, CA, USA). High resolution scans of the femurs of the mice were captured to evaluate the 3D structure. The BMDs of femur and spine were measured using the manufacturer's 3D analysis tools.
Cell preparation from spleen Spleens were excised from killed mice and placed immediately in RPMI 1640 media. Using the plunger end of a syringe, the spleen was mashed through the cell strainer into a 50 ml tube. After centrifugation of the tube at 1,500 rpm, 4℃ for 5 min, the samples containing white blood cells were treated with Ammonium-Chloride-Potassium Lysing Buffer (BioLegend, San Diego, CA, USA) for the lysis of red blood cells. After centrifugation at 1500 rpm, 4℃ for 5 min, the lymphocytes were stained with antibodies for analysis of T cell subtypes.

Statistical analysis
Experimental data are expressed as mean ± standard error of the mean (SEM). Differences between three groups were analyzed using the nonparametric Kruskal-Wallis test. If a statistical difference was detected (P < 0.05), post hoc pairwise group comparisons were performed using Dunn's test. Two-way analysis of the variance (ANOVA) was used to analyze TAK-242 effects on paw thickness and clinical score over time, following Turkey's post hoc test. Prism software v.5 (Graphpad Software, San Diego, CA, USA) was used for statistical analysis and graphing. Differences were considered statistically signi cant at P<0.05.

TAK-242 treatment prevented peripheral arthritis in SKG mice with SpA
To investigate whether the TLR4 inhibitor TAK-242 could ameliorate or prevent peripheral arthritic symptoms in the SpA mouse model, SKG mice were intraperitoneally injected with TAK-242 (3 mg/kg) one day before curdlan injection and then three times a week for 9 weeks. As shown in Fig. 1A and 1B, the clinical score and ankle width of hind leg was signi cantly reduced by TAK-242. However, the inhibitory effect of TAK-242 treatment was not stronger than that of anti-IL-23 Ab treatment. The inhibitory effect of TAK-242 and anti-IL-23 Ab was almost same after 8 weeks of treatment, suggesting that TAK-242 did not show a strong inhibitory effect at the initial stage of in ammation but may be as effective as anti-IL-23 Ab treatment at the late stage. The colon length was shortened in SKG mice with curdlan-induced SpA (Fig. 1C). The shortened colon length was recovered after the treatment of anti-IL-23 Ab or TAK-242 for 9 weeks.
To further examine the anti-in ammatory effect of TAK-242, the in ammation level of foot, ileum, lung and tail was investigated with H&E staining and IL-17 immunostaining. The level of tissue in ammation was assessed by the level of invasive immune cells and IL-17 expression (Fig. 2). Treatment of anti-IL-23 Ab decreased the in ammation level. TAK-242 treatment also reduced tissue in ammation as much as anti-IL-23 Ab treatment. Consistent with the level of in ammation observed by H&E staining, the level of IL-17 staining was signi cantly reduced by anti-IL-23 Ab or TAK-242 treatment (Fig. 3A). Furthermore, serum TNF-α level was also slightly decreased by treatment of anti-IL-23 Ab. In contrast, IL-6 level was signi cantly reduced by TAK-242 treatment (Fig. 3B). These results suggest that TAK-242 treatment inhibited the peripheral arthritis of SKG mice with SpA as effectively as anti-IL-23 Ab.
TAK-242 treatment recovered the BMD loss in femurs in SKG mice with SpA Next, we investigated the effect of TAK-242 on the bone health of SKG mice with SpA by micro-CT. The BMD was signi cantly decreased in femurs of SKG mice with curdlan-induced SpA (Fig. 4). Both anti-IL-23 Ab and TAK-242 signi cantly restored in ammation-mediated BMD loss in the femur but not in the spine. In particular, BMD was not decreased in the spine of curdlan-injected SKG mice. This result also indirectly suggests that the bone formation of ankylosing spondylitis is progressing in the spine, so this bone formation can mask the reduction of BMD in the spine.

TAK-242 treatment inhibited the proliferation of in ammatory T cells in spleen of SKG mice with SpA
The mice with SpA were sacri ced after 9 weeks of TAK-242 treatment. In the spleen, Th1 and Th17 cells of the CD4 + T cell subtype were analyzed by FACS. The population of Th1 and Th17 cells was signi cantly increased in SKG mice with SpA. Both TAK-242 and anti-IL-23 antibody treatment signi cantly inhibited the increased cell count of Th1 and Th17 (Fig. 5). This suggests that the inhibition of Th1 and Th17 cell proliferation by TAK-242 treatment may partially or signi cantly contribute to the inhibition of peripheral arthritis in SKG mice.

Discussion
TAK-242, a small molecule inhibitor of TLR4 signaling was developed for the treatment of sepsis. TAK-242 binds selectively to TLR4 and interferes with the interactions between TLR4 and its adaptor molecules [25]. However, TAK-242 failed in phase III clinical human trials for severe sepsis [7]. Another TLR4 antagonist, eritoran, also failed in clinical studies for severe sepsis [26]. We previously showed that the TLR4 inhibitor TAK-242 (resatorvid) inhibited arthritic disease in an AIA rat model [6]. In this study, we tested the potential anti-arthritis effect of TAK-242 in a curdlan-injected SKG mouse model with activated and proliferating autoimmune T cells. As shown in Fig. 1, curdlan injection induces severe in ammation in foot and ileum. TAK-242 treatment signi cantly inhibited the in ammation, reduced the increased ankle and increased the reduced ileum length.
SpA is chronic in ammatory disease, and SpA patients commonly have chronic gut in ammation. The links between bowel and joints could be explained by immunological mechanisms in which activated T cells migrate from gut into synovium. The migrated immune cells secrete excess pro-in ammatory cytokines such as IL-17 and IL-23 [27,28]. Thus, we examined the expression of IL-17 in foot and ileum by IL-17 immunostaining. The expression level of IL-17 in foot was decreased by the treatment of TAK-242 as well as anti-IL-23 antibody. However, the expression in ileum was not signi cantly inhibited by anti-IL-23 antibody, contrary to our expectations. The IL-23/IL-17 axis was shown to play important role in the pathogenesis of SpA through genetic, experimental and clinical studies [29]. IL-23 stimulates various cells to produce IL-17, which contributes to in ammation by increasing the production of in ammatory cytokines such as IL-6 and TNF-α. However, there is an inconsistent result against the hypothesis. For example, the IL-17A inhibitor ixekizumab effectively inhibited axial SpA. In contrast, inhibition of IL-12/IL-23p40 and IL-23p19 both failed to inhibit axial SpA but inhibited in ammatory bowel disease (IBD) which was associated with failed IL-17 inhibition in previous studies [30]. As shown in Fig. 3B, we examined cytokines such as IL-17A, TNF-α, IL-6, IL-2, IL-10, IFN-γ and IL-1β in serum. TAK-242 treatment also decreased the increased serum level of TNF-α and IL-6 in curdlan-induced in ammation. Only TNF-α and IL-6 were detectable in serum. Other cytokines may have altered levels of expression but were not measured.
As shown in Fig. 4, BMD was signi cantly decreased in femurs of SKG mice with curdlan-induced SpA. In ammation may induce osteoporosis, as it does to many other chronic in ammatory diseases such as rheumatoid arthritis, psoriasis, ankylosing spondylitis, systemic lupus erythematosus, multiple sclerosis, in ammatory bowel diseases, pemphigus vulgaris and others [31]. Bone loss is tightly related to immune system activation or in amm-aging in the form of acute, chronic active or chronic blazing in ammation.
Increased pro-in ammatory cytokines could stimulate osteoclastogenesis. Brie y, TNF-α increases the expression of RANKL in osteoblasts, which induces osteoclast differentiation and stimulates bone resorbing activity [32]. TAK-242 treatment decreased systematic in ammation, which could contribute to inhibition of bone loss.
TLR activation is involved in the pathogenesis of arthritic diseases [1]. TL4 signaling in T cells seems to be important to induce autoimmune in ammation [33]. In particular, when TLR4 was knocked out in only CD4 + T cells, the disease symptoms were almost completely blocked. The suppression of disease symptoms was primarily through Th17 reduction, with fewer Th1 responses, suggesting that Th17 and Th1 cells are more important to induce autoimmune diseases than other cells. Likewise, activation of TLRs is involved in T cell development and differentiation. In particular, T cell receptor (TCR) activation in distinct T cell subsets with different TLRs could lead to different results. For example, TLR4 activation in regulatory T cells (Tregs) enhances the suppressive function of the cells, while TLR6 activation inhibits the suppressive function of Tregs [23]. In addition, TLR-2 and TLR-4 agonists induced signi cant cell proliferation and increased the production of IL-6, IL-17 and IL-21 from CD4 + T cells in neuromyelitis optica spectrum disorder patients, a severe humoral autoimmune disease of the central nervous system [24]. Thus, TLR4 signaling in T cells also plays an important role in promoting autoimmune in ammation through direct regulation of T cell activation and survival [25]. Therefore, TAK-242 treatment could inhibit the activated TLR4 signaling pathway of T cells in curdlan-induced in ammation. As shown in Fig. 5, TAK-242 treatment inhibited the increased level of Th1 and Th17 cells. The inhibition degree of TAK-242 was different from that of anti-IL-23 antibody treatment. Furthermore, TLR4 signaling is closely related to activation of STAT3 as a key event of TLR4 activation [34], which regulates the differentiation of CD4 + T cells into Th17 cells [35,36]. Therefore, targeting the STAT3 signaling pathway could be a promising therapeutic strategy for immune diseases as well as numerous cancers [37,38]. Novel STAT3 inhibitors are being developed [38,39]. These inhibitors could be tested as a treatment for arthritic disease in the future.

Conclusion
We previously demonstrated that TAK-242 has an anti-arthritic effect in AIA rats through the inhibition of NF-κB and AP-1 activation. In this study, we further established the anti-arthritic effect of TAK-242 in curdlan-injected SKG mice through the inhibition of proliferating Th1 and Th17 cells. To extend TLR4 inhibitors to clinical trials for patients with arthritic diseases, the mechanisms of the therapeutic effects should be clari ed in various arthritis animal models. However, some patients may be resistant to existing drugs and thus new drugs need to be developed for patients with lower responsiveness to existing drugs. The TLR4 inhibitor TAK-242 could have potential to be developed as a therapeutic agent against arthritic diseases.