Fatostatin ameliorates inflammation without affecting cell viability

The mature form of sterol regulatory element‐binding protein (SREBP)1 is a transcription factor involved in lipid synthesis, which participates in toll like receptor 4‐triggered inflammatory pathways during the resolution phase of inflammation in macrophages. SREBP1 has thus attracted interest as a candidate target molecule for ameliorating inflammation. Fatostatin is a small molecule that inhibits the maturation and function of SREBP, and its role in regulating inflammation is poorly understood. To evaluate the anti‐inflammatory effect of fatostatin, we compared body weight, footpad and hock dimensions, and arthritis scores between K/BxN serum‐induced arthritis mice treated with fatostatin and those treated with dimethyl sulfoxide as the vehicle control. We performed hematoxylin and eosin staining of joints of distal paws to assess tissue inflammation. Moreover, inflammatory cytokine production levels and cell viability were measured in lipopolysaccharide‐responsive human embryonic kidney 293 cells (293/hTLR4A‐MD2‐CD14 cells) after fatostatin administration. In K/BxN serum‐induced arthritis mice, fatostatin treatment significantly reduced the arthritis scores and hyperplasia. In vitro analysis revealed that fatostatin significantly inhibited the secretion of inflammatory cytokines from cells activated with lipopolysaccharide, without affecting cell viability. This is the first study to demonstrate that fatostatin is an anti‐inflammatory agent that modulates the processing of lipid transcription factors without affecting cell viability. Accordingly, the study reveals the potential of anti‐inflammatory therapeutics that link lipid regulation and inflammation.

The mature form of sterol regulatory element-binding protein (SREBP)1 is a transcription factor involved in lipid synthesis, which participates in toll like receptor 4-triggered inflammatory pathways during the resolution phase of inflammation in macrophages. SREBP1 has thus attracted interest as a candidate target molecule for ameliorating inflammation. Fatostatin is a small molecule that inhibits the maturation and function of SREBP, and its role in regulating inflammation is poorly understood. To evaluate the anti-inflammatory effect of fatostatin, we compared body weight, footpad and hock dimensions, and arthritis scores between K/BxN serum-induced arthritis mice treated with fatostatin and those treated with dimethyl sulfoxide as the vehicle control. We performed hematoxylin and eosin staining of joints of distal paws to assess tissue inflammation. Moreover, inflammatory cytokine production levels and cell viability were measured in lipopolysaccharide-responsive human embryonic kidney 293 cells (293/ hTLR4A-MD2-CD14 cells) after fatostatin administration. In K/BxN serum-induced arthritis mice, fatostatin treatment significantly reduced the arthritis scores and hyperplasia. In vitro analysis revealed that fatostatin significantly inhibited the secretion of inflammatory cytokines from cells activated with lipopolysaccharide, without affecting cell viability. This is the first study to demonstrate that fatostatin is an anti-inflammatory agent that modulates the processing of lipid transcription factors without affecting cell viability. Accordingly, the study reveals the potential of antiinflammatory therapeutics that link lipid regulation and inflammation.
The transcription factors sterol regulatory elementbinding proteins (SREBPs) and liver X receptors (LXRs) regulate fatty acid homeostasis and lipid synthesis [1]. Importantly, these transcription factors can also have antagonistic effects on immune responses [2].
of lipid metabolism, SREBP2 increases cellular cholesterol synthesis [5], whereas activation of LXRs increases cholesterol efflux from macrophages [6], and overexpression of SREBP-1a causes fatty liver in vivo [4]. Concerning their effects on immune responses, LXRs inhibit inflammation by reducing the activation of pro-inflammatory transcription factors in macrophages [1], whereas SREBP1 promotes inflammation by increasing the transcription of interleukin-1 beta (IL-1b) [7]. However, during the resolution phase of inflammation, nuclear factor-kappa B activation leads to a decrease in LXR expression and, instead, SREBP1 suppresses the production of proinflammatory cytokines [8]. Additionally, SREBP1 upregulates the expression of omega-3 fatty acids and 9Z palmitoleic acid regulators, which bind G-protein coupled receptors and mediate anti-inflammatory effects on macrophages [1,9].
The SREBP precursor in the endoplasmic reticulum is escorted by sterol regulatory element-binding protein cleavage-activating protein (SCAP) to the Golgi apparatus. At its membrane binding site on the Golgi apparatus, SREBP is modified by Site-1 protease (S1P) and Site-2 protease (S2P), and the N-terminus of the protein migrates to the nucleus to function as a transcription factor for the sterol regulatory element (Fig. 1) [10]. The small-molecule fatostatin competes with SREBP for SCAP, and thus interferes with the translocation of the SREBP precursor from the endoplasmic reticulum to the Golgi apparatus, thereby preventing the maturation and function of SREBP [10,11].
Remarkably, compared to sterols, fatostatin has a different binding site and a distinct effect on the SCAP function [11]. In addition, fatostatin inhibits cell proliferation in a lipid-and SCAP-independent manner [12].
However, the ability of fatostatin to regulate inflammation is not well understood.
In the present study, we aimed to ascertain the role of fatostatin in regulating inflammation. The effect of fatostatin on inflammation was studied in a K/BxN serum-induced arthritis mouse model. The K/BxN serum-induced arthritis model can be triggered only with injection of serum from arthritic transgenic K/BxN mice [13][14][15]. Because no immunostimulatory application is needed [15], inflammation occurs more naturally. The expression levels of Lxrb, Srebp1a and Srebp1c of K/BxN serum-induced arthritis mice were quantified in the presence or absence of fatostatin treatment. The scores for hematoxylin and eosin (H&E)-stained sections of the joints from each group were compared. To determine the effect of fatostatin on cell proliferation or survival rate, we also performed in vitro experiments using 293/hTLR4A-MD2-CD14 cells, comprising HEK293 cells modified to elicit an inflammatory response to lipopolysaccharide (LPS).

Materials and methods
In vivo experiments for SREBP1 inhibition

K/BxN serum-induced arthritis mouse model and fatostatin treatment
Six-week-old BALB/c male mice (n = 20) were used in the present study. A 14:10 h light/dark photocycle was used, and the supplementation of food and water was conducted in accordance with the instructions of Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University. Food was replenished and the cage was cleaned once a week, and the water supply was provided via a nozzle in each cage set into a shelf. With a floor area of Fig. 1. Molecular mechanism for fatostatin with respect to inhibiting production of the matured form of SREBP. Fatostatin targets SCAP and binds to a site that is distinct from the sterol-binding domain. The SREBP precursor in the endoplasmic reticulum is translocated by SCAP to the Golgi apparatus. At its membrane-binding site on the Golgi apparatus, SREBP is cleaved by S1P and S2P, and the Nterminus of the protein migrates to the nucleus to function as a transcription factor that binds to the sterol regulatory element). 451 cm 2 , each cage contained five mice, in accordance with the instructions of National Research Council [16]. All mice were intraperitoneally injected with 30 µL of K/BxN mouse serum, as described previously [13,14]. The injection day was designated as day 0. Mice were then divided into two groups: control and fatostatin-treated, and each group involved 10 mice. Mice from the control group were administered 80 µL of DMSO (#043-07216; FUJIFILM Wako Pure Chemicals Corp, Osaka, Japan), whereas those from the fatostatin group were injected with 0.6 mg of fatostatin (#13562; Cayman, Ann Arbour, MI, USA) in 80 µL of DMSO. Both groups were administered their respective injections for 3 days. To reduce suffering, intraperitoneal injections were performed as infrequently as possible.

Sample collection and arthritis score evaluation
From days 0 to 10, mice were weighed, their footpad and hock dimensions were measured, and arthritis scores were defined as 0 = no inflammation, 1 = minimal inflammation, 2 = mild inflammation, 3 = moderate inflammation and 4 = severe inflammation using the method defined by previous studies [17,18]. On day 12, mice were killed by cervical dislocation, and splenic F4/80 positive cells, blood plasma and the distal limbs were collected from each mouse.
Spleens of mice were homogenized using a 70-µm cell strainer and 2-mL syringe plungers, and thereafter centrifuged at 620 g for 3 min. The cell pellet was washed twice with Hanks' balanced salt solution (#14170112; Thermo Fisher, Waltham, MA, USA). To obtain F4/80 positive cells, the pellets were dissolved in a solution of 0.5 M EDTA in phosphate-buffered saline containing 0.1% BSA and centrifuged at 300 g for 10 min before being subjected to magnetic activated cell sorting, with anti-F4/80 MicroBeads UltraPure (#130-110-443; Miltenyi Biotec, Bergisch Gladbach, Germany). Distal limbs of the mice were detached using animal surgery scissors for phenotypic analysis via H&E staining of sections. Blood was collected by cardiac blood sampling and then centrifuged at 1200 g for 30 min at 4°C to isolate the plasma. Triglyceride and cholesterol concentrations in the blood plasma of mice were measured by SRL, Inc. (Tokyo, Japan).

Experimental methods
Quantitative real-time polymerase chain reaction RNA was isolated using Isogen (#315-02504; Nippon Gene, Tokyo, Japan) and the RNeasy Mini kit (#74104; Qiagen, Hilden, Germany). After DNase treatment (#M6101; Promega, Madison, WI, USA), the complementary DNA was prepared using an iScript cDNA Synthesis Kit (#1708890; Bio-Rad, Hercules, CA, USA), in accordance with the manufacturer's instructions. A quantitative real-time PCR (qRT-PCR) was performed using TB Green (Takara, Kusatsu, Japan) in a 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Conditions for qRT-PCR were: one cycle for the initial denaturation stage at 95°C for 30 s, 70 cycles for the PCR stage with denaturation at 95°C for 10 s and annealing at 60°C for 34 s, and the setting for the melt curve stage was in accordance with the instructions of the Takara TB Green for 7500 Real-Time PCR System. qRT-PCR primers for mouse samples are listed in Table 1 [20][21][22].

Gene
Forward Sequences AGATGACCACGATGTAGGCAG [22] cell lysate with radioimmunoprecipitation assay buffer (#08714-04; Nacalai Tesque, Kyoto, Japan). The protein concentration was measured using a BCA protein assay kit (#23227; Thermo Scientific) at 562 nm absorbance in conjunction with a NanoDrop 1000 Spectrophotometer (Thermo Scientific). To measure the SREBP1 levels in the cytoplasm, 12 lg of protein was applied for each sample and 5 lg of protein was applied for each sample to measure the levels of beta-actin in the cytoplasm. The gel was run for 45 min at 220 V. The membrane was washed six times with 1 9 Tris-buffered saline with 0.1% Tween Ò 20 detergent (0.05% Tween 20 in 1 9 Tris-buffered saline) and incubated with Bullet Blocking One for western blotting (#13779-14; Nacalai Tesque) at 23°C-25°C for 5 min. After washing the membrane six times, it was incubated with primary antibodies (SREBP1: clone, 2A4, #MA5-16124; Thermo Fisher; beta-actin: clone, 8H10D10, #3700; Cell Signaling Technology, Danvers, MA, USA), which were diluted 500 times and 15 000 times in Blocking One solution (#03953-95; Nacalai Tesque) at 23-25°C for 2-2.5 h with rotation. After six washes, the membrane was incubated with a conjugated secondary antibody diluted 3000 times and 10 000 times in Blocking One solution (#03953-95; Nacalai Tesque) at 23-25°C for 40 min. After six washes, the membrane was developed using SuperSignal (#34577; Thermo Scientific) for 5 min and exposed to ultraviolet light for 180 s using a Che-miDoc MP system (Bio-Rad). Images were analyzed using IMAGE LAB, version 4.0 software (Bio-Rad) and IMAGEJ (NIH, Bethesda, MD, USA).

Ethical declaration
The study was approved by the Animal Research Committee of the Graduate School of Medicine, Kyoto University, Kyoto, Japan (MedKyo 20115). All experimental procedures were performed in accordance with the ethical guidelines of the Kyoto University. The study was performed following COPE guidelines and was in compliance with ARRIVE guidelines 2.0 (https://arriveguidelines.org/arriveguidelines).

Results
Fatostatin has beneficial effects on mice with K/ BxN serum-induced arthritis Figure 2A shows a schematic diagram for fatostatin treatment and sample collection from K/BxN mice. Briefly, K/BxN serum and fatostatin were both administered via intraperitoneal injections. From days 0 to 10, body weight, footpad and hock dimensions, and arthritis scores of each mouse were evaluated. On day 12, splenic F4/80 positive cells, blood plasma and  2B) for mice from the fatostatin group were significantly lower than those of mice from the control group, which were administered only DMSO. The arthritis scores were assigned according to the scoring method defined in previous studies [17,18]. The histology of wild-type mice before arthritis induction can be found in a prior study [23], although representative histological analysis with H&E-staining also revealed severe hyperplasia in the DMSO-treated group (Fig. 2C) compared to the fatostatin-treated group (Fig. 2D). Although lymphocyte infiltration did not demonstrate any significance between the two groups ( Table 2), different infiltration patterns can be seen in Fig. 2C,D, where examples of lymphocyte models were provided in the previous literature [24,25]. Notably, all mice in the control group developed hyperplasia, whereas four mice from the fatostatin group did not (P = 0.0325, Fisher's exact test) ( Table 2).
To determine whether fatostatin affected the expression of key transcription factors related to lipid regulation, their expression in splenic F4/80 positive cells was monitored via qRT-PCR analysis. Moreover, Lxrb (Nr1h2) expression was significantly downregulated in the fatostatin group (P = 0.0288) (Fig. 2E) and Srebp1a and Srebp1c were expressed at lower levels in the fatostatin-treated group, although a significant difference was not detected among them (P = 0.2177 and 0.3527, respectively). However, fatostatin did not affect plasma triglyceride or cholesterol concentrations (Fig. 2F,G).

Fatostatin reduces cell proliferation in a dosedependent manner
Because fatostatin-treated mice showed lower levels of hyperplasia compared to the DMSO-treated mice, we next determined whether fatostatin treatment could affect cell division. Using LPS-responsive HEK293 cells, we investigated whether fatostatin can improve inflammation in cells expressing SREBPs in addition to Table 2. Mouse hind paw arthritis scores, determined using H&E-stained sections. 'N' refers to DMSO-treated mice and 'F' refers to fatostatin-treated mice. The scores were defined as: 0 = no inflammation, 1 = minimal inflammation, 2 = mild inflammation, 3 = moderate inflammation and 4 = severe inflammation. '1' and '0' indicate whether hyperplasia, cell infiltration into the bone marrow and vessels in stroma were observed or not in each H&E-stained section. P values for lymphocyte infiltration and fibrosis score were determined by Mann -Whitney U-tests. The P value for hyperplasia, cell infiltration into the bone marrow and vessels in stroma were determined by Fisher's exact test. Bold indicates statistical significance (P < 0.05).

Lymphocyte infiltration
Fibrosis score Lining hyperplasia Bone marrow infiltration Vessels in stroma monocytes and macrophages. To confirm whether fatostatin inhibits SREBP maturation, aliquots of cell lysates from HEK293 cells, which were cultured with 0, 10 and 20 lM fatostatin (in 0.5% DMSO) for 44 h, were used for western blot analysis (Fig. 3A). The other cells cultured under the same conditions as for the western blot were treated with CFSE for the cell proliferation assay (Fig. 3B). The SREBP1 antibody used in the western blot targets both the mature form (60-70 kDa) and the precursor form (125 kDa) of SREBP1 and we measured and quantified the bands at 125 kDa in four individual experiments. As shown in Fig. 3C,D, an increase in the concentration of fatostatin was associated with an increase in the intensity of the bands for the SREBP precursor, indicating that fatostatin reduced the maturation of SREBP. This was also confirmed by another study in which fatostatin was shown to block lipid synthesis by inhibiting the activation of SREBP [11]. To determine the effect of fatostatin on cell proliferation, CFSE analysis of HEK293 cells treated with different concentrations of fatostatin was performed (Fig. 3E). Red, blue, yellow and green histograms represent 0, 1, 2 and 3 divisions, respectively. Evidently, fatostatin treatment reduced cell proliferation in a dose-dependent manner.
Fatostatin treatment suppresses inflammation without affecting cell viability

Discussion
The present study suggests that treatment with fatostatin ameliorated inflammation in K/BxN serum-induced arthritis mice. Our in vitro experiments demonstrated that fatostatin treatment significantly reduced the release of IL-8 and contributed to the amelioration of inflammation without affecting cell viability. The inflammatory roles of SREBP1 and SREBP2 in macrophages have been reported elsewhere [1,26,27] and our study has demonstrated that the severity of inflammation in joints was decreased in K/BxN mice treated with fatostatin, as shown in Fig. 2B. However, the mechanism shown in Fig. 1 is only observed in the cultured cell models and does not necessarily reflect the results of the animal model used in the present study. Although the results of Fig. 2B and Table 2 may contradict each other with respect to the significance of the improvement, Table 2 indicates a localized change compared to that of Fig. 2B and may not reflect the average outcome. In H&E staining, lymphocyte infiltration was not affected by fatostatin, probably as a result of its non-rigorous quantitative nature, although the significantly ameliorated hyperplasia highlighted the effects of fatostatin in inflammation.
To further evaluate the role of fatostatin in inflammation, we performed in vitro experiments using 293/ hTLR4A-MD2-CD14 cells, comprising HEK293 cells modified to respond to LPS. Regarding the influence of fatostatin on cell division, the results shown in Fig. 3E contradict those of Fig. 4B. However, it is plausible that, during the 48 h of pre-cultivation for the ELISA experiments (time 0-48 h in Fig. 4A), cell proliferation was just before the plateau, such that the fatostatin-dependent suppression of cell division may not have an effect on cell number. Thus, it can be suggested that inhibition of the mature form of SREBP with certain concentrations of fatostatin suppresses inflammation without disturbing cell proliferation. On the other hand, fatostatin significantly reduced the expression of Lxrb in splenic F4/80 positive cells from K/BxN mice (Fig. 2E), without affecting triglyceride and cholesterol levels in the blood plasma (Fig. 2F,G). A plausible explanation for this observation may be that the blood plasma levels of triglycerides and cholesterol were conserved as a result of homeostasis. In splenic F4/80 positive cells, the downregulation of Lxrb expression may be associated with that of Srebp1c because SREBP1C can be directly activated by LXRs [28]. However, the precise mechanism remains to be clarified.
It has been demonstrated that fatostatin can inhibit the activation of both SREBP1 and SREBP2, which belong to the SREBP family [11]. SREBP2 has been reported to cause an increase in the synthesis of cellular cholesterol [5] and was shown to bind inflammatory and interferon response target genes to promote  [26]. Because our qRT-PCR data on SREBP2 analysis in a previous study (unpublished data but contributed by Shuhe Ma) did not demonstrate any significant expression, the levels of the SREBP1 precursor were measured by western blotting (Fig. 3C,D) and we observed an increase of the SREBP1 precursor in the cytoplasm after fatostatin treatment. It should be noted that, because no induction of SREBP1 precursor was involved, the precursor levels were not significantly increased as a result of inhibition of endoplasmic reticulum-to-Golgi translocation or by S1P/S2P downstream proteolytic cleavage as a result of fatostatin application, which was consistent with our findings shown in Fig. 3D. We also tested IL-6 and CXCL10 expression levels in splenic macrophages other than SREBP1A, SREBP1C and LXRb (data not shown); however, they did not show any significant differences compared to the DMSO group. This might be because fatostatin specifically functioned on cells in certain kinds of inflammatory environment in vivo.
The present in vitro study further demonstrates that fatostatin ameliorates inflammation without affecting cell viability. IL-8 was tested because IL-8 is a better nuclear factor-kappa B activation indicator than IL-6 in HEK293, and the response to TLR4 stimulation in 293/ hTLR4A-MD2-CD14 cells can be satisfactorily evaluated by IL-8. However, we cannot exclude the possibility that the off-target effects of fatostatin have not eliminated in the present study; thus, experiments utilizing Srefp1 knockout mice, as well as those evaluating the effects of fatostatin on inflammation of human arthritic tissue, should be performed in future studies.

Conclusions
Treating K/BxN arthritis mice with fatostatin leads to an amelioration of inflammation. Furthermore, fatostatin caused a significant reduction in inflammatory cytokine production in LPS-responsive HEK293 cells. The constant cell survival rate between the fatostatintreated and non-treated groups suggests its potential role as an anti-inflammatory agent, which links lipid regulation and inflammation.